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Microglia are the resident macrophages of the brain, and when activated, have functions including cytokine production, phagocytosis and antigen presentation. The class II MHC genes encode proteins that present antigenic peptides to helper T cells, leading to T cell activation and the development of an antigen-specific immune response. Class II MHC gene expression is strictly regulated by the class II transactivator (CIITA) transcription factor. In this study, we investigated the effects of various immunomodulatory cytokines on IFN-gamma induction of class II MHC and CIITA gene expression in microglia, both primary microglia and a murine microglial cell line, EOC 20. By flow cytometry analysis we show that IFN-gamma-induced surface expression of class II MHC molecules on EOC 20 cells can be inhibited by the cytokines TGF-beta1, IL-4 and IL-10, but not IL-13. Using a ribonuclease protection assay, we have found that TGF-beta1, IL-4 and IL-10 act by inhibiting the expression of IFN-gamma-induced CIITA mRNA and, in turn, class II MHC mRNA. TGF-beta1, IL-4, and IL-10 inhibition of IFN-gamma-induced CIITA mRNA accumulation was not due to destabilization of CIITA mRNA, suggesting an effect at the level of transcription. In primary murine microglia, IL-10 and TGF-beta1 inhibited IFN-gamma-induced CIITA and class II MHC expression. However, a discordant effect of IL-4 was noted in that IL-4 enhanced IFN-gamma-induced CIITA and class II MHC expression in primary microglia. Although some differences are observed between EOC 20 cells and primary microglia in terms of responsiveness to TGF-beta, IL-4 and IL-10, CIITA and class II MHC gene expression are coordinately modulated.

The largest subunit of RNA polymerase II has an intriguing feature in its carboxyl-terminal domain (CTD) that consists of multiple repeats of an evolutionary conserved motif of seven amino acids. CTD phosphorylation plays a pivotal role in controlling mRNA synthesis and maturation. In exponentially growing cells, the phosphate turnover on the CTD is fast; it is blocked by common inhibitors of transcription, such as 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole and actinomycin D. Transcription-independent changes in CTD phosphorylation are observed at critical developmental stages, such as meiosis and early development.

Acetylcholine mediates cell-to-cell communications in the skin. Human epidermal keratinocytes respond to acetylcholine via two classes of cell-surface receptors, the nicotinic and the muscarinic cholinergic receptors. High affinity muscarinic acetylcholine receptors (mAChR) have been found on keratinocyte cell surfaces at high density. These receptors mediate effects of muscarinic drugs on keratinocyte viability, proliferation, adhesion, lateral migration, and differentiation. In this study, we investigated the molecular structure of keratinocyte mAChR and their location in human epidermis. Polymerase chain reaction amplification of cDNA sequences uniquely present within the third cytoplasmic loop of each subtype demonstrated the expression of the m1, m3, m4, and m5 mAChR subtypes. To visualize these mAChR, we raised rabbit anti-sera to synthetic peptide analogs of the carboxyl terminal regions of each subtype. The antibodies selectively bound to keratinocyte mAChR subtypes in immunoblotting membranes and epidermis, both of which could be abolished by preincubating the anti-serum with the peptide used for immunization. The immunofluorescent staining patterns produced by each antibody in the epidermis suggested that the profile of keratinocyte mAChR changes during epidermal turnover. The semiquantitative analysis of fluorescence revealed that basal cells predominantly expressed m3, prickle cells had equally high levels of m4 and m5, and granular cells mostly possessed m1. Thus, the results of this study demonstrate for the first time the presence of m1, m3, m4, and m5 mAChR in epidermal keratinocytes. Because keratinocytes express a unique combination of mAChR subtypes at each stage of their development in the epidermis, each receptor may regulate a specific cell function. Hence, a single cytotransmitter, acetylcholine, and muscarinic drugs may exert different biologic effects on keratinocytes at different stages of their maturation.

Microglia are one of the major glial cell types within the central nervous system, and can function as immune effector cells upon activation. CD40 is a cell surface receptor belonging to the TNF receptor family that plays a critical role in the regulation of immune responses. In this study, we investigated the expression of CD40 on microglia, and the role of transforming growth factor-beta (TGF-beta), an immunosuppressive cytokine, in regulating CD40 expression. Microglia constitutively express very low levels of CD40, and IFN-gamma enhances CD40 mRNA and protein expression in these cells. IFN-gamma-induced CD40 mRNA expression is partially sensitive to the protein synthesis inhibitor puromycin, suggesting that ongoing protein synthesis is necessary for optimal induction of CD40 mRNA by IFN-gamma. TGF-beta inhibits IFN-gamma-induced CD40 protein and mRNA expression. Inhibition of IFN-gamma-induced CD40 mRNA levels by TGF-beta in microglia is not due to inhibition of CD40 transcription; rather, inhibition is due to enhanced degradation of CD40 mRNA. These results indicate that TGF-beta can inhibit expression of an immunologically important receptor, CD40, in microglia, and does so at the post-transcriptional level by destabilizing CD40 mRNA. TGF-beta inhibition of CD40 expression may be one of the mechanisms by which TGF-beta exerts its suppressive effects on immune responses.

