Moloney murine sarcoma virus MuSVts110 DNA: cloning, nucleotide sequence, and gene expression.

We have cloned Moloney murine sarcoma virus (MuSV) MuSVts110 DNA by assembly of polymerase chain reaction (PCR)-amplified segments of integrated viral DNA from infected NRK cells (6m2 cells) and determined its complete sequence. Previously, by direct sequencing of MuSVts110 RNA transcribed in 6m2 cells, we established that the thermosensitive RNA splicing phenotype uniquely characteristic of MuSVts110 results from a deletion of 1,487 nucleotides of progenitor MuSV-124 sequences. As anticipated, the sequence obtained in this study contained precisely this same deletion. In addition, several other unexpected sequence differences were found between MuSVts110 and MuSV-124. For example, in the noncoding region upstream of the gag gene, MuSVts110 DNA contained a 52-nucleotide tract typical of murine leukemia virus rather than MuSV-124, suggesting that MuSVts110 originated as a MuSV-helper murine leukemia virus recombinant during reverse transcription rather than from a straightforward deletion within MuSV-124. In addition, both MuSVts110 long terminal repeats contained head-to-tail duplications of eight nucleotides in the U3 region. Finally, seven single-nucleotide substitutions were found scattered throughout MuSVts110 DNA. Three of the nucleotide substitutions were in the gag gene, resulting in one coding change in p15 and one in p30. All of the remaining nucleotide changes were found in the noncoding region between the 5' long terminal repeat and the gag gene. In NIH 3T3 cells transfected with the cloned MuSVts110 DNA, the pattern of viral RNA expression conformed with that observed in cells infected with authentic MuSVts110 virus in that viral RNA splicing was 30 to 40% efficient at growth temperatures between 28 and 33 degrees C but reduced to trace levels above 37 degrees C.


A cysteine and a hydrophobic sequence in the noncleaved portion of the pre-C leader peptide determine the biophysical properties of the secretory core protein (HBe protein) of human hepatitis B virus.

The molecular basis of the biophysical and antigenic differences between the cellular core protein (HBc protein) and the secreted core protein (HBe protein) of human hepatitis B virus was examined. The data show that the properties which distinguish the HBe protein from the HBc protein are due mostly to the 10-amino-acid portion of the HBe leader sequence which remains attached to the HBe protein after cleavage. A cysteine located within this region determines the quaternary structure and the antigenicity of the HBe protein. If this cysteine is lacking, the HBe protein, which is predominantly a monomer with only HBe antigenicity, is expressed as a disulfide-linked homodimer showing both HBe and HBc antigenicity. However, dimerization of the HBe protein was found to be neither sufficient nor required for particle formation. In fact, aggregation of the HBe protein was found to be inhibited by the strongly hydrophobic tripeptide Trp-Leu-Trp, which is also located in the noncleaved portion of the signal sequence. If this tripeptide was converted into either Asp-Asn-Asn or Ala-Asp-Leu, the HBe protein assembled into particles, independent of the presence of the cysteine.


Proteolytic cleavage of the reovirus sigma 3 protein results in enhanced double-stranded RNA-binding activity: identification of a repeated basic amino acid motif within the C-terminal binding region.

The reovirus capsid protein sigma 3 was examined for double-stranded RNA (dsRNA)-binding activity by Northwestern (RNA-protein) blot analysis. Treatment of virion-derived sigma 3 protein with Staphylococcus aureus V8 protease led to an increase in the dsRNA-binding activity associated with the C-terminal fragment of the protein. Recombinant C-terminal fragments of the sigma 3 protein were expressed in Escherichia coli from the S4 cDNA of reovirus serotype 1. These truncated sigma 3 proteins displayed proteolytic processing and dsRNA-binding activity similar to those observed for native, virion-derived sigma 3 protein as measured by Northwestern blot analysis. Construction of a modified pET3c vector, pET3Exo, allowed the production of 3'-terminal deletions of the S4 cDNA by using exonuclease III and rapid screening of the induced truncated sigma 3 proteins. An 85-amino-acid domain within the C-terminal portion of the sigma 3 protein which was responsible for dsRNA-binding activity was identified. The 85-amino-acid domain possessed a repeated basic amino acid motif which was conserved in all three serotypes of reovirus. Deletion of one of the basic motifs, predicted to be an amphipathic alpha-helix, destroyed dsRNA-binding activity.


Vaccinia virus infection induces a stress response that leads to association of Hsp70 with viral proteins.

We studied the impact of vaccinia virus infection on stress protein gene expression in human cells and investigated the possibility that eukaryotic heat shock proteins interact with viral components during assembly. Infection of human monocyte-macrophages by vaccinia virus caused a dramatic decrease in levels of cellular mRNAs such as those encoding actin and tubulin. In contrast, infection did not cause a significant reduction in the levels of Hsp90 and Hsp60 mRNAs and led to substantially increased levels of Hsp70 mRNAs. The accumulation of these stress protein mRNAs was due both to increases in their transcription rate and to their stability relative to other cellular mRNAs. The relative levels of the heat shock proteins and the other cellular proteins reflected the relative levels of their mRNAs. These results indicate that stress protein gene expression is relatively refractory to the generally deleterious effects of vaccinia virus infection on host cell gene expression. The continued expression of some of these stress proteins may be beneficial to the virus; the observations that the levels of Hsp70 are greatest at the peak of viral gene expression and that a large fraction of cellular Hsp70 is associated with vaccinia virus proteins suggest that Hsp70 is involved in vaccinia virus assembly.


Cys residues of the hepatitis B virus capsid protein are not essential for the assembly of viral core particles but can influence their stability.

In the spherical capsid of hepatitis B virus (HBV), intermolecular disulfide bonds cross-link the approximately 180 p21.5 capsid protein subunits into a stable lattice. In this study, we used mutant capsid proteins to investigate the role that disulfide bonds and the four p21.5 Cys residues (positions 48, 61, 107, and 185) play in capsid assembly and/or stabilization. p21.5 Cys residues were either replaced by Ala or removed (Cys-185) by carboxyl-terminal truncation, creating Cys-minus mutants which were expressed in Xenopus oocytes via microinjected synthetic mRNAs. Fractionation of radiolabeled oocyte extracts on 10 to 60% sucrose gradients revealed that Cys-minus core proteins resolved into the nonparticulate and capsid forms seen for wild-type p21.5. On 5 to 30% sucrose gradients, nonparticulate Cys-minus core proteins sedimented as dimers of approximately 40 kDa. We conclude that Cys residues and disulfides are not required for the assembly of either HBV capsids or the dimers that provide the precursors for capsid assembly. Since assembly presumably demands an appropriate p21.5 tertiary structure, it is unlikely that Cys residues are required for proper p21.5 folding. However, Cys residues stabilize isolated p21.5 structures, as evidenced by the marked reduction in stability of Cys-minus dimers and capsids (i) in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and (ii) upon protease digestion. We discuss these results in the context of the HBV life cycle and the role of Cys residues in other proteins.


Infectious Japanese encephalitis virus RNA can be synthesized from in vitro-ligated cDNA templates.

Japanese encephalitis virus (JEV) is a positive-stranded enveloped RNA virus that belongs to the family Flaviviridae. Genomic JEV RNA is approximately 11 kb long and encodes 10 proteins, 3 structural and 7 nonstructural. A full-length cDNA copy of the JEV genome was constructed by in vitro ligation of two cDNA fragments which encode the 5' (nucleotide positions 1 to 5576) and 3' (nucleotide positions 5577 to 10976) halves of the genome. T7 RNA polymerase transcripts of the ligated full-length cDNA template were infectious when transfected into BHK-21 cells. To identify the recombinant virus, a silent mutation was introduced into the clone encoding the 3' half of the genome, which abolished an XbaI site at nucleotide position 9131. Virus recovered by transfection with the transcripts contained this silent mutation, confirming its identity. Recombinant and parent viruses were identical with respect to growth and plaque production in BHK-21 cells, envelope protein expression in C6/36 cells, and neurovirulence and immunogenicity in mice. Repeated attempts to obtain infectious RNA by transcription from full-length JEV genome cDNA templates cloned into plasmid vectors were unsuccessful. Synthesis of infectious JEV RNA from in vitro-ligated JEV cDNA templates will be useful for molecular and genetic studies of flavivirus replication and virulence.


The nef gene of simian immunodeficiency virus SIVmac1A11.

The role of the simian immunodeficiency virus (SIV) nef gene in viral replication was investigated in several tissue culture systems. SIVmac1A11 is a molecularly cloned virus which replicates in both peripheral blood mononuclear cells (PBMC) and macrophages, although no disease is observed in infected rhesus macaques. In this report, we demonstrate that SIVmac1A11 contains a full open reading frame for nef which specifies a 37-kDa protein. To investigate the effects of nef on viral replication, a 70-bp deletion was introduced into the nef gene of SIVmac1A11. Analysis of infected cell extracts by immunoblotting revealed that both SIVmac1A11 and nef deletion virus SIVmac1A11 delta nef produced the same viral proteins, except that Nef was absent in the mutant virus. The deletion mutation did not affect viral replication in PBMC, in monocyte-derived and alveolar macrophages obtained from rhesus macaques, and in human cell lines HUT-78 and CEMx-174. In addition, SIVmac1A11 and SIVmac1A11 delta nef exhibited similar patterns of cytopathologic changes and ultrastructural appearances in infected cells. SIVmac1A11 and SIVmac1A11 delta nef did not infect human tumor macrophage cell line U937, GCT, THP-1, or HL-60 cells, although virus was produced after these cells were transfected with either wild-type or nef mutant viral DNA. Similar levels of virus were recovered from U937 and THP-1 cells transfected with mutant and parental proviral DNAs. In transient expression assays in a T-cell line and a macrophage line, the nef protein of SIVmac1A11 did not significantly suppress or enhance expression of the chloramphenicol acetyltransferase reporter gene linked to the SIVmac long terminal repeat. Thus, abrogation of nef did not affect several in vitro properties of SIVmac1A11, including patterns of viral infection in rhesus PBMC, rhesus macrophages, or human T-cell lines.


