Identification and partial characterization of two classes of receptors for human hepatocyte growth factor on adult rat hepatocytes in primary culture.

To characterize the receptor(s) for human hepatocyte growth factor (hHGF), a physiological hepatotrophic factor involved in liver regeneration following hepatic injury, recombinant hHGF (rhHGF) was radioiodinated. The labeled rhHGF retained its full biological activity on adult rat hepatocytes in primary culture. The specific binding of [125I]iodo-rhHGF to hepatocytes reached a plateau within 240 min at 4 degrees C. Scatchard plot analysis of the binding data suggested the presence of two classes of high affinity binding sites for [125I]iodo-rhHGF. One of the sites had a dissociation constant (Kd) of about 4.6 pM with 300 sites/cell and the other has a Kd of about 275 pM with 15,160 sites/cell. Unlabeled rhHGF displaced cell surface-bound [125I]iodo-rhHGF in a dose-dependent manner as did native hHGF purified from plasma of patients with fulminant hepatic failure. However, other growth factors to rat hepatocytes in primary culture such as insulin and human epidermal growth factor, and proteins which have high amino acid sequence-homology to hHGF such as plasminogen and prothrombin, did not compete with [125I]iodo-rhHGF in the binding, which suggests the binding was specific to hHGF. Covalent cross-linking experiment of [125I]iodo-rhHGF to cell surface receptor(s) on hepatocytes showed there were two macromolecular species with apparent molecular weights of 330,000 and 230,000. Unlabeled rhHGF and native hHGF competed for the binding of [125I]iodo-rhHGF to the two macromolecular species, but insulin, human epidermal growth factor, plasminogen, and prothrombin did not. Based upon our estimated molecular weight of rhHGF = 84,000, these results suggest that hHGF specifically binds to two polypeptides of 246,000 and 146,000 daltons which are likely to represent the hHGF receptors of primary cultured rat hepatocytes.


The effect of low density lipoproteins, cholesterol, and 25-hydroxycholesterol on apolipoprotein B gene expression in HepG2 cells.

The purpose of the present study was to examine the effects of exogenous cholesterol on the apolipoprotein (Apo) B gene expression in HepG2 cells. Pure cholesterol had no significant effect on either the cellular content of cholesteryl esters or the net accumulation of neutral lipids and ApoB in the culture medium. By contrast, addition of 25-hydroxycholesterol increased the net accumulation of cholesteryl esters in cells and medium by 2-3-fold and decreased that of unesterified cholesterol by 50% in both compartments. A 33% reduction in the cellular content of triglycerides was commensurate with a 40% increase in their accumulation in the medium. A significant 3-fold increase in the net accumulation of ApoB in the medium was predominantly due to enhanced secretion of newly synthesized ApoB as established by pulse-chase studies. The stimulation in ApoB secretion was accompanied by a 55% increase in cellular ApoB mRNA. Under these experimental conditions, the low density lipoprotein receptor activity was decreased by only 12-20%. Addition of progesterone prevented the effects of 25-hydroxycholesterol. The changes in the concentration of neutral lipids and ApoB were reflected in the composition of secreted "low-density" lipoproteins. These particles had increased percentage contents of cholesteryl esters and ApoB and a decreased percentage content of unesterified cholesterol in comparison with lipoproteins produced by control cells. The rate of ApoB production was not correlated with the triglyceride mass in the cells but was positively correlated with the cellular and secreted cholesteryl esters and secreted triglycerides. With the exception of unchanged cellular unesterified cholesterol and ApoB mRNA levels, plasma low density lipoprotein had similar, although less pronounced, effects on the production of neutral lipids and ApoB. These results demonstrate that in HepG2 cells the synthesis and secretion of ApoB and cholesteryl esters are tightly coupled and that 25-hydroxycholesterol increased the concentration of ApoB-containing lipoproteins primarily by stimulating their production rather than reducing their catabolism.


Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydroxypyridine (MPTP) on ganglionic transmission.

1. The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydroxypyridine (MPTP) caused blockade of ganglionic transmission as evidenced by a marked decrease of the amplitude of evoked post-ganglionic compound action potential of the isolated superior cervical ganglion of rat. 2. This effect was not related to its dopaminergic neurotoxicity since pretreatment with pargyline did not prevent the ganglion-blocking action of MPTP. The effects of MPTP on ganglionic transmission were qualitatively similar to those of its narcotic analgesic analogue meperidine. The effects of both drugs were antagonized by the narcotic antagonist naloxone, indicating that the ganglion-blocking effect of MPTP also involved opiate receptors. 3. Like meperidine and morphine, the ganglion-blocking action of MPTP was reduced when the concentration of Ca2+ in the Locke solution was elevated. The blocking effect of methadone on transmission was not reduced by naloxone or elevated Ca2+. 4. The effect of MPTP on nerve action potential was minimal, indicating that the drug blocked synaptic transmission in the ganglion. 5. These results suggest that MPTP inhibits synaptic transmission in the isolated superior cervical ganglion of the rat, probably by interfering with Ca2+ function.


A simplified method for the detection of maedi-visna virus RNA by in situ hybridization.

A simplified in situ hybridization method for the detection of maedi-visna virus (MVV) RNA in cultured cells using 35S-labelled DNA probes is described. The protocol currently used in this laboratory for the in situ detection of MVV RNA involves paraformaldehyde fixation followed by extensive cellular pretreatment prior to hybridization. It was found that substitution of paraformaldehyde fixation with brief acetone treatment and the removal of subsequent pretreatment steps gave a similar level of hybridization signal to that of our standard protocol. Acetone fixed, non-pretreated samples were used to develop a double labelling procedure in which immunocytochemistry and in situ hybridization were combined to allow the simultaneous detection of visna virus antigens and RNA within the same cell.


Colchicine treatment in the preparation of varicella-zoster virus inocula.

Two methods, ultrasonic and colchicine treatment, for preparing varicella-zoster virus (VZV) inocula were compared. Ultrasonic treatment was unsuccessful in achieving the release of detectable, infectious, cell-free VZV from infected human foreskin fibroblast (SF) cells. Sonication of infected SF cells resulted in losses of virus infectivity in direct correlation with losses in cell viability (r = 0.82). In the second method of generating VZV inocula, VZV-infected SF cells were treated for 15 min with 15-20 ng of colchicine per cell. Colchicine treatment maintained short-term survival of virus infectivity, but resulted in a dramatic decrease in long-term cell viability. In addition, colchicine-treated cells could not support the replication of VZV.


A monoclonal antibody capture enzyme-linked immunosorbent assay for detecting antibodies to infectious bursal disease virus.

A monoclonal antibody capture enzyme-linked immunosorbent assay (mAb-ELISA) for antibodies to infectious bursal disease virus (IBDV) in chicken sera was developed and compared with conventional ELISA. When sera from farm chickens were tested by the two ELISAs and serum neutralization (SN), the correlation rate between SN and mAb-ELISA was 100% (49/49), and that between SN and conventional ELISA was 81.6% (40/49). In mAb-ELISA, all of the sera that were antibody-negative by SN had low absorbance values (below 0.05), and the absorbance values correlated closely with the SN titers. In the conventional ELISA, however, the sera antibody-negative by SN had various absorbance values ranging from 0.06 to 0.32. mAb-ELISA had much lower non-specific reactions than the conventional ELISA against sera from IBD-negative chickens.


A simple and rapid method of high quantity DNA isolation from cervical scrapes for detection of human papillomavirus infection.

Infection with human papillomavirus (HPV) is an important etiological factor in the development of cervical cancer, and detection of the viral genome is of prognostic importance, particularly for preneoplastic lesions. We developed a simple, easy and efficient non-organic method of DNA extraction from cervical scrapes for reliable detection of HPV DNA sequences. The method involves incubation of cell nuclei in higher concentration of proteinase K at 65 degrees C for 2.5 h. Following prolonged incubation at higher temperature, the enzyme is autoinactivated and the DNA isolated can be used directly for analysis without further purification. The recovery of DNA is more than 95% and it can be easily cleaved by restriction enzymes and is suitable for amplification by the polymerase chain reaction (PCR). The whole procedure is carried out in a single Eppendorf tube and a large number of specimens can be processed at a time without any error of handling. DNA extracted from a single smear sample is sufficient to conduct as many as four different molecular biology tests. This provides an opportunity for verification of sensitivity, specificity and reliability of each test for diagnosis of HPV infection without resorting to biopsy.


Tn9 CAT gene contains a promoter for vaccinia virus transcription: implications for reverse-genetic techniques.

Vaccinia virus-dependent CAT expression was observed in virus-infected cells cotransfected with a promoterless CAT gene. Restriction endonuclease resection of the CAT plasmid indicated that expression was due to recognition by vaccinia virus RNA polymerase of sequences within the CAT gene itself, probably located within the 5' untranslated region of the gene. This observation is relevant to the design of reverse-genetic systems which use CAT as a reporter gene to detect replication of negative-strand RNA virus pseudogenomes.


Lupus anti-DNA antibodies bearing the 8.12 idiotype appear to be somatically mutated.

Anti-DNA antibodies in systemic lupus erythematosus (SLE) sera were analyzed using an antiidiotype designated 8.12 which recognizes a determinant on lambda light chains highly expressed in SLE sera. Eight of ten normal individuals had peripheral blood lymphocytes which produced high-titered 8.12-positive antibodies, following transformation with Epstein Barr virus, implying that the 8.12-reactive sequence originates in the germline gene (GLG). Of 58 SLE sera, 32 contained elevated titers of 8.12-reactive antibodies. Twenty-three of these sera had 8.12-reactive anti-DNA antibodies, suggesting a strong correlation between 8.12 idiotype and DNA binding. Moreover, 20 of 26 8.12-reactive IgG antibodies and only 4 of 10 8.12-reactive IgM antibodies bound DNA (P less than 0.05). These observations strengthen our previous findings in myeloma sera that DNA binding is associated with IgG isotype in the 8.12 idiotype system and suggest that the acquisition of anti-DNA reactivity in antibodies bearing the GLG idiotype 8.12 is achieved by somatic mutation, a feature of an antigen-driven response.


Cefamandole-susceptible strains of Legionella pneumophila serogroup 1: implications for diagnosis and utility as an epidemiological marker.

The standard selective Legionella medium that contains cefamandole failed to grow legionella pneumophila serogroup 1, subtype Bellingham, from a sputum sample from a patient with nosocomial Legionnaires' disease; the isolate did grow on a similar selective medium that substitutes vancomycin for cefamandole. Two Bellingham isolates from this patient's hospital environment also failed to grow when tested on the cefamandole medium. We tested 106 additional L. pneumophila serogroup 1 isolates that belonged to nine different monoclonal antibody subtypes and demonstrated that susceptibility to cefamandole was rare (10%) and limited to the Bellingham subtype. The diagnosis of Legionnaires' disease may be missed unless the culture protocol includes both a nonselective medium and a selective medium that does not contain cefamandole. In vitro susceptibility to cefamandole also provided an epidemiologic marker that linked a water source for a patient to nosocomial Legionnaires' disease.


