Broadening the scope of Baeyer-Villiger monooxygenase activities toward α,ß-unsaturated ketones: a promising route to chiral enol-lactones and ene-lactones.

Three regiodivergent Baeyer-Villiger mono-oxygenases (enantioselectively) oxidized a series of cyclic α,ß-unsaturated ketones into (chiral) either enol-lactones or ene-lactones. An easy-to-use and efficient biocatalytic process based on a host-microorganism deprived of unwanted activities (knock-out mutant) was developed to enable the exclusive synthesis of unsaturated lactones.

Parathyroid hormone fragments inhibit active hormone and hypocalcemia-induced 1,25(OH)2D synthesis.

Carboxyl (C)-terminal fragments of parathyroid hormone (PTH) oppose the calcemic, phosphaturic, and bone-resorbing effects of active hormone. To study the action of these fragments on 1,25(OH)(2)D (1,25-dihydroxyvitamin D) synthesis, we infused parathyroidectomized rats with human or rat active 1-34 or 1-84 PTH at doses selected to produce similar calcemic responses. Human active PTH influenced neither phosphate nor 1,25(OH)(2)D concentrations. However, active 1-34 rat PTH decreased phosphate to the same level as vehicle-treated rats and increased 1,25(OH)(2)D to very high levels, whereas active 1-84 PTH decreased phosphate but maintained 1,25(OH)(2)D. As the latter effect could have been due to C-terminal fragment generation during its metabolic breakdown, we infused a mixture of rat C-terminal fragments alone or with rat 1-34. The C-terminal fragments decreased 1,25(OH)(2)D and prevented hypocalcemic-induced 1,25(OH)(2)D synthesis. When infused with active rat 1-34, they lowered the 1,25(OH)(2)D level to that seen with intact rat 1-84. The C-terminal fragments did not influence either basal or rat 1-34- or 1-84-induced CYP27B1 mRNA levels, suggesting that their inhibitory effects on 1,25(OH)(2)D synthesis appears to be post-transcriptional.

Comprehensive analysis of a large genomic sequence at the putative B-cell chronic lymphocytic leukaemia (B-CLL) tumour suppresser gene locus.

In many haematological diseases, and more particularly in B-cell chronic lymphocytic leukaemia (B-CLL), the existence of a tumour suppressor gene located within the frequently deleted region 13q14.3, has been put forward. A wide candidate region spanning from marker D13S273 to D13S25 has been proposed and an extensive physical map has been constructed by several teams. In this study, we sequenced a minimal core deleted region that we have previously defined and annotated it with flanking available public sequences. Our analysis shows that this region is gene-poor. Furthermore, our work allowed us to identify new alternative transcripts, spanning core regions, of the previously defined candidate genes DLEU1 and DLEU2. Since their putative involvement in B-CLL was controversial, our present study provide support for reconsidering the DLEU1 and DLEU2 genes as B-CLL candidate genes, with a new definition of their organisation and context.

A physical map of human chromosome 14.

We report the construction of a tiling path of around 650 clones covering more than 99% of human chromosome 14. Clone overlap information to assemble the map was derived by comparing fully sequenced clones with a database of clone end sequences (sequence tag connector strategy). We selected homogeneously distributed seed points using an auxiliary high-resolution radiation hybrid map comprising 1,895 distinct positions. The high long-range continuity and low redundancy of the tiling path indicates that the sequence tag connector approach compares favourably with alternative mapping strategies.

Extracellular calcium-sensing receptor is expressed in rat hepatocytes. coupling to intracellular calcium mobilization and stimulation of bile flow.