The prevalence of hepatitis B virus (HBV), hepatitis C virus (HCV), and GB virus C or hepatitis G virus (GBV-C/HGV) infections was determined in 289 patients with liver disease in Ho Chi Minh City and 890 healthy inhabitants of its rural area, Dalat City, Vietnam, respectively. Serum HCV RNA and GBV-C/HGV RNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). HBsAg, HCV antibodies, and GBV-C/HGV RNA were detected in 139 (47%), 69 (23%), and ten (3%) subjects, respectively, often accompanied by elevated serum levels of alanine aminotransferase. HBsAg and HCV antibodies or HCV antibodies and GBV-C/HGV RNA were detectable simultaneously in 8% and 2% of the patients, respectively. In the inhabitants, HBsAg, HCV antibodies, and GBV-C/HGV RNA were found in 51 (5.7%), nine (1.0%), and 11 (1.2%) subjects, respectively. Thus, the prevalence of HBsAg, HCV antibodies, and GBV-C/HGV RNA was significantly higher in liver disease patients than those in the general population. In the samples from 69 patients and nine inhabitants who were seropositive for HCV antibodies, HCV RNA was detectable in 42 (61%) and 4 (44%), respectively. In patients with liver disease, ten belonged to HCV genotype 1a, ten to HCV 1b, three to HCV 2a, four to HCV 2b, and two to HCV 3a by PCR with genotype-specific primers. Nine patients had mixed genotypes, and the remaining four were not classified. Of the GBV-C/HGV RNA-positive individuals, two patients and two inhabitants were positive for HBsAg, while none of the residents had HCV antibodies, although six HCV antibodies (60%) and four HCV RNA (40%) were found in patients. When a phylogenetic tree of GBV-C/HGV was constructed based on the nucleotide sequences, the 21 isolates were classified into at least two genotypes; four isolates belonged to G2, and 17 to G3. The results indicate that in Ho Chi Minh HCV infection prevails with broad distribution of genotypes together with HBV infection among patients with liver disease. This study suggests that GBV-C/HGV infection occurs independently in the two different districts in association with HCV infection.

The purpose of this study was to investigate the possibility of direct toxic effects of nicotine (Nic) on human bronchial epithelial cells (BEC) suggested by our previous findings of functional nicotinic acetylcholine receptors (nAChRs) in the epithelial cells lining mucocutaneous membranes. We now demonstrate for the first time that human and murine BEC both in vivo and in vitro express functional nAChRs, and that classic alpha 3, alpha 4, alpha 5 and alpha 7 subunits can contribute to formation of these acetylcholine-gated ion channels. In human bronchial and mouse lung tissues, and in cultures of human BEC, the nAChRs were visualized by subunit-specific antibodies on the cell membranes, particularly at the sites of cell-to-cell contacts. The epithelial cells of submucosal glands abundantly expressed alpha 7 nAChRs. Smoking significantly (p < 0.05) increased the relative numbers of nAChRs, and this effects could be reproduced in cultures of BEC exposed to 10 microM Nic. At a higher dose, Nic decreased the relative numbers of alpha 5-containing nAChRs, suggesting a role for receptor desensitization. The function of the nAChR channels expressed by BEC was demonstrated by biphasic increase in the concentrations of intracellular calcium ([Ca++]i) in response to activation of the channel by Nic and fluctuations of [Ca++]i due to channel blockade by mecamylamine (Mec). Long-term exposure to milimolar concentrations of Nic resulted in a steady increase of [Ca++]i, which may lead to cell damage. The biological roles of epithelial nAChRs apparently involve regulation of cell-to-cell communications, adhesion and motility, because Mec caused rapid and profound changes in these cell functions which were reversible by Nic. An over exposure of BEC to Nic, however, produced an antagonist-like effect, suggesting that the pathobiological effects of Nic toxicity might result from both activation of nAChR channels and nAChR desensitization. We conclude that medical consequences of smoking can be mediated by direct toxic effects of inhaled Nic on the respiratory tissues wherein Nic specifically binds to and activates the nicotinic ion channels present on the cell surfaces of BEC. We believe that outside the neural system Nic interferes with functioning of non-neuronal cholinergic networks by displacing from nAChR its natural ligand acetylcholine which acts as a local hormone or cytokine in a variety of non-neuronal locations.

Wilson disease (WD), an autosomal recessive disorder of copper transport, is characterized by impaired biliary excretion and by impaired incorporation of copper into ceruloplasmin. Toxic accumulation of copper causes tissue damage, primarily in the liver, brain, and kidneys. The gene for WD (ATP7B) has been cloned, and the protein product is predicted to be a copper-transporting P-type ATPase with high amino acid identity with that for Menkes disease, an X-linked disorder of copper transport. Mutation screening in WD patients has led to the identification of at least 40 mutations. In addition, haplotype analysis using three dinucleotide-repeat markers, D13S314, D13S301, and D13S316, has been a useful indicator of specific mutations. We have determined haplotypes for the patients and their parents and sibs, in 21 unrelated WD families from Japan. Twenty-eight different haplotypes were observed on 42 WD chromosomes. In all the patients, the ATP7B coding sequence, including the intron-exon boundaries, was screened for mutations, by SSCP, followed by direct-sequence analysis of the shifted fragments. We identified 13 mutations, of which 11 mutations are novel, including 7 mutations-1 insertion, 4 deletions, and 2 missense mutations-in the coding region. The mutations reported in previous studies are 2299insC and Arg778Leu. Two patients were shown to have the 2299insC mutation, which has occurred in many different haplotypes in several populations, indicating a mutation hot spot. Primer-extension analysis of ATP7B mRNA has revealed multiple transcription start sites. Four of the novel mutations (three 1-bp changes and one 5-bp deletion) occur in the 5' UTR and may result in altered expression of the WD gene.

We compared two models of cardiac fibrosis in which collagen synthesis is controlled at different levels. Regulation is pretranslational in aldosterone-salt-induced hypertension in young rats and posttranslational in 24-month-old rats. However, little is known about the role of matrix metalloproteinases (MMP) in fibrosis development. Ventricular MMP activities were studied by zymography, and MMP-2 and MMP-1 mRNA levels were determined using slot-blot and ribonuclease protection assay, respectively. After 1 month of aldosterone-salt treatment, proMMP-2, MMP-2, and proMMP-1 collagenolytic activities and their gene expression were unchanged compared with sham-operated rats. After 2 months, total MMP-2 activity was increased by 40% with parallel stimulation of its gene expression. These changes were localized by in situ zymography within the media of coronary vessels. These results suggest that MMP play a prominent role in vascular remodeling during the first steps of hypertension. During aging, however, there were 40% and 45% decreases in MMP-2 and proMMP-1 activity, respectively, with a corresponding down-regulation of MMP-2 mRNA. These observations suggest that depression of the degradative pathway is partly responsible for age-associated fibrosis. Thus, MMP have differing involvements in the cardiac remodeling associated with hypertension or aging.