Comparison of sensitivities of three commercial MRC-5 cell lines grown in shell vials to cytomegalovirus and responses to enhancing agents.

MRC-5 cells grown in shell vials from three suppliers (Whittaker Bioproducts, Bartels, and ViroMed) were evaluated for sensitivity to human cytomegalovirus. Previous studies done in our laboratory showed that treatment of the cells with dexamethasone (DEX) and dimethyl sulfoxide (DMSO) or these agents plus calcium improved the sensitivity of cells from Whittaker. In this study, we compared the response of the Whittaker cells with those of the cells from Bartels and ViroMed, both treated and untreated, when tested with strain AD169, a strain of wild-type virus, and 41 clinical specimens from various sources. A progressive improvement in sensitivity to AD169 and the wild-type virus was seen for all cell lines with DEX-DSMO and DEX-DMSO-calcium treatments, although even untreated, the Whittaker cells were much more sensitive. Detection of most of the positive clinical specimens was enhanced by both treatments with the Whittaker cells. DEX-DMSO had little or no effect on Bartels or ViroMed cells, while DEX-DMSO-calcium usually effected some increase in the number of inclusions. Six specimens were recovered only on Whittaker cells. Protein studies were done on all groups to determine the effect, if any, of the treatments on the metabolism of the cells.


Rapid method for epidemiological evaluation of gram-positive cocci by field inversion gel electrophoresis.

We report a rapid method for the isolation of intact chromosomal DNA from gram-positive cocci that is suitable for in situ restriction endonuclease digestion in agarose blocks. When combined with a rapid field inversion gel electrophoresis protocol, this approach allows the preparation and electrophoretic analysis of chromosomal restriction fragments produced by rare-cutting enzymes in a total time period of 2 days from start to finish. The utility of the method is demonstrated in the epidemiological evaluation of Staphylococcus epidermidis clusters from two hospitals as well as of additional representative staphylococci and enterococci.


Monoclonal antibody solution hybridization assay for detection of mouse hepatitis virus infection.

A monoclonal antibody solution hybridization (MASH) assay was developed to detect fecal excretion of mouse hepatitis virus (MHV). The assay used a biotinylated cDNA probe to detect viral RNA target sequences by hybridization in solution, capture of hybrids on the solid phase with antibiotin antibody, and immunoassay with an enzyme-labelled monoclonal antibody specific for DNA-RNA hybrids. The MASH assay was used to monitor the time course of enterotropic MHV excretion after oronasal inoculation. Infectivity of the inoculated mice was simultaneously monitored with sentinel animals. The MASH assay detected MHV excretion in all inoculated mice, with the highest mean excretion levels occurring from day 3 through day 9 postinoculation. Mean excretion then decreased gradually to below detection limits by day 21 postinoculation. Sentinels became infected on exposure to inoculated mice up to but not after day 21 postinoculation. Infected sentinel mice showed a time course of virus excretion similar to that of inoculated mice. These results indicate that the MASH assay is useful for rapid, sensitive, and specific detection of MHV in clinical specimens from laboratory mice.


Early detection of bovine leukemia virus by using an enzyme-linked assay for polymerase chain reaction-amplified proviral DNA in experimentally infected cattle.

Bovine leukemia virus is the causative agent of bovine leukosis and has been described in many countries throughout the world. We describe here a sensitive and readily applicable assay for the detection of bovine leukemia proviral DNA. Detection relies on initial amplification of proviral DNA by using polymerase chain reaction (PCR) followed by an enzyme-linked assay (PCR-ELA). Amplification is carried out by using one biotinylated primer and a second primer containing the GCN4 protein binding site. DNA is detected by a colorimetric assay after it is coupled to GCN4-coated plates and subsequently incubated with horseradish-streptavidin peroxidase and the appropriate substrate to produce a chromogenic reaction. It was possible to detect proviral DNA for all of eight bovine leukemia virus-infected calves by 2 weeks postinfection. Use of the more conventional agar gel immunodiffusion assay failed to reveal the presence of the virus in any of the animals up to 4 weeks postinfection. The PCR-ELA detected as little as 0.1 to 0.2 ng of amplified DNA per well, which compares very favorably with ethidium bromide staining of gels, by which 1 to 2 ng per lane was detected. This method lends itself to mass screening, is carried out in a similar way to an enzyme-linked immunosorbent assay, and does not require gel electrophoresis or the use of radioactive gene probes.


Detection of cytomegalovirus in urine samples by an enzyme-linked immunosorbent assay using a monoclonal antibody against the viral 150-kilodalton protein.

McKeating et al. (J.A. McKeating, P.D. Griffiths, and J.E. Grundy, J. Gen. Virol. 68:785-792, 1987; J. A. McKeating, J. E. Grundy, Z. Varghese, and P. D. Griffiths, J. Med. Virol. 18:341-348, 1986; J. A. McKeating, S. Stagno, P. R. Stirk, and P. D. Griffiths, J. Med. Virol. 16:367-373, 1985) reported previously that beta 2 microglobulin inhibits the detection of human cytomegalovirus (CMV) in urine specimens by an enzyme-linked immunosorbent assay (ELISA) with a monoclonal antibody against the glycoprotein of CMV. They postulated that beta 2 microglobulin binds to the viral glycoproteins and masks the antigenic determinants. We developed here an ELISA method for the detection of CMV in urine by using a monoclonal antibody against the viral 150-kDa protein to capture the viral antigen. This assay detected CMV both in culture medium and in urine specifically at concentrations higher than 10(3) PFU/ml and quantitatively at concentrations higher than 10(4) PFU/ml. The sensitivity of the ELISA increased about 10-fold when peroxidase-labeled F(ab')2 from goat anti-human immunoglobulin G was used as a secondary detecting antibody in combination with concentration of the virus in urine samples by ultracentrifugation. The inhibition of ELISA by beta 2 microglobulin was not observed in this ELISA system. When 56 urine specimens from renal transplant recipients were examined for CMV antigens, the ELISA system had a sensitivity of 78% and a specificity of 97%. The positive and negative predictive values of the assay were 95 and 86%, respectively. Furthermore, CMV antigens in urine were quantitated by the assay during the course of typical CMV disease of renal transplant recipient. These results suggest strongly that the measurement of CMV antigens in urine by our rapid and quantitative ELISA system provides very useful data for the monitoring of CMV infections in renal transplant recipients and making decisions about therapy.


Epidemiology of Yersinia pseudotuberculosis and Y. enterocolitica infections in sheep in Australia.

Infections with Yersinia pseudotuberculosis serotype III and Y. enterocolitica serotype O2,3 were found to be common in Australian sheep flocks. Transmission of Y. pseudotuberculosis occurred in late winter and early spring, while Y. enterocolitica transmission occurred from midwinter to early summer. Excretion of Y. pseudotuberculosis was limited to the winter and spring period and was particularly common in 1- and 2-year-old sheep. Infection persisted for up to 14 weeks. Y. pseudotuberculosis infection did not confer immunity to natural infection with Y. enterocolitica. Y. enterocolitica excretion occurred year-round, with the greatest prevalence being in summer and autumn. Infection persisted for up to 29 weeks. Sheep less than 1 year old were most commonly infected with Y. enterocolitica. Infection with either Y. pseudotuberculosis or Y. enterocolitica was rare in aged sheep. Restriction endonuclease analysis of Y. pseudotuberculosis serotype III from sheep, cattle, deer, and pigs showed that the bacterial isolates were genetically indistinguishable. Similarly, Y. enterocolitica isolates from sheep were indistinguishable from those isolated from goats and cattle.


Evaluation of a microtiter plate fluorescent-antibody assay for rapid detection of human cytomegalovirus infection.

The use of monoclonal antibody (MAb) p63-27, which is reactive with the major immediate-early human cytomegalovirus (HCMV) protein pp72, was explored for the rapid diagnosis of HCMV viruria. The rapid assay detected all but 1 of 19 specimens identified by standard virus isolation methods from 1,676 newborn urine specimens, achieving a sensitivity of 94.5% and a specificity of 100%. The monoclonal antibody recognized 260 randomly obtained clinical isolates of HCMV, indicating the suitability of this reagent for use in screening assays. The sensitivity of the microtiter plate method declined rapidly for specimen from older infants and children with congenital CMV infection and virus-infected children attending a day-care center and was judged to be unacceptable for screening populations in this age group.


Use of polymerase chain reaction-amplified Helicobacter pylori urease structural genes for differentiation of isolates.

Helicobacter pylori has been demonstrated as an etiologic agent of human gastritis and peptic ulcer formation. However, there is no straightforward basis to distinguish different isolates. We used the polymerase chain reaction (PCR) to amplify the urease structural subunit genes, ureA and ureB, which, when digested with appropriate restriction endonucleases, allow the differentiation of patterns on agarose gels. PCR amplification was possible with DNA rapidly extracted from H. pylori by alkaline lysis and phenol-chloroform. The 2.4-kb PCR products amplified from 22 clinical isolates and subjected to HaeII restriction endonuclease digestion produced 10 distinct patterns on agarose gels, with two patterns being shared between five and six strains. PCR amplification of the urease genes may enable the differentiation of closely related H. pylori strains by restriction digest analysis of PCR-amplified ureA and ureB genes.


Disk diffusion quality control guidelines for Haemophilus susceptibility tests using cefdinir, CI-960, fleroxacin, temafloxacin, and trospectomycin.

A multilaboratory study to determine disk diffusion quality control ranges for Haemophilus influenzae ATCC 49247 and five investigational drugs was performed. Multiple lots of Haemophilus Test Medium and antibiotic disks were used for replicate testing in conformance with the recommendations of the National Committee for Clinical Laboratory Standards. Quality control disk zone diameter ranges were proposed for cefdinir, CI-960, fleroxacin, temafloxacin, and trospectomycin.


Effects of anticoagulants and storage of blood samples on efficacy of the polymerase chain reaction assay for hepatitis C virus.

Blood samples from 11 patients with posttransfusion hepatitis C virus infection were collected. Each sample was divided into three fractions to obtain sera, sodium-citrated plasma, and heparinized plasma and then tested for HCV RNA by a nested polymerase chain reaction (PCR). Of them, eight sodium-citrated plasma samples, seven serum samples, and no heparinized plasma samples were PCR positive. Eight PCR-positive sodium-citrated plasma samples were exposed to different physical conditions and semiquantified for HCV RNA after serial dilutions. Samples stored at -70 degrees C showed the best preservation of HCV RNA, and storage at the other conditions resulted in only minimal loss of the PCR signal. Therefore, serum or sodium-citrated plasma specimens are satisfactory for detecting HCV RNA by PCR, but heparinized blood specimens are not.


