Delayed gastric emptying and altered antrum protein metabolism during activity-based anorexia.

BACKGROUND: Anorexia nervosa, a restrictive eating disorder, is often associated with gastrointestinal disorders, particularly a delayed gastric emptying. However, the mechanisms remained poorly documented. Thus, we aimed to evaluate gastric emptying and antrum protein metabolism in the Activity-Based Anorexia model (ABA). METHODS: Females C57Bl/6 mice were randomized into 3 groups: Control, ABA, and Limited Food Access (LFA). Food access has been progressively limited from 6 h/day at day 6 to 3 h/day at day 9 and until day 17. ABA mice had free access to an activity wheel. Gastric emptying was assessed. On gastric extracts, a proteomic analysis was performed, as well as an evaluation of protein synthesis and protein oxidation. KEY RESULTS: Both LFA and ABA mice exhibited a delayed gastric emptying compared with Controls (P < .05). Proteomic approach revealed 15 proteins that were differentially expressed. Among these proteins, we identified 2 clusters of interest contributing to (i) the organization of muscle fiber with ACTA2, VCL, KRT19, KRT8, and DES proteins and (ii) "heat shock proteins" with STIP1, HSPD1, and HSPA8 proteins. ABA mice specifically exhibited an increased rate of gastric oxidized proteins. CONCLUSIONS AND INFERENCES: Delayed gastric emptying observed in anorectic conditions appears to be secondary to malnutrition. However, an oxidative stress is specifically present in the stomach of ABA mice. Its role remains to be further studied.

Chronic delivery of α-melanocyte-stimulating hormone in rat hypothalamus using albumin-alginate microparticles: effects on food intake and body weight.

Chronic delivery of neuropeptides in the brain is a useful experimental approach to study their long-term effects on various biological parameters. In this work, we tested albumin-alginate microparticles, as a potential delivery system, to study if continuous release in the hypothalamus of α-melanocyte-stimulating hormone (α-MSH), an anorexigenic neuropeptide, may result in a long-term decrease in food intake and body weight. The 2-week release of α-MSH from peptide-loaded particles was confirmed by an in vitro assay. Then, daily food intake and body weight were studied for 18 days in rats injected bilaterally into the paraventricular hypothalamic nucleus with particles loaded or not with α-MSH. A decrease in body weight gain, persisting throughout the study, was found in rats injected with α-MSH-charged particles as compared with rats receiving non-charged particles and with rats injected with the same dose of α-MSH in solution. Food intake was significantly decreased for 3 days in rats receiving α-MSH-loaded particles and it was not followed by the feeding rebound effect which appears after food restriction. The presence of α-MSH-loaded particles in the hypothalamus was confirmed by immunohistochemistry. In conclusion, our study validates albumin-alginate microparticles as a new carrier system for long-term delivery of neuropeptides in the brain and demonstrates that chronic delivery of α-MSH in the hypothalamus results in a prolonged suppression of food intake and a decrease of body weight gain in rats.

Bacterial ClpB heat-shock protein, an antigen-mimetic of the anorexigenic peptide α-MSH, at the origin of eating disorders.

The molecular mechanisms at the origin of eating disorders (EDs), including anorexia nervosa (AN), bulimia and binge-eating disorder (BED), are currently unknown. Previous data indicated that immunoglobulins (Igs) or autoantibodies (auto-Abs) reactive with α-melanocyte-stimulating hormone (α-MSH) are involved in regulation of feeding and emotion; however, the origin of such auto-Abs is unknown. Here, using proteomics, we identified ClpB heat-shock disaggregation chaperone protein of commensal gut bacteria Escherichia coli as a conformational antigen mimetic of α-MSH. We show that ClpB-immunized mice produce anti-ClpB IgG crossreactive with α-MSH, influencing food intake, body weight, anxiety and melanocortin receptor 4 signaling. Furthermore, chronic intragastric delivery of E. coli in mice decreased food intake and stimulated formation of ClpB- and α-MSH-reactive antibodies, while ClpB-deficient E. coli did not affect food intake or antibody levels. Finally, we show that plasma levels of anti-ClpB IgG crossreactive with α-MSH are increased in patients with AN, bulimia and BED, and that the ED Inventory-2 scores in ED patients correlate with anti-ClpB IgG and IgM, which is similar to our previous findings for α-MSH auto-Abs. In conclusion, this work shows that the bacterial ClpB protein, which is present in several commensal and pathogenic microorganisms, can be responsible for the production of auto-Abs crossreactive with α-MSH, associated with altered feeding and emotion in humans with ED. Our data suggest that ClpB-expressing gut microorganisms might be involved in the etiology of EDs.

