Rat liver dipeptidylpeptidase IV contains competing apical and basolateral targeting information.

Madin-Darby canine kidney (MDCK) cells deliver endogenous apical and basolateral proteins directly to the appropriate domains. We are investigating the molecular signals on a model plasma membrane hydrolase, dipeptidylpeptidase IV (DPPIV). Most newly synthesized rat liver DPPIV is delivered directly to the apical surface of transfected MDCK cells; however, about 20% is delivered first to the basolateral surface and reaches the apical surface via transcytosis (Casanova, J. E., Mishumi, Y., Ikehara, Y., Hubbard, A. L., and Mostov, K. E. (1991) J. Biol. Chem. 266, 24428-24432). A soluble form of DPPIV (solDPPIV) containing only the lumenal domain of the protein was efficiently transported and secreted by stably transfected MDCK cells. If this domain contains apical sorting information, we would expect 80% of the soluble protein to be secreted apically. Surprisingly, 95% of the secreted solDPPIV was found in the apical medium. The high efficiency of apical secretion suggested that the transmembrane domain and cytoplasmic tail of DPPIV might contain competing basolateral targeting information. To test this hypothesis, we investigated the trafficking of a chimera in which the cytoplasmic tail and transmembrane domains of DPPIV were joined to lysozyme, an exogenous protein which should not contain sorting information. This protein was delivered predominantly to the basolateral surface. Our results suggest that the lumenal domain of DPPIV carries dominant apical sorting information while the transmembrane domain and cytoplasmic tail of the molecule contains competing basolateral sorting information.


Activation of Ca2+/calmodulin-dependent protein kinase II and protein kinase C by glutamate in cultured rat hippocampal neurons.

In cultured rat hippocampal neurons, glutamate elevated the Ca(2+)-independent activity of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) through autophosphorylation when the neurons were incubated in Mg(2+)-free buffer, and this response was blocked by specific antagonists of the N-methyl-D-aspartate (NMDA) receptor. In addition, glutamate stimulated the transient translocation of protein kinase C (PKC) from the cytosol to the membrane fraction. This effect was not blocked by NMDA receptor antagonists but was partially blocked by DL-2-amino-3-phosphonopropionate. Quisqualate or trans-1-amoinocyclopentane-trans1,3-dicarboxylate produced a similar effect on the translocation of PKC. In the experiments with 32P-labeled cells, the phosphorylation of microtuble-associated protein 2 and synapsin I, as well as autophosphorylation of CaM kinase II, were found to be stimulated by exposure to glutamate. These results suggest that glutamate can activate CaM kinase II through the ionotropic NMDA receptor, which in turn increases the phosphorylation of microtuble-associated protein 2 and synapsin I. PKC was activated through the metabotropic glutamate receptor in the hippocampal neurons.


A retinoic acid-inducible mRNA from human erythroleukemia cells encodes a novel tissue transglutaminase homologue.

A 1.9-kilobase (kb) cDNA for a new transglutaminase protein has been cloned and sequenced from retinoic acid-induced human erythroleukemia (HEL) cells. Full-length cDNA analysis reveals an open reading frame coding for a polypeptide of 548 amino acid residues with a molecular weight of 61,740. The deduced amino acid sequence exhibited 98% identity to the human cellular transglutaminase sequence. The cysteine at position 277 in the active site and the putative Ca(2+)-binding pocket at residues 446-453 of cellular transglutaminase are conserved. Such evidence predicts that the encoded protein product is likely to be a transglutaminase homologue (TGase-H). Immunoprecipitation of the in vitro translation products from a synthetic TGase-H mRNA and from total protein of cultured erythroleukemia HEL cells revealed a protein with a molecular weight of 63,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Northern blot analysis of HEL cells and normal human fibroblast cells WI-38 using a cellular TGase probe detected the 1.9- and 4.0-kb RNA species at a relative abundance of 1:3 and 1:7, respectively. The 3'-end of the human cellular transglutaminase mRNA was also cloned and sequenced to allow comparison to the 3'-end of TGase-H reported here. This new piece gives a full length of 4012 nucleotides (4.0 kb) for human cellular transglutaminase. Comparison of the 5'-end (bases 1-1747) of the 1.9- and 4.0-kb cDNA sequences revealed a very high degree of identity. Beginning with base 1748, the sequences diverge showing no homology. The divergence point correlates with known intron-exon consensus boundaries indicative of alternative splicing.


Oncogenic ras induces an inhibitor of double-stranded RNA-dependent eukaryotic initiation factor 2 alpha-kinase activation.

The interferon-inducible 68-kDa dsRNA-dependent eIF2 alpha-kinase (dsI) is a potent cellular antiviral enzyme which is activated by autophosphorylation in response to double-stranded RNA (dsRNA). Activated dsI has also been implicated as a second messenger for gene induction by platelet-derived growth factor (PDGF) and interferon (IFN). We have shown previously that introduction of a transforming ras gene into BALB/c-3T3 fibroblasts blocks induction of responsive genes by PDGF and IFN. We therefore investigated the effect of transforming ras genes on dsI activity in these cells. We report here that dsRNA-mediated activation of dsI is blocked in v-ras-containing cells in a manner specific to ras and not attributable to the transformed phenotype since: 1) a dexamethasone-inducible v-Ha-ras gene produced the effect within 18 h of induction; 2) morphologic reversion of ras-transformed cells with cAMP or the Krev-1 gene restored potential for dsI activation; and 3) transformation by v-mos or v-abl had no effect on dsI activation. Latent dsI levels were unaffected by v-ras. A heat-sensitive dsI inhibitory activity could be demonstrated in v-ras-containing cells which functioned in trans when mixed with untransformed cell extracts prior to stimulation with dsRNA. The inhibitory activity, which was destroyed by phenol-chloroform extraction, did not bind dsRNA.


Expression of plant chaperonin-60 genes in Escherichia coli.

We have examined the expression in Escherichia coli of genes encoding a plant chloroplast molecular chaperone, chaperonin-60. Purified plant chaperonin-60 is distinct in that it contains two polypeptides, p60cpn-60 alpha and p60cpn-60 beta, which have divergent amino acid sequences (Hemmingsen, S. M., and Ellis, R. J. (1986) Plant Physiol. 80, 269-276; Martel, R., Cloney, L. P., Pelcher, L. E., and Hemmingsen, S. M. (1990) Gene (Amst.) 94, 181-187). The precise polypeptide composition(s) of the active tetradecameric specie(s) (cpn60(14)) has not been determined. Genes encoding the mature forms of the Brassica napus chaperonin polypeptides have been expressed separately and in combination in E. coli to produce three novel strains: alpha, beta, and alpha beta. The plant cpn60 polypeptides accumulated in soluble forms and to similar high levels in each. There was no conclusive evidence that p60cpn-60 alpha assembled into cpn60(14) species in alpha cells. In beta and alpha beta cells, the plant gene products assembled efficiently into cpn60(14) species. Thus, the assembly of p60cpn-60 alpha required the presence of p60cpn-60 beta, whereas the assembly of p60cpn-60 beta could occur in the absence of p60cpn-60 alpha. Significant proportions of the endogenous groEL polypeptides were not assembled into tetradecameric groEL14 in beta and alpha beta cells. Analysis of the tetradecameric species that did form indicated the presence of novel hybrid cpn6014 species that contained both plant and bacterial cpn60 polypeptides.


Assessment of plant chaperonin-60 gene function in Escherichia coli.

Brassica napus chaperonin-60 alpha and chaperonin-60 beta genes expressed separately and in combination produce three novel Escherichia coli strains: alpha, beta, and alpha beta. In beta and alpha beta cells, the plant gene products assemble efficiently into tetradecameric cpn60(14) species, including novel hybrids containing both bacterial and plant gene products. The levels of authentic groEL14 are reduced in these cells (Cloney, L. P., Wu, H. B., and Hemmingsen, S. M. (1992) J. Biol. Chem. 267, 23327-23332). The assembly of cyanobacterial ribulose-P2 carboxylase (rubisco) in E. coli requires the activities of the endogenous chaperonin proteins. Furthermore, the extent to which assembly occurs is limited by the normal levels of expression of the groE operon (Goloubinoff, P., Gatenby, A. A., and Lorimer, G. H. (1989) Nature 337, 44-47). We have now monitored the accumulation of cyanobacterial rubisco in E. coli alpha, beta, and alpha beta cells to assess the activity of the plant cpn60 gene products and effects on endogenous chaperonin functions. Expression of cpn-60 alpha alone did not enhance rubisco assembly, which is consistent with our previous observation that p60cpn-60 alpha required the presence of p60cpn-60 beta for assembly into cpn60(14) species. In contrast, expression of cpn-60 beta alone resulted in markedly enhanced rubisco assembly in cells that accumulated normal levels of both endogenous chaperonin polypeptides (groEL and groES). This demonstrates that assembled p60cpn-60 beta is functional as a chaperonin in E. coli. Co-expression of cpn-60 alpha and cpn-60 beta in cells with normal levels of expression of groES and groEL suppressed rubisco assembly. Increased expression of groES in cells in which cpn-60 alpha and cpn-60 beta were co-expressed relieved this suppression and resulted in enhanced rubisco assembly. Implications with respect to dependence of chloroplast cpn60 function on cpn10 are discussed.


A human polyadenylation factor is a G protein beta-subunit homologue.

Cleavage stimulation factor (CstF) is one of the multiple factors required for polyadenylation of mammalian pre-mRNAs in vitro. We have shown previously that this factor is composed of three distinct subunits of 77, 64, and 50 kDa, and that the 64-kDa subunit can be UV-cross-linked to RNA in a polyadenylation signal (AAUAAA)-dependent manner. By molecular cloning, the 64-kDa subunit was shown to contain a ribonucleoprotein-type RNA binding domain and a novel repeat structure. To study the functions of the other subunits, we have now isolated cDNAs encoding the 50-kDa subunit of human CstF. This subunit shares extensive homology with mammalian G protein beta-subunits and has a characteristic repeat structure (transducin repeat), in which an approximately 44-amino acid-long sequence is repeated seven times. To our knowledge, the 50-kDa subunit is the first example of a functional beta-subunit-like protein in vertebrates. Possible roles of the transducin repeat, both in CstF function specifically and in other beta-subunit homologues more generally, are discussed.


Involvement of sequences near both amino and carboxyl termini in the rapid intracellular degradation of tyrosine aminotransferase.

