Biomedical subjects

M Bernard

Publications and source records attributed to M Bernard.

At least 109 records · Page 6Linked to original sources

PubMed1997

Chick pineal clock regulates serotonin N-acetyltransferase mRNA rhythm in culture.

Melatonin production in the chick pineal gland is high at night and low during the day. This rhythm reflects circadian changes in the activity of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT; EC 2.3.1.87), the penultimate enzyme in melatonin synthesis. In contrast to the external regulation of pineal rhythms in mammals by the suprachiasmatic nucleus, rhythmic changes in AA-NAT activity in cultured chick pineal cells are controlled by an oscillator located in the pineal cells themselves. Here we present evidence that the chick pineal clock generates a rhythm in the abundance of AA-NAT mRNA in cultured cells that parallels the rhythm in AA-NAT activity. In contrast, elevating cAMP by forskolin treatment markedly increases AA-NAT activity without producing strong changes in AA-NAT mRNA levels, and lowering cAMP by norepinephrine treatment decreases enzyme activity without markedly decreasing mRNA. These results suggest that clock-controlled changes in AA-NAT activity occur primarily through changes at the mRNA level, whereas cAMP-controlled changes occur primarily through changes at the protein level. Related studies indicate that the clock-dependent nocturnal increase in AA-NAT mRNA requires gene expression but not de novo protein synthesis, and that AA-NAT mRNA levels are suppressed at all times of the day by a rapidly turning over protein. Further analysis of the regulation of chick pineal AA-NAT mRNA is likely to enhance our understanding of the molecular basis of vertebrate circadian rhythms.

Animals↗

PubMed1997

Inapparent polycythemia vera: an unrecognized diagnosis.

PURPOSE: The Polycythemia Vera Study Group (PVSG) has established useful criteria for the diagnosis of polycythemia vera. In some circumstances, an increase of plasma volume (PV) masks that of red cell mass (RCM), with hemoglobin (Hb) and hematocrit (Ht) remaining normal. This defines the concept of inapparent polycythemia. PATIENTS AND METHODS: One hundred and three patients seen in the hematology unit with the diagnosis of polycythemia vera were studied. There were 55 males and 48 females with a median age of 59 years. Ninety-five patients fulfilled the PVSG criteria. Spontaneous erythroid colonies and low serum erythropoietin level confirmed the diagnosis in the 8 other cases. Patients were classified according to Hb and Ht level. RESULTS: Group A consisted of 85 patients with increased Hb and Ht defined, respectively, by Hb > 18 g/dl, Ht > 0.52 in males and Hb > 16 g/dL, Ht>0.47 in females. Group B included 18 patients (17%) with inapparent polycythemia vera (IPV) defined by a normal Hb and Ht value at diagnosis. In this group, the reasons to perform RCM were as follows: splenomegaly associated with increased platelets and/or leucocytes counts (n = 8), portal vein thrombosis (n = 5), increased platelets or leucocytes counts without splenomegaly (n = 3), and isolated splenomegaly (n = 2). The two groups were balanced in terms of age, sex, leucocyte, serum iron, and platelet level. Hemoglobin and Ht levels were significantly different between the two groups. The difference between the PV was indeed highly significant. The mean PV increase was + 9.5% (nL < +20%) in group A versus + 36.3% in group B (P < 0.00005). Red cell mass was not different between the two groups. CONCLUSIONS: Increased Hb or Ht should constitute the sole criteria for RCM determination. In the context of portal vein thrombosis, isolated hyperleucocytosis, thrombocytosis, or splenomegaly, a RCM should be performed. The frequency of IPV remains to be specified but the diagnosis of polycythemia vera is probably underestimated.

Adult↗

PubMed1997

The effect of gamma radiation on the degradation of salbutamol.

The use of ionizing radiation for sterilization of pharmaceuticals is now a well established technology. Degradation of salbutamol was investigated after gamma irradiation using HPLC and ESR spectroscopy. HPLC evidenced the formation of radiolytic products after gamma irradiation. Salbutamol showed a degradation of nearly 2% at 25 kGy. Sterilization of salbutamol in the liquid state appeared not technically feasible. Simulation of the increase of free radicals versus dose was performed using linear and polynomial regression. These radicals could be detected even after a storage period of more than 12 months.

Albuterol↗

PubMed1997

ESR dosimetry of irradiated ascorbic acid.

