Does land-based exercise-therapy improve physical activity in people with knee osteoarthritis? A systematic review with meta-analyses.

PRIMARY OBJECTIVE: Investigate the effects of land-based exercise-therapy on physical activity in people with knee osteoarthritis (KOA). DESIGN: Systematic review and meta-analysis of randomised or quasi-randomised trials investigating land-based exercise-therapy on physical activity, fitness, and general health in people with KOA. We updated a 2013 Cochrane review search on exercise-therapy for KOA in April 2021 and applied the Cochrane Risk-of-Bias Tool 1.0 to included articles. Standardised mean differences (SMDs) and 95% confidence intervals (CI) were calculated. GRADE was used to assess certainty of the evidence. RESULTS: Twenty-eight randomised controlled trials (2,789 participants) evaluating the effects of resistance-training (n = 10), walking (n = 6) and mixed-exercise programs (n = 7) were identified. Low to moderate certainty evidence indicated small increases in physical activity for exercise-therapy compared to non-exercise interventions in the short-term (SMD, 95% CI = 0.29, 0.09 to 0.50), but not the medium- (0.03, -0.11 to 0.18) or long-term (-0.06, -0.34 to 0.22). Low certainty evidence indicated large increases in physical activity for walking programs (0.53, 0.11 to 0.95) and mixed-exercise programs (0.67, 0.37 to 0.97) compared to non-exercise interventions in the short-term. Low certainty evidence indicated moderate and small increases in physical activity for resistance-training combined with education focused on pain coping skills and self-efficacy compared to education alone at medium-term follow-up (0.45, 0.19 to 0.71). CONCLUSION: Walking and mixed-exercise, but not resistance-training, may improve physical activity in people with KOA in the short-term. Combining resistance-training with education may increase physical activity in the medium-, but not the long-term, highlighting the potential importance of developing more effective longer-term interventions for people with KOA. Future studies evaluating land-based exercise-therapy are encouraged to include physical activity outcomes and longer-term follow-up to increase the certainty of evidence.

Many physiotherapists lack preparedness to prescribe physical activity and exercise to people with musculoskeletal pain: A multi-national survey.

OBJECTIVES: Determine physiotherapists' (i) awareness of physical activity, and exercise prescription guidelines; (ii) perceived role, knowledge, confidence, skills and training in prescribing and progressing aerobic exercise and resistance training to people with musculoskeletal pain; (iii) professional development preferences; and (iv) perceived influences of external factors on exercise prescription for people with musculoskeletal pain. DESIGN: Multi-national cross-sectional survey. METHODS: An open online survey was developed specifically for this study. RESULTS: 1,352 physiotherapists from 56 countries participated. The majority of respondents correctly stated physical activity guidelines for adults (60%) and children (53%), but only 37% correctly stated guidelines for older adults. Eleven percent and 16% could name an accepted guideline for aerobic exercise and resistance training, respectively. Most agreed their role included prescribing aerobic exercise (75%) and resistance training (89%). Fewer reported they had the confidence, training or skills to prescribe aerobic exercise (38-50%) and resistance training (49-70%). Workshops were the most preferred (44%) professional development option. Most respondents believed appointment scheduling and access to equipment and professional development (62-79%) affected their ability to prescribe effective exercise. CONCLUSION: Many physiotherapists lack knowledge and training to provide physical activity advice, and to prescribe aerobic exercise and resistance training to people with musculoskeletal pain.

MicroRNAs are differentially abundant during Aedes albopictus diapause maintenance but not diapause induction.