The degradation of rat liver tyrosine aminotransferase has been studied after transfection of suitable expression vectors into mammalian cells in culture. A normal rapid rate of degradation (half-life about 6 h) was observed in cells under stable transfection conditions. However, the higher enzyme levels produced during transient transfections or after amplification with methotrexate caused the apparent half-life of degradation to increase substantially. Analysis of expression in Chinese hamster ovary (CHO)-DG44 cells from vectors with deletions near either end of the tyrosine aminotransferase coding sequence showed that approximately the first 40 and the last 12 amino acid residues are not required to obtain normal catalytic function. When catalytically active deletion mutants were examined for effects on tyrosine aminotransferase degradation in stably transfected CHO-DG44 cell populations, short sequences near each end of the protein were found to be necessary for rapid degradation. The required sequence near the amino terminus is located between amino acids 30 and 40 and includes the highly basic region RKKGRKAR, a potential ubiquitin attachment site. The other essential sequence (EECDK) is located at the very COOH terminus of the 454-amino acid chain and is part of an acidic domain rich in cysteines and having PEST characteristics (rich in Pro, Glu, and Thr). Ser448, a potential casein kinase II phosphorylation site, is not required for activity or rapid degradation of tyrosine aminotransferase. No correlation was observed between the intracellular degradation rates of the various mutant proteins and their heat stabilities in vitro.


Factors determining the specificity of signal transduction by guanine nucleotide-binding protein-coupled receptors. III. Coupling of alpha 2-adrenergic receptor subtypes in a cell type-specific manner.

A number of diverse signaling pathways can be activated by G-protein coupled receptors. However, the factors involved in selection of a particular transduction pathway by a single receptor are not well understood. We are attempting to address this issue utilizing the alpha 2-adrenergic receptor (alpha 2-AR) subfamily as a representative model system. In this report, we demonstrate that the cellular response mediated by an alpha 2-AR subtype is cell-specific and thus depends on its environment. Receptor coupling to adenylylcyclase was determined following stable expression of the rat alpha 2B- and alpha 2D-AR subtypes in three functionally distinct cell types (NIH-3T3 fibroblasts, DDT1 MF-2 smooth muscle cells, and the pheochromocytoma cell line PC-12). When the receptor subtype gene is expressed in NIH-3T3 and DDT1 MF-2 cells, receptor activation inhibits basal and forskolin-induced increases in cellular cAMP. However, in PC-12 transfectants the same receptor subtype actually increases basal cAMP and augments the effect of forskolin. Potentiation of the forskolin effect in PC-12 cells is insensitive to pertussis toxin but is blocked by loading the cells with BAPTA (bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid) which minimizes changes in Ca2+i by calcium chelation. These data and the functional demonstration of a Ca2+/calmodulin-sensitive adenylylcyclase in PC-12 but not NIH-3T3 and DDT1 MF-2 cells, suggests that the cell-specific effects of epinephrine are due to receptor coupling to both different G-proteins and types of adenylylcyclase.


Beta-adrenergic agonists that down-regulate receptor mRNA up-regulate a M(r) 35,000 protein(s) that selectively binds to beta-adrenergic receptor mRNAs.

In an effort to explore the molecular basis for agonist-induced destabilization of beta-adrenergic receptor mRNA, we investigated the nature of RNA-binding proteins both in untreated and agonist-treated DDT1-MF2 smooth muscle cells. Messenger RNAs for the alpha 1b-, beta 1-, and beta 2-adrenergic receptors as well as for beta-globin were transcribed in vitro, incubated with cytosolic fractions, covalently cross-linked by short-wave UV light, and analyzed by SDS-polyacrylamide gel electrophoresis. A prominent M(r) 35,000 radiolabeled protein(s) with the following characteristics was identified: (i) binds selectively to beta 1- and beta 2-adrenergic receptor mRNAs, both of which undergo agonist-induced down-regulation; (ii) does not bind to either alpha 1b-adrenergic receptor mRNA, which does not undergo agonist induced down-regulation, or to beta-globin mRNA; (iii) displays binding to beta 2-adrenergic receptor mRNA that is selectively competed by poly(U) RNA, but not poly(A), -(C), or -(G) RNA; and (iv) displays binding to receptor mRNA that can be competed by RNA harboring destabilizer sequences that are AU-rich and AUUUA pentamer-rich. The abundance of the M(r) 35,000 RNA-binding protein selective for beta-adrenergic receptor message, a factor we term beta ARB protein, varies inversely with the level of receptor mRNA, being induced by agonists that down-regulate receptor mRNA.


Regulation of ADP-ribosylation factor (ARF) expression. Cross-species conservation of the developmental and tissue-specific alternative polyadenylation of ARF 4 mRNA.

ADP-ribosylation factors (ARFs), approximately 20-kDa guanine nucleotide-binding proteins, are involved in protein trafficking and enhance cholera toxin ADP-ribosyltransferase activity. Expression of six ARF genes was examined in mammalian tissues; only ARF 4 mRNA was detected in rat testis in forms considerably shorter than those in other tissues. Testis-specific expression of short forms of ARF 4 mRNA was observed in several mammalian species. On Northern analysis of the developmental expression of rat ARF 4 mRNA, appearance of the shorter species was consistent with its involvement in a late stage of spermatogenesis. Sequences of products of rapid amplification of cDNA ends (RACE-polymerase chain reaction) of rat ARF 4 mRNA revealed that different mRNAs resulted from the use of three polyadenylation signals, one AUUAAA and two AAUAAA. Sequences of 3'-untranslated regions of rat and human ARF 4 mRNA were very similar with identical polyadenylation signals at similar positions. Of the ARF 4 mRNAs identified by RACE-PCR, with sizes of 1.1, 1.3, and 1.8 kb, the 1.1-kb mRNA was predominant in adult testis. By in situ hybridization, the 1.1-kb mRNA was identified primarily in mature sperm, consistent with the developmental studies. Shorter mRNAs, thought to be more stable, may compensate for cessation of transcription at late stages of spermatogenesis.


Functional involvement of alpha 1- and alpha 2-adrenoceptors in 86Rb efflux from liver slices and lipolysis in guinea-pig isolated adipocytes.

1. The application of an alpha 1-adrenoceptor agonist, amidephrine, to guinea-pig liver slices increases glucose release and 86Rb efflux. Since prazosin was more potent than yohimbine in inhibiting both responses, alpha 1-adrenoceptors seem to be involved in the effects evoked by the agonist. 2. Clonidine (an alpha 2-adrenoceptor agonist) at doses unable to activate liver glycogenolysis increased 86Rb release and potentiated isoprenaline in promoting 86Rb efflux. Since yohimbine antagonized clonidine in promoting 86Rb efflux, alpha 2-adrenoceptors also seem to control plasmalemmal permeability to 86Rb. 3. The liver slice responses resulting from alpha 1- and alpha 2-adrenoceptor stimulation required extracellular calcium. Calcium absence or the administration of D-600 attenuated the effects of amidephrine on glucose release and 86Rb outflow and Ca2+ excess re-established both responses. D-600 and apamin blocked clonidine-induced 86Rb efflux, suggesting that alpha 2-adrenoceptor stimulation activates calcium dependent K+ channels. 4. alpha 2-adrenoceptors do not appear to mediate antilipolytic effects in guinea-pig fat cells.


Effect of quinpirole on neurogenic vasoconstriction in the in situ autoperfused hindquarters and renal vascular beds of the rat.

1. The effects of local administration of quinpirole were studied in the in situ autoperfused hindquarters and renal vascular beds, in order to assess whether presynaptic dopamine receptors in these vascular systems could play a role in the hypotensive effect of this agonist. 2. In both preparations, local injection of quinpirole did not alter perfusion pressure, but reduced the pressor response to electrical stimulation of the sympathetic innervation. Increases in perfusion induced by local administration of noradrenaline were not altered by quinpirole. 3. The inhibitory effect of quinpirole on the stimulation-evoked pressor responses was completely antagonized by intravenous administration of the DA2-receptor antagonist domperidone (0.5 mg kg-1) but not by the DA1-receptor antagonist SCH 23390 (0.3 mg kg-1) or the alpha 2-adrenoceptor antagonist idazoxan (0.3 mg kg-1). 4. The results indicate that quinpirole inhibits neurally induced pressor responses in the autoperfused hindquarters and renal vascular beds of the rat by stimulation of presynaptic dopamine receptors. These receptors may be involved in the hypotensive action of quinpirole in the rat.


Biphasic dose-dependent effects of dopamine and involvement of dopamine autoreceptors on intra-ocular pressure in the rabbit.

1. This work was conducted to provide new data concerning the possible dose-dependent activity of dopamine (DA) after ocular instillation. Experiments were done in rabbits with normal intraocular pressure (IOP), or after transitory induced ocular hypertension in water-loaded animals. 2. In ocular normotensive animals, a biphasic dose-dependent activity is observed, with no significant effect for 0.001% and 0.003% DA, a decrease in IOP after 0.005% and 0.01% DA instillation, then an important increase in IOP at concentrations from 0.05% onwards. 3. During transitory ocular hypertension, this phenomenon was confirmed, with a marked ocular hypotensive activity for 0.01% DA, no effect after 0.005% DA, then an important ocular hypertension with 0.05% and 0.5% DA as compared to the control group (0.9% NaCl). 4. An immediate and similar ocular hypertensive effect with DA could be reproduced by a subsequent instillation at high concentration (1%), while the hypotensive activity induced at low concentration (0.01%) is followed by a long-lasting refractory period (about 18 h). 5. Such a dose-dependent biphasic effect was also observed with N-methyl-dopamine (NMDA) after ocular instillation. The effects of instilled dopaminergic compounds were tested and ocular hypotensive activities of the S(-)enantiomer of the DA analogues 3-(3-hydroxyphenyl)-N-propylpiperidine (3-PPP), and of thiothixene (TIX) were also demonstrated. 6. The possible relationships to DA1 and DA2 receptors of the dual effect is discussed.


Decreased tissue reaction to bioprosthetic heart valve material after L-glutamic acid treatment. A morphological study.

Degenerative alterations of two different glutaraldehyde (GA)-fixed bioprosthetic heart valve materials were investigated in subcutaneous rat implants: Bovine pericardium, prepared according to clinically used bioprosthetic heart valve material (BHV) was compared to alternatively preserved pericardium (APHV), which was fixed in GA and treated with L-glutamic acid. Following 63 days of subcutaneous implantation, calcification of APHV implants was significantly lower as compared to BHV implants (13 +/- 6 versus 158 +/- 18 micrograms Ca/mg dry weight tissue; p less than 0.05). In BHV implants ultrastructural investigations showed nucleation of plate-shaped hydroxyapatite crystals at the surface of collagen fibrils and in remnants of connective tissue cells; no signs of calcification could be detected in APHV implants. The time-course of the inflammatory reaction was determined by quantification of immunohistochemical stained mononuclear host-cells invading the implants. In both preparation groups inflammatory reaction reached maximum 42 days after implantation. However, infiltration rate of inflammatory cells was markedly decreased in APHVs as compared to BHVs (p less than 0.05).


Argyrophilic nucleolar organizer region counts and proliferating cell nuclear antigen scores are two reliable indicators of survival in pharyngeal carcinoma.