PURPOSE: As an alternative to heat and gas exposure sterilization, ionizing radiation is gaining interest as a sterilization process for medicinal products. The aim of this work was to develop equations to describe the ESR curves versus dose and storage time after gamma irradiation of ascorbic acid. Several ESR data sets previously acquired in this laboratory were adopted to check the performance of the models. RESULTS: Limit of detection and limit of discrimination are respectively 0.5 kGy and 2 kGy for ascorbic acid. Linear regression is applicable for doses lower than 25 kGy. Since the radiation dose selected must always be based upon the bioburden of the products and the Degree of Sterility required (ANSI/AAMI/ISO 11137), doses in the range 5-25 kGy could be investigated and linear regression would appear to be the least expensive route to follow and gives good results. Quadratic fit, power function, exponential function and bi-exponential functions are of more general applicability to predict irradiation dose. Decay kinetics for radicals versus storage were considered. Nonhomogeneous kinetics with time-dependent rate (diffusion-controlled second-order reaction) and bi-exponential function appeared valid to reproduce the experimental data. Discrimination between irradiated and unirradiated ascorbic acid is possible after a storage of 800 days. CONCLUSIONS: It is worth noting that, at present, ESR is the only technique which proves to be suitable for identification and quantification purposes in irradiated pharmaceuticals. Moreover, other features such as sensitivity, precision, ease and non-destructive readout make ESR superior to other proposed analytical techniques.

Ascorbic Acid↗

PubMed1997

Radiation sterilization of formoterol.

Chromatography, High Pressure Liquid↗

PubMed1997

Radiation effects on dopamine and norepinephrine.

PURPOSE: Radiation sterilization is becoming increasingly popular for the sterilization of many pharmaceutical products. We have investigated the gamma radiation induced effects on dopamine and norepinephrine by ESR spectroscopy. RESULTS AND DISCUSSION: Equations to describe the evolution of the ESR curves versus doses and time of storage are presented. Linear regression is, for dopamine hydrochloride, applicable for doses ranging from 10 to 25 kGy. Since the radiation dose selected must always be based upon the bioburden of the products and the degree of sterility required, doses in the range 10-25 kGy could be investigated and linear regression would appear to be the least expensive route to follow and gives good results. The comportment of noradrenaline bitartrate is more complex and the use of linear regression would appear more hazardous especially for low doses. For doses higher than 25 kGy, a more general equation is required. Power function using only 2 parameters could give good results but must be validated. Decay kinetics for radicals versus storage were considered. Non-homogeneous kinetics with time dependent rate constant and bi-exponential function appeared valid to reproduce the decay of radicals for, respectively, dopamine and norepinephrine. CONCLUSIONS: It is worth noting that, at present, ESR is the only technique which proved to be suitable for identification and quantification purposes in irradiated pharmaceuticals. Moreover, other features such as sensitivity, precision, ease and non-destructive readout make ESR superior to other proposed analytical techniques.

Dopamine↗

PubMed1997

Simultaneous SIL-TAL1 RT-PCR detection of all tal(d) deletions and identification of novel tal(d) variants.

Site-specific deletions of the 5' part of the TAL1 gene (tal(d)) are among the most frequent non-random genetic abnormalities in T-cell acute lymphoblastic leukaemia (T-ALL). They are usually detected by PCR from DNA with several primer pairs or by Southern blot analysis. Since tal(d) lead to expression of a SIL-TAL1 fusion transcript, irrespective of the genomic breakpoint, we have used a single monoplex RT-PCR reaction to screen 55 T-ALL patients at diagnosis. SIL-TAL1 transcripts were demonstrated in 12 (22%) cases, including 7/27 (26%) children <15 years of age, 2/8 (25%) adolescents and 2/17 (12%) adults aged >20 years. SIL-TAL1 RT-PCR was preferrable to tal(d) DNA PCR since it allowed the simultaneous detection of tal(d), tal(d2) and two previously undescribed tal(d) variants. SIL-TAL1 RT-PCR screening should therefore increase the detection rate of tal(d) by approximately 15-20%, with an at least comparable sensitivity to tal(d) genomic PCR, and represents a more practical and economic alternative to multiple DNA PCRs or Southern blotting when incorporated into molecular screening for multiple transcripts at diagnosis.

Adolescent↗

PubMed1997

Avian melatonin synthesis: photic and circadian regulation of serotonin N-acetyltransferase mRNA in the chicken pineal gland and retina.

