Daily and circadian regulation of 2-[125I]iodomelatonin binding in the chick brain. The pineal gland and its hormone melatonin are important in the control of circadian rhythms in birds. Recent research has indicated the presence of high affinity melatonin-binding sites in a wide array of avian cerebral structures. In this study melatonin-binding sites were localized and characterized in the brains of 2-week-old chicks using in vitro autoradiography and image analysis of 2-[125I]iodomelatonin (IMEL) binding. The IMEL binding predominated in the major components of the chick's visual and auditory systems. These brain binding sites showed a daily variation in IMEL binding, with a higher density of binding during the daytime. A significant peak in IMEL binding was observed in the late afternoon, Zeitgeber time 10. This rhythm of IMEL binding continued in constant darkness, with the peak at circadian time 10. The amplitude of the peak was increased in constant darkness in all structures, with the exception of the ectostriatum and neostriatum. Scatchard analysis of the binding revealed an average increase of 45 +/- 5% in the number of binding sites in the daytime vs. nighttime samples, with little change in the binding affinities, indicating that the rhythm in binding is caused by an increase in the total number of binding sites available in the daytime tissue, rather than an increase in the affinity of the binding sites. The data suggest that a rhythm of melatonin sensitivity in a diverse set of cerebral structures may regulate a temporal control of their function.


Transient down-regulation of androgen receptor messenger ribonucleic acid (mRNA) expression in Sertoli cells by follicle-stimulating hormone is followed by up-regulation of androgen receptor mRNA and protein. Cooperative actions of FSH and androgens on initiation, maintenance, and restoration of spermatogenesis have been described. In the present experiments the regulatory effects of FSH on androgen receptor (AR) gene expression in Sertoli cells were studied. In immature rats injection of FSH (1 microgram/g BW, ip) resulted in a rapid down-regulation of testicular AR mRNA expression (4 h), followed by recovery to the control level (10 h). Using cultured immature Sertoli cells, a similar transient effect on AR mRNA expression was observed after the addition of FSH (500 ng/ml) or (Bu)2cAMP (0.5 mM). Cycloheximide treatment of the cells did not prevent the rapid FSH-induced down-regulation of AR mRNA expression, indicating that de novo protein synthesis is not required for this effect. Furthermore, using a transcriptional run-on assay, no marked decrease in the rate of AR gene transcription was found upon treatment of the cultured Sertoli cells with FSH for 2 or 4 h. This demonstrates that the short term effect of FSH or AR mRNA expression reflects a change in mRNA stability. The AR protein level was not markedly affected by the transient decrease in AR mRNA expression. When immature Sertoli cells were incubated with FSH for longer time periods (24-72 h), both AR mRNA and protein expression were increased. In Sertoli cells isolated from 15-day-old rats, this increase was higher (mRNA, 2- to 3-fold; protein, 2-fold) than in Sertoli cells isolated from 25-day-old animals. The results indicate that FSH plays a complex role in the regulation of AR expression in immature rat Sertoli cells.


Actions of endothelin-1 on swine ovarian (granulosa) cells. We have investigated the regulatory actions of endothelin-1 (ET-1) on inositol phosphate accumulation, cytosolic free Ca2+ ion concentrations ([Ca2+]i), and basal and FSH-stimulated progesterone and cAMP accumulation by swine granulosa cells in serum-free cultures. ET-1 induced a rapid stimulation of phosphoinositide hydrolysis in populations of granulosa cells, as inferred by the rapid appearance of soluble inositol polyphosphates in response to ET-1 exposure. At the single cell level, fura-2 videomicroscopy was used to measure [Ca2+]i in individual granulosa cells. We observed cell-cell variability in the threshold concentration of ET-1 required to induce a rise in [Ca2+]i. More than 75% of granulosa cells responded to maximal doses of ET-1. The following parameters of [Ca2+]i were influenced by ET-1 concentration: percentage of responding cells, lag time for the onset of response, amplitude, and kinetics of the response. Two types of ET-1-mediated [Ca2+]i rises were observed. One type exhibited rapid Ca2+ kinetics, reaching at least a 2-fold increase above basal (spike phase) within 1-10 sec and returning to a new steady state (plateau phase) 2 min after onset. The other mode of response had slower [Ca2+]i kinetics, in which 50 sec or more were required to double [Ca2+]i, which remained at this level throughout the observation period (2.5 min). These responses to ET-1 were specific and were not initiated by vasopressin or tumor necrosis factor-alpha. In cell population studies using monolayer cultures of swine granulosa cells, ET-1 inhibited FSH-stimulated accumulation of progesterone and cAMP. The ET-1-mediated inhibition of FSH-stimulated accumulation of progesterone required at least 4 h of ET-1 exposure. The ET-1-mediated inhibition of both the FSH-stimulated accumulation of progesterone and cAMP after 24-h incubation was mimicked by an activator of protein kinase-C, phorbol 12-myristate 13-acetate, but not by an inactive phorbol. These observations in either single cells or populations of swine ovarian (granulosa) cells are consistent with a possible regulatory role of an ET-1-activated intracellular signaling pathway involving inositol phosphates, [Ca2+]i, and protein kinase-C in the mammalian granulosa cell.


Protein kinase-A inhibits phospholipase-C activity and alters protein phosphorylation in rat myometrial plasma membranes. Our previous studies implicated the involvement of protein kinase-A in the inhibitory effects of isoproterenol and relaxin on oxytocin-stimulated phosphoinositide turnover in rat myometrium. To understand the possible mechanisms involved, the properties and regulation of phospholipase-C (PLC) in purified myometrial plasma membranes from estrogen-primed rats were studied. The PLC activity measured with exogenous [3H]phosphatidylinositol 4,5-bisphosphate as substrate was Ca2+ dependent. The nonhydrolyzable GTP analog guanosine 5'-(3-O-thio)triphosphate stimulated PLC activity with a ED50 of 1.6 microM and shifted the calcium dependence curve to the left. Guanosine 5'-(3-O-thio)triphosphate-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis was inhibited by activation of endogenous and exogenous cAMP-dependent protein kinase (PKA). The effects of endogenous and exogenous PKA were significantly reversed by IP20, a potent synthetic peptide inhibitor of PKA. In the presence of [gamma-32Pi]ATP and exogenous PKA, 32Pi was incorporated in an IP20-sensitive manner into major bands at approximately 17,000, 20,000-24,000, 33,000, 38,000, 40,000-44,000, and other higher mol wt. These data indicate that one or more GTP-binding proteins mediate activation of membrane-bound PLC in rat myometrium. Phosphorylation of one or more membrane-associated proteins by PKA may regulate myometrial PLC activity and play a role in the inhibitory effects of isoproterenol and relaxin.


Transforming growth factor-beta modulates receptor binding of calciotropic hormones and G protein-mediated adenylate cyclase responses in osteoblast-like cells. Transforming growth factor-beta (TGF beta) produced by osteoblasts is present in high levels in bone and influences bone formation, replication of bone cells, and expression of osteoblast protein products. Interactions between bone active hormones and locally released and activated TGF beta were studied by examining the influence of TGF beta preincubation on PTH, calcitonin (CT), and vitamin D receptors in an osteoblastic cell line (UMR 106-06). Preincubation of UMR 106-06 cells with 1 ng/ml TGF beta for 3 days increased specific binding of [125I]PTH-related protein (PTHrP)(1-84) to 140% of that in control cells, but [125I]salmon CT binding decreased to 50% of controls. Binding isotherms indicated that the changes in binding were due to altered receptor numbers since affinities for 125I-labeled PTH and CT remained unchanged. The effect on receptor levels was time dependent, requiring 24 h preincubation with TGF beta for measurable changes, and dose dependent, with maximal effects seen with 1 ng/ml TGF beta. Binding of [3H]1,25(OH)2 vitamin D3 was increased to 130% of control in cytosolic extracts of UMR 106-06 cells pretreated for 3 days with 1 ng/ml TGF beta. Scatchard plots suggested an increase in receptor number without change in affinity. The adenylate cyclase response to PTH increased to 150% of control cells after 3 days of treatment with 1 ng/ml TGF beta; however, the adenylate cyclase response to CT was little changed. Forskolin- and cholera toxin-stimulated adenylate cyclase responses were increased by TGF beta treatment to 130-160% of control, indicating an increase in the stimulatory subunit of the G protein. Increased abundance of both Gs and Gi proteins were indicated by increased cholera toxin- or pertussis toxin-dependent [32P] NAD ribosylation of 47-kilodalton (kDa) and 42-kDa or 40-kDa proteins, respectively, in TGF beta-treated cells. Our data support a complex regulatory effect of TGF beta on UMR 106-06 cells with increases in PTH receptors, vitamin D receptors, and G proteins, whereas there is an apparent down-regulation of CT receptors. TGF beta might induce a more differentiated osteoblast phenotype of these cells, which already express differentiated features such as high alkaline phosphatase activity, PTH and vitamin D receptors, and collagenase production. Since low doses of PTH stimulate bone formation in vivo, TGF beta released or activated at sites of new bone formation might locally modulate PTH activity be allowing increased PTH receptor and postreceptor effectiveness.


Evidence that activation of protein kinase-C can stimulate 1,25-dihydroxyvitamin D3 secretion by rat proximal tubules. PTH stimulates mammalian renal proximal tubule cell synthesis and secretion of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] by a Ca-dependent process. In the present study regulation of 1,25-(OH)2D3 secretion by PTH, phorbol ester 12-O-tetradecanoylphorbol 13-acetate, the Ca ionophore A23187, and calcitonin was evaluated in perifused rat proximal tubule cells isolated by collagenase digestion and centrifugation through Percoll. Tubules from rats fed a low Ca diet secreted 1,25-(OH)2D3 at a rate 2.5 times that of tubule cells from rats fed a normal Ca diet. Perifusion of tubules with human PTH-(1-34) (10(-7) M) induced an immediate and sustained increase in 1,25-(OH)2D3 secretion. Perifusion with either A23187 or 12-O-tetradecanoylphorbol 13-acetate caused transient increases in hormone secretion, while both agents perifused simultaneously resulted in a sustained increase in 1,25-(OH)2D3 secretion. Perifusion of tubule cells with the protein kinase-C (PKC) inhibitor staurosporine blocked the PTH-induced increase in 1,25-(OH)2D3 secretion. Calcitonin had no effect on 1,25-(OH)2D3 secretion rates. The results of the present studies show that an activator of PKC increases 1,25-(OH)2D3 secretion by mammalian proximal tubule cells and suggest that the phospholipase-C/PKC signalling system may mediate PTH stimulation of 1,25-(OH)2D3 secretion.