Diapause is a programmed dormancy that allows organisms to tolerate predictable periods of unfavourable conditions by temporarily halting development and reducing metabolism. Diapause is widespread amongst insects and is crucial for allowing organisms to coordinate their growth and reproduction with favourable environmental conditions. Although the adaptive significance of diapause is well understood, the molecular mechanisms underpinning diapause remain unresolved. We performed high-throughput sequencing to investigate the role of microRNAs (miRNAs) in the diapause of the Asian tiger mosquito, Aedes albopictus. We first investigated miRNAs in diapause induction by characterizing maternally provisioned miRNAs in mature oocytes of Ae. albopictus under diapause-inducing and diapause-averting conditions. Second, we investigated miRNAs in diapause maintenance by characterizing miRNAs in diapause and nondiapause pharate larvae. We identified 162 miRNAs, 152 previously known and 10 putatively novel. We identified no differentially abundant miRNAs in mature oocytes and seven differentially abundant miRNAs in pharate larvae. The predicted targets of differentially abundant miRNAs include genes affecting several processes related to diapause maintenance including ecdysone regulation, immune response, lipid metabolism and regulation of development. Our results suggest that Ae. albopictus does not maternally provision a unique set of miRNAs during diapause induction but miRNAs are a component of diapause maintenance in this species.

Laccase wiring on free-standing electrospun carbon nanofibres using a mediator plug.

Electrospun carbon nanofibres (CNFs) containing CNTs were produced by electrospinning and subsequent thermal treatment. This material was evaluated as a bioelectrode for biofuel cell applications after covalent grafting of laccase. Bis-pyrene-modified ABTS was used as a plug to wire laccase to the nanofibres leading to a maximum current density of 100 µA cm(-2).

Non-covalent double functionalization of carbon nanotubes with a NADH oxidation Ru(II)-based molecular catalyst and a NAD-dependent glucose dehydrogenase.

We report the double functionalization of multiwalled carbon nanotube electrodes by two functional pyrene molecules. In combination, an immobilized Ru(II)-based NADH oxidation catalyst and glucose dehydrogenase achieve highly efficient glucose oxidation with low overpotential of -0.10 V and high current densities of 6 mA cm(-2).

Interest of rehabilitation in healing and preventing recurrence of ankle sprains.

UNLABELLED: To assess the impact of rehabilitation on healing and recurrence rate of ankle sprain, 1year apart, 111 patients, who suffered an ankle sprain (67 men and 44 women; 17 mild sprains, 67 medium and 27 severe), were included by emergency physicians of four emergency rooms (ER) of Finistère. The physician was free to prescribe, or not, further investigations. He prescribed systematically to patients RICE (rest, ice, compression, elevation) protocol, put an ankle brace, and gave a prescription of standardized rehabilitation. The prescription was the same for the four ER. All patients were recalled to 1year. Of the 111 patients initially included, 21 patients were excluded for lack of response after three phone calls. In the end, 90 patients were assessable (56 men and 34 women), mean age 31.4±12.6years (range 15-55) at the time of initial trauma. Emergency physicians had diagnosed, initially, 16 mild sprains (17.8%), 56 medium sprains (62.2%) and 18 severe sprains (20%). Of the 90 patients, 73 patients have been rehabilitated (81.1%). Of the 44 accidents of everyday life, 31 were rehabilitated (70.5%). Of the 27 sports accidents, 25 were rehabilitated (92.6%). Of the 19 work-related injuries, 17 were rehabilitated (89.5%). There is no significant relationship between rehabilitation and no recurrence (P=0.45) nor between rehabilitation and full recovery of the ankle (P=0.59). CONCLUSION: We find no association between rehabilitation and prevention of recurrence, nor between rehabilitation and healing of patients. However, our study is limited by the small size of the non-rehabilitated group.

Single glucose biofuel cells implanted in rats power electronic devices.

We describe the first implanted glucose biofuel cell (GBFC) that is capable of generating sufficient power from a mammal's body fluids to act as the sole power source for electronic devices. This GBFC is based on carbon nanotube/enzyme electrodes, which utilize glucose oxidase for glucose oxidation and laccase for dioxygen reduction. The GBFC, implanted in the abdominal cavity of a rat, produces an average open-circuit voltage of 0.57 V. This implanted GBFC delivered a power output of 38.7 µW, which corresponded to a power density of 193.5 µW cm(-2) and a volumetric power of 161 µW mL(-1). We demonstrate that one single implanted enzymatic GBFC can power a light-emitting diode (LED), or a digital thermometer. In addition, no signs of rejection or inflammation were observed after 110 days implantation in the rat.