The circadian rhythms in melatonin production in the chicken pineal gland and retina reflect changes in the activity of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase; AA-NAT; EC 2.3.1.87). Here we determined that the chicken AA-NAT mRNA is detectable in follicular pineal cells and retinal photoreceptors and that it exhibits a circadian rhythm, with peak levels at night. AA-NAT mRNA was not detected in other tissues. The AA-NAT mRNA rhythm in the pineal gland and retina persists in constant darkness (DD) and constant lighting (LL). The amplitude of the pineal mRNA rhythm is not decreased in LL. Light appears to influence the phase of the clock driving the rhythm in pineal AA-NAT mRNA in two ways: The peak is delayed by approximately 6 h in LL, and it is advanced by > 4 h by a 6-h light pulse late in subjective night in DD. Nocturnal AA-NAT mRNA levels do not change during a 20-min exposure to light, whereas this treatment dramatically decreases AA-NAT activity. These observations suggest that the rhythmic changes in chicken pineal AA-NAT activity reflect, at least in part, clock-generated changes in mRNA levels. In contrast, changes in mRNA content are not involved in the rapid light-induced decrease in AA-NAT activity.

Amino Acid Sequence↗

PubMed1997

Evolution of the large-subunit ribosomal RNA binding site for protein L23/25.

The region of the large-subunit rRNA encompassing the D7 divergent domain is organized within eukaryotes in a patchwork of short conservative secondary-structure features interspersed with more rapidly evolving sequences. It contains the attachment site of protein L25 (E. coli L23), which binds rRNA in the first stages of ribosome assembly, suggesting a crucial importance of this region in ribosome elaboration and functioning. A better understanding of its roles requires a good knowledge of its mode of structural variation during the course of evolution. With this aim, we sequenced the D7 region for 24 new invertebrate species belonging to annelids, molluscs, arthropods, and eight other deep-branching invertebrate phyla. Their comparison allowed us to propose refinements in previous eukaryotic folding models. A detailed analysis of the pattern of variation at each position both within the D7 region and along the L23/25 sequence by reference to previous heterologous binding experiments gives new insight into the rRNA-protein contacts. We identified in the D7 region and L23/25, respectively, six and five positions presenting a pattern of variation compatible with experimental results, three of which show coincident variations which support their possible involvement in the rRNA-L23/25 binding.

Amino Acid Sequence↗

PubMed1997

Specificity of cardiac markers troponin I and T in excluding postoperative myocardial infarction.

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Aged↗

PubMed1997

Femoral insertion of the ACL. Radiographic quadrant method.

The optimal femoral insertion or footprint for an anterior cruciate ligament (ACL) graft is the anatomic site. This study was designed to determine the radiographic localization of the femoral insertion of the ACL on a lateral roentgenogram using a quadrant method. Ten human cadaveric knees with intact ACL were dissected. The most anterior, posterior, proximal, and distal borders of the femoral insertion of the ACL were marked with K-wires that were shortened at the bone level of the intercondylar fossa. A roentgenogram was obtained in the strictly lateral position. The end of the K-wires determined the projection of the femoral ACL insertion on the lateral roentgenogram. The center of the radiographically marked area was defined as point K, then four distances were measured on the lateral roentgenogram: distance t (representing the total sagittal diameter of the lateral condyle measured along Blumensaat's line), distance h (representing the maximum intercondylar notch height), distance a (representing the distance of point K from the most dorsal subchondral contour of the lateral femoral condyle), and distance b (representing the distance of point K from Blumensaat's line). Distance a is a partial distance of t and distance b is a partial distance of h, and distances a and b are expressed as length ratios of t and h. The center of the femoral insertion of the ACL was located at 24.8% of the distance t measured from the most posterior contour of the lateral femoral condyle and at 28.5% of the height h measured from Blumensaat's line. Based on these results, the ACL can be found just inferior to the most superoposterior quadrant, which means in anatomic terms it is localized from the dorsal border of the condyle at approximately a quarter of the whole sagittal diameter of the condyle and from the roof of the notch at approximately a quarter of the notch height. By using this radiographic quadrant method combined with fluoroscopic control during surgery, we were able to reinsert the ACL at its anatomic insertion site. This method is independent of variation in knee size or film-focus distance, easy to handle, and reproducible.

Anterior Cruciate Ligament↗

PubMed1997

The melatonin rhythm-generating enzyme: molecular regulation of serotonin N-acetyltransferase in the pineal gland.

