Temporal and spatial heterogeneity in milk and immune-related gene expression during mammary gland involution in dairy cows.

The aim of this study was to investigate heterogeneity in tissue morphology, milk protein and immune-related gene expression, and apoptosis of epithelial cells in the lactating and involuting mammary glands of the dairy cow. Mammary tissue from different regions of the gland (alveolar, cisternal, and peripheral) was collected postmortem from nonpregnant, pasture-fed, Holstein-Friesian primiparous cows in mid-lactation that were killed at different time points postmilking: 0, 6, 12, 18, 24, 36, and 72 h (n = 6 per time point). The CSN1NS1 and LALBA mRNA was decreased in alveolar, cisternal, and peripheral tissue by 12 to 36 h postmilking. In contrast, lactoferrin (LF) and mammary serum amyloid A3 (M-SAA3) mRNA was increased in these regions by 36 to 72 h. During lactation, more variability was present in gene expression in alveolar tissue between cows and between quarters within a cow, than within quarters. Histological analysis indicated the alveolar tissue from lactating cows was mostly uniform in structure; however, in situ hybridization indicated that although most of the alveolar tissue expressed milk proteins, the level of expression varied within and between alveoli. This heterogeneity became more pronounced with involution and with increasing regions of alveoli expressing lactoferrin, indicating that alveoli enter involution asynchronously. The peripheral and cisternal tissue had more variability in gene expression between cows compared with the alveolar tissue. The M-SAA3 signal was more intense in the cisternal tissue and less intense than the peripheral compartment compared with LF particularly in the earlier time points. In addition, between cows within the later time points, differences were observed in tissue morphology, the levels of milk protein and immune-related gene expression, and phosphorylated signal transducer and activator of transcription (STAT) 5-P and STAT3-P proteins, and degree of apoptosis, indicating that involution of the mammary gland occurs at different rates between cows. Understanding the mechanisms initiating the process of involution of the mammary gland provides an opportunity for enhancing milk production of the dairy cow.

Cell survival signaling in the bovine mammary gland during the transition from lactation to involution.

In dairy cows, mammary gland involution, and thus a decline in milk production, occurs following peak lactation. To examine the cell signaling pathways regulating involution of the mammary gland, signal transducer and activator of transcription factors (STAT5 and 3), suppressors of cytokine signaling (SOCS1-3 and CIS), insulin-like growth factors (IGF1 and 2), and protein kinase B (Akt) were examined. Mammary involution was induced by termination of milking, and alveolar tissue was collected from 52 nonpregnant, primiparous, mid-lactation Holstein-Friesian cows killed at 0, 6, 12, 18, 24, 36, 72, and 192h postmilking. Qualitative immunohistochemistry showed that activated (phosphorylated) STAT5-P was localized in nuclei of mammary epithelial cells at the early time points, with detection levels decreasing by 24h postmilking. In contrast, STAT3-P was barely detectable at the early time points, with detection levels increasing following longer postmilking periods. This was supported by Western analysis, which showed a decline in STAT5 and STAT5-P protein levels by 24h postmilking, no change in STAT3 levels, and an increase in STAT3-P protein (barely detectable at the early time points) by 72h postmilking. Quantitative real-time reverse transcription PCR analysis showed SOCS1 and SOCS3 mRNA increased by 72h postmilking compared with 6h postmilking. The SOCS2 mRNA remained unchanged across the time series, whereas CIS decreased by 18h postmilking and remained lower compared with that at 6h postmilking until 72h postmilking. The IGF1 mRNA increased by 192h postmilking, whereas IGF2 mRNA decreased by 18h postmilking compared with 6h postmilking. The IGFBP5 mRNA and protein levels of Akt and Akt-P remained unchanged over the time series. These results show that reciprocal activation of STAT5 and STAT3 occurs at the onset of mammary gland involution in the bovine, albeit at a slower rate than in rodents. Mathematical modeling of the pathways indicated that activated STAT3 could block the STAT5 pathway by upregulating SOCS3. The regulation of IGF1-Akt signaling suggests that by 192h postmilking in dairy cows, the involution process is still in the reversible phase, with quiescent mammary epithelial cells not yet in the senescent phase.

cDNA microarray analysis reveals that antioxidant and immune genes are upregulated during involution of the bovine mammary gland.