Developmental and epigenetic anomalies in cloned cattle.

Many of the developmental anomalies observed in cloned animals are related to foetal and placental overgrowth, a phenomenon known as the 'large offspring syndrome' (LOS) in ruminants. It has been hypothesized that the epigenetic control of imprinted genes, that is, genes that are expressed in a parental-specific manner, is at the root of LOS. Our recent research has focused on understanding epigenetic alterations to imprinted genes that are associated with assisted reproductive technologies (ART), such as early embryo in vitro culture (IVC) and somatic cell nuclear transfer (SCNT) in cattle. We have sought and identified single nucleotide polymorphisms in Bos indicus DNA useful for the analysis of parental-specific alleles and their respective transcripts in tissues from hybrid embryos derived by crossing Bos indicus and Bos taurus cattle. By analysing differentially methylated regions (DMRs) of imprinted genes SNRPN, H19 and the IGF2R in cattle, we demonstrated that there is a generalized hypomethylation of the imprinted allele and the biallelic expression of embryos produced by SCNT when compared to the methylation patterns observed in vivo (artificially inseminated). Together, these results indicate that imprinting marks are erased during the reprogramming of the somatic cell nucleus during early development, indicating that such epigenetic anomalies may play a key role in mortality and morbidity of cloned animals.

Adherence and effectiveness of rehabilitation in acute ankle sprain.

OBJECTIVE: To estimate adherence to and effectiveness of rehabilitation after acute ankle sprain. METHOD: Patients with acute ankle sprain attending four emergency departments were recruited between February and July 2009. After the initial examination (classification of the severity of the sprain), each patient received an Aircast(®) ankle brace and the same, standardized rehabilitation program. Between two and three months later; the patient was contacted by phone (always by the same investigator) in order to find out whether he/she had performed the prescribed rehabilitation, establish whether the physiotherapist had complied with the prescribed rehabilitation programme and assess subjective recovery. If a patient failed to respond to three phone calls, he/she was excluded from the study. RESULTS: Of the 245 patients initially included, 111 (67 men and 44 women; 17 mild sprains, 67 moderate sprains and 27 severe sprains) answered the "phone questionnaire". In terms of treatment adherence by the patient, 92 patients (82.9%) performed their rehabilitation (beginning an average of 13.8 days after the injury). In terms of prescription compliance by the physiotherapist, 88 patients (95.6%) received massage, 71 (77.2%) underwent physiotherapy, 83 (90.2%) performed weight training and 87 (94.5%) received proprioceptive training. Eighty-two patients said that they had received manipulative therapy that was not part of the prescribed programme. Impact on recovery: 61 patients (55%) considered that their injury had healed (10 mild, 42 medium and nine severe sprains), whereas 50 had not healed (seven mild, 25 medium and 18 severe sprains). There was no statistically significant association between recovery and compliance with rehabilitation. However, the application of massage (p=0.004) and proprioceptive training (p=0.017) were significantly associated with recovery, while physiotherapy, weight training and manipulative therapy were not. CONCLUSION: In acute ankle sprain, adherence with rehabilitation is good and the treating physiotherapists comply with the prescription. However, there was no statistically significant link between rehabilitation compliance and subjective recovery at 3 months. Revaluation of these patients at one year may be necessary for estimating the impact of rehabilitation on ankle function and the rate of injury recurrence.

Loss of methylation at H19 DMD is associated with biallelic expression and reduced development in cattle derived by somatic cell nuclear transfer.