Differential effects of the 3',5'-cyclic adenosine monophosphate and protein kinase C pathways on the response of isolated rat osteoclasts to calcitonin. Calcitonin (CT) activates both the cAMP and the protein kinase C (PKC) pathways in the kidney cell line LLC-PK1. Although CT also activates cAMP in osteoclasts, its effects on PKC in this cell type are unknown. In order to determine whether the response of osteoclasts to CT also involves the PKC pathway, the effects of activators and inhibitors of PKC on bone resorption and cell surface area were analyzed in isolated rat osteoclasts. As expected, CT inhibited in a dose-dependent manner bone resorption by rat osteoclasts cultured for 24 h on devitalized bovine bone slices and this effect could be mimicked by cAMP. The inhibitory effect of CT could however also be mimicked by phorbol-12,13-dibutyrate (PDBu) and blocked by the PKC inhibitor sphingosine, as well as by the less specific inhibitors H7 and H8, none of which had detectable effects in the absence of CT. No changes in the number of attached osteoclasts were observed under any of these conditions. These results indicate that CT activates PKC in osteoclasts and that this activation, like the activation of cAMP-dependent protein kinase, leads to an inhibition of bone resorption. Quantitative time-lapse videomicroscopy showed that the CT-induced retraction of osteoclasts also involved activation of the PKC pathway and could therefore be induced by phorbol esters. In contrast, (Bu)2 cAMP (1-200 microM) failed to induce rapid cell retraction. It is concluded that, in osteoclasts, CT receptors are coupled to both the cAMP-dependent protein kinase and the PKC pathways. Although these two second messengers can have additive inhibitory effects on bone resorption, only activation of the PKC pathway induces rapid cell retraction. These two effects of calcitonin on osteoclasts are therefore independent and may be functionally unrelated.


Rat epididymal retinoic acid-binding protein: development of a radioimmunoassay, its tissue distribution, and its changes in selected androgen-dependent organs after orchiectomy. Rat epididymal retinoic acid-binding protein (EP-RABP) has been purified to apparent homogeneity from extracts of the epididymis by HPLC. N-Terminal amino acid sequence analysis revealed that the HPLC-purified protein consisted of two molecular variants, in that one has three extra N-terminal amino acids of NH2-TEG. These two molecular variants were subsequently separated by high performance electrophoresis chromatography. A specific and sensitive RIA has been developed to quantify this protein in various organ extracts of both male and female rats. Rat EP-RABP is a male-specific protein, since it was detected in male organ extracts, including epididymis, testis, prostate, seminal vesicles, liver, spleen, and brain, with a negligible concentration in the female liver and spleen. It was noted that this protein was absent in the systemic circulation of both male and female rats. It was first detected in the epididymis and testis of rats at 22 and 27 days of age, respectively. Both the concentrations (micrograms per g tissue) and the organ content (micrograms per organ pairs) of this protein in the testis and epididymis increased progressively with an increase in age and reached the highest levels at 60 and 120 days of age, respectively. At 120 days of age, its concentrations in all organs examined, with the exception of the epididymis, showed a dramatic decrease compared to levels in rats at 60 days of age. Orchiectomy decreased its concentrations in the caput, corpus, and cauda epididymis and in the ventral prostate, but had no apparent effect on seminal vesicles. Administration of dihydrotestosterone to castrated rats restored only 30% of the level of this protein in the caput epididymis compared to that in intact animals, but had no apparent effect on the corpus, cauda epididymis, or ventral prostate. These observations suggest that this protein is under multihormonal control in the epididymis and selected androgen-dependent organs.


Plasticity of adrenoceptor responsiveness on irANP secretion and pro-ANP mRNA expression in hypothalamic neuron cultures: modulation by dexamethasone. Atrial Natriuretic Peptide (ANP) or its smaller congeners are produced and secreted from the rat hypothalamus. Whereas immunoreactive (ir)ANP secretion and proANP mRNA expression in hypothalamic cell cultures of neonatal rats were augmented by norepinephrine acting through its alpha 2-adrenoceptors (AR), in the perifusion studies of adult hypothalamic fragments beta-AR was involved in the upregulation of irANP release. Here, we report that dexamethasone (DM) modulates irANP secretion and pro-ANP mRNA expression from hypothalamic neurons in culture by switching the adrenoceptor responsiveness of the cells from alpha 2- to that of beta-AR. In long term cultures of hypothalamic cells, treatment with clonidine (alpha 2-AR agonist) increased irANP secretion in a dose related manner. This effect of clonidine was abolished by DM, a glucocorticoid which by itself had little effect on the basal release of irANP. In contrast, isoprenaline, a beta-AR agonist which was ineffective when applied alone, enhanced irANP secretion from hypothalamic cultures in the presence of DM. Concurrent incubation of DM (5 nM) and isoprenaline (10 microM) augmented irANP release approximately 3 fold above that of cultures treated with DM alone (22.6 +/- 2.2; mean +/- SE, n = 4). However, phenylephrine, an alpha 1-AR agonist alone or in the presence of DM failed to stimulate irANP release. These immunoassay findings were accompanied by corresponding changes in the abundance of pro-ANP mRNA in the cultures as examined by colorimetric Northern blot analysis employing a 30 mer oligonucleotide probe corresponding to the first 10 amino acid sequence of rANP1-28. We conclude from the above observations that glucocorticoids modulate irANP secretion and pro-ANP mRNA expression in hypothalamic neurons by altering the responsiveness of the cells from alpha 2-AR to that of beta-AR.


Physarum actin is phosphorylated as the actin-fragmin complex at residues Thr203 and Thr202 by a specific 80 kDa kinase. The Physarum EGTA-resistant actin-fragmin complex, previously named cap 42(a+b), is phosphorylated in the actin subunit by an endogenous kinase [Maruta and Isenberg (1983) J. Biol. Chem., 258, 10151-10158]. This kinase has been purified and characterized. It is an 80 kDa monomeric enzyme, unaffected by known kinase regulators. Staurosporine acts as a potent inhibitor. The actin-fragmin complex is the preferred substrate. The phosphorylation is inhibited by micromolar Ca2+ concentrations, but only in the presence of additional actin. Polymerized actin (vertebrate muscle and non-muscle isoforms) and actin complexes with various actin-binding proteins are poorly phosphorylated. The heterotrimer consisting of two actins and one fragmin, which is formed from cap 42(a+b) and actin in the presence of micromolar concentrations of Ca2+, is also a poor substrate. From the other substrates tested, only histones were significantly phosphorylated, in particular histone H1. In the same manner, casein kinase I could also phosphorylate the actin-fragmin complex. The major phosphorylation site in actin is Thr203. A second minor site is Thr202. These residues constitute one of the contact sites for DNase I [Kabsch et al. (1990) Nature, 347, 37-44] and are also part of one of the predicted actin-actin contact sites in the F-actin model [Holmes et al. (1990) Nature, 347, 44-49].


X-ray structure of nucleoside diphosphate kinase. The X-ray structure of a point mutant of nucleoside diphosphate kinase (NDP kinase) from Dictyostelium discoideum has been determined to 2.2 A resolution. The enzyme is a hexamer made of identical subunits with a novel mononucleotide binding fold. Each subunit contains an alpha/beta domain with a four stranded, antiparallel beta-sheet. The topology is different from adenylate kinase, but identical to the allosteric domain of Escherichia coli ATCase regulatory subunits, which bind mononucleotides at an equivalent position. Dimer contacts between NDP kinase subunits within the hexamer are similar to those in ATCase. Trimer contacts involve a large loop of polypeptide chain that bears the site of the Pro----Ser substitution in Killer of prune (K-pn) mutants of the highly homologous Drosophila enzyme. Properties of Drosophila NDP kinase, the product of the awd developmental gene, and of the human enzyme, the product of the nm23 genes in tumorigenesis, are discussed in view of the three-dimensional structure and of possible interactions of NDP kinase with other nucleotide binding proteins.


Restoration of a lost metal-binding site: construction of two different copper sites into a subunit of the E. coli cytochrome o quinol oxidase complex. The cupredoxin fold, a Greek key beta-barrel, is a common structural motif in a family of small blue copper proteins and a subdomain in many multicopper oxidases. Here we show that a cupredoxin domain is present in subunit II of cytochrome c and quinol oxidase complexes. In the former complex this subunit is thought to bind a copper centre called CuA which is missing from the latter complex. We have expressed the C-terminal fragment of the membrane-bound CyoA subunit of the Escherichia coli cytochrome o quinol oxidase as a water-soluble protein. Two mutants have been designed into the CyoA fragment. The optical spectrum shows that one mutant is similar to blue copper proteins. The second mutant has an optical spectrum and redox potential like the purple copper site in nitrous oxide reductase (N2OR). This site is closely related to CuA, which is the copper centre typical of cytochrome c oxidase. The electron paramagnetic resonance (EPR) spectra of both this mutant and the entire cytochrome o complex, into which the CuA site has been introduced, are similar to the EPR spectra of the native CuA site in cytochrome oxidase. These results give the first experimental evidence that CuA is bound to the subunit II of cytochrome c oxidase and open a new way to study this peculiar copper site.


The general mitochondrial processing peptidase from potato is an integral part of cytochrome c reductase of the respiratory chain. The major mitochondrial processing activity removing presequences from nuclear encoded precursor proteins is present in the soluble fraction of fungal and mammalian mitochondria. We found that in potato, this activity resides in the inner mitochondrial membrane. Surprisingly, the proteolytic activity co-purifies with cytochrome c reductase, a protein complex of the respiratory chain. The purified complex is bifunctional, as it has the ability to transfer electrons from ubiquinol to cytochrome c and to cleave off the presequences of mitochondrial precursor proteins. In contrast to the nine subunit fungal complex, cytochrome c reductase from potato comprises 10 polypeptides. Protein sequencing of peptides from individual subunits and analysis of corresponding cDNA clones reveals that subunit III of cytochrome c reductase (51 kDa) represents the general mitochondrial processing peptidase.


Human beta-globin mRNAs that harbor a nonsense codon are degraded in murine erythroid tissues to intermediates lacking regions of exon I or exons I and II that have a cap-like structure at the 5' termini. Previous studies have demonstrated that nonsense codons within beta zero-thalassemic or in vitro-mutagenized human beta-globin transgenes result in the production of mRNAs that are degraded abnormally rapidly in the cytoplasm of murine erythroid cells. As a consequence, three RNA degradative intermediates are formed that lack sequences from either exon I or exons I and II. We show here that the intermediates, like the full-length mRNA from which they derive and the endogenous murine beta maj-globin mRNA, bind to the anticap monoclonal antibody H-20 in a way that is competed by the cap analogue m7G and eliminated by prior exposure to tobacco acid pyrophosphatase. Furthermore, the intermediates, like the two full-length mRNAs, are resistant to a 5'----3' exonuclease activity isolated from HeLa cell nuclei that degrades uncapped but not capped ribopolymers. Based on these observations, the intermediates appear to possess a structure that is indistinguishable from the cap at the 5' end of mRNA, i.e. a methylated nucleoside that is linked to the RNA by a 5'-5' phosphodiester bond. Detection of the intermediates during murine development was concomitant with detection of full-length thalassemic mRNA. Intermediate production appears to be influenced by RNA structure as indicated by the products that derive from a beta zero-thalassemic beta-globin transgene harboring a structural alteration (a 4 bp deletion) that was larger than any of those previously studied.