Epithelioid cell histiocytoma. A clinicopathologic and immunohistochemical study of eight cases.

Epithelioid cell histiocytomas histologically resemble the intradermal form of Spitz nevus. We have studied eight patients with epithelioid cell histiocytoma. In addition to the routine stains, histologic sections were prepared by immunohistochemical techniques for S-100 protein, HMB-45, vimentin, alpha 1-antitrypsin, and common leukocyte antigen. Epithelioid cell histiocytoma is characterized by proliferation of angulated epithelioid cells with abundant eosinophilic cytoplasm. Immunostaining for S-100 protein and HMB-45 were negative. The majority of cells had positive reactions to staining for vimentin and alpha 1-antitrypsin. Approximately 20% to 25% of cells also gave positive reactions to common leukocyte antigen. Immunostainings are useful in the differential diagnosis of epithelioid cell histiocytoma from the intradermal form of Spitz nevus.


GABA and epilepsy: their complex relationship and the evolution of our understanding.

Because of its abundance in the brain, its ability to produce hyperpolarizing inhibition of almost all neurons, its association with benzodiazepines, and the discovery that many convulsants inhibited its synthesis, gamma-aminobutyric acid (GABA) has often appeared to be the key to epilepsy. Many assumed that "primary" or "genetic" epilepsy must be a disorder of GABA synapses and that GABA agonists would be universal anticonvulsants if permeability and drug metabolism were controlled. The GABA synthetic gene was a logical "candidate gene" for epilepsy. However, the GABA-deficiency theory of epilepsy is less convincing today. GABA agonists were found to intensify seizures in some rodent and human cases. Absence and other generalized seizures in humans often worsened when treated with GABA transaminase inhibitors such as gamma-vinyl-GABA. Surprisingly, the GABA transaminase inhibitors appear to be more useful in partial than in generalized epilepsies. Neuronal GABA uptake blockers are proconvulsant. GABA agonists aggravate seizures in several mutants, ranging from the photosensitive baboon to the genetically epilepsy-prone rat. How can this be understood? Muscimol injections into the pedunculopontine nucleus increase seizures due to systematically administered convulsants, while the receptor blocker bicuculline suppresses seizures after injection into several brain regions, including the striatum. The result of inhibiting inhibitory circuits is excitation. Studies with GABA uptake blockers and the GABAB agonist baclofen are presented in which their combined administration provoked seizures in rats. Baclofen was shown also to increase the incidence of seizures evoked by pentylenetetrazole without increasing seizures due to local injections of excitatory amino acids. Baclofen antagonized the myoclonic effect of 5-hydroxytryptophan in rats with serotonin lesions. Baclofen augments some seizures and inhibits others. Selective inhibition of a particular tract, whether GABAergic or not, may have convulsant or anticonvulsant effects, depending on its connections and the state of the organism. GABAA receptor stimulation is usually but not always anticonvulsant. GABAB receptor stimulation may facilitate absence seizures and related primary generalized seizures. GABAB receptors may be abnormal in some forms of nonfocal epilepsy seen in childhood. It is likely that mutations of GABA transporter and GABAA receptor genes will be found in humans but they will probably not be patients with "pure epilepsy."


A neurologist's approach to neuroblastoma.

Neuroblastoma is among the most common malignancies of childhood. Despite greatly improved therapy for some pediatric tumors, the prognosis for children with metastatic neuroblastoma has not changed significantly in the past 10 years. With conventional chemotherapy, radiation therapy, and surgery, children with metastatic neuroblastoma have a 20% long-term survival rate. We have pursued novel chemotherapeutic approaches to neuroblastoma that target the neurotransmitter receptors on the surface of these cells. Specificity for these neural crest tumor cells is effected by (1) selective protection of normal neuronal elements from toxicity, or (2) selective potentiation of toxicity for neural tumor cells. In the first instance, the oxygen radical-generating neurotransmitter analogue 6-hydroxydopamine is used as a neural crest-specific toxin. Normal neural crest cells are protected from this toxicity by oxygen radical-scavenging analogues of the compound WR2721, which is specifically taken up by nonneoplastic cells. In the second instance, neocarzinostatin, an antineoplastic natural product that must be activated by thiol groups to be toxic, is used in conjunction with 6-mercaptodopamine, a thiol-containing compound that gains specific entry into neural crest cells by virtue of its neurotransmitter-like structure. We have found that neocarzinostatin induces morphologic differentiation of neuroblastoma cells, and we are also currently characterizing the biochemical accompaniments of this morphologic change.


Creatine kinase in epithelium of the inner ear.

Epithelium of the inner ear in the gerbil and mouse was examined immunocytochemically for presence of creatine kinase (CK). Marginal cells of the cochlear stria vascularis and dark cells and transitional cells of the vestibular system were found to contain an abundance of the MM isozyme (MM-CK). CK in these cells concurs with that which is coupled to Na,K-ATPase in other cells and is considered to supply ATP for the Na,K-ATPase that mediates the high KCl of endolymph. Inner hair cells revealed content of the BB isozyme and in this respect resembled the energy-transducing photoreceptor cells in retina. In addition, outer phalangeal (Deiters') cells stained for both MM- and BB-CK whereas inner phalangeal cells evidenced content of only the BB isozyme. Immunolocalization of CK appeared similar in mouse and gerbil inner ear. Specificity of the staining was affirmed by observations in agreement with those reported for CK in various cell types and by staining with antisera from more than one source.


Distribution of 3-hydroxybutyrate dehydrogenase in primary lobules of rat liver.

In an attempt to establish the functional organization of the hepatic parenchymal unit, we used histo- and microchemical procedures to assess metabolic liver cell heterogeneity at the level of the primary lobule. Because of the close interrelation of glucogenesis and ketone body formation, and in view of the distinct regional differences of the in vivo activity of glucose-6-phosphatase (G6Pase), these techniques were used on livers from male rats to investigate the distribution of the ketogenic enzyme, 3-hydroxybutyrate dehydrogenase (3-HBDH), during the post-resorptive phase. A close reciprocity was found between the general increase in the activity of 3-HBDH and the decrease of the in vivo activity of G6Pase along the sinusoidal axis, and also with regard to enzyme gradients along sinusoids of different origin. The activity of the ketogenic enzyme was higher throughout septal than portal sinusoids, whereas the opposite applied to the glucogenic enzyme. Histo- and microchemical data support the concept of a lobular parenchymal unit composed of "primary lobules," and show also that hepatocyte function varies with cell location along the sinusoidal axis and with the origin of the sinusoids.


Intracellular amplification of proviral DNA in tissue sections using the polymerase chain reaction.

We developed a new method to amplify cell DNA in situ using the polymerase chain reaction (PCR). Proviral sequences of mouse mammary tumor virus (MMTV) contained in cultured cells and tissue sections were amplified intracellularly using a thermal cycler. Two techniques were employed to maintain the localization of the amplified DNA. First, complementary tails at the 5' ends of the oligonucleotide primers resulted in the synthesis of high molecular weight concatamers containing the target sequences. Second, the PCR was carried out in a thin film of agarose solidified over the tissue sections. The specifically amplified and localized DNA was then detected by in situ hybridization (ISH). Our results demonstrate that (a) DNA in tissue sections can serve as the target for the polymerase chain reaction in situ, (b) cell morphology is maintained, and (c) a target of 167 BP can be specifically detected in individual cells. This technique should be generally applicable to amplifying cellular DNA targets in tissue sections for detection in situ.


Double in situ hybridization for detection of Herpes simplex virus and cytomegalovirus DNA using non-radioactive probes.

We describe a double in situ hybridization assay for the simultaneous detection of Herpes simplex virus (HSV) and cytomegalovirus (CMV) DNA in infected cell cultures using non-radioactive-labeled probes. This work used a biotinylated HSV DNA probe, which can be revealed by an avidin-biotin-peroxidase complex and a digoxigenin-labeled CMV DNA probe, visualized by anti-digoxigenin F(ab) fragments conjugated with alkaline phosphatase. Light microscopy visualization was achieved by the contrasting colors of appropriate peroxidase and alkaline phosphatase reaction products (red and dark blue, respectively). The time required to perform the double hybridization assay was about 3 hr. This double hybridization assay proved to be sensitive, specific, and provided good resolving power.


Simultaneous assessment of TGFB and cell cycle kinetics using IUdR/BrdU infusions in human neoplasms from plastic-embedded tissue.

We describe an immunohistochemical technique that makes use of two monoclonal antibodies (MAb), one to detect the transforming growth factor B (TGFB) and another that reacts with iodo- and bromodeoxyuridine. The purpose of this technique is to determine the relationship between TGFB expression and the S-phase cells in human tumors. Since both can be distinctly identified in situ from tissue embedded in plastic, in assessment of the geographic orientation of S-phase cells in relation to such factors as TGFB, contiguity to blood vessels, nerve fibers, and macrophages can also be achieved.


Differential expression of the alpha 2 and beta messenger RNAs of Na,K-ATPase in developing brine shrimp as measured by in situ hybridization.

We used in situ hybridization histochemistry with synthetic oligonucleotide probes to localize the mRNAs encoding the alpha 2- and beta-mRNAs of Na,K-ATPase during development of the brine shrimp Artemia. The mRNAs of the alpha 2- and beta-subunit were of low abundance in the cysts; in addition, less mRNA of the beta-subunit was localized. During emergence (12 hr), there was an increase in alpha 2-subunit mRNA in the gut mucosa, but there was a burst in beta-subunit mRNA throughout. As development progressed, the mRNAs of both the alpha 2- and beta-subunits showed a distinct pattern of expression in which the mRNA in the salt gland was of greatest abundance, followed by epidermal cells and gut mucosa. After 36 hr the alpha 2-subunit mRNA began to decrease in all positive cells but still remained highest in the salt gland and the brain region, while the mRNA of the beta-subunit kept increasing in the gut mucosa. Finally, the greatest abundance of the beta-subunit mRNA shifted from the salt gland to the antenna gland and the epidermal cells in the tail region, but the alpha 2-subunit mRNA did not. The more widespread distribution of the beta-mRNA than alpha 2-mRNA at certain stages (e.g., there was no alpha 2-mRNA in the antenna gland at the adult stage) is in all likelihood due to the marked drop in the alpha 2-subunit and a rise in alpha 1-subunit previously seen by Peterson et al. on polyacrylamide gel electrophoresis, as development progresses.


Retrograde tracing of zinc-containing neurons by selenide ions: a survey of seven selenium compounds.

The autometallographic retrograde tracing of zinc-containing neurons by intracerebral injection of sodium selenite (Na2SeO3), introduced by Danscher in 1982, has recently been described in more detail. Intracerebral injections of both sodium selenide (Na2Se) and sodium selenite (Na2SeO3) have been successfully used; however, sodium selenite had a rather toxic effect on the injected tissue. In the present study, we tested seven different selenium compounds to find the most suitable compound for retrograde tracing of zinc-positive pathways. Among the tested compounds, sodium selenide (Na2Se) caused insignificant necrosis within the injection site and was easily transported retrogradely when handled anaerobically. Sodium selenide is therefore recommended as the compound of choice.