Liver cells respond to changes in Ca(2+)(o). The hepatic functions affected include bile secretion, metabolic activity, liver regeneration, and the response to xenobiotics. In the present study, we demonstrate the presence, in the liver, of the extracellular calcium-sensing receptor (CASR), described previously in the parathyroid and thyroid glands and kidney. CASR mRNA was specifically expressed in hepatocytes and was absent in nonparenchymal liver cells (stellate, endothelial, and Kupffer cells). Western blot analysis using a specific CASR antibody showed staining in both whole liver and hepatocyte extracts. Immunohistochemistry and in situ hybridization of rat liver sections showed expression of CASR protein and mRNA by a subset of hepatocytes. The known agonists of the CASR, gadolinium (Gd(3+); 0.5-3.0 mm) and spermine (1.25-20 mm), in the absence of Ca(2+)(o), elicited dose-related increases in Ca(2+)(i) in isolated rat hepatocytes loaded with Fura-2/acetoxymethyl ester. There was a greatly attenuated response to a second challenge with either agonist. The response was also abrogated when inositol 1,4,5-trisphosphate (IP(3))-sensitive calcium pools had been depleted by pretreatment with either thapsigargin or phenylephrine, an alpha(1)-adrenergic receptor agonist known to mobilize Ca(2+)(i) from IP(3)-sensitive pools. Addition of the deschloro-phenylalkylamine compound, NPS R-467, but not the S enantiomer, NPS S-467, increased the sensitivity of the Ca(2+)(i) mobilization response to 1.25 mm spermine. Bile flow ceased after Ca(2+)(o) withdrawal, and its recovery was enhanced by spermine in isolated perfused liver preparations. The CASR agonists Ca(2+) and Gd(3+) increased bile flow, and the response to a submaximal Ca(2+) concentration was enhanced by NPS R-467 but not the S compound. Thus, the data indicate that rat hepatocytes harbor a CASR capable of mobilizing Ca(2+)(i) from IP(3)-sensitive stores and that activation of the CASR stimulates bile flow.

Influence of the in vivo calcium status on cellular calcium homeostasis and the level of the calcium-binding protein calreticulin in rat hepatocytes.

Little attention has been given to the consequences of the in vivo calcium status on intracellular calcium homeostasis despite several pathological states induced by perturbations of the in vivo calcium balance. The aim of these studies was to probe the influence of an in vivo calcium deficiency on the resting cytoplasmic Ca2+ concentration and the inositol-1,4,5-trisphosphate-sensitive Ca2+ pools. Studies were conducted in hepatocytes (a cell type well characterized for its cellular Ca2+ response) isolated from normal and calcium-deficient rats secondary to vitamin D depletion. Both resting cytoplasmic Ca2+ concentration and Ca2+ mobilization from inositol-1,4,5-trisphosphate-sensitive cellular pools were significantly lowered by calcium depletion. In addition, Ca deficiency was shown to significantly reduce calreticulin messenger RNA and protein levels but calcium entry through store-operated calcium channels remained unaffected, indicating that the Ca2+ entry mechanisms are still fully operational in calcium deficiency. The effects of calcium deficiency on cellular calcium homeostasis were reversible by repletion with oral calcium feeding alone or by the administration of the calcium-regulating hormone 1,25-dihydroxyvitamin D3, further strengthening the tight link between extra- and intracellular calcium. These data, therefore, challenge the currently prevailing hypothesis that extracellular Ca2+ has no significant impact on cellular Ca2+ by demonstrating that despite the large Ca2+ gradient between extra- and intracellular Ca2+ concentrations, calcium deficiency in vivo significantly alters the hormone-sensitive cellular calcium homeostasis.

A comparison of standard cardiopulmonary resuscitation and active compression-decompression resuscitation for out-of-hospital cardiac arrest. French Active Compression-Decompression Cardiopulmonary Resuscitation Study Group.

BACKGROUND: We previously observed that short-term survival after out-of-hospital cardiac arrest was greater with active compression-decompression cardiopulmonary resuscitation (CPR) than with standard CPR. In the current study, we assessed the effects of the active compression-decompression method on one-year survival. METHODS: Patients who had cardiac arrest in the Paris metropolitan area or in Thionville, France, more than 80 percent of whom had asystole, were assigned to receive either standard CPR (377 patients) or active compression-decompression CPR (373 patients) according to whether their arrest occurred on an even or odd day of the month, respectively. The primary end point was survival at one year. The rate of survival to hospital discharge without neurologic impairment and the neurologic outcome were secondary end points. RESULTS: Both the rate of hospital discharge without neurologic impairment (6 percent vs. 2 percent, P=0.01) and the one-year survival rate (5 percent vs. 2 percent, P=0.03) were significantly higher among patients who received active compression-decompression CPR than among those who received standard CPR. All patients who survived to one year had cardiac arrests that were witnessed. Nine of 17 one-year survivors in the active compression-decompression group and 2 of 7 in the standard group, respectively, initially had asystole or pulseless electrical activity. In 12 of the 17 survivors who had received active compression-decompression CPR, neurologic status returned to base line, as compared with 3 of 7 survivors who had received standard CPR (P=0.34). CONCLUSIONS: Active compression-decompression CPR performed during advanced life support significantly improved long-term survival rates among patients who had cardiac arrest outside the hospital.

