Synthetic "interface" peptides alter dimeric assembly of the HIV 1 and 2 proteases. Retroviral proteases are obligate homodimers and play an essential role in the viral life cycle. Dissociation of dimers or prevention of their assembly may inactivate these enzymes and prevent viral maturation. A salient structural feature of these enzymes is an extended interface composed of interdigitating N- and C-terminal residues of both monomers, which form a four-stranded beta-sheet. Peptides mimicking one beta-strand (residues 95-99), or two beta-strands (residues 1-5 plus 95-99 or 95-99 plus 95-99) from the human immunodeficiency virus 1 (HIV1) interface were shown to inhibit the HIV1 and 2 proteases (PRs) with IC50's in the low micromolar range. These interface peptides show cognate enzyme preference and do not inhibit pepsin, renin, or the Rous sarcoma virus PR, indicating a degree of specificity for the HIV PRs. A tethered HIV1 PR dimer was not inhibited to the same extent as the wild-type enzymes by any of the interface peptides, suggesting that these peptides can only interact effectively with the interface of the two-subunit HIV PR. Measurements of relative dissociation constants by limit dilution of the enzyme show that the one-strand peptide causes a shift in the observed Kd for the HIV1 PR. Both one- and two-strand peptides alter the monomer/dimer equilibrium of both HIV1 and HIV2 PRs. This was shown by the reduced cross-linking of the HIV2 PR by disuccinimidyl suberate in the presence of the interface peptides. Refolding of the HIV1 and HIV2 PRs with the interface peptides shows that only the two-strand peptides prevent the assembly of active PR dimers. Although both one- and two-strand peptides seem to affect dimer dissociation, only the two-strand peptides appear to block assembly. The latter may prove to be more effective backbones for the design of inhibitors directed toward retroviral PR dimerization in vivo.


Electronic and vibrational spectroscopy of the cytochrome c:cytochrome c oxidase complexes from bovine and Paracoccus denitrificans. The 1:1 complex between horse heart cytochrome c and bovine cytochrome c oxidase, and between yeast cytochrome c and Paracoccus denitrificans cytochrome c oxidase have been studied by a combination of second derivative absorption, circular dichroism (CD), and resonance Raman spectroscopy. The second derivative absorption and CD spectra reveal changes in the electronic transitions of cytochrome a upon complex formation. These results could reflect changes in ground state heme structure or changes in the protein environment surrounding the chromophore that affect either the ground or excited electronic states. The resonance Raman spectrum, on the other hand, reflects the heme structure in the ground electronic state only and shows no significant difference between cytochrome a vibrations in the complex or free enzyme. The only major difference between the Raman spectra of the free enzyme and complex is a broadening of the cytochrome a3 formyl band of the complex that is relieved upon complex dissociation at high ionic strength. These data suggest that the differences observed in the second derivative and CD spectra are the result of changes in the protein environment around cytochrome a that affect the electronic excited state. By analogy to other protein-chromophore systems, we suggest that the energy of the Soret pi* state of cytochrome a may be affected by (1) changes in the local dielectric, possibly brought about by movement of a charged amino acid side chain in proximity to the heme group, or (2) pi-pi interactions between the heme and aromatic amino acid residues.


[Effect of ketotifen, nedocromil sodium and budesonide on non-specific bronchial hyperreactivity to histamine in patients with asthma]. The aim of the study was to evaluate the effect of ketotifen, nedocromil sodium and budesonide on non-specific bronchial hyperreactivity in asthmatic patients. The study was performed on a group of 40 patients treated with these drugs for a period of 4-12 weeks. Bronchial provocation with histamine was carried out in all patients before and after therapy. In subjects receiving ketotifen an additional provocation test was performed 3 days and 4 weeks after commencement of treatment. The results showed that drugs inhibiting the release of mediators from mast cells or diminishing the late phase of bronchial allergic reaction significantly reduce non-specific bronchial hyperreactivity.


Excision of a transposable element from a viral vector introduced into maize plants by agroinfection. The geminivirus maize streak virus (MSV) was used as a vector to introduce the maize transposable element Dissociation (Ds) and to study its excision in maize plants. MSV carrying Ds1 in its genome was introduced into maize plants by agroinfection. Excision of the Ds1 element from the MSV genome was detected only when functions from the transposable element Activator (Ac) were supplied in trans, either endogenously by the recipient maize plant or by co-transformation with Agrobacterium carrying a genomic Ac clone. The excision of Ds1 could easily be visualized by the appearance of viral symptoms induced by the revertant virus. The junction sequences left on the MSV genome after excision revealed 'footprints' typical of transposition as described for maize. From these results, we conclude that transposition functions in our system and that the use of the MSV replicon provides a rapid and simple tool for the investigation of the excision of transposable elements in maize plants.


The in-vitro synthesized tomato shikimate kinase precursor is enzymatically active and is imported and processed to the mature enzyme by chloroplasts. Full-length cDNA clones encoding shikimate kinase (EC 2.7.1.71), an enzyme of the central section of the shikimate pathway, have been isolated from tomato (Lycopersicon esculentum L., cv. UC82b). The open reading frame has the capacity to encode a peptide of 300 amino acids. The in-vitro synthesized peptide catalysed the phosphorylation of shikimate thus confirming the identity of the isolated cDNA clones. The N-terminal portion of the deduced amino acid sequence resembles known chloroplast-specific transit peptides. The existence of such a transit peptide was proven by the uptake of the in-vitro synthesized peptide as well as its processing by isolated chloroplasts. Multiple sites of polyadenylation were observed in shikimate kinase mRNAs. The results of Northern and Southern blot analyses are consistent with the existence of only one shikimate kinase gene per haploid genome in tomato. These results are discussed with respect to the dual pathway hypothesis of the shikimate pathway in higher plants.


[Construction of an EB virus based shuttle vector plasmid pZH32]. The shuttle vector can replicate in both bacteria and eukaryotic cells. We have constructed a shuttle vector pZH32 based on the EB virus. Three component parts of the plasmid come from pSV2gpt, pS189 and pMCi5. An E. coli tyrosine suppressor tRNA gene, supF, was used as a mutagenic target in this plasmid, and a gpt gene was used as a selectable marker gene for transformed cells. This pZH32 based on EBV replicates autonomously in the nuclei of human cells. It has a low background mutation frequency. The plasmid can be used to examine the mutagenic mechanism of potential mutagens and carcinogens in mammalian cells.


[An experimental and comparative study of chemical method for etching metal frameworks in adhesive bridge]. Authors studied the best dispensing and the time of nickel base alloy etching gel by using orthogonal design and the scanning electron microscope (SEM) evaluation. Shear bond strengths were compared after treating with metal etching gel, electro-etching, painting KH-570 on the surface of Ni-Cr alloy after treating with the gel. The results showed that the gel was found to be an aqua regia solute gel. The optimum time was 90 mins. Shear stress tests revealed that the highest shear bond strength can be obtained by synthetic methods.


Changes in phosphatidylinositol phospholipase C isoenzymes and in their association with GTP gamma S-binding activity in guinea pig uterine smooth muscle during pregnancy. The nature distribution and associated GTP gamma S binding activity of phosphatidylinositol phospholipase C (PI-PLC) has been studied in non-pregnant and pregnant guinea pig uterine smooth muscle. Cytosolic fractions partially purified by Q-Sepharose and heparin-Agarose chromatography show two isoenzyme forms, one with an apparent molecular weight of 58 kD that crossreacts with PI-PLC alpha and a has Km for phosphatidylinositol of 292 +/- 72.6 microM, designated alpha, and a form that has an apparent molecular weight of 86 kD and a substrate Km of 54 +/- 20 microM designated delta. Approximately 80% of the total PI-PLC activity was recovered in the cytosolic fraction and this increased 8-10 fold for both isoenzymes from the non-pregnant to the late pregnant uterus and the proportion of the alpha isoenzyme increased from approximately 40% to 55% of the total. PI-PLC alpha but not delta activity had GTP gamma S binding activity associated with it after Q-Sepharose or heparin-Agarose chromatography. This associated activity accounted for 2% of the total GTP gamma S-binding activity in the non-pregnant uterus and 31% of that in the near-term uterus. On separation of the PI-PLCa-GTP gamma S-binding complex by gel filtration on Sephacryl S200 gave two peaks one of 118 kD accounting for two-thirds of all the binding and two-thirds of the enzyme activity and a 58 kD peak. The 118 kD peak could not be separated by treatment with 0.5% cholate, but in this form enzyme activity was protected from detergent inactivation found with the 58 kD form. In sodium dodecyl sulphate polyacrylamide-gel electrophoresis PI-PLC alpha was released from the 118 kD complex and showed an apparent molecular weight of 61.5 kD. All the activity in the residual membrane fraction could be released by washing with buffer followed by, 2 M KCl and then 2 M KCl plus 0.5% cholate. This released isoenzyme forms that appeared identical to those in the cytosolic fraction and with GTP gamma S-binding activity associated with PI-PLC alpha. It is concluded that in the near term guinea pig uterus there is a dramatic increase in the capacity for inositol polyphosphate production. Moreover the dramatic increase in GTP gamma S-binding activity associated with PI-PLC alpha implies large changes in the extent and possibly nature of the putative G-protein activation of this pathway.(ABSTRACT TRUNCATED AT 400 WORDS)


Superior division paresis of the oculomotor nerve caused by cryptococcal meningitis. A case of cryptococcal meningitis with unilateral paresis of the superior division of the oculomotor nerve was reported. The ocular signs were completely improved by antifungal therapy. This case demonstrates that divisional oculomotor paresis occurred in the subarachnoid portion of the third cranial nerve before its anatomic bifurcation.


Role of the oxytocin receptor in the choice between cyclicity and gestation in ruminants. The endometrial oxytocin receptor occupies a central point in the choice between luteolysis and pregnancy in ruminants. Receptor expression determines the time at which luteolysis occurs in nonpregnant animals, and thereby determines the duration of the oestrous cycle. Inhibition of receptor expression by trophoblast interferon (IFN) blocks the process of luteal regression and leads to continued progesterone secretion and the successful growth of the conceptus. Trophoblast IFN is probably required from the time of normal luteolysis during the cycle until luteal oxytocin concentrations of pregnancy decline, thus removing the stimulatory mechanism for the release of the episodes of prostaglandin F2 alpha required for luteolysis.