Evolution of the primate lentiviruses: evidence from vpx and vpr. The genomes of the four primate lentiviral groups are complex and contain several regulatory or accessory genes. Two of these genes, vpr and vpx, are found in various combinations within the four groups and encode proteins whose functions have yet to be elucidated. Comparison of the encoded protein sequences suggests that the vpx gene within the HIV-2 group arose by the duplication of an ancestral vpr gene within this group. Evolutionary distance analysis showed that both genes were well conserved when compared with viral regulatory genes, and indicated that the duplication occurred at approximately the same time as the HIV-2 group and the other primate lentivirus groups diverged from a common ancestor. Furthermore, although the SIVagm vpx proteins are homologous to the HIV-2 group vpx proteins, there are insufficient grounds from sequence analysis for classifying them as vpx proteins. Because of their similarity to the vpr proteins of other groups, we suggest reclassifying the SIVagm vpx gene as a vpr gene. This creates a simpler and more uniform picture of the genomic organization of the primate lentiviruses and allows the genomic organization of their common precursor to be defined; it probably contained five accessory genes: tat, rev, vif, nef and vpr.


A single-stranded DNA binding protein required for mitochondrial DNA replication in S. cerevisiae is homologous to E. coli SSB. It has previously been shown that the mitochondrial DNA (mtDNA) of Saccharomyces cerevisiae becomes thermosensitive due to the inactivation of the mitochondrial DNA helicase gene, PIF1. A suppressor of this thermosensitive phenotype was isolated from a wild-type plasmid library by transforming a pif1 null strain to growth on glycerol at the non-permissive temperature. This suppressor is a nuclear gene encoding a 135 amino acid protein that is itself essential for mtDNA replication; cells lacking this gene are totally devoid of mtDNA. We therefore named this gene RIM1 for replication in mitochondria. The primary structure of the RIM1 protein is homologous to the single-stranded DNA binding protein (SSB) from Escherichia coli and to the mitochondrial SSB from Xenopus laevis. The mature RIM1 gene product has been purified from yeast extracts using a DNA unwinding assay dependent upon the DNA helicase activity of SV40 T-antigen. Direct amino acid sequencing of the protein reveals that RIM1 is a previously uncharacterized SSB. Antibodies against this purified protein localize RIM1 to mitochondria. The SSB encoded by RIM1 is therefore an essential component of the yeast mtDNA replication apparatus.


Characterization of SAF-A, a novel nuclear DNA binding protein from HeLa cells with high affinity for nuclear matrix/scaffold attachment DNA elements. We identified four proteins in nuclear extracts from HeLa cells which specifically bind to a scaffold attachment region (SAR) element from the human genome. Of these four proteins, SAF-A (scaffold attachment factor A), shows the highest affinity for several homologous and heterologous SAR elements from vertebrate cells. SAF-A is an abundant nuclear protein and a constituent of the nuclear matrix and scaffold. The homogeneously purified protein is a novel double stranded DNA binding protein with an apparent molecular weight of 120 kDa. SAF-A binds at multiple sites to the human SAR element; competition studies with synthetic polynucleotides indicate that these sites most probably reside in the multitude of A/T-stretches which are distributed throughout this element. In addition we show by electron microscopy that the protein forms large aggregates and mediates the formation of looped DNA structures.


Multiple sites for double-strand breaks in whole meiotic chromosomes of Saccharomyces cerevisiae. We present a scheme for locating double-strand breaks (DSBs) in meiotic chromosomes of Saccharomyces cerevisiae, based on the separation of large DNA molecules by pulsed field gel electrophoresis. Using a rad50S mutant, in which DSBs are not processed, we show that DSBs are widely induced in S. cerevisiae chromosomes during meiosis. Some of the DSBs accumulate at certain preferred sites. We present general profiles of DSBs in chromosomes III, V, VI and VII. A map of the 12 preferred sites on chromosome III is presented. At least some of these sites correlate with known 'hot spots' for meiotic recombination. The data are discussed in view of current models of meiotic recombination and chromosome segregation.


Binding of the IS903 transposase to its inverted repeat in vitro. We have purified the transposase of IS903 in three different ways. We find that transposase expressed as a fusion protein with either glutathione-S-transferase or maltose-binding protein is soluble and can be purified rapidly using affinity chromatography. The third purification requires extracting the native transposase from an insoluble pellet using an alkaline pH buffer. All three proteins bind specifically to the ends of IS903 and give identical patterns of protection when challenged with DNase I. We have used the more stable fusion proteins to examine transposase--DNA interactions in vitro. Methylation interference experiments have identified critical bases for transposase binding; methylated purines that inhibit binding all lie within the inner part of the 18 bp inverted repeat (bp 7-16). Moreover, the positions and identities of these purines suggest that the transposase interacts with base pairs in adjacent major and minor grooves. Binding assays with mutant inverted repeats confirm that transposase binding is sensitive to sequence changes only within this inner region. We propose that the transposase binding site is limited to this domain of the inverted repeat. These data are consistent with our previous analysis of the behaviour of mutant ends in vivo, from which we postulated that the inverted repeat was composed of two functional domains; an inner binding domain (bp 6-18), which included a region of minor groove interactions, and an outer domain that was involved in a step subsequent to transposase binding.


Presence of atrial natriuretic factor and cyclic guanosine monophosphate in saliva. Comparison of plasma and salivary concentrations during a head-down tilt. Using a specific and sensitive radio-immunoassay involving separation and extraction procedures, we measured the concentration of saliva and plasma atrial natriuric factor (ANF) and cyclic guanosine monophosphate (cGMP) in men before and during a 10 h head-down tilt at -6 degrees. Saliva values closely correlated with plasma for ANF (r = 0.7-0.95) and for cGMP (r = 0.65-1). During this dynamic test, the mean concentrations of ANF and cGMP were significantly higher after 15 and 45 min, respectively, this increase persisting for 3.5 h. We concluded that the concentration of ANF in saliva may be significantly affected by a marked fluid shift from the lower to the upper half of the body. This is the first time that the presence of ANF and cGMP has been demonstrated in saliva. The great advantage of studying saliva is that it can be obtained non-invasively in athletes or during space flight. This methodology will be used during the Soviet-French space flight (Antares Project) planned for 1992.


Hepatitis C virus antibodies in a long-term follow-up of beta-thalassaemic children with acute and chronic non-A non-B hepatitis. The presence of antibodies toward hepatitis C virus (HCV) was examined in 78 polytransfused beta-thalassaemic children. The anti-HCV status was correlated with acute and chronic non-A non-B (NANB) hepatitis that developed during a follow up of about 13 years. Anti-HCV was present in 83.3% of children with acute NANB hepatitis and in 82.9% of those with chronic NANB hepatitis. The percentage of chronic evolution was 56.7% for acute anti-HCV positive NANB hepatitis and 50.0% for anti-HCV negative NANB hepatitis. The long-term persistence of anti-HCV antibodies did not correlate with chronic evolution of liver infection in thalassaemic patients. Histological features of chronic hepatitis showed little or no difference between HCV associated or non-associated liver disease. The multifactorial liver injury in beta-thalassaemic children explains the high prevalence of cirrhosis (about 30%) observed in these patients with NANB hepatitis. On the other hand, independent of liver disease, some patients never seroconverted during the follow up in spite of the high number of transfusions suggesting the existence of "non-responders".


Diagnosis and treatment in a case of juvenile subacute necrotizing encephalopathy Leigh without cytochrome c oxidase deficiency. Subacute necrotizing encephalopathy (Leigh syndrome) is characterized by lactacidosis, seizures, ataxia, multiple cerebral hypervascularized lesions and mitochondrial oxidation defects. This is a report on a 21-year-old patient with proven Leigh syndrome, mild central and provokable peripheral lactacidosis, an extra-erythrocyte complex II defect, functionally reduced myokinase adenylate deaminase activity, but no ultrastructural mitochondrial changes. Determination of lactate, pyruvate and ammonia under ischemic conditions plus a pyruvate loading test were particularly useful. Oral flunarizine (Sibelium 30 mg/d) proved to be therapeutically effective.


No re-expression of high-affinity nerve growth factor binding sites in spinal motor neurons in amyotrophic lateral sclerosis. Autoradiographic studies of high-affinity nerve growth factor binding sites in the cervical human spinal cord revealed a high density of binding sites in the dorsal horn and in the tract of Lissauer, which is in agreement with investigations in nonprimates. Very low or no binding was found in the motor neuron areas of controls as well as in amyotrophic lateral sclerosis cases, which indicates that the degeneration of motor neurons in this disease is not accompanied by re-expression of high-affinity nerve growth factor receptors.


Increased visual impairment after exercise (Uhthoff's phenomenon) in multiple sclerosis: therapeutic possibilities. The Uhthoff symptom, a transient impairment of visual function after exercise, is demonstrated in 2 multiple sclerosis patients. Following exercise, impairment of visual function, as documented most clearly by the testing of contrast sensitivity, was less obvious after body surface cooling and after treatment with orally administered 4-aminopyridine. It is hypothesized that both treatment modalities improve the nerve conduction safety factor and thereby prevent the occurrence of a conduction block, which is believed to be the mechanism underlying the Uhthoff symptom.


Secretion of beta-2-microglobulin from human hepatoblastoma and hepatoma cells on stimulation with interleukin-6. beta 2-microglobulin (beta 2-M) was secreted in a dose- and time-dependent manner after interleukin-6 (IL-6) treatment of human hepatoblastoma (HuH-6) and hepatoma cells (HuH-7). Pancreatic secretory trypsin inhibitor, which is one of the acute phase proteins secreted in these cells, was also secreted dose- and time-dependently in HuH-6 cells and dose-dependently in HuH-7 cells. It is conceivable that IL-6 induces the production of beta 2-M as an acute phase protein in the liver.


[Age-related incidence of spontaneous tumors in SPF C57BL/6 and BDF1 mice]. Incidence of spontaneous tumors in C57BL/6 NCrj (CR), C5BL/6 CrSlc (SL) and B 6 Crj x DBA/2 NCrj F1 (BD) mice, which were reared under a barrier system and died natural death, were examined. Cohorts of mice in 200 to 300 each were purchased at 4 weeks of age and raised under SPF conditions. A large portion of the mice were used for various experiments between 3 and 30 months old while not a small number died before use and were autopsied. Median survival periods of the female and male were estimated at 697 and 680 days for CR, 764 and 806 days for SL and 866 and 929 days for BD, respectively. Incidence of spontaneous neoplastic lesions in the autopsied animals were 77.4% and 79.2% of 535 female and 590 male CR, 69.7% and 55.1% of 502 female and 463 male SL, and 75.8% and 78.0% of 298 female and 346 male BD, respectively. In CR, histiocytic sarcoma was the most predominant tumor, accounting for 72.1% of all tumors. In SL, malignant lymphoma was the most prevailing, forming 62.3%, and, in male BD, hepatocellular carcinoma was the most frequent, accounting for 41.8% of all tumors.


Application of dried whole blood collected on filter paper disks to ELISA for the detection of Sendai virus and mouse hepatitis virus antibodies in mice. This study was undertaken to simplify the preparation procedures for test specimens by applying whole blood collected on filter paper disks. The results of ELISA obtained using specimens collected in this way for the detection of Sendai virus and mouse hepatitis virus antibodies in mice were comparable to those for ordinary ELISA using serum samples.