Identification and characterization of fusion and processing domains of the human immunodeficiency virus type 2 envelope glycoprotein.

The envelope glycoprotein of the human immunodeficiency virus type 2 (HIV-2) is synthesized as a polyprotein precursor which is proteolytically processed to produce the mature surface and transmembrane envelope glycoproteins. The processed envelope glycoprotein species are responsible for the fusion between the viral envelope and the host cell membrane during the infection process. The envelope glycoprotein also induces syncytium formation between envelope-expressing cells and receptor-bearing cells. To characterize domains of the HIV-2 envelope glycoprotein involved in membrane fusion and in proteolytic processing, we introduced single amino acid mutations into the region of the HIV-2 surface glycoprotein corresponding to the principal neutralizing determinant (the V3 loop) of HIV-1, the putative HIV-2 envelope precursor-processing sequence, and the hydrophobic amino terminus of the HIV-2 transmembrane envelope glycoprotein. The effects of these mutations on syncytium formation, virus infectivity, envelope expression, envelope processing, and CD4 binding were analyzed. Our results suggest that the V3-like region of the HIV-2 surface glycoprotein and the hydrophobic amino terminus of the transmembrane glycoprotein are HIV-2 fusion domains and characterize the effects of mutations in the HIV-2 envelope glycoprotein precursor-processing sequence.


Activation of the human immunodeficiency virus type 2 enhancer is dependent on purine box and kappa B regulatory elements.

Human immunodeficiency virus type 2 (HIV-2) displays several features which distinguish it from HIV-1. Among the differences in these two viruses are the responses of their enhancer regions to T-cell activation. For example, stimulation of HIV-1 transcription is largely dependent on two kappa B regulatory elements. In contrast, the HIV-2 enhancer has a single kappa B site and contains additional cis-acting sequences responsive to induction. One of these sites, previously termed CD3R, is a purine-rich site, also called PuB1, which is responsive to stimulation of the CD3 component of the T-cell receptor complex and binds Elf-1, a member of the ets proto-oncogene family. In this report, we examine the interaction of the PuB1 site with other sites in the HIV-2 enhancer. We demonstrate that the PuB1 site confers responsiveness to T-cell activators only in cooperation with additional enhancer elements. Induction of the HIV-2 enhancer is dependent on at least two other cis-acting regulatory elements in addition to PuB1 and kappa B. One of these elements is another purine-rich site (PuB2), which also binds recombinant Elf-1. An adjacent region, proximal to the PuB2 ets (pets) site, shows protection in DNase footprinting experiments with extracts from Jurkat T cells. Mutation of either the kappa B, PuB1, PuB2, or pets site significantly reduces the response of the HIV-2 enhancer to T-cell stimulation, an effect which is mediated at the RNA level. Therefore, activation of the HIV-2 enhancer is dependent on at least four cis-acting elements, only one of which is found in HIV-1, which act in synergy with one another. Despite their sequence similarity, the organization and function of the HIV-2 enhancer have diverged considerably from those of HIV-1.


High level of surface CD4 prevents stable human immunodeficiency virus infection of T-cell transfectants.

CD4 is the principal receptor for the human immunodeficiency virus (HIV). We have isolated and studied CD4-expressing tumor cell clones made by expressing CD4 in the T-cell tumor line HSB. Two clones, one designated HSBCD4, a clone expressing low levels of CD4, and the other, HSB10xCD4, a high-expresser CD4+ clone, were studied for their ability to bind and replicate HIV. In contrast to many other CD4+ cells that down-modulate CD4 following HIV infection, the HSB10xCD4 clones continued to express high levels of surface CD4 following infection with HIV. Unlike infection of HSBCD4 or many other human CD4+ cells, HIV infection of HSB10xCD4 clone was short lived: p24 antigen, provirus, or coculturable virus was present for less than 14 days following infection with several strains of HIV-1 or with HIV-2. When infection was initiated by transfection of proviral DNA, high and low CD4 expressers initially produced p24 antigen at approximately the same level. However, high CD4 expressers produced coculturable virus only during the first few days following transfection, whereas low CD4 expressers transfected with HIV continued to produce virus beyond 6 weeks. Monoclonal antibody-mediated down-modulation of CD4 surface expression on HSB10xCD4 clones permitted these formerly HIV-resistant cells to become persistently infected with HIV. Thus, high concentrations of CD4 on the surface of an HIV-infected cell prevent persistent HIV infection of CD4+ cells.


Lack of correlation between soluble CD4-induced shedding of the human immunodeficiency virus type 1 exterior envelope glycoprotein and subsequent membrane fusion events.

The noncovalent association of the gp120 and gp41 envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) is disrupted by soluble CD4 binding, resulting in shedding of the gp120 exterior envelope glycoprotein. This observation has led to the speculation that interaction of gp120 with the CD4 receptor triggers shedding of the exterior envelope glycoprotein, allowing exposure of gp41 domains necessary for membrane fusion steps involved in virus entry or syncytium formation. To test this hypothesis, a set of HIV-1 envelope glycoprotein mutants were used to examine the relationship of soluble CD4-induced shedding of the gp120 glycoprotein to envelope glycoprotein function in syncytium formation and virus entry. All mutants with a threefold or greater reduction in CD4-binding ability exhibited marked decreases in gp120 shedding in response to soluble CD4, even though several of these mutants exhibited significant levels of envelope glycoprotein function. Conversely, most fusion-defective mutants with wild-type gp120-CD4 binding affinity, including those with changes in the V3 loop, efficiently shed gp120 following soluble CD4 binding. Thus, soluble CD4-induced shedding of gp120 is not a generally useful marker for conformational changes in the HIV-1 envelope glycoproteins necessary for the virus entry or syncytium formation processes. Some gp120 mutants, despite being expressed on the cell surface and capable of efficiently binding soluble CD4, exhibited decreased gp120 shedding. These mutants were still sensitive to neutralization by soluble CD4, indicating that, for envelope glycoproteins exhibiting high affinity for soluble CD4, competitive inhibition may be more important than gp120 shedding for the antiviral effect.


Site-specific mutagenesis of the 35-kilodalton protein gene encoded by Autographa californica nuclear polyhedrosis virus: cell line-specific effects on virus replication.

The gene encoding the 35-kDa protein (35k gene) located within the EcoRI-S genome fragment of Autographa californica nuclear polyhedrosis virus (AcMNPV) is transcribed early in infection. To examine its function(s) with respect to virus multiplication, we introduced specific mutations of this early gene into the AcMNPV genome. In Spodoptera frugiperda (SF21) culture, deletion of the 35K gene reduced yields of extracellular, budded virus from 200- to 15,000-fold, depending on input multiplicity. Mutant replication was characterized by dramatically diminished levels of late and very late (occlusion-specific) virus gene expression and premature cell lysis. In contrast, 35K gene inactivation had no effect on virus growth in cultured Trichoplusia ni (TN368) cells. Insertion of the 35K gene and its promoter at an alternate site (polyhedrin locus) restored virus replication to wild-type levels in SF21 culture. Subsequent insertion of 4 bp after codon 81 generated a frameshift mutant that exhibited a virus phenotype indistinguishable from that of 35K deletion mutants and demonstrated that the 35K gene product (p35) was required for wild-type replication in SF21 cells. Mutagenesis also indicated that the C terminus of p35, including the last 12 residues, was required for function. In complementation assays, wild-type virus bearing a functional 35K gene allele stimulated all aspects of 35K null mutant replication and suppressed early cell lysis. These findings indicated that p35 is a trans-dominant factor that facilitates AcMNPV growth in a cell line-specific manner.


Antibody-dependent transcriptional regulation of measles virus in persistently infected neural cells.

Application of neutralizing anti-hemagglutinin antibodies to mouse neuroblastoma cells (NS20Y/MS) persistently infected with measles virus (MV) leads to a significant reduction of viral structural proteins within 6 days. While the transcriptional gradient for MV-specific mRNAs remained unaffected upon antibody treatment, the total amount of MV-specific transcripts dropped by 80% after 24 h. The expression of genomic RNA was affected similarly, with slightly slower time kinetics. Both transcription and expression of the viral structural proteins could be completely reactivated when viral antibodies were removed from the tissue culture. The same findings could be obtained in rat glioma cells persistently infected with subacute sclerosing panencephalitis virus (C6/SSPE) but not in cells of nonneural origin. The data indicate that antibody-induced antigenic modulation affects the early stages of viral transcription within a few hours after the addition of antibodies and leads to an almost complete repression of viral gene expression in cells of neural origin.


Expression of the influenza A virus M2 protein is restricted to apical surfaces of polarized epithelial cells.

The M2 protein of influenza A virus is a small, nonglycosylated transmembrane protein that is expressed on surfaces of virus-infected cells. A monoclonal antibody specific for the M2 protein was used to investigate its expression in polarized epithelial cells infected with influenza virus or a recombinant vaccinia virus that expresses M2. The expression of M2 on the surfaces of influenza virus-infected cells was found to be restricted to the apical surface, closely paralleling that of the influenza virus hemagglutinin (HA). Membrane domain-specific immunoprecipitation indicated that the M2 protein was inserted directly into the apical membrane with transport kinetics similar to those of HA. In polarized cells infected with a recombinant vaccinia virus that expresses M2, we found that 86 to 93% of surface M2 was restricted to the apical domain compared with 88 to 90% of HA in a similar assay. These results indicate that the M2 protein undergoes directional transport in the absence of other influenza virus proteins and that M2 contains the structural features required for apical transport in polarized epithelial cells. The ultrastructural localization of the M2 protein in influenza virus-infected MDCK cells was investigated by immunoelectron microscopy using M2 antibody and a gold conjugate. In cells in which extensive virus budding was occurring, the apical cell membrane was labeled with gold particles evenly distributed between microvilli and the surrounding membrane. In addition, a significant fraction of the M2 label was apparently associated with virions. A monoclonal antibody specific for HA demonstrated a similar labeling pattern. These results indicate that M2 is localized in close proximity to budding and assembled virions.