Trophoblast interferons in early pregnancy of domestic ruminants. A type I interferon (IFN) secreted by the trophoblast of early sheep and cow embryos is thought to be responsible for the maternal recognition of pregnancy. The expression of trophoblast IFN is tissue specific and temporally controlled. However, the isolated bovine trophoblast IFN promoter did not confer tissue specificity on the expression of a bacterial chloramphenicol acetyl transferase (CAT) reporter gene, and could not be induced by virus, unlike other type I IFNs. Trophoblast IFN acts locally within the uterus to prevent luteolysis and prolong progesterone secretion. Endometrial IFN receptors are present, and trophoblast IFN decreases expression of endometrial oxytocin receptor and increases expression of endometrial beta 2-microglobulin, MHC class I antigens and Mx (a mediator of IFN antiviral activity) only in the pregnant horn of pregnant ewes with a transected uterus. The primary effect of trophoblast IFN during early pregnancy appears to be an inhibition of oxytocin receptor expression, although studies in ovariectomized ewes suggest that luteal oxytocin may be required to facilitate the inhibition of prostaglandin F secretion by trophoblast IFN. An investigation of the isolated oxytocin receptor promoter should confirm its critical role in the maternal recognition of pregnancy.


Neuroendocrine regulation of sheep fetuses. During fetal development the neuroendocrine system plays a pivotal role in the regulation of normal intrauterine development, growth and differentiation and the onset of birth. Studies on the ontogenic development of neuroendocrine function in sheep fetuses are discussed with particular reference to the differential regulation of the pituitary-gonadal and pituitary-adrenal axis. Fetal pituitary-gonadal activity increases to a maximum at mid-gestation and is suppressed just before birth. Using immunocytochemistry, we have examined the ontogeny of gonadotroph development in the pituitary of female sheep fetuses. At day 70 of gestation (term = 145 days) only immunopositive luteinizing hormone beta (LH beta) cells were present. The number and intensity of staining of these LH beta cells increased by day 100 and declined again by day 130. Immunopositive alpha-subunit and follicle-stimulating hormone beta (FSH beta) cells appeared by day 100 of gestation and had further increased in number and staining intensity by day 130. Treatment of fetuses with the gonadotrophin-releasing hormone (GnRH) agonist, buserelin, from day 70 of gestation results in desensitization of the fetal pituitary gonadotrophs, suppression of pituitary gonadotrophin mRNA and a reduction in the number of immunopositive gonadotrophin-containing cells. Thus, in sheep fetuses the development of cells containing LH and FSH depends critically on an appropriate GnRH signal from the fetal hypothalamus. In contrast, hypothalamo-pituitary-adrenal activity increases during gestation to reach a maximum before birth. This is characterized by a progressive increase in fetal plasma adrenocorticotrophic hormone (ACTH) and cortisol concentrations, and a high frequency of ACTH and cortisol pulses in the final hours before parturition. Steady state concentrations of pro-opiomelanocortin (POMC) mRNA increase throughout fetal development but decline dramatically in the final days before birth, when ACTH concentrations are at a maximum. This decline in POMC expression is probably the result of the negative feedback effects of high cortisol concentrations. The neuroendocrine mechanisms that mediate the pulsatile secretion of ACTH at this crucial time are complex and as yet incompletely defined. However, the opioid antagonist, naloxone, suppresses the secretion of ACTH during the final days before birth, thus providing evidence for the tonic regulation of ACTH secretion by stimulatory endogenous opioids. Prostaglandins that are secreted from the placenta during late gestation stimulate fetal ACTH, but not gonadotrophin secretion, whereas placental steroids are thought to inhibit fetal gonadotrophin secretion.(ABSTRACT TRUNCATED AT 400 WORDS)


Regulation of gene expression in the ovine fetus. Expression of genes encoding pro-opiomelanocortin (POMC), glucocorticoid receptors and 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) was studied in sheep fetuses during development. POMC mRNA was present in the anterior pituitary by day 60 of gestation (term approximately 145 days), and its relative amount did not change significantly until after days 125-130. The amount of POMC mRNA in the pituitary increased significantly at days 138-143, remained high at term and increased further in newborn lambs. In contrast, POMC mRNA could not be detected in the hypothalamus and adrenal glands of fetuses at all ages studied. These results suggest that the prepartum rise in plasma adrenocorticotrophin (ACTH) concentrations in sheep fetuses is due to increased expression of POMC gene in the pituitary. The number of glucocorticoid receptors, but not the amount of glucocorticoid receptor mRNA changed significantly with gestational age in the hypothalamus, anterior pituitary and adrenal glands of the fetus. Changes in glucocorticoid receptor content of fetal tissues may reflect alterations in translation of glucocorticoid receptor mRNA, subsequent modifications, or glucocorticoid receptor turnover or a combination of these factors. However, in newborn lambs, amounts of glucocorticoid receptor mRNA increased significantly in the hypothalamus and pituitary but decreased to undetectable amounts in the adrenal glands, indicating that tissue-specific factors may influence expression of glucocorticoid receptor gene in neonatal sheep. The interconversion of cortisol and cortisone requires 11 beta-HSD. Since cortisone is biologically inactive, 11 beta-HSD may regulate the activity of intracellular cortisol. We cloned and sequenced a cDNA encoding sheep 11 beta-HSD. By northern blot analysis, this cDNA detected a single 1.8 kb transcript in the fetal and adult sheep liver, lung, hypothalamus, anterior pituitary and placenta. This could not be detected in the adrenal glands and kidneys, but a smaller (1.5 kb) transcript was present in the fetal and adult kidneys. During fetal development, the relative amount of 11 beta-HSD mRNA did not change significantly in the kidney and lung, but increased in lungs from newborn lambs. In contrast, amounts of hepatic 11 beta-HSD mRNA not only increased significantly in the fetus at term but also displayed a further increase in the newborn. These results clearly indicate that expression of ovine 11 beta-HSD gene in the fetus and newborn is regulated in a tissue-specific and developmentally programmed manner.


[Metastasizing pleomorphic adenoma].

[Metastasizing pleomorphic adenoma]. A case of metastasizing pleomorphic adenoma is reported with a review of the literature. It's a enigmatic lesion of the parotid gland in with both the primary tumor and metastasis were composed of benign pleomorphic structures. We think that some intra or extra cellular factors mut be discovered to recognize the malignant potential of this tumors and further to understand all cancerous pathology.


[Nephrogenic nephroma].

[Nephrogenic nephroma]. Nephrogenic nephroma was individualized by A. Pagès in 1980 as a rare renal neoplasia that mainly occurs in adolescents and adults and presents a benign outcome. The tumor is composed of very small tubular, papillary, and glomeruloid structures. Immunohistochemistry and electron microscopy show that the tumor is a proliferation of primitive epithelial cells that mimic the embryonal nephron. This neoplastic entity has often been confused with a nephroblastoma, although it is a benign tumor that is cured by simple resection. Further studies are needed to define its exact incidence and its histogenesis.


[Cystic nephroma in adults].

[Cystic nephroma in adults]. The authors report a case of cystic nephroma in a 36 year-old woman. This benign lesion complies with strict histologic criteria and requires a non aggressive surgical treatment. Recent studies support the concept of pathogenesis of the tumor originating from collecting and distal tubules.


[Unusual renal tumor in a young woman: cellular or atypical mesoblastic nephroma with partially cystic form]. A case of partially cystic atypical or cellular variant of mesoblastic nephroma in a 27-year old woman is reported. Nephrectomy was the only treatment. Since her operation, our patient has become pregnant and given birth to a healthy daughter. The mother is well and free of disease more than 18 months after nephrectomy. We have only found eight other reports of adult mesoblastic nephroma. Ours is the first published case of the cellular or atypical variant of mesoblastic nephroma in an adult patient.


[Renal tumors in children].

[Renal tumors in children]. A review of renal tumors of childhood is presented with special emphasis on different patterns of nephroblastoma, the changes induced by preoperative chemotherapy and new related tumors. Their diagnosis and prognosis are discussed. A large place is given to the nephrogenic rests and nephroblastomatosis, in the light of the recent paper by Dr Beckwith.


[Nephroblastomatosis and Wilms' tumor. Apropos of a case]. This case report illustrates the different anatomopathologic patterns and evolutive potentials of perilobar nephroblastomatosis described by Beckwith. The latter differs from those of an intralobar nephroblastomatosis. The evolutive possibilities of nephroblastomatosis underline the importance of imaging technology in the diagnosis and control of such lesions, as well as the role of chemotherapy and above all the caution required in the indications for surgery.


[Atypical congenital mesoblastic nephroma (atypical Bolande's tumor)]. A case of atypical or cellular variant of mesoblastic nephroma is reported. This rare tumor, variant of the classical congenital mesoblastic nephroma, described in 1967 by Bolande, has no pejorative prognostic significance in the child under 3 months. It must be distinguished from the highly aggressive clear cell sarcoma of the kidney.


[Cytomegalovirus retinitis in AIDS. Effectiveness and complications of long-term therapy]. In a retrospective study of the records of 64 patients with AIDS and cytomegalovirus (CMV) retinitis, 47 patients were evaluated regarding the effectiveness of initial virustatic therapy, and 41 patients regarding the anatomical and functional long-term development (maintenance therapy, rate and intervals of recurrences, complications, adverse drug reactions, time of survival, and visual outcome). The average survival time of 45 treated patients was 226 days (31-717 days). More than 90% responded to initial therapy with ganciclovir or foscarnet. Forty-four percent remained free of recurrences until they died (for up to 15 months); 56% had 1 to 4 recurrences, altogether 35 episodes, of which 14 occurred after withdrawal of therapy. Recurrences were treated with high-dose reinduction therapy with good or partial response in more than 80% of cases. Nine patients showed "smouldering retinitis" at a late stage. Two out of 45 patients became blind shortly after diagnosis despite therapy; both showed a course mimicking acute retinal necrosis syndrome with histologically proven CMV disease. None of the 43 patients on maintenance therapy for up to 2 years became blind in both eyes. Only 2 out of 43 patients had a poor visual outcome < 40/100 in the better eye at a late disease stage. Of 60 affected eyes, 11 showed retinal detachment, 14 eyes developed optic atrophy and 12 eyes became blind. Our retrospective study shows that consistent maintenance therapy of patients with CMV retinitis prevents severe deterioration of visual acuity in most cases even in long disease courses and thus maintains quality of life.


P400 protein is one of the major substrates for Ca2+/calmodulin-dependent protein kinase II in the postsynaptic density-enriched fraction isolated from rat cerebral cortex, hippocampus and cerebellum. Concanavalin A-binding glycoprotein with 250 K M(r) found in the postsynaptic density (PSD)-enriched preparation (or synaptic cytoskeleton) from rat cerebellum was identified with P400 protein from the physicochemical properties and enrichment in the cerebellum. Proteins homologous to the cerebellar 250 K M(r) protein occurred in the PSD-enriched preparations from rat cerebral cortex and from hippocampus, although the contents in the preparations were very low. The 250 K M(r) proteins in the PSD-enriched preparations from cerebellum and from cerebrum were highly phosphorylated by Ca2+/calmodulin (CaM)-dependent protein kinase II. The protein of synaptic plasma membrane (SPM) and PSD-enriched fractions prepared from cerebral cortex were not phosphorylated by the cAMP-dependent protein kinase endogenous to the fractions, whereas the protein from cerebellum was done in SPM and PSD-enriched fractions. The facts suggest that P400 or P400-like protein is closely associated with Ca2+/CaM-dependent protein kinase II in the PSD-enriched preparations, especially in the preparation from cerebral cortex. Phosphorylation of the protein by Ca2+/CaM-dependent protein kinase II may play an important role in the postsynaptic function in both cerebellum and at least in some areas of cerebrum.