The proliferative activity of pharyngeal carcinoma has been investigated by means of monoclonal antibody PC10 against proliferating cell nuclear antigen (PCNA/cyclin) and argyrophilic nucleolar organizer region (AgNOR) analysis in formalin-fixed, paraffin-embedded biopsies from 45 primary squamous and undifferentiated carcinomas, prior to therapy. The correlation between AgNOR counts and PCNA(PC10) scores was highly significant (r = 0.73; P < 0.0001) as determined by Pearson's correlation coefficient. Moreover, the univariate Kaplan-Meier survival analysis showed a significant correlation between 3- and 5-year survival rates and the mean AgNOR number per tumour cell (P = 0.0003) or the percentage of PCNA(PC10)-positive cells (P = 0.0001). Our results indicate that both AgNOR counts and PCNA(PC10) scores are reliable markers of the proliferative activity of pharyngeal carcinoma in small, routinely processed biopsies, in which they can allow simultaneous evaluation of the histology and tumour cell kinetics.


Adriamycin binding assay: a valuable chemosensitivity test in human osteosarcoma.

The reliability of a simple method evaluating the pattern of subcellular binding of Adriamycin (Adriamycin binding assay, ABA) as an index of sensitivity was demonstrated in different primary cultures and in sensitive and resistant cell lines of human osteosarcoma. After exposure to Adriamycin (10 micrograms/ml for 30 min at 37 degrees C), living sensitive cells showed selective intranuclear uptake of the drug, whereas in resistant cells no distinct subcellular distribution was observed. The binding pattern of Adriamycin in sensitive and in highly resistant cells was inversely related to the expression of P-glycoprotein. However, low levels of resistance in vitro, not detectable by increased levels of expression of P-glycoprotein, were revealed by ABA. The use of ABA in combination with the estimate of P-glycoprotein expression is recommended in clinical practice as an accurate means for predicting the sensitivity of osteosarcoma to Adriamycin.


Sialosylated Lewis chi expression in CD30-positive anaplastic large-cell lymphomas.

The expression of sialosylated Lewis chi (SLEX), a ligand for endothelial leukocyte adhesion molecule 1 in malignant lymphomas, was immunohistochemically examined, using the monoclonal antibody, CSLEX1, which specifically reacts with SLEX. It was expressed in 6 out of 64 non-Hodgkin's lymphomas, which consisted of 1 nasal large-cell lymphoma and 5 of 8 (62%) Ki-1-positive anaplastic large-cell lymphomas (ALCL). One nasal lymphoma positive for SLEX co-expressed a T cell marker, cluster of differentiation (CD) 5, and natural killer (NK) cell markers such as CD56 and CD16, indicating that SLEX+ nasal lymphoma cells are possibly malignant counterparts of SLEX+ NK cells. SLEX did not react with 30 B cell lymphomas or most Hodgkin's disease lymphomas, though it did with one lymphocyte predominance type. Although SLEX+ ALCL exhibit T cell markers in some cases, some ALCL expressing SLEX may represent histiocytic differentiation of the neoplastic cells. The lymphoma cells of ALCL were preferentially positive for SLEX, in contrast to Hodgkin's disease cells, and thus CSLEX1 in conjunction with CD30 and CD15 should be of use for analyzing and making differential diagnoses of routine paraffin-embedded sections of ALCL.


Clathrin assembly protein AP-2 induces aggregation of membrane vesicles: a possible role for AP-2 in endosome formation.

We have examined the in vitro behavior of clathrin-coated vesicles that have been stripped of their surface coats such that the majority of the clathrin is removed but substantial amounts of clathrin assembly proteins (AP) remain membrane-associated. Aggregation of these stripped coated vesicles (s-CV) is observed when they are placed under conditions that approximate the pH and ionic strength of the cell interior (pH 7.2, approximately 100 mM salt). This s-CV aggregation reaction is rapid (t1/2 < or = 0.5 min), independent of temperature within a range of 4-37 degrees C, and unaffected by ATP, guanosine-5'-O-(3-thiophosphate), and in particular EGTA, distinguishing it from Ca(2+)-dependent membrane aggregation reactions. The process is driven by the action of membrane-associated AP molecules since partial proteolysis results in a full loss of activity and since aggregation is abolished by pretreatment of the s-CVs with a monoclonal antibody that reacts with the alpha subunit of AP-2. However, vesicle aggregation is not inhibited by PPPi, indicating that the previously characterized polyphosphate-sensitive AP-2 self-association is not responsible for the reaction. The vesicle aggregation reaction can be reconstituted: liposomes of phospholipid composition approximating that found on the cytoplasmic surfaces of the plasma membrane and of coated vesicles (70% L-alpha-phosphatidylethanolamine (type I-A), 15% L-alpha-phosphatidyl-L-serine, and 15% L-alpha-phosphatidylinositol) aggregated after addition of AP-2, but not of AP-1, AP-3 (AP180), or pure clathrin triskelions. Aggregation of liposomes is abolished by limited proteolysis of AP-2 with trypsin. In addition, a highly purified AP-2 alpha preparation devoid of beta causes liposome aggregation, whereas pure beta subunit does not, consistent with results obtained in the s-CV assay which also indicate the involvement of the alpha subunit. Using a fluorescence energy transfer assay we show that AP-2 does not cause fusion of liposomes under physiological solution conditions. However, since the fusion of membranes necessarily requires the close opposition of the two participating bilayers, the AP-2-dependent vesicle aggregation events that we have identified may represent an initial step in the formation and fusion of endosomes that occur subsequent to endocytosis and clathrin uncoating in vivo.


The alternatively spliced V region contributes to the differential incorporation of plasma and cellular fibronectins into fibrin clots.

During blood clot formation in vivo, plasma fibronectin (pFN) is cross-linked to fibrin by coagulation factor XIIIa. Cellular FN (cFN), which localizes to connective tissue, is distinguished from pFN by the inclusion of alternatively spliced segments. To determine if these two FNs are functionally equivalent in blood clotting, the cross-linking of rat pFN and cFN to fibrin was compared in an in vitro clotting assay. Fibrinogen and FN were incubated at physiological ratios in the presence of thrombin and factor XIIIa. Cross-linking of FN to fibrin was monitored by SDS-PAGE and immunoblotting. Over 24 h, cFN was incorporated at a significantly slower rate than pFN and was not completely cross-linked to fibrin at a temperature that favors this interaction (0 degrees C). This difference was observed with purified fibrinogens from human, rat, and bovine and with rat plasma and was maintained even after incubation of pFN with rat fibroblasts for several days. Using the same assay, purified recombinant V(+)-V0 and V(+)-V+ FN dimers resembling pFN and cFN, respectively, showed a similar difference in cross-linking kinetics. These results suggest that the asymmetric distribution of the V region among pFN dimers plays a role in regulating its incorporation into blood clots. In fibrin clots, cFN was converted into a set of cross-linked intermediates distinct from those of pFN. For example, while pFN was initially cross-linked into a pFN-fibrin alpha heterodimer, this product was not a major intermediate in clots formed with cFN. This finding, in conjunction with evidence for the formation of factor XIIIa-catalyzed cFN-cFN cross-links, indicated that cFN molecules interact with each other, and with fibrin, differently from pFN. Together, these results show an important functional distinction between pFN and cFN.


Extracellular ATP and ADP stimulate proliferation of porcine aortic smooth muscle cells.

The mitogenic effect of extracellular ATP on porcine aortic smooth muscle cells (SMC) was examined. Stimulation of [3H]thymidine incorporation by ATP was dose-dependent; the maximal effect was obtained at 100 microM. ATP acted synergistically with insulin, IGF-1, EGF, PDGF, and various other mitogens. Incorporation of [3H]thymidine was correlated with the fraction of [3H]thymidine-labeled nuclei and changes in cell counts. The stimulation of proliferation was also determined by measurement of cellular DNA using bisbenzamide and by following the increase of mitochondrial dehydrogenase protein. The effect of ATP was not due to hydrolysis to adenosine, which shows synergism with ATP. ATP acted as a competence factor. The mitogenic effect of ATP, but not adenosine, was further increased by lysophosphatidate, phosphatidic acid, or norepinephrine. The inhibitor of adenosine deaminase, EHNA, stimulated the effect of adenosine but not ATP. The adenosine receptor antagonist theophylline depressed adenosine-induced mitogenesis. ADP and the non-hydrolyzable analogue adenosine 5'-[beta, gamma-imido]triphosphate (AMP-PNP) were equally mitogenic. Thus extracellular ATP stimulated mitogenesis of SMC via P2Y purinoceptors. The mechanism of ATP acting as a mitogen in SMC was further explored. Extracellular ATP stimulated the release of [3H]arachidonic acid (AA) and prostaglandin E2 (PGE2) into the medium, and enhanced cAMP accumulation in a dose-dependent fashion similar to ATP-induced [3H]thymidine incorporation. Inhibitors of the arachidonic acid metabolism pathway, quinacrine and indomethacin, partially inhibited the mitogenic effect of ATP but not of adenosine. Pertussis toxin inhibited ATP-stimulated DNA synthesis, AA release, PGE2 formation, and cAMP accumulation. Down-regulation of protein kinase C (PKC) by long-term exposure to phorbol dibutyrate (PDBu) partially prevented stimulation of DNA synthesis and activation of the AA pathway by ATP. The PKC inhibitor, staurosporine, antagonized mitogenesis stimulated by ATP. No synergistic effect was found when PDBu and ATP were added together. Therefore, a dual mechanism, including both arachidonic acid metabolism and PKC, is involved in ATP-mediated mitogenesis in SMC. In addition, ATP acted synergistically with angiotensin II, phospholipase C, serotonin, or carbachol to stimulate DNA synthesis. Finally, the possible physiological significance of ATP as a mitogen in SMC was further studied. The effect of endothelin and heparin, which are released from endothelial cells, on ATP-dependent mitogenesis was investigated. Extracellular ATP acted synergistically with endothelin to stimulate a greater extent of [3H]thymidine incorporation than was seen with PDGF plus endothelin. Heparin, believed to have a regulatory role, partially inhibited the stimulation of DNA synthesis caused both by ATP and PDGF.(ABSTRACT TRUNCATED AT 400 WORDS)


Evaluation of the role of electrostatic residues in human epidermal growth factor by site-directed mutagenesis and chemical modification.

Four residues in the carboxy-terminal domain of human epidermal growth factor (hEGF), glutamate 40, glutamine 43, arginine 45, and aspartate 46 were targeted for site-directed mutagenesis to evaluate their potential role in epidermal growth factor (EGF) receptor-ligand interaction. One or more mutations were generated at each of these sites and the altered recombinant hEGF gene products were purified and evaluated by radioreceptor competition binding assay. Charge-conservative replacement of glutamate 40 with aspartate resulted in a decrease in receptor binding affinity to 30% relative to wild-type hEGF. On the other hand, removal of the electrostatic charge by substitution of glutamate 40 with glutamine or alanine resulted in only a slightly greater decrease in receptor binding to 25% relative receptor affinity. The introduction of a positive charge upon substitution of glutamine 43 with lysine had no effect on receptor binding. The substitution of arginine 45 with lysine also showed no effect on receptor binding, unlike the absolute requirement observed for the arginine side-chain at position 41 [Engler DA, Campion SR, Hauser MR, Cook JS, Niyogi, SK: J Biol Chem 267:2274-2281, 1992]. Subsequent elimination of the positive charge of lysine 45 by reaction with potassium cyanate showed that the electrostatic property of the residue at this site, as well as that at lysine 28 and lysine 48, was not required for receptor-ligand association.(ABSTRACT TRUNCATED AT 250 WORDS)


Antennapedia homeobox as a signal for the cellular internalization and nuclear addressing of a small exogenous peptide.