Effect of reciprocal complementation of two defective human immunodeficiency virus type 1 (HIV-1) molecular clones on HIV-1 cell tropism and virulence.

Human immunodeficiency virus type 1 (HIV-1) displays both interstrain and intrastrain genetic variability. Virus populations with extensive microheterogeneity have been defined as swarms or quasispecies. Many of the genomes within HIV-1 swarms appear to be defective in one or more genes required for viral replication. It is unclear to what extent defective viruses play a role in the process of HIV-1 infection or in the pathogenesis of AIDS. We have isolated two biologically active HIV-1 clones: LW 12.3, which contains defects in the vif and vpr genes, and MN ST.1, which has a defect in the vpu gene. LW 12.3 is unable to replicate in peripheral blood mononuclear cells (PBMC). The growth of MN-ST.1 in SupT1 cells is marked by a 3-week lag in extracellular virus production and by the presence of unusually abundant viral buds. We demonstrate here that coinfection of PBMC with these two partially defective HIV-1 clones extends the cellular host range of LW 12.3, significantly increases the replication rate of both viral genomes, and eliminates the delay in production observed with the vpu-defective MN ST.1. When the lesions in vpr and vif of LW 12.3 are repaired, the resultant virus grows normally in PBMC. This is also the case when only vif is repaired, indicating that complementation of LW 12.3 in PBMC by MN ST.1 is mediated by vif in trans. The reciprocal complementation results in a dramatic increase of HIV-1 virulence. This two-component model represents a simplified version of the in vivo situation and illustrates one way in which interaction of defective viruses could increase the spread of infection and progression of disease.


In vivo binding of wild-type and mutant human immunodeficiency virus type 1 Rev proteins: implications for function.

The Rev transactivator protein of human immunodeficiency virus type 1 (HIV-1) is required for protein expression from the HIV-1 RNAs which contain a binding site for the Rev protein, termed the Rev-responsive element (RRE). This transactivator acts both at the level of splicing/transport of nuclear RNAs and at the level of translation of cytoplasmic RNAs. We used a monoclonal antibody specific for the HIV-1 Rev protein to immunoprecipitate cellular extracts from HIV-1-infected and -transfected cells. High levels of specific binding of wild-type Rev to the RRE-containing RNAs were found in cytoplasmic, but not nuclear, extracts from these cells. A Rev mutant which lacked both nuclear and cytoplasmic Rev function but retained RNA binding in vivo was generated. This binding was detectable with both nuclear and cytoplasmic extracts. These results verify the existence of direct binding of Rev to HIV-1 RNAs in vivo and conclusively prove that binding of Rev is not sufficient for nuclear or cytoplasmic Rev function. The results also support a direct role for Rev in the nuclear export and translation of HIV-1 RNAs.


Tat- and Rev-directed antisense RNA expression inhibits and abolishes replication of human immunodeficiency virus type 1: a temporal analysis.

Replication of human immunodeficiency virus type 1 (HIV-1) was inhibited by stable intracellular expression of antisense RNA in the human T-lymphoid cell line Jurkat. When the viral subregion encoding the HIV-1 activator proteins was targeted, the extent of antisense RNA-mediated inhibition was greater than 97% during the first 2 weeks postinfection. Later in the time course, productive HIV-1 infection broke through at high initial infective doses. However, at initial multiplicities of infection equal to or smaller than 0.1, HIV-1 production was not detectable during the 5 weeks of observation. The results underline the effectiveness of stable intracellular antisense RNA expression in inhibiting HIV-1 replication.


Contribution of the TATA motif to Tat-mediated transcriptional activation of human immunodeficiency virus gene expression.

Tat-mediated transcriptional activation of human immunodeficiency virus (HIV) gene expression requires the presence of the cis-acting Tat-responsive element, TAR, and a functional enhancer-promoter element. The ability of Tat to function with heterologous enhancer sequences led us to examine the role of the minimal basal promoter for trans activation. Substitution of HIV TATA sequences (nucleotides -20 to -35) with TATA elements derived from other promoters had little effect on the basal level of transcription or the ability to activate the HIV long terminal repeat upon stimulation through upstream activation sequences. In contrast, minimal alterations within the TATA motif had a profound effect on trans activation, as demonstrated by the 3- to 10-fold reduction in activation following expression of Tat. Our findings suggest that minor changes in the TATA motif affect the composition of the initiation-elongation complex and that the composition of this complex is critical for Tat-dependent activation of gene expression.


Multimeric CD4 binding exhibited by human and simian immunodeficiency virus envelope protein dimers.

The envelope (Env) glycoproteins of human and simian immunodeficiency viruses (HIV and SIV) form noncovalently associated oligomers which mediate virus binding to the cell surface and fusion between the viral envelope and plasma membrane. A high-affinity interaction with CD4 is a critical step in this process. In this report, we show that Env protein dimers, but not monomers, can bind two CD4 molecules simultaneously. Multimeric CD4 binding may have important implications for Env protein-CD4 avidity, CD4-induced release of gp120, and subunit-subunit cooperativity during virus membrane fusion as well as for therapeutic strategies.


Nonhomogeneous distribution of human immunodeficiency virus type 1 proviruses in the spleen.

A nonhomogeneous spatial distribution of human immunodeficiency virus type 1 proviruses in an infected spleen was observed. Antigenic stimulation of infected cells might explain this partition.


Interactions of HTF4 with E-box motifs in the long terminal repeat of human immunodeficiency virus type 1.

We have identified three consensus E-box motifs in the long terminal repeat of human immunodeficiency virus type 1. One of these E boxes interacts selectively with representative members of the class A group of basic helix-loop-helix proteins, including HTF4, E47, and their heterodimers. Our analyses implicate the helix-loop-helix proteins in regulation of human immunodeficiency virus type 1 gene expression.


Examination of potato virus X proteins synthesized in infected tobacco plants.

Nucleotide sequence analysis of potato virus X (PVX) genomic RNA predicts five open reading frames (ORFs). Previous analysis of total RNAs from PVX-infected leaf tissue suggested that six subgenomic RNAs are synthesized during infection. However, the proteins encoded by the genomic RNA, the subgenomic RNAs, or the predicted ORFs have not been identified in vivo. To characterize the coding properties of the viral RNA, particularly to determine whether the five predicted ORFs function in vivo, total protein extracts prepared from PVX-infected leaf tissue were analyzed by using antibodies raised against virus-specific synthetic peptides and against the virus capsid protein. Dot blot analyses showed that these antibodies reacted to PVX-infected extracts, indicating in vivo expression of the five predicted ORFs. In addition, Western blot (immunoblot) analysis of the extracts showed that ORF 1, 2, 3, and 4 peptide antisera and coat protein antiserum detect predominantly a single protein.


Regulation of nuclear transport of a plant potyvirus protein by autoproteolysis.

The NIa proteinase encoded by tobacco etch potyvirus catalyzes six processing events, three of which occur by an autoproteolytic mechanism. Autoproteolysis is necessary to cleave the boundaries of both NIa and the 6-kDa protein, which is located adjacent to the N terminus of NIa in the viral polyprotein. As a consequence, NIa may exist in a free form or in a transient polyprotein form containing the 6-kDa protein. While the majority of NIa molecules localize to the nuclei of infected cells, a fraction of the NIa pool is attached covalently to the 5' terminus of genomic RNA in the cytoplasm. To determine whether the presence of the 6-kDa protein affects the nuclear transport properties of NIa, we have generated transgenic plants that express genes encoding a reporter enzyme, beta-glucuronidase (GUS), fused to NIa or NIa-containing polyproteins. The NIa/GUS fusion protein was detected by histochemical analysis in the nucleus. Similarly, an NIa/GUS fusion protein that arose by autoproteolysis of a 6-kDa/NIa/GUS polyprotein was found in the nucleus. In contrast, fusion protein consisting of 6-kDa/NIa/GUS, which failed to undergo proteolysis because of the presence of a Cys-to-Ala substitution in the proteolytic domain of NIa, was detected in the cytoplasm. The inhibition of NIa-mediated nuclear transport was not due to the Cys-to-Ala substitution, since this alteration had no effect on translocation in the absence of the 6-kDa protein. These results indicate that the 6-kDa protein impedes nuclear localization of NIa and suggest that subcellular transport of NIa may be regulated by autoproteolysis.


The C terminus of human immunodeficiency virus type 1 matrix protein is involved in early steps of the virus life cycle.

Deletion mutations at the C terminus of the matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) were generated by site-directed mutagenesis. The resultant mutant viruses had a severe defect in virus infectivity. This defect did not involve late steps of the virus life cycle, as the synthesis and processing of the Gag polyprotein and the assembly and release of mutant virions were not greatly affected. The incorporation of viral proteins and the viral RNA genome was similar for mutant and wild-type virions. In contrast, the early steps of the virus life cycle were severely affected, as the synthesis of viral DNA postinfection was dramatically reduced in mutant-virus-infected cells. One stretch of amino acids that was deleted in one of the mutants has significant homology with a region in VP1 of the picornavirus family. This region of VP1 is presumably involved in poliovirus penetration into cells. These results suggest that in addition to its functional role in virus assembly, the MA protein of HIV-1, and possibly of other retroviruses, plays an important role in virus entry.


The precore gene of the woodchuck hepatitis virus genome is not essential for viral replication in the natural host.

A number of naturally occurring hepatitis B virus mutants that cannot synthesize the virus precore protein have been identified. Such mutants have been associated with more severe forms of hepatitis, including fulminant hepatitis. The most common mutation observed is a substitution of G to A in the distal precore gene that converts a codon specifying Trp (TGG) to a termination codon (TAG). Using oligonucleotide-directed mutagenesis, we have produced the same point mutation in the precore gene of an infectious clone of woodchuck hepatitis virus (WHV). Transfection of mutant WHV DNA into the livers of adult woodchucks resulted in replication of the mutant in three of three susceptible animals. Levels of virus replication and transient elevations in liver enzymes in serum were similar to those of adult animals infected with wild-type WHV. Virions, found to possess mutant precore genes by polymerase chain reaction amplification and DNA sequencing, were recovered from the serum of one of the animals and inoculated subcutaneously into neonatal woodchucks. They produced infection in all five animals studied. The level of virus replication in neonatal animals infected with this mutant virus was comparable to that found in neonatal woodchucks infected with wild-type WHV, but none of five woodchucks infected with the precore mutant virus as neonates became chronic virus carriers. It was concluded that the precore gene of the WHV genome is not essential for virus replication in the natural host but may be important for chronic infection.