Thyrotropin releasing hormone (TRH) and a degradation stabilized analogue (RX77368) stimulate phosphoinositide turnover in cultured astrocytes in a regionally specific manner. Whilst the CNS effects of thyrotropin releasing hormone (TRH) may result from a direct action on neurones it is also a possibility that another cell type may mediate an indirect action. A potential candidate for such a role is the astrocyte. The ability of TRH and a stable analogue RX77368 (di-methyl proline TRH) to stimulate phosphoinositide turnover has been investigated in cultured astrocytes from a number of CNS regions. Significant increases in turnover were noted in three of the four regions studied. Percentage values were: in spinal cord, 33% TRH, 31% RX77368 (n = 15); in brain stem, 33% TRH, 37% RX77368 (n = 6); in cerebellum, 72% TRH, 73% RX77368 (n = 6); in cortex, 4% TRH, 13% RX77368 (n = 6). EC50 values for both peptides were in the picomolar range. These results indicate that some astrocytes in vitro possess functional TRH receptors linked to the phosphoinositide second messenger system and hence this cell type would potentially be capable of mediating an indirect action of the peptide. Also, because the response was limited to certain regions, heterogeneity in receptor expression is implied. Furthermore, in the light of other evidence to support astrocyte heterogeneity within a region, we suggest that certain characteristics of the response seen in our experiments may be the result of TRH receptors being restricted to a subpopulation of astrocytes within a given culture. Thus, our data show that astrocytes prepared from some CNS regions possess functionally coupled TRH receptors.


Antiganglioside antibodies in motor-neuron diseases and peripheral neuropathies: study by ELISA technique and immunodetection on thin-layer chromatography. We report here our studies on IgM reactivity towards peripheral nervous system gangliosides, in motor-neuron diseases (MND) without IgM gammopathies, and in peripheral neuropathies with IgM gammopathies. We showed by enzyme linked immunosorbent assay technique, that anti-GM1 IgM antibodies were often present at a low level in normal controls in contrast to anti-GD1b antibodies, which were never detected in control sera. We evidenced that several steps of the ELISA technique were critical such as the nonaddition of detergent in buffer solutions used for dilutions and for washing and the choice of the ELISA plates. We studied 50 cases of motor-neuron diseases, among which 40 typical cases of Amyotrophic Lateral Sclerosis, only a few had high anti-GM1 antibodies levels, which were always confirmed by immunodetection on thin-layer chromatography. These antibodies were generally directed against the oligosaccharide epitope present also in asialoGM1. No correlation has been as yet established in relation to the clinical state of the patients. In a few cases of polyneuropathies associated with IgM gammopathies, antiganglioside antibodies have been reported. We have found anti-GD1b antibodies to be present in a sensory-motor axonal neuropathy; axonal involvement was evidenced by electrophysiological study.


Extreme pKa displacements at the active sites of FMN-dependent alpha-hydroxy acid-oxidizing enzymes. Flavocytochrome b2 (or L-lactate dehydrogenase) from baker's yeast is thought to operate by the initial formation of a carbanion, as do the evolutionarily related alpha-hydroxy acid-oxidizing FMN-dependent oxidases. Previous work has shown that, in the active site of the unligated reduced flavocytochrome b2, the group that has captured the substrate alpha-proton has a high pKapp, calculated to lie around 15 through the use of Eigen's equation. A detailed inspection of the now known three-dimensional structure of the enzyme leads to the conclusion that the high pKa belongs to His 373, an active site group that plays the role of general base in the forward reaction and of general acid in the reverse direction. Moreover, consideration of the kinetics of proton transfer during the catalytic cycle suggests that the pKa of the reduced FMN N5 position should be lowered by several pH units compared to its pKa of 20 or more when free. The features of the three-dimensional structure possibly responsible for these pK shifts are analyzed; they are proposed to consist of a network of hydrogen bonds with the solvent and of a mutual electrostatic stabilization of anionic reduced flavin and the imidazolium ion. Finally, it is suggested that similar pK shifts affect the active sites of the alpha-hydroxy acid-oxidizing flavooxidases, which are homologous to flavocytochrome b2. The functional significance of these pK shifts in terms of catalysis and semiquinone stabilization is discussed.


Characterization of EntF as a serine-activating enzyme. EntF is the enzyme responsible for serine activation during the biosynthesis of enterobactin (a cyclic trimer of N-dihydroxybenzoyl serine) in Escherichia coli. EntF has been overexpressed and purified to > 90% homogeneity. The enzyme has been shown to complement the entF- MK1 strain in the synthesis of 2,3-dihydroxybenzoyl serine derivatives and exhibits L-serine-dependent ATP[32P] pyrophosphate exchange activity with a Km for serine of 260 mM and a turnover number of 760 min-1. Release of PPi during incubation of EntF with serine and ATP was observed, but with a low turnover number of 1.0 min-1. These results suggested the presence of an enzyme-bound intermediate, which has been shown by gel filtration analysis to be (L-serine)adenylate.


Cis proline mutants of ribonuclease A. I. Thermal stability. A chemically synthesized gene for ribonuclease A has been expressed in Escherichia coli using a T7 expression system (Studier, F.W., Rosenberg, A.H., Dunn, J.J., & Dubendorff, J.W., 1990, Methods Enzymol. 185, 60-89). The expressed protein, which contains an additional N-terminal methionine residue, has physical and catalytic properties close to those of bovine ribonuclease A. The expressed protein accumulates in inclusion bodies and has scrambled disulfide bonds; the native disulfide bonds are regenerated during purification. Site-directed mutations have been made at each of the two cis proline residues, 93 and 114, and a double mutant has been made. In contrast to results reported for replacement of trans proline residues, replacement of either cis proline is strongly destabilizing. Thermal unfolding experiments on four single mutants give delta Tm approximately equal to 10 degrees C and delta delta G0 (apparent) = 2-3 kcal/mol. The reason is that either the substituted amino acid goes in cis, and cis<==>trans isomerization after unfolding pulls the unfolding equilibrium toward the unfolded state, or else there is a conformational change, which by itself is destabilizing relative to the wild-type conformation, that allows the substituted amino acid to form a trans peptide bond.


Analysis of peptide mixtures by capillary high performance liquid chromatography: a practical guide to small-scale separations. Capillary HPLC is a very effective means of separating small amounts of peptides and proteins. Capillary columns ranging from 0.01 mm to 0.5 mm in diameter can be constructed using recycled supports and inexpensive fused silica capillary tubing. Commercial pumping systems and UV detectors can be readily converted for operation in the flow rate range of 0.5-50 microL/min. Detailed procedures are given for the construction of columns and UV detector flow cells. A mixture of peptides derived from the endo Lys C digest of horse heart cytochrome c was used to illustrate various aspects of capillary chromatography of peptides and compares the performance of various-sized capillary columns and UV detector flow cell types.


Differentiation between transmembrane helices and peripheral helices by the deconvolution of circular dichroism spectra of membrane proteins. The interpretation of the circular dichroism (CD) spectra of proteins to date requires additional secondary structural information of the proteins to be analyzed, such as X-ray or NMR data. Therefore, these methods are inappropriate for a CD database whose secondary structures are unknown, as in the case of the membrane proteins. The convex constraint analysis algorithm (Perczel, A., Hollósi, M., Tusnády, G., & Fasman, G. D., 1991, Protein Eng. 4, 669-679), on the other hand, operates only on a collection of spectral data to extract the common spectral components with their spectral weights. The linear combinations of these derived "pure" CD curves can reconstruct the original data set with great accuracy. For a membrane protein data set, the five-component spectra so obtained from the deconvolution consisted of two different types of alpha helices (the alpha helix in the soluble domain and the alpha T helix, for the transmembrane alpha helix), a beta-pleated sheet, a class C-like spectrum related to beta turns, and a spectrum correlated with the unordered conformation. The deconvoluted CD spectrum for the alpha T helix was characterized by a positive red-shifted band in the range 195-200 nm (+95,000 deg cm2 dmol-1), with the intensity of the negative band at 208 nm being slightly less negative than that of the 222-nm band (-50,000 and -60,000 deg cm2 dmol-1, respectively) in comparison with the regular alpha helix, with a positive band at 190 nm and two negative bands at 208 and 222 nm with magnitudes of +70,000, -30,000, and -30,000 deg cm2 dmol-1, respectively.


Chimeric prion protein expression in cultured cells and transgenic mice. The efficient expression of exogenous prion protein (PrP) molecules in mouse neuroblastoma cells that are chronically infected with murine scrapie prions (ScN2a cells; Butler, D.A., et al., 1988, J. Virol. 62, 1558-1564) and in transgenic mice is described. This technology allows investigation of the PrP molecule for structural regions involved in determining species specificity, as well as ablation experiments designed to address the functionality of particular regions of the PrP molecule. Previous reports demonstrated that the PrP gene specifies the host range for susceptibility of transgenic animals to prions (Scott, M., et al., 1989, Cell 59, 847-857; Prusiner, S.B., et al., 1990, Cell 63, 673-686). Consistent with these results, we showed that Syrian hamster (SHa) PrP is ineligible for efficient conversion to PrPSc in ScN2a cells. By constructing a series of chimeric mouse (Mo)/SHaPrP genes, we developed an epitopically tagged functional variant of the MoPrP gene, which can efficiently form protease-resistant PrP molecules upon expression in ScN2a cells. The presence of a defined epitope for an SHa-specific monoclonal antibody allows the products of this chimeric gene to be discriminated from endogenous MoPrP and creates a useful reagent for exploring structure/function relationships via targeted mutagenesis. In addition, we developed a transgenic mouse expression vector by manipulation of an SHaPrP cosmid clone. This vector permits the efficient expression of foreign PrP genes in the brains of transgenic animals, enabling pathological consequences of in vitro mutagenesis to be studied.


Active site mutants of human cyclophilin A separate peptidyl-prolyl isomerase activity from cyclosporin A binding and calcineurin inhibition. Based on recent X-ray structural information, six site-directed mutants of human cyclophilin A (hCyPA) involving residues in the putative active site--H54, R55, F60, Q111, F113, and H126--have been constructed, overexpressed, and purified from Escherichia coli to homogeneity. The proteins W121A (Liu, J., Chen, C.-M., & Walsh, C.T., 1991a, Biochemistry 30, 2306-2310), H54Q, R55A, F60A, Q111A, F113A, and H126Q were assayed for cis-trans peptidyl-prolyl isomerase (PPIase) activity, their ability to bind the immunosuppressive drug cyclosporin A (CsA), and protein phosphatase 2B (calcineurin) inhibition in the presence of CsA. Results indicate that H54Q, Q111A, F113A, and W121A retain 3-15% of the catalytic efficiency (kcat/Km) of wild-type recombinant hCyPA. The remaining three mutants (R55A, F60A, and H126Q) each retain less than 1% of the wild-type catalytic efficiency, indicating participation by these residues in PPIase catalysis. Each of the mutants bound to a CsA affinity matrix. The mutants R55A, F60A, F113A, and H126Q inhibited calcineurin in the presence of CsA, whereas W121A did not. Although CsA is a competitive inhibitor of PPIase activity, it can complex with enzymatically inactive cyclophilins and inhibit the phosphatase activity of calcineurin.


