Quantitative encapsulation and retention of 227Th and decay daughters in core-shell lanthanum phosphate nanoparticles.

Targeted alpha therapy (TAT) offers great promise for treating recalcitrant tumors and micrometastatic cancers. One drawback of TAT is the potential damage to normal tissues and organs due to the relocation of decay daughters from the treatment site. The present study evaluates La(227Th)PO4 core (C) and core +2 shells (C2S) nanoparticles (NPs) as a delivery platform of 227Th to minimize systemic distribution of decay daughters, 223Ra and 211Pb. In vitro retention of decay daughters within La(227Th)PO4 C NPs was influenced by the concentration of reagents used during synthesis, in which the leakage of 223Ra was between 0.4 ± 0.2% and 20.3 ± 1.1% in deionized water. Deposition of two nonradioactive LaPO4 shells onto La(227Th)PO4 C NPs increased the retention of decay daughters to >99.75%. The toxicity of the nonradioactive LaPO4 C and C2S NP delivery platforms was examined in a mammalian breast cancer cell line, BT-474. No significant decrease in cell viability was observed for a monolayer of BT-474 cells for NP concentrations below 233.9 µg mL-1, however cell viability decreased below 60% when BT-474 spheroids were incubated with either LaPO4 C or C2S NPs at concentrations exceeding 29.2 µg mL-1. La(227Th)PO4 C2S NPs exhibit a high encapsulation and in vitro retention of radionuclides with limited contribution to cellular cytotoxicity for TAT applications.

Modular microfluidics for point-of-care protein purifications.

Biochemical separations are the heart of diagnostic assays and purification methods for biologics. On-chip miniaturization and modularization of separation procedures will enable the development of customized, portable devices for personalized health-care diagnostics and point-of-use production of treatments. In this report, we describe the design and fabrication of miniature ion exchange, size exclusion and affinity chromatography modules for on-chip clean-up of recombinantly-produced proteins. Our results demonstrate that these common separations techniques can be implemented in microfluidic modules with performance comparable to conventional approaches. We introduce embedded 3-D microfluidic interconnects for integrating micro-scale separation modules that can be arranged and reconfigured to suit a variety of fluidic operations or biochemical processes. We demonstrate the utility of the modular approach with a platform for the enrichment of enhanced green fluorescent protein (eGFP) from Escherichia coli lysate through integrated affinity and size-exclusion chromatography modules.

SAR216471, an alternative to the use of currently available P2Y12 receptor inhibitors?

P2Y12 antagonism is a key therapeutic strategy in the management and prevention of arterial thrombosis. The objective of this study was to characterize the pharmacodynamic (PD) and pharmacokinetic (PK) properties of SAR216471, a novel P2Y12 receptor antagonist. SAR216471 blocks the binding of 2MeSADP to P2Y12 receptors in vitro (IC50=17 nM). This inhibition was shown to be reversible. It potently antagonized ADP-induced platelet aggregation in human and rat platelet-rich plasma (IC50=108 and 62 nM, respectively). It also inhibited platelet aggregation when blood was exposed to collagen or thromboxane A2. Its high selectivity was demonstrated against a large panel of receptors, enzymes, and ion channels. Despite its moderate bioavailability in rats, oral administration of SAR216471 resulted in a fast, potent, and sustained inhibition of platelet aggregation where the extent and duration of platelet inhibition were directly proportional to its circulating plasma levels. Pre-clinical study of SAR216471 in a rat shunt thrombosis model demonstrated a dose-dependent antithrombotic activity after oral administration (ED50=6.7 mg/kg). By comparison, ED50 values for clopidogrel, prasugrel and ticagrelor were 6.3, 0.35 and 2.6 mg/kg, respectively. Finally, the anti-hemostatic effect of SAR216471 and its competitors was investigated in a rat tail bleeding model, revealing a favorable safety profile of SAR216471. Together, these findings have established a reliable antiplatelet profile of SAR216471, and support its potential use in clinical practice as an alternative to currently available P2Y12 receptor antagonists.

Past climate changes explain the phylogeography of Vitellaria paradoxa over Africa.