The downstream regulatory sequence of the adenovirus type 2 major late promoter is functionally redundant.

Mutagenesis of promoter sequences and oligonucleotide competition assays have been used to demonstrate the late-phase-specific stimulation of the adenovirus type 2 major late promoter is mediated by functionally redundant elements located between positions +75 and +125. These octamer motif-related sequences are recognized by multiple factors.


Interference established in mice by infection with Friend murine leukemia virus.

Retroviral interference is manifested in chronically infected cells as a decrease in susceptibility to superinfection by virions using the same cellular receptor. The pattern of interference reflects the cellular receptor specificity of the chronically infecting retrovirus and is mediated by the viral envelope glycoprotein, which is postulated to bind competitively all cellular receptors available for viral attachment. We established retroviral interference in mice by infecting them with Friend murine leukemia virus and them measured susceptibility to superinfection by challenging the mice with the erythroproliferative spleen focus-forming virus. Infection of approximately 10% of nucleated splenocytes rendered mice 1% as susceptible to superinfection as untreated controls. The magnitude of this effect was the same in mice incapable of producing neutralizing antibodies or genetically deficient for T cells. The results indicated that retroviral interference in vivo was established rapidly with infection of a fraction of the host cell population and that the decrease in susceptibility to superinfection occurred without a detectable contribution by immunologic factors.


Effect of a media blitz and a threatened lawsuit on stimulant treatment.

To enumerate and evaluate changes in the rate of medication treatment for hyperactive/inattentive students subsequent to negative media publicity about methylphenidate (Ritalin) and related lawsuits threatened or initiated from late 1987 to early 1989. Biennial 1971 to 1991 school nurse surveys of medication treatment for hyperactive/inattentive students; a 1989 school nurse questionnaire on parent attitudes about medication; annual 1984 through 1991 hyperkinetic clinic treatment data; annual 1986 through 1990 Drug Enforcement Administration estimates of retail sales of methylphenidate, nationally and locally. PRIMARY SETTING: All public and private elementary and secondary schools in Baltimore County, Maryland. Students receiving medication for hyperactivity/inattentiveness. Whereas the medication rate for the treatment of hyperactive/inattentive students in Baltimore County doubled every 4 to 7 years from 1971 through 1987, it declined 39% in the 1989 and 1991 surveys from its 1987 peak. This drop occurred after the 1987 through 1989 media blitz against methylphenidate and after a well-publicized threatened lawsuit locally. Parents became fearful of media-reported medication "side effects" and school staff hesitated to refer restless, impulsive, and inattentive students to physicians. Most inhibited from the prospect of medication treatment were less affluent parents and parents of hyperactive/inattentive elementary schoolchildren who had never received medication. Drug Enforcement Administration data revealed that the Baltimore metropolitan area had a far greater decline in methylphenidate use than that which occurred nationally. Strong circumstantial evidence suggests that the prominent 1989 and 1991 declines in the initiation of stimulant medication for hyperactive/inattentive students were related to the apprehension of parents and involved professionals generated by the methylphenidate media blitz and the threatened lawsuit.


Survey of advertising for nutritional supplements in health and bodybuilding magazines.

The use of food supplements by the general public is poorly quantified, and little information on this subject is available in the medical literature. We surveyed 12 recent issues of popular health and bodybuilding magazines (1) to quantify the number of advertisements for food supplements, the number of products advertised, and the number and type of ingredients in these products; (2) to identify the purported health benefits of these products; and (3) as a preliminary effort to identify areas for future research. We counted 89 brands, 311 products, and 235 unique ingredients, the most frequent of which were unspecified amino acids; the most frequently promoted health benefit was muscle growth. We also found many unusual or unidentifiable ingredients, and 22.2% of the products had no ingredients listed in their advertisements. Health professionals may not be aware of how popular food supplements are or of a particular supplement's potential effects or side effects. In addition, patients may be reluctant to discuss their use of these products with traditional medical practitioners. We recommend that routine history taking include specific questions about patients' use of food supplements and that any possible adverse effects or side effects be reported to public health authorities.


The definition of alcoholism. The Joint Committee of the National Council on Alcoholism and Drug Dependence and the American Society of Addiction Medicine to Study the Definition and Criteria for the Diagnosis of Alcoholism.

To establish a more precise use of the term alcoholism, a 23-member multidisciplinary committee of the National Council on Alcoholism and Drug Dependence and the American Society of Addiction Medicine conducted a 2-year study of the definition of alcoholism in the light of current concepts. The goals of the committee were to create by consensus a revised definition that is (1) scientifically valid, (2) clinically useful, and (3) understandable by the general public. Therefore, the committee agreed to define alcoholism as a primary, chronic disease with genetic, psychosocial, and environmental factors influencing its development and manifestations. The disease is often progressive and fatal. It is characterized by impaired control over drinking, preoccupation with the drug alcohol, use of alcohol despite adverse consequences, and distortions in thinking, most notably denial. Each of these symptoms may be continuous or periodic.


Multiple false-positive serologic tests for HIV, HTLV-1, and hepatitis C following influenza vaccination, 1991.

(1) To assess factors associated with the occurrence of multiple false-positive viral enzyme-linked immunosorbent assays (ELISAs) for human immunodeficiency virus (HIV), human T-cell lymphotrophic virus type 1 (HTLV-1), and hepatitis C virus (HCV) among individual blood donors and (2) to determine the frequency and time course of this phenomenon. Case-control study. A regional blood center. Blood donors found to have multiple false-positive viral ELISAs (case donors) and randomly selected seronegative controls (control donors) who donated between October 31, 1991, and December 15, 1991. An additional random sample of 262 donation records was reviewed to calculate the proportion of donors who received influenza vaccine. Multiple false-positive viral ELISAs, receipt of influenza vaccination formulated for the 1991-1992 influenza season, and follow-up ELISA results on serum samples obtained from case donors. Among 17,941 donors, 10 case donors were identified. Nine of the 10 case donors received influenza vaccine, compared with three of 30 control donors (odds ratio [OR] = 81; 95% confidence interval [CI], 6 to 3670; P less than .001). Among nine case donors, the mean time between vaccination and blood donation was 26 days (range, 9 to 68 days). Follow-up ELISAs of serum samples from seven case donors obtained 52 to 130 days (mean, 75 days) after vaccination demonstrated reversion to HIV and HTLV-1 seronegativity in all but one specimen, with persistence of positive HCV ELISAs in four specimens. We estimate between 0.6% and 1.7% of blood donors who received influenza vaccine this season had multiple false-positive viral ELISAs. The occurrence of multiple false-positive viral ELISAs among blood donors was associated with influenza vaccination, but was infrequent among vaccinees. This phenomenon is of short duration for HIV and HTLV-1, but may persist longer for HCV. We recommend influenza vaccinees not be deferred from blood donation. Blood donors with multiple false-positive viral ELISAs should be considered for future reentry as blood donors.


A prospective study of cigarette smoking and risk of cataract in men.

To examine the association between cigarette smoking and the incidence of cataract. The design was a prospective cohort study using data from the Physicians' Health Study, a randomized trial of aspirin and beta carotene among 22,071 US male physicians aged 40 to 84 years that began in 1982. This analysis includes the 17,824 physicians who did not report cataract at baseline and did provide complete risk factor information. Based on information reported at baseline, 10% were current smokers, 39% were past smokers, and 51% were never smokers. An incident cataract was defined as a self-report confirmed by medical record review to have been first diagnosed after randomization, age-related in origin, and responsible for a decrease in best corrected visual acuity to 20/30 or worse. During 60 months of follow-up, 557 incident cataracts among 371 participants were confirmed. Compared with never smokers, current smokers of 20 or more cigarettes per day had a statistically significant increase in the risk of cataract (relative risk [RR], 2.16; 95% confidence interval [Cl], 1.46 to 3.20; P less than .001). Similar results were obtained after simultaneously controlling for other potential cataract risk factors in a logistic regression model (RR, 2.05; 95% Cl, 1.38 to 3.05; P less than .001). Among the 557 eyes with cataract, nuclear sclerotic changes were present in 442 while posterior subcapsular changes were present in 204. After controlling for other potential cataract risk factors, current smokers of 20 or more cigarettes per day had statistically significant increases in nuclear sclerosis (RR, 2.24; 95% Cl, 1.47 to 3.41; P less than .001) and posterior subcapsular (RR, 3.17; 95% Cl, 1.81 to 5.53; P less than .001) cataract. Past smokers had an elevated risk of posterior subcapsular (RR, 1.44; 95% Cl, 0.97 to 2.13; P = .07) but not nuclear sclerosis cataract. For current smokers of fewer than 20 cigarettes per day, no increased risks were observed of total, nuclear sclerosis, or posterior subcapsular cataract. These data provide support for the hypothesis that cigarette smoking increases the risk of developing both nuclear sclerosis and posterior subcapsular cataract.


A prospective study of cigarette smoking and risk of cataract surgery in women.

To examine prospectively the association between cigarette smoking and the risk of cataract extraction in women. Prospective cohort study beginning in 1980 with 8 years of follow-up. United States, multistate. Registered nurses 45 to 67 years of age and free of diagnosed cancer. There were 50,828 women included in 1980 and others were added as they became 45 years of age. Incidence of senile cataract extraction. We documented 493 incident senile cataracts diagnosed and extracted during 470,302 person-years of follow-up. The age-adjusted relative risk (RR) among women who smoked at least 65 pack-years was 1.63 (95% confidence interval [Cl], 1.18 to 2.26; P for trend, .02). A similar increase in RR was noted when smoking was assessed in terms of number of cigarettes currently smoked or number of cigarettes smoked by past smokers. Results were also similar after adjusting for other potential risk factors for cataract. Smoking was also strongly associated with posterior subcapsular cataract specifically (RR, 2.59; 95% Cl, 1.49 to 4.50 for greater than or equal to 65-pack-year smokers relative to nonsmokers. Smoking appears to increase the risk of cataract extraction.