Although cloning of mammals has been achieved successfully, the percentage of live offspring is very low because of reduced fetal size and fewer implantation sites. Recent studies have attributed such pathological conditions to abnormal reprogramming of the donor cell used for cloning. The inability of the oocyte to fully restore the differentiated status of a somatic cell to its pluripotent and undifferentiated state is normally evidenced by aberrant DNA methylation patterns established throughout the genome during development to blastocyst. These aberrant methylation patterns are associated with abnormal expression of imprinted genes, which among other genes are essential for normal embryo development and gestation. We hypothesized that embryo loss and low implantation rates in cattle derived by somatic cell nuclear transfer (SCNT) are caused by abnormal epigenetic reprogramming of imprinted genes. To verify our hypothesis, we analyzed the parental expression and the differentially methylated domain (DMD) methylation status of the H19 gene. Using a parental-specific analysis, we confirmed for the first time that H19 biallelic expression is tightly associated with a severe demethylation of the paternal H19 DMD in SCNT embryos, suggesting that these epigenetic anomalies to the H19 locus could be directly responsible for the reduced size and low implantation rates of cloned embryos in cattle.

[Assessment of the contribution of the immunochromatographic pneumococcal urinary antigen test to the etiological diagnosis of pneumonia in hospitalized adults]. / Bilan de la contribution de l'antigénurie pneumocoque par test immunochromatographique au diagnostic étiologique des pneumonies chez l'adulte hospitalisé.

AIM OF THE STUDY: To assess the usefulness and prescription practices of the Binax Now Streptococcus pneumoniae urinary antigen test in hospitalized adults. PATIENTS AND METHODS: The results of the pneumococcal urinary antigen tests (UAT) performed from January 2002 to September 2004 were related to that of microbiological cultures, and in positive patients to radiographic findings and C-reactive protein (CRP) levels. The evolution of the number of prescriptions and positivity rate in 2007 versus 2002-2004 was analyzed. RESULTS: The pneumococcal UAT was positive in 32 of the 278 patients included from 2002 to 2004 (11.5%). Results were concordant with that of microbiological cultures in 90% of the 247 documented cases. Pneumococcal etiology was considered to be definite in 19 patients (isolation of S. pneumoniae from blood, 17 patients; or pleural fluid, two patients), of whom 15 had a positive UAT (sensitivity: 79%); to be probable in 22 patients (positive UAT, 17 patients and/or isolation of S. pneumoniae from respiratory samples, six patients), and was retained in 39 of the 41 patients (positive predictive value: 93.7%). CRP was greater than 100mg/L in 34 of 39 documented patients and lobar alveolar radiographic opacities observed in 25 of 28 documented patients. In 2007, the dramatic increase in the number of UAT prescriptions and the diversification of prescribing units were associated to a decreased positivity rate (8.1%). CONCLUSION: Whereas the pneumococcal UAT clearly increases etiological diagnosis, pneumococcal pneumonia cannot be ruled out if negative. Indications for its use need to be refined to improve the cost-effectiveness of this test.

Prolonged progesterone treatment of endometrial epithelial cells modifies the effect of estradiol on their sensitivity to oxytocin.

Estradiol (E2), progesterone (P4), and oxytocin (OT) are important for the initiation of luteolysis in ruminants but the mechanisms involved are still poorly understood. The objective of this study was to determine if duration of exposure of bovine endometrial epithelial cells to P4 affected the response of the cells to E2. Endometrial epithelial cells, from cows at Days 1-3 of the estrous cycle, were cultured for 10, 17, and 21 days in the presence or absence of P4 (100 ng ml(-1)). After culture, each group of cells was incubated for a further 6, 12, 24 or 48 h with or without E2 (100 pg ml(-1)) and then incubated for 6 h with different doses of OT (2, 20, and 200 ng ml(-1)). E2 enhanced OT-stimulated PGF2 alpha secretion in cells cultured with P4 for 17 or 21 days, with a maximum effect after 24-h exposure, but not in cells cultured with P4 for 10 days. To determine the mechanism of action of E2, COX-1 and COX-2 were measured by Western blotting and OTR number was measured by saturation analysis. OT increased COX-2 (P<0.05), but there was no significant effect of E2 on the expression of either COX-1 or COX-2. E2 did, however, increase (P<0.001) the OTR number in cells cultured with P4 for 21 days, whereas it inhibited OTR in cells cultured for 10 days. These data show that E2 can stimulate PGF2 alpha secretion by increasing OTR expression in bovine endometrial cells in vitro, but only after exposure to P4.