Alpha-Amanitin is a well-known specific inhibitor of RNA polymerase II (RNAPII) in vitro and in vivo. It is a cyclic octapeptide which binds with high affinity to the largest subunit of RNAPII, RPB1. We have found that in murine fibroblasts exposure to alpha-amanitin triggered degradation of the RPB1 subunit, while other RNAPII subunits, RPB5 and RPB8, remained almost unaffected. Transcriptional inhibition in alpha-amanitin-treated cells was slow and closely followed the disappearance of RPB1. The degradation rate of RPB1 was alpha-amanitin dose dependent and was not a consequence of transcriptional arrest. Alpha-Amanitin-promoted degradation of RPB1 was prevented in cells exposed to actinomycin D, another transcriptional inhibitor. Epitope-tagged recombinant human RPB1 subunits were expressed in mouse fibroblasts. In cells exposed to alpha-amanitin the wild-type recombinant subunit was degraded like the endogenous protein, but a mutated alpha-amanitin-resistant subunit remained unaffected. Hence, alpha-amanitin did not activate a proteolytic system, but instead its binding to mRPB1 likely represented a signal for degradation. Thus, in contrast to other inhibitors, such as actinomycin D or 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole, which reversibly act on transcription, inhibition by alpha-amanitin cannot be but an irreversible process because of the destruction of RNAPII.

Pseudomonas aeruginosa has an anabolic (ArgF) and a catabolic (ArcB) ornithine carbamoyltransferase (OTCase). Despite extensive sequence similarities, these enzymes function unidirectionally in vivo. In the dodecameric catabolic OTCase, homotropic cooperativity for carbamoylphosphate strongly depresses the anabolic reaction; the residue Glu1O5 and the C-terminus are known to be essential for this cooperativity. When Glu1O5 and nine C-terminal amino acids of the catabolic OTCase were introduced, by in vitro genetic manipulation, into the closely related, trimeric, anabolic (ArgF) OTCase of Escherichia coli, the enzyme displayed Michaelis-Menten kinetics and no cooperativity was observed. This indicates that additional amino acid residues are required to produce homotropic cooperativity and a dodecameric assembly. To localize these residues, we constructed several hybrid enzymes by fusing, in vivo or in vitro, the E. coli argF gene to the P. aeruginosa arcB gene. A hybrid enzyme consisting of 101 N-terminal ArgF amino acids fused to 233 C-terminal ArcB residues and the reciprocal ArcB-ArgF hybrid were both trimers with little or no cooperativity. Replacing the seven N-terminal residues of the ArcB enzyme by the corresponding six residues of E. coli ArgF enzyme produced a dodecameric enzyme which showed a reduced affinity for carbamoylphosphate and an increase in homotropic cooperativity. Thus, the N-terminal amino acids of catabolic OTCase are important for interaction with carbamoylphosphate, but do not alone determine dodecameric assembly. Hybrid enzymes consisting of either 26 or 42 N-terminal ArgF amino acids and the corresponding C-terminal ArcB residues were both trimeric, yet they retained some homotropic cooperativity. Within the N-terminal ArcB region, a replacement of motif 28-33 by the corresponding ArgF segment destabilized the dodecameric structure and the enzyme existed in trimeric and dodecameric states, indicating that this region is important for dodecameric assembly. These findings were interpreted in the light of the three-dimensional structure of catabolic OTCase, which allows predictions about trimer-trimer interactions. Dodecameric assembly appears to require at least three regions: the N- and C-termini (which are close to each other in a monomer), residues 28-33 and residues 147-154. Dodecameric structure correlates with high carbamoylphosphate cooperativity and thermal stability, but some trimeric hybrid enzymes retain cooperativity, and the dodecameric Glu1O5-->Ala mutant gives hyperbolic carbamoylphosphate saturation, indicating that dodecameric structure is neither necessary nor sufficient to ensure cooperativity.

Upon heat stress, the cell physiology is profoundly altered. The extent of the alterations depends on the severity of the stress and may lead to cell death. The heat shock response is an array of metabolic changes characterized by the impairment of major cellular functions and by an adaptative reprogramming of the cell metabolism. The enhanced synthesis of the HSPs is a spectacular manifestation of this reprogramming. Numerous post translational modifications of proteins occur in response to heat stress and can be related to altered cellular functions. Some proteins are heat-denatured and temporarily inactivated. Heat-denaturation is reversible, chaperones may contribute to the repair. The extent of heat-denaturation depends on the cell metabolism: (a) it is attenuated in thermotolerant cells or in cells overexpressing the appropriate chaperones (b) it is enhanced in energy-deprived cells. Covalent modifications may also rapidly alter protein function. Changes in protein glycosylation, methylation, acetylation, farnesylation, ubiquitination have been found to occur during stress. But protein phosphorylation is the most studied modification. Several protein kinase cascades are activated, among which the various mitogen activated protein kinase (MAP kinase) cascades which are also triggered by a wide range of stimuli. As a possible consequence, stress modifies the phosphorylation status and the activity of components from the transcriptional and translational apparatuses. The same kinases also target key enzymes of the cellular metabolism. Protein denaturation results in constitutive hsp titration, this titration is a signal to trigger the heat-shock gene transcription and to activate some of the protein kinase cascades.