The evolution of the savanna biome has been deeply marked by repeated contraction/expansion phases due to climate perturbations during the Quaternary period. In this study, we investigated the impact of the last glacial maximum (LGM) on the present genetic pattern of Vitellaria paradoxa (shea tree), a major African savanna tree. A range-wide sampling of the species enabled us to sample 374 individuals from 71 populations distributed throughout sub-Sahelian Africa. Trees were genotyped using 3 chloroplasts and 12 nuclear microsatellites, and were sequenced for 2 polymorphic chloroplast intergenic spacers. Analyses of genetic diversity and structure were based on frequency-based and Bayesian methods. Potential distributions of V. paradoxa at present, during the LGM and the last interglacial period, were examined using DIVA-GIS ecological niche modelling (ENM). Haplotypic and allelic richness varied significantly across the range according to chloroplast and nuclear microsatellites, which pointed to higher diversity in West Africa. A high but contrasted level of differentiation was revealed among populations with a clear phylogeographic signal, with both nuclear (F(ST) = 0.21; R(ST) = 0.28; R(ST) > R(ST) (permuted)) and chloroplast simple sequence repeats (SSRs) (G(ST) = 0.81; N(ST) = 0.90; N(ST) > N(ST) (permuted)). We identified a strong geographically related structure separating western and eastern populations, and a substructure in the eastern part of the area consistent with subspecies distinction. Using ENM, we deduced that perturbations during the LGM fragmented the potential eastern distribution of shea tree, but not its distribution in West Africa. Our main results suggest that climate variations are the major factor explaining the genetic pattern of V. paradoxa.

Apelin/APJ signaling system: a potential link between adipose tissue and endothelial angiogenic processes.

Adipose tissue is an active endocrine organ that produces a variety of secretory factors involved in the initiation of angiogenic processes. The bioactive peptide apelin is the endogenous ligand of the G protein-coupled receptor, APJ. Here we investigated the potential role of apelin and its receptor, APJ, in the angiogenic responses of human endothelial cells and the development of a functional vascular network in a model of adipose tissue development in mice. Treatment of human umbilical vein endothelial cells with apelin dose-dependently increased angiogenic responses, including endothelial cell migration, proliferation, and Matrigel(R) capillary tubelike structure formation. These endothelial effects of apelin were due to activation of APJ, because siRNA directed against APJ, which led to long-lasting down-regulation of APJ mRNA, abolished cell migration induced by apelin in contrast to control nonsilencing siRNA. Hypoxia up-regulated the expression of apelin in 3T3F442A adipocytes, and we therefore determined whether apelin could play a role in adipose tissue angiogenesis in vivo. Epididymal white adipose tissue (EWAT) transplantation was performed as a model of adipose tissue angiogenesis. Transplantation led to increased apelin mRNA levels 2 and 5 days after transplantation associated with tissue hypoxia, as evidenced by hydroxyprobe staining on tissue sections. Graft revascularization evolved in parallel, as the first functional vessels in EWAT grafts were observed 2 days after transplantation and a strong angiogenic response was apparent on day 14. This was confirmed by determination of graft hemoglobin levels, which are indicative of functional vascularization and were strongly increased 5 and 14 days after transplantation. The role of apelin in the graft neovascularization was then assessed by local delivery of stable complex apelin-targeting siRNA leading to dramatically reduced apelin mRNA levels and vascularization (quantified by hemogloblin content) in grafted EWAT on day 5 when compared with control siRNA. Taken together, our data provide the first evidence that apelin/APJ signaling pathways play a critical role in the development of the functional vascular network in adipose tissue. In addition, we have shown that adipocyte-derived apelin can be up-regulated by hypoxia. These findings provide novel insights into the complex relationship between adipose tissue and endothelial vascular function and may lead to new therapeutic strategies to modulate angiogenesis.

Reversible biotinylated oligosaccharides: a new approach for a better management of anticoagulant therapy.

