Dramatic changes of sulfated proteoglycans composition in a tumorigenic SV-40-transformed renal proximal-tubule cell line. To characterize the sulfated proteoglycans (PGs) alterations associated with malignant transformation of epithelial cells in vitro, the localization, charge, size, and composition of cell-associated and secreted sulfated PGs have been compared in rabbit renal proximal-tubule cells in primary culture (Ronco et al., 1990) and in a derived SV-40 transformed cell line (RC.SV1) exhibiting a proximal phenotype and high tumor-inducing ability (Vandewalle et al., 1989). Both normal and transformed cells incorporated PGs into a thick basement membrane layer as shown by ruthenium red staining and immunodetection with a monoclonal antibody raised against the core protein of the bovine basement membrane heparan sulfate-PG (HS-PG). In primary cultures of normal cells, cell-associated PGs were almost identical to those extracted from renal tubule fractions in vivo by their size (Kav = 0.27 vs. 0.26 on Sepharose CL-6B) and composition characterized by the exclusive presence of heparan sulfate glycosaminoglycan (HS-GAG) chains. In addition, the cells secreted a HS-PG with similar biochemical characteristics (Kav = 0.29; 100% HS-GAG chains). The SV-40-transformed RC.SV1 cells also synthesized and secreted a unique PG with the same charge and Kav values and apparently the same core protein (35 kDa) as in nontransformed cells, but three major differences were observed: (i) an increased proportion of PG-associated [35S]sulfate radioactivity released into the culture medium (36 vs. 21%), (ii) the emergence of free GAG chains unincorporated into PGs and detected only in the cell-associated fraction, and (iii) a dramatic change in the composition of GAG chains in which chondroitin sulfate replaced heparan-sulfate. The latter finding is in keeping with the known chondroitin sulfate increase and heparan-sulfate decrease in epithelial tumors. The alterations of PGs observed in this study may play a role in the acquisition and/or maintenance of the malignant phenotype.


Activation of the Na+/H+ antiporter during cell volume regulation. Evidence for a phosphorylation-independent mechanism. A variety of cell types regulate their volume in anisotonic media by stimulating Na+/H+ exchange. Like growth factors, osmotic challenge activates the Na+/H+ antiport by increasing its sensitivity to intracellular [H+]. To investigate the molecular mechanism underlying this shift in pH sensitivity, the antiporter of 32P-labeled human bladder carcinoma cells and of Chinese hamster ovary cells was immunoprecipitated using antibodies raised against the cytosolic domain of the NHE-1 isoform of the Na+/H+ exchanger. Unlike the effects of growth promoters, activation of the antiport during volume regulation was not associated with increased phosphorylation. The possible coexistence of multiple antiporter isoforms was considered. The cytosolic alkalosis normally elicited by hypertonic media was found to be absent in Na+/H+ exchange-deficient fibroblasts. Responsiveness to osmotic challenge was restored by stable transfection of these cells with the cDNA encoding NHE-1. In these transfectants, phosphorylation of the antiporter was also unaffected during osmotic activation. The unchanged phosphate content of the antiporter might be explained by dephosphorylation of one site with concomitant phosphorylation at a different site. However, this possibility appears unlikely since phosphoamino acid analysis revealed that serine was the only residue phosphorylated in immunoprecipitated antiports of both control and osmotically stimulated cells. Moreover, phosphopeptide maps of control and hypertonically activated antiports were identical. These findings reveal a novel mode of activation of Na+/H+ exchange not requiring direct phosphorylation of the antiporter. We propose the existence of dual control of Na+/H+ exchange by phosphorylation-dependent and -independent mechanisms.


Inhibition of Acanthamoeba myosin I heavy chain kinase by Ca(2+)-calmodulin. The actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosins I depends on phosphorylation of their single heavy chains by myosin I heavy chain kinase. Kinase activity is enhanced > 50-fold by autophosphorylation at multiple sites. The rate of kinase autophosphorylation is increased approximately 20-fold by acidic phospholipids independent of the presence of Ca2+ and diglycerides. We show in this paper that Ca(2+)-calmodulin inhibits phospholipid-stimulated autophosphorylation of myosin I heavy chain kinase and hence also inhibits the catalytic activity of unphosphorylated kinase in the presence of phospholipid. Ca(2+)-calmodulin does not inhibit kinase activity in the absence of phospholipid. Micromolar Ca(2+)-calmodulin also inhibits binding of myosin I heavy chain kinase to phospholipid vesicles and purified plasma membranes. Proteolytic removal of a 7-kDa NH2-terminal segment from the 97-kDa kinase prevents binding of both calmodulin and phospholipid; therefore, we propose that they bind to the same or overlapping sites. These data provide a mechanism by which Ca2+ could inhibit the actin-activated Mg(2+)-ATPase activity of the myosin I isozymes in vivo and thus regulate myosin I-dependent motile activities.


Characterization of the phosphotransferase domain of casein kinase II by site-directed mutagenesis and expression in Escherichia coli. The catalytic alpha subunit of casein kinase II contains the 11 conserved domains characteristic of all protein kinases. Domain II and VII are involved in nucleotide binding and phosphotransfer. Two residues of the alpha subunit, Val-66 (in domain II) and Trp-176 (in domain VII), were changed to Ala-66 and Phe-176, the residues present in more than 95% of the identified protein kinase sequences. These changes altered the selectivity of the alpha subunit for ATP and GTP. The Ala-66 mutant showed an increase in the Km value for GTP from 45 to 71 microM, while the Km value for ATP decreased from 13 to 9 microM. The Km value for ATP with the Phe-176 mutant showed a decrease from 13 to 7 microM. A double mutant of Ala-66/Phe-176 showed the combined effects, with a Km of 6 microM for ATP and 70 microM for GTP. Alteration of Trp-176 to Lys-176, an amino acid which is not present in the corresponding position of any known protein kinase, resulted in a lack of phosphotransferase activity. The mutations, Val-66 to Ala-66 and Trp-176 to Phe-176, also altered the interaction of the alpha subunit with the regulatory beta subunit. In contrast to the wild-type alpha subunit, which was stimulated 4-fold by addition of the beta subunit, the Ala-66 and Ala-66/Phe-176 mutants were not stimulated by the beta subunit, while the Phe-176 mutant was stimulated only 2.5-fold. All of the reconstituted holoenzymes were similar in molecular weight to the native holoenzyme. The stimulation of the phosphotransferase activity toward beta-casein B by spermine and polylysine, which is mediated by the beta subunit, was similar for holoenzymes reconstituted with either wild-type or mutant alpha subunits. Therefore, binding of the beta subunit appears to alter the active site of the alpha subunit directly or indirectly by inducing a conformational change. Ala-66 and Phe-176 mutations appear to change the structure of the alpha subunit sufficiently so that interaction of the subunits is altered and the stimulatory effect of the beta subunit is reduced or eliminated.


Cross-linking of the erythrocyte (Na+,K+)-ATPase. Chemical cross-linkers induce alpha-subunit-band 3 heterodimers and do not induce alpha-subunit homodimers. Earlier studies (Periyasamy, S. M., Huang, W.-H., and Askari, A. (1983) J. Biol. Chem. 258, 9878-9885) suggested that Cu2+ and o-phenanthroline induced the formation of cross-linked homodimers between alpha-subunits of the erythrocyte (Na+,K+)-ATPase. This was interpreted as indicating that alpha-subunits existed in close proximity in native erythrocyte membranes. The alpha-subunit and band 3 monomers have similar molecular weights (M(r) approximately 100,000) and exist in the membrane in molar ratios of approximately 1:3000 alpha-subunit:band 3. We explored the possibility that alpha-subunit and band 3 could be induced to form heterodimeric structures in the presence of cross-linking reagents. Using methods similar to those employed in the above-cited reference we demonstrated that cross-linked dimers containing phosphorylated alpha-subunits had proteolytic sensitivity that was inconsistent with the formation of alpha-subunit homodimers and fully consistent with heterodimer formation between alpha-subunit and band 3. The data also indicated that alpha-subunit-band 3 heterodimer formation is dependent on the conformational state of the (Na+,K+)-ATPase. Using the appropriate reagents we obtained cross-linked products which were consistent with heterodimer formation between alpha- and beta-subunits of the (Na+,K+)-ATPase. Our data argue against a close association between pairs of (Na+,K+)-ATPase alpha-subunits in the human red cell membrane.


Growth-associated glycosylation of transferrin secreted by HepG2 cells. Growth-associated alterations in the oligosaccharide structures of transferrin secreted by HepG2 cells were examined by concanavalin A-crossed affinoimmuno-electrophoresis. Slowly dividing, confluent cultures produced transferrin with a approximately 4.5-fold greater proportion of biantennary complex-type oligosaccharides than rapidly dividing, subconfluent cultures. The activity of N-acetylglucosaminyltransferase V (GlcNAc-T V) and galactosyltransferase were measured in cell extracts from subconfluent and confluent cultures. While the activity of galactosyltransferase remained relatively constant, the activity of GlcNAc-T V was approximately 3.2-fold lower in confluent cultures than in subconfluent cultures. These results suggest that the growth-associated alteration in the oligosaccharides of transferrin is due, at least in part, to the regulation of GlcNAc-T V activity.


Interaction between the p21ras GTPase activating protein and the insulin receptor. We investigated the involvement of the p21ras-GTPase activating protein (GAP) in insulin-induced signal transduction. In cells overexpressing the insulin receptor, we did not observe association between GAP and the insulin receptor after insulin treatment nor the phosphorylation of GAP on tyrosine residues. However, after insulin treatment in the presence of the phosphotyrosine phosphatase inhibitor phenylarsine oxide (PAO), 5-10% of GAP was found to be associated with the insulin receptor, and, in addition, a fraction of total GAP was phosphorylated on tyrosine. Using in vitro binding we showed that the N-terminal part of GAP containing the src-homology domains 2 and 3 (SH2-SH3-SH2 region) is involved in binding to the autophosphorylated insulin receptor beta-chain. In vitro binding between GAP and the autophosphorylated insulin receptor occurred independently of PAO pretreatment. These results suggest that GAP can transiently interact with the insulin receptor after insulin treatment, and this interaction is arrested after PAO pretreatment.