In a previous study we demonstrated that a homeobox peptide corresponding to the 60 amino acid long DNA-binding region of the Drosophila antennapedia homeo-protein was capable of crossing the plasma membrane of cells in culture. This finding has led us to investigate whether chimeric molecules encompassing the homeobox would behave in a similar manner. We demonstrate here that a peptide of 93 amino acids composed of the homeobox and of the C terminus of Rab3, a small GTP-binding protein, crosses the membrane of myoblasts, myotubes and neurons and is conveyed to their nuclei. This transport is highly efficient, is observed in all the cells present in the culture and occurs at 37 degrees C and 12 degrees C without quantitative peptide degradation. Beyond its theoretical implications for our current views on cellular interactions, this finding could have technical repercussions on the development of drugs with intracellular targets.


Force contribution of the LFA-1/ICAM-1 complex to T cell adhesion.

Little is known in quantitative terms about forces between cells generated during adhesion and recognition, or about the contribution of any one set of molecular associations to the development of these forces. To determine the forces involved in adhesion dependent on lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1), we have measured the junctional avidity between single cell pairs consisting of a cloned T cell that expresses LFA-1 and a fibroblast cell that expresses MHC class II molecules and ICAM-1 after transfection. Micromanipulation was used to induce conjugation of cell pairs and to determine the force required to separate the conjugate. T cell adhesion to three related fibroblast cell lines was compared: the parent line that does not express ICAM-1 or other LFA-1 counter-receptors, and two transfectants that have high and moderate levels of surface ICAM-1 expression. The force needed to separate the conjugates varied with the fibroblast ICAM-1 expression levels. The T cell adhesion to ICAM-1-expressing fibroblasts was strong, and the critical separation stresses measured for the three cell lines were 1.4 x 10(3) dyn/cm2 (1 dyn=10(-5) N) for the ICAM-1-negative fibroblast, 4.98 x 10(3) dyn/cm2 for the fibroblast with a moderate level of ICAM-1 expression, and 6.25 x 10(3) dyn/cm2 for the fibroblast line with the highest ICAM-1 expression. The dependence of adhesion strength on the LFA-1/ICAM-1 complex was confirmed by the use of blocking antibodies, which showed the contribution from the interaction of CD4/MHC class II to be negligible.


Intranuclear co-location of newly replicated DNA and PCNA by simultaneous immunofluorescent labelling and confocal microscopy in MCF-7 cells.

The intranuclear distribution of newly replicated DNA and of the proliferating cell nuclear antigen (PCNA) was mapped by confocal laser scanning microscopy after simultaneous immunofluorescent labelling of incorporated bromodeoxyuridine (BrdUrd) and PCNA. A mild hydrolysis with HCl followed by an enzymic digestion of DNA was used to produce single-stranded DNA required for BrdUrd immunorevelation, since this procedure preserves PCNA antigenicity. Optical sections obtained with a laser scanning microscope clearly showed a similar distribution of PCNA and BrdUrd within the nuclei, thus confirming previous observations on parallel labelled synchronized cultures. The intranuclear distribution of PCNA and BrdUrd varies concomitantly during the S phase of MCF-7 cells.


["In-dartos" orchidopexy in the treatment of cryptorchism].

Maldescended testis is a common congenital abnormality. The main complications are infertility and venue of malignant testicular tumor. If early orchiopexy can prevent infertility, it remains a high risk of venue of a malignant tumor. So, the best technique of orchiopexy is that which allows and easy clinical follow-up of the maldescended testis after surgical treatment, and doesn't involve hematoseminal barrear. From the analysis of fourty cases of scrotal pouch orchiopexy and a review of the literature, it appears that this procedure is efficient and gives more than 90% of good results.


Sensitive determination of phenothiazines in body fluids by gas chromatography with surface ionization detection.

Fourteen phenothiazine derivatives were tested for their detection by gas chromatography (GC) with surface ionization detection (SID). The sensitivity of GC-SID was highest with trimeprazine and levomepromazine, which contain aliphatic tertiary amino side chains, and lowest with thiethylperazine and thioproperazine, which contain sulphur residues. Chlorpromazine, trimeprazine and promazine showed excellent linearity between the SID response and the drug amount in the range 0.25-3.0 pmol on-column. Their detection limits were as low as ca. 5-10 pg (15-30 fmol) on-column (250-500 pg per ml of body fluid). A detailed procedure for isolation of phenothiazines from human whole blood and urine using Sep-Pak C18 cartridges, before the GC with SID, is also presented. The recoveries of the drugs (100 pmol), which were added to 1 ml of whole blood or urine, were more than 79%. The baselines remained steady as the column temperature was increased.


Selective system of identification and determination of antidepressants and neuroleptics in serum or plasma by solid-phase extraction followed by high-performance liquid chromatography with photodiode-array detection in analytical toxicology.

A selective off-line solid-phase isolation of antidepressants, neuroleptics and other structurally related basic drugs from plasma or serum prior to high-performance liquid chromatography was tested and optimized for general use in toxicological analyses where concomitant drugs can be encountered. The sequential elution preseparated drug mixtures and simplified the subsequent analytical steps. High isolation efficiencies of cyano-bonded silica cartridges from Baker for fifteen amine drugs were determined. Isocratic chromatography on octadecylsilica proved to be very suitable for broad practical applications in complicated cases. The identification of an unknown peak was supported by photodiode-array detection in the range 200-400 nm with a resolution of 2 nm. The linearity of the assay from therapeutic to toxic concentrations was attained. Sufficient sensitivities covering low therapeutic levels of parent drugs and their demethylated metabolites were reached. The system is flexible and allows various methods of quantitative assay to be devised according to the conditions of a particular case in clinical or forensic toxicology.


Antibodies to Puumala virus in humans determined by neutralization test.

An assay for detection of neutralizing antibodies to Puumala virus using 96-well microtiter plates (NT-ELISA) was developed and evaluated. The test proved to have similar sensitivity and specificity as an IgG ELISA and indirect immunofluorescence test, when screening 187 sera (with an antibody prevalence rate of 19%) from normal populations in an endemic area of Nephropathia epidemica (NE) in Sweden. NE-patients monitored for 2 years had neutralizing antibodies in early sera collected 1-4 days after the onset of disease with a continuous increase in neutralizing antibodies with time. Furthermore, high titers of neutralizing antibodies were detected 10-20 years post-infection. This neutralization assay was also evaluated as a screening method in the production of monoclonal antibodies. The format of the NT-ELISA makes it feasible to screen a large number of specimens with results similar to the standard plaque or focus-reduction neutralization tests.


Clonal origins of adrenocorticotropin-secreting pituitary tissue in Cushing's disease.

It is unclear whether Cushing's disease results from a primary pituitary disorder or arises in response to abnormal hypothalamic control of the pituitary gland. Clonal analysis can provide information as to whether neoplastic tissue is derived from a monoclonal proliferation of a genetically altered cell or from a polyclonal expansion of a group of cells affected by a common stimulus. We used X-linked restriction fragment length polymorphisms at the phosphoglycerate kinase, hypoxanthine phosphoribosyltransferase, and DXS255 loci in 11 women with biochemically and pathologically confirmed Cushing's disease to determine the clonal origins of corticotroph adenomas and corticotroph hyperplasia. Tumor tissue from all 10 women with morphologically and immunohistochemically confirmed ACTH-secreting pituitary microadenomas demonstrated a monoclonal pattern. Pathologically confirmed corticotroph hyperplasia in a patient with a CRH-secreting bronchial carcinoid was found to be polyclonal. We conclude that corticotroph microadenomas in Cushing's disease are monoclonal, supporting the theory that a spontaneous somatic mutation is the primary pathogenetic mechanism in this disorder. In addition, the demonstration of polyclonality in corticotroph hyperplasia implies that excess of hypothalamic hormones is an etiologic mechanism in cases of Cushing's syndrome associated with ectopic CRH-secreting tumors.


Chronic somatostatin analog administration in patients with alpha-subunit-secreting pituitary tumors.

Glycoprotein hormone-producing (GPH) pituitary adenomas represent approximately 25% of all pituitary tumors. Elevated serum levels of intact GPHs or their free alpha- and beta-subunits have been demonstrated in patients with such tumors, and isolated hypersecretion of alpha-subunit has been reported to occur in 7% of patients. Somatostatin has been shown to decrease GPH subunit levels in cultured adenoma cells in vitro, and somatostatin receptors have been identified on the cell membranes of these tumors. We, therefore, investigated the effect of chronic somatostatin analog administration on hormone production and tumor size in six patients with GPH-producing macroadenomas and elevated serum alpha-subunit levels. Patients initially received native somatostatin as an iv 250-micrograms bolus at 0800 h, followed by a constant infusion of 2 mg over 4 h, and serum alpha-subunit concentrations were measured at 30-min intervals after baseline sampling for a total of 9 h. Patients then received a somatostatin analog, octreotide (100 micrograms, twice daily, sc) for 8 weeks. Serum alpha-subunit levels were determined weekly at 30-min intervals before and for 4 h after the 0800 h octreotide dose. Pituitary magnetic resonance imaging scans and visual field testing were assessed before and after the study. During the 4-h somatostatin infusion, four patients had a significant decrease in alpha-subunit levels (P < 0.05). During the 8-week chronic octreotide administration period, two patients had significant decreases in alpha-subunit levels of 34.6% and 26.7% (P = 0.03 and 0.01, respectively). One of these two patients had a small reduction in tumor size. Two patients whose serum alpha-subunit level did not significantly change while receiving octreotide had a reduction in tumor size or definite improvement in visual field abnormalities. Three patients received a maximum octreotide dose of 250 micrograms, three times daily. In one patient, there was a significant decrease in alpha-subunit levels by 45% (P = 0.0001) in association with a marked improvement in visual field abnormalities. In another such patient, continued administration of octreotide to a maximum dose of 250 micrograms, three times daily, was associated with a marked reduction in tumor size. Of the four patients who demonstrated significant decreases in alpha-subunit concentrations during the initial somatostatin infusion, three patients had a significant reduction in alpha-subunit levels while receiving octreotide. One patient who did not have a decrease in alpha-subunit levels during the somatostatin infusion demonstrated a small decrease in tumor size during higher dose octreotide treatment.(ABSTRACT TRUNCATED AT 400 WORDS)


Amplification of unknown DNA sequences by sequence-independent nested polymerase chain reaction using a standardized adaptor without specific primers.