Non-founder mutations in the MEFV gene establish this gene as the cause of familial Mediterranean fever (FMF).

Familial Mediterranean fever (FMF) is an autosomal recessive disorder characterized by recurring attacks of fever and serositis. It affects primarily North African Jews, Armenians, Turks and Arabs, in which a founder effect has been demonstrated. The marenostrin-pyrin-encoding gene has been proposed as a candidate gene for the disease ( MEFV ), on the basis of the identification of putative mutations clustered in exon 10 (M680V, M694I, M694V and V726A), each segregating with one ancestral haplotype. In a search for additional MEFV mutations in 120 apparently non-founder FMF chromosomes, we observed eight novel mutations in exon 2 (E148Q, E167D and T267I), exon 5 (F479L) and exon 10 (I692del K695R, A744S and R761H). Except for E148Q and K695R, all mutations were found in a single chromosome. Mutation E148Q was found in all ethnic groups studied and in association with a novel ancestral haplotype in non-Ashkenazi Jews (S2). Altogether, these new findings definitively establish the marenostrin/pyrin-encoding gene as the MEFV locus.

A transcriptional Map of the FMF region.

Familial Mediterranean fever (FMF) is a recessively inherited disorder characterized by attacks of fever and serositis, which affects primarily non-Ashkenazi Jews, Armenians, Turks, and Arabs. We present here a transcriptional map covering the FMF locus that we constructed in the course of the positional cloning of the gene responsible for this disease. This map was established from a contig constructed with YAC, BAC, and cosmid clones and covers about 500 kb of 16p13.3. It contains nine transcriptional units corresponding to known genes or to genes belonging to known gene families, 23 gene fragments characterized by partial sequences, and an endogenous retrovirus sequence. It thus considerably increases the number of genes in this interval and improves our knowledge concerning some of the genes or gene families present in this region. Data accumulated in this region were also used in a comparative study of different methods of exon detection.

Influence of late reopening of the infarct-related artery on left ventricular remodelling after myocardial infarction. IRIS Study Group.

AIMS: This trial was undertaken to assess the impact of late reopening of the infarct-related artery on left ventricular remodelling in post-myocardial infarction patients. METHODS: One hundred and fifty seven patients with recent myocardial infarction were routinely submitted to delayed (second week) catheterization. They also underwent systematic angioplasty of a significantly narrowed infarct-related artery with a suitable anatomy, or reopening of a totally occluded infarct-related artery, and repeat follow-up catheterization after 4 months. Changes in left ventricular ejection fraction, left ventricular volumes, and percent of regional hypokinesia were assessed over the study period. RESULTS: One hundred and thirty-two patients had two interpretable left ventriculograms and two interpretable coronary angiograms. At initial angiography, 56 out of 96 patients with a patent infarct-related artery were successfully submitted to percutaneous coronary angioplasty, of whom 25 had restenosis and eight had total reocclusion at follow-up angiography. Percutaneous transluminal coronary angioplasty was not attempted in the remaining 40 patients due to unsuitable anatomy in 18 or a nonsignificant lesion in 22. The infarct-related artery was totally occluded in 36 patients at initial angiography, and successfully reopened by means of angioplasty in 19, of whom seven showed a reocclusion at follow-up angiography. The independent predictors of left ventricular enlargement, identified by means of multivariate regression analysis, were initial stroke volume index < 40 ml.m-2 (odds ratio = 6.3, 95% confidence interval = [2.5; 16.6]), initial end-systolic volume index > 50 ml.m-2 (odds ratio = 7.1, 95% confidence interval = [1.5; 25.8]), anterior infarct location (odds ratio = 4.1, 95% confidence interval = [1.4; 11.5]) and reocclusion of the infarct-related artery (odds ratio = 7.3, 95% confidence interval = [1.3; 27.3]). Angioplasty of a patent but significantly narrowed infarct-related artery was not found predictive. CONCLUSIONS: This study demonstrates that reocclusion of a previously open infarct-related artery, as well as the initial low stroke volume index, enlarged end-systolic volume index and anterior infarct location are independent predictors of long-term left ventricular enlargement. These results emphasize the impact of long-term sustained patency of the infarct-related artery on the prevention of left ventricular dysfunction. The need for a larger randomized trial is recognised.

Hypocalcemia modifies the intracellular calcium response to the alpha 1-adrenergic agent phenylephrine in rat hepatocytes.