Site-directed mutational analysis of human tumor necrosis factor-alpha receptor binding site and structure-functional relationship. In order to define the receptor binding site and the structure-functional relationship of tumor necrosis factor (TNF), single amino acid substitutions were made by site-directed mutagenesis at selected residues of human tumor necrosis factor, using a phagemid mutagenesis/expression vector. The recombinant TNF mutants were compared to the wild type TNF in assays using crude bacterial lysates, for protein yield, solubility, subunit trimerization, receptor binding inhibition activity, and in vitro cytotoxic activity. All mutants which did not form cross-linkable trimer also showed little cytotoxic activity or receptor binding inhibition activity, indicating that trimer formation is obligatory for TNF-alpha activity. Most mutations of internal residues yielded no cross-linkable trimer, while most mutations of surface residues yielded cross-linkable trimer. Mutations at surface residues Leu29, Arg31, and Ala35 yielded cross-linkable trimers with good activities, except proline substitutions which may cause conformational changes in the polypeptide chain. This suggested that these residues are near the receptor binding site. Mutations at other strictly conserved internal residues such as Ser60, His78, and Tyr119 form cross-linkable trimer with little activity. These mutations may indirectly affect the receptor binding site by forming trimers with undetectable abnormalities. Mutants of surface residues Tyr87, Ser95, Ser133, and Ser147 affect receptor binding and cytotoxic activity but not trimer formation, suggesting that these residues are involved directly in receptor binding. The fact that residues Arg31, Ala35, Tyr87, Ser95, and Ser147, located on the opposite sides of a monomer, are clustered at the intersubunit grooves of TNF trimer supports the current notion that TNF receptor binding sites are trivalent and are located at the three intersubunit grooves. However, our finding that Ser133, which is outside the groove, can also be involved directly in receptor binding suggested that the receptor binding sites of TNF may not be confined to the intersubunit grooves, but extended to include additional surface residues.


Purification and characterization of a soluble phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae. A phosphatidylinositol (PI) 4-kinase was purified 25,000-fold from the cytosolic fraction of extracts from the yeast Saccharomyces cerevisiae. The purification consisted of an ammonium sulfate fractionation followed by chromatography on sulfonated-agarose (S-Sepharose), phosphocellulose, threonine-agarose, and quaternary amino (Mono Q), and sulfonated (Mono S) beads. Major contaminants in the purification, Hsc82 and Hsp82 (yeast homologs of the mammalian heat shock protein Hsp90), were eliminated by using a combination of molecular genetics (to construct a null mutation in HSC82), altered growth conditions (to minimize expression from the inducible HSP82 gene), and high ionic strength fractionation conditions (to remove the residual Hsp82). The purified enzyme had an apparent subunit molecular weight of 125,000, much larger than any other well characterized PI-4-kinase reported previously. Like mammalian PI-4-kinases, the yeast enzyme specifically phosphorylated PI on position 4 of the inositol ring and was stimulated by Triton X-100. However, activity was not inhibited by adenosine, a potent inhibitor of certain (type II) mammalian PI-4-kinases. The enzyme displayed typical Michaelis-Menten kinetics with apparent Km values of 100 microM for ATP and 50 microM for PI. To date, this yeast enzyme is the first soluble PI-4-kinase purified from any source.


Purification and characterization of the mouse mammary tumor virus protease expressed in Escherichia coli. The mouse mammary tumor virus (MMTV) protease gene was cloned into pGEX-2T, an Escherichia coli expression vector containing the glutathione S-transferase coding region of Schistosoma japonicum. The chimeric protein was formed by fusion of the glutathione S-transferase with a hexapeptide which contains a thrombin cleavage site, followed by the MMTV protease. Affinity chromatography on a glutathione-Sepharose 4B column was used to isolate the chimeric protein. After thrombin cleavage, the glutathione S-transferase and the protease were separated by gel filtration chromatography on a Sephadex G-75 column. The overall yield of the protease purification procedure was about 1 mg of protease/liter of culture, and the specific activity was 380 pmol/min.micrograms of enzyme. Like other retroviral proteases, the MMTV enzyme was active as a dimer, showed maximum activity at pH between 4 and 6, and could be inhibited by pepstatin A and a phosphinic acid derivative HIV-1 protease inhibitor. Enzymatic characterization of this protease reveals its broad specificity, showing a clear preference for the oligopeptide substrate mimicking the cleavage site at the amino-terminal end of the capsid protein (kcat/Km = 9725.5 M-1.s-1). The chimeric protein was also an active dimer and showed a similar Km (17 microM) for such an oligopeptide, although its kcat was about 10 times smaller. Autocatalytic processing of the MMTV protease was observed after expression of clones containing the natural cleavage site, as it occurs at the amino-terminal end of the viral protease, instead of the thrombin-sensitive sequence.


Inhibition of field stimulation-induced contractions of rat cauda epididymis by purinoceptor agonists but not by adrenoceptor agonists. 1. Adenosine, AMP, ADP, and ATP were approximately equipotent in inhibiting contractions evoked by field stimulation (10 Hz, 60 V, 1 ms, 10 s) of isolated preparations of rat cauda epididymis. Adenosine had no significant effect on contractions induced by the exogenous application of either noradrenaline or ATP. 2. The effects of adenosine and AMP were markedly potentiated by the adenosine uptake inhibitor, dipyridamole. In contrast there was only a two-fold potentiation of the effects of ATP and no potentiation of the effects of ADP by dipyridamole. 3. Inhibitory responses to both adenosine and ATP were blocked by 8-phenyltheophylline. Neither the P2Y-purinoceptor antagonist, reactive blue 2, nor P2X purinoceptor antagonist alpha,beta-methylene ATP, blocked the inhibitory effects of ATP. 4. Several stable analogues of adenosine, namely 5'-(N-ethyl) carboxamidoadenosine (NECA), N6-cyclohexyl-adenosine (CHA), L-N6-(2-phenyl-isopropyl)adenosine (L-PIA), D-N6-(2-phenyl-isopropyl)adenosine (D-PIA) and 2-chloroadenosine (CADO) also inhibited nerve stimulation-induced contractions. NECA was the most potent. L-PIA and D-PIA were approximately equipotent, except in the presence of dipyridamole, when the potency of L-PIA exceeded that of D-PIA. 5. Field stimulation-induced contractions of the rat cauda epididymis were unaffected by the alpha 2-adrenoceptor agonists clonidine and xylazine; isoprenaline in high concentrations produced phentolamine-sensitive inhibition of contractions evoked by field stimulation and exogenous application of noradrenaline. 6. These findings taken together are consistent with the possibility that prejunctional purinoceptors, which are atypical in that they are directly activated by both ATP and adenosine, mediate inhibition of neurotransmission in this tissue. No evidence for the presence of functional alpha 2-adrenoceptors modulating neurotransmission was obtained.


Evaluation of hydroxylapatite/poly(L-lactide) composites: mechanical behavior. By mixing hydroxylapatite (HA) into L(-)-dilactide monomer, prior to polymerization to poly(L-lactide) (PLLA), hydroxylapatite filled poly(L-lactide) composites were obtained. This study reports about the mechanical properties of these composites compared with unfilled PLLA. It was concluded that a 30 wt% HA/PLLA composite has better compressive and tensile strengths, higher stiffness and Vickers hardness number than unfilled PLLA (Mv: 125-150,000). Gas sterilization (ethylene oxide) affects molecular weight and flexural strength significantly. Implantation studies revealed loss of 50% of initial flexural strength within 3 weeks, and a faster decline of flexural strength was observed in phosphate buffered saline than in the subcutis of goats. From a mechanical point of view storage at -20 degrees C proved to be a safe method. In its current state HA/PLLA composites can not be used as implant materials that have to resist major forces. However, such composites might be useful in non-loadbearing applications in orthopedic or maxillofacial surgery.


Mechanism and strength of bonding between two bioactive ceramics in vivo. A study was conducted to examine the mechanism and strength of bonding between two bioactive ceramic plates in vivo. Rectangular plates (15 mm X 10 mm X 2 mm) of Bioglass, apatite-wollastonite-containing glass ceramic (designated A-W.GC), and two types of hydroxyapatite sintered at 900 degrees C and 1200 degrees C (designated HA900 and HA1200) were prepared. Two plates of the same materials tied together with silk thread were implanted subcutaneously into rats. The force required to detach the mutually bonded bioactive ceramic plates was measured 4, 8, 12, and 24 weeks after implantation. The interface between the two bonded plates was examined by SEM-EPMA and thin-film x-ray diffraction analysis. At 24 weeks after implantation, the mutual bonding of Bioglass and A-W.GC was stronger than that of the two HA types. SEM-EPMA and thin-film x-ray diffraction analysis of the bonded area of Bioglass and A-W.GC plates showed bonding zones with apatite in the margins, and a bonding zone with calcite in the center. The greater strength of bonding of Bioglass and A-W.GC plates compared with the two types of HA plate 24 weeks after implantation is explained by the wider bonding zone provided by the calcite layer formed in the center of the plates, which is considered to have been perfused with PO4-poor body fluids resulting from PO4 consumption for apatite formation in the margins.


The ultrastructure of the bone-hydroxyapatite interface in vitro. Rat bone marrow cells were cultured on plasma-sprayed hydroxyapatite (HA). The cells formed a mineralized extracellular matrix (ECM) that exhibited several characteristics of bone tissue. The interface between this mineralized ECM and the HA was studied at the ultrastructural level with scanning and transmission electron microscopy and x-ray microanalysis. Initially, the deposition of a globular, afibrillar matrix was observed on HA. This was followed by the integration of collagen fibers in this matrix and their subsequent mineralization. At the bone-HA interface two distinctly different interfacial structures were observed. An electron-dense layer with a thickness of 20-60 nm was regularly present, which contained both organic and inorganic material and was rich in glycosaminoglycans. The interfaces differed however, in the presence or absence of an amorphous zone which was free of collagen fibers and had an average thickness of 0.7-0.8 microns. It was frequently seen interposed between the electron-dense layer and the hydroxyapatite. Similar interfacial structures have also been described in the in vivo environment, where they were referred to as lamina limitans-like or cement linelike. From the results of this study, it can be concluded that the described in vitro system is a suitable model to study bone-biomaterial interactions.


Biomaterial-induced alterations of neutrophil superoxide production. Because periprosthetic infection remains a vexing problem for patients receiving implanted devices, we evaluated the effect of several materials on neutrophil free radical production. Human peripheral blood neutrophils were incubated with several sterile, lipopolysaccharide (LPS)-free biomaterials used in surgically implantable prosthetic devices: polyurethane, woven dacron, and velcro. Free radical formation as the superoxide (O2-) anion was evaluated by cytochrome c reduction in neutrophils that were exposed to the materials and then removed and in neutrophils allowed to remain in association with the materials. Neutrophils exposed to polyurethane or woven dacron for 30 or 60 min and then removed consistently exhibited an enhanced release of O2- after simulation via receptor engagement with formyl methionyl-leucyl-phenylalanine. Enhanced reactivity to stimulation via protein kinase C with phorbol myristate acetate, however, was not consistently observed. The cells evaluated for O2- release during continuous association with the biomaterials showed enhanced metabolic activity during short periods of association (especially with polyurethane and woven dacron). Although O2- release by neutrophils in association with these materials decreased with longer periods of incubation, it was not obliterated. These studies, therefore, show that several commonly used biomaterials activate neutrophils soon after exposure and that this activated state diminishes with prolonged exposure but nevertheless remains measurable. The diminishing level of activity with prolonged exposure, however, suggests that ultimately a depletion of reactivity may occur and may result in increased susceptibility to periprosthetic infection.