A remarkably constant feature of vertebrate physiology is a daily rhythm of melatonin in the circulation, which serves as the hormonal signal of the daily light/dark cycle: melatonin levels are always elevated at night. The biochemical basis of this hormonal rhythm is one of the enzymes involved in melatonin synthesis in the pineal gland-the melatonin rhythm-generating enzyme-serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT, E.C. 2.3.1.87). In all vertebrates, enzyme activity is high at night. This reflects the influences of internal circadian clocks and of light. The dynamics of this enzyme are remarkable. The magnitude of the nocturnal increase in enzyme activity ranges from 7- to 150-fold on a species-to-species basis among vertebrates. In all cases the nocturnal levels of AA-NAT activity decrease very rapidly following exposure to light. A major advance in the study of the molecular basis of these changes was the cloning of cDNA encoding the enzyme. This has resulted in rapid progress in our understanding of the biology and structure of AA-NAT and how it is regulated. Several constant features of this enzyme have become apparent, including structural features, tissue distribution, and a close association of enzyme activity and protein. However, some remarkable differences among species in the molecular mechanisms involved in regulating the enzyme have been discovered. In sheep, AA-NAT mRNA levels show relatively little change over a 24-hour period and changes in AA-NAT activity are primarily regulated at the protein level. In the rat, AA-NAT is also regulated at a protein level; however, in addition, AA-NAT mRNA levels exhibit a 150-fold rhythm, which reflects cyclic AMP-dependent regulation of expression of the AA-NAT gene. In the chicken, cyclic AMP acts primarily at the protein level and a rhythm in AA-NAT mRNA is driven by a noncyclic AMP-dependent mechanism linked to the clock within the pineal gland. Finally, in the trout, AA-NAT mRNA levels show little change and activity is regulated by light acting directly on the pineal gland. The variety of mechanisms that have evolved among vertebrates to achieve the same goal-a rhythm in melatonin-underlines the important role melatonin plays as the hormonal signal of environmental lighting in vertebrates.

Amino Acid Sequence↗

PubMed1996

Circadian regulation of hydroxyindole-O-methyltransferase mRNA in the chicken pineal gland in vivo and in vitro.

The production of the pineal hormone melatonin displays circadian variations with high levels at night. The last enzyme involved in melatonin biosynthesis is hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4). The expression of the mRNA encoding chicken HIOMT was investigated in vivo and in vitro throughout the light/dark cycle, in constant darkness and with light interruption of the dark phase. The stability of HIOMT mRNA was also examined. A day/night rhythm of HIOMT mRNA levels, with a peak at the midlight phase, was observed in vivo as well as in vitro. Constant darkness did not abolish this rhythm in vivo. One cycle of the HIOMT mRNA rhythm could be observed in constant darkness in vitro. In addition, a stimulatory effect of light on HIOMT mRNA levels during the dark phase could be observed in vivo as well as in vitro. HIOMT mRNA stability was not affected by light or dark conditions, as demonstrated by chase experiments with actinomycin D. The results indicate that the daily changes in HIOMT mRNA concentration reflect transcriptional regulation by circadian oscillators and photosensory mechanisms that are endogenous to the pineal gland.

Acetylserotonin O-Methyltransferase↗

PubMed1996

Hydroxyindole-O-methyltransferase in Y-79 cells: regulation by serum.

Hydroxyindole-O-methyltransferase (HIOMT) catalyzes the last step in the synthesis of melatonin. In the present study, the regulation of HIOMT expression was examined in the human Y-79 retinoblastoma cell line. Cells were grown in suspension culture using medium supplemented with 10% fetal calf serum (FCS). HIOMT activity and mRNA were strongly reduced when FCS was substituted with 0.1% bovine serum albumin (BSA), and were restored by addition of FCS. The effect of FCS on HIOMT expression was relatively selective, because the abundance of mRNA encoding actin, G3PDH or interphotoreceptor retinoid-binding protein did not change following serum deprivation. However, S-antigen (arrestin) mRNA was regulated by serum coordinately with HIOMT mRNA, suggesting that S-antigen expression is also controlled by a serum factor. The effect of serum on HIOMT expression was not duplicated by treatment with a series of known differentiating factors, nor was it reduced by dialysis or stripping procedures which remove steroids, growth factors and thyroid hormones.

Acetylserotonin O-Methyltransferase↗

PubMed1996

The human serotonin N-acetyltransferase (EC 2.3.1.87) gene (AANAT): structure, chromosomal localization, and tissue expression.

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT, HGMW-approved symbol AANAT; EC 2.3.1.87) is the penultimate enzyme in melatonin synthesis and controls the night/day rhythm in melatonin production in the vertebrate pineal gland. We have found that the human AA-NAT gene spans approximately 2.5 kb, contains four exons, and is located at chromosome 17q25. The open reading frame encodes a 23.2-kDa protein that is approximately 80% identical to sheep and rat AA-NAT. The AA-NAT transcript (approximately 1 kb) is highly abundant in the pineal gland and is expressed at lower levels in the retina and in the Y79 retinoblastoma cell line. AA-NAT mRNA is also detectable at low levels in several brain regions and the pituitary gland, but not in several peripheral tissues examined. Brain and pituitary AA-NAT could modulate serotonin-dependent aspects of human behavior and pituitary function.

