ABSTRACT
At the onset of lactation in dairy cows, inflammation and oxidative stress may occur and result in a risk of pathologies and lower milk yield. To propose an innovative management strategy for cows during this period, it is essential to better understand these physiological variations. Our objective was to evaluate the metabolic, redox and immune status of 7 primiparous and 8 multiparous Holstein cows during late gestation and the first months of lactation. Blood samples were collected between 3 weeks before calving until 12 weeks postpartum. Milk samples were also collected, but only at the time points after calving. The metabolic (nonesterified fatty acids (NEFA), BHB, glucose, urea, calcium) and redox (reactive oxygen metabolites (ROM), oxidative stress index (OSI), glutathione peroxidase activity, vitamin E) statuses were analyzed in plasma or erythrocytes. The expression of genes related to antioxidant functions was determined in leukocytes collected from milk. For immune status, plasma cytokine levels and the production of reactive oxygen species (ROS) in classical and regulatory neutrophils were measured in 2 whole blood ex vivo challenges. The data were analyzed using a mixed model that included the fixed effects of parity and week and their interaction. Milk yield, plasma NEFA and BHB in wk 2 and 4 after calving were higher in multiparous cows than in primiparous cows, whereas glucose and calcium tended to be lower. Plasma ROM and OSI levels in wk 8 were higher in multiparous than in primiparous cows. Multiparous cows also displayed higher glutathione peroxidase activity in erythrocytes, and antioxidant transcription factor and superoxide dismutase-1 expression levels in milk leukocytes. Moreover, multiparous cows had higher plasma concentrations of vitamin E but lower plasma levels of cytokines CXCL10, CCL2, IL1Rα and IFNγ. Following ex vivo whole blood stimulation with Escherichia coli, lower IL1α and TNFα levels were measured in multiparous than in primiparous cows. Intracellular ROS production by neutrophils was lower in multiparous than in primiparous cows. These results thus indicated marked physiological changes in wk 8 compared with wk 2 and 4 of lactation. These differences in the physiological status of primiparous and multiparous cows offer interesting perspectives for potential dietary strategies to prevent pathologies which take account of parity and week relative to calving.
ABSTRACT
Mastitis is among the main reasons women cease breastfeeding, which leads to them supplementing breast milk with artificial formula. In farm animals, mastitis results in significant economic losses and the premature culling of some animals. Nevertheless, researchers do not know enough about the effect of inflammation on the mammary gland. This article discusses the changes to DNA methylation in mouse mammary tissue caused by lipopolysaccharide-induced inflammation (4 h post-injection of lipopolysaccharide). We analysed the expression of some genes related to mammary gland function, epigenetic regulation, and the immune response. The analysis focused on three comparisons: inflammation during the first lactation, inflammation during second lactation with no history of inflammation, and inflammation during second lactation with previous inflammation. We identified differentially methylated cytosines (DMCs), differentially methylated regions (DMRs), and some differentially expressed genes (DEGs) for each comparison. The three comparisons shared some DEGs; however, few DMCs and only one DMR were shared. These observations suggest that inflammation is one of several factors affecting epigenetic regulation during successive lactations. Furthermore, the comparison between animals in second lactation with and without inflammation, with no inflammation history during first lactation showed a different pattern compared to the other conditions in this experiment. This indicates that inflammation history plays an important role in determining epigenetic changes. The data presented in this study suggest that lactation rank and previous inflammation history are equally important when explaining mammary tissue gene expression and DNA methylation changes.Abbreviations: RRBS, reduced representation bisulfite sequencing; RT-qPCR, real-time quantitative polymerase chain reaction; MEC, mammary epithelial cells; TSS, transcription start site; TTS, transcription termination site; UTR, untranslated region; SINE, short interspersed nuclear element; LINE, long interspersed nuclear element; CGI, CpG island; DEG, differentially expressed gene; DMC, differentially methylated cytosine; DMR, differentially methylated region; GO term, gene ontology term; MF, molecular function; BP, biological process.