In vivo infection of marrow stromal fibroblasts by feline leukemia virus. Marrow stromal fibroblasts (FBs) likely play an important role in the regulation of hematopoiesis within the marrow microenvironment. Infection of these cells by feline leukemia virus (FeLV) might not only contribute to the pathogenesis of FeLV-induced hematologic diseases, but could provide a reservoir for virus in the infected cat. To determine the frequency of FeLV infection among marrow FB precursor cells (fibroblast colony-forming units, CFU-F) of cats viremic with FeLV-C/Sarma and FeLV-A/61E, marrow FBs and FB cell clones were isolated and assayed for expression of FeLV gag protein. From 30% to 86% and 64% to 88% of marrow FB precursors were infected with FeLV-C/Sarma and FeLV-A/61E, respectively. CFU-F from a cat viremic with FeLV-A/61E were not affected by exposure to antibody against FeLV envelope glycoprotein gp70 and heterologous complement, whereas similarly treated hematopoietic progenitors (erythroid colony-forming units, CFU-E; erythroid burst-forming units, BFU-E; and granulocyte-macrophage colony-forming units, CFU-GM) and culture-propagated, FeLV-infected marrow FBs were effectively lysed, suggesting that infected CFU-F within the marrow microenvironment do not express a significant amount of gp70 on their cell membranes. Thus, marrow FB precursor cells appear to be a major target for FeLV in vivo. Furthermore, the low level of gp70 antigen expression on the surface of these cells in vivo may allow them to escape immune surveillance and provide a reservoir of virus during active or latent infection.


ACE-inhibitors and atherosclerosis.

ACE-inhibitors and atherosclerosis. The involvement of the circulating and local renin-angiotensin system in atherosclerotic process has been hypothesized on the basis of experimental data showing presence and specific actions of the components of this system in the vascular wall. In particular, angiotensin II may participate in well known events in atherogenesis as the control of smooth muscle cell growth and proliferation. Recent studies have shown an effect of angiotensin converting enzyme (ACE) inhibition on the development of atherosclerosis in animal models. Captopril and cilazapril prevent myointimal proliferation after vascular injury in rat. Captopril reduces aortic cholesterol content and percentage intimal aortic surface covered by lesions in Watanabe heritable hyperlipidemic rabbits. Captopril also significantly reduces the progression of carotid and coronary lesions in monkeys fed a high cholesterol diet. In addition, a role for converting enzyme inhibitors in reducing aortic and microvascular growth either in hypertensive or normotensive rats has been demonstrated. It is possible that ACE-inhibitors prevent angiotensin II-induced vascular proliferation and thereby suppress the development of atherosclerosis in animals. It is also conceivable that the blood pressure effects of ACE-inhibitors could play a role in the antiatherosclerotic effect shown by these drugs, even though this explanation cannot be addressed by studies dealing with normotensive animals. Then, other mechanisms could be involved, including hypothesized effects of blockade of the renin-angiotensin system on sympathetic nervous system activity, regulation of vascular growth factors and insulin sensitivity. The clinical significance of these experimental findings is unknown.(ABSTRACT TRUNCATED AT 250 WORDS)


DNA unwinding activity of replication protein A. Replication protein A (RP-A) is a heterotrimeric complex conserved in eukaryotic cells. It binds to single-stranded DNA and is essential for initiation and elongation of DNA replication. In this communication we give evidence that this protein can unwind DNA independent of magnesium and ATP, two essential cofactors for bona fide DNA helicase activity. RP-A can unwind up to at least 350 basepairs and appears to be required in stoichiometric amounts. The reaction is extremely sensitive to NaCl and MgCl2. This activity of RF-A is suggestive for a possible unwinding function in initiation of DNA replication in eukaryotes.


Non-equivalent natures of the coordinated imidazole rings of cytochrome c3 from D. vulgaris Miyazaki F as studied by 1H NMR. All of the C2 proton signals of the coordinated histidine residues in the 1H NMR spectrum of cytochrome c3 from D. vulgaris Miyazaki F were assigned by specific deuteration. They appeared at extremely high fields and scattered in a wide range from -4 to -22 ppm. This clearly shows that the chemical properties of the imidazole groups are quite different from one another. The extremely high-field shift of the C2 signal indicates that some of them must carry the imidazolate-like nature to some extent. This might be responsible for the extremely low redox potentials of the four hemes. On changing temperature, most of them showed Curie-type change. All of the C2 signals showed a small p2H dependence in the range of p2H 4.8-10.0.


The protein phosphatase inhibitor, okadaic acid, potentiates the stimulatory effect of phorbol ester on phosphatidylcholine synthesis, but not on phospholipid hydrolysis, in fibroblasts. The potent protein phosphatase inhibitor, okadaic acid, was used to determine the possible role of protein phosphorylation reaction(s) in phorbol ester-induced synthesis and hydrolysis of phosphatidylcholine (PtdCho) in NIH 3T3 fibroblasts. Okadaic acid (2 microM) was found to enhance the stimulatory effects of lower concentrations (2.5-25 nM) of phorbol 12-myristate 13-acetate (PMA) on PtdCho synthesis, but not on PtdCho hydrolysis, after treatments for 30-60 min. These data support a view that in fibroblasts PMA stimulates only PtdCho synthesis, and not PtdCho hydrolysis, by a protein phosphorylation-dependent mechanism.


Activation of protein kinase C attenuates the cyclic GMP responses to C-type natriuretic peptide in cultured mouse astrocytes. C-type natriuretic peptide (CNP), a recently discovered natriuretic peptide, has a potent stimulatory effect on cyclic GMP (cGMP) formation in cultured mouse astrocytes. Pretreatment of astrocytes with phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), attenuated CNP-induced cGMP responses in a dose-dependent manner, with a half-maximal inhibitory concentration of 6 nM, whereas the inactive phorbol ester analog, 4 alpha-phorbol 12,13-didecanoate, was without effect. In the presence of staurosporine, a PKC inhibitor, the inhibitory effect of PMA on CNP-stimulated cGMP production was reversed. These results suggest that PKC is an inhibitory modulator of CNP-stimulated cGMP responses in astrocytes and that CNP may interact with neuropeptides which stimulate PKC.


Zymosan induces selective release of arachidonic acid from rabbit alveolar macrophages via stimulation of a beta-glucan receptor. Zymosan, which is composed primarily of alpha-mannan and beta-glucan polymers, is a well recognized activator of macrophages. The type receptor by which unopsonized zymosan induces arachidonic acid release was investigated. It was found that particulate beta-glucan and zymosan stimulated an identical dose-dependent release of arachidonic acid. This release of arachidonic acid by zymosan was blocked by soluble beta-glucans whereas soluble mannan had no effect. This inhibition was not due to a general toxic effect of the soluble beta-glucans as they had no effect on calcium ionophore-induced release of arachidonic acid. Beta-glucan-induced fatty acid release from these cells was shown to be fairly specific for arachidonic acid. These data reveal that zymosan stimulates the specific release of arachidonic acid from rabbit alveolar macrophages, at least in part, via a beta-glucan receptor.


The formate complex of the cytochrome bo quinol oxidase of Escherichia coli exhibits a 'g = 12' EPR feature analogous to that of 'slow' cytochrome oxidase. The cytochrome bo quinol oxidase of Escherichia coli is homologous in sequence and in structure to cytochrome aa3 type cytochrome oxidase in subunit I, which contains the catalytic core. The cytochrome bo enzyme forms a formate complex which exhibits 'g = 12' and 'g = 2.9' EPR signals at X band; similar signals have previously been observed only in association with the 'slow' and formate-ligand states of cytochrome oxidase. These signals arise from transitions within integral spin multiples identified with the homologous heme-copper binuclear catalytic centers in both enzymes.


Stimulation of SV40 DNA replication by the human c-myc enhancer. In earlier studies we had shown that a transcriptional enhancer sequence exists about 2 kb upstream of the human c-myc gene. The core sequence necessary for enhancer activity was defined therein as a 21 bp nucleotide element, which also showed autonomous replicating activity [EMBO J. (1988) 7, 3135-3142; EMBO J. (1989) 8, 4273-4279]. Recently, several reports have substantiated the notion that transcription and replication can be concertedly regulated in a larger number of cases than expected. In this report, we took the simian virus 40 (SV 40) ori/promoter as a model system. The SV40 enhancer is known to enhance transcription from its ori/promoter, but to reduce its replication (probably due to a negative feedback). The SV40 enhancer was replaced by the c-myc enhancer core in order to see its effect upon SV40 DNA replication and transcription. The results showed that besides stimulating transcription, the c-myc enhancer promoted SV40 DNA replication in monkey CosI cells. Stimulation was only observed when the c-myc enhancer was inserted in the 'up-to-down' orientation to the SV40 promoter. The promoting function of the c-myc enhancer on DNA replication correlated with the transcriptional activation function, as determined by systematic point mutations introduced within the 21 bp core sequence.


Identification of heme macrocycle type by near-infrared magnetic circular dichroism spectroscopy at cryogenic temperatures. The electron paramagnetic resonance (EPR) and near-infrared magnetic circular dichroism (MCD) spectra of the azide and cyanide adducts of nitrimyoglobin and hydroperoxidase II from Escherichia coli have been measured at cryogenic temperatures. For the first time, ligand-to-metal charge-transfer transitions in the near-infrared have been observed for an Fe(III)-chlorine system. It is shown that near-ultraviolet-to-visible region electronic spectra of 'green' hemes such as these are an unreliable indicator of macrocycle type. However, the combined application of EPR and near-infrared MCD spectroscopies clearly distinguishes between the porphyrin-containing nitrimyoglobin and the chlorine-containing hydroperoxidase II.


The angiotensin AT2 receptor modulates T-type calcium current in non-differentiated NG108-15 cells. We report here that angiotensin II (AII) and the AT2 receptor-selective ligand, CGP 42112, modulate the T-type calcium current in non-differentiated NG108-15 cells, which express only AT2 receptors. Both peptides decrease the T-type calcium current at membrane potentials above -40 mV and shift the current-voltage curve at lower potentials with maximal effect between 5 and 10 min after application. These data describe a new cellular response to AII and suggest that the AT2 receptor mediates certain neurophysiological actions of this hormone.


Antibody to the inositol trisphosphate receptor blocks thimerosal-enhanced Ca(2+)-induced Ca2+ release and Ca2+ oscillations in hamster eggs. The sulfhydryl reagent thimerosal enhanced the sensitivity of hamster eggs to injected inositol 1,4,5-trisphosphate (InsP3) or Ca2+ to generate regenerative Ca2+ release from intracellular pools. A monoclonal antibody (mAb) to the InsP3 receptor blocked both the InsP3-induced Ca2+ release (IICR) and Ca(2+)-induced Ca2+ release (CICR). The mAb also blocked Ca2+ oscillations induced by thimerosal. The results indicate that thimerosal enhances IICR sensitized by cytosolic Ca2+, but not CICR from InsP3-insensitive pools, and causes repetitive Ca2+ releases from InsP3-sensitive pools.