Medical researchers and the media. Attitudes toward public dissemination of research.

To study the experience of recently published authors with the news media. A self-administered questionnaire. All first authors of scientific articles published in JAMA and The New England Journal of Medicine during a 6-month period. Of 397 surveyed, 92% responded. Researchers were asked about (1) their experience with the news media, (2) their attitudes toward the dissemination of health-related research to the general public, and (3) their attitudes toward the lay press. Of respondents, 65% stated that their research was discussed in the lay press, and 60% reported that they were directly contacted by the press. Researchers had positive attitudes toward the press; 86% reported that news reports based on their research were accurate and 44% felt that media coverage would help them achieve their overall professional goals. Positive aspects of media coverage most frequently endorsed were that (1) it improves the image of the profession, (2) it informs the professional community of their research, and (3) it allows the public to understand the topic better. Negative aspects of media coverage were (1) it gives the impression that the researcher is seeking publicity, (2) it creates jealousy among colleagues, and (3) it takes too much time. Researchers were not eager to change the existing dissemination process, yet they endorsed the need for uniform standards concerning relations with the press. The majority of first authors in two leading medical journals reported substantial media coverage of their research, expressed generally positive sentiments about the press coverage of their work, and expressed a need for consensus on interactions involving the press.


Educational programs in US medical schools.

Trends of the past few years indicate that the 1990s will be a time of intense activity in medical education reform. A number of areas described in this annual review of medical education are grounds for optimism, tempered, however, by caution. The applicant pool has been increasing rapidly over the past 2 years and has reached the levels of the early 1980s. The average proportion of women and some minorities also has been rising. While these are positive signs, efforts to ensure diversity in the student population should not be abandoned. The number of faculty members continues to rise, especially in the clinical disciplines. The increases, occurring in the context of stable medical student enrollments, raise questions about the various roles and responsibilities of medical school faculty. Many medical schools are in the process of curriculum review and revision; while these changes respond to identified problems, they may have implications for faculty and other resources. External financial support fueled previous waves of curriculum innovation, and some of these gains could not be maintained when that support was withdrawn. The revisions in the examinations of the NBME are being well received, and the single pathway to licensure through USMLE has been initiated. This system does, however, affect graduates' options for licensure. Finally, the increased interest in program evaluation, especially the definition of goals and the measurement of educational outcomes to assess their attainment, demonstrates that medical schools are serious about educational accountability. Some schools also are being asked to address externally imposed objectives, related specifically to specialty choice, creating a potential for conflict between the objectives that the medical school sets for itself and those mandated by its external constituencies. While this analysis may imply that medical education is now in a "good news/bad news" situation, the message is that planning and careful assessment of options are perhaps even more important today than they were in the past. Change has its costs and its implications, but it must nonetheless be undertaken.


Evidence for the effectiveness of CME. A review of 50 randomized controlled trials.

To assess the impact of diverse continuing medical education (CME) interventions on physician performance and health care outcomes. Using continuing medical education and related phrases, we performed regular searches of the indexed literature (MEDLINE, Social Science Index, the National Technical Information Service, and Educational Research Information Clearinghouse) from 1975 through 1991. In addition, for these years, we used manual searches, key informants, and requests to authors to locate other indexed articles and the nonindexed literature of adult and continuing professional education. From the resulting database we selected studies that met the following criteria: randomized controlled trials; educational programs, activities, or other interventions; studies that included 50% or more physicians; follow-up assessments of at least 75% of study subjects; and objective assessments of either physician performance or health care outcomes. Studies were reviewed for data related to physician specialty and setting. Continuing medical education interventions were classified by their mode(s) of activity as being predisposing, enabling, or facilitating. Using the statistical tests supplied by the original investigators, physician performance outcomes and patient outcomes were classified as positive, negative, or inconclusive. We located 777 CME studies, of which 50 met all criteria. Thirty-two of these analyzed physician performance; seven evaluated patient outcomes; 11 examined both measures. The majority of the 43 studies of physician performance showed positive results in some important measures of resource utilization, counseling strategies, and preventive medicine. Of the 18 studies of health care outcomes, eight demonstrated positive changes in patients' health care outcomes. Broadly defined CME interventions using practice-enabling or reinforcing strategies consistently improve physician performance and, in some instances, health care outcomes.


Polyamine excretion in depleted patients with gastrointestinal malignancy: effect of perioperative nutrition and tumor removal.

Polyamines, synthesized by all mammalian cells, are involved in protein and energy metabolism. We measured urinary excretion of polyamines, putrescine, spermidine, spermine, and their metabolites N1-acetylspermidine and N8-acetylspermidine, resting energy expenditure, and nitrogen excretion in 12 depleted patients with gastrointestinal malignancy during preoperative and postoperative parenteral nutrition and in 7 patients with multiple trauma receiving similar parenteral nutrition. During preoperative nutrition support, the excretion of putrescine (p less than .05) and total polyamines (p less than .01) increased by 420% and 60%, respectively. Increases in energy balance and resting energy expenditure during nutrition could entirely explain the observed changes in polyamine excretion. Preoperatively, the excretion of N1-acetylspermidine (p less than .05), N8-acetylspermidine (p less than .001) and total polyamines (p less than .05) was higher in patients with a surgically noncurable tumor than in those with a surgically curable tumor. The energy balance and resting energy expenditure could also explain the differences in polyamine excretion between patients with surgically curable and noncurable disease, excluding the increased N8-acetylspermidine. Postoperatively, the excretion of N8-acetylspermidine in patients with multiple trauma without malignancy and in patients with palliative operation was similar, and was higher than in patients with a totally resected malignancy (p less than .01). Our results suggest that the excretion of polyamines reflects the activity of energy metabolism in general and that polyamine excretion is not specific for any particular disease.


Interaction of protein and zinc malnutrition with the murine response to infection.

Malnutrition increases the host's susceptibility to infection. However, the mechanisms are not well understood. This study examined the interaction of protein and zinc underfeeding in mice before challenge with an intracellular pathogen, Salmonella typhimurium. C3H/HeN mice (n = 68) were weighted and placed on one of four diets: 20% ovalbumin with adequate zinc (20% NL), 20% ovalbumin without zinc (20% LO), 1% ovalbumin with zinc (1% NL), and 1% ovalbumin without zinc (1% LO). At the end of 6 weeks they were again weighed and then challenged with 10(4) S. typhimurium intraperitoneally. Mortality was recorded over the next 2 weeks. Although weight loss was markedly affected by protein malnutrition (two-way analysis of variance: p = .0001), there was no independent effect by zinc (p = .3459). Similarly, protein malnutrition alone affected mortality rates (chi 2: p = .0001), whereas zinc had no independent effect (p = .7166). However, both protein and zinc underfeeding shortened the length of survival (Mann-Whitney U test: p less than .001). We conclude that protein malnutrition is the dominant factor influencing weight loss and mortality in this model. However, zinc malnutrition does shorten the length of survival and may contribute to the global immunosuppression noted in malnourished subjects.


Metabolic effect of short-term total parenteral nutrition highly enriched with leucine or valine in rats recovering from severe trauma.

The metabolic impact of infusing a large amount of leucine (Leu) or valine (Val) was examined with regard to the corrective effect of total parenteral nutrition (TPN). Rats recovering from severe sepsis received either Leu- or Val-enriched TPN solution for 30 hours. The in vivo behavior of the amino acids administered was explored by a pulse injection of 14C-labeled Leu or Val. The recovery of 14CO2 from Leu increased by 64% in the septic rats of Leu-TPN group (41% of dose; p less than .01), as compared with control rats receiving the same TPN solution, whereas no significant rise in the 14CO2 recovery from Val occurred in the septic rats given Val-TPN (45% of dose) in comparison with the corresponding controls. The enhancement of Leu catabolism to CO2 in the Leu-TPN group was compatible with the alterations of urinary nitrogen excretion, plasma Leu level, and metabolite contents of liver and muscle. The only difference in metabolite levels observed between the two TPN groups was in hepatic total adenine nucleotides. Plasma amino acid levels were largely unaffected by infusion of these TPN solutions highly enriched with branched-chain amino acids (45%), except for an approximately threefold elevation of the Val level in Val-TPN rats. Thus, when administered in a large quantity during such short-term TPN, Leu can exert its metabolic effect without causing an imbalance in plasma amino acids under severe catabolic conditions.


Thiamine, riboflavin, folate, and vitamin B12 status of low birth weight infants receiving parenteral and enteral nutrition.

Thirty infants were randomly assigned to receive either 3 mL of MVI-Pediatric supplement (PAR3 group, parenterally fed) or 2 mL (PAR2 group, parenterally fed). For the first week, 100% received total parenteral nutrition (TPN), 50% by the second, and less than 33% by the third. Eighteen control infants received enteral feeds of infant formula. Baseline (before TPN) and subsequent weekly blood samples, dietary data, and 24-hour urine collections were obtained. The adequacies of thiamine and riboflavin were assessed by the thiamine pyrophosphate effect and erythrocyte glutathione reductase activity, respectively. Urinary thiamine and riboflavin levels were measured by fluorometry. Plasma folate, red blood cell folate, urinary folate, and plasma vitamin B12 concentrations were determined by radioassay. No differences between groups were observed in thiamine pyrophosphate effect, erythrocyte glutathione reductase activity, urinary B1 or B2, or red blood cell folate levels at any time. Plasma folate differed (p less than .05) among the PAR3 group (24 +/- 7 ng/mL), and both the PAR2 (13 +/- 5 ng/mL) and enterally fed (ENT) groups (16 +/- 3 ng/mL) before the initiation of feeds, at week 1 (PAR3 = 32 +/- 15 ng/mL; PAR2 = 18 +/- 4 ng/mL; ENT = 19 +/- 9, ng/mL) and between the PAR3 (30 +/- 16 ng/mL) and PAR2 (16 +/- 4 ng/mL) infants at week 2. Plasma vitamin B12 levels differed among the ENT groups (551 +/- 287 pg/mL) and both the parenteral groups (PAR2 = 841 +/- 405 pg/mL; PAR3 = 924 +/- 424 pg/mL) at week 1 and between the ENT (530 +/- 238 pg/mL) and PAR3 (999 +/- 425 pg/mL) groups at week 2.(ABSTRACT TRUNCATED AT 250 WORDS)


Muscle protein degradation in severely malnourished patients with chronic obstructive pulmonary disease subject to short-term total parenteral nutrition.