Compositional dependence of calcium phosphate layer formation in fluoride Bioglasses. Bioglasses form a double layer composed of apatite and a silica-rich layer when placed in a simulated physiological solution as well as in living tissue [A.E. Clark, C.G. Pantano, and L. L. Hench, "Auger spectroscopic analysis of bioglass corrosion films," J. Am. Ceram. Soc., 59(1-2), 37-39 (1976).]. In the present work, the mechanisms of the calcium phosphate layer and the silica-rich layer formation of fluoride Bioglasses in Tris-buffer solution are studied as a function of the SiO2 content. Fourier Transform Infrared Reflection Spectroscopy (FTIRS) is used to investigate the mechanism of formation of calcium phosphate and silica-rich layers on the glass surface. Ion concentration in reacted solution and elemental depth profiles are obtained by Induced Coupled Plasma Atomic Emission Spectrometry (ICP) and Auger Electron Spectroscopy (AES), respectively. Si--O bonds with one nonbridging oxygen and Si--O--Si bonds form at the early stage of reaction. Strong phosphorus ion uptake occurs when an amorphous calcium phosphate layer crystallizes. Glasses with high silica content (conventional glass) form the silica-rich layer first followed by a calcium phosphate layer on top. However, glasses with low silica content (invert glass) form both layers simultaneously. The rate of apatite formation decreases with increasing SiO2 content, especially in the region of conventional glass compositions. Ion release rates decreases as SiO2 content increases, with a significant change occurring at the compositional boundary between invert and conventional glasses.


Protein interaction with tantalum: changes with oxide layer and hydroxyapatite at the interface. For metallic implants the surface nature is extremely important because blood and tissue interactions with metal depend upon it. Protein adsorption is the initial reaction that takes place when an implant comes in contact with blood or tissue. We attempted to coat different thicknesses of oxide layers and hydroxyapatite on tantalum and examined the changes in water contact angle and adsorption of albumin and fibrinogen. Protein adsorption studies were performed with 125I-labeled proteins. A decrease in water contact angle was observed as the oxide layer thickness of tantalum increased. Fibrinogen adsorption increased on oxide layer coated and hydroxyapatite coated surfaces, compared to bare tantalum.


Ca2+ and pH determine the interaction of chromaffin cell scinderin with phosphatidylserine and phosphatidylinositol 4,5,-biphosphate and its cellular distribution during nicotinic-receptor stimulation and protein kinase C activation. Nicotinic stimulation and high K(+)-depolarization of chromaffin cells cause disassembly of cortical filamentous actin networks and redistribution of scinderin, a Ca(2+)-dependent actin filament-severing protein. These events which are Ca(2+)-dependent precede exocytosis. Activation of scinderin by Ca2+ may cause disassembly of actin filaments leaving cortical areas of low cytoplasmic viscosity which are the sites of exocytosis (Vitale, M. L., A. Rodríguez Del Castillo, L. Tchakarov, and J.-M. Trifaró. 1991. J. Cell. Biol. 113:1057-1067). It has been suggested that protein kinase C (PKC) regulates secretion. Therefore, the possibility that PKC activation might modulate scinderin redistribution was investigated. Here we report that PMA, a PKC activator, caused scinderin redistribution, although with a slower onset than that induced by nicotine. PMA effects were independent of either extra or intracellular Ca2+ as indicated by measurements of Ca2+ transients, and they were likely to be mediated through direct activation of PKC because inhibitors of the enzyme completely blocked the response to PMA. Scinderin was not phosphorylated by the kinase and further experiments using the Na+/H+ antiport inhibitors and intracellular pH determinations, demonstrated that PKC-mediated scinderin redistribution was a consequence of an increase in intracellular pH. Moreover, it was shown that scinderin binds to phosphatidylserine and phosphatidylinositol 4,5-biphosphate liposomes in a Ca(2+)-dependent manner, an effect which was modulated by the pH. The results suggest that under resting conditions, cortical scinderin is bound to plasma membrane phospholipids. The results also show that during nicotinic receptor stimulation both a rise in intracellular Ca2+ and pH are observed. The rise in intracellular pH might be the result of the translocation and activation of PKC produced by Ca2+ entry. This also would explain why scinderin redistribution induced by nicotine is partially (26-40%) inhibited by inhibitors of either PKC or the Na+/H+ antiport. In view of these findings, a model which can explain how scinderin redistribution and activity may be regulated by pH and Ca2+ in resting and stimulated conditions is proposed.


Evidence for functional homology in the F-actin binding domains of gelsolin and alpha-actinin: implications for the requirements of severing and capping. The F-actin binding domains of gelsolin and alpha-actinin compete for the same site on actin filaments with similar binding affinities. Both contain tandem repeats of approximately 125 amino acids, the first of which is shown to contain the actin-binding site. We have replaced the F-actin binding domain in the NH2-terminal half of gelsolin by that of alpha-actinin. The hybrid severs filaments almost as efficiently as does gelsolin or its NH2-terminal half, but unlike the latter, requires calcium ions. The hybrid binds two actin monomers and caps the barbed ends of filaments in the presence or absence of calcium. The cap produced by the hybrid binds with lower affinity than that of gelsolin and is not stable: It dissociates from filament ends with a half life of approximately 15 min. Although there is no extended sequence homology between these two different F-actin binding domains, our experiments show that they are functionally equivalent and provide new insights into the mechanism of microfilament severing.


Research priorities for burn nursing: rehabilitation, discharge planning, and follow-up care. Fourteen of the 101 research questions that were proposed in the Burn Nursing Delphi study by Marvin et al. (J Burn Care Rehabil 1991;12:190-7) were concerned with the rehabilitation, discharge planning, and follow-up care of patients with burns. Questions that were ranked as top priorities for patient welfare related to control of postburn itching, prevention of contractures, and effective means of community-based follow-up to meet physical, social, and emotional needs of patients and families. Burn nurses identified the study of nursing interventions that are the most effective means for preparing patients, families, and community nurses for posthospitalization care as the research priority that has the greatest potential impact on the profession of burn nursing. Respondents to the Delphi study favored a collaborative approach to research for most of the questions in the rehabilitation subgroup. Suggestions are made for nurses who wish to take the lead in designing and implementing qualitative and quantitative studies that relate to the increasingly prevalent problems that are encountered by survivors of burn injury.


Platelet-activating factor stimulates multiple signaling pathways in cultured rat mesangial cells. We have previously reported that platelet-activating factor (PAF) elevates cytosolic free calcium concentration ([Ca2+]i) in fura-2-loaded glomerular mesangial cells. To confirm that this increase in [Ca2+]i is a result of receptor-mediated activation of phospholipase C, we investigated hydrolysis of phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2) in PAF-treated mesangial cells. PAF (10(-7) M) stimulated a rapid and transient formation of inositol trisphosphate. In concomitant experiments, PAF stimulated a biphasic accumulation of 3H-arachidonate-labeled 1,2-diacylglycerol (DAG). The secondary elevation in DAG was coincident with a rise in 3H-phosphorylcholine (PC) and 3H-phosphorylethanolamine (PE) suggesting that PAF stimulates delayed phospholipase activities which hydrolyze alternate phospholipids besides the polyphosphoinositides. This PAF-stimulated elevation in 3H-water soluble phosphorylbases was seen at 5 min but not at 15 sec suggesting that the initial rise in DAG as well as the initial elevation in [Ca2+]i are due primarily to PtdIns-4,5-P2 hydrolysis. PAF also stimulated PGE2 as well as 3H-arachidonic acid and 3H-lyso phosphatidylcholine (PtdCho) formation. We suggest that arachidonate released specifically from PtdCho via phospholipase A2 is a source of this PAF-elevated PGE2. It has been postulated that anti-inflammatory prostaglandins may antagonize the contractile and proinflammatory effects of PAF via activation of adenylate cyclase. Surprisingly, exogenous PAF reduced basal and receptor-mediated cAMP concentration indicating that PAF-stimulated transmembrane signaling pathways may oppose receptor-mediated activation of adenylyl cyclase. We have taken advantage of the different sensitivities of phospholipases A2 and C(s) to PMA, EGTA, and pertussis toxin to dissociate phospholipase A2 and C activities. Acute PMA-treatment enhanced PAF-stimulated PGE2 formation, reduced PAF-induced elevations in [Ca2+]i and had no effect upon PAF-stimulated 3H-PE. We have also demonstrated that phospholipase A2, but not PtdIns-specific phospholipase C, was sensitive to external calcium concentration. The role of a GTP-binding protein to couple PAF-receptors to the PtdIns-specific phospholipase C was confirmed as GTP gamma S synergistically elevated PAF-stimulated inositol phosphate formation. We also demonstrated that pertussis toxin ADP-ribosylates a single protein of an apparent 42 kD mass and that PAF pretreatment reduced subsequent ADP-ribosylation in a time-dependent manner. However, pertussis toxin had no effect upon phospholipase C-generated water soluble phosphorylbases or inositol phosphates. In contrast, PAF-stimulated phospholipase A2 and PAF-inhibited adenylyl cyclase activities were sensitive to pertussis toxin.(ABSTRACT TRUNCATED AT 400 WORDS)


Transforming growth factor beta 1 and adrenocorticotropin differentially regulate the synthesis of adrenocortical cell heparan sulfate proteoglycans and their binding of basic fibroblast growth factor. Adrenocortical differentiated functions are under the control of both endocrine hormones such as ACTH and local factors such as transforming growth factor beta (TGF beta) or basic fibroblast growth factor (bFGF). Besides their regulatory actions on the synthesis of corticosteroids, these two classes of factors also exert some important effects on the cellular environment. We have examined here the regulation by ACTH and TGF beta of adrenocortical cell proteoglycan synthesis and secretion. Under basal conditions, adrenocortical cells synthesized and secreted several species of sulfated proteoglycans, 80% of them being recovered in solution in the culture medium. When analyzed by ion exchange chromatography, the cell extracts and the media from cells metabolically labeled with 35S-sulfate were found to contain two and three species of radioactive sulfated proteoglycans, respectively. All species were proteoheparan-sulfates. Treatment of adrenocortical cells with TGF beta 1 or ACTH resulted in a significant increase of the incorporation of 35S into both secreted and cell-associated proteoglycans. ACTH stimulated more than three times the amount of secreted proteoglycans eluting from DEAE-Trisacryl as peak B, whereas TGF beta preferentially increased the amount of peak C. No important modification of the size of the synthesized proteoglycans was observed. The subpopulation of heparan sulfate proteoglycans capable to bind bFGF was also largely increased after ACTH or TGF beta treatment and paralleled the variation in overall proteoheparan sulfate synthesis. Thus those effects of TGF beta and ACTH on proteoglycan synthesis may participate in an increased ability of adrenocortical cells to bind and respond to bFGF.