Protein denaturation and aggregation are most likely the cause for the noxious effects of heat shock. There are some indications that the nucleus is one of the most sensitive cellular compartments. To test the possibility that the intranuclear microenvironment might be detrimental to the heat stability of proteins, we compared the in situ thermal stability of a reporter protein localized in the nucleus or in the cytoplasm. A recombinant firefly (Photynus pyralis) luciferase carrying a point mutation in the C-terminal domain remains in the cytoplasm (cyt-luciferase). A nuclear localization sequence was fused to the N-terminal domain of cyt-luciferase; the resulting nuc-luciferase was efficiently targeted to the cell nucleus. In both cases, decreased luciferase activity and solubility were found in lysates from heat-shocked cells. These characteristics were taken as an indication of thermal denaturation in situ. The heat-inactivated luciferases were partially reactivated during recovery after stress, indicating the capacity of both the cytoplasmic and nuclear compartments to reassemble proteins from an aggregated state. Although both the nuc- and the cyt-luciferases were heat inactivated at similar rates in vitro, nuc-luciferase was more susceptible to thermal denaturation in situ compared to cyt-luciferase. This observation suggests that the microenvironment of an intracellular compartment may modulate the thermal stability of proteins. The local concentration might be one element of this microenvironment affecting the heat-stability of proteins. In cells made thermotolerant by a priming shock, the thermal inactivation of the recombinant luciferases occurred at a slower rate during a second challenging stress. However, this decreased thermal sensitivity was less pronounced for the nuc-luciferase (threefold) than for the cyt-luciferase (sevenfold). The nuclear luciferase might become a useful tool to investigate the action of molecular chaperones in the nucleus.

RNA polymerase (RNAP) II is a multisubunit enzyme composed of several different subunits. Phosphorylation of the C-terminal domain (CTD) of the largest subunit is tightly regulated. In quiescent or in exponentially growing cells, both the unphosphorylated (IIa) and the multiphosphorylated (IIo) subunits of RNAP II are found in equivalent amounts as the result of the equilibrated antagonist action of protein kinases and phosphatases. In Drosophila and mammalian cells, heat shock markedly modifies the phosphorylation of the RNAP II CTD. Mild heat shocks result in dephosphorylation of the RNAP II CTD. This dephosphorylation is blocked in the presence of actinomycin D, as the CTD dephosphorylation observed in the presence of protein kinase inhibitors. Thus, heat shock might inactivate CTD kinases which are operative at normal growth temperatures, as some protein kinase inhibitors do. In contrast, severe heat shocks are found to increase the amount of phosphorylated subunit independently of the transcriptional activity of the cells. Mild and severe heat shocks activate protein kinases, which then phosphorylate, in vitro and in vivo, the CTD fused to beta-galactosidase. Most of the heat-shock-activated CTD kinases present in cytosolic lysates co-purify with the activated mitogen-activated protein (MAP) kinases, p42mapk and p44mapk. The weak CTD kinase activation occurring upon mild heat shock might be insufficient to compensate for the heat inactivation of the already existing CTD kinases. However, under severe stress, the MAP kinases are strongly heat activated and might prevail over the phosphatases. A survey of different cells and different heat-shock conditions shows that the RNAP II CTD hyperphosphorylation rates follow the extent of MAP kinase activation. These observations lead to the proposal that the RNAP II CTD might be an in vivo target for the activated p42mapk and p44mapk MAP kinases.

Massage and Acupression have a history of many years of use by the Vietnamese people in the treatment of diseases, and they can give wonderful therapeutic effects in painful syndromes and chronic diseases, etc. On the other hand, some methods of Chrono-Acupuncture based on chronobiological theory and the holistic concept of traditional medicine are studied and applied in clinical applications. This paper presents the therapy advice system based on Chrono-Massage and Acupression using the method of ZiWuLiuZhu called CHROMASSI. The system includes four major parts. Massage and Acupression Teaching: This part can provide the user with some background in Massage and Acupression theory such as the pathology of the meridians, the classification of points and their function, the therapeutic properties of points, the methods of Massage and Acupression (including Pression, Friction, Rubbing, Light Massage, Petrissage, Rolling and Rubbing, Percussion and Vibration), and the direction of the meridians circulation, displaying AcuPoints represented by color pictures of the 12 main meridians and 2 vessels. More than 330 popular AcuPoints are used in the system. Open AcuPoint Calculating: This module can help us to calculate open AcuPoints based on data about days, months, years and hours using the special method of ZiWuLiuZhu. The Points adopted by ZiWuLiuZhu are the Five Shu Points and Source Points including 66 points (all of them are located below the elbows and knees). The effectiveness of these points becomes particularly evident when they are needled or punctured at optimum time intervals. For example, at 9:00 a.m., September 22, 1994, the open Points by the ZiWuLiuZhu method will be the points K2 (Nhien Coc) and K10 (Am Coc). According to the chronotherapeutic method, first we have to pressure (or puncture) the above points in order to attain the sensation RDac KhiS (arrival of energy), then pressure the other treating points as in ordinary Massage and Acupression. Therapy Consultation: Knowledge of the system was provided by Prof. Nguyen Van Thang and Doctor Nguyen Nhu Oanh at the Vietnam National Institute of Oriental Medicine. CHROMASSI is able to advise on ways to treat about 153 diseases and symptoms in the following fields: Aches and Pains, Insomnia, Common Cold and Influenza, Sexual Disturbances, Medical Aesthetics in Face, Breast and Buttock, Hygiene, Cardio-Vascular Tract, Digestive Tract, Urinary Tract, Respiratory Tract, Genital Tract, Ear-Nose-Throat Tract, Nervous Tract. The system can provide information about Remarks, Acupoints formulas for treating by Massage and Acupression with colour pictures of meridians. Explanation: The CHROMASSI system can explain why the AcuPoints are used for treating diseases based on the theoretical bases of traditional Vietnamese medicine and on the meridians and collaterals system theory. The colour pictures representing the circulation of vital energy in the meridians are used for explanation. The CHROMASSI system was developed in TURBO-PROLOG and TURBO-PASCAL and can run on IBM PC/AT computers and compatibles. The system can be used for teaching and for clinics of Massage and Acupression combined with Chronotherapeutics. At present the system is used by some physicians for clinical applications. The first results indicate that, in 20 cases of generalized headache compared with the control group, the combining of chronoacupression using the ZiWuLiuZhu method and ordinary Massage and Acupression gave better effects than that obtained by either method alone.