A missense mutation in melanocortin 1 receptor is associated with the red coat colour in donkeys.

The seven donkey breeds recognised by the French studbook are characterised by few coat colours: black, bay and grey. Normand bay donkeys seldom give birth to red foals, a colour more commonly seen and recognised in American miniature donkeys. Red resembles the equine chestnut colour, previously attributed to a mutation in the melanocortin 1 receptor gene (MC1R). We used a panel of 124 donkeys to identify a recessive missense c.629T>C variant in MC1R that showed a perfect association with the red coat colour. This variant leads to a methionine to threonine substitution at position 210 in the protein. We showed that methionine 210 is highly conserved among vertebrate melanocortin receptors. Previous in silico and in vitro analyses predicted this residue to lie within a functional site. Our in vivo results emphasised the pivotal role played by this residue, the alteration of which yielded a phenotype fully compatible with a loss of function of MC1R. We thus propose to name the c.629T>C allele in donkeys the e allele, which further enlarges the panel of recessive MC1R loss-of-function alleles described in animals and humans.

[One-leg cycling aerobic training with the healthy leg in amateur soccer players after anterior cruciate ligament reconstruction]. / Entraînement en endurance à partir du membre inférieur sain chez des footballeurs amateurs opérés de ligamentoplastie de genou.

OBJECTIVE: To examine cardiorespiratory fitness changes in subjects having undergone knee surgery and to assess the benefits of one-leg cycling aerobic training program during the rehabilitation period. METHOD: Two groups of 12 patients took part in this study. The control group profited from a five weeks conventional rehabilitation in day hospital without cardiorespiratory training. The second group profited in supplement from a one-leg cycling aerobic training program with the valid leg. The subjects were trained for 21 min, by alternating 3 min at 70% and 3 min at 85% of VO(2 peak). They totaled 15 sessions spread over five weeks. The initial evaluation (T1) is carried out the first day of rehabilitation and the final evaluation (T2) at a distance within 35 days. The evaluation consisted in realizing a maximal graded tests starting from the valid leg. RESULTS: After five weeks of conventional rehabilitation, we record a reduction of peak power output (W(peak)), peak oxygen uptake (VO(2 peak)) and peak minute ventilation (VE(peak)), respectively of 11, 12 and 13% for the control group. On the other hand, in T2, the training group has on average identical maximum values and some of them increased (W(peak): +14%; VE(peak): +15%). The first and second ventilatory thresholds appear with higher intensities of exercises. CONCLUSION: After knee surgery, conventional rehabilitation does not limit cardiorespiratory deconditioning. One leg cycling appears to be an adapted method to stop the effects of hypoactivity.

In vitro and in vivo evaluation of the haematopoietic potential of skeletal muscle in a non-human primate model.

This study was aimed at evaluating the in vitro and in vivo haematopoietic potential in macaque skeletal muscle cells. Biopsy samples showed the presence of CD34(+) (7.6%), CD90(+) (8.4%), CD117(+), CD31(+), side population (SP) cells (7-10%) and a low number of CD45(+) cells. In clonogenic and long-term culture-initiating cell assays, no haematopoietic potential could be detected in either total mononuclear cells or SP cells. Regarding in vivo studies, two animals were transplanted with unfractionated fresh muscle cells after lethal irradiation. Both animals died early after transplant without any evidence of haematopoietic reconstitution. In two other monkeys, harvested muscle cells were frozen and secondarily marked using a green fluorescent protein (GFP)-lentiviral vector. After sublethal irradiation, both animals were transplanted with GFP-expressing muscle cells followed by a bone marrow rescue. Both animals had haematopoietic reconstitution at days 22 and 25, but no GFP-expressing haematopoietic cells could be detected by flow cytometry, either in the blood or in clonogenic cells from marrow aspirates. Using PCR assays, GFP(+) cells were detected in a single marrow sample of one animal at 41 days after transplantation. These results strongly suggest that as opposed to murine muscle, the non-human primate skeletal muscle does not harbour cells with a straightforward haematopoietic potential.