The role of the C-terminal region of rat brain natriuretic peptide in receptor selectivity. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) have different C-terminal tail structures compared with the rather conservative ring structures which consist of 17 amino acid residues. To examine the different effects of the tail structures of ANP and BNP on their interaction with receptors, we synthesized several peptide analogs and measured their biological actions in three different assay systems. Deletion of the C-terminal tail from rat BNP did not effect the vasorelaxation activity against rat aorta, but it promoted cGMP production in cultured rat aortic smooth muscle cells (RASMC). Deletion of the C-terminal tail from rat ANP diminished both vasorelaxant and cGMP producing activities. In a binding competition assay with RASMC and [125I]rat ANP-(1-28), the competition activities of both ANP and BNP were greatly reduced by C-terminal deletion. In addition, we obtained agonists with novel receptor selectivity.


DNA base modifications in chromatin of human cancerous tissues. Free radical-induced damage to DNA in vivo is implicated to play a role in carcinogenesis. Evidence exists that DNA damage by endogenous free radicals occurs in vivo, and there is a steady-state level of free radical-modified bases in cellular DNA. We have investigated endogenous levels of typical free radical-induced DNA base modifications in chromatin of various human cancerous tissues and their cancer-free surrounding tissues. Five different types of surgically removed tissues were used, namely colon, stomach, ovary, brain and lung tissues. In chromatin samples isolated from these tissues, five pyrimidine-derived and six purine-derived modified DNA bases were identified and quantitated by gas chromatography/mass spectrometry with selected-ion monitoring. These were 5-hydroxy-5-methylhydantoin, 5-hydroxyhydantoin, 5-(hydroxymethyl)uracil, 5-hydroxycytosine, 5,6-dihydroxycytosine, 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, xanthine, 2-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine. These compounds are known to be formed typically by hydroxyl radical attack on DNA bases. In all cases, elevated amounts over control levels of modified DNA bases were found in cancerous tissues. The amounts of modified bases depended on the tissue type. Lung tissues removed from smokers had the highest increases of modified bases above the control levels, and the highest overall amounts. Colon cancer tissue samples had the lowest increases of modified bases over the control levels. The results clearly indicate higher steady-state levels of modified DNA bases in cancerous tissues than in their cancer-free surrounding tissues. Some of these lesions are known to be promutagenic, although others have not been investigated for their mutagenicity. Identified DNA lesions may play a causative role in carcinogenesis.


Alterations of ion-dependent ATPase activities in the brain of Singi fish, Heteropneustes fossilis (Bloch), by triiodothyronine. The activities of Na+K(+)- and Mg(2+)-ATPases in mitochondrial, microsomal, and cytosolic fractions of Singi fish (Heteropneustes fossilis Bloch) brain were investigated after injections of various doses (0.012, 0.025, 0.05, and 0.10 micrograms/g) of triiodothyronine (T3) for 3 consecutive days. Both ATPases were found in the mitochondrial and microsomal fractions. The cytosolic fraction showed only Mg(2+)-ATPase activity. Mitochondrial Na+K(+)-ATPase activity increased to almost the same level in fish treated with 0.025, 0.05, or 0.10 micrograms of T3/g, while the T3 dose of 0.012 micrograms/g was ineffective in this respect. Microsomal Na+K(+)-ATPase activity increased to about the same level with all of the doses of T3 used. No detectable amount of Na+K(+)-ATPase was found in the brain cytosolic fraction. Mitochondrial Mg(2+)-ATPase activity was enhanced with 0.025, 0.05, and 0.10 micrograms of T3/g. The last dose, however, produced a higher increase in activity than the other two doses. Surprisingly, microsomal and cytosolic Mg(2+)-ATPase activity was not increased by T3 treatment. Although T3 concentrations rose sharply after each T3 injection, the serum T3 level in T3-injected fish was not different from that in the control as observed on the fourth day. The T3-induced rise of Na+K(+)- and Mg(2+)-ATPase activities was inhibited by cycloheximide treatment. Immersion of Singi fishes in thiourea significantly reduced brain Na+K(+)-ATPase activity in microsomal and mitochondrial fractions but decreased Mg(2+)-ATPase activity only in the mitochondrial fraction. Three consecutive daily injections of T3 (0.10 micrograms/g) into the thiourea-treated fishes increased their ATPase activities even beyond the control level.(ABSTRACT TRUNCATED AT 250 WORDS)


A pSC101-par sequence-mediated study on the intracellular state of supercoiling of the pBR322 genome in Escherichia coli DNA topoisomerase I deletion mutant. In Escherichia coli DNA topoisomerase I deletion mutant DM800, transcription of the tetracycline-resistance gene (tet) in the pBR322 genome is thought to create and maintain two domains of positive supercoils ahead, and negative supercoils behind, the transcription complex. To assess the actual intracellular state of twin-supercoiled domains, par sequence (365 bp) of plasmid pSC101, which shows a high affinity for DNA gyrase, was inserted into the EcoRI site upstream, or the AvaI site downstream, of the tet gene on the pBR322 genome. Analysis of the oxolinic acid-induced sites of cleavage by gyrase in DM800 revealed that the pBR322 derivatives are highly preferentially cleaved at the par sequence of the EcoRI site as well as the AvaI site and efficiently linearized when compared with pBR322. Assessment of the state of negative supercoiling of the pBR322 derivatives isolated suggested that the DNA (containing the AvaI site) ahead of the tet transcripts, is not so positively supercoiled and preferential interaction of gyrase with the EcoRI-par sequence does not result in removing negative superhelical turns so effectively as DNA topoisomerase I does on pBR322 DNA in the isogenic wild-type cells.


Distribution of insertion sequence IS1 in multiple-antibiotic resistant clinical Enterobacteriaceae strains. The presence of insertion sequence IS1 in 70 multiple-antibiotic resistant clinical strains was determined. This 70-strain collection comprised 46 Escherichia coli, 18 Salmonella and 6 Shigella strains. The presence of IS1 was detected in the chromosome and plasmids of 73% and 63% of the strains, respectively, and 51% of the strains carried IS1 in both. The frequency of IS1 was higher in Salmonella than in E. coli and Shigella strains. A total of 31 strains carried large plasmids with IS1; 10 of these strains (32.3%) were able to transfer all or some of the antibiotic resistance markers to E. coli K12 or S. typhimurium recipient strains. Resistance markers of all clinical strains were maintained stably after several generations of growth. The presence of IS1 in a relatively high percentage of plasmids of multiple-antibiotic resistant clinical isolates, suggests a role for this sequence in the dissemination of genes which code for antibiotic resistance.


Altered substrate specificity by substitutions at Tyr218 in bacterial aminoglycoside 3'-phosphotransferase-II. Mutant aminoglycoside 3'-phosphotransferase II enzymes were produced in which Tyr218 was changed to serine, aspartic acid, or phenylalanine. In each case the mutation resulted in increased bacterial susceptibility to neomycin and kanamycin, while simultaneously increasing the Km values for these substrates. For the Ser and Asp mutants, bacterial resistance to amikacin increased, with a concomitant increase in affinity for this drug. Initial velocity studies indicated that the wild-type and mutant enzymes all followed Michaelis-Menten kinetics. Although these mutagenic substitutions changed the substrate specificity of these enzymes they did not alter the enzyme affinity for Mg(2+)-ATP.


Free radical generation and coupled thiol oxidation by lactoperoxidase/SCN-/H2O2. The lactoperoxidase-catalyzed oxidation of glutathione (GSH) and thiocyanate (SCN-) was studied. Oxidation of SCN- was recorded by ultraviolet spectroscopy and by electron spin resonance (ESR). Consumption of GSH was measured by amperometric titration. One or two moles of GSH was oxidized per mole of H2O2 added, depending on the reaction conditions. Omission of SCN- prevented the oxidation of GSH. The oxidation of GSH required only catalytic amounts of SCN-, which was therefore recycled. Iodide (I-) could replace SCN-, while chloride or bromide were ineffective. The apparent Michaelis constant for SCN- was 17 microM. Oxidation of SCN- gave rise to two reactive intermediates, one stable and one unstable. The stable intermediate (-OSC. = N-(?)) decayed by a second-order reaction with a rate constant of 1.1 M-1 s-1. The decay of the unstable radical was very fast. The data (a) explain the short- and long-term antibacterial effects of lactoperoxidase-halide-H2O2 system, (b) point to possible deleterious effects due to glutathione depletion, (c) are of relevance for free radical diseases involving sulphur-centered free radicals, and (d) support previous observations on lipid peroxidation/halogenation in biological membranes, liposomes, and unsaturated fatty acids.


Scavenger and antioxidant properties of ten synthetic flavones. To study the effect of the hydroxyl groups on biological activities of flavones, we synthesized 10 polyhydroxyflavones with varied substitution patterns. The abilities of the 10 compounds to act as radical scavengers were investigated using chemiluminescence in two biological models: the xanthine/xanthine oxidase system and the oxidative burst of rat alveolar macrophages. Stable radical formation was observed by electron spin resonance (ESR) spectroscopy. We found that the presence of the pyrogallol moiety in the B component of flavones gave rise to radical scavenger activity and that C-6 substituted hydroxyl group may also provide the basis for biological activity. Furthermore, compounds with a hydroxyl at C-7 position appeared to be xanthine oxidase inhibitors. One particular compound exhibited radical scavenger activity and xanthine oxidase inhibition. This type of compound should prove to be useful in the treatment of ischemia, for which both properties were required.


Photosensitized formation of ascorbate radicals by chloroaluminum phthalocyanine tetrasulfonate: an electron spin resonance study. The chloroaluminum phthalocyanine tetrasulfonate sensitized photooxidation of ascorbic acid to ascorbate radical (A.-) was followed by electron spin resonance (ESR) spectroscopy. In air saturated aqueous media, steady-state amounts of A.- are rapidly established upon irradiation. The ESR signal disappears within a few seconds after the light is extinguished--more slowly under constant irradiation as oxygen is depleted. No photooxidation was observed in deaerated media. The effect of added superoxide dismutase, catalase, desferrioxamine, and singlet oxygen scavengers (NaN3 and tryptophan) was studied, as was replacement of water by D2O and saturation with O2. The results are indicative of free radical production by direct reaction between ascorbate ion and sensitized phthalocyanine (a Type I mechanism) in competition with the (Type II) reaction of HA- with singlet oxygen, a reaction which does not produce ascorbate radical intermediates.