A new procedure for sequence-independent PCR amplification of DNA fragments is described. DNA from pUC18 plasmid was used as a test DNA. It was digested with a frequently cutting restriction enzyme (Sau3A), generating sticky ends. The DNA was ligated to a synthetic, non-phosphorylated adaptor and subsequently amplified in a nested PCR using two oligonucleotides with sequences derived from the adaptor. As little as 1 fg of pUC18 DNA could be detected by this procedure. The product was analyzed on a gel and hybridized with a pUC18-specific probe. The sequence-independent nested PCR was repeated with different amounts of pUC18 DNA in the presence of an excess of non-specific DNA. In these experiments, pUC18 DNA fragments were amplified in a concentration-dependent manner. After hybridization with a digoxigenin dUTP-labelled pUC18 DNA probe, 1 fg of pUC18 DNA could still be detected. This method allows rapid screening of blood for low titred and mutated viruses in which primer binding sites are not conserved.


Analysis by the polymerase chain reaction of histocompatibility leucocyte antigen-DR9-linked susceptibility to insulin-dependent diabetes mellitus.

DNA sequence analysis of class II HLA from Caucasian and black patients with type 1 (insulin-dependent) diabetes mellitus has suggested that aspartic acid at position 57 (Asp 57) of the DQ beta chain provides protection against insulin-dependent diabetes mellitus (IDDM). In contrast, most Japanese patients with IDDM have Asp 57-positive alleles. To determine the reason for the differences and to localize the HLA-linked diabetogenic gene in Japanese, we studied the DQA1 and DQB1 genes of Japanese patients with IDDM and control subjects by the polymerase chain reaction in combination with restriction fragment length polymorphism analysis. Associations of DQA1*0301 and DQB1*0303 with IDDM were observed. DQA1*01 was associated negatively with IDDM. The HLA-DR9 haplotype, which is associated positively with IDDM in Japanese, was associated with DQA1*0301 and DQB1*0303, indicating that the Japanese DR9 haplotype is the same as that in caucasians but different from that in blacks. Of the loci on Japanese DR9 haplotypes, the DQA1*0301 allele showed the highest association with IDDM. DQB1*0303 was also positively associated with IDDM. Since DQB1*0303 is identical to DQB1*0302 except that it contains Asp 57, the data suggests that an Asp 57-positive allele confers susceptibility to IDDM when the whole molecule of the DQ beta chain is similar to other susceptible DQ beta chains. DQA1*0301 appears to be a marker of IDDM in all these populations: Japanese, caucasian, and black.


In vivo models of human lymphopoiesis and autoimmunity in severe combined immune deficient mice.

The discovery of the SCID mouse mutation has been an important advance for the study of human lymphopoiesis and autoimmunity. Further work in the SCID mouse models described in this review should yield important new information related to transplantation of human hematopoietic stem cells across HLA barriers, characterization of hematopoietic development in vivo, and identification of pathogenic human T cell clones in organ-specific autoimmune diseases. If pluripotent hematopoietic stem cells and pathogenic autoimmune T cells can be defined using SCID mouse recipients, this would pave the way for development of novel strategies for bone marrow transplantation and for interventional immunotherapy of autoimmune diseases targeted at the T cell receptor (99).


Secretory immunoglobulins in serum from human immunodeficiency virus (HIV)-infected patients.

Infection by the human immunodeficiency virus is associated with polyclonal B cell activation and increased levels of serum IgA. In order to characterize the molecular species of serum IgA, we have measured total IgA, IgA1, and IgA2 in sera from 60 HIV-1-infected patients and 40 healthy controls. In addition, secretory IgA (S-IgA), secretory IgM (S-IgM), free immunoreactive secretory component (SC), and the distribution of monomeric and polymeric IgA were determined. The data confirm the elevation of total serum IgA levels in HIV-1-infected patients, and both IgA1 and IgA2 concentrations are elevated. Furthermore, the data show a substantial increase in serum levels of both monomeric and polymeric IgA. Serum S-IgA levels were significantly increased in CDC group II patients versus controls and more frequently elevated in CDC group IV patients. The highest S-IgA levels were found among patients with the lowest blood CD4+ cell counts. Serum S-IgA levels were not correlated with serum levels of either total IgA or polymeric IgA. Serum S-IgM levels were also increased in HIV-1-infected patients and positively correlated with serum S-IgA levels. Conversely, serum levels of free SC were not altered. An increase in serum S-IgA was not related to human hepatitis B virus infection and/or to hepatic dysfunction or to diarrhea or overt intestinal infection. The data indicate that secretory Ig (S-IgM and S-IgA), which are likely to be produced at mucosal sites, increase in the serum of HIV-1-infected patients.


Lymphocyte migration into the CNS modelled in vitro.

We report on a series of experiments which examines the factors controlling lymphocyte adhesion to brain endothelium in vitro and the factors which control cell migration across the endothelium, using a new migration assay. Although lymphocyte adhesion preceded migration across the brain endothelium, the two processes are not identical. We noted that activated CD4+ T cells were particularly good at migrating across endothelia. CD8+ T cells and B cells did not migrate but adhered well to endothelia. Moreover, the endothelium maintained high levels of cell traffic without being disrupted and without exhausting the molecular systems which allowed migration. From the viewpoint of migration of dividing cells, the state of lymphocyte activation appeared to be the most important controlling factor--these cells migrated equally well across endothelium activated with cytokines or untreated endothelium. The kinetics of adhesion suggested that the LFA-1/ICAM-1 and VLA-4/VCAM combinations of adhesion molecules were important in controlling migration. With antibody blocking studies, the role of the LFA-1/ICAM-1 system was equivocal. While anti-LFA-1 blocked lymphocyte adhesion, anti-ICAM-1 did not, suggesting that the level of ICAM-1 was not critical.


Developmental effects of basic fibroblast growth factor and platelet-derived growth factor on glial cells in a three-dimensional cell culture system.

In order to study peptide growth factor action in a three-dimensional cellular environment, aggregating cell cultures prepared from 15-day fetal rat telencephalon were grown in a chemically defined medium and treated during an early developmental stage with either bovine fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF homodimers AA and BB). A single dose (5-50 ng/ml) of either growth factor given to the cultures on day 3 greatly enhanced the developmental increase of the two glia-specific enzyme activities, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and glutamine synthetase (GS), whereas it had relatively little effect on total protein and DNA content. Distinct patterns of dose-dependency were found for CNP and GS stimulation. At low concentrations of bFGF (0.5-5 ng/ml) and at all PDGF concentrations applied, the oligodendroglial marker enzyme CNP was the most affected. A relatively small but significant mitogenic effect was observed after treatment with PDGF, particularly at higher concentrations or after repetitive stimulation. The two PDGF homodimers AA and BB were similar in their biological effects and potency. The present results show that under histotypic conditions both growth factors, bFGF and PDGF, promote the maturation rather than the proliferation of immature oligodendrocytes and astrocytes.


Recruitment of CD11b/CD18 to the neutrophil surface and adherence-dependent cell locomotion.

Chemotactic stimulation of neutrophils results in translocation of CD11b/CD18 (Mac-1) from intracellular storage pools to the cell surface. Though results from several laboratories indicate that the newly arrived surface Mac-1 is not involved in the adherence induced by the initial stimulus, the present study addresses the hypothesis that this Mac-1 plays a role in subsequent adherence-dependent functions. The response of human neutrophils to changing concentrations of a chemotactic stimulus was evaluated by determining the amount of newly arrived surface Mac-1, and Mac-1-dependent adhesion and locomotion. Small step-wise increases in the concentration of f-Met-Leu-Phe (FMLP) resulted in proportional stepwise increases in surface Mac-1 that plateaued within 2-4 min. This newly arrived Mac-1 supported adhesion to protein-coated surfaces only when the cells were exposed to an additional increase in the FMLP stimulus level. Adherence-dependent cellular locomotion was evaluated in chambers that allowed rapid changes in the stimulus concentration. Repeated small increments in the stimulus level at 200-s intervals resulted in significantly longer migration paths than a single-step increase in the stimulus. The results support the hypothesis that small increments in the chemotactic stimulus bring Mac-1 to the cell surface, and this newly mobilized Mac-1 is available for adherence-dependent locomotion with subsequent increases in the concentration of the stimulus.


Pharmacological tolerance to alpha 1-adrenergic receptor antagonism mediated by terazosin in humans.

Chronic administration of alpha 1-receptor antagonists is associated with loss of clinical efficacy, especially in congestive heart failure, although the mechanism is uncertain. To evaluate changes in venous alpha 1-adrenoceptor responsiveness during chronic alpha 1-adrenoceptor blockade, dose-response curves to phenylephrine and angiotensin II were constructed in 10 healthy subjects before, during, and after administration of terazosin 1 mg orally for 28 d. Terazosin initially shifted the dose-response curve of phenylephrine to the right, with a significant increase in ED50 for phenylephrine from a control value of 102 to 759 ng/min on day 1 of terazosin (P < 0.001). However, by day 28, the dose-response curve had shifted back towards baseline with an ED50 of 112 ng/min. After discontinuing terazosin, the ED50 for phenylephrine remained near the baseline value, indicating no evidence of supersensitivity to phenylephrine. There was no change in responsiveness to angiotensin II during the course of treatment with terazosin. Plasma terazosin concentrations were stable throughout the period of drug administration. The mean Kd of terazosin was estimated as 11 +/- 15 nM in the first few days of treatment. This study demonstrates that pharmacological tolerance to the alpha 1-adrenoceptor blocking action of terazosin occurs in man and may be responsible for loss in efficacy with chronic therapy.


Treatment of posttraumatic stress disorder with eye movement desensitization.

This case report describes the successful treatment of a posttraumatic stress disorder (PTSD) using eye movement desensitization (EMD). The client, a 40-year-old male, presented with an 8-year history of PTSD following an incident in which he was shot with a hand gun and left dying. Using EMD treatment, this trauma was quickly desensitized. Two earlier traumas with similar themes then emerged and they too were desensitized. Test results, taken pre-treatment and posttreatment, along with the client's verbatim account of cognitive and behavioral changes 8 months later, converged to document the successful treatment outcome.


Effects of transforming growth factor-beta on murine astrocyte glutamine synthetase activity. Implications in neuronal injury.