Effect of high-intensity interval training and detraining on extra VO2 and on the VO2 slow component.

To examine the effect of 6-week of high-intensity interval training (HIT) and of 6-week of detraining on the VO2/Work Rate (WR) relationship and on the slow component of VO2, nine young male adults performed on cycle ergometer, before, after training and after detraining, an incremental exercise (IE), and a 6-min constant work rate exercise (CWRE) above the first ventilatory threshold (VT1). For each IE, the slope and the intercept of the VO2/WR relationship were calculated with linear regression using data before VT1. The difference between VO2max measured and VO2max expected using the pre-VT1 slope was calculated (extra VO2). The difference between VO2 at 6th min and VO2 at 3rd min during CWRE (DeltaVO2(6'-3')) was also determined. HIT induced significant improvement of most of the aerobic fitness parameters while most of these parameters returned to their pre-training level after detraining. Extra VO2 during IE was reduced after training (130 +/- 100 vs. -29 +/- 175 ml min(-1), P = 0.04) and was not altered after detraining compared to post-training. DeltaVO2(6'-3') during CWRE was unchanged by training and by detraining. We found a significant correlation (r2 = 0.575, P = 0.02) between extra VO2 and DeltaVO2(6'-3') before training. These results show that an alteration of extra VO2 can occur without any change in the VO2 slow component, suggesting a possible dissociation of the two phenomena. Moreover, the fact that extra VO2 did not change after detraining could indicate that this improvement may remain after the loss of other adaptations.

Effect of prior heavy exercise on muscle deoxygenation kinetics at the onset of subsequent heavy exercise.

This study examines the effect of prior heavy exercise on muscle deoxygenation kinetics at the onset of heavy-intensity cycling exercise. Ten young male adults (20 +/- 2 years) performed two repetitions of step transitions (6 min) from 35 W to heavy-intensity exercise preceded by either no warm-up or by a heavy-intensity exercise. VO2 was measured breath-by-breath, and muscle deoxygenation (HHb) and total hemoglobin (Hb(tot)) were monitored continuously by near-infrared spectroscopy. We used a two-exponential model to describe the VO2 kinetics and a mono-exponential model for the HHb kinetic. The parameters of the phase II VO2 kinetics (TD1 VO2, tau1 VO2 and A1 VO2) were unaffected by prior heavy exercise, while some parameters of local muscle deoxygenation kinetics were significantly faster (TD HHb: 7 +/- 2 vs. 5 +/- 2 s; P < 0.001, MRT HHb: 20 +/- 3 vs. 15+/- 4 s; P < 0.05). Blood lactate, heart rate and Hb(tot) values were significantly higher before the second bout of heavy exercise. These results collectively suggest that the prior heavy exercise probably increased muscle O2 availability and improved O2 utilization at the onset of a subsequent bout of heavy exercise.

Respiratory muscle oxygenation kinetics: relationships with breathing pattern during exercise.

This work aimed to investigate accessory respiratory muscle oxygenation (RMO(2)) during exercise, using near-infrared spectroscopy, and to study relationships between RMO(2) kinetics and breathing parameters. Nineteen young males (19.3 +/- 1.5 years) performed a maximal incremental test on a cycle ergometer. Changes in breathing pattern were characterized by accelerated rise in the breathing frequency (f (Racc)), plateau of tidal volume (V (Tplateau)) and inflection point in the V. (E)/V (T) relationship (V. (E)/V (T inflection)). First and second ventilatory thresholds (VT1 and VT2) were also determined. RMO (2) kinetics were monitored by NIRS on the serratus anterior. During exercise, all subjects showed reduced RMO (2) (deoxygenation) with a breakdown (B-RMO(2)) at submaximal workload (86 % .VO(2max)). .VO(2) corresponding to B-RMO (2) and to f (Racc), V (Tplateau), .V(E)/V(T inflection), or VT2 were not different. Relationships were found between the .VO(2) at B-RMO(2) and the .VO(2) at f (Racc) (r = 0.88, p < 0.001), V (Tplateau) (r = 0.84, p < 0.001), V. (E)/V (T inflection) (r = 0.58, p < 0.05) or VT2 (r = 0.79, p < 0.001). The amplitude of RMO(2) at maximal workload was weakly related to .VO(2max) (r = 0.58, p < 0.05). B-RMO (2) seems to be due to the change in breathing pattern and especially to the important rise in breathing frequency at the VT2 exercise level. Moreover, subjects who exhibit higher .VO(2max) also exhibit a higher decrease in respiratory muscle oxygenation during exercise.