Tsg101 control of human immunodeficiency virus type 1 Gag trafficking and release.

The structural precursor polyprotein of human immunodeficiency virus type 1, Pr55(gag), contains a proline-rich motif (PTAP) called the "late domain" in its C-terminal p6 region that directs release of mature virus-like particles (VLPs) from the plasma membranes of gag-transfected COS-1 cells. The motif binds Tsg101 (vacuolar protein-sorting protein 23, or Vps23), which functions in endocytic trafficking. Here, we show that accumulation of the wild-type (wt) Gag precursor in a fraction of COS-1 cytoplasm enriched in multivesicular bodies and small particulate components of the plasma membrane (P100) is p6 dependent. Cleavage intermediates and mature CA mainly partitioned with more rapidly sedimenting larger material enriched in components of lysosomes and early endosomes (P27), and this also was p6 dependent. Expression of truncated or full-length Tsg101 proteins interfered with VLP assembly and Gag accumulation in the P100 fraction. This correlated with reduced accumulation of Gag tagged with green fluorescent protein (Gag-GFP) at the plasma membrane and colocalization with the tagged Tsg101 in perinuclear early endosomes, as visualized by confocal microscopy. Fractionation analysis and confocal examination both indicated that the N-terminal region of Tsg101, which contains binding sites for PTAP and ubiquitin (Ub), was required for Gag trafficking to the plasma membrane. Expression of FLAG-tagged Tsg101 with a deletion in the Ub-binding pocket inhibited VLP release almost completely and to a significantly greater extent than expression of the wt tagged Tsg101 protein or Tsg101-FLAG containing a deletion in the PTAP-binding region. The results demonstrate that Gag associates with endosomal trafficking compartments and indicate that efficient release of virus particles from the plasma membrane requires both the PTAP- and Ub-binding functions of Tsg101 to recruit the cellular machinery required for budding.

Hydrolysis of pectins with different degrees and patterns of methylation by the endopolygalacturonase of Fusarium moniliforme.

The mode of action of the endopolygalacturonase from Fusarium moniliforme was studied towards a series of pectins with different amounts and distribution patterns of methyl-ester groups. The enzyme hydrolysed the linkages between two galacturonic acid residues according to a multi-chain attack mechanism, at least at the early stage of the reaction. The final percentage of hydrolysis decreased with increasing the degree of methylation. The distribution pattern of the methyl groups affected the rate of hydrolysis as well as the final percentage of hydrolysis, a blockwise distribution being more favourable than a random one. The final products, as analysed by mass spectrometry, included methyl-esterified oligogalacturonates. The detailed analysis of the structure of the oligomers showed that the enzyme was able to accommodate methylated galacturonic acid in its active site, but that methyl-esterification negatively affected the affinity of the enzyme.

Molecular cloning and induction of bovine prostaglandin E synthase by gonadotropins in ovarian follicles prior to ovulation in vivo.