We have used cDNA microarray analysis to identify genes that play a role in bovine mammary involution. Involution was induced by termination of milking, and alveolar tissue was collected from 48 nonpregnant Friesian cows in mid lactation sacrificed at 0, 6, 12, 18, 24, 36, 72, and 192 h (n = 6/group) postmilking. The most highly upregulated genes were those associated with oxidative stress. Quantitative real-time reverse-transcription PCR analysis confirmed that mRNA expression of spermidine/spermine N(1)-acetyltransferase was increased by 24 h, superoxide dismutase 2 and metallothionein 1A by 36 h, and glutathione peroxidase by 72 h postmilking. The mRNA expression of the host defense proteins lactoferrin and lingual antimicrobial peptide were increased by 192 h postmilking. A dramatic increase in the protein expression of lactoferrin by 192 h postmilking was also detected by Western analysis. Decreased mRNA expression of the milk protein genes alpha(S1)-, beta-, and kappa-casein, and alpha-lactalbumin were early events in the process of involution occurring within 24 to 36 h postmilking, whereas beta-lactoglobulin mRNA was decreased by 192 h postmilking. Decreases in alpha-lactalbumin and beta-lactoglobulin protein levels in alveolar tissue occurred by 24 and 192 h postmilking, respectively, and the cell survival factors beta1-integrin and focal adhesion kinase were decreased by 72 and 192 h postmilking, respectively. The results demonstrate that in the bovine mammary gland, decreased milk protein gene expression and cell survival signaling are associated with multiple protective responses to oxidative stress that occur before the induction of immune responses and mammary epithelial cell apoptosis during involution.

Bovine parotid secretory protein: structure, expression and relatedness to other BPI (bactericidal/permeability-increasing protein)-like proteins.

Members of the family of BPI (bactericidal/permeability-increasing protein)-like proteins are as yet incompletely characterized, particularly in cattle, where full-length sequence information is available for only three of the 13 family members known from other species. Structural bioinformatic analyses incorporating bovine homologues of several members of the BPI-like protein family, including two forms of bovine parotid secretory protein (PSP), showed that this family is also present in cattle. Expression analyses of several members of the BPI-like protein family in cattle, including PSP (Bsp30), von Ebner's minor salivary gland protein (VEMSGP) and lung-specific X protein (LUNX), showed a restricted pattern of expression, consistent with earlier hypotheses that these proteins function in the innate immune response to bacteria. The possible role of bovine PSP in susceptibility to pasture bloat in cattle is discussed.

Behaviour of laying hens in two types of aviary systems on 25 commercial farms in Sweden.

1. Fifty-one flocks of laying hens in two high-density loose-housing systems were studied on 25 commercial farms in Sweden as part of a government test programme for evaluating new systems for laying hens. Six different hybrids were used in group sizes ranging from 250 to 5 000 birds. Stocking-densities varied from 10.2 to 19.1 birds per m2 floor area. No birds were beak trimmed. 2. The distribution of birds in the system, the frequency and location of aggressive pecks and feather pecks, the dust bathing activity and the birds' fear reaction to the keeper and to a novel object were measured. Direct behaviour observations were carried out twice per flock, at weeks 35 and 55. 3. The proportion of birds at the different locations was relatively constant across the 8-h observation period in the tiered system, but changed over time in the perch system, which may reflect a difference in access to resources between the systems. At night the top perches/tiers were preferred although when stocking-density increased, other sites were also used. 4. Aggression occurred mainly on the litter or in the nest areas. It did not differ between hybrids, but increased with age in the tiered system. Feather pecks occurred mainly on the litter. Brown hybrids feather pecked more than white ones, while white hybrids reacted more both to the keeper and to a novel object than did the brown hybrids. 5. It was concluded that access to nests was insufficient in both systems, as was litter space. Feed space was insufficient in the tiered system if food requirements increased. Design of the top perches, in the perch system, should be improved to allow birds to perch high up in the system without blocking access to feed etc. for others.

Space use and agonistic behaviour in relation to sex composition in large flocks of laying hens.