[Preliminary study of cardiorespiratory decondittioning in athletes after anterior cruciate ligament reconstruction]. / Etude préliminaire de la désadaptation cardiorespiratoire après une ligamentoplastie de genou chez le sportif.

AIM: The aim of this study was to analyze changes in cardiorespiratory fitness of athletes who had surgery following a lesion of the anterior cruciate ligament of the knee. METHODS: Two groups of 12 athletes at the regional level underwent surgical repair to rebuild the external anterior crossed ligament of the knee (central third bone patellar tendon bone autograft and doubled semitendinosus/doubled gracilis autograft techniques). All subjects were evaluated before and after surgery within 7 days: the first group underwent maximal incremental tests with the upper limbs, and the second group measurement of resting cardiac volumes. RESULTS: Surgery followed by a few days of confinement generated a quick and significant reduction in the maximal oxygen consumption (-7%, P<0.05) and peak aerobic power (-8%, P<0.05). End diastolic volume and stroke volume were reduced, by 23% and 27% respectively (P<0.05). A significant reduction of ejection fraction was also observed (P<0.05). The mean left ventricular ejection fraction was 65% before the surgery 60% after 7 days' of hospitalization. CONCLUSION: In sportsmen, 7 days of hospitalization due to surgery of the knee led to resting cardiac unsuitability characterized by a significant reduction in the stroke volume. These elements could involve decreased aerobic fitness and should encourage the hospital practitioner to propose a program of aerobic training in addition to conventional rehabilitation.

Effect of prior exercise on the VO2/work rate relationship during incremental exercise and constant work rate exercise.

The disproportionate increase in VO2 ("extra VO2) reported at elevated intensity during incremental exercise (IE) might result from the same physiological mechanisms as the VO2 slow component observed during heavy constant work rate exercise (CWRE). Moreover, it has been demonstrated that prior heavy exercise can diminish the VO2 slow component. The aim of this study was to evaluate whether prior heavy exercise also alters the "extra VO2" during IE. Ten trained sprinters performed three tests on a cycle ergometer: Test 1 was an IE; Test 2 consisted of six minutes of a CWRE (90% of VO2max) followed by six minutes at 35 W and by an IE and Test 3 was composed of two CWRE of six minutes separated by six minutes of exercise at 35 W. For each IE, the slope and the intercept of the VO2/work rate relationship were calculated by linear regression using data before the first Ventilatory Threshold (pre-VT1 slope). The difference between VO2max measured and VO2max expected using the pre-LT slope was calculated (deltaVO2). We also calculated the difference between VO2 at min five and VO2 at min three during CWRE of Test 3 (deltaVO2(5' - 3')). VO2max was significantly higher than VO2exp during IE of Test 1 and Test 2. deltaVO2 during IE did not differ between Test 1 and Test 2 (+ 259 +/- 229 ml x min(-1) vs. + 222 +/- 221 ml x min(-1)). During Test 3, six subjects achieved five minutes of exercise during the second CWRE and deltaVO2(5' - 3') was significantly decreased during the second CWRE (338 +/- 65 ml x min(-1) vs. 68 +/- 98 ml x min(-1), n = 6). These results demonstrate that the amplitude of the "extra VO2"during IE was not affected by prior exercise, whereas the slow component of VO2 evaluated by deltaVO2(5' - 3') during CWRE was lowered. This implies that prior exercise does not have the same effect on the slow component of VO2 and on the "extra VO2". Therefore we were unable to demonstrate a relationship between the VO2 slow component and the extra-VO2 phenomenon during IE.

MhcDRB-sequences from cynomolgus macaques (Macaca fascicularis) of different origin.