Atypical receptor-mediated signal transduction events in the EGF-dependent growth-inhibited cell line, MDA-468. It is now generally considered that early signalling from tyrosine kinases that induce mitogenesis is initiated through the formation of heteromeric complexes consisting of the autophosphorylated tyrosine kinase and a number of tyrosylphosphorylated proteins, including phospholipase C-gamma (PLC-gamma) and GTPase activating protein (GAP). However, since much of this work has been performed on proliferative, chimeric cell lines expressing heterologous receptor molecules, we examined the nature of the epidermal growth factor receptor (EGFR) signalling complex formation in the human breast cancer cell line, MDA-468. This cell line has an amplified, native EGFR gene, correspondingly overexpresses the EGFR, and its growth in culture is inversely related to the EGF concentration. Our results indicate that in MDA-468 cells, both the EGFR and PLC-gamma are phosphorylated on tyrosine residues and can be co-immunoprecipitated. This occurs at both high and low EGF concentrations regardless of the proliferative endpoint. The molecular association is correlated with a significant increase in total inositol phosphates formed in response to the growth factor treatment. In contrast, however, there is no evidence that GAP is either phosphorylated on tyrosine residues or forms a complex with the activated EGFR in EGF-treated MDA-468 cells. These observations suggest that as a model for growth factor action, the formation of heteromeric protein signalling complexes may demonstrate considerable diversity depending upon both cell type and physiology.


Reversible hyperphosphorylation and reorganization of vimentin intermediate filaments by okadaic acid in 9L rat brain tumor cells. Okadaic acid (OA), a protein phosphatase inhibitor, was found to induce hyperphosphorylation and reorganization of vimentin intermediate filaments in 9L rat brain tumor cells. The process was dose dependent. Vimentin phosphorylation was initially enhanced by 400 nM OA in 30 min and reached maximal level (about 26-fold) when cells were treated with 400 nM OA for 90 min. Upon removal of OA, dephosphorylation of the hyperphosphorylated vimentin was observed and the levels of phosphorylation returned to that of the controls after the cells recovered under normal growing conditions for 11 h. The phosphorylation and dephosphorylation of vimentin induced by OA concomitantly resulted in reversible reorganization of vimentin filaments and alteration of cell morphology. Cells rounded up as they were entering mitosis in the presence of OA and returned to normal appearance after 11 h of recovery. Immuno-staining with anti-vimentin antibody revealed that vimentin filaments were disassembled and clustered around the nucleus when the cells were treated with OA but subsequently returned to the filamentous states when OA was removed. Two-dimensional electrophoresis analysis further revealed that hyperphosphorylation of vimentin generated at least seven isoforms having different isoelectric points. Furthermore, the enhanced vimentin phosphorylation was accompanied by changes in the detergent-solubility of the protein. In untreated cells, the detergent-soluble and -insoluble vimentins were of equal amounts but the solubility could be increased when vimentins were hyperphosphorylated in the presence of OA. Taken together, the results indicated that OA could be involved in reversible hyperphosphorylation and reorganization of vimentin intermediate filaments, which may play an important role in the structure-function regulation of cytoskeleton in the cell.


Differential transport and processing of variant mouse mammary tumor virus glycoproteins. The transport and proteolytic processing of two individual gene isolates of the mouse mammary tumor virus (MMTV) glycoprotein were compared in transfected rat HTC hepatoma cells. Plasmids were constructed such that the MMTV glycoprotein genes were constitutively expressed from the promoter within the Rous Sarcoma Virus 5' Long Terminal Repeat in the absence of other MMTV proteins. An isolate of the GR strain MMTV glycoprotein was efficiently transported and processed resulting in the localization of MMTV glycoproteins at the cell surface and in the extracellular environment. Moreover, the kinetics of acquisition of endoglycosidase H resistant oligosaccharide side chains and the rate of endoproteolytic cleavage of the glycosylated polyprotein expressed in transfected cells were virtually identical to that observed in viral-infected rat hepatoma cells. In contrast, a natural variant of the C3H strain MMTV glycoprotein expressed in transfected cells was retained in an intracellular compartment by a heavy chain binding protein (BiP)-independent pathway in an endoglycosidase H sensitive and uncleaved form. This MMTV glycoprotein isolate was retained early in the exocytic pathway and displayed a half-life of approximately 45 min in transfected cells. Only a minor fraction of the expressed C3H variant glycoprotein was detected at the cell surface but was not externalized. Our results suggest that the variant C3H MMTV glycoprotein contains one or more mutations that preclude its efficient transport through the exocytic pathway.


Interaction of MAR-sequences with nuclear matrix proteins. The recent discovery of DNA sequences responsible for the specific attachment of chromosomal DNA to the nuclear skeleton (MARs/SARs) was an important step towards our understanding of the functional and structural organization of eukaryotic chromatin [Mirkovitch et al.: Cell 44:273-282, 1984; Cockerill and Garrard: Cell 44:273-282, 1986]. A most important question, however, remains the nature of the matrix proteins involved in the specific binding of the MARs. It has been shown that topoisomerase II and histone H1 were capable of a specific interaction with SARs by the formation of precipitable complexes [Adachi et al.: EMBO J8:3997-4006, 1989; Izaurralde et al.: J Mol Biol 210:573-585, 1989]. Here, applying a different approach, we were able to "visualize" some of the skeletal proteins recognizing and specifically binding MAR-sequences. It is shown that the major matrix proteins are practically the same in both salt- and LIS-extracted matrices. However, the relative MAR-binding activity of the individual protein components may be different, depending on the method of matrix preparation. The immunological approach applied here allowed us to identify some of the individual MAR-binding matrix proteins. Histone H1 and nuclear actin are shown to be not only important components of the matrix, but to be involved in a highly efficient interaction with MAR-sequences as well. Evidence is presented that proteins recognized by the anti-HMG antibodies also participate in MAR-interactions.


Fluorescence cytometry of microtubules and nuclear DNA during cell-cycle and reverse-transformation. Synchronized CHO-K1 cells and their dibutyryl c-AMP treated counterparts have been characterized by means of static and flow fluorescence cytometry at the level of nuclear DNA and cytoplasmic microtubules. In order to confirm earlier findings on synchronized population, Carnoy fixed and hydrolyzed, several new findings are here reported at the level of single intact cell. The fluorescence intensity of DAPI-stained glutaraldehyde fixed 2C cells correlates well with the average absorbance of the corresponding Feulgen-stained cells, thereby appearing also to be a measure of chromatin condensation during the G1 phase. In the early part of G1, the drastic alteration in anti-beta tubulin immunostaining is shown to parallel microtubule depolymerization induced by calcium or colcemide. The known 1-2 h lengthening of the G1 period after reverse-transformation appears to correlate with a similar delay in the abrupt chromatin decondensation. The above results are discussed in terms of the role of microtubules and nuclear morphometry (and their coupling) in the control of cell cycle progression of transformed vs. fibroblast-like cells.


Cholera toxin potentiates TPA-induced mitogenesis and c-fos expression in BALB/c-3T3-derived proadipocytes. Treatment of quiescent density-arrested A31T6 proadipocytes with medium supplemented with either 12-O-tetradecanoylphorbol-13-acetate (TPA), insulin, or cholera toxin alone did not stimulate G0/G1 traverse and initiation of DNA synthesis. Combinations of either TPA and cholera toxin or insulin and cholera toxin caused a small stimulation of proliferation. Addition of medium supplemented with TPA and insulin caused a marked stimulation of cell cycle traverse which was significantly potentiated by the coaddition of cholera toxin. The actions of cholera toxin were mimicked by forskolin. Expression of c-fos was regulated in a manner that reflected the results of the mitogenic experiments. TPA caused a marked induction of expression, while only a small increase in transcript levels was seen after treatment with cholera toxin. Addition of a combination of cholera toxin and TPA caused a synergistic induction of c-fos expression. The model system described in this paper allows a detailed analysis of the regulation, by independent second messenger systems, of the transcription of a gene in a mitogenically relevant manner.


Changes in the metaphase transit times and the pattern of sister chromatid separation in stamen hair cells of Tradescantia after treatment with protein phosphatase inhibitors. Stamen hair cells from the spiderwort plant, Tradescantia virginiana, exhibit remarkably predictable metaphase transit times, making them uniquely suitable for temporal studies on mitotic regulation. In this study, we describe two kinds of experiments that test whether protein phosphatase activity is a necessary prerequisite for entry into anaphase in living, mitotic cells. We treated cells at specific points during prophase, prometaphase and metaphase with the broad-spectrum protein phosphatase inhibitor, alpha-naphthyl phosphate (administered by microinjection), or with the naturally occurring, potent phosphatase inhibitors okadaic acid, microcystin-LR or microcystin-RR (administered by perfusion), and we have observed changes in the metaphase transit time that are primarily dependent on the time of initial exposure to the inhibitor. Maximal extensions of the metaphase transit time result from alpha-naphthyl phosphate microinjections initiated in mid-metaphase, 10-20 min after nuclear envelope breakdown. Perfusions with okadaic acid started during a specific interval in mid-metaphase, 15-20 min after nuclear envelope breakdown, resulted in a statistically significant extension of the metaphase transit time. Perfusions with either microcystin-LR or microcystin-RR initiated 15-26 min after nuclear envelope breakdown extended the metaphase transit times significantly. Treatments of cells with okadaic acid or with either of the microcystins initiated outside this mid-metaphase interval either were without effect or, alternatively, resulted in a significant shortening of the metaphase transit time. In addition to their effects on the timing of anaphase onset, treatments with these protein phosphatase inhibitors also resulted in a remarkable change in the way in which these cells enter anaphase. Sister chromatid separation in stamen hair cells typically requires only 5 seconds, but after treatment with any of these inhibitors some, but not all, of the chromatids split apart at anaphase onset. Those that split begin to migrate toward the spindle pole regions, while those that fail to split remain at the metaphase plate. Later, more of the paired chromatids split apart and begin moving toward the spindle pole regions. Those that fail to separate remain at the metaphase plate. This process can be repeated several times before all of the chromatids have separated. Thus, entry into anaphase becomes extremely asynchronous, and as much as 30 min can transpire between the centromeric separation of the first and last chromosomes. Some of the chromosomes complete their anaphase movements before others have even split apart at the metaphase plate. Asynchronous separation did not result in a permanent segregation anomaly.(ABSTRACT TRUNCATED AT 400 WORDS)


Phosphorylated baculovirus p10 is a heat-stable microtubule-associated protein associated with process formation in Sf9 cells. Insect ovarian Sf9 cells extend processes with complex morphologies when infected with a recombinant baculovirus encoding the catalytic subunit of protein kinase A. Within the shafts of the processes are abundant microtubules, which, in contrast to those in Sf9 cells expressing the microtubule-associated protein tau, are generally not organized into parallel bundles. During infection the late viral polypeptide p10 becomes phosphorylated by the protein kinase A catalytic subunit at its penultimate residue, Ser92. The expression or phosphorylation of other major host cell or viral polypeptides does not change, compared with polypeptides from a wild-type viral infection. Once phosphorylated, p10 associates with microtubules in the infected cells and may thereby play a role in process formation.