Some authors have found indications of subgroup formation when domestic fowl are forced to live together in large flocks, while others have not. In this study experiments were carried out to test the hypothesis that hens in large flocks have home ranges in parts of the pen and that they form subgroups. We also studied if this is influenced by males. In a tiered aviary system (density averaged 16 hens/m(2) of floor area) eight flocks of 568+/-59 ISA Brown laying hybrids were kept in pens. Half of the pens contained 1 male per on average 24 females (mixed flocks). At peak production (36-53 weeks of age) four females roosting closely together for about 14 days and four females roosting far apart from each other were taken out from each flock and put together in separate groups in small pens. Their agonistic behaviour was studied for 2 days before they were put back. This was repeated with new birds, resulting in 16 small sample groups being studied. At 70 weeks, three groups of 10 females per flock roosting closely together in different parts of the pen were dyed with different colours and their locations were observed for 2 nights and 2 days.The incidence of aggressive pecks during day 1 among birds that had been roosting close to each other tended to be lower (P=0.05) than among birds that had been roosting far apart. This effect was not significant among birds from all-female flocks, but among birds from mixed flocks (P<0.05). However, this indicates a recognition of roosting partners and possibly also a rebound effect of the males' reduction of female aggressiveness towards strangers. Irrespective of sex composition in the flocks, birds marked while roosting at the ends of the pens were significantly more often observed within these areas than in other areas of the pen during daytime and came back to the same roosting sites at night (P<0.05-P<0.001). This was not the case for birds from the middle of the pens, where the distribution in the pen in most cases did not differ from random. These results show that laying hens in large groups are rather constant in their use of space, which indicate the presence of home ranges. However, environmental features that facilitate localisation may be important. In summary, we think that these findings indicate the existence of subgroup formation.

Impaired awareness and employment outcome after traumatic brain injury.

Inaccurate self-awareness is a common finding after traumatic brain injury. Such impaired awareness has been hypothesized to limit patients' eventual functional outcomes by decreasing motivation for treatment and resulting in selection of inappropriate long-term goals. Previous investigations of the association between impaired awareness and employment outcome have produced inconsistent results. The present article reviews these studies and presents the results of our new investigation of this issue. In addition, we studied the comparability of two methods of measuring impaired awareness. Results provided strong support for a positive relationship between accurate self-awareness and favorable employment outcome at follow-up.

Rehabilitation of the post-cardiac surgery stroke patient: analysis of cognitive and functional assessment.

OBJECTIVE: Determine whether, as expected, patients sustaining post-cardiac surgery stroke (PCS) (n = 19) differ from other stroke (OS) patients (n = 216). DESIGN: A total of 235 stroke patients were surveyed. Therapist ratings of Functional Independence Measure (FIM) on admission to and discharge from a rehabilitation unit were compared. Cooperation with formal neuropsychologic evaluation was assessed. SETTING: The rehabilitation unit of a tertiary care hospital. PARTICIPANTS: Medical records for consecutive stroke patients were reviewed (January 1994 to December 1995). Groups did not differ in age, gender, or admission FIM. INTERVENTIONS: Standardized neuropsychologic evaluation of seven cognitive domains was attempted for each patient referred to the neuropsychology service. All of the patients received FIM ratings on admission to and discharge from the rehabilitation unit. OUTCOME MEASURES: Gain in FIM per week of rehabilitation unit stay (FIM efficiency) and discharge destination. RESULTS: Contrary to expectations, PCS patients did not differ significantly from OS patients in FIM efficiency or discharge destination. However, PCS patients were significantly less able to cooperate with formal neuropsychologic testing, possibly secondary to their physical condition, higher-level cognitive deficits, or both. CONCLUSION: Although PCS patients may sustain medical and cognitive deficits that interfere with exhaustive neuropsychologic evaluation, these deficits do not significantly interfere with functional progress in rehabilitation and should not make PCS patients ineligible for rehabilitation services.

Assessment and treatment of impaired awareness after brain injury: implications for community re-integration.

Impaired self-awareness of deficits is a common finding in patients who have suffered traumatic brain injury. Impaired awareness can limit motivation for treatment and contribute to poor outcome. Consequently, it is important for brain injury rehabilitation professionals to understand this phenomenon and utilize treatment approaches that may improve patient awareness. The present article reviews the existing literature on measurement of impaired awareness, characteristics of impaired awareness, the relationship of impaired awareness to functional outcome, possible treatment approaches for impaired awareness and empirical investigations of interventions to improve awareness. The treatment strategies we use to address impaired awareness in our community re-integration program for brain injury survivors are described in detail. These approaches include: establishment of the therapeutic alliance, family interventions, peer feedback, education, roleplaying, videotape feedback, real world experiences, therapeutic milieu and psychotherapy.