Subject(s)
DNA Methylation , Mastitis , Humans , Female , Mice , Animals , Epigenesis, Genetic , Lipopolysaccharides/toxicity , Lactation/genetics , Mastitis/genetics , Gene ExpressionABSTRACT
This study aimed to determine the effect of lipopolysaccharide (LPS)-induced mastitis with or without nonsteroidal anti-inflammatory drug (NSAID) on dairy cows' clinical, physiological, and behavioral responses in the milking parlor and freestalls as well as the specificity (Sp) and sensitivity (Se) of behavioral responses in detecting cows with LPS-induced mastitis. Twenty-seven cows received an intramammary infusion of 25 µg of Escherichia coli LPS in 1 healthy quarter. Following LPS infusion, 14 cows received a placebo (LPS cows), and 13 cows received 3 mg/kg of body weight of ketoprofen i.m. (LPS+NSAID cows). Cow response to the challenge was monitored at regular intervals from 24 h before to 48 h postinfusion (hpi) through direct clinical observations, markers of inflammation in milk, and via point-in-time direct behavioral observations in the barn and at milking. In LPS cows, infusion induced a significant increase of plasma cortisol levels at 3 and 8 hpi, milk cortisol levels at 8 hpi, somatic cell counts from 8 to 48 hpi, IL-6 and IL-8 at 8 hpi, milk amyloid A (mAA) and haptoglobin at 8 and 24 hpi, rectal temperature at 8 hpi, and respiratory rate at 8 hpi. Their rumen motility rate decreased at 8 and 32 hpi. Compared with before the challenge, significantly more LPS cows stopped feeding/ruminating and pressed their tail between their legs at 3 and 5 hpi, increased feeding/ruminating at 24 hpi, and had the tendency to be less responsive, dropping their head, and dropping their ears at 5 hpi. At milking, compared with before challenge, significantly more LPS cows lifted their hooves at forestripping at 8 hpi. The 2 groups showed similar patterns of response for milk cortisol, somatic cell count, respiratory rate, mAA, haptoglobin, and IL-6, IL-1ß, and IL-8. Compared with LPS cows, LPS+NSAID cows had significantly lower plasma cortisol levels at 3 hpi, their rectal temperature decreased at 8 hpi, their rumen motility rate increased at 8 and 32 hpi, and their heart rate increased at 32 hpi. Compared with LPS cows, a significantly larger proportion of LPS+NSAID cows were feeding/ruminating, a lower proportion had ears down at 5 hpi, and a larger proportion lied down at 24 hpi. At milking, whatever the phase of milking, for "hoof to belly," 9 out of 14 cows did not show this behavior before infusion (Sp = 64%) and 14/14 did not kick during pre-infusion milking (Sp = 100%). Regarding sensitivity, at maximum, 5 cows out of 14 (Se = 36%) displayed "hoof to belly" after infusion. For "lifting hoof," 14/14 did not show hoof-lifting before infusion (Sp = 100%) and 6/14 displayed it after infusion (Se = 43%) at forestripping only. In the freestall barn, 9 behaviors had a Sp >75% (at minimum, 10/14 did not show the behavior) whatever the time point but Se < 60% (at maximum, 8/14 displayed the behavior). Finally, "absence of feeding and ruminating" had Sp of 86% (12/14 ate/ruminated) and Se of 71% (10/14 did not eat/ruminate) at 5 hpi. This study shows that feeding/ruminating, tail position, and reactivity at forestripping could be used as behavioral indictors for early detection of mastitis-related pain in dairy cows.
Subject(s)
Cattle Diseases , Mastitis, Bovine , Female , Cattle , Animals , Lipopolysaccharides/pharmacology , Interleukin-8/adverse effects , Hydrocortisone , Haptoglobins , Behavior Observation Techniques , Interleukin-6 , Mastitis, Bovine/drug therapy , Pain/drug therapy , Pain/veterinary , Milk , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Escherichia coli , Serum Amyloid A Protein/therapeutic use , Cattle Diseases/drug therapyABSTRACT
Mastitis is among the main reasons women cease breastfeeding. In farm animals, mastitis results in significant economic losses and the premature culling of some animals. Nevertheless, the effect of inflammation on the mammary gland is not completely understood. This article discusses the changes to DNA methylation in mouse mammary tissue caused by lipopolysaccharide-induced inflammation after in vivo intramammary challenges and the differences in DNA methylation between 1st and 2nd lactations. Lactation rank induces 981 differential methylations of cytosines (DMCs) in mammary tissue. Inflammation in 1st lactation compared to inflammation in 2nd lactation results in the identification of 964 DMCs. When comparing inflammation in 1st vs. 2nd lactations with previous inflammation history, 2590 DMCs were identified. Moreover, Fluidigm PCR data show changes in the expression of several genes related to mammary function, epigenetic regulation, and the immune response. We show that the epigenetic regulation of two successive physiological lactations is not the same in terms of DNA methylation and that the effect of lactation rank on DNA methylation is stronger than that of the onset of inflammation. The conditions presented here show that few DMCs are shared between comparisons, suggesting a specific epigenetic response depending on lactation rank, the presence of inflammation, and even whether the cells had previously suffered inflammation. In the long term, this information could lead to a better understanding of the epigenetic regulation of lactation in both physiological and pathological conditions.Abbreviations: RRBS, reduced representation bisulphite sequencing; RT-qPCR, real-time quantitative polymerase chain reaction; MEC, mammary epithelial cells; MaSC, mammary stem cell; TSS, transcription start site; TTS, transcription termination site; UTR, untranslated region; SINE, short interspersed nuclear element; LINE, long interspersed nuclear element; CGI, CpG island; DEG, differentially expressed gene; DMC, differentially methylated cytosine; DMR, differentially methylated region; GO term, gene ontology term; MF, molecular function; BP, biological process.