OBJECTIVE: In order to obtain a neutralizable antithrombotic, a chimeric molecule (SSR126517E) containing the sequence of a long-lasting antithrombin (AT)-dependent anti-factor Xa pentasaccharide, idraparinux, linked to a biotin molecule was synthesized and tested for anticoagulant and antithrombotic activity. METHODS: SSR126517E was tested in several models in vitro and in vivo for its pharmacological properties as well as its ability to be neutralized by avidin. RESULTS: SSR126517E displayed exactly the same properties as idraparinux. In vitro, SSR126517E had a very high affinity for AT (K(d) < 1 nm) and showed a potent anti-FXa effect and inhibition of thrombin generation with IC(50) values similar to those of idraparinux. Ex vivo, after intravenous administration to rats, SSR126517E produced a potent and long-lasting anti-FXa effect comparable to that obtained with idraparinux; as with idraparinux, the subcutaneous bioavailability was 100%. In vivo, SSR126517E was a potent antithrombotic in rat and mouse venous and arterial thrombosis models. Direct comparison in rats showed that SSR126517E was as active as idraparinux, when administered at the same molar dose. Furthermore, injection of avidin triggered the immediate elimination of SSR126517E from the bloodstream, resulting in complete neutralization of the antithrombotic activity of SSR126517E. CONCLUSIONS: These results show for the first time that coupling an oligosaccharide with biotin has no effect on the former's pharmacokinetic and pharmacologic properties and renders neutralization easy by injection of avidin.

Application of Single Strand Conformation Polymorphism --PCR method for distinguishing cheese bacterial communities that inhibit Listeria monocytogenes.

The aim of this study was to compare the microbial communities of different cheeses where Listeria monocytogenes either grew or did not grow. For this purpose, (i) isolates from the most inhibitory cheese ecosystem were identified and their ability to produce anti-Listeria substances was determined, (ii) bacterial communities of cheeses with and without L. monocytogenes growth were compared using the Single Strand Conformation Polymorphism method. The study showed SSCP to be an effective tool for differentiating between the bacterial communities of different cheeses manufactured with the same technology. All the cheeses with the lowest L. monocytogenes counts on day 8 were distinguished by the dominance in their SSCP profiles, after amplification of the V2 region of the 16S rRNA gene, of 3 peaks whose nucleotide sequences comigrated with Enterococcus faecium and Enterococcus saccharominimus, Chryseobacterium sp and Corynebacterium flavescens, Lactococcus garvieae and Lactococcus lactis respectively. However, no anti-Listeria compounds were produced under our experimental conditions. These six bacterial species were inoculated, separately or together, into pasteurised milk and their anti-listerial activity in cheese was evaluated. The area of inhibition between the control and trial curves confirmed that L. monocytogenes is inhibited by E. saccharominimus, C. flavescens, L. lactis, L. garvieae and the mixture of all six bacterial strains. Further studies should be performed to determine the metabolites involved in L. monocytogenes inhibition.

From surveillance to action: early gains from the National Violent Death Reporting System.

OBJECTIVES: Drawing from the experiences of individual state programs that currently participate in the National Violent Death Reporting System (NVDRS), this article reviews some of the practical benefits that may accrue from the introduction of violent death surveillance systems. DESIGN: As a state-based surveillance system that uses multiple data sources and relies upon multiple stakeholders, the NVDRS program has fostered an array of initiatives within and among individual state programs. State-based initiatives highlighted in this article were selected on the basis of a purposive sampling strategy intended to illustrate key aspects of program development. SETTING: The NVDRS state programs are in Alaska, California, Colorado, Georgia, Kentucky, Maryland, Massachusetts, New Jersey, New Mexico, North Carolina, Oklahoma, Oregon, Rhode Island, South Carolina, Utah, Virginia, and Wisconsin. RESULTS: The NVDRS has helped to build alliances and collaborative efforts between key stakeholders, facilitated the recognition of violent death as a public health problem through outreach and media attention, acted as a catalyst for new projects, enhanced surveillance of special populations and utility for evaluation, and identified key circumstances that will target interventions in state prevention planning. CONCLUSIONS: The NVDRS has implemented data collection efforts and is beginning to produce and analyze findings. In the process of implementing the data collection system and publicizing findings, state NVDRS programs are realizing other gains that strengthen their surveillance efforts. The use of data for prevention purposes will be the ultimate indicator of program success.