Prostaglandin E(2) (PGE(2)) is thought to be an ultimate prostaglandin effector during the ovulatory process, and the objectives of this study were to clone bovine PGE synthase (PGES) and to characterize its regulation by gonadotropins in preovulatory follicles in vivo. The bovine PGES complementary DNA (cDNA) was shown to contain a 5'-untranslated region of eight base pairs (bp), an open reading frame of 462 bp and a 3'-untranslated region of 406 bp. The putative bovine PGES open reading frame encodes a 153-amino acid protein that is 85, 78, and 78% identical to the human, rat, and mouse PGES homologs, respectively. The regulation of PGES during ovulation was studied using three different models in vivo: 1) human chorionic gonadotropin (hCG)-induced ovulation during a normal estrous cycle; 2) hCG-induced ovulation following ovarian hyperstimulation; and 3) spontaneous ovulation during natural estrus. Results from semi-quantitative reverse transcription-polymerase chain reaction/Southern blotting analyses showed that the hCG/luteinizing hormone surge caused a significant increase in PGES mRNA. Levels of PGES transcripts were low or undetectable prior to hCG/luteinizing hormone but increased markedly 18-24 h after hCG in models 1 and 2, and 18-24 h after the onset of natural estrus in model 3 (p < 0.05). Analyses on isolated preparations of granulosa and theca interna cells indicated that the granulosa cell layer was the predominant site of follicular PGES expression. The regulation of the protein was studied in the same models using a specific antibody raised against a fragment of bovine protein (Delta PGES; from Glu(49) to Val(146)). Results from immunoblots showed an induction of bovine PGES (M(r) = 17,000) 18-24 h after hCG treatment or onset of estrus (p < 0.05). The protein was detected in extracts of granulosa cells but not in theca interna. Collectively, these results demonstrate that the ovulatory process is associated with a gonadotropin-dependent induction of PGES in granulosa cells of ovarian follicles in vivo, thus establishing for the first time the regulation of the enzyme in a physiological context.

Study of the mode of action of endopolygalacturonase from Fusarium moniliforme.

One endopolygalacturonase from Fusarium moniliforme was purified from the culture broth of a transformed strain of Saccharomyces cerevisiae. Its kinetic parameters and mode of action were studied on galacturonic acid oligomers and homogalacturonan. The dimer was not a substrate for the enzyme. The enzyme was shown to follow Michaelis-Menten behaviour towards the other substrates tested. Affinity and maximum rate of hydrolysis increased with increasing chain length, up to the hexamer or heptamer, for which V(max) was in the same range as with homogalacturonan. The enzyme was demonstrated to have a multi-chain attack mode of action and its active site included five subsites ranging from -3 to +2. The final products of hydrolysis of homogalacturonan were the monomer and the dimer of galacturonic acid.

Addition of a glycophosphatidylinositol to acetylcholinesterase. Processing, degradation, and secretion.

We introduced various mutations and modifications in the GPI anchoring signal of rat acetylcholinesterase (AChE). 1) The resulting mutants, expressed in transiently transfected COS cells, were initially produced at the same rate, in an active form, but the fraction of GPI-anchored AChE and the steady state level of AChE activity varied over a wide range. 2) Productive interaction with the GPI addition machinery led to GPI anchoring, secretion of uncleaved protein, and secretion of a cleaved protein, in variable proportions. Unproductive interaction led to degradation; poorly processed molecules were degraded rather than retained intracellularly or secreted. 3) An efficient glypiation appeared necessary but not sufficient for a high level of secretion; the cleaved, secreted protein was possibly generated as a by-product of transamidation. 4) Glypiation was influenced by a wider context than the triplet omega/omega + 1/omega + 2, particularly omega - 1. 5) Glypiation was not affected by the closeness of the omega site to the alpha(10) helix of the catalytic domain. 6) A cysteine could simultaneously form a disulfide bond and serve as an omega site; however, there was a mutual interference between glypiation and the formation of an intercatenary disulfide bond, at a short distance upstream of omega. 7) Glypiation was not affected by the presence of an N-glycosylation site at omega or in its vicinity or by the addition of a short hydrophilic, highly charged peptide (FLAG; DYKDDDDK) at the C terminus of the hydrophobic region.

Role of the major homology region in assembly of HIV-1 Gag.