To investigate the nature of the genetic component controlling susceptibility to leprosy and its subtypes, 402 nuclear families were ascertained through a leprosy patient followed at the Dermatology Hospital in Ho Chi Minh City, Vietnam; 285 families were of Vietnamese origin and 117 were of Chinese origin with a higher proportion of lepromatous forms among Chinese patients. Segregation analyses were conducted using the model developed by Abel and Bonney [(1990) Genet Epidemiol 7:391-407], which accounted for variable age of onset and time-dependent covariates. Three phenotypes were considered: leprosy per se (all forms of leprosy together), nonlepromatous leprosy, and lepromatous leprosy. For each of this phenotype, analyses were performed on the whole sample and separately on the Vietnamese and the Chinese families. The results showed that a single Mendelian gene could not account for the familial distributions of leprosy per se and its two subtypes in the whole sample. However, these results were different according to the ethnic origin of the families. In the Vietnamese subsample, there was evidence for a codominant major gene with residual familial dependences for the leprosy per se phenotype, and borderline rejection of the Mendelian transmission hypothesis for the nonlepromatous phenotype. In Chinese families, strong rejection of Mendelian transmission was obtained in the analysis of leprosy per se, and no evidence for a familial component in the distribution of the nonlepromatous phenotype was observed. For the lepromatous phenotype, the discrimination between models was poor, and no definitive conclusion could be reached. Referring to immunological data, we suggest that these results could be explained by a heterogeneity in the definition of the lepromatous phenotype. It is likely that progress in the understanding of the genetic components involved in the expression of leprosy will come from a better definition of the phenotype under study, and immunological studies are ongoing in this population to investigate this hypothesis.

Pseudomonas aeruginosa has a pair of distinct ornithine carbamoyltransferases. The anabolic ornithine carbamoyltransferase encoded by the argF gene catalyzes the formation of citrulline from ornithine and carbamoylphosphate. The catabolic ornithine carbamoyltransferase encoded by the arcB gene promotes the reverse reaction in vivo; although this enzyme can be assayed in vitro for citrulline synthesis, its unidirectionality in vivo is determined by its high concentration at half maximum velocity for carbamoylphosphate ([S]0.5) and high cooperativity toward this substrate. We have isolated mutant forms of catabolic ornithine carbamoyltransferase catalyzing the anabolic reaction in vivo. The corresponding arcB mutant alleles on a multicopy plasmid specifically suppressed an argF mutation of P. aeruginosa. Two new mutant enzymes were obtained. When methionine 321 was replaced by isoleucine, the mutant enzyme showed loss of homotropic cooperativity at physiological carbamoylphosphate concentrations. Substitution of glutamate 105 by lysine resulted in a partial loss of the sigmoidal response to increasing carbamoylphosphate concentrations. However, both mutant enzymes were still sensitive to the allosteric activator AMP and to the inhibitor spermidine. These results indicate that at least two residues of catabolic ornithine carbamoyltransferase are critically involved in positive carbamoylphosphate cooperativity: glutamate 105 (previously known to be important) and methionine 321. Mutational changes in either amino acid will affect the geometry of helix H2, which contains several residues required for carbamoylphosphate binding.

The largest subunit of RNA polymerase (RNAP) II contains at it C-terminus an unusual domain comprising tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This C-terminal domain (CTD) can undergo phosphorylation at multiple sites giving rise to a form of the enzyme designated RNAP IIO. The unphosphorylated form is designated RNAP IIA. The largest subunits of RNAPs IIO and IIA are designated IIo and IIa, respectively. In quiescent NIH 3T3 fibroblasts, subunits IIo and IIa are present in comparable amounts. Upon serum stimulation, the amount of subunit IIo increases markedly and remains elevated for several hours. The increase of subunit IIo also occurs in transcription-inhibited cells and, therefore, is not a consequence of serum-activated transcription. This observation suggests that serum stimulation activates a CTD kinase and/or inhibits a CTD phosphatase. This hypothesis is supported by the finding that serum stimulates phosphorylation of a beta-galactosidase-CTD fusion protein expressed in these cells. Furthermore, an enhanced CTD kinase activity was discovered in lysates from serum-stimulated fibroblasts and was found to copurify with MAP kinases on a Mono Q column and to bind to anti-MAP kinase antibodies. The idea that MAP kinases phosphorylate the CTD in vivo is supported by the observation that subunit IIa, but not subunit IIb which lacks the CTD, is phosphorylated at multiple sites by purified MAP kinase. Consequently, the MAP kinases are a new class of CTD kinases which appear to be involved in the phosphorylation of RNAP II following serum stimulation. This phosphorylation may contribute to the transcriptional activation of serum-stimulated genes.

Appendicitis is a frequently encountered surgical emergency which can be diagnosed by clinical and laboratory criteria. But there are some atypical cases that ultrasound can help to diagnose. From April the 9th 1992 to April the 11th 1993, we have examined by ultrasound 465 patients entered in our hospital for right lower quadrant pain. 190 patients have been diagnosed as appendicitis and operated (true positive = 124 cases, false negative = 54 cases and false positive = 12 cases). 275 patients have been diagnosed as negative and they are not operated (true = 273 cases, false positive = 2 cases). So in our study, the sensitivity is 70%, the specificity is 95% and the accuracy is 85%. Our scientific report has the objective to show the usefulness and the limits of ultrasound in the diagnosis of appendicitis.