Three-dimensional structure of the Fab fragment of a neutralizing antibody to human rhinovirus serotype 2. The crystal structure of the antigen-binding fragment of a monoclonal antibody (8F5) that neutralizes human rhinovirus serotype 2 has been determined by X-ray diffraction studies. Antibody 8F5, obtained by immunization with native HRV2 virions, cross-reacts with peptides of the viral capsid protein VP2, which contribute to the neutralizing immunogenic site B in this serotype. The structure was solved by the molecular replacement method and has been refined to an R-factor of 18.9% at 2.8 A resolution. The elbow angle, relating the variable and constant modules of the molecule is 127 degrees, representing the smallest elbow angle observed so far in an Fab fragment. Furthermore, the charged residues of the epitope can be well accommodated in the antigen-binding site. This is the first crystal structure reported for an antibody directed against an icosahedral virus.


A study of the effects of rifabutin on isoniazid pharmacokinetics and metabolism in healthy volunteers. The effect of repeated administration of rifabutin on the pharmacokinetics and metabolism of isoniazid was evaluated in 6 healthy volunteers. The subjects received on day 1 and 9 a single oral dose of 300 mg isoniazid and from day 2 to 8 a single daily oral dose of 300 mg rifabutin. Two out of 6 subjects were shown to be rapid acetylators. No significant modification of the plasma pharmacokinetic profiles of isoniazid and acetylisoniazid was found. Evidence exists in the present study for autoinduction of rifabutin metabolism; this is shown by the lower plasma concentrations obtained 24 h after the seventh dose as compared to the theoretical concentrations.


[Establishing orthotopic transplanted models of human pancreatic cancer in nude mice and study on their biological properties]. Two cell lines of human pancreatic cancer have been established, which can be successfully transplanted into pancreas of nude mice, the first of this kind of cell lines in China. Fresh specimens human of pancreatic cancer taken surgically were transplanted in the pancreas of pure line BALB/C-nu/nu nude mice. The transplanted tumours grew and reproduced successfully, and were named PINMP-1 and PINMP-2, respectively. So far, 9 generations of PINMP-1 and 6 generations of PINMP-2 were obtained. Their biological properties, ways of invasion and metastasis and morphological characteristics under light and electron microscope were studied. The results showed a 95%-100% transplanting success rate, with the success rate of transplanting from tissues revivified from the liquid nitrogen preservation being 100%. Both of the lines could produce large amount of CEA, and chromosome analysis confirmed that they had retained a karyotype of the human cancer cells. In nude mice transplanted with the tumours, metastasis could be found in the lymph nodes, lungs and livers. Metastasis via lymphatic channels and blood vessels were also demonstrated. The pathological and ultrastructural examination confirmed that the transplanted tumours had identical characteristics as their donor tumours. The transplanted cells grew independently in the pancreas of the nude mice, making a better model for study on tumour invasion and metastasis than subcutaneously transplanted tumours. This indicated that the microenvironment in the transplantation site had certain influence on the biological behavior of the transplanted tumours. The models could be used in the study on the invasion, metastasis and experimental therapy of pancreatic carcinomas.


[Evaluation of radioimmunotherapy in the multimodality treatment of hepatocellular carcinoma (HCC)]. The evaluation of radioimmunotherapy using 131I-anti HCC isoferritin IgG antibody in the multimodality treatment of HCC was reported. Forty three patients with surgically verified unresectable HCC have been treated by radioimmunotherapy as a part of multimodality treatment during 1985-1990. The short-term responses and prolong survival were compared with that in control group of 39 patients with HCC receiving conventional multimodality treatment. The rates of tumor shrinkage, AFP level decline and second resection in radioimmunotherapy group were 67.4% (29/43), 69.6% (16/23) and 30.2% (13/43) respectively, significantly higher than those in control group 23.1% (15/39), 40.0% (8/20) and 10.3% (4/39) respectively. The 1, 3, 5-year survival rates were 61.5%, 40.4% and 35.5% in radioimmunotherapy group, however, in control group were 51.3%, 20.1% and 15.5%, respectively. The results suggested that radioimmunotherapy is one of modalities of choice, particularly for the treatment of unresectable HCC in the multimodality treatment regimen.


[The treatment of small cell lung cancer]. Forty-nine cases with small cell lung cancer (SCLC) treated with chemotherapy and radiotherapy were analyzed. Eight of them were of limited disease (LD), and 41(83.7%) had extensive disease (ED). Forty six were treated on a protocol comprising the induction with cyclophosphamide 1g i.v. on days 1st and 8th, vincristine 1-2mg, i.v. on days 1st and 8th, etoposide 100mg i.v. on days 3rd-7th and methotrexate 20mg i.v. on day 3rd, 5th, 9th, 11th and than repeated with the second course of treatment after 21 days. Eight patients (17.4%) responded completely. There were 29 cases with partial response (63%). The total response rate was therefore 80.4%. Then radiotherapy (50 GY Over 4 weeks) was administered to primary cancer area in 36 patients of good response. The CR was increased from 19.4% to 55.6%. Twenty patients had CR. These patients were treated by late intensification with average of six cycles. The median survival period was 10 months. 21 patients (58.3%) were alive more than 1yr. 10 patients (27.8%) survived more than 2 yrs. Six patients (24%) were alive more than 3 yrs. One patient survived over 7 yrs. It was suggested that combined therapy is important for achieving a better response. CR is beneficial for long term remission. Late intensification is a kind of treatment with minimal toxicity and longer remission. Metastases to the central nervous system usually caused death, so prophylactic CNS irradiation (PCI) should be used in patients with ED for achieving a longer remission.


[Two-stage resection for advanced hepatocellular carcinoma--preliminary results of 13 cases]. We performed two-stage resection for thirteen patients with advanced hepatocellular carcinoma from January 1987 to January 1991. All patients underwent various surgical therapies prior to resection which included gauze packing hemostasis in 1 case, hyperthermia plus radiotherapy in 1, hepatic arterial ligation in 2, operative hepatic arterial embolization in 1, and transcatheter embolization in 8. The median interval between the first therapy and tumor resection was 54 days with a range of 29-769 days and the median diameter of tumors decreased from 10.5 cm to 7.5 cm. The majority of procedures on two-stage resection were irregular hepatectomy or lobectomy under occlusion of porta hepatis. Regular hepatectomies were done in 4 cases. Pathological examination showed complete coagulation necrosis in 3 specimens. However, in the others were still found residual viable tumor cells outside or beneath the tumor capsule with varying degrees of necrosis within the tumors. Survival periods of the patients who received two-stage resection were from 4 months to 4 years except 2 operative death. The significance, possibility as well as methods of two-stage resection were discussed.


[Computed tomography of retroperitoneal neoplasms]. CT findings of retroperitoneal neoplasma in 50 cases (51 tumors) were reviewed. There were 28 (55%) malignant tumors and 23 (45%) benign ones. MFH and liposarcoma were the most common malignant tumors and neural origin tumors were the most common benign ones. Differentiation is difficult on the basis of CT features alone. Benign tumors were usually smooth and well defined, and malignant ones ill-defined, irregular in shape, heterogenous in density with massive necrosis. The characteristic CT appearance of liposarcoma is the CT attenuation value by fat density. Neural origin tumors are usually located near the spine. They may have thick wall cystic appearance or are dumbbell shaped, showing expansion or extrinsic pressure to the adjacent bone structures. MFH, hemangiopericytoma and other malignant tumors may have marked enhancement after contrast administration. Non-resectability is shown as: 1. big vessels encased by tumor over 90 degrees, 2. adjacent organs or structures invaded by tumor, 3. multiple masses, and 4. huge tumor invading into the pelvis. Local recurrence is common after surgery. Follow-up CT scans every 6 months in a 2 year period is suggested for early detection of recurrence.


[Surgical treatment of lung cancer in young adult]. Ninety-three young adult patients (< 40 years old) with lung cancer were operated and pathologically confirmed during 1977. 1-1990. 12. The first misdiagnostic rate was 74.2%. The resectable rate was as low as 76.3%. The incidence of SCLC in this group was 17.2%. Pneumonectomy rate was 23.9%. But lower mortality rate was showed in this group since young patients are usually with better reservation of heart and lung functions. The five-year survival rate in this group was 33.3%. It is emphasized that in case of a young adult suspected of suffering from lung cancer, chest films, and if necessary, sputum cytology, bronchoscopy, standard tomography, CT and transthoracic needle biopsy should be taken. Early diagnosis and treatment are of great importance. In authors' opinion, patients with SCLC should also be treated surgically combined with chemotherapy or radiotherapy.


[The prognostic significance of plasma cyclic nucleotides in patients with congestive heart failure]. The relationship between plasma levels of cyclic nucleotides and mortality was studied in 108 patients with congestive heart failure (CHF) in whom plasma cAMP and cGMP, were determined by radioimmunoassay and were followed-up for 36 to 48 months. 29 patients died. The plasma concentrations of cAMP and cGMP in the dead group were significantly higher than those in the survival group (27.41 +/- 6.11 and 31.11 +/- 18.23 vs 21.56 +/- 5.37 and 17.45 +/- 9.35 nmol/L, respectively, mean +/- s, P < 0.001). Compared with patients with cAMP concentrations below the median value of 22.31 nmol/L, those with higher levels of cAMP had a higher mortality rate (46.3% vs 7.4%, P < 0.001). Patients with cGMP levels above the median value of 17.24 nmol/L also had a higher mortality rate than those with lower levels (40.7% vs 13.0%, P < 0.01). Multivariate stepwise regression analysis including age, cardiac function classification, heart rate, serum potassium and sodium, plasma cAMP, cGMP and cAMP/cGMP was carried out and revealed that only plasma cAMP and cGMP could provide independent prognostic information. Thus plasma levels of cyclic nucleotides was considered the excellent predictor for patients with CHF.


[ELISA of serum isoferritin and its clinical application]. Serum isoferritin levels were detected by ELISA in 96 normal, 11 cases of hepatocellular carcinoma (HCC), 28 breast cancer (BC), 31 lung cancer (LC), 26 breast fibroma, 11 pneumonia and 11 tuberculosis. The results reveal significant differences of serum isoferritin levels between the normals and the patients, and between the malignant cases and benign cases (P < 0.01). Serum isoferritin demonstrates higher sensitivity in detecting HCC, LC and BC and thus is of great value in the differential diagnosis of these cancers.