Cytokines have been implicated in the pathogenesis of a number of brain diseases in which neurological dysfunction has been attributed to a change in amino acid neurotransmitter metabolism. In the present in vitro study, we investigated the effects of cytokines on astrocyte glutamine synthetase (GS) activity and subsequently on N-methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity. Proinflammatory cytokines IL-1 alpha, IL-1 beta, and IL-6 at a concentration of 20 ng/ml did not affect GS activity; however, tumor necrosis factor-alpha inhibited this activity by 20% in mixed neuronal/astrocyte cultures. Treatment for 24 h with transforming growth factor (TGF)-beta 1 or -beta 2 inhibited up to 60% GS activity. TGF-beta 2 also inhibited GS in enriched astrocyte cultures with an ED50 of 10 pg/ml. Antibodies specific to TGF-beta 2 blocked this effect. Treatment of astrocytes with TGF-beta 2 (250 pg/ml) resulted in markedly dilated rough endoplasmic reticulum. Since astrocyte GS may play a protective role in NMDA receptor-mediated neurotoxicity, we treated mixed neuronal/astrocyte cultures with TGF-beta 2 (250 pg/ml) and found a threefold potentiation of NMDA receptor-mediated neurotoxicity. These data suggest that TGF-beta impairs astrocyte GS function and enhances neurotoxicity, thus providing insight into understanding one mechanism of cytokine-mediated central nervous system disease.


The site of gastrointestinal absorption of gepirone in humans.

This study was conducted in seven healthy male subjects and was performed over four sessions with a 1-week washout between sessions. It was designed to compare the bioavailability of an oral 20-mg gepirone dose (treatment 1) with that obtained after application of the same dose by gastric intubation to the distal (treatment 2) and proximal (treatment 3) regions of the small intestine, and after 4 consecutive 5-mg gepirone doses given orally at hourly intervals (treatment 4). Serial blood samples were taken over 24 hours after dose after each treatment. Plasma concentrations of gepirone and 1-(2-pyrimidinyl)-piperazine (1-PP), a metabolite of gepirone, were quantitated by gas chromatography-mass spectrometry. Mean gepirone time to reach peak concentration (tmax) after treatments 1, 2, and 3 ranged between 0.57 and 1.07 hours. There were no significant differences between sites and treatments for gepirone t1/2, which ranged between 2.8 and 3.3 hours. The mean gepirone maximum peak plasma concentration (Cmax) was significantly higher (P less than .05) after treatment 2 (12.92 +/- 7.24 ng/mL) compared with treatment 1 (6.79 +/- 3.54 ng/mL) or treatment 3 (6.33 +/- 2.26 ng/mL). Gepirone area under the curve (AUCinf) was also significantly higher (P less than .05) after treatment 2 (29.83 +/- 17.42 ng.h/mL) compared with treatment 1 (18.07 +/- 6.10 ng.h/mL) or treatment 3 (17.74 +/- 7.69 ng.h/mL). There were no significant differences in gepirone AUCinf between treatments 1 and 4.(ABSTRACT TRUNCATED AT 250 WORDS)


Double-blind placebo-controlled trial of terazosin effect on blood pressure and urinary output of dopamine in hypertensive patients.

In a parallel, double-blind study, 12 untreated hypertensive patients received terazosin (2-4 mg/day for 4 weeks), and 12 received placebo during the same period. Systolic and diastolic blood pressure decreased significantly in the terazosin group, from 150 +/- 5.0 mmHg systolic and 99.6 +/- 2.0 diastolic before treatment, to 134.0 +/- 7.0 systolic and 85.6 +/- 3.0 mmHg diastolic at week 4 of treatment. No significant blood pressure changes occurred in the placebo group. Blood pressure decrease showed a positive correlation (r = .62 and r = .52 for systolic and diastolic blood pressure, respectively) with the patient's age (P less than .05). Total plasma cholesterol decreased 18% in the terazosin group (P less than .05) and 9% in the placebo group (P greater than .05). Urinary dopamine excretion decreased significantly from 692.8 +/- 180.0 to 330.5 +/- 52.0 micrograms/24 hours in the terazosin group (P less than .05) and showed a nonsignificant increase in the placebo group. Compared with 22 age- and sex-matched healthy volunteers, urinary dopamine excretion in the hypertensive group before treatment was not statistically different (779.3 +/- 83.1 micrograms/24 hours). Dopamine excretion was higher in untreated hypertensive men and in male healthy volunteers compared with women. The decrease of urinary dopamine excretion observed under terazosin treatment could be due to a decrease of kidney dopamine synthesis or release induced by blood pressure reduction, or secondarily to the blockade of kidney alpha 1-receptors, modulating dopamine excretion. No significant changes were observed in urinary excretion of noradrenaline and adrenaline.


Hypothyroidism complicated by angina pectoris: therapeutic approaches.

The association of hypothyroidism and coronary artery disease is not uncommon. The precipitation of angina pectoris, cardiac arrhythmia, and even myocardial infarction may occur in patients when initiating rapid replacement therapy for hypothyroidism. This is particularly true when replacement therapy is instituted in elderly persons or in patients with preexisting coronary artery disease. A starting daily dose of 12.5 to 25 micrograms and increments of 25 micrograms every 2 to 3 weeks is recommended. Close monitoring of cardiac symptoms is essential to avoid side effects. Medical management of angina pectoris includes administration of beta-blockers, nitrates, or at times combination antianginal therapy may be most effective. Persistence of angina in these patients may require coronary angiography with subsequent angioplasty or coronary artery bypass surgery.


Long distance directed axonal growth from human dopaminergic mesencephalic neuroblasts implanted along the nigrostriatal pathway in 6-hydroxydopamine lesioned adult rats.

Dissociated ventral mesencephalon of 6 to 8-week-old human embryos were implanted by stereotaxic injection at different sites along the nigrostriatal pathway in adult rats, previously subjected to a 6-hydroxydopamine lesion of the intrinsic mesotelencephalic dopamine pathways. The recipients were immunosuppressed by daily injections of cyclosporin A to prevent rejection. At 13-20 weeks after transplantation, the implanted human neurons and their associated fiber outgrowths were visualized with a species-specific antibody recognizing human, but not rat, intermediary neurofilaments (HNF). From implants placed in the host rostral mesencephalic region, HNF-positive axonal projections were seen to extend in large numbers rostrally along the medial forebrain bundle and the internal capsule, and ramify within the caudate putamen, the ventral striatum and the amygdaloid nuclei (a distance of about 5-6 mm), and more sparsely in the frontal cortex and the olfactory bulb (a distance of about 10 mm). From implants placed in the internal capsule, abundant HNF-positive axons extended in the rostral, but not caudal, direction along the myelinated fiber bundles into the caudate putamen and the ventral striatum. Tyrosine hydroxylase (TH) immunohistochemistry revealed that the vast majority of the rostrally projecting HNF-positive axons were also TH-positive, and that the graft-derived axons gave rise to dense TH-positive terminal networks, above all in large areas of the previously denervated caudate putamen. From control implants of cortical neuroblasts, axonal projections were seen along the medial forebrain bundle and the internal capsule, but the axons were TH-negative and showed only sparse projections to the striatal areas. Instead, dense projections were seen, e.g., in the frontal cortex. The results demonstrate a remarkable ability of human mesencephalic neuroblasts to extend axons along the trajectories of the nigrostriatal and mesolimbocortical pathways to reach and innervate the principal striatal and limbic target areas in the forebrain. This shows that the basic requirements for the formation of long axonal pathways may be present in the adult mammalian central nervous system (CNS) at least for certain types of projection neurons. Furthermore, it implies that the developing human neuroblasts can escape the inhibitory features known to be present along myelinated growth trajectories in the adult mammalian brain. In addition, the present approach may offer new possibilities for functional neural grafting in the rat Parkinson model, since transplanted nigral neurons placed in their natural position within the rostral mesencephalon could provide an anatomically and functionally more integrated system than the standard model with ectopically placed intrastriatal nigral grafts.


Pemphigus vulgaris in siblings: HLA-DR4 and HLA-DQw3 and susceptibility to pemphigus.

The association of pemphigus vulgaris with the HLA serotypes, DR4 and DRw6, and with the DQ-beta chain alleles, DQw1 and DQw3, suggests that there is a genetic predisposition to this disease. However, familial cases of pemphigus vulgaris are exceedingly rare. We studied two siblings with pemphigus vulgaris and three unaffected family members to determine whether an HLA allele was associated with the development of pemphigus vulgaris in this family. We utilized restriction fragment length polymorphism methods using the HLA-DR beta 1, HLA-DQ alpha, and HLA-DW beta 1 genes as cDNA probes. We found that the affected siblings share the haplotype HLA-DR4 and the DQw.3.2 allele. Our findings of gene sharing among siblings with pemphigus vulgaris lend support to previous studies of unrelated Caucasian patients, which implicated DQw3 allele polymorphisms in conferring susceptibility to pemphigus.


Response of aged versus young skin to intradermal administration of interferon gamma.

Interferon gamma (IFN-gamma) induces the interaction of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen type 1 expression, and of HLA-DR antigens by keratinocytes. The aim of the present study was to determine the potential ability of aged versus young skin to respond to intradermal administration of IFN-gamma, as an in vivo immunologic stimulus. For 3 consecutive days elderly and young volunteers were injected with 10 micrograms of recombinant IFN-gamma diluted in 0.1 ml of sterile water. On day 5, punch biopsy specimens were obtained from the injected sites. Histologic and immunohistochemical stainings were performed on all sections. ICAM-1 was expressed by keratinocytes in both aged and young skin. An impairment was manifested mainly by the reduced accumulation of mononuclear cells throughout the dermis, the absence of HLA-DR expression by keratinocytes in 7 of 10 elderly volunteers, and the absence of an effect on the Langerhans cell population. This observation shows a diminished immune response in aged skin.


Minocycline hyperpigmentation: model for in situ phagocytic activity of factor XIIIa positive dermal dendrocytes.

Pigmentary disorders resulting from the prolonged use of the semisynthetic tetracycline derivative antibiotic, minocycline, are rare but well recognized sequelae of ingestion of this drug. We present two cases of women with bullous pemphigoid who developed blue-black pigmentation in areas of scarring on the legs after oral minocycline therapy. On histologic examination, the deposited pigment was localized within dendritic cells limited to the upper dermis. Immunohistochemical analysis revealed these cells to be factor XIIIa positive dermal dendrocytes which was confirmed by transmission electron microscopy. We believe these cases demonstrate, in-situ, the phagocytic capabilities of this recently described cell population.


Repeated administration of antidepressant drugs reduces regional somatostatin concentrations in rat brain.

A possible role for somatostatin in affective disorders is suggested by its low concentration in cerebrospinal fluid of patients with depression. Therefore, we studied the regional effects of antidepressant drugs and antimanic agents on somatostatin concentrations in rat brain. Repeated, but not acute, administration of clomipramine, a specific serotonin uptake inhibitor, caused a highly significant, widespread reduction in somatostatin levels. Somatostatin content was similarly reduced in the hypothalamus, and midbrain and thalamus following repeated administration of zimelidine, another specific serotonin uptake inhibitor. Repeated administration of either imipramine, maprotiline, mianserin, carbamazepine or zotepine were without effect on somatostatin levels. These results suggest that somatostatin in the brain might be involved in therapeutic effects of some of antidepressant drugs.