Control of Listeria monocytogenes in raw-milk cheeses.

The development of Listeria monocytogenes in cheeses made with raw-milk originating from six different farms and according to the Saint-Nectaire cheesemaking technology was studied. Milk was inoculated with two strains of L. monocytogenes at 5 to 10 CFU/25 ml. Microbial and chemical analyses were carried out at appropriate intervals during ripening. L. monocytogenes did not grow in the cores of cheeses prepared with milk originating from three farms. That inhibition could be partially attributed to the pH values and L-lactate content. There was no growth in cheeses with pH below 5.2 and lactate content around 14 mg/g. In all cheeses, L. monocytogenes stopped growing in the cores of cheeses after eight days and some other factors may be involved in the inhibition. No relation was found between L. monocytogenes count and other microbial counts. Growth occurred on cheese surfaces between eight and eighteen days, when the pH significantly increased. The lowest L. monocytogenes growth was found on the surface of cheeses with the lowest pH and without any core growth. Further studies will be performed to clarify the involvement of the microbial community in L. monocytogenes inhibition, in particular during the ripening period.

SSR182289A enhances thrombolysis induced by fibrinolytic agents in rabbit models of venous and arterial thrombosis.

BACKGROUND AND OBJECTIVES: The purpose of this work was to investigate whether thrombolysis induced by recombinant tissue plasminogen activator (rt-PA) or streptokinase (SK) was enhanced in different rabbit models of thrombolysis by SSR182289A, a novel synthetic direct thrombin inhibitor which has been shown to possess potent antithrombotic properties in several experimental animal models. METHODS AND RESULTS: Human rt-PA alone (0.125 mg kg(-1) h(-1) for 2 h) induced a significant thrombolysis (18%, P < 0.05) of a venous-type thrombus in the rabbit jugular vein. Under these conditions, SSR182289A (3 mg kg(-1) i.v. bolus) inhibited 125I-fibrin accretion onto a preformed thrombus in the rabbit jugular vein by 72%, but was unable to induce thrombolysis on its own. However, coadministration of SSR182289A and rt-PA strongly enhanced rt-PA-induced thrombolysis (38.4%, P < 0.01). The effect of SSR182289A was further assessed in a model of thrombolysis of an electrical injury-induced, stable (occlusion duration > 2 h) thrombus formed in the rabbit femoral artery. Whereas local intra-arterial infusion of high doses of SSR182289A (3 mg kg(-1) h(-1) for 1 h) alone was able to restore flow, SK (12,000 U kg(-1) h(-1)) and a low dose of SSR182289A (0.3 mg kg(-1) h(-1)) were ineffective. However, intra-arterial coadministration of SSR182289A (0.3 mg kg(-1) h(-1)) and SK (12,000 U kg(-1) h(-1)) induced significant reflow (time to reflow was shortened by 34.7 +/- 7.5 min, P < 0.05). In the same model, systemic i.v. administration of high doses of SSR182289A (10 mg kg(-1) i.v. bolus) and rt-PA (1 mg kg(-1) h(-1)) alone did not induce any thrombolysis. However, the association of both compounds quickly (30 +/- 6 min) restored and maintained flow (duration > 2 h) in all animals. CONCLUSIONS: The present results show that bolus i.v. injection of SSR182289A is able to potentiate thrombolysis induced by two fibrinolytic agents whether the thrombus is of venous or arterial origin, thus suggesting that SSR182289A may be of use as an adjunct to thrombolysis.

Effect of SanOrg123781A, a synthetic hexadecasaccharide, on clot-bound thrombin and factor Xa in vitro and in vivo.