[Effects of fibronectin on cytodifferentiation and cell cycle in a human hepatoma cell line QGY-7703]. The effects of fibronectin (FN) on cytodifferentiation and cell cycle in a human hepatoma cell line QGY-7703 were studied by flow cytometry, electron microscopy, etc. The results indicated that FN inhibited cell proliferation and mitosis; significant differences were noted between the control group and the experimental group (P < 0.01). FN blocked the cell cycle in phase S; cell spreading improved with less cell overlapping, and the growth of monolayer cell was partially restored. The number of microvilli and marginal ruffles decreased and that of mitochondria, Golgi complex, endoplasmic reticulum increased. The findings suggested that FN could partially restore QGY-7703 cell normal phenotype and probably be useful for the treatment of tumor.


[Hepatic carcinoma treated by hepatic arterial embolization using 131I and chemotherapeutic agent gelatin microspheres: report of 9 cases]. Nine patients with inoperable hepatoma were treated by using hepatic arterial embolization 131I and chemotherapeutic agent gelatin microsphere (131I-CA-GM). The emission CT after operation detected that the microspheres were concentrated on tumor area. The ratio between the radioactivity in tumor and that in liver was 4.1:1. A case died of ictopic embolization; the others survived 3, 4, 5, 19, 24, 7, 8, and 12 months respectively. Three of them were still alive. 131I-CA-GM has triple anticarcinogenic actions, including the arterial occlusion, targeting chemotherapy and internal radiation. The microspheres can selectively accumulate in the tumor artery and can be easily traced by gamma-camera or emission CT. 131I-CA-GM is a hopeful embolic agent for the treatment of liver cancer, but some problems about ectopic arterial embolization should be further studied.


[Regional vascular occlusion at hepatic hila for the resection of hepatic tumor--report of 63 cases]. A new operative procedure for hepatic segmentectomy was performed by dissecting the hepatic hila to occlude blood vessels to and from hemihepatis, one lobe or one segment which bore hepatoma. Forty-three liver cancers with 33 of them in stage IV, 4 metastatic liver cancers and 16 hemangiomas were resected by this method. The types of hepatic segmentectomy consisted of monosegmentectomy, multisegmentectomy, peripheral and central segmentectomy, segmentectomy after hepatic arteria embolization and resegmentectomy for recurrent hepatic cellular carcinoma etc. The vascular occlusion lasted as long as 120 minutes for the right portal vein and 70 minutes for the left. Intravenous infusion of extract from Salive mliltiorrhiza before vascular occlusion could prevent hepatic cells from reperfusion injury. The mentioned maneuvers resulted in mild postoperative liver dysfunction, rapid recovery and hepatocytes regeneration. Two patients died within 30 days postoperatively, constituting an operative mortality of 3.1%.


Interleukin-1 beta and beta-endorphin circadian rhythms are inversely related in normal and stress-altered sleep. Normal sleep is associated to physiological nocturnal rises in Interleukin 1 beta (IL 1 beta) secretion. The 24 h pattern of IL 1 beta, beta-Endorphin (beta-EPH), ACTH and cortisol (F) production was evaluated in four male healthy volunteers. Two subjects were unable to sleep, due to the stress of the experiment; in these cases, no detectable plasma IL 1 beta secretion, both diurnal and nocturnal, was present, beta-EPH plasma levels were significantly higher (p < 0.01) than in the subjects who slept regularly and, in one case, increased F plasma levels were also reported. A strong negative correlation between IL 1 beta and beta-EPH plasma levels was present in all the cases. In conclusion, stress-induced sleep alterations might deeply affect both diurnal and nocturnal IL 1 beta plasma secretion, probably due to the hypothalamus-pituitary-adrenal axis (HPAA) activation, and beta-EPH might be the reliable marker of the stress-induced HPAA activation level.


The role of cholinergic neurons in stress-induced increase of interictal discharges in hippocampal-kindled rats. This study aims to clarify the role of cholinergic neurons in the occurrence of epileptic seizures when emotional stress is loaded. The effects of stress and drugs on hippocampal interictal discharges were compared among groups of fully kindled male Wistar rats intraperitoneally given atropine, physostigmine and saline with and without immobilization, or pituitary-adrenocortical hormones without immobilization. Hippocampal interictal discharges increased during immobilization, because hippocampal cholinergic neurons were activated by emotional stress. Pituitary-adrenocortical hormones had no effects on the discharges.


An evaluation of the staphylococcal co-agglutination test for the detection of group A rotavirus in human faeces. The group A rotavirus staphylococcal co-agglutination test was evaluated and its sensitivity and specificity compared with an in-house enzyme-linked immunosorbent assay (ELISA) and a commercial latex agglutination test (Rotalex). In addition, the storage stability of the staphylococcal reagents was ascertained. Examination of 136 clarified suspensions of diarrhoeal faeces by the staphylococcal co-agglutination test revealed a high proportion of false positives (26%) and uninterpretable results (34%) due to non-specific agglutination. Non-specific agglutination could be removed effectively by prior absorption of the clarified faecal specimens with unsensitized staphylococci. The staphylococcal co-agglutination test was less sensitive and specific than the in-house enzyme-linked immunosorbent assay but was comparable to the Rotalex slide latex agglutination test. The staphylococcal reagents have a shelf life of at least 29 weeks.


Wilms' tumour in Malaysian children: a histopathological study of cases encountered at the University Hospital, Kuala Lumpur over a 22-year period. Formerly thought to have a constant incidence rate throughout the world, Wilms' tumour (nephroblastoma) has been shown to be less common among Asian children. A retrospective demographic and morphological study of Wilms' tumour histologically diagnosed over a 22-year period at the Department of Pathology, University Hospital, Kuala Lumpur was conducted to assess for inherent demographic and morphological differences between tumours in Malaysian children and those of Western populations. Thirty-seven cases of histologically proven Wilms' tumour qualified for inclusion in this study. 19 patients were Chinese, 13 Malay, 4 Indian and 1 Anglo-asian. 21 were male and 16 were female (M:F ratio = 1.3:1). Their ages ranged from 1 month to 4 years. 70.3% of the patients were below 2 years of age. 36 cases had unilateral and 1 bilateral tumours. Of unilateral tumours, 19 involved the left kidney and 17 the right. Histological assessment, based on criteria of the National Wilms' Tumor Study Group, revealed 20 (52.6%) tumours with a mixed pattern while 8 (21.1%) showed epithelial, 7 (18.4%) blastemal and 3 (7.8%) stromal-predominant patterns. Anaplasia was observed in only 2 tumours (5.3%). There was no obvious difference in age range and sex distribution, laterality of tumours and incidence of anaplasia between this and Western studies. No ethnic predilection was observed. A notably larger percentage of cases were below 2 years of age. Also, a larger proportion of epithelial-predominant and a lower proportion of blastemal-predominant tumours was observed compared with patterns reported from Western populations.


[Malignant primary intrathoracic histiocytofibroma]. Malignant primary intrathoracic fibrous histiocytoma (MFH) is a rare tumour: since it was first described in 1979, hardly more than 80 cases have been published. We present a large MFH which had started in the pleura and was intrathoracic. The tumour was typical, being comprised of malignant fibroblastic and histiocytoid cells in storiform arrangement. It was revealed by spinal bone metastases which multiplied over the whole skeleton in spite of chemotherapy. The patient died of cerebral metastasis developed 8 months after the onset of the disease. The cases found in the literature are gathered together and commented.


[Lambert-Eaton syndrome. Apropos of 2 cases]. Lambert-Eaton syndrome is a myasthenia-like syndrome of paraneoplastic origin which is often associated with anaplastic small-cell lung cancer. It seems to be an autoimmune disease responsible for a deficit of acetylcholine ejection in the motor end plate. On the occasion of two recent cases, we review the clinical, physiopathological and diagnostic aspects of this paraneoplastic syndrome.


Detection of human papillomavirus DNA in normal epithelium and in squamous metaplasia of the uterine cervix by the polymerase chain reaction. The presence of human papillomavirus (HPV) 16 was studied by using the polymerase chain reaction (PCR) directly on sections histologically defined as normal squamous epithelium and metaplastic squamous tissue of the uterine cervix. Ten specimens of normal epithelium were obtained at hysterectomy from women with uterine leiomyoma. Six specimens of squamous metaplasia were adjacent to the areas of dysplastic epithelium in which HPV 16 DNA had been detected previously by PCR. HPV 16 DNA was amplified specifically and detected in two of 10 normal cervical epithelium specimens and in all of 6 squamous metaplasia specimens adjacent to dysplastic lesions. However, HPV DNA could not be detected in the metaplasia by in situ hybridization. These results suggest that metaplastic squamous tissue adjacent to dysplastic lesions harbors fewer copies of HPV DNA than the dysplastic area and the carcinoma and that the HPV copy number per cell may be relevant to the pathogenesis of cervical carcinoma.


Effects of massive small bowel resection on metabolism of bile acids and vitamin D3 and gastrin release in dogs. The effects of ursodeoxycholic acid (UDCA) and 1 alpha-hydroxyvitamin D3 on pathophysiological changes following massive resection of the distal small bowel (75%) were investigated by using adult beagle dogs. After surgery, body weight decreased, watery diarrhea occurred, and the transit time of the alimentary tract shortened. These undesirable consequences lessened markedly after oral administration of UDCA, though 1 alpha-hydroxyvitamin D3 was not effective. Plasma levels of both 25-hydroxyvitamin D3 and 24, 25-dihydroxyvitamin D3 decreased after surgery, while plasma 1 alpha, 25-dihydroxyvitamin D3 concentrations remained unchanged during the observation period of six months. Although fasting plasma concentrations of total bile acid were not reduced, the integrated response to a meal decreased significantly after surgery in spite of the administration of UDCA. The concentration of UDCA in the gallbladder bile increased markedly in dogs which received UDCA. Taurine-conjugated bile acids accounted for more than 90% of the gallbladder bile. Postprandial hypergastrinemia occurred following the massive small bowel resection in the control group and in the group which received 1 alpha-hydroxyvitamin D3 alone, while it did not occur in the group given UDCA together with 1 alpha-hydroxyvitamin D3. These results indicate that administration of UDCA after massive resection of the small intestine is effective in maintaining good nutritional state.


Prevalence of antibodies to selected viruses in bovine embryo donors and recipients from Brazil, and its implications in international embryo trade. The prevalence of serum antibody to enzootic bovine leukosis (EBL), blue-tongue (BT), bovine herpesvirus 1 (BHV 1) and bovine virus diarrhoea (BVD) viruses in bovine embryo donors (D) and recipients (R) from Minas Gerais State, Brazil was investigated. Of 451 sera (130 D plus 321 R) tested for antibodies against EBL virus 104 (23.1%) were positive. Of 410 sera (130 D plus 280 R) tested for antibody to BT, BHV 1 and BVD viruses the respective number of positive sera were 313 (76.3%), 209 (51.0%) and 153 (37.3%). Donors had significantly (Chi-square test, p < 0.05) higher prevalence rates of antibody than recipients to EBL and BVD viruses. The donors were 60 Bos indicus and 70 Bos taurus purebred cows. Antibody to EBL virus was significantly less common among Bos indicus.