Independent control of locomotion and orientation during Dictyostelium discoideum chemotaxis. Chemotaxis is cell movement in the direction of a chemical and is composed of two component: movement and directionality. The directionality of eukaryotic chemotaxis is probably derived from orientation: the detection of the spacial gradient of chemoattractant over the cell length. Chemotaxis was investigated in eukaryotic Dictyostelium discoideum cells that were permeabilized by high-voltage discharges. These permeable cells respond chemotactically to extracellular cAMP. However, locomotion is impaired if the Ca2+ concentration is clamped at submicromolar concentrations; interestingly, these non-motile cells still form pseudopodia and elongate in the direction of the cAMP gradient. These results imply that locomotion and orientation during Dictyostelium chemotaxis are independently regulated.


Inhibitors of topoisomerases do not block the passage of human lymphocyte chromosomes through mitosis. Cultured human lymphocytes have been treated with a number of topoisomerase inhibitors, to see whether topoisomerase II is involved in the process of chromosome segregation at anaphase. Results were assessed by examination of cytogenetical preparations of spread chromosomes. Four effects were observed, although no inhibitor produced all four effects. These effects were: inhibition of entry into mitosis; chromosome breakage and rearrangement; inhibition of chromosome condensation; and inhibition of chromosome segregation. Evidence for the last was ambiguous. Although there was evidence that separation of chromatids was affected when cells were treated with colchicine as well as topoisomerase II inhibitors (most notably with nalidixic acid, which resulted in complete fusion of the chromatids), no evidence was obtained to show that, in the absence of colchicine, cells treated with inhibitors could not proceed through anaphase normally. The topoisomerase I inhibitor, camptothecin, differed from the topoisomerase II inhibitors in not showing any effect on chromosome condensation or any significant effect on segregation.


Effects of okadaic acid on mitotic HeLa cells. Mitotic HeLa cells were treated with different concentrations of okadaic acid (OA), known to inhibit phosphatase 1 and 2A activities. The cytological effects on the course of mitosis were studied at the light microscopic, immunoflourescence and electron microscopic levels. At the lowest concentration used (1 nM), OA did not show any effect on mitosis, but at higher concentrations it showed pronounced effects. The mitotic chromosomes became scattered, the mitotic spindle became deranged and the cells failed to enter anaphase. At the electron microscopic level formation of isolated microtubules and regular trilaminar kinetochores were observed. An extensive growth of the endoplasmic reticulum could be noted in these cells. Decondensation of chromatin and nuclear envelope re-formation could be seen only after withdrawal of OA. A high frequency of multinucleate cells could be found after 24 h of recovery. Cells treated with 100 nM OA for 3 hours showed diplochromosomes in over 50% of mitotic cells after 24 h recovery. These were presumably formed due to the failure of sister chromatid separation in the earlier mitosis in the presence of OA. At the electron microscopic level the diplochromosomes showed a quadruplet structure. The role of phosphatase 1 in controlling some late mitotic events, i.e. sister chromatid separation, MPF-inactivation and nuclear envelope re-formation etc., is discussed.


The distribution of the fiber cell intrinsic membrane proteins MP20 and connexin46 in the bovine lens. MP20 is an intrinsic membrane protein previously identified in mammalian lens fiber cells. To identify a possible role for this protein in the lens, the distribution of MP20 and connexin46 has now been examined. Western immunoblotting with an anti-peptide antibody generated to the C-terminal 8 amino acids of MP20 confirmed the presence of this protein in the lens of several different mammalian species. A monoclonal antibody 5H1 was prepared that, in Western blots of bovine lesn membranes, recognized the same component as an antibody to rat connexin46 (Cx46). The apparent molecular mass of this component decreased from 59 kDa to 55 kDa following treatment of lens membranes with alkaline phosphatase. A monoclonal antibody to connexin-related MP70 recognized a 70 kDa component in bovine lens membranes confirming the presence of these two different connexin proteins in bovine lens membranes. To localize MP20 and Cx46 in the bovine lens membrane, lens fiber cell bundles were immunofluorescently labeled with both the MP20 antibody, and the monoclonal antibody to Cx46. Cx46 was identified in large plaques on the broad faces of the lens fiber cells throughout the outer 1 mm of the lens cortex. MP20 colocalized with Cx46 only in a restricted area 0.5 mm to 1.0 mm into the lens. In other regions of the lens, MP20 appeared more diffusely distributed over the fiber cell surface, although apparently concentrated in the ball-and-socket regions at the corners of the narrow side of the inner cortical lens fiber cells. These inner cortical regions were devoid of Cx46. A difference in distribution of these two proteins was confirmed in studies of immunofluorescently labeled lens cryosections. Furthermore, immunogold electron microscopy of purified lens membranes identified MP20 in both junctional regions (with Cx46) and in single membranes. These results provide evidence for a role for MP20 in mammalian lens fiber cell junctional formation or organization.


Examination of transcellular membrane protein polarity of bovine aortic endothelial cells in vitro using the cationic colloidal silica microbead membrane-isolation procedure. In this report we describe a rapid, high-yield protocol for the isolation of apical (AP) and basolateral (BL) plasma membrane domains from monolayers of bovine aortic endothelial cells (BAECs) grown on tissue culture dishes as well as microcarrier beads. Using a modified cationic colloidal silica microbead membrane-isolation procedure, which deposits a uniform silica-polyacrylate pellicle over the entire AP membrane surface, a 4- to 9.6-fold relative enrichment of AP membrane and a 3.55- to 3.67-fold relative enrichment of BL membrane was obtained when the isolated domains were examined for silica and Na+/K(+)-ATPase, respectively. Immunoblotting of the isolated membrane domains displayed the presence of angiotensin-converting enzyme (ACE) exclusively in the AP domain and collagen receptors (CRs) highly enriched in the BL membrane domain when monolayers were grown on a gelatin substratum.


Cell-, age- and stage-dependent distribution of connexin43 gap junctions in testes. Immunocytochemical data demonstrate that the distribution of gap junction connexin43 (Cx43) in rodent testes is dependent on cell type, testis maturation, and stage of the mature seminiferous epithelium. Western blotting and indirect immunofluorescence microscopy using anti-peptide antisera to Cx43 revealed abundant Cx43 in rat and mouse testes and mouse TM3 and TM4 cells. Cx43 mRNA was detected in rat testes and mouse TM4 cells by Northern blot analysis. Cx43 was localized by immunogold electron microscopy to gap junctions on Sertoli cells and Leydig cells. A punctate distribution of Cx43 was observed on peritubular cell surfaces following indirect immunofluorescence of detergent-permeabilized tubule segments. In cryosections from testes of immature (to 30 days) rats, and mature rats and mice, Leydig cells showed a punctate surface distribution of Cx43 following indirect immunofluorescence. A diffuse cytoplasmic fluorescence was also seen in spermatocytes and spermatogonia. Cx43 staining associated with Sertoli cells was age- and stage-dependent. Over 90% of the tubules in immature tests (22-30 days) contained Cx43 in the region of Sertoli-Sertoli occluding junctions and in the adluminal compartment. In mature rat testes, however, Cx43 immunostaining was detected in only 60% of 1195 tubule sections where it was abundant proximal to the Sertoli cell occluding junctions. All strongly stained tubules were from stages I-VIII, while negatively stained tubules were at stages IX-XIV. Cx43 immunostaining in mature mouse testes was also stage-dependent with all positive tubules at stages VI-VIII. In contrast to Cx43, Cx26 and Cx32 were detected by immunofluorescence only in the apical regions of the seminiferous epithelia in 90% of tubules from mature rats. Consistent with the observed Cx43 immunostaining, octanol-sensitive in situ dye-coupling was observed between Leydig cells, between peritubular cells and between Sertoli cells, suggesting the occurrence of functional gap junctions in these cell types. These observations provide evidence for extensive gap junction-mediated communication between a variety of testis cell types important to the support of spermatogenesis.


The periodic paralyses.

The periodic paralyses. These mysterious attacks of muscle weakness are difficult to treat because the different forms are hard to define, and potassium plays a different role in each one. New genetic probes should finally resolve these issues. In the meantime, attacks can be prevented in at least some patients. A multicenter study is now under way to determine the best treatment for each subtype.


The colorectal cancer gene hunt: current findings. Identification of four genes, each with its own clinical significance, promises probes for early diagnosis of colorectal cancer in the near future. It also suggests possibilities for new forms of chemotherapy to offset the effects of mutated or lost genes. Given the "moving target" of metastasis, this is likely to be a more fruitful approach than gene replacement--and a more immediate one.


Determination of sparfloxacin in serum and urine by high-performance liquid chromatography. A specific and sensitive analytical method for the determination of sparfloxacin in serum and urine is described. Serum proteins are removed by precipitation with acetonitrile after the addition of ofloxacin as an internal standard. The supernatant solvent is evaporated in a vacuum concentrator and the dry residue is redissolved in the mobile phase. Separation is performed on a cation-exchange column (Nucleosil 100 5SA, 125 x 4.0 mm I.D., 5 microns particle size) protected by a guard column (Perisorb RP-18, 30 x 4.0 mm I.D., 30-40 microns particle diameter). The mobile phase consisted of 750 ml of acetonitrile and 250 ml of 100 mmol/l phosphoric acid (v/v) to which sodium hydroxide had been added. The final concentration of sodium was 23 mmol/l and the pH was 3.82. Sparfloxacin and ofloxacin were determined by spectrofluorimetry (excitation wavelength 295 nm; emission wavelength 525 nm). The flow-rate was 1.5 ml/min and the retention times were 4.7 (sparfloxacin) and 8.0 (ofloxacin) min. Validation of the method yielded the following results for serum: detection limit 0.05 mg/l; precision between series 10.4-3.6%; recovery 99.5-100.0%; comparison with a microbiological assay c(bioassay) = 1.035c(HPLC) - 0.06. The test organism was Bacillus subtilis ATCC 6633. For urine the results were: detection limit 0.5 mg/l; precision between series 7.8-5.0%; recovery 97.0-97.8%; method comparison c(bioassay) = 1.092c(HPLC) - 1.09. No interferences were observed in human volunteers. The method can also be applied to stool samples.


Confirmation testing of 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid in urine with micellar electrokinetic capillary chromatography. The major urinary metabolite of the most commonly abused psychotropic drug, delta-9-tetrahydrocannabinol, is 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH). With basic hydrolysis, extraction and concentration, this compound can easily be determined using micellar electrokinetic capillary chromatography with on-column multi-wavelength detection. After solid-phase extraction of 5 ml of urine, drug concentrations down to about 10 ng/ml can be unambiguously monitored. Peak assignment is achieved through comparison of the retention time and absorption spectrum of the eluting THC-COOH peak with those of computer-stored model runs. The effectiveness of the approach is demonstrated with data obtained from urine samples from different patients which tested positively for cannabinoids using a fluorescence polarization immunoassay.