Free radical formation induced by ultrasound and its biological implications. The chemical effects of ultrasound in aqueous solutions are due to acoustic cavitation, which refers to the formation, growth, and collapse of small gas bubbles in liquids. The very high temperatures (several thousand K) and pressures (several hundred atmospheres) of collapsing gas bubbles lead to the thermal dissociation of water vapor into .OH radicals and .H atoms. Their formation has been confirmed by electron spin resonance (ESR) and spin trapping. The sonochemistry of aqueous solutions of gases and of volatile and nonvolatile solutes is reviewed. The similarities and differences between sonochemistry and radiation chemistry of aqueous solutions are explained. Some unusual characteristics of aqueous sonochemistry can be understood by considering the properties of supercritical water. By the use of rare gases with different thermal conductivities, it is possible to distinguish between temperature-dependent processes such as redox reactions initiated by .OH radicals and .H atoms and pressure-dependent processes which lead to polymer degradation and cell lysis. The evidence for free radical formation in aqueous solutions by pulsed ultrasound is discussed. This subject is of interest because it is related to the possible deleterious effects of ultrasonic diagnostic devices. The role of free radicals and of mechanical effects induced by ultrasound in DNA degradation, inactivation of enzymes, lipid peroxidation, and cell killing is reviewed.


The role of free radicals in brassica-induced anaemia of sheep: an ESR spin trapping study. The formation of reactive free radical species in sheep erythrocytes challenged with dimethyldisulphide, a brassica-derived haemolysin, has been investigated by electron spin resonance spectroscopy using the spin trap alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone. Erythrocytes exposed to this agent undergo a burst of free radical activity as demonstrated by the appearance of a spin adduct. The results suggest that haemolytic anaemia which can occur in sheep grazing forage brassicas is a consequence of oxidative stress.


Detection of the ethyl- and pentyl-radical adducts of alpha-(4-pyridyl-1- oxide)-N-tert-butylnitrone in rat-liver microsomes treated with ADP, NADPH and ferric chloride. HPLC-EPR analyses of the reaction mixtures of microsomal suspensions incubated with ADP, ferric chloride, NADPH and alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) were performed. In the elution pattern of the reaction mixture, three peaks (peaks 1, 2 and 3) were detected. The radical adducts (1 and 3) were identified as being the pentyl- and ethyl-radical adducts of 4-POBN by comparing their retention times with those of the authentic radical adducts.


Endogenous digoxin-like immunoreactivity measured in seminal fluid from a normal male population. Endogenous digoxin-like immunoreactivity (EDLI) has been detected in different biological fluids and in several pathophysiological conditions. In this study, using radioimmunoassay we reported for the first time the existence of bound and unbound EDLI in normal seminal fluid. The unusual finding was the detection of unbound EDLI in the seminal fluid, while this reactivity was undetected in plasma. Two main hypotheses are presented: (1) local secretion of unbound EDLI and/or (2) passive diffusion from plasma to the seminal fluid of unbound EDLI and subsequent local concentration.


[Sutureless bowel anastomosis with a biofragmentable ring in Crohn's disease]. A biofragmentable anastomosis ring has recently been developed and is used in end-to-end intestinal anastomosis. It is composed of 2 circular polyglycolic acid (Dexon) components which are interdigitated to form a sutureless anastomosis. We used it in an ileo-ileal and 2 ileo-colic end-to-end anastomoses in 3 patients with Crohn's disease in the past 6 months. Postoperative recovery of bowel function and oral feeding were comparable to that in patients after conventional anastomoses with sutures or staplers. Experience accumulated in 2 large multicentric comparative studies proved that the ring is a safe, fast and effective method for bowel anastomosis. We found it to be safe even in Crohn's disease.


Clinical application of peroperative ultrasonography in liver surgery. This communication outlines the findings at peroperative ultrasonography in patients who had resectional liver surgery because of primary hepatocellular carcinoma. Peroperative ultrasonography revealed the accurate location of the tumor, its relation with locoregional vascular structures, presence of tumor metastases and tumor thrombus. Additionally, ultrasonography provided useful guidance for tumor resection by making possible tumor staining via echo-guided puncture of the feeding vessel of the tumor.


In situ hybridization studies in hepatitis A infection. An in situ hybridization method using radiolabeled oligonucleotide probes was developed to study primary sites of hepatitis A virus replication in an experimental animal model of infection. Hepatitis A genomic sequences were demonstrated in hepatocytes of four marmosets with acute hepatitis A by use of antisense probes. In two of these animals, staining was also found when a sense probe was used, which is consistent with active replication in the hepatocytes. The specificity of the hybridization signal was confirmed by neutralization with "cold" (i.e., unlabeled) probes and by absence of hybridization with non-A hepatitis and reverse antisense probes. The hepatocyte appeared to be the only cell type showing staining. No hybridization was found in other organs, including the intestine (n = 4) and, in one animal, the kidney and spleen.


Limitations of current preoperative liver imaging techniques for intrahepatic metastatic nodules of hepatocellular carcinoma. To determine the limitations of the latest techniques in preoperative liver imaging for hepatocellular carcinoma, 20 patients with histologically proven intrahepatic metastatic tumors were studied. In 32 masses, we were able to assess the relationship between these intrahepatic metastatic tumors and the findings of preoperative imaging individually. Six intrahepatic metastatic tumors not exceeding 5 mm in diameter were missed in all the imaging examinations. The detection rate was 60% in 13 intrahepatic metastatic tumors of 5 to 10 mm, 77% in 10 intrahepatic metastatic tumors of 10 to 20 mm and 100% in 3 intrahepatic metastatic tumors exceeding 20 mm. In total, computed tomographic imaging during arterial portography demonstrated the highest rate of detection (40%) of all the studies performed. Magnetic resonance imaging, which was recently introduced, was rather disappointing (31%) in this series. In two patients, intrahepatic metastatic tumors were only histologically confirmed. Our study suggests a low rate of detection of intrahepatic metastatic tumors with current preoperative imaging modalities. For improvement of prognosis after hepatectomy for hepatocellular carcinoma adjuvant therapy and extended hepatectomy seem necessary if the functional capacity of the remaining liver permits.


Comparison of hepatectomy and transcatheter arterial chemoembolization for the treatment of hepatocellular carcinoma: necessity for prospective randomized trial. Transcatheter arterial chemoembolization is now widely used in cases of surgically unresectable hepatocellular carcinoma. However, it is unclear whether patients with surgically resectable hepatocellular carcinoma should always be treated with hepatectomy as opposed to transcatheter arterial chemoembolization. Sixty-six patients with hepatocellular carcinoma underwent hepatectomy, whereas 29 patients with more advanced hepatocellular carcinoma were treated with transcatheter arterial chemoembolization at our hospital from 1984 to 1990. All cases were associated with cirrhosis of Child class A or B. All of them underwent hepatectomy or transcatheter arterial chemoembolization for the first time. Their outcomes were determined on March 31, 1991. The backgrounds and survival curves for hepatectomy and transcatheter arterial chemoembolization were compared in both Child A and Child B patients. For both Child A and B patients, no significant difference was found between hepatectomy and transcatheter arterial chemoembolization with respect to age, sex, cause of underlying cirrhosis, liver function assessed by indocyanine green test and maximum diameter of the main tumor. The incidence of multiple hepatocellular carcinoma, more advanced hepatocellular carcinoma (TNM stage III or IV) or both was significantly higher in the transcatheter arterial chemoembolization group than in the hepatectomy group for both Child A and Child B patients. The survival curves of both the hepatectomy and the transcatheter arterial chemoembolization groups showed no significant difference for both Child A and Child B patients. A prospective study is therefore warranted to elucidate whether hepatectomy or transcatheter arterial chemoembolization is more effective for treating resectable hepatocellular carcinoma associated with cirrhosis.


High concentrations of soluble tumor necrosis factor receptors in ascites. Ascites and plasma concentrations of soluble tumor necrosis factor receptors p55 and p75 were measured in a prospective study in 34 patients (35 occasions of ascites) with hepatic (5 infected and 21 uninfected) and malignancy-related (9) ascites. All patients had high concentrations of both soluble tumor necrosis factor receptors in ascites and plasma; these were about 500 times higher than the corresponding tumor necrosis factor-alpha concentrations. Ascites levels of soluble tumor necrosis factor receptors p55 and soluble tumor necrosis factor receptors p75 were significantly elevated in patients with malignancy-related (p55: 26.0 +/- 8.6 ng/ml; p75: 20.5 +/- 17.4 ng/ml; mean +/- S.D.) and infected ascites (p55: 25.1 +/- 10.9 ng/ml, p75: 22.6 +/- 11.0 ng/ml) compared with patients with uncomplicated hepatic ascites (p55: 10.1 +/- 4.4 ng/ml; p75: 6.0 +/- 2.6 ng/ml). Patients with infected or malignancy-related ascites also showed higher soluble tumor necrosis factor receptor concentrations in plasma than did patients with plain hepatic ascites. Successful antibiotic treatment of peritonitis reduced soluble tumor necrosis factor receptor p55 and p75 ascites levels in three patients from 24.2 +/- 15.2 ng/ml to 10.7 +/- 1.9 ng/ml and from 20.2 +/- 14.4 ng/ml to 7.5 +/- 1.8 ng/ml, respectively. Soluble tumor necrosis factor receptors p55 and p75 at cutoff levels of 16.5 ng/ml and 9.5 ng/ml, respectively, differentiated between infected or malignant and plain hepatic ascites with diagnostic accuracies of 94% and 89%, respectively. They did not differentiate between infected and malignant ascites. The concentrations of soluble tumor necrosis factor receptor p55 were usually higher in ascites than in plasma in all subgroups of patients.(ABSTRACT TRUNCATED AT 250 WORDS)


Influence of dietary zinc on hepatic collagen and prolyl hydroxylase activity in alcoholic rats. The effects of dietary zinc on hepatic collagen and prolyl hydroxylase activity in normal and alcoholic rats has been investigated in four groups of pair-fed male Wistar rats given either liquid ethanol or a control diet for 12 wk. Each group of pair-fed animals received a diet with a different zinc concentration (standard diet, 7.6 mg/L; low-zinc diet, 3.4 mg/L; zinc-supplemented diet, 76 mg/L; and zinc-extrasupplemented, 300 mg/L. There were no significant differences in hepatic collagen concentration and prolyl hydroxylase activity between alcoholic and normal rats receiving a standard diet (collagen, 77 +/- 5 and 73 +/- 6 micrograms/mg protein; and prolyl hydroxylase; 37 +/- 26 and 36 +/- 22 cpm/mg protein). Alcoholic rats fed a low-zinc diet showed increased prolyl hydroxylase activity (75 +/- 10 cpm/mg protein, p less than 0.05), although no changes in hepatic collagen (77 +/- 10 micrograms/mg protein) were observed in comparison with rats fed a standard alcoholic diet. By contrast, hepatic collagen was significantly lower in alcoholic rats fed a zinc-supplemented diet (66 +/- 4 and 63 +/- 3 micrograms/mg protein, p less than 0.05 and p less than 0.01, respectively), and hepatic prolyl hydroxylase activity was particularly lower in rats receiving zinc 300 mg/L (18 +/- 20 cpm/mg protein). Similar effects were observed in normal rats. We conclude that dietary zinc influences hepatic prolyl hydroxylase activity and collagen deposition in alcoholic rats, and in consequence, the control of dietary zinc is necessary to assess the effects of alcohol on collagen metabolism in rats.