Factor (F)Xa and thrombin bound to the clot during its formation contribute to the propensity of thrombi to activate the coagulation system. The aim of this work was to study the inhibition of clot-bound FXa and clot-bound thrombin by SanOrg123781A, a synthetic hexadecasaccharide that enhances the inhibition of thrombin and FXa by antithrombin (AT). SanOrg123781A, designed to exhibit low non-specific binding to proteins other than AT, was compared with heparin. In buffer, heparin and SanOrg123781A inhibited FXa and thrombin at similar concentrations [concentration inhibiting 50% (IC50) of Xa and IIa activity were, respectively: heparin 120 +/- 7 and 3 +/- 1 ng mL-1; SanOrg123781A 77 +/- 5 and 4 +/- 1 ng mL-1]. In human plasma, the activity of both compounds was reduced, although the activity of heparin was much more affected than that of SanOrg123781A (IC50 values for inhibition of FXa and FIIa activity were, respectively: heparin 100 +/- 5 and 800 +/- 40 ng mL-1; SanOrg123781A 10 +/- 5 and 30 +/- 3 ng mL-1). We demonstrated, in agreement with our previous results, that the procoagulant activity of the clot is essentially due to clot-bound FXa and to some extent to clot-bound thrombin. We showed that heparin and SanOrg123781A were able to inhibit fragment F1+2 generation induced by clot-bound FXa with IC50 values of 2 +/- 0.5 micro g mL-1 and 0.6 +/- 0.2 micro g mL-1, respectively. Both compounds also inhibited clot-bound thrombin activity, the IC50 values of heparin and SanOrg123781A being 1 +/- 0.01 micro g mL-1 and 0.1 +/- 0.1 micro g mL-1, respectively. Moreover, both heparin and SanOrg123781A significantly inhibited fibrinopeptide A generated by the action of clot-bound thrombin on fibrinogen but also by free thrombin generated from prothrombin by clot-bound FXa with IC50 values of 4 +/- 0.6 and 1 +/- 0.1 micro g mL-1, respectively. As with clot-bound enzymatic activities, SanOrg123781A was three times more active than heparin in vivo on fibrinogen accretion onto a pre-existing thrombus and as activators of recombinant tissue-type plasminogen activator-induced thrombolysis. In conclusion, due to the specific activities of SanOrg123781A, this compound is much more active than heparin in the presence of plasma proteins, on clot-bound enzymes and in in vivo models of thrombosis/thrombolysis.

Antithrombotic properties of SSR182289A, a new, orally active thrombin inhibitor.

N-[3-[[[(1S)-4-(5-Amino-2-pyridinyl)-1-[[4-difluoromethylene)-1-piperidinyl]carbonyl]butyl]amino]sulfonyl][1,1'-biphenyl]-2-yl]acetamide hydrochloride (SSR182289A) is a novel, potent, and selective thrombin inhibitor. We have examined the antithrombotic properties of SSR182289A administered by i.v. and p.o. routes in several different animal thrombosis models in comparison with reference antithrombotic agents. Oral administration of SSR182289A produced dose-related antithrombotic effects in the following models; rat venous thrombosis (ED(50) 0.9 mg/kg p.o.), rat silk thread arterio-venous (AV) shunt (ED(50) 3.8 mg/kg p.o.), rat thromboplastin-induced AV shunt (ED(50) 3.1 mg/kg p.o.), rat carotid artery thrombosis (ED(200) 5.9 mg/kg p.o.), and rabbit venous thrombosis (ED(50) 7.5 mg/kg p.o.). Administered as an i.v. bolus, SSR182289A showed antithrombotic activity in the above models with ED(50)/ED(200) values in the range of 0.2 to 1.9 mg/kg i.v. SSR182289A increased rat tail transection bleeding time at doses > or =10 mg/kg p.o. In the rat thromboplastin-induced AV shunt model, SSR182289A 10 mg/kg p.o. produced marked antithrombotic effects at 30, 60, 120, and 240 min after administration. Hence, SSR182289A demonstrates potent oral antithrombotic properties in animal venous, AV-shunt, and arterial thrombosis models.

Fibrolytic activities and cellulolytic bacterial community structure in the solid and liquid phases of rumen contents.