Crystallization and preliminary X-ray diffraction analysis of the ArsC protein from the Escherichia coli arsenical resistance plasmid, R773.

Diffraction data to 3.0 A resolution were collected on crystals of ArsC protein from the conjugative resistance factor R773 which mediates arsenical resistance in the Gram negative bacterium Escherichia coli. The crystal system is tetragonal, a = 116.2 A, c = 145.0 A and the space group is either P4(1)2(1)2 or P4(3)2(1)2. The most probable range for the contents of the asymmetric unit is four to eight ArsC molecules (M(r) = 15,811).

Properties of the arsenate reductase of plasmid R773.

Resistance to toxic oxyanions in Escherichia coli is conferred by the ars operon carried on plasmid R773. The gene products of this operon catalyze extrusion of antimonials and arsenicals from cells of E. coli, thus providing resistance to those toxic oxyanions. In addition, resistance to arsenate is conferred by the product of the arsC gene. In this report, purified ArsC protein was shown to catalyze reduction of arsenate to arsenite. The enzymatic activity of the ArsC protein required glutaredoxin as a source of reducing equivalents. Other reductants, including glutathione and thioredoxin, were not effective electron donors. A spectrophotometric assay was devised in which arsenate reduction was coupled to NADPH oxidation. The results obtained with the coupled assay corresponded to those found by direct reduction of radioactive arsenate to arsenite. The only substrate of the reaction was arsenate (Km = 8 mM); other oxyanions including phosphate, sulfate, and antimonate were not reduced. Phosphate and sulfate were weak inhibitors, while the product, arsenite, was a stronger inhibitor (Ki = 0.1 mM). Arsenate reductase activity exhibited a pH optimum of 6.3-6.8. These results indicate that the ArsC protein is a novel reductase, and elucidation of its enzymatic mechanism should be of interest.

Arsenate reduction mediated by the plasmid-encoded ArsC protein is coupled to glutathione.

Resistance to arsenate conferred on Escherichia coli by the ars operon of plasmid R773 requires both the product of the arsC gene and reduction of arsenate to arsenite. A genetic analysis was performed to identify the source of reducing potential in vivo. In addition to the ars genes, arsenate resistance required the products of the gor gene for glutathione reductase and the gshA and gshB genes for glutathione synthesis. Mutations in the trx and grx genes for thioredoxin and glutaredoxin, respectively, had no effect on arsenate resistance. Although resistance required the arsC gene, the rate of reduction of arsenate to arsenite was nearly the same in cells lacking the ars operon. In strains deficient in glutathione biosynthesis this endogenous reduction was greatly diminished, and cells exhibited increased sensitivity to arsenate. When glutathione was supplied exogenously to such mutants, resistance was restored only to cells expressing the ars operon, and only such cells had detectable arsenate reduction after addition of glutathione. Since ArsC-catalysed reduction of arsenate provides high level resistance, physical coupling of the ArsC reaction to efflux of the resulting arsenite is hypothesised.

Energetic efficiency of Escherichia coli: effects of mutations in components of the aerobic respiratory chain.

The aerobic respiratory chain of Escherichia coli can function with either of two different membrane-bound NADH dehydrogenases (NDH-1 and NDH-2) and with either of two ubiquinol oxidases (bd-type and bo-type). The amounts of each of these enzymes present in the E. coli membrane depend on growth conditions in general and particularly on the dissolved oxygen concentration. Previous in vitro studies have established that NDH-1 and NDH-2 differ in the extent to which they are coupled to the generation of an energy-conserving proton motive force. The same is true for the two ubiquinol oxidases. Hence, the bioenergetic efficiency of the aerobic respiratory chain must depend on the electron flux through each of the specific enzyme components which are being utilized. In this work, the specific rates of oxygen consumption for cells growing under glucose-limited conditions are reported for a series of isogenic strains in which one or more respiratory components are genetically eliminated. The results are compatible with the proton translocation values of the various components reported from in vitro measurements. The data show that (i) the bd-type oxidase is less efficient than is the bo-type oxidase, but the former is still a coupling site in the respiratory chain; and (ii) under the conditions employed, the wild-type strain uses both the NDH-1 and NDH-2 NADH dehydrogenases to a significant degree, but most of the electron flux is directed through the bo-type oxidase.