Peripheral blood mononuclear cells of the elderly contain varicella-zoster virus DNA.

Peripheral blood mononuclear cells (PBMC) from humans of different ages were analyzed for DNA sequences specific for varicella-zoster virus (VZV) genes 29 and 62 by polymerase chain reaction (PCR). Neither VZV gene was detected in DNA from umbilical cord blood PBMC of 10 infants or from blood PBMC of two 3-year-old children. In 22 humans less than 60 years old, gene 29 was not detected, and gene 62 was detected in only one subject. In 33 humans greater than 60 years old, including patients with postherpetic neuralgia, PBMC from 4 subjects contained gene 29, 4 contained gene 62, and 1 contained both genes. The presence of VZV DNA correlated significantly with age (P less than .05, chi 2 and logistic regression analysis), but not with gender or postherpetic neuralgia.


Prevention of human rotavirus-induced diarrhea in gnotobiotic piglets using bovine antibody.

The efficacy of passively administered bovine antibody for preventing human rotavirus (HRV)-induced diarrhea was investigated using a gnotobiotic pig model. Cows were immunized with inactivated HRV serotypes 1 (Wa) and 2 (S2) and simian rotavirus serotype 3 (SA11), and immune colostrum and milk were collected. Antibody concentrates derived from these materials were fed to germ-free piglets that were subsequently inoculated with HRV Wa. Both viral shedding and diarrhea were effectively reduced or eliminated in a dose-dependent manner as a result of HRV immune antibody feeding. A quantitative virus-neutralizing (VN) antibody method permitted assessment of the functional antibody dose required to achieve a 50% reduction of disease (PD50). PD50 dose levels of 15.8 and 19.5 x 10(6) VN antibody units were determined for inhibition of diarrhea and viral shedding, respectively. Studies reported here provide new information on the quantitative relationship between protective antibody dose and diarrheal disease response.


Effect of high-dose dexamethasone on the outcome of acute encephalitis due to Japanese encephalitis virus.

Death due to Japanese encephalitis usually occurs in the first 5 days of hospitalization as a result of deepening coma with respiratory arrest. Death may result from edema-induced increases in intracranial pressure that might be reduced by the administration of steroids. Sixty-five patients presenting in Thailand to four hospitals with a diagnosis of acute Japanese encephalitis were randomized in a double-masked fashion and stratified by initial mental status into a placebo group (saline) or a treatment group (dexamethasone 0.6 mg/kg intravenously as a loading dose followed by 0.2 mg/kg every 6 h for 5 days). Fifty-five of the 65 had confirmed Japanese encephalitis as demonstrated by detection of virus or by Japanese encephalitis virus-specific IgM antibody. Important outcome measures included mortality (24%, treatment group; 27%, control group), days to alert mental status (3.9 vs. 6.2), and neurologic status 3 months after discharge (45% abnormal in each group). No statistically significant benefit of high-dose dexamethasone could be detected.


High-level quinolone resistance in clinical isolates of Campylobacter jejuni.

During a recent clinical trial of ciprofloxacin in the therapy of acute diarrhea, two subjects infected with Campylobacter jejuni who received ciprofloxacin failed microbiologically and one also failed clinically. Although both pretreatment isolates were susceptible to ciprofloxacin, the posttreatment isolates were resistant to ciprofloxacin (MIC = 32 micrograms/ml) and to other quinolones. The posttreatment isolates remained susceptible to nonquinolone antimicrobials. DNA gyrase holoenzyme was isolated from one of the resistant posttreatment isolates and was 8- to 16-fold less sensitive to inhibition by ciprofloxacin than was the gyrase from the paired pretreatment susceptible isolate. Ciprofloxacin accumulation was diminished in the two resistant posttreatment isolates. These results show that mutation in C. jejuni can occur in vivo and is associated with clinically significant resistance to the newer quinolones.


Mother-to-child transmission of hepatitis C virus detected by nested polymerase chain reaction.

Serum samples from eight pregnant women and their offspring were studied by nested polymerase chain reaction (PCR) for detection of hepatitis C virus (HCV) RNA to evaluate mother-to-child transmission of this virus. The mothers were all infected with human immunodeficiency virus (HIV); none showed symptoms of HCV infection. Anti-HCV antibodies were tested for by recombinant immunoblot assay. HCV viral sequences were found in five of the mothers and four of eight children, three of them at birth. Viremia was persistent in one infant who had chronic transaminase elevation and persistently remained anti-HCV-positive. The other three babies had intermittent viremia; all were asymptomatic and lost anti-HCV antibodies during follow-up. This loss of antibodies was also observed in PCR-negative infants. Thus, these results demonstrate transmission of HCV from mother to child by women coinfected with HCV and HIV. They indicate the usefulness of PCR for direct and early detection of HCV viremia in neonates.


Oral bioavailability and anti-simian varicella virus efficacy of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU) in monkeys.

BV-araU (1-beta-D-arabinofuranosyl-E-5-[2-bromovinyl]uracil) has potent antiviral activity against varicella zoster virus in cell culture and is undergoing clinical evaluation. In the present study, pharmacokinetic parameters and the efficacy of BV-araU against infection with simian varicella virus (SVV) were evaluated in African green monkeys. Pharmacokinetic parameters were determined by analysis of the BV-araU content of sera obtained after oral and intravenous administration to normal monkeys. Peak serum concentrations showed dose proportionality, with the 0.1 mg/kg dose resulting in a peak serum concentration of 0.05 micrograms/ml, the approximately ED50 value for the SVV inoculum in cell culture. BV-araU administered orally twice daily at 0.1 mg/kg for 10 days starting 48 h after intratracheal SVV infection prevented vesicular rash development and suppressed viremia. Effective therapy could be initiated 96 h after infection. Taken together, these results indicate that BV-araU is effective oral therapy at doses that achieve peak serum levels equivalent to the ED50 for SVV in cell culture.


A phorbol diester resistant monocytic leukemia cell line is PKC deficient.

MIA C51 is a rat monocytic leukemia cell line which exhibits undifferentiated monocytic phenotype in culture. The proliferation of MIA C51 cells was not inhibited by the addition of 12-O-tetradecanoyl-phorbol-13-acetate (TPA, 1 microgram/ml) or phorbol 12, 13 dibutyrate (PDBu, 10 micrograms/ml). Comparison of MIA C51 cells to a phorbol diester-sensitive human monoblastoid U-937 cell line demonstrated that MIA C51 cells contained significantly lower number of PDBu receptors, protein kinase C (PKC) activity, and PKC protein level. Further experiments demonstrated that addition of TPA to MIA C51 cells did not induce the expression of c-fos proto-oncogene; whereas incubation of MIA C51 cells with N6, O2-dibutyryl cyclic adenosine 3',5' monophosphate (Bt2cAMP) resulted in a rapid increase of c-fos mRNA level. Thus, this cell line provides a new system for studying the signal transduction mechanisms in induced monocytic differentiation.


Regulation of guinea pig plasma low density lipoprotein kinetics by dietary fat saturation.

Dietary fat saturation has been shown to affect hepatic apoB/E receptor expression and to modify low density lipoprotein (LDL) composition and density in guinea pigs. The current studies were designed to investigate the independent and interactive effects of dietary fat saturation alterations in apoB/E receptor expression and LDL composition on in vivo LDL turnover kinetics, both receptor-mediated and receptor-independent. Guinea pigs were fed semi-purified diets containing 15% fat, either polyunsaturated corn oil (CO), monounsaturated olive oil (OL), or saturated lard, and injected with radioiodinated LDL isolated from animals fed the homologous diet. Blood samples were obtained over 33 h to determine apoLDL fractional catabolic rates (FCR) and flux rates. Compared to animals fed OL- or lard-based diets, intake of the CO-based diet resulted in a 50% decrease in LDL apoB pool size associated with a twofold increase in receptor-mediated FCR (P less than 0.001) and a 28% decrease in flux rate (P less than 0.05). Maximal LDL binding capacity of hepatic apoB/E receptors, determined in vitro, was twofold higher for animals fed the CO-based diet compared to guinea pigs fed the OL- and lard-based diets (P less than 0.01). There was a significant correlation between hepatic apoB/E receptor number and in vivo receptor-mediated LDL FCR (r = 0.987). Significant differences in LDL turnover were related to the source of LDL. When injected into animals fed a nonpurified commercial diet, the smaller, cholesteryl ester-depleted LDL isolated from animals fed the CO-based diet had a twofold higher FCR compared to larger LDLs from guinea pigs fed the OL- and lard-based diets, which had similar turnover rates. When LDL from animals fed the commercial diet was radiolabeled and injected into animals fed the three types of dietary fat, significant differences in LDL turnover were observed in the order CO greater than lard greater than OL, suggesting that intravascular processing and tissue uptake of the smaller LDL from animals fed the commercial diet varies depending on the dietary fat saturation fed to the recipient animals. These studies demonstrate that guinea pigs fed polyunsaturated fat diets lower plasma LDL levels in part by an increase in apoB/E receptor-mediated fractional LDL turnover and a decrease in apoLDL flux. In addition, fat saturation alters LDL composition and size which independently affect LDL turnover rates in vivo.


The insulin-like growth factor I receptor is overexpressed in psoriatic epidermis, but is differentially regulated from the epidermal growth factor receptor.

Insulin-like growth factor I (IGF-I)/somatomedin C is an important mediator of keratinocyte growth in vitro, and the expression of IGF-I receptors in the basal layer of normal epidermis suggests that this growth pathway may function in the regulation of keratinocyte growth in vivo as well. The pattern of IGF-I receptor expression in normal skin is distinct from that of the epidermal growth factor (EGF) receptor, suggesting that these receptors might be differentially regulated. The purpose of this study was to obtain a better understanding of IGF-I receptor function in the skin by examining IGF-I receptor expression in psoriatic epidermis and in cultured human keratinocytes. Our findings indicate that IGF-I receptor expression is increased in psoriasis as measured by protein tyrosine kinase assays of biopsy extracts and by immunohistochemical staining with an IGF-I receptor-specific monoclonal antibody. Unlike EGF receptor expression, which is also increased in psoriatic epidermis, the pattern of IGF-I receptor expression corresponds closely with the increased size of the keratinocyte proliferative compartment in psoriasis. Biochemical agents that diminish EGF receptor ligand binding (phorbol ester or calcium ionophore treatment) produce opposite effects on the IGF-I receptor. These results suggest that cellular expression and differential regulation of both growth factor receptor systems may control critical aspects of epidermal proliferation or function.


CD28 and T cell antigen receptor signal transduction coordinately regulate interleukin 2 gene expression in response to superantigen stimulation.