Four sheep were fed an alfalfa hay diet. Rumen content samples were collected three hours after feeding in order to total microorganism population (TP), solid attached population (SAP) and solid attached firmly population (SAFP). Fibrolytic specific activities (xylanase, CMCase and beta-glycosidases) were estimated by the amount of reducing sugars or p-nitrophenol released from the appropriate substrate. The distribution of the three main cellulolytic bacterial species (Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens) was quantified by dot-blot hybridisation using specific 16S-rRNA-targeting probes. Specific activities of polysaccharidase enzymes were higher in SAP than in TP, and in SAFP than in SAP. The sum of RNA of the three cellulolytic bacterial species represented on average 9% of the total bacterial RNA, and increased after filtration. In all samples, the relative population size of F. succinogenes was higher than that of R. albus and of R. flavefaciens. These results demonstrate that the most active enzymes are secreted by the particle-associated microorganisms. The differences in composition of the microflora between the solid and liquid phase suggest that bacteria are not equally distributed throughout the rumen content: the cellulolytic species are present in a higher proportion in the solid phase of rumen contents.

[Effect of nutrition on adipose tissue metabolism in humans]. / Vliv výzivy na metabolismus tukové tkáne u cloveka.

Lipolysis in adipose tissue and balance between energy intake and expenditure are involved in the regulation of adipose tissue mass. Several recent findings suggest that alterations in the regulation of lipolysis and/or energy balance might contribute to the development of obesity. Hormone-sensitive lipase and uncoupling proteins play important role in regulation of lipolysis in adipose tissue as well as in the regulation of energy balance of various tissues. Mechanisms of the control of expression of genes coding synthesis of these proteins are poorly known. A brief overview of the present knowledge of the effects of nutritional intervention on the regulation of lipolysis in adipose tissue and on the expression of genes of hormone-sensitive lipase and that of uncoupling proteins is given in this article. Results of the authors' studies on the effect of calorie restriction on gene expression in adipose tissue are presented.

Cereal supplementation modified the fibrolytic activity but not the structure of the cellulolytic bacterial community associated with rumen solid digesta.

4 ruminally cannulated cows were fed a forage diet (93% hay + 7% straw) and a mixed diet (33 % hay + 7% straw + 40% barley) in a 2 x 2 crossover experimental design. In sacco degradation of forage, fibrolytic activities (polysaccharidases and glycosidases) of the solid-associated bacteria (SAB), and distribution of the 3 main cellulolytic bacterial species (Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens) were determined for both diets. Barley supplementation decreased the hay degradation rate and mainly the polysaccharidase activities of the SAB (30% on average). The sum of rRNA of the 3 cellulolytic bacterial species represented on average 17% of the total bacterial signal and R. albus was the dominant cellulolytic bacterial species of the 3 studied. Barley supplementation did not modify the proportion of the 3 cellulolytic bacteria attached to plant particles. The negative effect of barley on the ruminal hay degradation rate is due to a decrease in fibrolytic activity of the SAB, and not to a modification of the balance of the three cellulolytic bacterial species examined.

Lack of skeletal muscle uncoupling protein 2 and 3 mRNA induction during fasting in type-2 diabetic subjects.

Skeletal muscle uncoupling protein 2 and 3 (UCP-2 and UCP-3) mRNA levels are increased during calorie restriction in lean and nondiabetic obese subjects. In this work, we have investigated the effect of a 5-day hypocaloric diet (1,045 kJ/day) on UCP-2 and UCP-3 gene expression in the skeletal muscle of type-2 diabetic obese patients. Before the diet, UCP-2 and UCP-3 mRNA levels were more abundant in diabetic than in nondiabetic subjects. The long (UCP-3(L)) and short (UCP-3(S)) forms of UCP-3 transcripts were expressed at similar levels in nondiabetic subjects, but UCP-3(S) transcripts were twofold more abundant than UCP-3(L) transcripts in the muscle of diabetic patients. Calorie restriction induced a two- to threefold increase in UCP-2 and UCP-3 mRNA levels in nondiabetic patients. No change was observed in type-2 diabetic patients. Variations in plasma nonesterified fatty acid level were positively correlated with changes in skeletal muscle UCP-3(L) (r = 0.6, P < 0.05) and adipose tissue hormone-sensitive lipase (r = 0.9, P < 0.001) mRNA levels. Lack of increase in plasma nonesterified fatty acid level and in hormone-sensitive lipase upregulation in diabetic patients during the diet strengthens the hypothesis that fatty acids are associated with the upregulation of uncoupling proteins during calorie restriction.