Amino Acid Sequence↗

PubMed1996

Purification and properties of human urinary beta-D-mannosidase.

Two forms of the lysosomal enzyme beta-mannosidase were identified and purified from human urine. The purification strategy employed allowed sufficient quantities of both forms to be obtained for subunit analysis and for further characterizations. The two beta-mannosidases were identified as beta-mannosidase B and A, in order of their elution from an ion-exchange column. In all samples examined, the A form was predominant, and the B/A ratio was consistently 0.14. The two forms displayed the same optimum pH (i.e., 4.3) and both were retained by a Concanavalin-A Sepharose column, but showed different isoelectric points, molecular masses and subunit compositions. Native- and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of pure beta-mannosidases B and A suggest that active protein B (160 kDa) consists of three subunits, one 75 kDa and two 49 kDa subunits. Protein A is smaller and appears to be composed of three subunits of 75 kDa, 49 kDa and 37 kDa. Two forms of beta-mannosidase, exhibiting a chromatographic behaviour comparable to the urinary forms, were also detected in human kidney. Nevertheless, in this tissue their relative distribution was different, the B/A ratio being 19.

Chromatography, Agarose↗

PubMed1996

Reactions of meniscal tissue after arthroscopic laser application: an in vivo study using five different laser systems.

In many clinical and in vitro studies, the effect of laser radiation on meniscal tissue was examined. Clinical studies referred to clinical criteria like swelling, effusion, and pain to evaluate laser effects. In vitro studies showed the laser effect in the moment of cutting the tissue. But the effect of laser radiation on biological tissue also depends on the vital reaction of the tissue. So, the real extent of tissue damage caused by laser irradiation can only be examined in long-term in vivo studies. This was the purpose of this study. Seventy-two knees of pigs underwent arthroscopic meniscal cuts in the anterior horn of the medial meniscus. The pigs were divided into 6 groups: The first 5 groups were operated with 5 different laser systems: Neodym: YAG 1,440-nm wavelength; Nd:YAG 1,064-nm wavelength, Excimer, Holmium:YAG, and CO2. The sixth group was operated with mechanical punches. From each group, the menisci of the pigs were examined macroscopically and by light-microscope after survival periods of 0, 2, 6, 12 weeks. Results were as follows. (1) All laser systems caused greater damage to the meniscal tissue than mechanical instruments. (2) This damage was a biological reaction of the tissue, characterized by a necrotic zone surrounding the meniscus cut. (3) This necrotic zone was not visible intraoperatively but only 2, 6 and 12 weeks after operation. The diameter of the necrotic zone ranged between 1.5 nm and 9 mm. (4) Meniscus cuts with mechanical instruments showed no necrotic zone in the surrounding tissue. (5) Laser cuts in the meniscus caused more extensive healing reaction than cuts with mechanical instruments. (6) The quality of this healing reaction varied with the different laser systems: the Nd:YAG 1,064-nm, Ho:YAG, and CO2 laser caused only an incomplete healing because the tissue repair showed by tissue growing from the synovial edge into the defect only. The Nd:YAG 1,440-nm wavelength and Excimer led to tissue growing from the synovial edge and to remodeling of original meniscal tissue, recognizable by reduction of the necrotic zone. Arthroscopic surgeons should be aware that the damage to meniscus tissue caused by a laser is much greater than can be seen intraoperatively and is much greater than the damage caused by mechanical punches. The healing reaction of the tissue is more extensive after laser application than after use of mechanical instruments. Results of in vitro studies on the tissue damage caused by lasers are insufficient to describe the whole extent of laser effects on living tissue.

Animals↗

PubMed1996

Retinoic acid increases hydroxyindole-O-methyltransferase activity and mRNA in human Y-79 retinoblastoma cells.

Hydroxyindole-O-methyltransferase (HIOMT) plays an important role as the final enzyme in the synthesis of melatonin. Here we present the first evidence that retinoic acid (RA) stereoisomers are potent regulators of HIOMT in the human retinoblastoma-derived Y-79 cell line. Treatment with all-trans-, 13-cis-, and 9-cis-RA induced a gradual 10-fold increase in HIOMT activity and mRNA, without changing the levels of mRNA encoding glyceraldehyde-3-phosphate dehydrogenase, actin, S-antigen, and interphotoreceptor retinoid-binding protein. These findings point to the possibility that RA may play a physiological role in the regulation of human HIOMT.

Acetylserotonin O-Methyltransferase↗