Historical progress and the future of human cell culture research. Progress in human cell culture research is discussed based primarily on our hematopoietic cell culture studies. The article includes a historical background of Burkitt lymphoma cell lines, discovery of EBV, normal B-lymphoblastoid cell lines with EBV, a variety of leukemia, lymphoma, and myeloma cell lines, clinical and theoretical contributions made by studies of T-cell leukemia cell lines, the discovery and clinical relevance of HTLV, HIV and HBLV, early attempts at adoptive immunotherapy of patients with cancer, and the future of human cell culture research. Despite the fact that current cell culture methods permit maintenance of only limited cell types of both normal and malignant origins, biotechnological advances such as hybridoma and recombinant DNA technologies should continue to provide unlimited research opportunities in all fields.


[Free radicals and their biological significance: present and future]. Free radicals are usually active species which have unpair electron (S) in molecules or Atomic groups. Of the free radicals, O2- and .OH could easily be produced in mammalian cells, by oxidation and reduction cycle catalyzed by fravoproteins and by iron + H2O2 reaction, respectively. Other free radicals would also be produced in mammalian cell, such as amino acid radicals, semiquinone radicals and flavine radicals etc. In general, free radicals cause cell injury though membrane lipid peroxidation and DNA strand cleavage and some other mechanisms.


Self propagation of injured tissue by localized changes of free radicals in the central nervous system. To examine the role of free radicals in the pathogenesis of injured tissue in the central nervous system (CNS), we developed a new technique for mapping superoxide free radicals, vascular permeability, and energy metabolism simultaneously. The distribution of superoxide anions in the CNS is based upon the 380 nm chemiluminescence of 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2a] pyrazin-3-one (CLA-phenyl) when it reacts with superoxide anions in frozen tissue sections. This new CLA-phenyl hybrid--paper technique show clear relationships between the regional production of superoxide free radicals, increased vascular permeability, and changes of energy metabolism in the self propagating phenomena occurring in the various lesions in the CNS.


[Human lung cancer cell lines in our laboratory: establishment of three large cell carcinoma cell lines and their biological characterization]. Three large cell carcinoma cell lines were established from tumors of lung cancer patients. The cell lines were named NUTLC-2, NUTLC-4 and NUTLC-5 and they were found to have the following biological characterization. 1) By chromosomal analysis, the tumor cells of two of the cell lines (NUTLC-2 and NUTLC-5) were human-origin cells. Numbers of chromosomes of these cells ranged from 52 to 59 in NUTLC-2 and from 68 to 75 in NUTLC-5, with the modal numbers of 56 and 71, respectively. 2) Primary tumor resected from the patient with lung cancer was heterotransplanted into the subcutis of a nude mouse. NUTLC-4 cell line was established in vitro from the tumor in nude mouse and the tumor cells were found to be mouse-origin cells by chromosomal analysis. Human DNA was not detected by Alu analysis. 3) The tumor cells of three cell lines could be heterotransplanted subcutaneously into nude mice. However, no natural distant metastasis in nude mouse was observed. 4) Drug sensitivity to NUTLC-2, NUTLC-4 and NUTLC-5 tumor cells differed individually according to MTT colorimetric assay, and characteristic drug sensitivity was not noted in histological types of lung cancer.


An unusual case of regional odontodysplasia. A case is presented of a 3-year-old child with regional odontodysplasia affecting all erupted and developing mandibular teeth. Maxillary teeth were unaffected. This pattern of distribution does not appear to have been reported previously and the extent of involvement poses problems for the long-term dental management of the patient.


[Comparison of 2 cisplatin and etoposide dosages in relapsing small cell lung cancer]. The synergistic combination of cisplatinum and etoposide appears as the best second line treatment in patients relapsing from small cell lung carcinoma (SCLC). In order to test the dose-effect relationship of cisplatinum and etoposide in this situation, we have performed a randomised phase II trial comparing 2 five-day regimens: cisplatinum 20 mg/m2/day+etoposide 60 mg/m2/day (arm A) versus cisplatinum 40 mg/m2/day+etoposide 100 mg/m2/day (arm B) every 4 weeks. Thirty-seven patients were included (arm A: 18, arm B: 19), and 32 were considered to be eligible (arm A: 15, arm B: 17). Eight patients were non evaluable, five of them because of toxic death occurring prior to the second course (arm A: one from neutropenia; arm B: three from neutropenia and one from thrombopenia). The two groups were well balanced with regard to the main prognostic factors (age, sex, performance status, LDH level, response to induction chemotherapy). An objective response was observed in 10/24 evaluable patients (arm A: 4, arm B: 6) and was considered as complete in one patient in arm A and in 2 pts in arm B; these two patients presented with cerebral metastases and their response lasted 9 and 15 weeks respectively. The mean duration of response was 11 weeks in arm A and 10.5 weeks in arm B. The median actuarial survival of the overall population of eligible patients was 15 weeks: 13 weeks in arm A and 16.5 weeks in arm B. The study was discontinued because of the 23.5% toxic deaths rate in the high doses arm in this heavily pre-treated population of patients. However, the high response rate (54% overall, 35% considering toxic death as a failure) is impressive and presents evidence for the dose/effect relationship in SCLC.


Biochemical changes in sciatic nerve of hens treated with tri-o-cresyl phosphate: increased phosphorylation of cytoskeletal proteins. Calcium- and calmodulin-regulated protein phosphorylation has been suggested to play a role in the pathogenesis of organophosphorus compound-induced delayed neurotoxicity (OPIDN). This condition is characterized by ataxia that progresses to paralysis concurrent with a central-peripheral distal axonopathy after a delay period of 1-2 weeks following exposure to an organophosphorus compound causing delayed neurotoxicity, such as tri-o-cresyl phosphate (TOCP). Calcium/calmodulin (CaM) kinase II is involved in the increased phosphorylation of brain microtubule and spinal cord neurofilament triplet proteins following treatment of animals with organophosphorus compounds that are capable of producing OPIDN. In this study, chickens were given a single oral neurotoxic dose of 750 mg TOCP/kg body weight and killed after 1, 6, 14 or 21 days following treatment. Protein kinase-mediated phosphorylation of cytoskeletal proteins was studied in proximal and distal parts of sciatic nerves of control and treated hens. Peripheral nerve proteins were phosphorylated in vitro using [gamma-32P]ATP as a phosphoryl group donor. Phosphorylated proteins were separated by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein phosphorylation was detected by autoradiography and quantified by laser microdensitometry. The extent of Ca2+-calmodulin dependent phosphorylation of five cytoskeletal proteins was significantly increased in TOCP treated animals, particularly at 1 and 6 days after treatment, in both the proximal and distal portion of the nerve. The identity of these proteins was confirmed by 2-D PAGE as tubulin, the neurofilament triplet proteins and microtubule associated protein-2 (MAP-2). These results confirm earlier observation of the close temporal relationship between increased cytoskeletal protein phosphorylation and the development and OPIDN.


The postnatal development of benzodiazepine receptor sites in the chick optic lobe is modulated by environmental lighting. The present paper describes the ability of benzodiazepine receptor sites to undergo light mediated-plastic changes during the early postnatal development of the chick optic lobe. The postnatal development pattern of these receptors was studied under different levels of light stimulation, i.e. normal-, light-and dark-rearing. At hatching the specific binding of [3H]Flunitrazepam was 0.23 +/- 0.01 pmol/mg protein. The developmental profile shows a sharp and transient peak of receptor overexpression between the 1st and the 2nd postnatal day in three experimental groups. Between the 2nd and the 6th day significant differences were found between the three groups, being this difference maximal during the peak of overexpression. In fact, on the 2nd day the specific [3H]Flunitrazepam binding showed an increase of 17% (P < 0.0005) and a decrease of 34% (P < 0.0005) for light- and dark-reared animals as compared with normally-reared ones. The changes in receptor density were transient since from the 6th day onward they gradually disappeared, being almost identical in the three groups by the day 15. At this moment the number of benzodiazepine receptor sites stabilized at the adult level. Scatchard analysis at the 2nd postnatal day revealed that the differences observed in the high affinity benzodiazepine binding sites between the three groups were due to modifications in the total number of binding sites while the affinity remained unchanged. The maximal number of binding sites were: 2.76 +/- 0.03, 3.40 +/- 0.01 and 1.46 +/- 0.11 pmol/mg protein in normally-, light- and dark-reared chicks, respectively; while the apparent dissociation constants were unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of prostaglandin E2 (PGE2) and cyclic adenosine monophosphate (cAMP) upon actin cytoskeleton in human pulmonary fibroblasts (ICP-23) infected by measles virus. The evolution of the actin cytoskeleton after trypsinization and recultivation as well as the effect of the PGE2 modulator and that of the secondary messenger, the cyclic AMP upon the same cytoskeletal proteins in human pulmonary fibroblasts (ICP-23) were studied. The substances were administered simultaneously and after one hour of viral adsorption. Using epifluorescence for pointing out filamentous actin the modifications occurring in this cytoskeletal protein when contacting trypsin and the virus and when PGE2 and cAMP are administered in the experimental variants are observed. Actin arrangement is obviously modified by the viral infection but the restrictive effect of PGE2 and cAMP upon virus replication is correlated with modifications occurring in the actin cytoskeleton.