Uncoupling protein-2 (UCP2) and uncoupling protein-3 (UCP3) expression in adipose tissue and skeletal muscle in humans.

Uncoupling protein-2 (UCP2) and uncoupling protein-3 (UCP3) are mitochondrial proteins that may play a role in the control of energy expenditure by uncoupling respiration from ATP synthesis. The present review focuses on data obtained in humans. UCP2 is widely expressed in the body, whereas UCP3 expression is restricted to skeletal muscle. Positive correlations have been reported between UCP2 mRNA concentrations in adipose tissue, UCP3 mRNA concentrations in skeletal muscle, and components of the metabolic rate. Fasting induces an up-regulation of UCP2 and UCP3 mRNA expression. In vivo and in vitro studies suggest that fatty acids could modulate uncoupling protein gene expression. The putative relationship between obesity, energy expenditure and uncoupling protein expression, and the unexpected rise in UCP2 and UCP3 mRNA concentrations during short-term fasting, are discussed in view of the recent data obtained in rodents and cell lines.

Identification of Ruminococcus flavefaciens as the predominant cellulolytic bacterial species of the equine cecum.

Detection and quantification of cellulolytic bacteria with oligonucleotide probes showed that Ruminococcus flavefaciens was the predominant species in the pony and donkey cecum. Fibrobacter succinogenes and Ruminococcus albus were present at low levels. Four isolates, morphologically resembling R. flavefaciens, differed from ruminal strains by their carbohydrate utilization and their end products of cellobiose fermentation.

Identification of Staphylococcus carnosus and Staphylococcus warneri isolated from meat by fluorescent in situ hybridization with 16S rRNA-targeted oligonucleotide probes.

16S rRNA targeted oligonucleotide probes were designed by sequence analysis of an rRNA database to discriminate S. carnosus, S. warneri, and S. saprophyticus species. After establishing hybridization conditions by RNA dot blot hybridization with reference species, our probes were shown to be specific. By in situ hybridization only S-S-S.carno-0440-a-A-23 and S-S-S.war-0180-a-A-23 can specifically detect S. carnosus and S. warneri, respectively. The detection of old cells of S. carnosus 833 was more limited by the permeabilisation than by the low rRNA content. One day old cells could be permeabilized with lysostaphin, whereas young cells were permeabilized with lysozyme.

mRNA expression of the long and short forms of uncoupling protein-3 in obese and lean humans.

Uncoupling protein-3 (UCP3) is a mitochondrial protein expressed in skeletal muscle, an important site of thermogenesis in humans. By uncoupling respiration from ATP synthesis, UCP3 might be involved in the control of energy expenditure. Two transcripts encoding long (UCP3L) and short (UCP3S) form are generated from the human UCP3 gene. UCP3S is predicted to encode a protein which lacks the C-terminus of UCP3L, a region which contains motifs critical for uncoupling activity. We have investigated the regulation of UCP3L and UCP3S mRNAs in lean and obese humans. A specific reverse transcription-competitive polymerase chain reaction assay was developed to separately quantify the two mRNAs. Each transcript represents half of total UCP3 mRNA in 16 vastus lateralis muscle samples. The amounts of UCP3L and UCP3S mRNAs did not differ between obese and lean subjects. The effect of fasting was studied in six lean and seven obese subjects maintained on a hypocaloric diet (1045 kJ/d) for 5 days. Calorie restriction results in an approximately threefold increase of UCP3L and UCP3S mRNA levels. The induction was similar in lean and obese subjects. The data suggest that there is no major alteration of UCP3 gene expression and regulation at the level of transcription and alternative splicing in skeletal muscle of obese subjects.