Extracellular matrix in hepatoblastoma: an immunohistochemical investigation. Eleven hepatoblastomas of various subtypes and normal liver tissue were investigated with antibodies against collagen types I-VI, laminin, fibronectin and endothelial and macrophage-associated antigens. Epithelial hepatoblastoma cells, unlike non-neoplastic hepatocytes, exhibited intracellular immunoreactivity for various extracellular matrix proteins (depending on the subtype: laminin, fibronectin and collagen types III, IV and V). The intracellular expression of extracellular matrix proteins by the tumour cells increased from the fetal subtype, through the embryonal subtype, to the small cell subtype. The epithelial tumours exhibited sinusoid-like blood vessels in numbers that varied according to the subtype. These contained Kupffer cells and exhibited greater amounts of the basement membrane components collagen type IV and laminin in the perisinusoidal space than those in the normal liver. The small cell hepatoblastoma exhibited smaller numbers of sinusoids, pronounced intracellular expression of extracellular matrix proteins and large numbers of fibres immunoreactive for collagen type III. In the mixed hepatoblastomas, the extracellular matrix of the osteoid was most strongly immunoreactive for collagen type I and that of the spindle cell areas for collagen type III.


Eight novel inactivating germ line mutations at the APC gene identified by denaturing gradient gel electrophoresis. Familial adenomatous polyposis (FAP) is a dominantly inherited condition predisposing to colorectal cancer. The recent isolation of the responsible gene (adenomatous polyposis coli or APC) has facilitated the search for germ line mutations in affected individuals. Previous authors have used the RNase protection assay and the single-strand conformation polymorphisms procedure to screen for mutations. In this study we used denaturing gradient gel electrophoresis (DGGE). DGGE analysis of 10 APC exons (4, 5, 7, 8, 9, 10, 12, 13, 14, and part of 15) in 33 unrelated Dutch FAP patients has led to the identification of eight novel germ line mutations resulting in stop codons or frameshifts. The results reported here indicate that (1) familial adenomatous polyposis is caused by an extremely heterogeneous spectrum of point mutations; (2) all the mutations found in this study are chain terminating; and (3) DGGE represents a rapid and sensitive technique for the detection of mutations in the unusually large APC gene. An extension of the DGGE analysis to the entire coding region in a sufficient number of clinically well-characterized, unrelated patients will facilitate the establishment of genotype-phenotype correlations. On the other hand, the occurrence of an extremely heterogeneous spectrum of mutations spread throughout the entire length of the large APC gene among the FAP patients indicates that this approach may not be useful as a rapid presymptomatic diagnostic procedure in a routine laboratory. Nevertheless, the above DGGE approach has incidentally led to the identification of a common polymorphism in exon 13. Such intragenic polymorphisms offer a practical approach to a more rapid procedure for presymptomatic diagnosis of FAP by linkage analysis in informative families.


The gene for the dihydropyridine-sensitive calcium channel alpha 2 subunit (CCHL2A) maps to the proximal region of mouse chromosome 5. A rat brain cDNA probe for the gene encoding the alpha 2 subunit of the dihydropyridine-sensitive L-type calcium channel was used as a hybridization probe for the Southern blot analysis of Chinese hamster x mouse somatic cell hybrids and the progeny of an intersubspecies backcross. This gene, termed Cchl2a, was mapped near the centromeric end of the Chromosome 5 linkage group with gene order: centromere-Pgy-1-Cchl2a-Il-6-Pgm-1.


Colocalization of the gene for nephrogenic diabetes insipidus (DIR) and the vasopressin type 2 receptor gene (AVPR2) in the Xq28 region. The gene for nephrogenic diabetes insipidus (DIR) and the vasopressin type 2 receptor gene (AVPR2) have both been localized in the Xqter region by genetic mapping and functional expression studies, respectively. In this paper genetic evidence that the DIR locus is localized distal to the DXS305 locus and that the functional gene for the V2 receptor is localized between the markers DXS269 and F8 is presented. These further refinements in the localization of both genes strengthen the assumption that both genes are identical and provide a rationale for cloning the gene by reversed genetics strategies.


Altered kinetic attributes of Na(+)+K(+)-ATPase activity in kidney, brain and erythrocyte membranes in alloxan-diabetic rats. Effects of alloxan-diabetes on kinetic attributes of Na(+)+K(+)-ATPase were examined in rat kidney, brain and erythrocyte membranes. The enzyme activity decreased significantly from 60-80% in the three membrane systems as a result of diabetic state. Kinetic analysis revealed that Km of the enzyme increased by 5- and 10-fold respectively in the kidney and brain membranes while registering a 50-60% decrease in Vmax. Ouabain binding studies revealed that in the kidney membranes the I50 value increased by 150-fold in diabetic animals with a significant decrease in number of ouabain molecules bound; at concentrations beyond 10(-7) M de-binding of ouabain occurred. For the brain membranes the I50 values for ouabain increased even more significantly (2000-fold increase) without any change in Hill coefficient for ouabain binding. Glycosylation studies revealed that its extent was highest for the brain and least for the kidney membranes which correlated to some extent with the I50 and Km values but not with Vmax. The results thus suggest that glycosylation in critical domains of the membrane and/or enzyme structure may play an important regulatory role. Physiological significance of these findings is discussed.


Protection of bone marrow of Swiss albino mouse against whole body gamma irradiation by WR-2721. Preinjected with a radioprotective drug, WR-2721, the Swiss albino mice were whole body irradiated with 5 Gy of 60Co gamma rays. The animals were sacrificed at different intervals and bone marrow films were prepared for differential counting of lymphocytes, pronormoblasts and normoblasts and granulocytes. The results indicated significant protection of the bone marrow cells by the drug against radiation induced damage. It is therefore concluded that WR-2721 protects all types of cells including as sensitive ones as lymphocytes, pronormoblasts and normoblasts.


Effects of combinations of a prostacyclin analogue (Cicaprost) with different antithrombotic agents in a rat microcirculatory thrombosis model. The antithrombotic effects of the stable PGI2-analogue Cicaprost, acetylsalicylic acid (Aspisol), the low molecular weight heparin CY 216 (Fraxiparin) and Molsidomin (Corvaton) have been investigated alone and in combinations in a thrombosis model in rats in which mesenteric venules with a diameter of 20-30 microns were injured by defined argon laser lesions. In this animal thrombosis model all agents showed a significant and dose-dependent antithrombotic effect. A strong additive effect was observed when Cicaprost was infused together with an i.v. dose of 0.1 mg/kg Molsidomin. A similar additive effect was observed after infusion of minimal effective doses of Cicaprost together with Fraxiparin. The combination of Cicaprost and Aspisol did not have any additive effect. Our results suggest that combinations of antithrombotic agents which show additive effects in animal thrombosis model may lead to a more effective prophylaxis and treatment of human venous or arterial thrombosis.


[A special form of Bazex acrokeratosis in small cell bronchial cancer]. A 66-year-old pensioner developed distinct, erythematosquamous and keratonic lesions on the hands and feet within 2 months, and also a progressive red-bluish discoloration of the whole integument. Clinical and X-ray exploration revealed a still asymptomatic small-cell bronchial carcinoma, so that the otherwise inexplicable skin lesions made an acrokeratotic paraneoplastic syndrome of the Bazex type seem most likely. This very rare syndrome has hitherto been observed only in patients with carcinomas of either the bronchial or the upper digestive tract, with or without cervical and mediastinal lymph node metastases. We report on our third patient with Bazex-type acrokeratosis, mainly because of the uncommon distribution and severity of his otherwise typical lesions. In addition, recent reports on this syndrome in the literature are reviewed.


Short sequence in L1 region of human papillomaviruses correlates with clinical pictures and grouping by cross-hybridization. A consensus primer-mediated polymerase chain reaction devised to amplify a short sequence in L1 region (L1-PCR) efficiently detected genital human papillomaviruses (HPVs) in clinical materials. Nucleotide sequencing of the amplified fragment showed that L1-PCR is also applicable to hitherto unsequenced HPVs. By comparing the amplified 210 nucleotides HPVs were classified into six groups, which are consistent with clinical pictures and a grouping based on cross-hybridization under the stringent condition.


Direct detection of Epstein-Barr virus DNA from a single Reed-Sternberg cell of Hodgkin's disease by polymerase chain reaction. Eleven cases of Hodgkin's disease (HD) were examined for the presence of the Epstein-Barr virus (EBV) genome, using the polymerase chain reaction (PCR) to detect EBV DNA in whole paraffin-embedded tissue specimens and in single cells picked out from the specimens with a micromanipulator. The EBV genome was detected in 5 of the 11 cases by conventional PCR. Single cell PCR demonstrated the EBV genome in Reed-Sternberg cells from all the EBV-positive cases, but not from any of the EBV-negative cases. Background lymphocytes and lysozyme-positive histiocytes from EBV-positive cases did not contain the EBV genome. These results indicate an etiological association of EBV with some cases of HD.


Fetal development in cattle with multiple ovulations. Treatment of lactating and nonlactating parous cows (n = 379) with 12 mg of FSH-P to evaluate development of multiple bovine fetuses resulted in ovulation rates ranging from 1 to 27 corpora lutea (CL). Fertilization rate (i.e., ova fertilized at 6 to 8 d postmating, 80.0%) was not affected by ovulation rate. The percentage of fetuses developing normally at 51 to 53 d postmating decreased (P less than .01) as ovulation rate increased; 1 CL, 100.0%; 2 CL, 100.0%; 3 CL, 66.7%; 4 CL, 45.8%; 5 CL, 33.3%; 6 to 10 CL, 13.6%; and greater than 10 CL, 8.9%. Of the 86 cows permitted to calve, 47 produced singles, 22 twins, 9 triplets, 7 quadruplets, and 1 quintuplets. Calf birth weight and gestational length decreased (P less than .01) as the number of calves born increased from one to two to three. Smaller decreases (P less than .05) in birth weight occurred among triplets, quadruplets, and quintuplets, whereas gestational length did not differ (P greater than .1) among these groups. Systemic progesterone concentrations in the dam were proportional (P less than .01) to the number of fetuses in utero between d 126 and 266 for dams gestating one, two, or three or more fetuses; estrone sulfate was lower (P less than .01) in dams with one than in those with two or more fetuses. Placental weight (i.e., cotyledons plus intercotyledonary membranes) per fetus at 52 +/- 1 d of gestation and at term decreased as the number of fetuses increased. The chorioallantoic membranes were often fused among multiple fetuses and contained either all viable or all dead fetuses, but not both, within the same anastomosed placental unit. These results suggest that ovulation rate is the first limiting factor to increasing cow productivity for beef cattle because some bovine females had the capacity to gestate up to three fetuses per uterine horn, or a total of five fetuses, above which pregnancy was terminated.