Patients with chronic obstructive pulmonary disease (COPD) often lose weight and muscle mass with progression of the disease. Muscle protein degradation in patients with COPD has never been examined before and during hypercaloric feeding. Eight severely malnourished patients with COPD were examined at home consuming their usual intake, in the hospital after 3 days of a meat-free regular oral diet (period B), and during a hypercaloric (55 kcal/kg) high-lipid (55%) parenteral formula (total parenteral nutrition [TPN]). During period B, 8 well-nourished patients and 10 malnourished cancer patients were used as control groups. Measurements included plasma assays, leg blood flow, leg exchange (of 3-methylhistidine [3MeH], glucose, lactate, and oxygen) and urinary measures of 3MeH, creatinine, and nitrogen. During period B, net release of 3MeH across the leg in patients with COPD was similar to that in well-nourished control subjects and cachectic cancer patients. In COPD patients, there was only a transient decrease in leg exchange values of 3MeH with administration of TPN. COPD patients demonstrated a reduction (p less than .01) in urinary 3MeH excretion and an increase in nitrogen balance (p less than .01) with TPN compared with period B. The decrease in muscle protein degradation with administration of TPN accounts for about 50% of the increase in nitrogen retention in patients with COPD. These data suggest that in severely malnourished patients with COPD the weight loss is not dependent on increased rates of skeletal muscle protein degradation; nevertheless, degradation rates attenuate with a positive nitrogen balance during nutrition repletion.


Effects of pectin and cellulose on fat absorption after massive small-bowel resection in weanling rats.

The effects of pectin and cellulose on fat absorption after massive small-bowel resection were studied in rats. Weanling Wistar rats underwent 80% resection of the small intestine with gastrostomy, and fat emulsion labeled with 14C-linoleate was given via a gastrostomy tube for the fat absorption test on the 10th postoperative day. They were classified into three groups by diet as follows: E group, a fiber-free, chemically defined diet (CDD); EP group, CDD with 2% pectin; and EC group, CDD with 2% cellulose. The cumulative and hourly output of 14C in the expired air, the absorption rate of 14C in the intestine, and the metabolic utilization rate of 14C were calculated. The cumulative 14CO2 output was significantly lower in the EC group than in the E and EP groups. However, there was no significant difference in the 14CO2 output between the E and EP groups. The highest hourly 14CO2 output in the E and EP groups was observed during the first 2 hours. However, in the EC group, this fluctuated at low levels. No significant difference in the 1-14C-linoleate absorption rate was observed between the E and EP groups, whereas it was significantly lower in the EC group than in the E and EP groups. The metabolic utilization rate of 1-14C-linoleate showed no significant difference among the three groups. These data suggest that an addition of pectin to CDD does not impair small intestinal absorption of 1-14C-linoleate after massive small-bowel resection, and an addition of cellulose decreases absorption of 1-14C-linoleate.


Effects of amino acids in mixtures given by enteral or parenteral route on intestinal morphology and hydrolases in rats.

This study compares the effects of amino acid addition to an elemental liquid diet containing carbohydrates and triglycerides given either intragastrically or intravenously on the morphology and on hydrolase activities in the jejunum and ileum of adult rats. The isocaloric mixtures were administered for 4 days and control rats received an isocaloric laboratory diet orally. Independent of their content in amino acid, all mixtures given intravenously caused a drop in mucosal weight and a shortening of the height of the villi in both the jejunum and ileum. By enteral route, the addition of amino acids to a carbohydrate-triglyceride liquid diet led to the maintenance of normal villus height (this effect being prominent in the ileum) and to a significant increase of jejunal sucrase and aminopeptidase activities when compared with the carbohydrate-triglyceride mixture. Feeding the mixtures by parenteral route caused a significant drop of both enzyme activities. In contrast, lactase activity was generally not modified by the route of nutrient administration or by the composition of the diets. However, the absence of amino acid in the mixture given intravenously caused a specific drop of lactase activity in the ileum. Ileal sucrase activity was lowered dramatically by intragastric or intravenous feeding of the elemental diets. This effect was not modulated by the presence of amino acids. The presence of amino acids caused a significant drop of aminopeptidase activity in the ileum independently of the route of administration when compared with animals receiving the carbohydrate-triglyceride liquid diet.(ABSTRACT TRUNCATED AT 250 WORDS)


Duplex Doppler ultrasound identifies veins suitable for insertion of central feeding catheters.

Central venous access for feeding catheters may prove difficult in patients who have had numerous previous central line insertions or complications. Duplex Doppler ultrasound was used to identify the anatomy and patency of major central veins in 11 patients in whom attempts at obtaining central venous access by an experienced operator had failed at least once and in 40 control subjects. Doppler ultrasound demonstrated the subclavian veins (diameter 12.5 +/- 3.5 mm, mean +/- SE) and internal jugular veins (11 +/- 3.5 mm) in all the control subjects. In the patients, 18 of 44 veins were patent, 11 were small or had low blood flow, and 15 were thrombosed. In 7 patients who required central feeding catheter insertion, a suitable vein was identified and the catheter suitably placed, even in 3 subjects where no central vein was considered normal. Duplex Doppler ultrasonography is a useful technique for identifying veins suitable for the insertion of central venous lines when access has previously proved difficult.


Metabolic clearance of a fat emulsion containing medium-chain triglycerides in cirrhotic patients.

Medium-chain triglycerides (MCTs) may be advantageous in cirrhotic patients, but data on the clearing capacity are lacking. Intravenous fat tolerance test (IVFTT) was performed on 28 cirrhotic patients (Child's A, n = 9; Child's B, n = 13; Child's C, n = 6) to determine the clearance rate of Lipofundin MCT 20%, which contains a physical mixture of MCT and long-chain triglycerides (LCT). The mean fractional elimination rate (k2) of MCT/LCT physical blend in the cirrhotic patients (7.72%/min) was comparable with that of the healthy control subjects (n = 9, k2 = 5.43%/min). Significantly higher serum free fatty acid (FFA) levels were found in the cirrhotic patients during IVFTT. The difference could be accounted for by their higher basal levels. In all patients, serum FFA concentrations returned to basal value at the end of 1 hour. Prompt appearance of glycerol and beta-hydroxybutyrate was seen during IVFTT, indicating that hydrolysis of triglycerides and release of ketone bodies from the liver were rapid. It was concluded that the ability to eliminate MCT/LCT physical blend was not impaired in the cirrhotic patients.


Inadvertent arterial administration of parenteral hyperalimentation solution resulting in generalized seizure activity.

Serious complications may arise from improper central venous catheter placement. In this case report, seizures occurred as a result of inadvertent arterial administration of parenteral hyperalimentation. There is a need for radiographic confirmation of central venous catheter position by qualified personnel before catheter use.


Subcutaneous jejunostomy.

When the surgeon must obviate to an impending obstruction because of an unresectable tumor of the supramesocolic space and an intestinal bypass is not feasible, a jejunostomy is usually indicated. We describe a new method to provide enteral nutrition via a subcutaneous jejunostomy without any external device, which can be used only when the patient is at risk for developing an obstruction or dysphagia. The feeding tube is inserted into the jejunum and then connected to a Port-a-Cath lodged in a subcutaneous pocket. Subsequently, the nutrition can be delivered via a Huber needle inserted in the port, or, to minimize the need for strict aseptic surveillance, the tube can be exteriorized from the pocket and used as the usual tube jejunostomy. This procedure allows placing a precautionary jejunostomy without distortion of the body-image.


Different quantities of two commercial liquid diets consumed by weight-losing cancer patients.

Twenty ambulatory, weight-losing patients with advanced cancer of the lung, breast, or ovary were randomized to supplement their diet for 2 months with either of two commercial complete liquid diets, one containing intact milk proteins and the other partially hydrolyzed soy proteins. Both products were prescribed as sip feeds in addition to normal food. The patients consumed more of the hydrolysate-containing product than of that with intact (milk) protein. The difference was significant and was maintained during both months of the study. An increase in total energy and protein was obtained in both groups, but was significant only with the hydrolysate product. At the end of the study there was no group difference in measures of nutritional status, but weight loss was halted in both groups.


Vancomycin stability in heparin and total parenteral nutrition solutions: novel approach to therapy of central venous catheter-related infections.

To facilitate therapy of central venous catheter-related Gram-positive bacterial infection in patients who require total parenteral nutrition (TPN) therapy, we studied the stability of vancomycin in a commonly used TPN solution (V-TPN) at final concentrations of 0.5 mg/mL and 1.0 mg/mL and in heparin (100 U/mL in 0.9% NaCl) at 25 micrograms/mL (V-H). Vancomycin concentrations in V-TPN and V-H after storage at 4 degrees C over 35 and 14 days, respectively, were stable (within 10% of the prestorage vancomycin concentration). After 14 days at 4 degrees C heparin activity in V-H solution was 100 +/- 4% of that noted initially. Vancomycin remained stable (100 +/- 6% of the original vancomycin concentration) when the previously refrigerated V-TPN was held for an additional 24 hours at 22 degrees C. When the previously refrigerated V-H was held for an additional 24 hours at 37 degrees C, vancomycin concentrations decreased to 78 +/- 9% of the baseline concentrations (p less than .001). The stability of vancomycin in this TPN solution allows the daily dose of vancomycin to be mixed with the solution and then infused over 10 hours. As shown with pharmacokinetic modeling, this form of therapy will achieve serum vancomycin concentrations within the therapeutic range throughout a 24-hour period. The relative stability of vancomycin in a heparin line-flush solution allows vancomycin concentration in the lumen of the catheter to be maintained at greater than or equal to 15 micrograms/mL during the interval between catheter flushing and the subsequent TPN infusion. A simplified method of administering vancomycin to patients receiving concurrent TPN is possible.