Subject(s)
DNA Methylation , Mastitis , Female , Animals , Mice , Humans , Epigenesis, Genetic , Lactation , Inflammation , Cytosine , Gene ExpressionABSTRACT
The objective was to assess effects of experimentally induced undernutrition on responses to an intramammary lipopolysaccharide (LPS) challenge in early-lactation cows. Starting at 24 ± 3 d in milk, multiparous Holstein cows either received a ration containing 48% straw for 96 h to restrict nutrient intake (REST, n = 8) or were allowed ad libitum intake of a lactation diet (CONT, n = 9). After 72 h on diet or after an equivalent period for CONT, 50 µg of LPS (Escherichia coli 0111:B4) was injected into one healthy rear mammary quarter to induce an acute inflammation response. Blood samples were collected weekly until 7 wk of lactation, daily during feed restriction (or control), before and at 1, 2, 4, 6, 10, and 24 h relative to LPS injection. Foremilk quarter samples were collected before and at 4, 6, 10, and 24 h after LPS injection. Dry matter intake, milk yield, energy balance, plasma glucose, nonesterified fatty acids (NEFA), and ß-hydroxybutyrate (BHB) concentrations did not differ between CONT and REST immediately before nutrient restriction in REST (least squares means at d -1 were 21.8, 39.0 kg/d, -2.5 MJ/d, and 3.78, 0.415, 0.66 mM, respectively) but were significantly altered at 72 h of nutrient restriction (9.8, 28.3 kg/d, -81.6 MJ/d, and 2.77, 1.672, and 2.98 mM, respectively), when the LPS challenge was performed. The rectal temperature increment from baseline values in response to LPS did not differ, but cortisol increment was greater and cortisol response area under the curve (AUC) tended to be greater [202 vs. 122 (ng/mL) × 10 h] for REST than CONT. No treatment differences were observed in foremilk IL-8, IL-1ß, tumor necrosis factor-α, and chemokine (C-X-C motif) ligand 3 concentrations in response to LPS injection. Composite milk somatic cell count per milliliter (6.919 × 106 vs. 1.956 × 106 cells/mL) and total number of somatic cells secreted in milk per day were greater for REST than CONT during the day following LPS. Plasma glucose, urea, and insulin concentrations increased after the LPS challenge, suggesting establishment of insulin resistance and modifications of glucose metabolism to support acute inflammation in both CONT and REST. Nonetheless, nutrient-restricted cows had delayed plasma insulin and glucose responses to LPS, smaller insulin AUC but greater glucose AUC compared with CONT, despite the limited nutrient availability to sustain an inflammation response. Undernutrition altered peripheral metabolic responses to an intramammary LPS challenge but had limited effects on selected indicators of inflammation response in early-lactation cows.