Isolation and characterization of a new class of cytochrome d terminal oxidase mutants of Escherichia coli.

Cytochrome d terminal oxidase mutants were isolated by using hydroxylamine mutagenesis of pNG2, a pBR322-derived plasmid containing the wild-type cyd operon. The mutagenized plasmid was transformed into a cyo cyd recA strain, and the transformants were screened for the inability to confer aerobic growth on nonfermentable carbon sources. Western blot analysis and visible-light spectroscopy were performed to characterize three independent mutants grown both aerobically and anaerobically. The mutational variants of the cytochrome d complex were stabilized under anaerobic growth conditions. All three mutations perturb the b595 and d heme components of the complex. These mutations were mapped and sequenced and are shown to be located in the N-terminal third of subunit II of the cytochrome d complex. It is proposed that the N terminus of subunit II may interact with subunit I to form an interface that binds the b595 and d heme centers.

Genomic replacement in Escherichia coli K-12 using covalently closed circular plasmid DNA.

A number of gene replacements at different loci were constructed using covalently closed circular (ccc) plasmid DNA in the recB21 recC22 sbcB15 sbcC201 mutant of Escherichia coli (JC7623). Selected constructs representing deletions and insertion mutations formed from double-crossover events involving the ccc plasmid molecules and the genome were confirmed by Southern blots, and the frequency of double-crossover events was evaluated. It is reported that such mutants may be constructed without linearizing plasmid DNA, as described previously.

S-adenosylmethionine synthetase in cultured normal and oncogenically-transformed human and rat cells.

We have investigated the enzymatic formation of S-adenosylmethionine in extracts of a variety of normal and oncogenically-transformed human and rat cell lines which differ in their ability to grow in medium in which methionine is replaced by its immediate precursor homocysteine. We have localized the bulk of the S-adenosylmethionine synthetase activity to the post-mitochondrial supernatant. We show that in all cell lines a single kinetic species exists in a dialyzed extract with a Km for methionine of about 3-12 microM. In selected lines we have demonstrated a requirement for Mg2+ in addition to that needed to form the Mg X ATP complex for enzyme activity and have shown that the enzyme can be regulated by product feedback inhibition. Because we detect no differences in the enzymatic ability of these cell extracts to utilize methionine for S-adenosylmethionine formation in vitro, we suggest that the failure of oncogenically-transformed cell lines to grow in homocysteine medium may result from the decreased methionine pools in these cells or from the loss of ability of these cells to properly metabolize homocysteine, adenosine, or their cellular product S-adenosylhomocysteine.

S-adenosyl-L-methionine synthetase from human erythrocytes: role in the regulation of cellular S-adenosylmethionine levels.

The properties of human erythrocyte S-adenosyl-L-methionine synthetase (ATP:L-methionine S-adenosyltransferase, EC 2.5.1.6) were studied with respect to the role of S-adenosylmethionine in transmethylation reactions. Kinetic values obtained with both a cytosolic and a 350-fold purified preparation of enzyme were compared with measured intracellular concentrations of substrates and products. This analysis revealed that effective regulation of enzyme activity and product concentration can occur through feedback inhibition by S-adenosylmethionine (Ki = 2.0-2.9 microM; the endogenous concentration is 3.5 microM). This enzyme can be distinguished from S-adenosylmethionine synthetases found in other tissues and appears to be specialized for its role in erythrocyte methyl group metabolism, especially with regard to protein carboxyl methyl-transfer reactions.

A minimum-stress procedure for repeated measurements of nociceptive threshold and analgesia.

A procedure was developed which allows repeated measurement of threshold responses to a thermal pain stimulus administered to an unrestrained animal. The apparatus consists of a metal plate whose temperature can be rapidly incremented and decremented. The device was tested with rats using two experimental procedures. In a titration procedure, temperature was raised gradually until the subject licked its paws. At this time, the temperature was gradually decreased, with the amount of decrement determined by the duration of the paw licking. In a discrete trials procedure, temperature was increased rapidly until paw licking occurred, and then returned rapidly to room temperature. Morphine reliably reduced the responses to heat. The effect was dose-dependent and diminished with repeated doses.