[Hemodynamics of intrahepatic portal vein studied in healthy subjects and liver cirrhosis by pulsed Doppler method].

The hemodynamics of the intrahepatic portal vein in 35 healthy subjects and 74 patients with liver cirrhosis was studied by measuring blood flow velocity with pulsed Doppler ultrasound technique. The flow velocity of portal vein decreased from the portal trunk to the first branches and to the second branches in the right and left lobes. Especially, an abrupt decrease of the flow velocity in the umbilical part of the left portal vein was characteristic of hemodynamics in the intrahepatic portal vein. The flow velocity in the third branches decreased more than that of the second branch in the right portal vein, but did not exist in the left. And the velocity was almost equal in all the subsegments. Blood clots of the left portal vein in 25 of 36 lesions were found in 31 patients with intrahepatic portal vein thrombus. This characteristic hemodynamics of the left portal vein was thought to be the main cause for the formation of blood clots. In the group with liver cirrhosis, the flow velocity was lower than in healthy subjects in the portal trunk, bilateral first branches and right second branches, but did not exist in the left second branch and bilateral third branches. No interrelationship between the intrahepatic portal flow velocity and the severity of liver cirrhosis and portal hypertension was observed.


[Clinical utility of receptor imaging in the assessment of liver function].

Technetium-99m diethylene triamine pentaacetic acid-galactosyl human serum albumin (TcGSA) is a newly developed receptor-binding radiopharmaceutical, specific for the asialoglycoprotein receptor, which resides exclusively on the plasma membrane of hepatocytes. Clinical utility of TcGSA was evaluated in 3 control subjects with normal livers and in 54 patients with various liver diseases. The parameter, Receptor Index, was derived from liver and heart time-activity data and is the ratio of radioactivity of the liver over the radioactivity of the liver plus heart at 15 min after the intravenous injection of 3 mg of TcGSA. Receptor concentration ([R]o) was obtained by kinetic analysis of liver and heart time-activity data using pharmacokinetic nonlinear modeling. Values for the Receptor Index and [R]o was statistically different in the control subjects and in patients with mild, moderate, and severe liver diseases. Good correlations were obtained between the Receptor Index, [R]o and conventional liver function tests, such as Child-Turcotte criteria score, prothrombin time, and indocyanine green test. Receptor Index and [R]o were properly estimated even in patients with obstructive jaundice or remarkable portocaval shunt. These data suggest that the receptor imaging as well as its parameters, Receptor Index and [R]o, is a potentially practical and reliable diagnostic method for estimating the functioning hepatocyte mass and for assessing liver function.


Prognostic indicators in prostatic cancer.

For therapeutic decisions it is essential to have an evaluation of the tumor volume and the grade of dedifferentiation. Measurement of PSA gives a good additional guide to the tumor volume and to dissemination of the disease. Investigation of the DNA ploidy values can offer further important information on the aggressiveness of the tumor and be helpful for our understanding of the process of tumor propagation. However, DNA studies can still not be regarded as being standard in the clinical work-up of these patients. They are optional but they have a definite place in the research on prostatic cancer. The various methods to study tumor growth by analysis of the S phase fraction are interesting new contributions but still belong to the research laboratories. When we consider prognostic indicators we have to take into account the biologic character of carcinogenesis. Modern research has shown that the development and the progression of cancer is not an instantaneous and solitary reaction. It is a series of events and a net-work reaction between growth-regulating factors, stimulating and inhibiting, a step-wise alteration of the genome. We must recognize that what we are observing is the condition at the present time, and, of course, the observation must be evaluated together with the whole clinical scenario, the man's age, his general condition etc. But still the series of diagnostic procedures presented here will give a rather solid ground for both our therapeutic decisions and for evaluation of the results of treatment.


Thromboxane synthesis and action within the kidney.

PGH2 and TxA2 exert their actions via tissue specific, receptor isoforms. PGH2/TxA2-dependent platelet aggregation and contraction of vascular and bronchial smooth muscle and of glomerular mesangial cells occur via receptors linked to activation of phospholipase C. Although PGH2/TxA2 appear to be of little importance in the maintenance of renal function under physiological circumstances, increased renal TxA2 biosynthesis has been documented in a variety of animal models of renal disease and in some clinical disorders (Table 2). The effects of this eicosanoid on renal tissues in vitro and of pharmacological manipulation of TxA2 synthesis and action in vivo suggest that such interventions will provide new drugs for the treatment of human kidney disease.


Acute phosphate depletion and in vitro rat proximal tubule injury: protection by glycine and acidosis.

The effects of phosphate (PO4) removal from Krebs Henseleit buffer on freshly isolated rat proximal tubules (rPT) were assessed by measuring Ca2+ uptake (nmol/mg protein), cellular adenosine triphosphate (ATP) (nmol/mg), tissue K+ content (nmol/mg) and lactate dehydrogenase (LDH) as an index of cell integrity. Ca2+ uptake increased by 50% in rPT incubated in zero PO4 medium as compared to control (2.6 +/- 0.1 vs. 3.9 +/- 0.19, P less than 0.001) and LDH release increased 2.5-fold from 14.2 +/- 0.6 to 31.6 +/- 1.6%, P less than 0.001. Neither verapamil (200 microM) nor mepacrine (50 microM) reduced Ca2+ uptake or decreased LDH release suggesting that the increased Ca2+ uptake was not occurring through potential operated channels and that phospholipase-induced cell injury was not the cause of increased LDH release. Either glycine (2 mM) or extracellular fluid acidosis (pH 7.06), however, significantly diminished rPT injury and Ca2+ uptake. Specifically, as compared to the increased LDH released in untreated. PO4-depleted rPT, LDH release was diminished significantly by glycine treatment (31.0 +/- 0.9 vs. 15.5 +/- 1.6%, P less than 0.001) or acidosis (30.3 +/- 0.04 vs. 19.2 +/- 0.9%, P less than 0.01). Ca2+ uptake did not increase in glycine treated tubules (2.6 +/- 0.1 vs. 2.8 +/- 0.2 nmol/mg, NS) or in the presence of acidosis (2.6 +/- 0.1 vs. 2.97 +/- 0.17 nmol/mg, NS). ATP concentrations were markedly reduced by PO4 depletion (2.8 +/- 0.2 vs. 4.8 +/- 0.3 nmol/mg, P less than 0.001) and remained at low levels during either acidosis or glycine-induced protection.(ABSTRACT TRUNCATED AT 250 WORDS)


Impaired phagocytosis in chronic renal failure is mediated by secondary hyperparathyroidism.

Patients with chronic renal failure (CRF) display impaired phagocytosis by the polymorphonuclear leucocytes (PMNL), and these cells have elevated basal levels of cytosolic calcium ([Ca2+]i) and reduced ATP content. It has been suggested that these changes in PMNL metabolism and function are mediated by the state of secondary hyperparathyroidism of CRF. To examine the role of excess PTH in these derangements of PMNL, we studied [Ca2+]i, ATP and phagocytic ability of PMNL in five groups of rats including: CRF, CRF normocalcemic parathyroidectomized (CRF-PTX), CRF and normal animals treated with verapamil (CRF-V), and normal-V, respectively. The level of [Ca2+]i in the PMNL of CRF rats (149 +/- 2.7 nM) was significantly (P less than 0.01) higher and the ATP content (4.2 +/- 0.17 nmol/5 x 10(6) PMNL) significantly lower (P less than 0.01) than in normal (108 +/- 2.4 nM; 9.5 +/- 0.15 nmol/5 x 10(6) PMNL), CRF-PTX (103 +/- 2.9 nM; 9.2 +/- 0.19 nmol/5 x 10(6) PMNL), CRF-V (107 +/- 2.2 nM; 9.0 +/- 0.2 nmol/5 x 10(6) PMNL) and normal-V (106 +/- 1.8 nM; 9.2 +/- 0.2 nmol/5 x 10(6) PMNL), despite sustained elevation in blood PTH in the CRF-V group. Phagocytosis was significantly (P less than 0.01) impaired in CRF animals (5.6 +/- 0.25 micrograms oil/10(7) PMNL/min) but was normal in CRF-PTX (9.3 +/- 0.21 micrograms oil/10(7) PMNL/min) and CRF-V (9.5 +/- 0.22 micrograms oil/10(7) PMNL/min) rats. The values of phagocytosis in normal and normal-V rats were 9.6 +/- 44 and 9.6 +/- 0.18 micrograms oil/10(7) PMNL/min, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


Culture of endothelial cells from baboon and human glomeruli.

Whereas mesangial and epithelial cells from glomeruli are commonly grown in vitro, there has been a failure to isolate and propagate human glomerular capillary endothelial cells. This study defines the conditions for the reproducible isolation and growth of homogeneous monolayers of primate (baboon and human) glomerular capillary endothelial cells. Using selective media and growth factors, the following criteria were identified to optimize the isolation and proliferation of glomerular endothelial cells: (1) collagenase treatment of isolated glomeruli; (2) requirement for 20% serum, endothelial cell growth factor and heparin; (3) requirement of fibronectin as surface matrix; and (4) isolation from donors less than 60 years old, as premature senescence was directly proportional to the age of the human kidney donor. Under these conditions, primary cultures with an endothelial cell composition greater than 70% were reproducibly obtained. Homogeneous endothelial monolayers were developed from 20 of 23 human kidneys, and maintained for 5 to 10 passages, depending on the age of the kidney donor. Purification to homogeneity was achieved by patch cloning or by fluorescence-activated cell sorting. Glomerular capillary endothelial cells exhibited a cobblestone morphology at confluence, expressed factor VIII-related antigen, angiotensin converting enzyme activity, and endocytosed acetylated low-density lipoproteins. Electron microscopy revealed the presence of intracellular Weibel-Palade bodies and caveolae and microvillous projections on the luminal surface. Glomerular cells also stained positive for Ulex europaeus, a lectin characteristic of human endothelial cells. In addition, preliminary results indicate that human glomerular endothelial cells increase intracellular cyGMP in response to alpha-human 5 to 28 atrial natriuretic peptide and intracellular free calcium in response to thrombin.