Subject(s)
Energy Metabolism/drug effects , Inflammation/veterinary , Malnutrition/veterinary , Milk/metabolism , 3-Hydroxybutyric Acid/blood , Animals , Blood Glucose/drug effects , Cattle , Diet/veterinary , Eating/drug effects , Escherichia coli/chemistry , Fatty Acids, Nonesterified/blood , Female , Inflammation/chemically induced , Inflammation/metabolism , Insulin/blood , Insulin Resistance , Lactation/drug effects , Lipopolysaccharides/adverse effects , Malnutrition/metabolismABSTRACT
An extended milking interval of 24 h (24-h milking interval (24h-MI)) constitutes the acute phase of cow adaptation to once-daily milking (ODM). A recent trial including 724 24h-MI challenges demonstrated that milk yield responses to this acute phase of ODM are highly variable (from+22% to -52% of milk yield when switching to the 24h-MI, mean=-25.3%) and that factors such as stage of lactation parity and milk yield level influenced cows' responses but did not account for all individual variability. Additional traits related to physiological, immune and behavioural adaptation were measured on a subset (96 observations) of this data set. This study aimed to determine (1) the relationship of these traits with cows' milk yield responses, (2) their ability - combined with previously identified traits - to help predict milk yield responses to 24h-MI (adaptive profiles). The 24h-MI challenge consisted of three successive periods: one control week of twice-daily milking (cTDM), one single day of 24h-MI and then 13 days of TDM (pTDM). Milk yield responses to the 24h-MI (corrected for effects of stage of lactation, parity, milk yield level and milk yield) were related to physiological traits measured during cTDM (milk flow rate, presence or absence of interleukin-8) and to their changes during the 24h-MI (absolute increase in milk flow rate and relative udder distension). Analysis of associations between milk yield responses, stage of lactation, parity, milk yield level, proteolysis, udder expansion and immune traits found three adaptive cow profile clusters. Cows in cluster 1 had a less compliant udder than cows in cluster 2, and they lost more milk during the 24h-MI than cluster-2 and cluster-3 cows. After resuming twice daily-milking (TDM), cluster-2 cows fully recovered the milk they had lost during the 24h-MI. On the opposite, cluster-3 cows did not recover the milk they lost, likely due to udder inflammation during cTDM, as suggested by elevated concentrations of interleukin-8 in their milk. These results combining new traits with stage of lactation, parity and milk yield level constitute a first step towards predicting individual cow responses to a 24h-MI.
Subject(s)
Cattle/physiology , Dairying/methods , Interleukin-8/metabolism , Mammary Glands, Animal/physiology , Milk/chemistry , Animals , Cattle/immunology , Female , Mammary Glands, Animal/immunology , Mastitis, Bovine/immunology , Proteolysis , Time FactorsABSTRACT
Bovine mastitis, an inflammatory disease of the mammary gland often associated to bacterial infection, is the first cause of antibiotic use in dairy cattle. Because of the risk of antibioresistance emergence, alternative non-antibiotic strategies are needed to prevent or to cure bovine mastitis and reduce the antibiotic use in veterinary medicine. In this work, we investigated Lactococcus lactis V7, a strain isolated from the mammary gland, as a probiotic option against bovine mastitis. Using bovine mammary epithelial cell (bMEC) culture, and two representative strains for Escherichia coli and for Staphylococcus aureus, two major mastitis pathogens, we investigated L. lactis V7 ability to inhibit cell invasion (i.e. adhesion and internalization) of these pathogens into bMEC. L. lactis V7 ability to modulate the production of CXCL8, a key chemokine IL-8 responsible for neutrophil influx, in bMEC upon challenge with E. coli was investigated by an ELISA dosage of CXCL8 in bMEC culture supernatants. We showed that L. lactis V7 inhibited the internalisation of both E. coli and S. aureus strains into bMEC, whereas it inhibited the adhesion of only one out of the two S. aureus strains and of none of the E. coli strains tested. Investigation of the bMEC immune response showed that L. lactis V7 alone induced a slight increase in CXCL8 production in bMEC and that it increased the inflammatory response in bMEC challenged with the E. coli strains. Altogether these features of L. lactis V7 make it a potential promising candidate for a probiotic prevention strategy against bovine mastitis.
Subject(s)
Antibiosis , Bacterial Adhesion , Endocytosis , Epithelial Cells/microbiology , Escherichia coli/physiology , Lactococcus lactis/physiology , Staphylococcus aureus/physiology , Animals , Cattle , Cell Line , Enzyme-Linked Immunosorbent Assay , Interleukin-8/analysis , Mastitis, Bovine/prevention & control , Mastitis, Bovine/therapy , Probiotics/pharmacologyABSTRACT
Mastitis pathogens belonging to Escherichia coli species are often considered as environmental opportunistic pathogens that invade the udder and are rapidly killed by the immune system of cows. However, several studies have reported that some of these strains are able to persist in the udder for prolonged periods or to adhere and invade mammary epithelial cells, suggesting that they might possess some specific properties or genes that could be involved in their capacity to provoke mastitis. The aim of this work was to search for such specific genes in the E. coli strain P4, which was isolated from a case of severe mastitis and is often used to induce experimental mastitis. We established that this strain belongs to phylogenetic group A of the E. coli species, and that its core genome is very similar to that of the commensal nonpathogenic strain E. coli K-12 MG1655. Seventeen transfer RNA loci, known to be frequently associated with genomic islands, were screened and an altered structure was detected for 7 of them. The partial characterization of 5 of these loci (asnT, leuX, pheV, serU, and thrW) and the complete characterization of 1 (argW) revealed the presence of genomic islands that differ from those already described in pathogenic or nonpathogenic E. coli strains.