Restriction endonuclease patterns of adenovirus type 12 and 18. Restriction endonuclease analysis using 10 restriction enzymes was performed on six and three wild isolates of adenovirus (Ad) type 12 and 18, respectively. Among the Ad12 strains, five DNA variants could be identified. The degree of pairwise comigration of restriction fragments suggests a high degree of genomic diversity within Ad12. The wild isolates of Ad18, on the other hand, displayed a low degree of genetic variability and comprised one DNA variant closely related to the prototype strain. The BglII, BstEII, and HindIII restriction endonuclease patterns of Ad18 were inconsistent with those originally presented. Identical RE-patterns among Ad18 prototype strains (DC) obtained from four different sources, including directly from the American Type Culture Collection, verify that the genuine Ad18 prototype was used in the present study. Using the revised restriction patterns of BglII, BstEII, and HindIII, a proper identification of Ad18 will be facilitated.


Detection of flavivirus antigens in purified infected Vero cell plasma membranes. The types of Kunjin virus-specified proteins present in purified Vero cell plasma membrane were studied. Immunofluorescence of unfixed Kunjin virus-infected whole cell monolayers, indicated that two structural proteins (envelope and prM) and three non-structural proteins (NS1, 3 and 5) were found at the plasma membrane. There was no obvious progressive accumulation of the observed antigens over the time periods between 8 to 24 h p.i. Thus SDS-PAGE analysis was performed using purified radiolabelled Vero cell plasma membranes. From the protein profiles, all five antigens detected by immunofluorescent staining were also present. In addition, two smaller molecular weight non-structural proteins NS4B and NS2B were also observed. Generally, all the non-structural proteins found in the purified plasma membranes were of the same molecular weights as those found in infected whole cell lysate. Interestingly, both the structural proteins, i.e., envelope (E) and prM proteins in the plasma membrane sample were of higher molecular weights as compared to the counterparts in the infected whole cell lysate. The envelope protein of purified extracellular Kunjin virus was also lower in molecular weight compared to the same protein in the plasma membrane.


Assessment of inactivation of hepatitis A vaccine by compound PCR. Assuring the complete inactivation of hepatitis A virus (HAV) vaccine commonly requires prolonged tissue culture amplification, followed by detection of virus antigen in cell lysates. A reliable, but faster, alternative procedure is highly desirable since it will permit the prescreening of experimental batches of killed HAV, prior to tissue-culture amplification. We established experimental conditions for simultaneous, polymerase chain reaction (PCR)-based amplification of viral and cellular mRNA sequences from infected cell RNA (compound PCR). Under these conditions, the presence of virus-specific amplified sequences, as detected by Southern blot, allows the identification of incompletely inactivated vaccine batches with a threshold practically identical to that of the more time-consuming subculture and ELISA. Compound PCR is, by its nature flexible enough for adaption to different requirements and it should prove useful for rapid prescreening of vaccine batches and pilot studies for improvement of inactivation protocols.


A liquid-hybridization method for typing the Vp4 and Vp7 genes of bovine rotaviruses. A simple liquid-hybridization assay was developed which allows assessment of the degree of hybridization between the two serotype-determining genes of the bovine rotavirus strain UK and the homologous genes of the isolate under test. 32P-labelled transcription probes were produced from cloned complementary DNA (cDNA) copies of UK gene segments 4 and 8 and hybridized to double stranded RNA (dsRNA) extracted from rotavirus-positive field samples. Subsequent treatment with ribonuclease A (RNase A), separation of the RNase A-resistant hybrid fragments by polyacrylamide gel electrophoresis (PAGE) and autoradiography yielded a specific, reproducible banding pattern for each isolate. A total of 74 field samples was tested by both the hybridization assay and by an enzyme-linked immunosorbent assay (ELISA) using serotype-specific monoclonal antibodies (Mabs). The results obtained were in excellent agreement and confirmed that serotype G6 rotaviruses predominated. Hybridization of these G6 viruses with the gene 4 probe suggested that viruses with Vp4s related to that of UK rotavirus are also common. The hybridization assay was more sensitive than the ELISA.


An enzyme-linked immunosorbent assay for detection of porcine parvovirus in fetal tissues. An enzyme-linked immunosorbent assay for porcine parvovirus was developed for laboratory detection of parvovirus antigen in fetal tissues and compared with the fluorescent antibody and haemagglutination tests. The ELISA proved to be superior in terms of specificity, sensitivity, speed of performance and ease of interpretation.


Comparison of three immunoassays for the detection of anti-HHV6. Sera from 96 blood donors were tested for antibody to human herpesvirus 6 by indirect immunofluorescence (IF), circle immunoassay (CIA) and competitive radioimmunoassay (RIA). The correlation between the three assays was good but the CIA and competitive RIA were more sensitive for the detection of HHV6 antibody than indirect IF. The crossreaction of HHV6 antibody with that to the other human herpesviruses was also studied in this blood donor group. No correlation was found between antibody to human herpesvirus 6 by any of the methods described and antibody to any of the other human herpesviruses in these sera.


Generation of macaque B lymphoblastoid cell lines with simian Epstein-Barr-like viruses: transformation procedure, characterization of the cell lines and occurrence of simian foamy virus. Two simian Epstein-Barr-like viruses, a rhesus Epstein-Barr virus and Herpesvirus papio, were used to transform B cells from rhesus or cynomolgus macaques. The resulting cell lines exhibited predominantly a B lymphocyte phenotype and expressed Epstein-Barr virus antigens. The majority of B lymphoblastoid cell lines from macaques, which were seropositive for simian foamy virus, developed giant cells in culture. The cytopathic agent was identified as a foamy virus and was transmissible to human embryonal fibroblasts. Treatment of cell cultures with AZT abolished giant cell formation.


An in vitro system for studying the initial stages of cottontail rabbit papillomavirus infection. Cottontail rabbit papillomavirus (CRPV) infection of an established cottontail epidermal cell line (Sf1Ep) resulted in the production of CRPV-specific transcripts without concomitant morphological transformation. The most abundant transcripts corresponded in size to those of the E6 and E7 open reading frames (ORFs), which are also among the commonest in domestic and cottontail rabbit papillomas. CRPV RNA production was both time- and dose-dependent, with RNA production diminishing with decreasing viral dose and increasing culture passage. Infected cultures contained episomal CRPV DNA, which did not appreciably change in abundance with time but was significantly reduced with culture passage. All features of in vitro infection, especially RNA production, were inhibited by CRPV-neutralizing, but not HPV-11-neutralizing, monoclonal antibodies. Much of this inhibition could be attributed to a blockage of viral penetration, as indicated by the reduction of CRPV DNA within virus-neutralized cultures. The results indicate that, although CRPV infection of Sf1Ep cells was abortive, it serves as a useful model for analysis of early infection events.


Detection of Junin virus by the polymerase chain reaction. Argentine hemorrhagic fever is an often fatal human disease caused by Junin virus, an RNA-containing virus and member of the Arenavirus family. This virus was detected in vitro by the polymerase chain reaction (PCR) procedure. A pair of Junin virus-specific PCR DNA oligonucleotide primers and an oligonucleotide probe were designed from a known portion of the viral RNA sequence. RNA was isolated from Junin virus-infected monkey kidney cells and used to produce complementary DNA (cDNA) by reverse transcription. A DNA segment, 151 +/- 24 bp long, was amplified from this cDNA and characterized by agarose gel electrophoresis and Southern blot hybridization with the Junin virus-specific DNA probe. Sensitivity experiments showed that Junin virus could be detected with nanogram quantities of RNA isolated from virus-infected cells. The rapid and sensitive assay described here may contribute towards the development of a procedure for the early diagnosis of Argentine hemorrhagic fever.


Enhanced detection of respiratory viruses using the shell vial technique and monoclonal antibodies. The shell vial technique using A549 and MDCK cells, coupled with the use of Bartels respiratory viral monoclonal antibodies, was evaluated initially for the detection of 28 previously isolated respiratory viruses. All viruses were recovered and correctly identified. The shell vial-monoclonal antibody technique was then evaluated for virus isolation from 338 respiratory specimens and compared with the conventional tube method. Both methods gave rise to a total of 83 virus isolates. Of these isolates, 68 (20.1%) were isolated and identified by the shell vial-monoclonal method; 60 (17.8%) were culture-positive by the conventional tube method; forty-five (13.3%) were positive by both methods. The shell vial-monoclonal antibody method yielded 12 isolates of influenza A, two isolates of parainfluenza type 3 and one each of parainfluenza types 1 and 3, which were missed by the conventional tube method, indicating the superior sensitivity and specificity of the shell vial-monoclonal antibody method (Chi-square analysis, P = 0.001) for the detection of these viruses. Of the 50 RSV isolates, 29 were detected by both methods and there were 21 discrepancies between the two methods. The shell vial-monoclonal antibody method also improved the turn-around time for the respiratory virus groups.


Examination of the buoyant density of hepatitis C virus by the polymerase chain reaction. Sucrose and cesium chloride density gradients were used to fractionate hepatitis C virus (HCV) infectious chimpanzee plasma. The fractionated plasma was then evaluated for HCV RNA sequences using cDNA synthesis and the polymerase chain reaction (cDNA/PCR). cDNA/PCR detectable HCV RNA was identified repeatedly in two regions. One region was at the top of the gradients with a buoyant density of < or = 1.03 g/cm3, the other at a density of approximately 1.18-1.21 g/cm3.


Rapid detection of equine herpesvirus type-1 antigens in nasal swab specimens using an antigen capture enzyme-linked immunosorbent assay. An antigen capture enzyme-linked immunosorbent assay (ELISA) was developed for the detection of equine herpesvirus type-1 (EHV-1) antigens in nasal swab specimens. The test was designed as a solid phase, amplified sandwich assay in which an EHV-1 specific monoclonal antibody was used to capture virus antigen and polyclonal antisera used to detect antigen bound to the test plates. Eight monoclonal antibodies were tested for their ability to capture virus antigen and one was selected for routine use. The sensitivity and specificity of the ELISA was compared with that of virus isolation using swabs from ponies which were experimentally infected with EHV-1. Of 72 nasal swabs collected, 32 were found to be positive by both virus isolation (VI) and ELISA, a further 15 samples were positive by VI alone, but none of the samples were positive by ELISA and negative by VI. This yielded an overall assay sensitivity of 68% and specificity of 100%. The assay proved useful for diagnosis since virus antigen was detected during the first four days post-infection which corresponded to the acute phase of disease when some clinical symptoms were apparent. In addition, the assay could be completed within one day when antibody coated plates were available.