Activation of an immune response requires intercellular contact between T lymphocytes and antigen-presenting cells (APC). Interaction of the T cell antigen receptor (TCR) with antigen in the context of major histocompatibility molecules mediates signal transduction, but T cell activation appears to require the induction of a second costimulatory signal transduction pathway. Recent studies suggest that interaction of CD28 with B7 on APC might deliver such a costimulatory signal. To investigate the role of CD28 signal transduction during APC-dependent T cell activation, we have used Staphylococcal enterotoxins (SEs) presented by a B7-positive APC. We used anti-B7 monoclonal antibodies and a mutant interleukin 2 (IL-2) promoter construct, unresponsive to CD28-generated signals, in transient transfection assays to examine the contribution of the CD28-B7 interaction to IL-2 gene activation. These studies indicate that the CD28-regulated signal transduction pathway is activated during SE stimulation of T cells and plays an important role in SE induction of IL-2 gene expression through its influence upon the CD28-responsive element contained within the IL-2 gene promoter. This effect is particularly profound in the activation of the IL-2 gene in peripheral blood T cells.


Adult adrenoleukodystrophy: the clinical spectrum in a large Dutch family.

A large family with adrenoleukodystrophy is described and the case histories of two clinically symptomatic and related male patients are presented. Clinical, biochemical and genetic screening of their family demonstrated two clinically affected males, one biochemically affected male and five carrier females. Two women were symptomatic; one suffered an acute exacerbation. One female was diagnosed as a carrier, based on genetic analysis and the family history only. Endocrinological screening was performed in the five affected males, demonstrating an elevated adrenocorticotrophic hormone level and a normal cortisol level in two, as evidence of compensated adrenocortical failure.


Detection of varicella-zoster virus DNA by polymerase chain reaction in cerebrospinal fluid of patients with herpes zoster meningitis.

In three of five patients with herpes zoster meningitis, varicella-zoster virus (VZV) DNA was detected by the polymerase chain reaction (PCR) in the initial samples of cerebrospinal fluid. DNA fragments of group A or B, following classification of VZV strains by the size of the variable region IV of VZV genome, were found at the 7th, 10th and 24th illness day in the three positive cases: one of these cases did not have skin lesions. These results suggest that the detection of VZV DNA by PCR is useful for the diagnosis of herpes zoster meningitis, as well as for its molecular epidemiology.


A double-blind placebo-controlled trial of L-threonine in amyotrophic lateral sclerosis.

Fifteen patients with the unequivocal diagnosis of amyotrophic lateral sclerosis (ALS) completed a 1-year randomized double-blind placebo-controlled trial of L-threonine (2 g daily). During the study, patients in the placebo group showed a decline in functional status consistent with the natural history of ALS, which was not statistically different from outcome in the patients in the L-threonine group.


Multifocal peripheral neuropathy in eosinophilic fasciitis.

Polyneuropathy may complicate eosinophilia-associated connective tissue disease. We report a multi-focal neuropathy in a patient with eosinophilic fasciitis proven by demonstrating an eosinophilic cellular infiltrate in a fascial biopsy specimen from the forearm. Sural nerve biopsy revealed lymphocytic cuffing of epineural arterioles. Although described in the L-tryptophan-related eosinophilia-myalgia syndrome, peripheral neuropathy with these features has not been previously noted in a patient with eosinophilic fasciitis who had not consumed L-tryptophan.


Calcium-dependent potassium currents in neurons from cat sensorimotor cortex.

1. Ca(2+)-dependent K+ currents were studied in large pyramidal neurons (Betz cells) from layer V of cat sensorimotor cortex by use of an in vitro brain slice and single microelectrode voltage clamp. The Ca(2+)-dependent outward current was taken as the difference current obtained before and after blockade of Ca2+ influx. During step depolarizations in the presence of tetrodotoxin (TTX), this current exhibited a fast onset of variable amplitude and a prominent slowly developing component. 2. The Ca(2+)-dependent outward current first appeared when membrane potential was stepped positive to -40 mV. Downsteps from a holding potential of -40 mV revealed little or no time-, voltage-, or Ca(2+)-dependent current. When membrane potential was stepped positive to -40 mV, a prolonged Ca(2+)-dependent outward tail current followed repolarization. The decay of this tail current at -40 mV was best described by a single exponential function having a time constant of 275 +/- 75 (SD) ms. The tail current reversed at 96 +/- 5 mV in 3 mM extracellular K+ concentration ([K+]o) and at more positive potentials when [K+]o was raised, suggesting that it was carried predominantly by K+. 3. The Ca(2+)-dependent K+ current consisted of two pharmacologically separable components. The slowly developing current was insensitive to 1 mM tetraethylammonium (TEA), but a substantial portion was reduced by 100 nM apamin. Most of the remaining current was blocked by the addition of isoproterenol (20-50 microM) or muscarine (10-20 microM). 4. The time courses of the apamin- and transmitter-sensitive components were similar when activated by step depolarizations in voltage clamp, but they were quite different when activated by a train of action potentials. Applying the voltage clamp at the end of a train of 90 spikes (evoked at 100-200 Hz) resulted in an Ca(2+)-dependent K+ current with a prominent rapidly decaying portion (time constant approximately 50 ms at -64 mV) and a smaller slowly decaying portion (time constant approximately 500 ms at -64 mV). The rapidly decaying portion was blocked by apamin (50-200 nM), and the slowly decaying portion was blocked by isoproterenol (20-50 microM). 5. When recorded with microelectrodes containing 2 mM dimethyl-bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (dimethyl-BAPTA), which causes prolonged afterhyperpolarizations, the Ca(2+)-dependent K+ current evoked by step depolarizations had an extremely slow onset and decay. The current recorded after a train of evoked spikes had a similar slow decay.(ABSTRACT TRUNCATED AT 400 WORDS)


Tertiary and quaternary local anesthetics protect CNS white matter from anoxic injury at concentrations that do not block excitability.

1. Anoxic injury in CNS white matter was studied using the in vitro rat optic nerve preparation. Optic nerves were subjected to 60 min of anoxia, and functional recovery was assessed using the area under the compound action potential (CAP). In normal cerebrospinal fluid, CAP area recovered to 33.5 +/- 9.3% (SD) of control. 2. Lidocaine and procaine (0.1 or 1.0 mM), applied beginning 1 h before anoxia, significantly improved postanoxic recovery of CAP area. However, both agents also depressed the preanoxic CAP. Procaine generally allowed greater recovery with less depression compared with lidocaine. 3. The quaternary derivatives QX-314 (0.1-1.0 mM) and QX-222 (0.3-3.0 mM) resulted in more complete recovery of the CAP area from anoxia, with less depression of preanoxic excitability, compared with the tertiary compounds. At 0.3 mM, QX-314 reduced the preanoxic CAP very little (to 94.4 +/- 14% of control CAP area), yet allowed the postanoxic CAP area to recover to 99.6 +/- 19%. 4. We conclude that quaternary local anesthetics are more effective at protecting CNS white matter tracts from anoxia than tertiary compounds and that these agents can result in markedly improved recovery even at concentrations that do not block conduction. Moreover, given the relative specificity of QX-314 for noninactivating Na+ channels, we hypothesize that this channel subtype plays an important role in mediating anoxic injury in central myelinated axons.


High myocardial accumulation of radioiodinated digoxin derivative: a possible Na,K-ATPase imaging agent.

In view of the high binding ability of cardiac glycosides to the myocardial Na,K-ATPase, radioiodinated digoxin derivatives were surveyed as candidates for myocardial imaging, with particular emphasis on the noninvasive monitoring of cardiac glycoside therapy. Among the radioiodinated digoxin derivatives surveyed, 125I-digoxin-iodohistamine(bis(O-carboxymethyloxime)) showed the highest accumulation in the myocardium and similar binding ability to Na,K-ATPase as digoxin itself against ouabain displacement, as indicated by in vivo and in vitro studies. Based on these results, 123I labeling of digoxin-histamine(bis(O-carboxymethyloxime)) and imaging in a dog demonstrated uptake in the myocardium.


Effects of structured controversy on students' perceptions of their skills in discussing controversial issues.

The teaching strategy that educators use when addressing controversial issues can help students develop the skills they need to deal effectively with controversial issues. Structured controversy is designed to allow students to practice these skills. This study measured the change in students' perceptions of their skills in perspective-taking, logical arguing, and reaching consensus after using structured controversy on two controversial issues. Results of two-factor analyses of variance and comparison of means indicate significant positive changes in 51 senior nursing students' perceptions of their skills in perspective-taking (less than .001), logical arguing (less than .01), and reaching consensus (less than .01). These results are consistent with findings of previous research studies on the effectiveness of structured controversy.


Participation of nursing faculty in university governance.

It has been suggested that faculty participation in governance in American colleges is low, and that faculty in schools of nursing are particularly unlikely to be involved in governance activities. This study was designed to determine actual and ideal levels of nursing faculty participation in five areas of governance: academic, student, personnel, public, and financial affairs. A survey of nursing faculty suggested that they were involved substantially in academic affairs, but less involved in the other areas of governance. Generally, the faculty indicated satisfaction with their high level of participation in academic affairs, and with their lower level of participation in student affairs, personnel affairs, and public affairs; the faculty did indicate dissatisfaction with their low level of participation in financial affairs.


Power and change in health care: challenge for nursing.

This article proposes the need for consideration of status and power issues in nursing research, in theory, and at all levels of nursing education. Including the concept of power in nursing curricula will better prepare nurses to participate in social and political decisions affecting health care. By recognizing the role nurses have played, often in the face of opposition, nursing students will gain a sense of the potential power and strength of nurses. Curricular reform and revision of nursing theories to emphasize the sociopolitical context of nursing may change the socialization of student nurses by stressing autonomy, independence, and confidence. These changes may have a positive impact on recruitment, retention, and on efforts to change the public's perception of nursing.


Empowerment through teaching.

Empowerment through teaching is built on the feminist belief that successful and effective teaching is a cointentional process, emerging from meaningful connections between students and faculty. Empowerment results from teaching characterized by caring, commitment, creativity, interaction, and a recognition of the humanity of both teacher and students. Teaching is conceptualized as a directional process emerging from the energy of both student and teacher. There must be continued, intense, and frequent exchange of these energies. The tools that students and teacher must possess or acquire for empowerment include positive self-concept, creativity, resources, information, and support. This perspective permits a new understanding of teaching that results in empowerment of both parties involved in the process of learning.


The professional self-concepts of nursing undergraduates and their perceptions of influential forces.

This article explores the professional self-concepts of senior nursing undergraduates and their perceptions of the most significant forces that influenced the process. The method was qualitative; a grounded theory approach was used. The sample included 23 senior baccalaureate nursing students in their final clinical rotation prior to graduation. Audiotaped interviews were analyzed through the constant comparative method. Informants overwhelmingly perceived themselves as caring. They valued professional competence and many identified a lack of confidence in their skills. Caring qualities, caring role models, and qualities of the role models are listed.