In vivo, extracellular calcium ([Ca2+]e) homeostasis is maintained within a very narrow range by the calcium regulating hormones. At the cellular level, the response to many agents is transduced by changes in cytosolic Ca2+ ([Ca2+]i) which involves both mobilization of cellular pools and entry of [Ca2+]e through plasma membrane channels. To investigate the cellular effects of chronic hypocalcemia (Ca-) on [Ca2+]i homeostasis, hepatocytes, a cell type well characterized for its [Ca2+]i response, were used. Data indicate that Ca- leads to a significant shift to the left in the basal resting cytosolic Ca2+ concentration distribution curve with half-maximum cumulative frequency of 119 versus 149 nM in Ca- and normal rats (N) respectively (P < 0.0001). The response to the alpha 1-adrenergic agonist phenylephrine (Phe) was also influenced by Ca- with a dampening of the dose-response curve, a significant decrease in the frequency of sustained responses (P < 0.001), and significant changes in the oscillation pattern. Indeed, hepatocytes obtained from Ca- exhibited a higher frequency of large amplitude, low frequency oscillations than N most particularly at the 2 and 5 microM Phe dose while N predominantly exhibited low amplitude, high frequency oscillations on sustained plateaus (P < 0.001). IP3 receptor (IP3R) binding studies and Ca2+ mobilization from IP3-sensitive pools showed that IP3R was highly sensitive to the prevailing Ca2+ with, in the range of resting [Ca2+]i, R affinity significantly lower in Ca- than in N. Upon exposure of permeabilized cells to 25 microM IP3, Ca2+ mobilization from the IP3-sensitive intracellular pool was significantly reduced by Ca- (P < 0.05) suggesting a decrease in the IP3-mobilizable Ca2+ pool in Ca-. Our results indicate that hypocalcemia significantly alters [Ca2+]i signalling by perturbing the initial response to agonist and the [Ca2+]i response pattern. In addition, the decrease in Ca2+ mobilization from IP3-sensitive pools suggests that hypocalcemia may also lead to a decrease in the Ca2+ content of intracellular pools.

Effect of phalloidin on cholestasis, hemodynamics, and microcirculation in isolated perfused rat liver.

In this study, the possible role of the hepatic microcirculation in phalloidin-induced cholestasis and hepatotoxicity was examined in isolated perfused rat livers (IPRL). Administration of a phalloidin bolus (1 mg/kg body weight) through the portal vein induced an immediate reduction of bile flow. In 16.9 minutes, bile flow was 50% lower than basal values. Portal pressure was only increased in 60 minutes after phalloidin injection and increased sharply from this time up to the end of perfusion (90 minutes). Under these conditions, phalloidin did not induce liver cell cytolysis, as assessed by aspartate transaminase (AST) and lactate dehydrogenase (LDH) release in the perfusate effluent. Under electron microscopy, hepatocytic vacuolization was mild 15 minutes after phalloidin administration but increased with time. At the end of perfusion, the hepatic architecture was markedly altered; erythrocyte accumulation was observed in both sinusoids and hepatocyte vacuoles. Evaluation by multiple indicator dilution curves showed that extravascular volume (EVV) was significantly affected by phalloidin. It was augmented in 30 minutes after phalloidin administration with values increasing gradually over time. Neither vascular nor cellular volume was altered. The hepatic swelling may be attributed to enlargement of the extravascular space of the liver. These results indicate that changes in the liver microcirculation are not the primary cause of phalloidin-induced cholestasis in the IPRL.

The NACP/synuclein gene: chromosomal assignment and screening for alterations in Alzheimer disease.

The major component of the vascular and plaque amyloid deposits in Alzheimer disease is the amyloid beta peptide (A beta). A second intrinsic component of amyloid, the NAC (non-A beta component of amyloid) peptide, has recently been identified, and its precursor protein was named NACP. A computer homology search allowed us to establish that the human NACP gene was homologous to the rat synuclein gene. We mapped the NACP/synuclein gene to chromosome 4 and cloned three alternatively spliced transcripts in lymphocytes derived from a normal subject. We analyzed by RT-PCR and direct sequencing the entire coding region of the NACP/synuclein gene in a group of patients with familial early onset Alzheimer disease. No mutation was found in 26 unrelated patients. Further studies are required to investigate the implication of the NACP/synuclein gene in Alzheimer disease.

Propranolol metabolism by isolated hepatocytes from normal and cirrhotic rat livers: the effect of albumin.