Effect of recombinant porcine somatotropin on energy and protein utilization by growing pigs: interaction with capacity for lean tissue growth. Three litters of four pigs from each of four different groups were used to evaluate the effect of porcine somatotropin (pST) on growth performance, body gain composition, energy and N metabolism, and in vitro cytochrome oxydase (final enzyme of the respiratory chain) activity of tissues. The four groups included boars from a synthetic line (SG1) or the Large White breed (SG2) and barrows from the Large White breed (SG3) or crossbred between Large White and Meishan breeds (SG4). Inherent capacity for daily lean tissue growth (LTG) decreased from SG1 to SG4. Within a litter, one pig was slaughtered and dissected at the beginning of the experiment (55 kg BW) and the three others were fed the same daily supply of protein and amino acids (26 g of lysine/d) but relative daily energy levels were either 113 (without pST: E1/0), 100 (3 mg of pST/d: E2/pST) or 87 (3 mg of pST/d: E3/pST). The 100 energy level corresponded to the ad libitum intake of E2/pST pigs. Two energy and N balances were carried out in respiration chambers during the experimental period. Pigs were slaughtered and dissected at approximately 95 kg BW and composition of gain was estimated using the comparative slaughter technique. In E1/0 pigs, daily BW, lean, and N gain were affected (P less than .01) by group; 566, 471, 374, and 315 g/d of lean tissue gain in SG1, SG2, SG3, and SG4 pigs, respectively. At high ME intake (E2/pST vs E1/0), pST increased daily BW (+14%), lean (+27%), or N (+26%) gain and reduced adipose tissue (-50%) gain, but the pST effect was inversely related to LTG: for N, the improvement was 2.8, 7.1, 7.0, and 11.1 g/d in SG1, SG2, SG3, and SG4 groups, respectively. Energy restriction (E3/pST vs E2/pST) reduced (P less than .001) adipose tissue gain in all groups but did not affect lean tissue or N gains in SG1, SG2, and SG3 pigs. In the pST-treated pigs of the SG4 group, the lean tissue or N gains were reduced (P less than .01) by energy restriction. Energy restriction combined with pST treatment (E3/pST) led to negligible amounts of fat deposited (40 g/d for SG1 + SG2 + SG3 pigs) and a gain:feed ratio higher than 500 g/kg (580 in SG1 pigs). The increased heat production measured in pST-treated pigs was due to its maintenance component: 275 vs 257 kcal of ME.kg BW-.60.d-1 (P less than .01).(ABSTRACT TRUNCATED AT 400 WORDS)


Technical note: mineral deposits on Dacron bags during ruminal incubation. Extensive DM contamination was found on Dacron bags that were incubated for prolonged periods of time in the rumen of steers fed alfalfa hay. The ash content of the contaminant was high, and most of it was acid-soluble X-ray analysis indicated the presence of hydroxylapatite and synthetic calcium magnesium phosphate or whitlockite. The contaminant appeared as a smooth coating on the Dacron fiber, suggesting that contamination was a gradual process rather than the result of entrapment of dislodged crystals from plant material. Contamination seemed to occur exponentially within the range of observations (0 to 42 d). Contamination also occurred in steers fed orchardgrass, although to a lesser extent than in steers fed alfalfa hay. The DM contamination was less than .04 g per bag (average bag weight was 1.2 g) during the first 10 d of incubation. However, correction for contamination might be required for studies involving longterm incubation or mineral digestion.


Efficacy and safety of itraconazole in the long-term treatment of onychomycosis. Sixty-one patients with a clinical diagnosis of onychomycosis in finger or toe nails were treated with itraconazole 100 mg/day or griseofulvin 500 mg/day for six to nine months. The infective causes were Trichophyton rubrum, Trichophyton mentagrophytes, or Trichophyton violaceum, and in two cases Candida albicans. A total of 27 finger and 390 toe nails were infected. Statistically significant intragroup reductions from baseline symptom severity values were seen at endpoint (month 6 or 9) for both treatment groups for all parameters: colour change, thickness, brittleness and unaffected area. No clinically or statistically significant differences between the treatment groups were seen at endpoint. However, the itraconazole group continued to improve during the follow-up, while the mean symptom severity ratings remained the same in the griseofulvin group. All itraconazole patients and 85% of griseofulvin patients were rated as cured or markedly improved at endpoint. Nineteen out of 26 evaluable itraconazole patients (73%) remained cured during the three month follow-up period, compared with 12 out of 17 griseofulvin patients (71%). The rather large number of drop-outs, especially among griseofulvin patients, makes it difficult to draw definitive conclusions of the symptom recurrence. Two itraconazole patients stopped medication due to an adverse event, compared to four patients in the griseofulvin group. The clinical laboratory data on itraconazole-treated patients did not show any statistically or clinically significant changes. In conclusion, itraconazole was at least as effective as griseofulvin in the treatment of onychomycosis. The itraconazole group continued to improve after the treatment was stopped. The results show that itraconazole 100 mg/day is safe and efficient in the long-term treatment of fungal nail infections.


The in-vitro activity of two new quinolones: rufloxacin and MF 961. The in-vitro activity of two new quinolone antimicrobials, rufloxacin and MF 961, together with the desmethylated metabolite of rufloxacin (MF 922) were compared with other orally administered agents against 622 bacterial strains. Against Enterobacteriaceae and Pseudomonas aeruginosa rufloxacin was generally active (MIC90 1-8 mg/L) with the exception of Klebsiella and Serratia spp. (MIC90 32 mg/L and Enterobacter spp. (MIC90) 64 mg/L. The respiratory pathogens Haemophilus influenzae and Moraxella catarrhalis were susceptible to rufloxacin (MIC90 0.5 and 1 mg/L respectively) but Streptococcus pneumoniae was less susceptible (MIC90 32 mg/L). Staphylococcus aureus were susceptible to rufloxacin (MIC90 2 mg/L). The rufloxacin metabolite MF 922 was generally as active as its parent. MF 961 was usually two-fold more active than rufloxacin. All three compounds were four to 16 times less active than norfloxacin, but rufloxacin was as active or somewhat more active than norfloxacin against Staphylococcus spp. Any strains showing decreased susceptibility to other quinolones exhibited cross resistance to these new agents. The MBC of rufloxacin and MF 922 was within one dilution of the MIC and human serum had little effect upon the activity of both agents. The protein binding of rufloxacin and MF 922 at 1 and 10 mg/L were 55% and 63.8% and 30.3% and 32.6% respectively. The activity of rufloxacin against four strains of Chlamydia trachomatis and one strain of Chlamydia pneumoniae was determined. The MICs for C. trachomatis were 4-8 mg/L and 4 mg/L for C. pneumoniae.


Effect of rufloxacin on in-vitro proliferation and differentiation of human mononuclear cells. We investigated the effects of rufloxacin, a new, long acting fluoroquinolone, on the growth and differentiation of human peripheral blood mononuclear cells (MNC) stimulated with T- and B-cell mitogenic agents. Rufloxacin inhibited 3H-thymidine incorporation into MNC stimulated with phytohaemagglutinin (PHA) and pokeweed mitogen (PWM) in a dose-dependent manner. The concentrations of rufloxacin required to inhibit 1/2 maximal proliferation of T- and B-cells were 62 and 33.5 mg/L respectively. Rufloxacin, at clinically achievable serum levels (less than 10 mg/L), was found not to inhibit PHA-induced T-cell differentiation as assessed by IL-2 production, IL-2 receptor expression and the expression of cell differentiation markers (CD4 and CD8). However, higher concentrations of rufloxacin (10 and 50 mg/L) markedly inhibited B-cell differentiation in-vitro as determined by the measurement of immunoglobulin production by MNC stimulated with PWM. The clinical relevance of our in-vitro findings remains to be elucidated.


Evaluation of temafloxacin in a rat model of intra-abdominal abscess. The in-vitro activity of temafloxacin, a new fluoroquinolone, was evaluated in an experimental model of intra-abdominal abscess in rats. Mixed aerobic-anaerobic infection was induced by intraperitoneal implantation of gelatin capsules containing Bacteroides fragilis, Escherichia coli, and sterile rat faeces. Temafloxacin was highly active with a 90.9% cure rate in comparison with no treatment (no cures; P less than 0.0001), and as active as a combination of clindamycin and gentamicin (100% cure rate; P greater than 0.05). Temafloxacin (12 mg/dose) gave rise to serum concentrations that exceeded the MIC values of both microorganisms for at least 8 h.


Ethanol inhibits ligand-activated Ca2+ channels in human B lymphocytes. Ethanol reportedly is immunosuppressive, interfering with lymphocyte proliferation. To investigate the basis for this immunosuppression, the effects of acute treatment with ethanol were studied on Ca2+ mobilization in tonsillar B lymphocytes and the human lymphoblastoid B-cell line, Ramos. The level of intracellular Ca2+ was monitored in cells loaded with the fluorescent dye indo-1 following stimulation with either anti-IgM antibody or platelet activating factor. The effect of ethanol was also examined on the induction of the early proto-oncogene c-fos in these cells. Ethanol inhibited ligand-activated Ca2+ mobilization due to transmembrane influx but not intracellular store release, in a dose- and time-dependent manner. This inhibition was not due to the inability of anti-IgM to bind to its surface receptor nor to membrane depolarization induced by ethanol. Ethanol also inhibited the Ca2(+)-dependent induction by anti-IgM of c-fos in these cells. The inhibitory effects of ethanol on ligand-activated Ca2+ channels and subsequent induction of c-fos may provide the basis for its immunosuppressive action.


Selective regulation of beta 2-adrenergic receptor gene expression by interleukin-1 in cultured human lung tumor cells. The regulation of beta 1- and beta 2-adrenergic receptors (beta 1AR and beta 2AR) and receptor gene expression by interleukin-1 alpha (IL-1 alpha) was studied in cultured A549 human lung adenocarcinoma cells. The density and affinity of beta 1 AR and beta 2 AR were analyzed by computerized curve fitting of 125I-pindolol binding and its displacement by subtype selective antagonists. Steady state levels of receptor mRNAs were quantified by DNA excess solution hybridization assays. A549 cells in preconfluent cultures had fewer beta 1AR than beta 2AR (beta 1: 1.9 +/- 0.3 vs beta 2: 4.0 +/- 0.5 fmol/mg protein, means +/- SE), but lost most of their beta 2 AR upon reaching confluency (beta 1: 2.7 +/- 0.4, beta 2: 0.8 +/- 0.3 fmol/mg). Incubation of preconfluent cells for 24 hr with 20 pM of human recombinant IL-1 alpha did not modify the density of either of the beta AR subtypes. Similar incubations of confluent cells increased the density of beta 2 AR from 0.8 +/- 0.3 to 4.2 +/- 0.9 fmol/mg, while the density of beta 1 AR and the antagonist affinities of both receptors remained unaltered. The IL-1 alpha-induced increase in beta 2 AR density in confluent cells was antagonized in a concentration-dependent manner by a recombinant protein antagonist of type I IL-1 receptors (IC50: 0.2 nM). The IL-1 alpha-induced increase in beta 2AR density was preceded by an increase in the steady state level of beta 2AR mRNA, while levels of beta 1AR mRNA remained unchanged. IL-1 alpha increased the stability as well as the rate of transcription of beta 2AR mRNA. These findings demonstrate for the first time that activation of type I IL-1 receptors in A549 cells leads to a cell density-dependent, selective upregulation of beta 2AR, and that the mechanism of this effect involves increased formation and stability of the beta 2AR message.