Knowledge about aging and Alzheimer's disease among baccalaureate nursing students.

Alzheimer's disease (AD) is a debilitating disease that poses many physical, social, psychological, and management problems for family and professional caregivers. Nursing students' ability to meet this challenge was measured using Palmore's (1988) Facts on Aging Quiz, Version 2 (FAQ2) and the Alzheimer's Disease Knowledge (ADK) Test developed by Dieckmann, Zarit, Zarit, and Gatz (1988). No significant difference in ADK Test scores was found for students who had previous personal or educational experiences with AD. However, older students, seniors, and those who reported knowing more about AD had significantly better scores on the ADK Test. Knowledge about AD was not found to be related to knowledge of aging, and subjects as a group were found to exhibit a negative bias toward the elderly as measured by Palmore's FAQ2.


The influence of dietary fiber on proliferation of intestinal mucosal cells in miniature swine may not be mediated primarily by fermentation.

Cannulated miniature pigs were used as a model for evaluating the effects of fiber-containing diets on proliferation of colonic epithelial cells. Biopsy specimens taken from the mucosa of the cecum and distal colon were incubated in [3H]thymidine and processed using autoradiography. Digesta from the cecum was analyzed for concentrations of short-chain fatty acids (SCFA), pH and volume. Cellular proliferation at both intestinal sites was influenced by the source or quantity of dietary fiber. These dietary variables also influenced SCFA concentrations and pH of cecal digesta and the volume of contents in the fermentation chamber. Cellular proliferation was not significantly correlated, however, with the quantity of water-soluble dietary fiber consumed, with SCFA concentrations or pH of the cecal digesta, or with the volume of the fermentation chamber. We suggest that the influence of dietary fiber on intestinal cell proliferation cannot be predicted from markers of the fermentation capacity of specific diets, because fiber likely influences intestinal proliferation through several interactive mechanisms, some of which include luminal factors.


Occupationally-induced scleroderma.

Systemic sclerosis or scleroderma is an uncommon multisystem disease with a reported incidence of 2 to 12 cases per million people per year. The clinical and pathological features can be grouped into three main categories: those related to fibrosis, to vascular abnormalities, and to immunological abnormalities. Cutaneous features dominate the patients' appearance, and Raynaud's phenomenon is an early symptom. A possible association between scleroderma and workers exposed to hand-arm vibration and/or silica has been suggested by reports in the literature since the turn of the century. A further four patients with collagen disease are reported here. Three were occupationally exposed to both hand-arm vibration and silica, the fourth to hand-arm vibration alone. In conjunction with previously reported cases, this supports the hypothesis that collagen disease may be work attributable in hypersusceptible persons.


Outbreaks of human enteric adenovirus types 40 and 41 in Houston day care centers.

Human enteric adenovirus (EAd) types 40 and 41 cause diarrhea in young children, but little is known about their association with outbreaks of diarrhea in the child care setting. This study evaluated EAd as a cause of outbreaks of diarrhea among infants and toddlers in day care centers. Stool specimens were collected weekly regardless of symptoms during four periods from January 1986 to April 1991, from children 6 to 24 months of age enrolled in prospective studies of diarrhea in day care centers. All diarrhea stool specimens were tested for bacterial enteropathogens, rotavirus, and Giardia lamblia. A total of 131 outbreaks occurred during the study. No etiologic agent was identified in 77 outbreaks. Stool specimens from 75 of these 77 outbreaks and from another 21 outbreaks of diarrhea with a known cause were evaluated for EAd with a monoclonal antibody-based enzyme immunoassay. A total of 4402 stool specimens from 613 children from these 96 outbreaks was tested for EAd. The virus was detected in specimens collected during 10 outbreaks, 3 of which occurred in 1986, 3 in 1988, 1 in 1989, 1 in 1990, and 2 in 1991. Of 249 children, 94 (38%) in these 10 EAd outbreaks were infected with EAd. In 51 children (54%) the infection was symptomatic and in 43 (46%) it was asymptomatic. Outbreaks lasted 7 to 44 days (mean 24.5 days). Duration of EAd excretion ranged from 1 to 14 days (mean 3.9 days), with excretion occurring from 7 days (mean 2.6) before diarrhea began to 11 days (mean 5.3 days) after diarrhea stopped. Enteric adenovirus types 40 and 41 are an important cause of outbreaks of diarrhea among children attending day care centers, often involve children in more than one room, and frequently produce asymptomatic infection.


A multicenter study of the treatment of childhood chronic idiopathic thrombocytopenic purpura with anti-D.

We evaluated the effects of the intravenous administration of anti-D, an immune globulin directed at the D antigen on erythrocytes that is purified from plasma from sensitized persons, on patients with idiopathic thrombocytopenic purpura. To determine the most effective dose, the duration of response, and the side effects of this therapy in children, we performed a multicenter cohort study of escalating doses of intravenously administered anti-D in children aged 1 to 18 years with chronic idiopathic thrombocytopenic purpura, defined as idiopathic thrombocytopenic purpura persisting for more than 6 months with a platelet count of less than 50 x 10(9) cells/L. Twenty-five Rh-positive children received increasing doses of anti-D as follows: day 1, 25 micrograms/kg; day 2, 25 micrograms/kg; day 7, 35 micrograms/kg; day 14, 45 micrograms/kg; and day 21, 55 micrograms/kg. Administration of anti-D was stopped after day 21 or when the platelet count rose to greater than 150 x 10(9) cells/L or the hemoglobin level was 100 gm/L. Platelet count was less than 50 x 10(9) cells/L in all children before treatment. A response was defined as an increase in the platelet count to more than 50 x 10(9)/L and a doubling of the pretreatment platelet count. Of 25 children, 23 (92%) had responses by day 7 of the initial treatment protocol. Eighteen children (72%) had platelet counts greater than 150 x 10(9) cells/L by day 7 after two doses of anti-D. Median duration of response was 5 weeks (range 1 to 24 weeks). Average drop in hemoglobin level was 13.7 gm/L; in one child (a nonresponder) hemoglobin value fell to less than 100 gm/L. No other untoward side effects were seen. Of the 23 children who responded, 21 were retreated with one dose of anti-D when platelet counts returned to baseline values of less than 50 x 10(9) cells/L; all but three of the children who underwent retreatment showed a response the second time. Sixteen children continued to receive intermittent anti-D therapy after completion of the study, and all continued to have excellent responses. We conclude that anti-D is a safe, effective, and relatively inexpensive therapy for childhood chronic idiopathic thrombocytopenic purpura.


Persistent cerebrospinal fluid neutrophilia in delayed-onset neonatal encephalitis caused by herpes simplex virus type 2.

We describe an infant with three unusual features of perinatally acquired herpes simplex virus type 2 encephalitis: onset of illness at 34 days of age, absolute cerebrospinal fluid neutrophilia, and systemic viral dissemination after central nervous system disease. To provide early, effective antiviral therapy, clinicians should be aware of atypical presentations of serious herpes simplex virus infections.


Acyclovir treatment of varicella in otherwise healthy adolescents. The Collaborative Acyclovir Varicella Study Group.

To determine whether orally administered acyclovir is of therapeutic benefit for varicella in otherwise healthy adolescents, and to compare the severity of the disease in adolescents with that in younger children. Multicenter, randomized, placebo-controlled, double-blind trial. Patients' homes and university hospital clinics. Sixty-eight adolescents between 13 and 18 years of age with varicella entered the study. Of the 62 adolescents with laboratory-confirmed varicella who were included in the final analysis, 31 received acyclovir and 31 received placebo. Placebo or an 800 mg acyclovir tablet was given orally four times daily for 5 days, beginning within 24 hours of onset of rash. Acyclovir recipients had significant reductions in times to cessation of new lesion formation (p less than 0.001), maximum number of lesions (p = 0.019), and defervescence (p = 0.045). Mean constitutional illness score was significantly reduced on day 4 (0.5 vs 1.5, p = 0.05), as was the mean number of residual hypopigmented lesions present on 28-day follow-up examination (22.7 vs 92.7, p = 0.018). Two complications, both bacterial superinfections, occurred in placebo recipients. Adverse experiences and varicella-zoster virus antibody titers measured 28 days after enrollment were similar in both treatment groups. Comparison of placebo recipients with children 2 to 12 years of age participating in a companion study indicated that varicella is more severe in adolescents: mean maximum total lesions (421 vs 347, p = 0.003), mean maximum constitutional illness score (3.1 vs 2.2, p = 0.032), and mean number of residual lesions (92.7 vs 33.2, p = 0.01) were all greater in the adolescent population. Oral acyclovir therapy is safe and effective for treatment of varicella in otherwise healthy adolescents; this may be an appropriate subgroup for treatment with antiviral drugs because the disease is more severe in them than in younger children.


Gingival crevicular fluid concentration and side effects of minocycline: a comparison of two dose regimens.

The purpose of this study was to conduct a direct comparison of two dose regimens of minocycline to determine 1) whether they achieved crevicular fluid concentrations in a therapeutic range; and 2) the frequency of side effects. In a double-blind design, 30 patients divided into 2 groups were given either 100 mg minocycline or 200 mg minocycline per day for an 8-day period. The concentration of minocycline in the gingival clevicular fluid (GCF) at 8 days was 4.77 micrograms/ml for the 100 mg a day group and 5.97 micrograms/ml for the 200 mg a day group and at 15 days was 4.30 micrograms/ml for the 100 mg a day group and 4.17 micrograms/ml for the 200 mg a day group. There was no significant difference in the antibiotic concentration in the gingival crevicular fluid between the 2 groups. Reported adverse experiences to the minocycline were greater in the 200 mg a day group. Short-term changes in periodontal health as measured by plaque index, gingival index, pocket depth, and bleeding upon probing showed improvements in all parameters over the 15 day period. There were no significant differences in these parameters between the 100 mg a day and 200 mg a day group. At 8 days reduced levels of Porphyromonas gingivalis and Prevotella intermedia were achieved but they were not eliminated from infected subgingival sites in either group. Achieving bacteriostatic concentrations of GCF, fewer side effects, and the potential for better compliance suggests that a single daily dose of 100 mg minocycline should now be investigated for its efficacy in managing periodontal infections manifesting as periodontitis.


In vitro platelet serotonin secretion and inhibition on variously treated root surfaces.