Comparison of MRC-5 and continuous cell lines for detection of cytomegalovirus in centrifugation cultures. Continuous cell lines were assessed for use for rapid human cytomegalovirus (HCMV) detection procedures combining tissue culture, centrifugation, and immediate early antigen (IEA) immunostaining. Human cells (MRC-5 embryonic fibroblasts, U-373MG astrocytoma cells, differentiated teratocarcinoma (Tera-2) cells), murine cells (BALB/c-3T3 and Y-1 cells), BHK21 hamster cells, and mink lung (ML) cells were first inoculated with HCMV laboratory strain. IEA synthesizing cells were detected by immunoperoxidase assay using a monoclonal antibody. ML cells and differentiated Tera-2 cells exhibited more positive cells than MRC-5 cells. BHK21, and MRC-5 cells were equivalent in sensitivity whereas U-373MG, BALB/c-3T3, and Y-1 cells had only reduced IEA positive cells. When 63 urine specimens were inoculated onto MRC-5, ML and differentiated Tera-2 cells, 20 (31.7%) were positive in MRC-5 cells versus 18 (28.5%) in ML or Tera-2 cells. Moreover, greater numbers of infected cells were detected in MRC-5 cells than in these two cell lines. MRC-5 cells were superior for detection of HCMV in clinical samples by centrifugation cultures.


Detection of false seroconversions in cytomegalovirus seronegative platelet donors by serial complement fixation or IgG-specific radioimmunosorbent tests. A panel of 421 cytomegalovirus (CMV) seronegative-platelet donors was followed using complement-fixation (CFT) and IgG-specific radioimmunosorbent tests (RIST) to detect seroconversion. During 4623 person-months of observation 2452 serum specimens were tested, and the annual rate of seroreactivity detected by RIST was 56%. However, most of the positive RIST results were followed by negative results with later serum samples. CFT seroreactivity was a poor predictor of RIST seroreactivity. The high rate of transient seroreactivity detected by RIST may have reflected the lack of absolute specificity of the test. Alternatively, the results could illustrate the difficulty of determining whether an individual harbours latent CMV infection using antibody tests when some seronegative individuals appear to be latently infected. In the light of our observations recommendations for CMV antibody testing of blood donors are proposed.


A reverse polymerase chain reaction method for detection of human cytomegalovirus late transcripts in cells infected in vitro. A rapid and simple application of the polymerase chain reaction is described for the detection of human cytomegalovirus (HCMV) mRNAs in cells infected in-vitro. The method was first used to study the transcription of two HCMV genes, and confirm the link between the transcription of one, encoding for the major capsid protein, and viral replication. The oligonucleotides chosen in this region were specific for HCMV genome and sensitivity experiments showed that a single infected cell in 5 x 10(5) can be detected. Detection of this transcript should be suitable for diagnostic purposes, permitting the distinction between latency and active infection.


Cloning and expression of a cDNA encoding the Epstein-Barr virus thymidine kinase gene. A clone of the Epstein-Barr virus (EBV) thymidine kinase (TK) gene was derived from a cDNA library of P3HR1 cells. The gene product was expressed as a fusion protein in a procaryotic system by using T7 RNA polymerase. The recombinant TK showed a molecular mass of 67 kDa and was biologically active. Antiserum raised in mice immunized with partially purified TK recognized an antigen present in EBV-superinfected Raji cells using an indirect immunofluorescence assay.


Quantitative flow cytometry of mouse mammary tumor virus envelope glycoprotein (gp52): alternative measures of hormone-mediated change in a viral cell surface antigen. An immunofluorescence procedure with C3H mouse mammary tumor cells (Mm5mt/cl) has incorporated flow cytometry to provide a fluorescence-based measurement of changes in the mouse mammary tumor virus (MMTV) cell surface glycoprotein (gp52). A comparison of mean channel fluorescence intensity (delta mean) of cell populations stained with immune sera and NRS permitted a gp52-specific signal to be measured for controls and cells treated with 10(-6) M dexamethasone (Dex). Three different methods have been developed to quantitatively compare gp52-related fluorescence on control and hormone-treated cells. First, delta mean, measured as a gp52-specific difference in channel number was 169-209 for control cells and 299-341 for Dex-treated cells. These fluorescence measurements with 4 different sera demonstrated gp52-specific increases due to Dex treatment of 141, 130, 143, and 115 channels. A second method of gp52 quantitation determined the percentage shift in staining populations over NRS and specified channel intensity markers. Dex treatment resulted in a 6.9 to 32.4% shift over channel 508 (NRS marker) and a more marked shift of 45.5 to 49.2% over channel 676 (control cell marker). A third methodology utilized fluorescein bead standards to calculate molecules of equivalent soluble fluorescein (MESF). These MESF determinations permitted hormonal effects to be measured as fold increases over controls. Dex induction of gp52 for C3H and GR mammary tumor cells ranged from 1.5 to 9.1 fold increases. Alternative steroid treatments and antibody directed against the internal cytoplasmic MMTV P27 provided negative controls for measurements of changing gp52 levels.


Rapid DNA diagnosis of herpes simplex virus serotypes. The presence of nucleotide sequences specific for each of herpes simplex virus (HSV) serotypes was demonstrated. These sequences were applied for dot DNA-DNA hybridization and for PCR for rapid DNA diagnosis of HSV infections. These sequences were found by molecular cloning of HSV-DNA fragments after digestion of DNA by KpnI enzyme. The type 1-specific sequence was found around the 5' end of BamHI B-fragment in the L region of type 1 DNA (corresponds to alpha gene 27, promoter-regulatory region) and the type 2-specific sequence was around the junction region of the L and S of type 2 DNA (corresponds to a' sequence). Both simple dot blot hybridization and PCR of HSV DNA's, employing these type-specific nucleotide sequences, were proven to be much more useful than immunofluorescence in terms of type-specific diagnosis of HSV infections.


Blocking ELISA for distinguishing infectious bovine rhinotracheitis virus (IBRV)-infected animals from those vaccinated with a gene-deleted marker vaccine. A sensitive and specific blocking enzyme-linked immunosorbent assay (ELISA) was developed to distinguish infectious bovine rhinotracheitis virus (IBRV)-infected animals from those immunized with a glycoprotein gIII deletion mutant, IBRV(NG)dltkdlgIII. For this ELISA, undiluted test sera are used to block the binding of an anti-IBRV gIII monoclonal antibody (mAbgIII)-horseradish peroxidase (HRPO) conjugate to gIII antigen. TMB substrate is used for color development. Negative S/N values (defined as the absorbance at 650 nm of test sera/absorbance at 650 nm of negative control sera) of > 0.80 were obtained with immune sera from gnotobiotic cattle immunized with several bovine viruses, with bovine antisera to bovine herpesvirus-2, and vesicular stomatitis virus, with porcine antisera to pseudorabies virus and parvovirus, and with normal sera from heterologous species. Negative S/N values were also obtained with sera from rabbits twice vaccinated with IBRV(NG)dltkdlgIII. However, the S/N values became positive (S/N < 0.8) 10 to 17 days after the rabbits were challenge exposed to virulent IBRV(Cooper). Most of 116 sera (84%) from feedlot cattle with virus neutralization (VN) titers of < 1:2 or < 1:4 had negative S/N values > 0.8, but 18 sera with negative VN titers had positive S/N values, consistent with observations indicating that an IBRV outbreak was occurring in one of the feedlot herds. Thirty nine sera (98%) from feedlot cattle with VN titers of 1:2 to 1:128 had positive S/N values (< 0.8). One serum with a VN titer of 1:2 had a borderline (+/-) S/N value of 0.81. After immunization with a commercial gIII-positive IBRV vaccine, 115/116 sera with VN titers of 1:2 to 1:256 had positive S/N values (< 0.8). One serum with a VN titer of 1:2 had a negative S/N value of 0.83. Serum from one vaccinated animal that failed to seroconvert after vaccination (VN < 1:4) showed a strongly positive ELISA S/N of 0.48.


Processing procedures for recovering enteric viruses from wastewater sludges. A powdered beef extract specially formulated for recovering viruses from environmental samples and designated as beef extract V was evaluated using indigenous and viral seeded wastewater sludge samples. When beef extract V was used to process activated and aerobically digested sludge solids, virus recoveries were shown to be similar to other methods that used commercially available supplemented beef extract. When used to process other sludge solids (primary and activated without primary clarification), cytotoxicity resulted in the BGM cell line used for virus assay. When these sludge solids were processed with the supplemented commercially marketed beef extract cell toxicity did not occur. Metal concentrations in the processed sewage sludge eluates were analyzed, but based on the levels observed they could not be shown as the source of the cytotoxicity. This did not exclude possible synergistic cytotoxic effects or organometal complexes. The commercially marketed beef extract was supplemented with either a floccing aid (FeCl3), a filter aid (Celite) or a floc prepared from paste beef extract. The paste beef floc supplement proved to be the most useful and reliable method for processing for viruses from wastewater sludge solids.


Epidemiological evaluation of a monoclonal ELISA detecting bovine viral diarrhoea pestivirus antigens in field blood samples of persistently infected cattle. An enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies for capture and detection, was developed for detecting bovine viral diarrhoea virus (BVDV) antigens in blood samples. The test was evaluated using 761 field samples of known status (viraemic or not). When an appropriate cut-off value was chosen, the sensitivity, specificity, and predictive values of the assay were 100%, higher than the values obtained by classical virus isolation. Correlation with the latter technique exceeded 90%. The ELISA is a good candidate for replacing virus isolation as a reference method for BVDV antigen detection in persistently infected carriers. A method based on the mean of the standard deviation ratio can be used to choose the cut-off value in order to optimise reproducibility.


An M-antibody capture radioimmunoassay (MACRIA) for detection of JC virus-specific IgM. A solid-phase M-antibody capture radioimmunoassay (MACRIA) for detecting JC-specific IgM is described. The assay is based on a JC-specific monoclonal antibody (17.7.6) and Nonidet P40-treated, glycine-extracted antigen. MACRIA is more sensitive for JC IgM detection than haemagglutination inhibition (HI) following serum fractionation on a sucrose density gradient, and can be applied to large numbers of sera. The specificity of the assay was confirmed by examining sera from several acute virus infections and also those containing rheumatoid factor. Sera collected from renal transplant recipients with known active JC virus infection were found to contain more than 5 units of JC IgM. In this group of patients JC IgM represents either primary or reactivated JC infection. JC IgM was detected by MACRIA in 15 of 100 unselected blood donors, indicating that JC IgM is frequently produced in healthy seropositive individuals. Thirteen of the 15 sera positive from blood donors contained only low levels of JC IgM (< 5 units), but the specificity of all these results was confirmed in a blocking assay. It is suggested that these low levels of JC IgM may occur in up to 28% of seropositive individuals and result from active JC antigenic stimulation in healthy immunocompetent adults.