Subject(s)
Escherichia coli/classification , Escherichia coli/genetics , Genomic Islands , Mastitis, Bovine/microbiology , Animals , Cattle , Escherichia coli/isolation & purification , Female , Genome, Bacterial , Mammary Glands, Animal/microbiology , Mice , Models, Animal , Phylogeny , RNA, Transfer/genetics , Sequence Analysis, DNA , Sequence Analysis, ProteinABSTRACT
The M protein is an important surface-located virulence factor of Streptococcus pyogenes, the group A streptococcus (GAS). Expression of M protein is primarily controlled by Mga, a transcriptional activator protein. A recent report suggested that the sag locus, which includes nine genes necessary and sufficient for production of streptolysin S, another GAS virulence factor, is also needed for transcription of emm, encoding the M protein (Z. Li, D. D. Sledjeski, B. Kreikemeyer, A. Podbielski, and M. D. Boyle, J. Bacteriol. 181:6019-6027, 1999). To investigate this in more detail, we constructed an insertion-deletion mutation in sagA, the first gene in the sag locus, in the M6 strain JRS4. The resulting strain, JRS470, produced no detectable streptolysin S and showed a drastic reduction in cell surface-associated M protein, as measured by cell aggregation and Western blot analysis. However, transcription of the emm gene was unaffected by the sagA mutation. Detailed analysis with monoclonal antibodies and an antipeptide antibody showed that the M protein in the sagA mutant strain was truncated so that it lacks the C-repeat region and the C-terminal domain required for anchoring it to the cell surface. This truncated M protein was largely found, as expected, in the culture supernatant. Lack of surface-located M protein made the sagA mutant strain susceptible to phagocytosis. Thus, although sagA does not affect transcription of the M6 protein gene, it is needed for the surface localization of this important virulence factor.
Subject(s)
Antigens, Bacterial , Bacterial Outer Membrane Proteins/metabolism , Bacterial Proteins , Carrier Proteins/metabolism , Streptococcus pyogenes/metabolism , Streptolysins/metabolism , Bacterial Outer Membrane Proteins/biosynthesis , Carrier Proteins/biosynthesis , Cell Membrane/metabolism , Cysteine Endopeptidases/metabolism , Humans , Mutagenesis, Insertional , Phagocytosis/immunology , Sequence Deletion , Serotyping , Streptococcus pyogenes/genetics , Streptococcus pyogenes/immunology , Streptolysins/geneticsABSTRACT
TolQ, TolR, and TolA inner membrane proteins of Escherichia coli are involved in maintaining the stability of the outer membrane. They share homology with the ExbB, ExbD, and TonB proteins, respectively. The last is involved in energy transduction between the inner and the outer membrane, and its conformation has been shown to depend on the presence of the proton motive force (PMF), ExbB, and ExbD. Using limited proteolysis experiments, we investigated whether the conformation of TolA was also affected by the PMF. We found that dissipation of the PMF by uncouplers led to the formation of a proteinase K digestion fragment of TolA not seen when uncouplers are omitted. This fragment was also detected in Delta tolQ, Delta tolR, and tolA(H22P) mutants but, in contrast to the parental strain, was also seen in the absence of uncouplers. We repeated those experiments in outer membrane mutants such as lpp, pal, and Delta rfa mutants: the behavior of TolA in lpp mutants was similar to that observed with the parental strain. However, the proteinase K-resistant fragment was never detected in the Delta rfa mutant. Altogether, these results suggest that TolA is able to undergo a PMF-dependent change of conformation. This change requires TolQ, TolR, and a functional TolA N-terminal domain. The potential role of this energy-dependent process in the stability of the outer membrane is discussed.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/physiology , Escherichia coli Proteins , Membrane Proteins/chemistry , Periplasmic Proteins , Proteoglycans , Proton-Motive Force/physiology , Bacterial Outer Membrane Proteins/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carrier Proteins/genetics , Endopeptidase K/metabolism , Escherichia coli/metabolism , Escherichia coli/physiology , Lipoproteins/genetics , Membrane Proteins/metabolism , Membrane Proteins/physiology , Mutagenesis , Peptidoglycan/genetics , Protein ConformationABSTRACT
The Tol-Pal system of Escherichia coli is involved in maintaining outer membrane stability. Mutations in tolQ, tolR, tolA, tolB, or pal genes result in sensitivity to bile salts and the leakage of periplasmic proteins. Moreover, some of the tol genes are necessary for the entry of group A colicins and the DNA of filamentous bacteriophages. TolQ, TolR, and TolA are located in the cytoplasmic membrane where they interact with each other via their transmembrane domains. TolB and Pal form a periplasmic complex near the outer membrane. We used suppressor genetics to identify the regions important for the interaction between TolB and Pal. Intragenic suppressor mutations were characterized in a domain of Pal that was shown to be involved in interactions with TolB and peptidoglycan. Extragenic suppressor mutations were located in tolB gene. The C-terminal region of TolB predicted to adopt a beta-propeller structure was shown to be responsible for the interaction of the protein with Pal. Unexpectedly, none of the suppressor mutations was able to restore a correct association between Pal and peptidoglycan, suggesting that interactions between Pal and other components such as TolB may also be important for outer membrane stability.