Omega-agatoxins differentially block calcium channels in locust, chick and rat synaptosomes. Three toxins (omega-Agatoxins IA, IIA and IIIA) isolated from the venom of the funnel web spider, Agelenopsis aperta, differentially block depolarization-induced calcium influx in chick, rat and locust synaptosomes. In chick, this block of calcium influx is observed with omega-Agatoxins IIA and IIIA but not with omega-Agatoxin IA. Block by omega-Agatoxin IIA and IIIA is maximal at 70 and 82% respectively of the total depolarization-induced calcium influx; maximal suppression of calcium influx by omega-Conotoxin GVIA (omega-CgTx) is 100%. The IC50 for block with omega-Agatoxin IIA is ca 3 nM as compared with an IC50 of 38 nM for omega-CgTx. Incomplete block of calcium influx at saturating concentrations of omega-Agatoxins IIA and IIIA (above 100 nM) suggests that both omega-Agatoxin-sensitive and -insensitive calcium channels occur in chick brain synaptosomes. In rat cerebrocortical synaptosomes, omega-Agatoxins IA and IIA are only partially effective at blocking depolarization-induced calcium influx, as is omega-CgTx, whilst IIIA blocks 47% of this effective at blocking depolarization-induced calcium influx, as is omega-CgTx, whilst IIIA blocks 47% of this influx. In synaptosomes prepared from the CNS of adult locusts, omega-Agatoxins IA and IIA are most effective at blocking depolarization-induced calcium influx; omega-CgTx and omega-Agatoxin IIIA are ineffective. Block of depolarization-induced calcium influx in chick brain synaptosomes by omega-Agatoxins IIA, IIIA and omega-CgTx suggests that the spider toxin interacts directly with the voltage-dependent calcium channel.(ABSTRACT TRUNCATED AT 250 WORDS)


[The effect of different body allowances of aldosterone on serotonin and cAMP metabolism in the tissues of rats with a disordered trophic function of the nervous system]. Effect of aldosterone on the content of serotonin, cAMP, and the activity of 5-hydroxytryptophane decarboxylase was studied in different rat tissues (hypothalamus, kidneys, tibial muscles) in norm and following the lesion of sciatic nerve in nonadrenalectomized and adrenalectomized animals, that is in conditions of severe deficiency of endogenous aldosterone. In nonadrenalectomized rats, exogenous aldosterone exerted either very little effect or no effect on the tissues metabolism of serotonin and cAMP. Aldosterone induced changes in the content of considered substances against the background of adrenalectomy in animals with intact sciatic nerve. These changes were observed usually only in hypothalamus and kidneys and reached their peak 60 or 90 min following the aldosterone administration. The changes in serotonin and cAMP tissues metabolism under the chronic stimulation of nervous system periphery with aldosterone may be an additional factor which enables further aggravation of trophic state of tissues and organs and alteration of their sensitivity to hormones, in particular, aldosterone.


Opioid receptor dependent long-term potentiation: peptidergic regulation of synaptic plasticity in the hippocampus. Rapid progress has been made towards understanding the synaptic physiology of excitatory amino acid transmission in the hippocampus. by comparison, the function of opioid peptides localized to some of the same pathways which use glutamate for fast excitation is poorly understood. Here I consider new evidence specifically implicating opioid peptides in long-term potentiation (LTP) induced by high-frequency stimulation of pathways which combine glutamate and opioid neurotransmission. This form of LTP is unique in that it depends on activation of opioid receptors, and unlike many excitatory systems in brain, it does not require activation of the N-methyl-D-aspartate (NMDA) type of glutamate receptor. Thus one of the main functions of opioids in the hippocampus may be to regulate activity-dependent changes in synaptic strength and neuronal excitability. At another level, "opioid" LTP may provide basic insights into peptidergic transmission and its functional interactions with classical neurotransmitters in the brain.


Strychnine-insensitive glycine receptors in embryonic chick retina: characteristics and modulation of NMDA neurotoxicity. In the mammalian brain, the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is coupled to a cation channel and a strychnine-insensitive glycine receptor. The present paper demonstrates the presence of NMDA receptor-coupled strychnine-insensitive glycine receptors in embryonic chick retina. Both glycine and 1-aminocyclopropanecarboxylic acid (ACPC) exhibited similar potencies (271 +/- 39 vs 247 +/- 39 nM, respectively) as inhibitors of strychnine-insensitive [3H]glycine binding to retinal membranes. Moreover, glycine and ACPC enhanced [3H]MK-801 binding to sites within the NMDA-coupled cation channel in retinal membranes with potencies comparable to those reported in rat brain. While the potency of ACPC was significantly higher than glycine (EC50 54 +/- 12 vs 256 +/- 57 nM, P < 0.02) in this measure, there were no significant differences in the maximum enhancement (efficacy) of [3H]MK-801 binding by these compounds. Since glycine appears to be required for the operation of NMDA-coupled cation channels, we examined the effects of glycine and ACPC on NMDA-induced acute excytotoxicity in the 14-day embryonic chick retina. Histological evaluation of retina revealed that either ACPC (10-100 microM) or glycine (200 microM) attenuated NMDA-induced (200 microM) retinal damage and a combination of these agents produced an enhanced protection against acute NMDA toxicity. ACPC (100 microM), but not MK-801 (1 microM) also afforded a modest protection against kainate-induced (25 microM) retinal damage. These findings demonstrate that while strychnine-insensitive glycine receptors are present in embryonic chick retina, occupation of these sites does not augment the cytotoxic actions of NMDA. Moreover, the ability of ACPC and glycine to attenuate NMDA-induced cytotoxicity does not appear to be mediated through occupation of these sites.


Modulation of rat brain insulin receptor kinase activity in diabetes. Insulin receptors from rat brain regions were studied for insulin binding and receptor associated kinase activity, in alloxan induced short-term and long-term diabetes, and insulin induced hypoglycemia. Insulin receptor activity was assessed by [125I]insulin binding, and basal as well as insulin stimulated kinase activity of the receptor, expressed as phosphorylation of the synthetic peptide poly (Glu-Tyr (4:1)). Regional distribution pattern elicited the highest binding and kinase activity in the olfactory bulb. Diabetes caused a significant increase in the kinase activity. The data suggests that brain insulin receptor kinase is regulated differently compared to peripheral tissues and supports the concept of an active brain insulin receptor in vivo.


Synthesis and release of GABA in cerebral cortical neurons co-cultured with astrocytes from cerebral cortex or cerebellum. Cerebral cortical neurons were co-cultured for up to 7 days with astrocytes after plating on top of a confluent layer of astrocytes cultured from either cerebral cortex or cerebellum (sandwich co-cultures). Neurons co-cultured with either cortical or cerebellar astrocytes showed a high stimulus coupled release of gamma-aminobutyric acid (GABA), which is the neurotransmitter of these neurons. When the astrocyte selective GABA uptake inhibitor 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol was added during the release experiments, an increase in the stimulus coupled GABA release was seen, indicating that the astrocytes take up a large fraction of GABA released from the neurons. The activity of the GABA synthesizing enzyme glutamate decarboxylase, which is a specific marker of GABAergic neurons, was markedly increased in sandwich co-cultures of cortical neurons and cerebellar astrocytes compared to neurons cultured in the absence of astrocytes whereas in co-cultures with cortical astrocytes this increase was less pronounced. Pure astrocyte cultures did not show any detectable glutamate decarboxylase activity. The astrocyte specific marker enzyme glutamine synthetase (GS) was present at high activity in a glucocorticoid-inducible form in pure astrocytes as well as in co-cultures regardless of the regional origin of the astrocytes. When neurons were cultured on top of the astrocytes, the specific activity of GS was lower compared to astrocytes cultured alone, a result compatible with the notion that neurons are devoid of this enzyme. The results show that cortical neurons develop and differentiate when seeded on top of both homotypic and heterotypic astrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)


Differences in hydrogen exchange behavior between the oxidized and reduced forms of Escherichia coli thioredoxin. Amide proton exchange of thioredoxin is used to monitor the structural effects of reduction of its single disulfide. An effective 3-5-proton difference between the oxidized and reduced protein form is observed early in proton out-exchange of the whole protein, which is independent of temperature in the range of 5-45 degrees C, indicating that redox-sensitive changes are probably not due to low-energy structural fluctuations. Medium resolution hydrogen exchange experiments have localized the redox-sensitive amide protons to two parts of the sequence that are distant from each other in the three-dimensional structure: the active-site turn and the first beta-strand. The sum of the proton differences observed in the peptides from these regions is equal to that of the whole protein, indicating that all redox-sensitive hydrogen exchange effects are observed in the peptide experiments. A model combining structural changes within the protein matrix with changes in the surface hydration properties is proposed as a mechanism for the communication between distant sites within the protein. Sound velocity and density measurements of reduced and oxidized thioredoxin are presented in the accompanying paper (Kaminsky, S.M. & Richards, F.M., 1992, Protein Sci. 1, 22-30).


Brønsted analysis of aspartate aminotransferase via exogenous catalysis of reactions of an inactive mutant. Primary amines functionally replace lysine 258 by catalyzing both the 1,3-prototropic shift and external aldimine hydrolysis reactions with the inactive aspartate aminotransferase mutant K258A. This finding allows classical Brønsted analyses of proton transfer reactions to be applied to enzyme-catalyzed reactions. An earlier study of the reaction of K258A with cysteine sulfinate (Toney, M.D. & Kirsch, J.F., 1989, Science 243, 1485) provided a beta value of 0.4 for the 1,3-prototropic shift. The beta value reported here for the transamination of oxalacetate to aspartate is 0.6. The catalytic efficacy of primary amines is largely determined by basicity and molecular volume. The dependence of the rate constants for the reactions of K258A and K258M on amine molecular volume is nearly identical. This observation argues that the alkyl groups of the added amines do not occupy the position of the lysine 258 side chain in the wild type enzyme. Large primary C alpha and insignificant solvent deuterium kinetic isotope effects with amino acid substrates demonstrate that the amine nitrogen of the exogenous catalysts directly abstracts the labile proton in the rate-determining step.


Calculation of the free energy of association for protein complexes. We have developed a method for calculating the association energy of quaternary complexes starting from their atomic coordinates. The association energy is described as the sum of two solvation terms and an energy term to account for the loss of translational and rotational entropy. The calculated solvation energy, using atomic solvation parameters and the solvent accessible surface areas, has a correlation of 96% with experimentally determined values. We have applied this methodology to examine intermediates in viral assembly and to assess the contribution isomerization makes to the association energy of molecular complexes. In addition, we have shown that the calculated association can be used as a predictive tool for analyzing modeled molecular complexes.


The calmodulin-binding site of the plasma membrane Ca2+ pump interacts with the transduction domain of the enzyme. Calpain proteolysis of the plasma membrane Ca2+ pump removes a C-terminal 14-kDa portion which includes the calmodulin-binding domain. This produces a fully activated 124-kDa fragment, which can be inhibited by synthetic versions of the calmodulin-binding domain. The inhibition is strongest when Trp-8 in the latter domain is replaced by a Tyr residue (Falchetto, R., Vorherr, T., Brunner, J., & Carafoli, E., 1991, J. Biol. Chem. 266, 2930-2936). In the present study, the N-terminus of the 28-residue synthetic calmodulin-binding domain was acetylated with 3H-acetic anhydride, and Phe in position 25 was replaced by a phenylalanine derivatized with a diazirine-based, photoactivatable carbene precursor. This peptide (C28WC*) inhibited the fully active 124-kDa fragment of the pump and became cross-linked to it upon photolysis. After proteolysis of the fragment with Asp-N or Staphylococcus aureus V8 (Glu-C) protease, labeled peptides were isolated by reversed-phase high-performance liquid chromatography and subjected to Edman sequence analysis. The peptides originated from a region of the pump located within the unit protruding into the cytoplasm between transmembrane domain two and three. This unit has been proposed to be the site of the energy transduction domain, which would couple the ATP hydrolysis to Ca2+ translocation.