Heat shock proteins (HSP) belong to a family of highly homologous proteins that are encoded by several genes and function as protein chaperones. A number of HSP genes have been described in the major histocompatibility complex (MHC) on chromosome 6. We have cloned and mapped one chromosome 14 HSP gene (HSPA2) using a genomic probe (pH2.3) derived from one of the HSP genes present in the MHC, which was previously shown to also detect sequences on chromosome 14. Screening of a chromosome 14-specific cosmid library yielded one relevant clone that contains an HSP-like sequence, showing a high degree of identity with the HSP genes from the MHC and with HSP70.2 (Hspa2) in the mouse. This new HSP gene is expressed abundantly in muscle, heart, oesophagus and brain, and to a lesser extent in testis. Its localization to 14q22 was established by using a somatic cell hybrid panel and FISH analysis. A pentanucleotide repeat motif, showing seven alleles, was also identified in the cosmid clone.

The long arm of human chromosome 14 (14q), an acrocentric chromosome, is estimated to be approximately 93 Mb in length. Genetic maps published to date have included mostly random DNA markers. The map we present here includes 13 genes, as well as 29 random DNA segments, based on typing of the CEPH families. Our map has been placed in context with other published maps. The resulting map of 42 loci has markers on average 5 cM apart and has a total length of 163 cM. The map order differs in the telomeric region from that of other maps previously published. 14q has homology, throughout approximately half its length, with mouse chromosome 12 and has a proximal region of homology with mouse chromosome 14.

The RNA polymerase IIO and IIA differ by the extent of phosphorylation in the carboxyl-terminal domain (CTD) of the largest subunit. It has been proposed that the IIA form of RNA polymerase II interacts with the promoter to form a stable preinitiation complex whereas the IIO form would be generated upon entry into initiation of transcription. Phosphorylation of the CTD might be required to release the interaction between the polymerase and the promoter binding factors. In this paper, we show that in the presence of actinomycin D, the phosphorylated IIO form accumulates. In contrast, the dephosphorylated IIA form accumulates while the amount of phosphorylated IIo form decreases in cells treated with CTD-kinase inhibitors such as the nucleoside analog, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole or the isoquinoline sulfonamide derivatives H-7* or H-8. These changes are fast and suggest a very rapid phosphate turnover on the CTD. Transcription is inhibited in intact cells by drug concentrations that are effective in altering CTD phosphorylation, although no causal relationship is established yet. These effects do not concern other cellular functions such as protein synthesis. Thus isoquinoline sulfonamide derivatives might be helpful to further dissect the role of CTD phosphorylation in transcription.

In an attempt to understand the influence of the intracellular environment on protein stability, the thermal denaturation of various reporter proteins was examined within cultured mammalian cells. Loss of solubility and of enzymatic activities were taken as indicators of thermal denaturation. Photinus pyralis luciferase, Escherichia coli beta-galactosidase, the 70-kDa constitutive heat-shock proteins and the 68-kDa dsRNA-dependent protein kinase are found mostly in the supernatant fractions of centrifuged lysates from control unshocked mammalian cells. However, when cells are lysed after heat shock, a proportion of the reporter molecules is found to be aggregated to the nuclear pellets. This insolubilization does not affect all cellular proteins; many of them remain unaffected by heat shock. The heat-induced insolubilization of all four reporter proteins is markedly enhanced when the intracellular ATP concentration is drastically decreased after inhibition of both oxidative phosphorylation and glycolysis. Although ATP molecules bind to luciferase and protect it from thermal inactivation in vitro, the consequences of strong ATP depletion on luciferase thermal stability within the cells are found to be much greater than expected from in vitro data. The 70-kDa constitutive heat-shock proteins and the 68-kDa protein kinase are ATP-binding proteins but ATP depletion also considerably increases the aggregation of beta-galactosidase to the nuclear pellets, although this enzyme is not known to be an ATP-binding molecule. Insolubilization of all four reporter proteins occurs in ATP-depleted cells even at normal growing temperatures (37 degrees C). Protein denaturation may be enhanced either by the aggregation and disappearance of the intracellular 'free' chaperones or by the trapping of unfolded protein molecules on chaperones; the chaperone/unfolded protein complexes could not dissociate in the absence of ATP. Enhanced protein denaturation due to ATP depletion is proposed to account for the greater heat sensitivity of ATP-depleted cells and for the ability of mitochondrial uncouplers to trigger a heat-shock response in some cells.

Since 1990, the creation of a Rehabilitation Center for Vietnamese leprosy patients under the aegis of "Oeuvres Hospitalières Françaises de l'Ordre de Malte" is the result of emphasized collaboration between governmental and non-governmental organizations, and between medical and paramedical specialists. This humanitarian action is not "a present home delivery". The end of this action is to set progressively a realist enterprise that depends on preliminary epidemiologic investigations on the spot to analyse means and necessities. Frequency of disabilities (49.6%) and predominance of grades 1 and 2 (83.5%) require aids. The realisation of technology transfer at all medical care levels is necessary. But at the same time, it is essential to build surroundings adapted infrastructure, to equip with effective material giving comfort and security for patients, and even to supply with pharmaceutic drugs in order to continue rehabilitation's activities. In short range, patient's selection and regular control give first objective results.