Cynomolgus macaques are frequently used in biomedical research. However, in contrast to their closest relative, the rhesus macaque, little is known about their Mhc genes except for the DQB1 locus. In this study, 33 DRB-sequences belonging to 17 allelic lineages were detected in a total of 68 macaques, 58 originating from Mauritius and 10 from China. The majority of the sequences were detected in the few macaques from China, confirming the low degree of genetic variation in macaques from Mauritius. In summary, the DRB region in cynomolgus macaques is polymorphic. The sequences belong in general to the same allelic lineages as in their closest relative, the rhesus macaque. Two exon 2 DNA sequences were identical in both species and may represent a trans-species origin. In addition, protein sequences of members of the DRB*W1 lineage seem to be rather conserved in the three macaque species examined so far. Six DRB-haplotypes were detected in the macaques from Mauritius. While single DRB-alleles or some protein sequences seemed to be conserved among macaque species, we could not detect any evidence for a trans-species conservation of a complete DRB region. Overall, the data indicate that reorganization of the DRB region by recombination is a major force in creating diversity in cynomolgus macaques as it is in rhesus macaques.

In vivo behaviour of hydroxyapatite coatings on titanium implants: a quantitative study in the rabbit.

The aim of this study was to evaluate quantitatively the behaviour of in vivo hydroxyapatite coated implants (HA) in the rabbit over time, and to compare the results with observations made on titanium plasma spray implants (TPS). Results were analysed according to the percentage of bone contact. Eighteen HA cylindrical implants (3.25 x 8 mm) and 6 TPS cylindrical implants from Steri-Oss were placed in the epiphysis of the femur in 24 white rabbits. Each rabbit received one implant. Three rabbits with one HA implant (n = 3) and 1 rabbit with one TPS implant (n = 1) were sacrificed after implantation periods of 2, 4, 6, 8, 10 and 12 months. Implants were cut along the long axis and prepared for histological and histomorphometrical evaluations. Measurements of coating thickness and percentage of bone contact were performed with scanning electron microscopy analysis on the sides of the implant, in 3 different types of bone, namely cortical, trabecular and marrow. In cortical bone, dense bone was apposed to the HA implants: from 92.3 +/- 5.5% at 2 months to 89.6 +/- 6.5% at 1 year, with no significant regression of HA thickness (P = 0.37). TPS coating showed less bone contact, but thickness was stable (P = 0.46). In trabecular zone, where bone contact was less pronounced, a significant regression of HA coatings thickness (P < 0.05) was observed. Nevertheless TPS coatings were stable (P = 0.81). Histomorphometrical results demonstrated that a highly significant regression (P < 0.0001) of HA thickness was observed in the marrow area, where the bone-to-implant contact never exceeded 7.6% from 2 to 12 months. TPS coating did not reveal any sign of resorption (P = 0.88), despite a rare bone contact. Histological analysis revealed inflammatory and giant cells, principally in the marrow area in contact with HA coating, but always in restrictive numbers. We conclude that bone contact protected the HA coating from resorption.

Proteome analysis of Aspergillus fumigatus identifies glycosylphosphatidylinositol-anchored proteins associated to the cell wall biosynthesis.

Previous studies in Aspergillus fumigatus (Mouyna I., Fontaine T., Vai M., Monod M., Fonzi W. A., Diaquin M., Popolo L., Hartland R. P., Latgé J.-P, J. Biol. Chem. 2000, 275, 14882-14889) have shown that a glucanosyltransferase playing an important role in fungal cell wall biosynthesis is glycosylphosphatidylinositol (GPI) anchored to the membrane. To identify other GPI-anchored proteins putatively involved in cell wall biogenesis, a proteomic analysis has been undertaken in A. fumigatus and the protein data were matched with the yeast genomic data. GPI-anchored proteins of A. fumigatus were released from membrane preparation by an endogenous GPI-phospholipase C, purified by liquid chromatography and separated by two-dimensional electrophoresis. They were characterized by their peptide mass fingerprint through matrix-assisted laser desorption/ionization-time of flight-(MALDI-TOF)-mass spectrometry and by internal amino acid sequencing. Nine GPI-anchored proteins were identified in A. fumigatus. Five of them were homologs of putatively GPI-anchored yeast proteins (Csa1p, Crh1p, Crh2p, Ecm33p, Gas1p) of unknown function but shown by gene disruption analysis to play a role in cell wall morphogenesis. In addition, a comparative study performed with chitin synthase and glucanosyl transferase mutants of A. fumigatus showed that a modification of the growth phenotype seen in these mutants was associated to an alteration of the pattern of GPI-anchored proteins. These results suggest that GPI-anchored proteins identified in this study are involved in A. fumigatus cell wall organization.