Bronchodilator and bronchoprotective effects of salmeterol in young patients with asthma.

In adults with asthma, the selective beta 2-adrenergic agonist salmeterol has a prolonged bronchodilator and bronchoprotective effect. To date, there are few published studies of salmeterol in children. We compared the bronchodilator and bronchoprotective effects of salmeterol, 25 and 50 micrograms, with salbutamol, 200 micrograms, and with placebo, administered via metered-dose inhaler, in a randomized, double-blind, within-patient, four-way crossover, single-dose study in 20 children. Mean baseline forced expiratory volume in 1 second (FEV1) and PC20 methacholine were not significantly different (p > 0.05) on the 4 study days, and did not change significantly after placebo. FEV1 increased significantly from 5 to 30 minutes after salbutamol, and from 5 minutes to 12 hours after 25 micrograms or 50 micrograms salmeterol, compared with placebo. After 25 micrograms or 50 micrograms salmeterol, FEV1 was significantly lower than after salbutamol at 5 and 10 minutes, did not differ from salbutamol at 30 minutes, and was significantly greater than after salbutamol from 3 to 12 hours. No significant difference occurred between the effect of 25 micrograms salmeterol and the effect of 50 micrograms salmeterol on FEV1. After salbutamol, there was a significant increase in PC20 only at 30 minutes. After 25 micrograms or 50 micrograms salmeterol, PC20 increased significantly from 30 minutes to 12 hours. Salmeterol, 25 micrograms and 50 micrograms provided significantly greater bronchoprotection than salbutamol from 3 to 12 hours and from 30 minutes to 12 hours, respectively. Salmeterol, 50 micrograms, provided significantly better bronchoprotection than 25 micrograms salmeterol from 30 minutes to 12 hours. The amount of change in PC20 accounted for by change in FEV1 varied from 14% to 28%, indicating that protection against bronchoconstriction was not entirely dependent on bronchodilation. Salmeterol is a potent, long-acting bronchodilator, with a slower onset of bronchodilation than salbutamol. It provides significantly greater and longer-lasting protection against bronchoconstriction than salbutamol.


Spinal cord blood flow decreases following chemical stimulation of the rostral ventrolateral medullary pressor area in anesthetized rats.

In urethane-anesthetized, paralyzed and artificially ventilated rats, the neurons in the rostral ventrolateral medullary pressore area (VLPA) were chemically stimulated by microinjections of L-glutamate (1.7-5.0 nmol in 100 nl of 0.9% sodium chloride solution) and the spinal cord blood flow (SCBF) was determined using a combination of labeled microspheres (57Co, 113Sn and 46Sc). In order to measure SCBF at normotension, moderate hypotension within the lower limit of spinal cord autoregulation was induced by controlled hemorrhage (n = 12). Unilateral chemical stimulation of the VLPA in these rats increased arterial blood pressure (ABP) but it remained within normotensive range. The SCBFs of cervical, thoracic and lumbar cord decreased significantly from 27 +/- 3 (mean +/- S.E.M.) to 20 +/- 2 (P less than 0.01), from 22 +/- 1 to 17 +/- 2 (P less than 0.05), and from 41 +/- 5 to 26 +/- 3 (P less than 0.05) ml.min-1.(100 g)-1, respectively. The spinal cord vascular resistances (SCVRs) of cervical, thoracic and lumbar cord increased significantly from 3.7 +/- 0.4 to 5.0 +/- 0.6 (P less than 0.05), from 4.2 +/- 0.2 to 5.9 +/- 0.7 (P less than 0.05), and from 2.5 +/- 0.2 to 3.8 +/- 0.4 (P less than 0.05) mmHg per [ml.min-1.(100 g)-1], respectively. During the chemical stimulation of the VLPA, SCBF increased in response to the changes in arterial PaCO2 indicating that the reactivity of spinal cord vasculature was intact (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of nucleus tractus solitarius lesions on cardiovascular responses elicited from the caudal ventrolateral medulla.

Neurons in the caudal ventrolateral medulla (CVLM) which inhibit sympathetic vasomotor tone may have reciprocal connections with the nucleus tractus solitarius (nTS). This study determined whether changes in arterial pressure elicited by chemical excitation or inhibition of neurons in the vasodepressor region of the rabbit CVLM depend on the integrity of the nTS. Unilateral injections of L-glutamate (10 pmol to 100 nmol), or bilateral injections of GABA (1 nmol to 125 nmol), were made into the CVLM, and dose-response effects on arterial pressure determined. The nTS was then bilaterally cauterized, or inhibited by local injections of muscimol, and the dose-response curves were repeated. Neither cauterization nor injection of muscimol significantly altered the slope of the log dose-response curves for L-glutamate, but nTS muscimol increased the fall in arterial pressure for each dose of L-glutamate (P less than 0.01). Cauterization of the nTS significantly (P less than 0.01) increased the slope of the curve relating dose of GABA to rise in arterial pressure observed, after injection of GABA into the CVLM. This increase in slope was similar to the increase observed when GABA is injected into the CVLM in baroreceptor-denervated rabbits. We conclude that neither the depressor nor the pressor response evoked by stimulation or inhibition of the CVLM is dependent on the integrity of the nTS. Inactivation of the nTS tends to increase the magnitude of the CVLM responses, possibly by removal of baroreceptor-mediated buffering of the responses.


Generalized thyroid hormone resistance: identification of an arginine to cystine mutation in codon 315 of the c-erb A beta thyroid hormone receptor.

The present report studies a large kindred (WR) with generalized thyroid hormone resistance that has varying degrees of neuropsychological dysfunction, hyperactivity, poor attention span, decreased IQ and/or abnormalities in spatial perception. In this kindred, there has been found tight linkage of the syndrome with the c-erb A beta gene. The present study was performed to identify the presence of a possible gene mutation as a cause for this syndrome. DNA from peripheral leukocytes was isolated from 15 unaffected and 8 affected individuals from the kindred. Primers encompassing exons 9 (nucleotides 1171-1429) and 10 (nucleotides 1430-1698) were synthesized and used in PCR reactions to amplify these exons. Direct sequencing revealed a consistent substitution in each affected subject, but in none of the unaffected individuals, of a C to T change in one allele from nucleotide 1243, resulting in an arg to cys change in codon 315. The mutant and wild-type human beta 1 receptors were prepared and their translated proteins were analyzed for T3 binding. The WR T3 receptor from affected patients had reduced T3 binding affinity, with values approximately 2.5 x 10(10) M-1 compared to about 5 x 10(10) M-1 in normals. In summary, we have: i) identified a consistent and reproducible mutation of a C to T change in nucleotide 1243 in each of the affected but in none of the unaffected individuals of a large well characterized kindred with generalized thyroid hormone resistance; and ii) noted that the WR allele causes an approximate 50% decrease in the T3 binding affinity.(ABSTRACT TRUNCATED AT 250 WORDS)


Longitudinal study of psychotropic drug use by elderly nursing home residents.

In this longitudinal study of patterns of use of psychotropic drugs by a cohort of elderly nursing home residents (N = 5,752), drug use was examined upon admission, 3 months later, and at discharge/end of study. At each time point, 17% of the cohort used neuroleptics. Half of the subjects discontinued neuroleptics at each time point; however, a similar number were initiated on the drug. Benzodiazepines were used by 21%, 15%, and 15% at each of the three time points, respectively. Twice as many people were taken off benzodiazepines as initiated on them following admission. The 5% rate of antidepressant use was constant across the three time periods, although only half of those who took antidepressants upon admission were also taking them upon discharge/end of study. The amount of change due to discontinuation of these drugs and adjustment in dosage levels challenges the stereotype of the "neglected psychotropic drug user" in nursing homes.


Application of an optimized immunostaining technique to evaluate the heterogeneous secretory response from porcine somatotropes by cell blotting.

The objective of the present study was to quantify the absolute hormone release from individual porcine pituitary cells incubated on polyvinylidene difluoride (PVDF) transfer membranes (cell-blot assay). After immunoperoxidase staining, growth hormone (GH) release from isolated somatotrope cells appeared like a colored zone of secretion surrounding the cell. Optical densities of these secretion zones were quantitated by computerized image analysis and translated into picograms by means of an appropriate standard curve. As a prior step, the staining method and the optimal immunocytochemical conditions were selected by applying purified porcine growth hormone (pGH) to the transfer membranes. The avidin-biotin-peroxidase nickel-intensified (ABC-Ni) method produced a better resolution than the peroxide-anti-peroxidase (PAP) method, although both techniques were similar with regard to background, sensitivity, and range of quantitation. The amount of GH released from single porcine somatotropes was highly heterogeneous, although the cells were treated under the same conditions. Moreover, this fact was consistent with the stimulation of the average release of GH by GH-releasing factor (GHRF) but not by GHRF+somatostatin (SRIF). Our results confirm the availability of the recently developed cell-blot assay and support the concept of functional heterogeneity in anterior pituitary cell populations.


Antibiotic resistance associated with selective decontamination of the digestive tract.

Selective decontamination of the digestive tract (SDD) appears to reduce infection, particularly pneumonia, in intensive care, and some patients benefit markedly. Gram-positive overgrowth and antibiotic resistance in both Gram-positive and Gram-negative organisms has been recorded. However, the clinical and epidemiological significance of these observations is still debated. Future studies will need to be of sufficient size and duration to provide good quality data on which the safety and efficacy of SDD can be properly judged.


Clinically significant coagulase-negative staphylococci: identification and resistance patterns.

Coagulase-negative staphylococci (CNS) of clinical significance, isolated from 131 patients, were investigated during the period 1989-90 in northern Norway. The staphylococci were isolated from blood cultures (68; 51.9%), vascular catheters (6), osteomyelitis foci (13), postoperative and other wounds (15), and urine samples (29). The use of Gram-positive Identification Card (Vitek) and 'Staph-zym' (Rosco) both gave a primarily correct species identification in 95% of the cases. Staphylococcus epidermidis was the predominant species (72.3%). Methicillin-resistance was found in 40 of 131 (30.5%) of all CNS and in 34 of 96 (35.4%) of S. epidermidis. Methicillin-resistant (MR) S. epidermidis strains were usually resistant to gentamicin, tetracycline, chloramphenicol and trimethoprim. MR strains were, however, less resistant to sulphonamides than methicillin-sensitive strains (10 out of 34 vs. 55 out of 62). Methicillin-resistance implied resistance to all beta-lactam antibiotics, including imipenem. Among S. epidermidis, MR isolates increased from 10% in 1987 to 35.4% in 1989-90. All strains were sensitive to vancomycin and rifampicin.


Plasmid-pattern analysis in the differentiation of Staphylococcus epidermidis isolates.

Plasmids carried by 29 Staphylococcus epidermidis isolates from two different origins (invasive and carrier, non-hospital-associated strains) were analysed by agarose gel electrophoresis in order to evaluate any association with virulence. Plasmid patterns were found to be characteristic of individual strains and no particular molecular size band was found to correlate with either of the two categories of isolates. It is concluded that the plasmid pattern analysis carried out could not differentiate between carrier and invasive S. epidermidis, but did provide a useful tool for epidemiological investigation.