From November 1990 to June 1991, 33 cases of acute melioidosis were diagnosed in tropical Northern Territory, Australia during an exceptionally wet monsoon. Eighteen (55%) were alcoholic, 16 (48%) diabetic and only 4 (12%, all survivors) had no risk factors. Twenty-seven (82%) were considered recent infection, with an incubation period of 3-21 days (mean 14) documented in eight cases with presumed cutaneous inoculation. Fourteen patients presented with pneumonia (4 septicemic) and of 11 others with septicemia 4 had genitourinary foci. Three of 4 with splenic abscesses required splenectomy. Three had only skin/soft tissue infection. One patient with brainstem encephalitis needed prolonged ventilation. Overall mortality was 36% (12 cases, including three relapses), despite therapy with ceftazidime and intensive care facilities. Pseudomonas pseudomallei is the commonest diagnosed cause of fatal bacteremic pneumonia at Royal Darwin Hospital and emphasis is placed on early appropriate antibiotic therapy and compliance with maintenance therapy for at least three months.

The substitution of alanine for glutamate at position 105 (E105A) of the allosteric ornithine carbamoyltransferase (OTCase) of Pseudomonas aeruginosa abolishes the carbamoylphosphate (CP) cooperativity observed in the wild-type enzyme. A kinetic analysis of [E105A]OTCase was performed in order to determine the mechanism of the reaction. The results of initial velocity and inhibition studies are consistent with an ordered mechanism with CP as the first substrate to add to the enzyme. In addition, similar studies have been made using the wild-type enzyme in the presence of the activator, phosphate. The results are similar to those obtained with [E105A]OTCase indicating that the residue E105 is critical for the allosteric transition of the wild-type enzyme. The activities of the wild-type allosteric OTCase and of [E105A]OTCase have been studied in the pH range 5.8-8.2 in the absence and in the presence of positive and negative effectors. The sigmoid saturation of OTCases by CP has been analyzed according to the Hill equation. At low pH values, CP cooperativity is low in the wild-type enzyme but cooperativity and [S]CP0.5 values increase markedly with pH. For [E105A]OTCase, the saturation by CP is hyperbolic at all pH values; in this modified enzyme, the presence of spermidine, an allosteric inhibitor of the wild-type enzyme, results in an inhibition which induces CP cooperativity. Thus, the ionization of the residue E105 apparently results in a conformational change in the wild-type enzyme which modifies the catalytic site. Since the [E105A] enzyme retains the heterotropic effects of the wild-type enzyme, other structural features are required for the allosteric transition in the wild-type catabolic OTCase.

Comparative trials of pirantel analogs--combatrin (Pfizer, Philippines) and embovin (Russia) were carried out in a hyperendemic focus of intestinal nematodoses in Vietnam. The drugs were prescribed in a single dose of 10 mg/kg. No significant differences in the efficacies of the drugs were revealed: combatrin and embovin efficacies in ascariasis were 94.3 and 98.0%, respectively (p > 0.05), in ankylostomiasis 76.3 and 58.3%, respectively (p > 0.05).

Analysis of the stress response of closely related rat hepatoma clones revealed that the major inducible heat-shock protein 68 (HSP68) was only slightly inducible upon stress in the glucocorticoid-resistant, dedifferentiated clone-2 cells, but strongly activated in the differentiated, glucocorticoid-sensitive Faza 967 cells from which clone 2 was derived. The decreased inducibility of HSP68 in clone-2 cells was not the consequence of altered kinetics of protein synthesis recovery, was not correlated with the deficient inducibility of other major heat-shock proteins and had no effect on the heat sensitivity of the cells. This deficiency was observed after treatment with mild and strong heat and various chemicals. The results of nuclear run-on experiments suggested that the impairment of HSP68 mRNA induction most likely occurs at the transcriptional level and is probably specific for the corresponding gene. In Faza 967 and clone-2 cells, stress activated comparable levels of heat-shock-factor binding to the heat-shock element, and the expression of a reporter gene under the control of murine HSP70.1 promoter was strongly stimulated in both cells. Therefore, our results raise the possibility that the deficient stress inducibility of HSP68 is due to some specific regulation of the endogeneous HSP68 gene, rather than to a deficiency of the heat-shock factor or mutation of the corresponding gene.

An intensive focus of intestinal nematodiases in a foothill province of Vietnam is described; the incidence of ascaridiasis there was found to be 81.8%, trichuriasis 78.1%, and ankylostomiasis 12.3%. Polyinvasions were detected in more than 80% of the examinees. Analysis of five schemes of medamin therapy, used in three provinces in 595 patients with ascaridiasis, 490 ones with trichuriasis, and 323 ones with ankylostomiasis has shown that the treatment efficacy was reducing with the invasion intensity augmentation. In ascariasis the 3- and 5-day therapeutic schemes were the most effective, their efficacies being 68.4 and 82.0%, respectively; in trichuriasis and ankylostomiasis a 3-day scheme was the best, its efficacies being 74.0 and 71.9%, respectively. Retrograde ascarid migration occurred in 1.2% of 595 ascaridiasis patients after medamin therapy.

The interaction between human corticosteroid binding globulin and activated leukocytes is restricted to the granulocyte population, and is characterized by specific proteolytic cleavage of corticosteroid binding globulin which markedly reduces its steroid binding activity. A direct interaction between corticosteroid binding globulin and the activated cells appears to enhance this event, and does not involve cellular internalization of corticosteroid binding globulin or its proteolytic degradation products, which resemble those obtained after incubation of corticosteroid binding globulin with neutrophil elastase. These data suggest that corticosteroid binding globulin interacts with elastase on the surface of activated neutrophils, and may promote glucocorticoid delivery to these cells during inflammation.

The efficacy of and tolerance to fancimef in 49 patients was compared to the efficacy of and tolerance to quinine in combination with fansidar in 33 patients with moderate P. falciparum malaria. A good tolerance to and a high efficacy of fancimef have been shown, which was manifested in rapid fever arrest, disappearance of parasitemia, absence of the disease relapses, which were observed in 2 patients on quinine combined with fansidar. The advantage of fansimef is one-time administration of the total course dose.