Sugar-mediated crosslinking of alpha-biotinylated-Lys to cysteamine-agarose support: a method to isolate Maillard Lys-Lys-like crosslinks.

Advanced glycation end products (AGEs) and, specifically, protein-protein AGE crosslinks have long been studied for their potential role in aging, diabetic complications and Alzheimer disease. With few exceptions, the chemical nature of these structures remains unknown. We report here a simple approach that allows the preparation and isolation of milligram quantities of sugar-mediated AGE Lys-Lys-like crosslinks from glycation mixtures. The method is based on a sugar-dependent incorporation of N(alpha)-biotinyl-L-Lys into cysteaminyldisulfide Sepharose 6B (AE-S-S-Sepharose 6B). Glycation mixtures with six different sugars showed a time- and sugar-dependent decrease in the concentration of the support-bound primary amino groups and accounted for almost 90% loss of cysteaminyl amino groups at the end of the various incubation periods. 4-Hydroxyazobenzene-2-carboxylic acid-avidin assays indicated the incorporation of N(alpha)-biotinyl-L-Lys equal to 8% of the total support amino groups with methylglyoxal after 7 d and 1% with fructose and glucose after 1 mo of incubation. Treatment of the washed, sugar-modified supports with 2-mercaptoethanol released the bulk of the bound AGE modifications and the crosslinks. Subsequent fractionation of these preparations over a monomeric avidin column afforded a complete separation of sugar-mediated AGE modifications and the crosslinks. Depending on the sugar employed, micromolar amounts of biotinylated Lys-Lys-like crosslinks were generated by this two-step procedure from 8 mL of the original AE-S-S-Sepharose 6B.

Glucan synthase complex of Aspergillus fumigatus.

The glucan synthase complex of the human pathogenic mold Aspergillus fumigatus has been investigated. The genes encoding the putative catalytic subunit Fks1p and four Rho proteins of A. fumigatus were cloned and sequenced. Sequence analysis showed that AfFks1p was a transmembrane protein very similar to other Fksp proteins in yeasts and in Aspergillus nidulans. Heterologous expression of the conserved internal hydrophilic domain of AfFks1p was achieved in Escherichia coli. Anti-Fks1p antibodies labeled the apex of the germ tube, as did aniline blue fluorochrome, which was specific for beta(1-3) glucans, showing that AfFks1p colocalized with the newly synthesized beta(1-3) glucans. AfRHO1, the most homologous gene to RHO1 of Saccharomyces cerevisiae, was studied for the first time in a filamentous fungus. AfRho proteins have GTP binding and hydrolysis consensus sequences identical to those of yeast Rho proteins and have a slightly modified geranylation site in AfRho1p and AfRho3p. Purification of the glucan synthase complex by product entrapment led to the enrichment of four proteins: Fks1p, Rho1p, a 100-kDa protein homologous to a membrane H(+)-ATPase, and a 160-kDa protein which was labeled by an anti-beta(1-3) glucan antibody and was homologous to ABC bacterial beta(1-2) glucan transporters.

Vancomycin resistance is associated with serine-containing peptidoglycan in Enterococcus gallinarum.

In Enterococcus gallinarum SC1, a low-level vancomycin-resistant strain, only monomeric muropentapeptides with a C-terminal D-alanine were detected after growth without vancomycin. In contrast, in SC1 induced by vancomycin, as well as in AIB39, a constitutive vancomycin-resistant strain, monomeric and dimeric muropentapeptides with a C-terminal D-serine were detected.

Effect of nicotine on rat gingival fibroblasts in vitro.