The major homology region (MHR) is a highly conserved sequence in the gag gene of all retroviruses, including HIV-1. Its role in assembly is unknown, but deletion of the motif significantly impairs membrane binding and viral particle formation. To begin characterizing this defect, we have determined the contribution of this region to the energetics of the assembly process. Intrinsic fluorescence studies were conducted to determine the change in free energy associated with membrane and RNA binding using tRNA and large unilamellar vesicles of 1-palmitoyl-2-oleoylphosphatidylserine as models. For the wild-type protein, the change in free energy was within RT [600 cal/(mol.K)] whether Gag binds first to RNA or to the membrane. Thus, the initial binding of Gag can be to either substrate, but in vivo conditions favor initial association to RNA presumably due to its higher local concentration. After establishing the pattern of assembly, we compared the binding energy of Gag(WT) versus the deletion mutant, Gag(Delta)(MHR). Gag(WT) bound to membranes with a 2-fold higher affinity than Gag(Delta)(MHR), and the binding to RNA was similar for the two proteins. Gag prebound to RNA or to membrane exhibited approximately 2-4-fold greater binding affinity than Gag(Delta)(MHR) for binding the membrane or RNA, respectively. Most importantly, the mutant was significantly impaired in its ability to self-associate on RNA or on membrane surfaces. This key role of the MHR in promoting productive protein-protein interactions was also seen in altered amounts of cleavage products and the lack of membrane-bound, RNA-containing replication intermediates in infected cells. These results suggest that Gag first binds to RNA and then assembles into a multimeric complex with a large membrane-binding face that facilitates subsequent membrane binding. Deletion of the MHR disrupts the protein-protein interactions required to complete this process.

Protein synthesis during maturation of bovine oocytes, effect of epidermal growth factor.

The objective of this study was to determine whether epidermal growth factor (EGF) affected protein synthesis in oocytes during maturation. Initially, the effect of EGF on oocyte maturation was examined to ensure that there was a beneficial effect of EGF in the protein-free maturation medium used in these studies. Results showed that the presence of EGF during maturation significantly enhanced cleavage rate and development to the blastocyst stage. Development after maturation in the presence of EGF was similar to that seen in medium containing serum, luteinizing hormone, follicle-stimulating hormone and estradiol. Protein synthesis was examined in immature oocytes and after 16 or 24 h maturation. Oocytes from each group were labelled by incubation for 4 h with 35S-methionine, the proteins were then separated by two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Between 400 and 500 proteins could be separated using this method and marked changes in protein synthesis was observed during maturation. Changes in eight different proteins were observed when protein patterns from oocytes matured for 16 h with and without EGF were compared. These results suggest that EGF plays a physiological role in oocyte maturation and identification of the proteins induced by EGF could be important for improving our understanding of oocyte maturation in vitro.

The glial and the neuronal glycine transporters differ in their reactivity to sulfhydryl reagents.

The neuronal (GlyT2) and glial (GlyT1) glycine transporters, two members of the Na(+)/Cl(-)-dependent neurotransmitter transporter superfamily, differ by many aspects, such as substrate specificity and Na(+) coupling. We have characterized under voltage clamp their reactivity toward the membrane impermeant sulfhydryl reagent [2-(trimethylammonium)-ethyl]-methanethiosulfonate (MTSET). In Xenopus oocytes expressing GlyT1b, application of MTSET reduced to the same extent the Na(+)-dependent charge movement, the glycine-evoked current, and the glycine uptake, indicating a complete inactivation of the transporters following cysteine modification. In contrast, this compound had no detectable effect on the glycine uptake and the glycine-evoked current of GlyT2a. The sensitivities to MTSET of the two transporters can be permutated by suppressing a cysteine (C62A) in the first extracellular loop (EL1) of GlyT1b and introducing one at the equivalent position in GlyT2a, either by point mutation (A223C) or by swapping the EL1 sequence (GlyT1b-EL1 and GlyT2a-EL1) resulting in AFQ <--> CYR modification. Inactivation by MTSET was five times faster in GlyT2a-A223C than in GlyT2a-EL1 or GlyT1b, suggesting that the arginine in position +2 reduced the cysteine reactivity. Protection assays indicate that EL1 cysteines are less accessible in the presence of all co-transported substrates: Na(+), Cl(-), and glycine. Application of dithioerythritol reverses the inactivation by MTSET of the sensitive transporters. Together, these results indicate that EL1 conformation differs between GlyT1b and GlyT2a and is modified by substrate binding and translocation.