Several recent reports have shown that the hepatic uptake and subsequent elimination of some substrates is faster in the presence of albumin than in its absence, as if some of the substrate bound to albumin was also available for uptake. In the present study, we examined the effect of albumin on the clearance of propranolol by isolated rat hepatocyte suspensions. The clearance of total drug decreased progressively as albumin concentration increased. There was also a progressive decrease in the free fraction of propranolol and the net result was an increase in the clearance of unbound drug (+50% at 40 g/L albumin). This increase was not due to an oncotic pressure effect of albumin, nor to the presence of fatty acids bound to albumin. The clearance of propranolol by isolated hepatocytes from cirrhotic rats was decreased compared with controls (-50%), and albumin also increased propranolol free clearance, albeit to a lesser extent than in control animals. Our results indicate that albumin facilitates the elimination of propranolol by hepatocytes, possibly because of surface-mediated catalysis of the albumin-propranolol complexes.

[Epidemiology of severe cranial injuries in children and the prognosis of injured patients hospitalized in neurosurgery units]. / Epidémiologie des traumatismes crâniens graves chez l'enfant et prognostic des blessés hospitalisés en neurochirurgie.

Over five years (1979-1983), 1,058 children who sustained injuries in the Val de Marne District (near Paris) were treated by the Emergency and Intensive Care Mobile Unit. Among these patients, 17.8% had an isolated, severe head injury (Glasgow score less than 12) and were admitted to a neurosurgical unit; the age and sex distribution in this group was comparable to that of the entire group of injured children (2/3 boys). The severe head injury was caused by a motor vehicle accident in 47% of cases and a fall in 34% of cases. One half of patients had a skull fracture; all patients with an extra-dural (14 cases) or sub-dural (7 cases) hematoma had a skull fracture. Seventy per cent of patients had cerebral edema and 25% had a meningeal hemorrhage. Immediate severe neurologic disorders (Glasgow score less than 9) were present in 53% of cases and 27% of patients had focal neurologic signs. Mean duration of the stay in the neurosurgical unit was 6 to 15 days. Mortality was 15.3%; in most cases (75%) death occurred within the first 48 hours. One-year morbidity was very significant; 67% of surviving children had residual disease, and 40% had severe sequelae.

Pre hospital intensive care in multiple trauma children.

From January 1979 to December 1984, 1,272 calls, concerning injured children, aged 11 days to 15 years, justified the intervention of a Mobile Medical Emergency and Intensive Care Service, in the department of "Val-de-Marne" near Paris. Three hundred and twenty-two were very serious trauma children (25%); 45 were in cardiac arrest, and 41 died on the scene of the accident despite the intensive cares delivered by the anesthetists or pediatricians. Two hundred and eighty-one children were hospitalized in an intensive multiple trauma pediatric unit (97 cases) or in a neurosurgical pediatric unit (184 cases). The mode of accident was traffic accident (252), fall (48), fire arms (4), knife wounds (7), hanging or strangulation (9), others (2). They concerned 119 females and 203 males. 126 were multiple trauma children (40%). 37% of these accidents happened between May and July, and 40% occurred between 3 to 6 p.m. The 322 children immediately received medical care but 26% died during their hospitalization (17% in the first 24 hours). Thus mortality rate is 35% (114 cases).

Simple method for development of sensitive and specific antiinsulin antisera for laboratory use.

Commercial sources provide good, though expensive antiinsulin antisera. We describe here a simple, fast and inexpensive method for the production of antiinsulin antisera. Purified pork insulin (Lente) was injected subcutaneously in oil/water/complete Freund adjuvant mixture. Three guinea pigs received 0.25 mg of insulin and three received 0.5 mg of insulin. Subsequent injections of the same dose were done 40 and 60 days later. Five animals developed antisera with titers superior to 1:10,000 40 days after the primary inoculation. Four out of five guinea pigs improved further their antibody titer after the 2nd and 3rd injection (p less than 0.0005). Good sensitivity was associated with titers superior to 1:50,000 and appeared only after the 2nd injection to improve further after the 3rd. Thus, four out of six animals developed antiinsulin antisera suitable for the radioimmunoassay (RIA). The antisera bound proinsulin on an equimolar basis with insulin while glucagon was not bound up to 100 ng/ml. The minimum detectable insulin concentration was about 12 pg (0.3 microU) at the optimum antiserum dilution. Six animals given a small dose of insulin (0.06 mg) developed antisera of a low titer and sensitivity, unsuitable for the RIA.