Tobacco smoking is considered a major risk factor for the development and progression of periodontal diseases (Haber, J. and Wattles, J. (1994). J. Periodontol., 64, 16-23). The purpose of this study was to determine the effects of nicotine on rat gingival fibroblasts (RGF) cultured in vitro. After ether anesthesia, rat gingival tissues were obtained from the attached gingiva of a Wistar rat. Small fragments of gingiva were maintained in culture in Petri dishes. Fibroblasts developing from these explants were collected to obtain monolayer cultures. After the fourth passage (T4), cells were supplemented with nicotine at various concentrations. Control and treated cells were examined under phase contrast or transmission electron microscopy. They were compared as regards their DNA content, mitochondrial activity, collagen and protein synthesis, and cell death by apoptosis or necrosis. Nicotine from 0.05 microM to 1 mM did not affect the DNA content or protein and collagen synthesis. At concentrations between 3 and 5 mM, growth was significantly diminished and the survival rate reduced. Ultrastructural analysis revealed dilated mitochondria and vacuolization in treated cells, suggestive of necrosis, but increased apoptosis was also revealed by cytometry. On the basis of this in vitro study, it appears that tobacco, through its component nicotine, may directly affect various functions of RGF.

Novel mechanism of beta-lactam resistance due to bypass of DD-transpeptidation in Enterococcus faecium.

The peptidoglycan structure of in vitro selected ampicillin-resistant mutant Enterococcus faecium D344M512 and of the susceptible parental strain D344S was determined by reverse phase high performance liquid chromatography and mass spectrometry. The muropeptide monomers were almost identical in the two strains. The substantial majority (99.3%) of the oligomers from the susceptible strain D344S contained the usual d-alanyl --> d-asparaginyl (or d-aspartyl)-l-lysyl cross-link (d-Ala --> d-Asx-l-Lys) generated by beta-lactam-sensitive DD-transpeptidation. The remaining oligomers (0.7%) were produced by beta-lactam-insensitive LD-transpeptidation, because they contained l-Lys --> d-Asx-l-Lys cross-links. The muropeptide oligomers of the ampicillin-resistant mutant D344M512 contained only these l-Lys --> d-Asx-l-Lys cross-links indicating that resistance was due to the bypass of the beta-lactam-sensitive DD-transpeptidation reaction. The discovery of this novel resistance mechanism indicates that DD-transpeptidases cannot be considered anymore as the sole essential transpeptidase enzymes.

Interspecies complementation in Saccharopolyspora erythraea : elucidation of the function of oleP1, oleG1 and oleG2 from the oleandomycin biosynthetic gene cluster of Streptomyces antibioticus and generation of new erythromycin derivatives.

Two glycosyltransferase genes, oleG1 and oleG2, and a putative isomerase gene, oleP1, have previously been identified in the oleandomycin biosynthetic gene cluster of Streptomyces antibioticus. In order to identify which of these two glycosyltransferases encodes the desosaminyltransferase and which the oleandrosyltransferase, interspecies complementation has been carried out, using two mutant strains of Saccharopolyspora erythraea, one strain carrying an internal deletion in the eryCIII (desosaminyltransferase) gene and the other an internal deletion in the eryBV (mycarosyltransferase) gene. Expression of the oleG1 gene in the eryCIII deletion mutant restored the production of erythromycin A (although at a low level), demonstrating that oleG1 encodes the desosaminyltransferase required for the biosynthesis of oleandomycin and indicating that, as in erythromycin biosynthesis, the neutral sugar is transferred before the aminosugar onto the macrocyclic ring. Significantly, when an intact oleG2 gene (presumed to encode the oleandrosyltransferase) was expressed in the eryBV deletion mutant, antibiotic activity was also restored and, in addition to erythromycin A, new bioactive compounds were produced with a good yield. The neutral sugar residue present in these compounds was identified as L-rhamnose attached at position C-3 of an erythronolide B or a 6-deoxyerythronolide B lactone ring, thus indicating a relaxed specificity of the oleandrosyltransferase, OleG2, for both the activated sugar and the macrolactone substrate. The oleP1 gene located immediately upstream of oleG1 was likewise introduced into an eryCII deletion mutant of Sac. erythraea, and production of erythromycin A was again restored, demonstrating that the function of OleP1 is identical to that of EryCII in the biosynthesis of dTDP-D-desosamine, which we have previously proposed to be a dTDP-4-keto-6-deoxy-D-glucose 3, 4-isomerase.