Platelet degranulation can result in the release of a variety of factors which are chemotactic, mitogenic, and angiogenic, making platelets extremely important in the regulation of the repair process. This study examines how various types of root surfaces affect platelet deposition and the release of serotonin from dense granules. In addition, experiments were performed to evaluate the effects of the cyclo-oxygenase inhibitor, indomethacin, on platelet deposition and dense granule release. Roots from freshly extracted teeth from sites with periodontal disease (PD) and from healthy sites were sectioned and had the following surface conditions: 1) periodontal ligament present; 2) PD; 3) PD, root planed; 4) PD, root planed and demineralized; and 5) condition 4 treated with collagenase. In addition, rabbit calcaneal tendon collagen was used. All samples were incubated with platelets labeled with both 111Indium and 14C serotonin, with and without the addition of indomethacin. It was observed that the greatest number of platelets deposited on the tendon collagen. Furthermore, serotonin release occurred on all samples except PD and indomethacin partially inhibited platelet deposition on all samples except tendon collagen. Finally, indomethacin inhibited serotonin release on all surfaces. These results suggest that attachment of platelets to the root surface is facilitated by metabolism through the cyclo-oxygenase pathway and that limited platelet deposition can occur in the absence of dense body release.


Assessment of the relative cytotoxicity of Porphyromonas gingivalis cells, products, and components on human epithelial cell lines.

Established human cell lines derived from a transitional cell carcinoma (J82), a squamous carcinoma (SCaBER), and a normal urothelium (HCV-29) were used to assess the relative cytotoxicity and tissue specificity of putative virulence determinants of P. gingivalis W83. Intact cells of W83 had no effect on any of the cell lines, whereas disrupted cells caused extensive cytotoxicity particularly to monolayers of HCV-29 and J82. The purified cysteine proteinase, gingivain, caused marked disruption of the basement membrane of the SCaBER monolayers but had no cytotoxic effects. Use of the thiol-inhibitor, 2,2'-dipyridyl disulphide revealed that the effects observed with the vesicles and the culture supernatant were due to the presence of the cysteine proteinase. The attachment of vesicles to the SCaBER cells was evident in electron micrographs. Short-chain volatile fatty acids added in concentrations equivalent to those present in the culture supernatant had no effect on any of the cell lines tested. Culture supernatants obtained from high speed centrifugation (150,000 x g) showed no cytotoxic effects. This was in marked contrast to the supernatant obtained by lower sedimentation (18,000 x g), which damaged all monolayers tested. These results suggest that these cell lines are potentially useful for assessing putative virulence determinants of P. gingivalis and other periodontal pathogens.


Microbiological and clinical results of metronidazole plus amoxicillin therapy in Actinobacillus actinomycetemcomitans-associated periodontitis.

We report on the microbiological and clinical effects of mechanical debridement in combination with metronidazole plus amoxicillin therapy in 118 patients with Actinobacillus actinomycetemcomitans-associated periodontitis. Patients were categorized into 3 groups: 28 had localized periodontitis; 50 had generalized periodontitis, and 40 had refractory periodontitis. After initial treatment and metronidazole plus amoxicillin therapy 114 of 118 (96.6%) patients had no detectable A. actinomycetemcomitans. Significant reduction in pocket probing depth and gain of clinical attachment were achieved in almost all patients. Four patients were still positive for A. actinomycetemcomitans after therapy. Metronidazole resistance (MIC greater than 25 micrograms/ml) was observed in 2 of 4 strains from these patients. Patients still positive for A. actinomycetemcomitans or Porphyromonas gingivalis showed a significant higher bleeding tendency after therapy. It was concluded that mechanical periodontal treatment in combination with the metronidazole plus amoxicillin therapy is effective for subgingival suppression of A. actinomycetemcomitans in patients with severe periodontitis.


Dentifrice abrasivity and cervical dentinal hypersensitivity. Results 12 weeks following cessation of 8 weeks' supervised use.

Following an 8-week controlled investigation of 2 strontium chloride hexahydrate dentifrices (SCH) of differing abrasivity, 2 groups of 20 subjects each, with cervical dentinal hypersensitivity, were re-examined at 20 weeks; that is, 12 weeks after the active period. The examination procedures were conducted in the same manner as in the main clinical trial. Sensitivity levels were assessed by 2 instrument methods: tactile (Yeaple probe), and cold air (dental air syringe), and by subjective perception of pain by means of a Visual Analogue Scale. The results from these methods of assessment demonstrated that 12 weeks following the cessation of 8 weeks' controlled use of standard and low abrasive SCH dentifrices, sensitivity levels reversed on slightly in both groups and, overall, sensitivity remained significantly lower than at baseline. The abrasivity of the dentifrice did not affect the desensitivity activity.


Periodontal repair in dogs: effect of a composite graft protocol on healing in supraalveolar periodontal defects.

This study evaluated the effect of a composite graft as an adjunct to gingival flap surgery in induced chronic supraalveolar periodontal defects in the mandibular premolar region in beagle dogs. The vertical dimension of the defects, measured from the cemento-enamel junction to the alveolar bone, approximated 5 mm. Root surface treatment in quadrants receiving the graft protocol included conditioning with both critic acid and tetracycline. The composite graft (including: hydroxyapatite, freeze-dried decalcified bone, tetracycline, and fibronectin) was then fitted to the defects. Flaps were placed and sutured to cover most of the crowns of the teeth but the tips of the cusps. The root surfaces in contralateral jaw quadrants were conditioned with critic acid and the flaps similarly placed and sutured. Dogs were sacrificed 6 weeks after surgery and tissue blocks including teeth and surrounding structures processed for histometric analysis. Connective tissue repair to the root surface in teeth treated with the graft protocol approximated 60% of the defect height. Connective tissue repair in teeth treated with citric acid only was significantly greater and averaged 98% of the defect height (P less than or equal to 0.01). Cementum formation was limited following both treatments (approximately 6% of the defect height). Bone regeneration was significantly smaller in grafted sites than in sites treated with citric acid only (approximately 2% and 10% of the defect height, respectively; P less than or equal to 0.05). Root resorption was observed in almost all teeth. Ankylosis was present in two citric acid-treated specimens, both from the same dog.(ABSTRACT TRUNCATED AT 250 WORDS)


Humoral antibody responses in periodontal disease.

Several forms of periodontal disease are considered to be infectious diseases with associated specific bacteria. This study examined the humoral antibody levels as assayed by ELISA to Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia in adult periodontitis (AP), localized juvenile periodontitis (LJP), rapidly progressive periodontitis (RPP), and in periodontally healthy subjects (HS). Sixty-two of the 64 (96.9%) patients had significantly elevated antibody levels to at least one of the three organisms. Elevated levels of antibodies to P. gingivalis occurred in 82.8% of the RPP, LJP, and AP patients with all 3 disease groups showing greater responses than HS controls. Antibodies to A. actinomycetemcomitans were found in 59.4% of the RPP, LJP, and AP patients and were significantly higher in both LJP and RPP patients. Only 21.9% of the RPP, LJP, and AP patients showed elevated levels to P. intermedia with only significantly higher levels in the RPP and LJP groups. Antibodies to A. actinomycetemcomitans and P. intermedia were rarely found alone (only 5.1% and 2.6% of the patients respectively) but were usually accompanied by P. gingivalis. These results suggest that one or more combinations of these 3 bacteria may play a role in the pathogenesis of these forms of periodontal disease.


Evaluation of oral bacteria as risk indicators for periodontitis in older adults.

The prevalence of people and sites with attachment loss, pocket depth, Actinobacillus actinomycetemcomitans, Prevotella intermedia, and Porphyromonas gingivalis are described for a random sample of 366 black and 297 white community-dwelling adults, aged 65 or over, residing in five counties in North Carolina. In addition, relationships between sites harboring these microorganisms and loss of attachment (LA) and pocket depth (PD) are presented in a manner that considers the lack of independence of sites within each person. Pocket depths and recession were measured on all teeth by trained examiners during household visits. Immunofluorescent assays for A. actinomycetecomitans, P. intermedia, and P. gingivalis were conducted on subgingival plaque samples obtained from the mesiobuccal aspect of the four first molar teeth using paper points. The prevalences of A. actinomycetemcomitans, P. intermedia, and P. gingivalis were greater in blacks than in whites. The most striking difference was seen for P. gingivalis, which was found in 38.8% of blacks and 9.4% of whites. Similar relationships were found when the percent of sites with these organisms were assessed. Blacks with P. gingivalis or P. intermedia had a higher prevalence of sites with LA greater than or equal to 7 mm as compared to blacks not infected with P. gingivalis or P. intermedia. The same was true for whites. Similar relationships between P. gingivalis or P. intermedia and PD greater than or equal to 6 mm were found for both blacks and whites.(ABSTRACT TRUNCATED AT 250 WORDS)


CoMFA analysis of the interactions of antipicornavirus compounds in the binding pocket of human rhinovirus-14.

A CoMFA analysis of eight compounds related to disoxaril whose X-ray structures bound to HRV-14 had been determined resulted in a strong positive correlation of activity with steric effects of the compounds, particularly toward the pore end of the compound binding site, and no correlation with electrostatic effects. These results confirm what had been previously found, that the activity of these compounds was highly dependent upon their hydrophobic nature as expressed by log p. The CoMFA study also confirmed the results from the comparison of a series of active and inactive compounds using volume maps which showed that bulk at the pore end of the molecule was conducive to high levels of antiviral activity while excessive bulk around the ring led to poor activity.


Conformational effects on the activity of drugs. 13. A revision of previously proposed models for the activation of alpha- and beta-adrenergic receptors.

The alpha 1-, alpha 2-, beta 1-, and beta 2-adrenergic properties of the 2-(3,4-dihydroxyphenyl)morpholines 3 and 4 (2-DPMs), of the 3-(3,4-dihydroxyphenyl)-3-piperidinols 5 and 6 (3-DPPs), and of the trans-2-amino-5,6-dihydroxytetrahydronaphthalen-1-ols 7 and 8 and the trans-2-amino-6,7-dihydroxytetrahydronaphthalen-1-ols 9 and 10 (2-ADTNs) were evaluated in vitro both by radioligand binding assays and by functional tests on isolated preparations and compared with those of norepinephrine (NE, 1) and isoprenaline (ISO, 2). Through a comparison of the stereostructures of the compounds examined with their biopharmacological properties, it was possible to revise previously proposed molecular models for the direct activation of alpha- and beta-adrenergic receptors. The revised models (A-C) provided information about the conformational requirements of adrenergic drugs, which substantially fit in with the results of several published studies involving conformationally-restricted adrenoceptor agonists. The different position of the catecholic hydroxyl groups in model B, which refers to the alpha 2 receptors, and in model C, which refers to the beta receptors, confirms the importance of the rotameric position of the aromatic ring of catecholamines in the interaction with the alpha- and beta-adrenergic receptor.


(H+,K+)-ATPase inhibiting 2-[(2-pyridylmethyl)sulfinyl]benzimidazoles. 4. A novel series of dimethoxypyridyl-substituted inhibitors with enhanced selectivity. The selection of pantoprazole as a clinical candidate.