Residual adrenocortical function after bilateral adrenalectomy for pituitary-dependent Cushing's syndrome. The incidence of residual, functioning adrenal tissue in patients treated by total bilateral adrenalectomy for Cushing's disease is not known. Between 1962 and May 1988 50 patients with Cushing's disease were treated by bilateral adrenalectomy. Of these patients, 29 underwent surgery between 1962 and 1980, when bilateral adrenalectomy was the treatment of first choice in our hospital in patients with pituitary-dependent Cushing's syndrome. In 37 patients the presence or absence of cortisol-producing tissue could be evaluated (follow-up period, median and range, 8.3 and 0.1-18.9 yr). Evidence of functioning cortisol-producing tissue (plasma cortisol level > 50 nmol/L after stopping glucocorticoid and mineralocorticoid substitution therapy for 24 h) was found in 9 patients (24%). Plasma cortisol levels in these 9 patients varied between 60 and 330 nmol/L (mean +/- SD, 180 +/- 100 nmol/L). Signs and symptoms of recurrent Cushing's syndrome were present in only 1 patient. There was no difference in plasma ACTH levels and duration of follow-up between the patients with and without evidence of functioning cortisol-producing tissue. In all 9 patients detectable aldosterone levels indicated endogenous mineralocorticoid production, whereas in only 1 patient adrenaline was detectable in the circulation. In 8 of the 9 patients suspected of functioning cortisol-producing tissue we performed a stimulation test with synthetic ACTH (1-24). In 2 patients plasma cortisol levels rose, in 6 they remained virtually unchanged. Although we found clinically relevant signs and symptoms of Cushing's syndrome in only 1 of the 50 patients, the relatively high incidence of residual, functioning adrenal tissue after "total" adrenalectomy for pituitary-dependent Cushing's syndrome necessitates continuous surveillance for recurrent Cushing's syndrome. There is no place for routine administration of full replacement doses of glucocorticoids after total adrenalectomy.


Iodine-induced subclinical hypothyroidism in euthyroid subjects with a previous episode of amiodarone-induced thyrotoxicosis. Amiodarone-induced thyrotoxicosis (AIT) occurs most frequently in patients with underlying thyroid disease and is generally believed to be due to the iodine contamination of amiodarone and iodine released by the metabolism of the drug. We and others have suggested that the thyrotoxicosis may also be secondary to amiodarone-induced thyroiditis. To further determine the etiology of AIT, we administered large doses of iodides [10 drops saturated solution of potassium iodide (SSKI) daily] to 10 euthyroid patients long after an episode of AIT believed to be due at least in part to amiodarone-induced thyroiditis. Six of these 10 patients had an abnormal iodide-perchlorate discharge test before SSKI administration, indicating a subtle defect in the thyroidal organification of iodide. During SSKI administration, 6 patients developed marked iodine-induced basal and/or TRH-stimulated serum TSH elevations, 2 had suppressed basal and TRH-stimulated TSH values, and 2 had normal TSH responses compared to SSKI-treated euthyroid subjects with no history of amiodarone ingestion or thyroid disease. Serum T4 and T3 concentrations remained normal and unchanged during SSKI administration in both the AIT patients and control subjects. These results strongly suggest that excess iodine may not be the cause of the hyperthyroidism associated with amiodarone therapy, especially in those patients with probable amiodarone-induced thyroiditis. Furthermore, like patients with a previous history of subacute thyroiditis and postpartum thyroiditis, the present results suggest that some patients with a previous history of AIT may be at risk to develop hypothyroidism when given excess iodine.


Pulsatile human corticotropin-releasing hormone prevents dexamethasone-induced suppression of the plasma cortisol response to hypoglycemia in normal men. Insulin-induced hypoglycemia causes a sequential stimulation of all three components of the hypothalamic-pituitary-adrenal axis. States of acute glucocorticoid excess, such as the overnight (1 mg) dexamethasone suppression test (DST), inhibit both the basal cortisol level and the response to an insulin tolerance test (ITT). However, whether this negative feedback effect is exerted primarily at the hypothalamic or the pituitary level is not clear. To explore this question further we have examined the cortisol response to insulin-induced hypoglycemia in three experimental settings, in the following order: 1) a control ITT performed at 0900 h after an overnight hospital stay (cITT); 2) an ITT at 0900 h after oral dexamethasone, 1 mg, at 2300 h on the previous evening (DST + ITT); and 3) an ITT at 0900 h after dexamethasone, 1 mg, at 2300 h and hCRH, 1 microgram/kg iv, at 90 min intervals from 0100-0700 h (DST+hCRH + ITT). The response to ITT was defined as the peak cortisol increment (peak minus baseline). Since the study objective was to test whether overnight pulsatile hCRH could prevent dexamethasone-induced suppression of the response to a morning ITT, only subjects that demonstrated a greater than 25% decrease in the cortisol response to DST + ITT vs. cITT received the full protocol (five of nine normal men). Basal ACTH and cortisol secretion remained suppressed throughout the night during both the Dex + ITT and Dex + hCRH + ITT studies when compared to the control study (cITT, P < 0.05). However, the cortisol response to hypoglycemia during DST + hCRH + ITT was significantly greater than during DST+ITT (P < 0.05) and was similar to the cITT response. Thus, pulsatile hCRH, administered during the 10 h between dexamethasone and the subsequent hypoglycemic stimulus, prevented acute suppression by dexamethasone of the cortisol response to hypoglycemia. We conclude that the dexamethasone-induced inhibition of the cortisol response to hypoglycemia results primarily from suppression by dexamethasone of basal hypothalamic corticotropin-releasing factor and the consequent impairment of corticotroph responsiveness to exogenous and endogenous corticotropin-releasing factor.


Inhibition of ovulation by low-dose mifepristone (RU 486). Mifepristone (RU 486) is a potent antigestagen and antiglucocorticoid which when given at a dose of 25-600 mg disrupts folliculogenesis, inhibits ovulation and induces menses in healthy women. This study reports the effects of much lower doses of mifepristone than used previously, given for the duration of a complete menstrual cycle. Healthy female volunteers (n = 11) with regular menstrual cycles were given mifepristone at a daily dose of 5 mg (n = 6) or 2 mg (n = 5) for 30 days, beginning immediately after an ovulatory placebo cycle. Mifepristone prevented menstruation for the duration of the treatment period, with recurrence of menses 15-29 days after replacement of mifepristone with placebo. Daily mifepristone given in either 5 mg or 2 mg doses inhibited ovulation, as indicated by the lack of a rise in urinary pregnanediol excretion. The excretion of oestrone glucuronide in urine rose during treatment, suggesting ovarian follicular development. Inhibition of ovulation appeared to result from a failure of the positive feedback effect of oestradiol on the hypothalamo-pituitary axis, as no surges of luteinizing hormone were seen despite pre-ovulatory levels of oestrone glucuronide being measured during exposure to mifepristone. The cycle immediately following treatment was shorter than the pre-treatment cycle, with lower peak levels of pregnanediol glucuronide, suggesting an inadequate luteal phase. Recovery from the effects of mifepristone treatment was more rapid after 2 mg than after 5 mg and one subject conceived in the immediate post-treatment phase, indicating adequate ovulation and luteinization.(ABSTRACT TRUNCATED AT 250 WORDS)


Inflammatory mediators in demyelinating disorders of the CNS and PNS. Work in both experimental models and human disorders of the central and peripheral nervous system has delineated multiple effector mechanisms that operate to produce inflammatory demyelination. The role of various soluble inflammatory mediators generated and released by both blood-borne and resident cells in this process will be reviewed. Cytokines such as interleukin (IL)-1, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha are pivotal in orchestrating immune and inflammatory cell-cell interactions and represent potentially noxious molecules to the myelin sheath, Schwann cells, and/or oligodendrocytes. Arachidonic acid metabolites, synthesized by and liberated from astrocytes, microglial cells and macrophages, are intimately involved in the inflammatory process by enhancing vascular permeability, providing chemotactic signals and modulating inflammatory cell activities. Reactive oxygen species can damage myelin by lipid peroxidation and may be cytotoxic to myelin-producing cells. They are released from macrophages and microglial cells in response to inflammatory cytokines. Activation of complement yields a number of inflammatory mediators and results in the assembly of the membrane attack complex that inserts into the myelin sheath-creating pores. Activated complement may contribute both to functional disturbance of neural impulse propagation, and to full-blown demyelination. Proteases, abundantly present at inflammatory foci, can degrade myelin. Vasoactive amines may play an important role in breaching of the blood-brain/blood-nerve barrier. The importance of nitric oxide metabolites in inflammatory demyelination merits investigation. A better understanding of the multiple effector mechanisms operating in inflammatory demyelination may help to devise more efficacious antigen non-specific therapy.


Cytomegalovirus induction of tumor necrosis factor-alpha by human monocytes and mucosal macrophages. Cytomegalovirus (CMV) is a major cause of inflammatory organ disease in immunosuppressed persons. To elucidate the mechanisms of CMV-induced inflammation, we investigated whether tumor necrosis factor-alpha (TNF-alpha) was involved in the pathogenesis of CMV colitis in patients with AIDS. In in situ hybridization experiments, TNF-alpha mRNA was shown to be abundantly present in colonic mucosa from AIDS patients with CMV colitis but not in colonic mucosa from control (AIDS and normal) subjects. The TNF-alpha transcripts, identified in macrophage-like cells containing cytomegalic inclusions, were positively associated with CMV, but not HIV-1, within the mucosa. In in vitro experiments, a patient-derived isolate of CMV, but not HIV-1Ba-L, induced human monocytes to express TNF-alpha mRNA and to release increased levels of TNF-alpha peptide following stimulation. CMV induction of TNF-alpha may play a critical role in CMV-induced inflammation and, since TNF-alpha upregulates expression of HIV-1, offers a mechanism by which CMV could serve as a co-factor for HIV-1 expression without both viruses infecting the same cell.


Balloon angioplasty enhances the expression of angiotensin II AT1 receptors in neointima of rat aorta. Angiotensin II is a vasoactive peptide and may act as a growth factor in vascular smooth muscle cells. Experimental injury of the rat aorta causes rapid migration of medial smooth muscle cells and their proliferation resulting in the formation of neointima. We have examined, using quantitative autoradiography, the expression of angiotensin II receptor subtypes AT1 and AT2, and angiotensin-converting enzyme, in the neointima formed in the rat thoracic aorta 15 d after balloon-catheter injury. In contrast to the normal aortic wall, which contained both AT1 and AT2 receptors (80% and 20%, respectively), neointimal cells expressed almost exclusively angiotensin II AT1 receptors. The apparent number of these receptors was fourfold higher in the neointima compared to that in the normal aortic wall. The affinities of the neointimal receptors to angiotensin II or to the AT1 receptor antagonist, losartan, were not different from those in the normal aortic wall. Angiotensin-converting enzyme binding in the neointima was not different from that in the media of the uninjured aorta. Our data suggest that angiotensin II AT1 receptors may have a significant role in injury-induced vascular smooth muscle proliferation and migration.