Subject(s)
Bacterial Outer Membrane Proteins/genetics , Bacterial Proteins/genetics , Escherichia coli Proteins , Escherichia coli/genetics , Lipoproteins/genetics , Peptidoglycan/genetics , Periplasmic Proteins , Proteoglycans , Repressor Proteins/metabolism , Amino Acid Sequence , Molecular Sequence Data , Mutagenesis , Protein Binding , Sequence AlignmentABSTRACT
The Tol proteins of Escherichia coli are involved in outer membrane stability. They are also required for the uptake of the group A colicins and the translocation of filamentous phage DNA into the cytoplasm. The tol-pal genes constitute two operons in the E. coli genome, orfltolQRA and tolBpalorf2. The TolQ TolR TolA proteins form a complex in the cytoplasmic membrane, while TolB and Pal interact near the outer membrane. Most of the amino acid residues of TolA, TolB, TolR and Pal are localized in the periplasm. Recent advances in the knowledge of interactions of Tol-Pal proteins with other envelope components, or with group A colicins, are presented, together with current hypotheses about the role of the Tol proteins in outer membrane stability.
Subject(s)
Bacterial Proteins/metabolism , Colicins/metabolism , DNA, Viral/metabolism , Escherichia coli/physiology , Membrane Proteins/metabolism , Biological Transport , Lipoproteins/metabolism , Macromolecular Substances , Peptidoglycan/metabolismABSTRACT
The Tol-Pal proteins of Escherichia coli are involved in maintaining outer membrane integrity. They form two complexes in the cell envelope. Transmembrane domains of TolQ, TolR, and TolA interact in the cytoplasmic membrane, while TolB and Pal form a complex near the outer membrane. The N-terminal transmembrane domain of TolA anchors the protein to the cytoplasmic membrane and interacts with TolQ and TolR. Extensive mutagenesis of the N-terminal part of TolA was carried out to characterize the residues involved in such processes. Mutations affecting the function of TolA resulted in a lack or an alteration in TolA-TolQ or TolR-TolA interactions but did not affect the formation of TolQ-TolR complexes. Our results confirmed the importance of residues serine 18 and histidine 22, which are part of an SHLS motif highly conserved in the TolA and the related TonB proteins from different organisms. Genetic suppression experiments were performed to restore the functional activity of some tolA mutants. The suppressor mutations all affected the first transmembrane helix of TolQ. These results confirmed the essential role of the transmembrane domain of TolA in triggering interactions with TolQ and TolR.
Subject(s)
Bacterial Proteins/genetics , Escherichia coli Proteins , Escherichia coli/genetics , Membrane Proteins/genetics , Suppression, Genetic , Amino Acid Sequence , Bacterial Proteins/metabolism , Binding Sites , Escherichia coli/metabolism , Membrane Proteins/metabolism , Molecular Sequence Data , MutagenesisABSTRACT
The Tol-Pal proteins of Escherichia coli are involved in maintaining outer membrane integrity. Transmembrane domains of TolQ, TolR and TolA interact in the cytoplasmic membrane, while TolB and Pal form a complex near the outer membrane. TolB and the central domain of TolA interact in vitro with the outer membrane porins. In this study, both genetic and biochemical analyses were carried out to analyse the links between TolB, Pal and other components of the cell envelope. It was shown that TolB could be cross-linked in vivo with Pal, OmpA and Lpp, while Pal was associated with TolB and OmpA. The isolation of pal and tolB mutants disrupting some interactions between these proteins represents at first approach to characterizing the residues contributing to the interactions. We propose that TolB and Pal are part of a multiprotein complex that links the peptidoglycan to the outer membrane. The Tol-Pal proteins might form transenvelope complexes that bring the two membranes into close proximity and help some outer membrane components to reach their final destination.
Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Bacterial Proteins/metabolism , Carrier Proteins/metabolism , Escherichia coli Proteins , Escherichia coli/metabolism , Lipoproteins/metabolism , Membrane Proteins/metabolism , Peptidoglycan/metabolism , Periplasmic Proteins , Proteoglycans , Amino Acid Sequence , Bacterial Outer Membrane Proteins/biosynthesis , Bacterial Outer Membrane Proteins/genetics , Bacterial Proteins/genetics , Cross-Linking Reagents , Escherichia coli/genetics , Lipoproteins/genetics , Molecular Sequence Data , Mutagenesis , Peptidoglycan/genetics , Sequence Homology, Amino AcidABSTRACT
Rhodococcus strains were screened for their ability to degrade the herbicide atrazine. Only rhodococci that degrade the herbicide EPTC (s-ethyl-dipropylthiocarbamate) metabolized atrazine. Rhodococcus strain TE1 metabolized atrazine under aerobic conditions to produce deethyl- and deisopropylatrazine, which were not degraded further and which accumulated in the incubation medium. The bacterium also metabolized the other s-triazine herbicides propazine, simazine, and cyanazine. The N dealkylation of triazine herbicides by Rhodococcus strain TE1 was associated with a 77-kb plasmid previously shown to be required for EPTC degradation.
Subject(s)
Atrazine/metabolism , Rhodococcus/metabolism , Bacteriological Techniques , Biodegradation, Environmental , Culture Media/chemistry , Plasmids , Rhodococcus/geneticsABSTRACT
During a three-year period, complete revascularization of diffusely diseased left anterior descending (LAD) coronary arteries was accomplished by extensive endarterectomy in conjunction with bypass grafting in 37 patients in whom conventional bypass was not feasible. This group constituted 7.0% of all patients undergoing nonemergency coronary revascularization during this period. The left internal mammary artery was used to bypass the endarterectomized LAD artery in 22 patients. There was 1 (2.7%) operative death and 1 perioperative myocardial infarction. At follow-up, which was 100% with a mean of 41.4 months, all endarterectomy patients were in New York Heart Association Functional Class I or II. Twenty-four endarterectomy patients underwent first-pass radionuclide angiographic stress testing 20 months after operation. Twenty patients (83%) had excellent postoperative exercise tolerance, achieving 5 to 7 mets on treadmill testing. Left ventricular functional reserve was preserved, as evidenced by an increase of global ejection fraction from 48 +/- 15% at rest to 59 +/- 18% (p less than 0.005) with exercise. A similar increase was measured in the proximal and distal anterior wall segmental ejection fractions. No difference in response to exercise was found between the internal mammary artery and the vein graft groups. Thus, complete revascularization of the diffusely diseased LAD artery can be accomplished by adjunct endarterectomy without added morbidity or mortality and with excellent functional results.
Subject(s)
Coronary Disease/surgery , Coronary Vessels/surgery , Endarterectomy/methods , Adult , Aged , Cardioplegic Solutions/administration & dosage , Coronary Artery Bypass , Coronary Disease/diagnostic imaging , Coronary Disease/physiopathology , Coronary Vessels/diagnostic imaging , Coronary Vessels/physiopathology , Evaluation Studies as Topic , Exercise Test , Female , Follow-Up Studies , Humans , Hypothermia, Induced , Male , Middle Aged , Myocardial Revascularization , Radionuclide Imaging , Recurrence , Saphenous Vein/transplantation , Sodium Pertechnetate Tc 99m , Stroke VolumeABSTRACT
Forty consecutive asymptomatic patients with chronic aortic regurgitation who underwent three serial yearly rest and postexercise radionuclide angiograms were compared with 27 consecutive patients with chronic aortic regurgitation and aortic valve replacement who were studied preoperatively, 3 and 15 months postoperatively. Patients were divided into four subgroups based upon the resting left ventricular ejection fraction and the functional reserve on the initial study. Of the 40 medically treated patients, 19 (47.5%) and 24 (60%) demonstrated a response at least one type lower at 12 months and 24 months, respectively. Initial functional reserve, initial duration of exercise, and the change in exercise duration during the 24 months was not associated with changes in resting or postexercise left ventricular ejection fraction. A seesaw pattern was observed between the resting and the postexercise left ventricular ejection fraction as ventricular function deteriorated. We observed in the surgical groups a reversal of the seesaw interaction between the resting and postexercise ejection fraction seen in the medical patients. In the surgical groups the left ventricular end-diastolic pressure, initial functional reserve, initial duration of exercise, and change in exercise duration postoperatively were not predictors of improvement in left ventricular function at 15 months. Comparing medical and surgical serial data, we suggest yearly radionuclide angiographic determination of rest left ventricular ejection fraction in asymptomatic patients with chronic aortic regurgitation. When the rest ejection fraction is less than 50%, exercise angiography should be performed to determine functional reserve. When functional reserve is also abnormal, surgery should be recommended.