The cardiovascular effects of the deproteinized blood extract were investigated in anaesthetized dogs: respiration, general metabolism and systemic haemodynamics were tested and the changes in these parameters after the administration of the drug were studied. At 2 ml kg-1 i.v., the deproteinized blood extract induced a transient drop in arterial blood pressure and bradycardia. On the other hand, the total peripheral resistance and the elastic resistance, of the arteries were decreased and this effect was of longer duration. Moreover, the deproteinized blood extract stimulated the myocardium contractile force and dc/dt and increased the cardiac performances. The mechanism of action of this drug is discussed.

In the isolated Rabbit heart, high pressure, on the one hand, inhibits the noradrenaline release, and on the other hand, induces both the biosynthesis and release of PGs(PGE2,PGF2 alpha) from the auricles and mainly from the right auricle i.e. where the sympathetic nerve endings are the most numerous. Therefore, high pressure, by a negative feedback process, plays a great part in moderating the adrenergic neurotransmission responsible for the hypertension. These effects result from a supplementary pressure which acts on the walls of coronary arteries and arterioles and modifies their distensibility. It thus appears that in the walls of arteries and arterioles there are baroreceptors which are sensitive to the pressure variations in order to regulate adrenergic neurotransmission, by means of a reflex path still unknown.

Thymectomy is often successful as treatment for the autoimmune disease myasthenia gravis. One complication of the operation on such patients is respiratory difficulty especially post-operatively, due to interference with the already disturbed transmission of signals along damaged nerve-endings. Acupuncture anesthesia offers a solution to that problem. In this series of 90 patients, the results of operation under conventional general anesthesia were compared with those under acupuncture anesthesia. It was found that patients in the latter group suffered fewer complications.

Rabbit corticosteroid-binding globulin (CBG) from the serum of pregnant and nonpregnant females differs in terms of charge microheterogeneity, and both forms were, therefore, radiolabeled and injected iv into 23- to 27-day pregnant rabbits (n = 6) to assess their biological half-lives and possible transfer to the fetal compartment. After an initially rapid distribution phase, the serum half-lives of both forms of [125I]CBG were essentially identical (approximately 13 h) and did not vary at different gestational ages. There was also no difference in the transfer of either form of [125I]CBG from maternal to fetal compartments in any of the animals studied. Moreover, [125I]CBG showed no sign of degradation and retained its steroid-binding activity in fetal urine and amniotic fluid. Twenty-two hours after administration of [125I]CBG to rabbits (n = 2) at 23 days gestation, its mean level in fetal urine (7 cpm/microliter) and amniotic fluid (2.5 cpm/microliter) was much higher than that in fetal blood (0.6 cpm/microliter). More importantly, the specific activities of [125I]CBG in fetal urine and amniotic fluid were comparable to that in maternal serum, and approximately 2 orders of magnitude higher than that in fetal serum. Taken together, these results suggest that CBG in fetal urine and amniotic fluid is largely of maternal origin, and that maternal CBG crosses the fetal kidney preferentially.

A procedure for isolation of NAD-kinase from rabbit liver resulting in 4000-fold purification and the activity yield of 50-60% is described. The molecular weight of the NAD-kinase subunit determined by SDS electrophoresis is 30 000. The purified enzyme is a dimer. Partially purified preparations of NAD kinase contain multiple forms with mol. Weights ranging from 650 000 to 180 000 and have complex kinetic behaviour. A thermostable activator of NAD-kinase which, when added to the homogeneous enzyme preparation, destroys the linear dependence of the enzyme specific activity on concentration, was detected. The nature of multiple forms of NAD-kinase and the possible role of the activator in their formation is discussed.

The firefly enzyme luciferase catalyzes the luminescent reaction of luciferin with ATP and oxygen. The luciferase gene has recently been cloned and proposed as a reporter gene in procaryotic and eucaryotic cells. We present here a luciferase activity assay which relies on luminescence detection using a standard scintillation counter. This technique is simple, fast, inexpensive, and still very sensitive: as little as 0.02 pg (250,000 molecules) of enzyme is readily detected. The technique is optimized for the luciferase assay in mammalian cell lysates. Thus, the luciferase gene may become a very useful tool for gene regulation studies.

Two hundred cases of lethal outcomes in schizophrenic patients (who died at one of the Moscow hospitals within a period of 1971 to 1978) are analyzed. These were patients with various forms of the disease: continuously progressing (91 cases), paroxysmal progressing (83 cases), and recurrent (26 cases). It was found that the age distribution of the deceased was close to that indicated by world statistics for developed countries with a shift to the left, mainly, at the expense of males who die at a younger age than females. By the time of the death the ratios between the disease forms changed: the percentages of the continuously progressing and paroxysmal progressing forms in the hospital patients got closer (45.5 and 41.5%, respectively); this brought the distribution to the disease forms in those patients nearer to that observed in schizophrenic outpatients. The somatic causes of the death were similar to those characteristic for the general population: these were cardiovascular diseases (43.0%), malignant neoplasms (15.5%), respiratory diseases (11.5%). The underlying psychic disease was the cause of the death in 22% of the cases. It manifested in the forms of terminal hypostatic pneumonia (54.4%); cachexy (25%); an additional psychic disease (13.7%), and a febrile attack (6.8%).U

The effects of a deproteinized blood extract on the modifications of respiration, general metabolism and systemic haemodynamics induced by hypoxia were studied. Under the experimental conditions adopted, the deproteinized blood extract moderated the reactions of the organism which are exacerbated by hypoxia, that is increase in oxygen consumption and stimulation of the cardiac performances. The mechanism of action of the deproteinized blood extract has been discussed.