[(Pyridylmethyl)sulfinyl]benzimidazoles 1 (PSBs) are a class of highly potent antisecretory (H+,K+)-ATPase inhibitors which need to be activated by acid to form their active principle, the cyclic sulfenamide 4. Selective inhibitors of the (H+,K+)-ATPase in vivo give rise to the nonselective thiophile 4 solely at low pH, thus avoiding interaction with other thiol groups in the body. The propensity to undergo the acid-catalyzed transformation is dependent on the nucleophilic/electrophilic properties of the functional groups involved in the formation of 2 since this step is both rate-determining and pH-dependent. The aim of this study was to identify compounds with high (H+,K+)-ATPase inhibitory activity in stimulated gastric glands possessing acidic pH, but low reactivity (high chemical stability) at neutral pH as reflected by in vitro (Na+,K+)-ATPase inhibitory activity. The critical influence of substituents flanking the pyridine 4-methoxy substituent present in all derivatives was carefully studied. The introduction of a 3-methoxy group gave inhibitors possessing a combination of high potency, similar to omeprazole and lansoprazole, but increased stability. As a result of these studies, compound 1a (INN pantoprazole) was selected as a candidate drug and is currently undergoing phase III clinical studies.


Susceptibility of selected strains of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) to chikungunya virus.

The relative susceptibility of selected strains of Aedes aegypti (L.) and Aedes albopictus (Skuse) fed on a viremic monkey to infection with chikungunya virus was determined. Infection rates were consistently higher in 10 strains of Ae. albopictus tested than in 7 strains of Ae. aegypti tested, regardless of the geographic location from which the strains originated or the dose of virus ingested. Similarly, virus dissemination rates were higher in the Ae. albopictus strains compared with the Ae. aegypti strains. For nearly all (11 of 12) strains tested of both species, groups of mosquitoes with one or more females with a disseminated infection transmitted virus by bite to weanling mice. Based on these studies, Ae. albopictus appears to be a more competent laboratory vector of chikungunya virus than does Ae. aegypti.


An intrachromosomal insertion causing 5q22 deletion and familial adenomatous polyposis coli in two generations.

We report familial adenomatous polyposis coli (FAPC) with epidermoid cysts, osteomata, and areas of congenital hypertrophy of the retinal pigment epithelium (CHRPEs) in a male patient and his maternal aunt, both of whom suffered a mild to moderate degree of mental handicap. Both had an interstitial deletion of the long arm of chromosome 5 (del(5)(q22q23.2)). Two other normal family members had the underlying direct insertion of chromosome 5(dir ins(5)(q31.3q22q23.2)). Molecular genetic and fluorescent hybridisation studies have shown that loci D5S37 and D5S98 are outside the deletion whereas loci detected by probes EF5.44 and YN5.48 are lost. As expected, the molecular analyses indicate loss of one allele at the MCC and APC loci. The APC gene is located within band 5q22. Familial direct insertions should be considered as a cause of recurrent microdeletion syndromes.


Regulation of endogenous and expressed Na+/K+ pumps in Xenopus oocytes by membrane potential and stimulation of protein kinases.

Modulation of the current generated by the Na+/K+ pump by membrane potential and protein kinases was investigated in oocytes of Xenopus laevis. In addition to a positive slope region in the current-voltage (I-V) relationship of the Na+/K+ pump, a negative slope region has been described in these cells (Lafaire & Schwarz, 1986) and has been attributed to a voltage-dependent apparent Km value for pump stimulation by external [K+] (Rakowski et al., 1991). To study this feature in more detail, Xenopus oocytes were used for comparative analysis of the negative slope of the I-V relationship of the endogenous Na+/K+ pump and of the Na+/K+ pump of the electric organ of Torpedo californica expressed in the oocytes. The effects of stimulation of protein kinases A and C on the negative slope were also analyzed. To investigate the negative slope over a wide potential range, experiments were performed in Na(+)-free solution and in the presence of high concentrations of Ba2+ and tetraethylammonium, to block all nonpump related K(+)-sensitive currents. Pump currents and pump-mediated fluxes were determined as differences of currents or fluxes in solutions with and without extracellular K+. The voltage dependence of the Km value for stimulation of the Na+/K+ pump by external [K+] shows significant species differences. Over the entire voltage range from -140 to +20 mV, the Km value for the Na+/K+ pump of Torpedo electroplax is substantially higher than for the endogenous pump and exhibits more pronounced voltage dependence. For the Xenopus pump, the voltage dependence can be described by voltage-dependent stimulation by external [K+] and can be interpreted by voltage-dependent K+ binding, assuming that an effective charge between 0.37 and 0.56 of an elementary charge is moved in the electrical field. An analogous evaluation of the voltage dependence of the Torpedo pump requires the assumption of movement of two effective charges of 0.16 and 1.0 of an elementary charge. Application of 1,2-dioctanoyl-sn-glycerol (diC8, 10-50 microM), which is known to stimulate protein kinase C, reduces the maximum activity of the Xenopus pumps in the oocyte membrane by 40% and modulates the voltage dependence of K+ stimulation. For the endogenous Xenopus pump, the apparent effective charge increased from 0.37 to 0.51 of elementary charge and the apparent Km at 0 mV increased from 0.46 to 0.83 mM. For the Torpedo pump, one of the apparent effective charges increased from 1.0 to 2.5 of elementary charge.(ABSTRACT TRUNCATED AT 400 WORDS)


Electrogenic proton secretion in the hindgut of the desert locust, Schistocerca gregaria.

The cellular mechanisms responsible for rectal acidification in the desert locust, Schistocerca gregaria, were investigated in isolated recta mounted as flat sheets in modified Ussing chambers. Previous studies conducted in the nominal absence of exogenous CO2 and HCO3- suggested that the acidification was due to a proton-secretory rather than bicarbonate-reabsorptive mechanism (Thomson, R.B., Speight, J.D., Phillips, J.E. 1988. J. Insect Physiol. 34:829-837). This conclusion was confirmed in the present study by demonstrating that metabolic CO2 could not contribute sufficient HCO3- to the lumen to account for the rates of rectal acidification observed under the nominally CO2/HCO3(-)-free conditions used in these investigations. Rates of luminal acidification (JH+) were completely unaffected by changes in contraluminal pH, but could be progressively reduced (and eventually abolished) by imposition of either transepithelial pH gradients (lumen acid) or transepithelial electrical gradients (lumen positive). Under short-circuit current conditions, the bulk of JH+ was not dependent on Na+, K+, Cl-, Mg2+, or Ca2+ and was due to a primary electrogenic proton translocating mechanism located on the apical membrane. A small component (10-16%) of JH+ measured under these conditions could be attributed to an apical amiloride-inhibitable Na+/H+ exchange mechanism.


Serum independence of low K+ induction of Na,K-ATPase: possible role of c-fos.

Cultured ARL15 cells respond to abnormally low extracellular K+ concentrations by increasing the abundance of Na,K-ATPase (the Na/K pump). This response is preceded by significant increases in the mRNAs of the alpha 1 and beta 1 subunits of this enzyme, implying transcriptional or post-transcriptional regulation in the response. The present study concerned the possible participation of serum factors in low K+ induction of Na,K-ATPase. In normal K+ (4.5 mM) or low K+ (0.68 mM) the presence of 10% calf serum had no effect on Na,K-ATPase activity. The serum independence of the response to low K+ raised the possibility that low K+ may itself elicit a "growth" response. Accordingly, the effect of low K+ on mRNA abundances of four proto-oncogenes (c-fos, c-myc, c-jun and c-ski) was evaluated in the early phase of the response by quantitative Northern blot analysis. The mRNA for c-fos was transiently elevated by low K+, with a peak at 30 min. In contrast, low K+ had no measurable effect on the abundances of c-myc, c-jun and c-ski, for up to 2 hr of exposure. The early elevation of c-fos mRNA makes it a candidate mediator in this signal-transduction pathway. Induction of c-fos mRNA by the phorbol ester, PMA, or by dioctanoyl glycerol, however, had no effect on Na,K-ATPase activity. These results indicate that an increase in c-fos mRNA alone is not sufficient to induce Na,K-ATPase. Whether induction of c-fos is necessary for the response to low K+ remains to be determined in future studies.


Prevention of urinary tract infection and bacteremia following transurethral surgery: oral lomefloxacin compared to parenteral cefotaxime.

A multicenter, randomized, open label study compared the safety and efficacy of a single dose of oral lomefloxacin, a broad-spectrum antimicrobial agent of the quinolone class, to a single parenteral dose of cefotaxime, a third generation cephalosporin, for prophylaxis in transurethral surgery. Of the 230 patients initially recruited 182 were considered evaluable: 92 in the lomefloxacin group and 90 in the cefotaxime group. Both study groups were well balanced with respect to demographics and transurethral procedures. Efficacy and safety were evaluated with urine cultures, clinical laboratory evaluations and monitoring of adverse events. The success rate among the lomefloxacin patients was 98% versus 94% in the cefotaxime patients. The difference was not statistically significant. Adverse events, regardless of attributability, were reported by 16% of the lomefloxacin patients and 17% of the cefotaxime patients, respectively. Our results indicate efficacy and safety profiles of lomefloxacin equivalent to cefotaxime. Lomefloxacin has the economic advantage of an oral route of administration compared to the parenteral route of cefotaxime for prophylaxis in transurethral genitourinary procedures.


Aplastic anemia and viral hepatitis. Non-A, Non-B, Non-C?

To test the hypothesis that the rare, often fatal, syndrome of hepatitis-associated aplasia is associated with hepatitis C virus infection. Case series. Tertiary referral centers in the United States, Japan, Italy, and Germany. Twenty-eight patients with onset of aplastic anemia within 90 days after seeking medical attention for jaundice, or having serum transaminase levels 150% or more of normal (hepatitis-associated aplasia patients) and three patients who developed aplastic anemia following liver transplantation for non-A, non-B hepatitis. Presence of hepatitis C in serum, bone marrow, and liver samples, detected by the polymerase chain reaction; antibody testing; and percentage of activated peripheral cytotoxic T lymphocytes determined by immunophenotyping. Hepatitis ribonucleic acid was present in the serum samples of 10 (36%) patients with hepatitis-associated aplasia. However, hepatitis C virus viremia was associated with transfusions received after the onset of aplasia: seven (58%) of 12 patients with hepatitis-associated aplasia who had received 21 or more units of blood products at the time of serum sampling were viremic, compared with only three (19%) of 16 patients with hepatitis-associated aplasia who had received 20 or less units of blood products (P less than .05). Hepatitis C virus was not found in blood and bone marrow samples of three National Institutes of Health case patients tested at the time of diagnosis. None of three livers from non-A, non-B hepatitis patients who developed aplastic anemia after liver transplantation contained hepatitis C virus ribonucleic acid. Activated CD8+ T lymphocytes were elevated three- to 20-fold early in the course of hepatitis-associated aplasia. Our results implicate a novel, non-A, non-B, and non-C agent in both hepatitis-associated aplasia and fulminant hepatitis.