Subject(s)
Aortic Valve Insufficiency/diagnostic imaging , Cardiac Output , Heart Valve Prosthesis , Myocardial Contraction , Physical Exertion , Adolescent , Adult , Aged , Aortic Valve Insufficiency/surgery , Child , Chronic Disease , Coronary Circulation , Female , Heart Rate , Heart Ventricles/diagnostic imaging , Humans , Male , Middle Aged , Postoperative Complications/diagnostic imaging , Radionuclide Imaging , Sodium Pertechnetate Tc 99mABSTRACT
Employing rest and exercise first-pass radionuclide angiography before and 3 months after surgery, we studied patients with hemodynamically stable left ventricular aneurysm (LVA) undergoing both coronary artery bypass surgery to relieve angina pectoris and elective aneurysmectomy. There were 15 patients, 14 men and 1 woman with a mean age of 54 +/- 7 years. All patients had anterior and/or apical LVA. After surgery the postexercise mean left ventricular ejection fraction (LVEF) for the whole group improved significantly (p less than 0.004) compared with the preoperative value, but the resting LVEF did not change. The duration of exercise improved (p less than 0.01) after surgery, but not the double product. However, based upon the preoperative LVEF response to exercise, two groups were seen: Group A (n = 5) had greater than or equal to 5% increase in their LVEF with exercise versus Group B (n = 10), who had less than 5% increase or a decrease in their LVEF. Postoperatively, Group A decreased their LVEF with exercise and failed to improve exercise capacity or double product. Postoperatively, Group B increased the LVEF by greater than or equal to 5% as well as increasing exercise capacity (p less than 0.01), and double product (p less than 0.03). Group A had lower preoperative LVEF than Group B (p less than 0.01) and larger LVA. Patients with hemodynamically stable LVA who require coronary artery bypass surgery for angina should not have aneurysmectomy. The presence of hemodynamically stable LVA is not a contraindication to deriving benefit from myocardial revascularization.
Subject(s)
Heart Aneurysm/surgery , Myocardial Contraction , Adult , Aged , Angina Pectoris/surgery , Cardiac Output , Coronary Artery Bypass , Electrocardiography , Exercise Test , Female , Heart Ventricles/surgery , Humans , Male , Middle Aged , Myocardial Infarction/surgery , Postoperative Complications/diagnosisABSTRACT
First pass radionuclide angiography (RA) was used to assess its ability to detect and quantitate the presence of left-sided regurgitation due to isolated mitral regurgitation (MR). In this study the nuclear regurgitant fraction (NRF) obtained from first pass RA was correlated with the results of contrast ventriculography (CV) in 50 patients, 18 with and 32 without MR. The correlation between CV and RA in the confirmation and quantification of isolated MR revealed a sensitivity of 100 percent and a specificity of 97 percent in patients with LVEF greater than or equal to 35 percent.
Subject(s)
Mitral Valve Insufficiency/diagnostic imaging , Adult , Aged , False Positive Reactions , Female , Heart Ventricles/diagnostic imaging , Heart Ventricles/physiopathology , Humans , Male , Middle Aged , Mitral Valve Insufficiency/physiopathology , Radiography , Radionuclide Imaging , Stroke VolumeABSTRACT
To evaluate right ventricular function following mitral valve replacement, we studied 84 patients with isolated mitral valve disease with the use of first-pass radionuclide angiography before, 1 week after, and up to 1 year after operation. The right ventricular ejection fraction for the entire group improved from 29% +/- 11% to 43% +/- 10% (p less than 0.001) at 1 week. This increase was maintained at 3 months (41% +/- 10%) and up to year after operation (40% +/- 12%). The improvement was found not to be influenced by either the type of valvular lesion or the presence and/or level of pulmonary hypertension. When the patients were grouped according to the type of prosthetic valve placed at operation, the right ventricular ejection fraction increased in all patients within 1 week of operation, with sustained improvement at 3 months postoperatively. Thereafter, it began to decline in patients receiving a Carpentier bioprosthesis while being maintained in those patients who received disc valves. Further analysis revealed that those patients who receiving the larger Carpentier bioprostheses had a greater deterioration of right ventricular function than those receiving the smaller Carpentier valves. Left ventricular function in the entire group was normal preoperatively (62% +/- 16%) and was unchanged at 1 week (60% +/- 16%) and a 1 year (59% +/- 16%) after operation.