Stein and Moore Award address. Reconstructing history with amino acid sequences. The main goal of the protein evolutionist is the reconstruction of past events leading to the structures of contemporary proteins. The common strategy is to align amino acid sequences and make inferences about matters of common ancestry. The rate of change of amino acid sequence varies greatly from protein to protein, and this naturally affects how far back a given protein's ancestry can be traced. Happily, the rate of change of many proteins is slow enough that very ancient events can be inferred. Many mainstream metabolic enzymes, for example, are 40-50% identical in prokaryotes and eukaryotes, groups that diverged from a common ancestor more than 1.5 billion years ago. Moreover, some eukaryotic proteins like actin and tubulin change so slowly that they are seldom less than 60% identical, no matter from what source they are drawn. As it happens, prokaryotic counterparts for many eukaryotic cytoskeletal proteins are unknown. A recent exception involves the finding that a heat shock protein cognate is a relative of actin. The gene duplication that gave rise to these two proteins must have been an ancient event. The more recent invention of other proteins whose distribution is restricted to one or the other of the major kingdoms may be easier to trace. Among the factors that can confound the reconstruction of events, however, are occasional horizontal gene transfers and exon shuffling. The latter has led to a number of mosaic proteins, many of which contain various combinations of a relatively small set of modules like the epidermal growth factor domain.


Substrate polarization by residues in delta 5-3-ketosteroid isomerase probed by site-directed mutagenesis and UV resonance Raman spectroscopy. delta 5-3-Ketosteroid isomerase (KSI: EC 5.3.3.1) of Pseudomonas testosteroni catalyzes the isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by the stereospecific transfer of the steroid 4 beta-proton to the 6 beta-position, using Tyr-14 as a general acid and Asp-38 as a base. Ultraviolet resonance Raman (UVRR) spectra have been obtained for the catalytically active double mutant Y55F + Y88F, which retains Tyr-14 as the only tyrosine residue (referred to as the Y14(0) mutant), and the Y14F mutant, which has 50,000-fold lower activity. The UVRR results establish that binding of the product analog and competitive inhibitors 19-nortestosterone or 4-fluoro-19-nortestosterone to the Y14(0) mutant does not result in the formation of deprotonated Tyr-14. The UVRR spectra of the steroid inhibitors show large decreases in the vinyl and carbonyl stretching frequencies on binding to the Y14(0) enzyme but not on binding to the Y14F enzyme. These changes cannot be mimicked by protonation of the steroids. For 19-nortestosterone, the vinyl and carbonyl stretching frequencies shift down (with respect to the values in aqueous solution) by 18 and 27 cm-1, respectively, on binding to Y14(0) KSI. It is proposed that the changes in the steroid resonance Raman spectrum arise from polarization of the enone moiety via the close proximity of the charged Asp-38 side chain to the vinyl group and the directional hydrogen bond between Tyr-14 and the 3-carbonyl oxygen of the steroid enone. The 230-nm-excited UVRR spectra do not, however, show changes that are characteristic of strong hydrogen bonding from the tyrosine hydrogen. It is proposed that this hydrogen bonding is compensated by a second hydrogen bond to the Tyr-14 oxygen from another protein residue. UVRR spectra of the Y14(0) enzyme obtained using 200 nm excitation show enhancement of the amide II and S Raman bands. The secondary structure of KSI was estimated from the amide II and S intensities and was found to be low in alpha-helical structure. The alpha-helix content was estimated to be in the range of 0-25% (i.e., 10 +/- 15%).


Effect of acute footshock stress on the responsiveness of the isolated rat tail artery to phenylephrine and epinephrine. The effects of acute footshock stress on the sensitivity of the isolated rat tail artery were studied. Footshock stress applied to male Wistar rats (200-300 g) causes subsensitivity to the vasoconstrictor effects of phenylephrine and epinephrine. No significant changes in the pA2 values of prazosin were detected, using epinephrine as the agonist. Footshock stress-induced subsensitivity to epinephrine was not affected by the calcium entry blocker nifedipine. However, nifedipine significantly depressed the maximum response to epinephrine in tail arteries isolated from acute footshock-stressed rats. The present results suggest that acute footshock stress-induced reduced sensitivity to phenylephrine and epinephrine may not be only related to events at the alpha-adrenoceptor level. The nifedipine-induced depression of the maximum response to epinephrine suggests a role for the calcium mobilization processes in the vascular responsiveness during acute stress.


[Function of lymphocyte beta-adrenergic receptor in depression]. Lymphocyte beta-adrenergic receptor function of 20 depressed patients and 18 healthy volunteers were measured using radioligand binding technique. It was found that the affinity and sensitivity of this receptor were significantly higher in endogenous depressed patients than those in normal controls. After the electro-acupuncture-treatment (EAT), lymphocyte beta-receptor function decreased in patients who responded well to EAT, but still not reached normal level.


[A study of cyclic nucleotide acids in the serum in patients with myasthenia gravis]. The levels of cAMP and cGMP in serum of 100 patients with myasthenia gravis have been measured by 125I-RIA. The results showed that cAMP and cGMP were higher in the patients than in the controls significantly (P < 0.001). In 33 cases of these patients after using anticholinesterase drugs and prednisone, the levels of cAMP and cGMP reduced significantly. According to our study, we presumed that there would be cyclic nucleotide acid metabolic disturbances in patients with MG and cAMP might play a regulatory role in morbidity of MG.


Dexamethasone-induced adrenal cortex atrophy and recovery of the gland from partial, steroid-induced atrophy. This study aims to examine the effects of low dexamethasone (DX) doses on cellular and functional changes in the rat adrenal cortex and to observe the recovery of the gland from DX-induced partial atrophy. Within seven days doses of 15, 30 and 60 micrograms DX/100 g/day resulted in a dose dependent decrease in body and adrenal gland weight. Within two weeks the lowest DX dose tested caused further decrease in adrenal weight. DX markedly lowered plasma ACTH and corticosterone (B) level, B content in the gland and B output by adrenal slices. Adrenocortical atrophy induced by 15 micrograms DX was dependent upon the linear decrease in the volume of fasciculata and reticularis zones, in the average volume of the fasciculata cell and in the number of adrenocortical cells in the entire cortex. This dose of DX resulted in a prompt and potent inhibition of proliferative activity of adrenocortical cells as assessed by the counting of metaphases per adrenal section. Partial adrenal atrophy evoked by 7 day DX-treatment was reversible within seven days of the discontinuation of the steroid treatment. Recovery of the gland depended mainly on the increases in the volume of all adrenocortical zones, in the average volume of fasciculata cell and in the number of parenchymal cells in the gland. Seven days after discontinuation of DX administration plasma ACTH was significantly higher than in controls. Plasma B and B secretion by adrenal slices were similar to the control group while B content in the gland was still depressed.(ABSTRACT TRUNCATED AT 250 WORDS)


Influence of the electrical stimulation of the medial amygdala on adrenocortical sensitivity to adrenocorticotrophin in hypophysectomized rats. The effects of electrical stimulation of medial amygdala on the adrenocortical sensitivity to adrenocorticotrophin (ACTH) were investigated in hypophysectomized rats. The intravenous injection of ACTH increased the rates of 14C transfer from 14C-l-acetate into corticosterone and cortisol in the adrenal slices of hypophysectomized rats. The electrical stimulation of the medial amygdala produced a further increase in the rates of 14C transfer from 14C-l-acetate into corticosterone and cortisol. From these results, it might be suggested that the medial amygdala was capable of enhancing the adrenocortical steroidogenesis in response to ACTH.


[Activity of acidic granulocyte phosphatase and myeloperoxidase among women with EPH gestosis]. Among 30 pregnant women with EPH-gestosis activity of myeloperoxidase was significantly decreased and activity of acid phosphatase was not much increased. Results of scientific researches can confirm the fact of weakness of anti-infection immunity among women with EPH-gestosis in compare with healthy pregnant women.


Agents associated with neonatal diarrhoea in Ethiopian dairy calves. Faeces samples collected from diarrhoeic dairy calves in the first 8 weeks of life were examined for the presence of 5 enteropathogens. The majority of the 108 diarrhoea cases occurred in the first 5 weeks of life and a commercial ELISA kit detected bovine enteric coronavirus (BEC) in 38.9%, serogroup A rotavirus (RV) in 16.7% and K99 (F5) fimbrial adhesin-positive Escherichia coli (K99 ETEC) in 11.1 per cent. Concurrent infections of these enteropathogens were detected in 14.8% of samples (30.8% of samples positive for these agents). No evidence of cryptosporidial infection was found using a differential staining method on faecal smears nor was salmonella excretion detected. On 2 of the 8 farms only BEC was present; the other 6 farms were positive for all 3 agents. It is concluded that BEC is the major infectious cause of neonatal calf diarrhoea in the Ethiopian dairy herds studied with RV and K99 ETEC also contributing to morbidity, either alone or as mixed infections.


Prevalence of subclinical infectious bursal disease and its significance in India. From a total of 32 poultry flocks, 1,176 serum samples were screened by the agar gel precipitation test and 314 (26.7%) were positive for antibodies to infectious bursal disease virus (IBDV). Prevalence of infection on the farms ranged from 8.88 to 53.84 per cent. The prevalence was highest (61.82%) in chickens between 7 and 11 weeks old and lowest (3.92%) in those above 22 weeks of age. In commercial broilers and layers 51.61 and 17.78% respectively were seropositive reactors. The high prevalence of subclinical IBD and its economic significance are discussed.


[Antibodies against hepatitis C virus. Study in voluntary blood donors. Cuba 1991]. Four hundred and sixty one samples randomly selected among blood donors from three blood banks in Havana City were investigated for HCV antibodies. Samples repeatedly reactive to Organcon Tecknica immunoenzymatic assay were considered positives and were further confirmed by an Ortho test. An epidemiological survey was conducted among all donors included in the sample to assess infection risk factors. Seven subjects (1.5%) resulted repeatedly reactive to the test, slightly higher than in developed countries. Mean age in positive patients was seven years higher than in negative subjects (39 vs 31.5), but the Student's test did not show any significant difference. Seropositivity was higher in males (1.7 vs 0.9) and very similar as to the color of skin (1.9 mulattos, 1.5 whites and 1.0 blacks) but it was not statistically significant (p > 0.05). Among risk factors with higher odd ratios were to have more than five sexual partners in the last five years and having worked in a health care institution, but this was not statistically significant (p > 0.05). The probability of receiving one unit of HCV infected blood was of 1.5% and 45% when receiving 40 units. Given the importance of the virus, the need of establishing specific control measures in the shortest period of time for HCV detection in blood is under-lined.


Comparison of modified HAVAB and ELISA for determination of vaccine-induced anti-HAV response. An inhibition ELISA was compared with a modification of the HAVAB assay for measuring antibodies induced by a killed HAV vaccine. GMT's expressed in mIU/ml were higher by ELISA than by modified HAVAB, especially after the first and second doses of vaccine but seroconversion rates were very similar and a good correlation was found between both assays. Because of its higher sensitivity and specificity, the ELISA assay was preferred to modified HAVAB for the evaluation of a Hepatitis A vaccine in human volunteers.