The relative importance of the routes and sources of wound contamination during general surgery. II. Airborne.

The influence of airborne bacteria on wound contamination during biliary surgery was studied. When bacteria grew in the bile they accounted for most of the bacteria in the wound but when the wounds were free of bile bacteria many of the bacteria came from the patient's skin. It was only in wounds with little contamination from non-airborne routes that it was possible to demonstrate an effect of airborne contamination. In such a situation it was estimated that a reduction in the airborne bacteria in the operating room of about 13-fold would reduce the wound contamination by about 50%. The contamination of patient drapes from various sources and its relationship to wound contamination was studied. It was demonstrated that in areas away from the wound, the bacterial concentration on the drape surface was significantly affected only by airborne bacteria. In the area close to the wound, airborne bacteria and bacteria from the wound significantly affected drape contamination. However, it was found that more bacteria transferred from the wound to the drape surface than vice versa. Punctured gloves, impervious gowns and the number of bacteria on the patient's skin did not significantly affect the counts on the drapes' surfaces.


Colonization of burns and the duration of hospital stay of severely burned patients.

In a retrospective study the influence of several factors on the length of hospital stay of severely burned patients (at least 24% total body surface area) has been investigated. The influence of these factors was studied by means of the Cox model survival analysis with time-varying covariates. Seventy-one patients were included in this study. The mean age was 32 years (range 1-82 years), the mean total body surface area burned 40% (range 24-80%) and the mean full-thickness area burned 32% (range 10-70%). The length of hospital stay was positively correlated with the extent of the burned area and with the age of the patient. Wound colonization with Enterobacteriaceae or with a combination of Pseudomonas spp. and Staphylococcus aureus was also associated with a prolonged stay in hospital.


An outbreak of Candida tropicalis peritonitis in patients on intermittent peritoneal dialysis.

An outbreak of Candida tropicalis peritonitis in intermittent peritoneal dialysis patients during a 6-week period is reported from a general hospital. Five patients were involved and there were three deaths. The strains recovered from affected patients were identical to those recovered from the water baths on the basis of biotyping, antimicrobial susceptibility pattern and chromosomal DNA fingerprints. No further cases were identified on subsequent surveillance after the prohibition of wet-warming of peritoneal dialysate in the hospital.


A novel electrical method for the prevention of microbial colonization of intravascular cannulae.

An electrical device designed to prevent microbial attachment to plastic intravascular cannulae is described. The device produced an electrical current (10 microA) in both carbon-impregnated and 'Hydrocath' cannulae. In-vitro models were utilized to demonstrate that the current can block several mechanisms by which microorganisms gain initial access to intravascular devices, including extra- and intra-luminal routes. The electrical system prevented organisms from traversing along both the external and internal surfaces of the cannulae. Effective prevention of cannula colonization was demonstrated subsequent to challenge with Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecalis, Proteus mirabilis, Pseudomonas aeruginosa and Candida albicans. The findings suggest that the electrical device may offer in-vivo cannula protection from microbial attachment and colonization, thereby reducing the potential for infection.


Costs of an outbreak of wound infections in an orthopaedic ward.

An outbreak of serious wound infections occurring in an orthopaedic ward is reported. The outbreak involved 10 patients, all of whom had undergone clean, orthopaedic operative procedures. The outbreak was eventually terminated by discarding five damaged and contaminated mattresses. The costs incurred during this outbreak were calculated using a retrospective comparative study. The infected patients had an average increased hospital stay of 17 days and average increased costs of 2220 pounds. The outbreak demonstrated the high costs of hospital-acquired infection and the need for further investment in infection control programmes.


A new approach to the management of Broviac catheter infection.

Infection continues to be a major complication of the use of indwelling venous catheters. In an attempt to avoid removal of the catheter and to minimize the systemic side-effects of antibiotics, the potential value of in-situ treatment of confirmed Broviac catheter infection was assessed in carefully selected patients attending an oncology unit. Fourteen episodes from 11 children were included in the study. A variety of organisms were encountered. Infective episodes were divided into two categories: (a) those occurring in patients with negative peripheral blood cultures and neutrophil count greater than 1.5 x 10(9) l-1 which were treated only by local instillation of heparinized antibiotic 8-hourly for 7-14 days (N = 8); (b) those occurring simultaneously with positive peripheral blood culture (or peripheral blood culture not performed) regardless of neutrophil count, or infection restricted to Broviac catheter but with a neutrophil count of less than 1.5 x 10(9) l-1; these were treated, with one exception, as above with the addition of systemic antibiotics (N = 6). Treatment was successful in 100% of infective episodes with negative cultures achieved between 5 and 12 days. Catheters remained in use a mean of 118 days following treatment of infection. This approach has obvious advantages but requires careful patient selection and monitoring. It prolongs the catheter life, obviates the need for systemic antibiotics for a local infection, and with appropriate instruction to parents and family practitioner, treatment may be administered on an outpatient basis.


Virulence factors in Escherichia coli from urinary tract infections in patients with spinal injuries.

A collection of 70 strains of Escherichia coli from urinary tract infections in spine-injured patients undergoing long-term bladder catheterization were tested for characteristics that have been associated with the ability to produce pyelonephritis. The incidence of the virulence factors were: mannose-resistant haemagglutinins (30%), P-fimbriae (17%), haemolysin (27%), K-antigens (28%) and aerobactin (by bioassay 33%, by gene probe 39%). Only 54% of the strains belonged to the O-serotypes usually associated with urinary tract infections. E. coli carrying the full complement of virulence factors were rare in the urinary tract of the spinal patients and were not associated with episodes of symptomatic pyelonephritis. It is clear that the neuropathic bladder and the presence of the catheter permits a wide variety of bacterial types to colonize the urinary tract and cause infection of the kidney. The identification of host markers rather than bacterial factors is suggested as a more fruitful approach to the early detection of cases likely to progress to pyelonephritis in this group of patients.


Antibiotic resistance of urinary pathogens isolated from patients attending the Toronto Hospital between 1986 and 1990.

A study was carried out on 1523 urinary isolates obtained at The Toronto Hospital, Canada's largest tertiary care establishment, over three 1-month periods in 1986, 1987 and 1990. Escherichia coli was the most frequently isolated organism, with Enterococcus spp. the second most common isolate in 1986 and 1987, and Streptococcus spp. in 1990. Pseudomonas aeruginosa isolates were found to be resistant to many of the antimicrobial agents tested. Resistance patterns were found to commonly prescribed ampicillin, co-trimoxazole and, to some extent, the new fluoroquinolones, ciprofloxacin and norfloxacin. These results are relevant to the treatment and management of urinary tract infections in patients attending a tertiary care hospital.


A study of glutaraldehyde disinfection of fibreoptic bronchoscopes experimentally contaminated with Mycobacterium tuberculosis.

Mycobacteria are difficult to inactivate, and concern about the spread of tuberculosis at bronchoscopy has a major influence on infection control practices. Recommendations from the UK Department of Health are based largely on in-vitro mycobactericidal assays which do not take into account the particular conditions encountered in endoscopy units. In this applied study cleaning and disinfection methods were examined using five bronchoscopes that were heavily contaminated with a recent isolate of Mycobacterium tuberculosis in sputum. Cleaning reduced contamination by a mean 3.5 log(10) colony forming units (cfu) per ml; all bronchoscopes were free of detectable mycobacteria after 10 min in 2% alkaline glutaraldehyde (AG). It is recommended that all bronchoscopes be thoroughly pre-cleaned and disinfected in 2% AG for 20 min as part of a uniform policy of infection control.


An examination of needlestick injury rates, hepatitis B vaccination uptake and instruction on 'sharps' technique among medical students.

A 12-question survey designed to examine venepuncture techniques and instruction and uptake of hepatitis B vaccination was completed by 172 of the 275 medical students to whom it was distributed (a response rate of 62.5%). Seventy-five injuries were reported, at an average of 0.3 per student per year. Of the respondents, 63% resheathed needles after use, a practice frequently cited as a cause of needlestick injury. However, in this sample resheathing was not significantly associated with a higher or lower injury rate (chi 2 = 2.07, P > 0.1). Of the respondents from the most recent intake, only 20 out of 57 had completed a course of hepatitis B vaccinations prior to the commencement of venepuncture duties. There was almost universal ignorance concerning the correct course of action following 'sharps' injury. Recommendations are made concerning hepatitis B vaccination and teaching strategies for medical students.


An evaluation of the Hamo LS-76 washing, drying and disinfecting machine for anaesthetic equipment.

This report evaluates a machine designed to wash, disinfect and dry anaesthetic equipment. Following adjustments to the program these objectives could be reliably achieved within a reasonable time. Multifunction machines should be considered for future use in Sterile Service Departments, although a careful assessment of the workload capacity should be made before purchase.


Biochemical mis-identification of Brucella melitensis and subsequent laboratory-acquired infections.

Brucella species are mis-identified in the API 20NE system as Moraxella phenylpyruvica (profile number 1200004). Since some Brucella spp. grow readily in routine blood culture medium and may be isolated from patients without clinically obvious brucellosis, the risk of laboratory-acquired brucellosis exists. We describe two such cases.


Clostridium difficile infection: responses, relapses and re-infections.

Clostridium difficile infection is a common and potentially lethal complication of antibiotic usage. Since the aetiology of antibiotic-associated colitis was discovered 14 years ago, two antibiotics in particular, metronidazole and vancomycin, have been used to treat C. difficile infection. Studies comparing the efficacy of these antibiotics are reviewed. It is now apparent that many of the so-called 'relapses' of C. difficile infection following antibiotic treatment are, in fact, re-infections. Such findings have major infection control implications.


In-vitro efficacy of a central venous catheter ('Hydrocath') loaded with teicoplanin to prevent bacterial colonization.

A technique is described by which a central venous catheter ('Hydrocath') is loaded with the glycopeptide teicoplanin for the prevention of catheter infection. Catheters are immersed in teicoplanin solution and, due to the hydrophilic surface coating of the 'Hydrocath' catheter, teicoplanin is absorbed by the surface layer. The catheter loading is influenced by the experimental conditions and is assessed by measuring teicoplanin elution from the catheter using a bioassay. Increasing the antibiotic concentration, incubation time and temperature leads to the binding of higher amounts of teicoplanin to the catheter, resulting in a higher teicoplanin release from the catheter. Experiments on in-vitro bacterial adherence to teicoplanin-loaded and unloaded catheters reveal that the initial bacterial adhesion is not prevented. However, in the case of the teicoplanin-loaded catheter initially adherent bacteria are eliminated from the catheter surface, thus preventing catheter colonization by bacteria for at least 48 h. Such loaded catheters could be suitable for inhibiting early-onset, catheter-related infections.