Depletion of CD8 positive cells in nephrotoxic serum nephritis of WKY rats.

Following a small dose of nephrotoxic serum (NTS) WKY rats demonstrated crescentic glomerulonephritis, which was characterized by the early infiltration of CD8 positive cells in glomeruli. In vivo depletion of CD8 positive cells from WKY rats completely prevented proteinuria (4.6 +/- 4.8 mg/day vs. 105.3 +/- 11.6 mg/day on day 10; N = 19, P less than 0.001) and crescent formation (2.7 +/- 2.9% vs. 94.3 +/- 2.6%; P less than 0.001). Immunofluorescence revealed complete inhibition of the influx of CD8 positive cells and subsequent reduction of the infiltration of macrophages in the glomeruli. Glomerular binding of 125I-anti-rat glomerular basement membrane antibodies, host anti-rabbit IgG production and the C3 level in the circulation were the same as in the control. These data indicate that CD8 positive cells play a key role in glomerular injury and crescent formation. This model provides a useful system for studying the cellular mechanisms that lead to glomerular injury and subsequent crescent formation.


Effects of salt restriction on renal growth and glomerular injury in rats with remnant kidneys.

Male Munich-Wistar rats underwent right nephrectomy and infarction of two thirds of the left kidney. Rats were randomly assigned to ingest standard chow (REM) or a moderately salt restricted chow (LS). A third group of rats were fed the low salt diet and were injected with an androgen (LSA). Eight weeks after ablation, glomerular volume and glomerular capillary radius were markedly increased in REM. This increase was prevented by the low salt diet, however, the antihypertrophic effect of the diet was overcome by androgen. Values for glomerular volume and capillary radius were similar in LSA and REM. Morphologic studies revealed that approximately 25% of glomeruli were abnormal in REM. Much less injury was observed in salt restricted rats, however, the protective effect of the low salt diet was significantly abrogated when renal growth was stimulated in salt restricted rats by androgen. Micropuncture studies revealed that glomerular pressure was elevated in all three groups and not affected by diet or androgen. Serum cholesterol was also similar in the three groups. These findings indicate that renal and glomerular hypertrophy are correlated with the development of glomerular injury after reduction in renal mass and suggest that dietary salt restriction lessens renal damage, at least in part, by inhibiting compensatory renal growth.


Chronic metabolic acidosis accelerates whole body proteolysis and oxidation in awake rats.

Previous work has documented an acceleration of proteolysis and branched-chain amino acid oxidation when muscles from rats with chronic metabolic acidosis were incubated in vitro. The present study examines the impact of chronic metabolic acidosis on whole body amino acid turnover and oxidation in chronically catheterized awake male Sprague-Dawley rats using stochastic modeling and a primed continuous infusion of L-[1-14C] leucine. Whole body protein turnover was accelerated by acidosis as reflected in a 70% increase in proteolysis and a 55% increase in protein synthesis. Amino acid oxidation was increased 145% in rats with chronic metabolic acidosis relative to control rats receiving diets identical in protein and calories based on a reciprocal pool model and plasma alpha-ketoisocaproate specific radioactivity. These changes were accompanied by a 104% increase in liver branched-chain ketoacid dehydrogenase (BCKAD) activity in rats with acidosis, similar to previously documented increases in skeletal muscle BCKAD activity caused by acidosis. In contrast, kidney BCKAD activity was decreased 38% by acidosis, illustrating the tissue-specificity of the changes that were present. We conclude that chronic metabolic acidosis accelerates whole body protein turnover and affects the reincorporation of amino acid into body proteins by accelerating amino acid oxidation.


Importance of NO/EDRF for glomerular and tubular function: studies in the isolated perfused rat kidney.

The effect of the addition of N omega-nitro-L-arginine (L-NNA, 10 and 100 microM) to isolated rat kidneys perfused with a complex medium containing 21 amino acids has been studied. A cyclooxygenase inhibitor was added throughout to block prostaglandin synthesis. L-NNA caused significant reductions in renal perfusion flow rate (PFR, 9.8 +/- 1.4 vs. 15.9 +/- 1.1 ml.min-1.g kidney wt-1, P less than 0.0001), glomerular filtration rate (GFR, 566 +/- 57 vs. 705 +/- 47 microliters.min-1.g kidney wt-1, P less than 0.05) and an increase in the relative filtration fraction (%FF, 7.0 +/- 0.6 vs. 5.2 +/- 0.4%, P less than 0.05) compared to control kidneys. L-NNA perfused kidneys had a lower absolute sodium (72 +/- 9 vs. 88 +/- 4 mumol.min-1.g kidney wt-1, P less than 0.05) and glucose reabsorption (3.5 +/- 0.5 vs. 5.4 +/- 0.4 mumol.min-1.g kidney wt-1, P less than 0.05), corresponding mainly to a lower sodium and glucose filtration. However, the relative reabsorption of sodium and glucose in the presence of L-NNA was attenuated, too (82.8 +/- 2.0 vs. 87.0 +/- 3.3% P less than 0.05 and 91.3 +/- 1.1 vs. 94.1 +/- 0.5%, P less than 0.05). Potassium handling and protein excretion were not changed significantly; fractional protein excretion increased slightly with the addition of L-arginine (47 +/- 5 vs. 55 +/- 7 ng.microliters-1, P less than 0.05). The differences between control and L-NNA treated kidneys (with the exception of differences in FRGluc) could be fully (L-NNA, 10 microM) or partially (L-NNA 100 microns) reversed by adding L-arginine (1 mM) to the perfusion medium. The observed results could be obtained in two different rat strains (Sprague-Dawley and Wistar). Only L-NNA and L-arginine caused the observed changes, while D-NNA and D-arginine were without effect. It is concluded that NO/EDRF is basally released from the isolated perfused rat kidney, and is of importance not only in the regulation of renal hemodynamics but also in the regulation of renal tubular function.


Long term effects of morphine on mesangial cell proliferation and matrix synthesis.

Since focal glomerulosclerosis is the predominant glomerular lesion in heroin nephropathy and since mesangial expansion is considered to be a precursor of glomerulosclerosis, we have evaluated the effect of opiates on mesangial cell (MC) proliferation and matrix synthesis. We showed, using a fluorometric assay, that MC are not capable of metabolizing heroin to its active metabolite morphine. Cells exposed to morphine (10(-5) M or 10(-4) M) in prolonged cultures either continuously (Group A) or intermittently (Group B) showed enhanced incorporation of [3H]thymidine when compared to control cells (control, 88600 +/- 26303 cpm/well vs. morphine 10(-4) M-Group A, 321203 +/- 52867, P less than 0.001; control vs. morphine 10(-4) M-Group B, 223126 +/- 46866 cpm/well, P less than 0.01; control, 107593 +/- 42284 cpm/well vs. morphine 10(-5) M - Group A, 267108 +/- 41866 cpm/well, P less than 0.001; control vs. morphine 10(-5) M - Group B, 202317 +/- 24325 cpm/well, P less than 0.05). However, MC incubated with a lower concentration of morphine (10(-6) M) enhanced DNA synthesis when exposed intermittently only (control, 107593 +/- 42284 cpm/well vs. Group B, 219164 +/- 15552 cpm/well, P less than 0.05). This growth stimulating effect of morphine (10(-6) M and 10(-5) M) was also observed at earlier time points, that is, one- and one-and-a-half-week old cultures. However, in one-week-old cultures. morphine in a higher concentration (10(-4) M) showed a suppressive effect (P less than 0.05) on MC proliferation (morphine, 3620 +/- 220 cpm/well vs. control, 4668 +/- 410 cpm/well). This effect not only subsided by one and a half weeks but morphine (10(-4) M) treated cells enhanced MC proliferation. An opioid antagonist, naloxone attenuated the effect of morphine in one and half week old cultures. Morphine at 10(-6) M to 10(-4) M concentrations enhanced incorporation of [3H]proline in the extracellular proline pool (a component of mesangial matrix) when compared to control (control, 309661 +/- 3992 vs. morphine 10(-4) M, 363104 +/- 10539 cpm/well, P less than 0.05 or morphine 10(-5) M, 397954 +/- 31008 cpm/well, P less than 0.001 or morphine 10(-6) M, 384630 +/- 26369 cpm/well, P less than 0.01). In addition, MC incubated with morphine (10(-6) M and 10(-4) M) also enhanced (P less than 0.001) synthesis of laminin.(ABSTRACT TRUNCATED AT 250 WORDS)


Direct amphotericin B-mediated tubular toxicity: assessments of selected cytoprotective agents.

Amphotericin B (AB) may induce acute renal failure by vasoconstrictive and tubulo-toxic effects. Although mannitol, Ca2+ channel blockers, and lipid-based AB preparations have been suggested to mitigate in vivo AB nephrotoxicity, whether they confer direct tubular cytoprotection has not been defined. Therefore, this study assessed the impact of mannitol, verapamil/extracellular Ca2+, and cholesteryl sulfate (CS) AB binding on AB cytotoxicity, employing an isolated rat proximal tubular segment (PTS) preparation. After 30 to 60 minutes of incubation, 0.2 mg/ml of AB (Fungizone) caused marked toxicity, as assessed by LDH release (29 to 44%) and ATP depletion (greater than 90%). Approximately 40% of the LDH release could be attributed to deoxycholate, the standard AB (Fungizone) solubilizing agent. Both 100 mM mannitol and 100 mM glucose decreased AB-mediated LDH release, despite having a quantitatively trivial impact on ATP concentrations (increments of less than or equal to 1% at normal values). Dimethylthiourea (25 mM; equipotent to 100 mM mannitol/glucose as a hydroxyl radical scavenger) did not decrease LDH release. Neither verapamil addition (100 microM) nor Ca2+ removal from the PTS buffer had a protective effect. CS binding completely eliminated AB's toxicity (no LDH or ATP losses). The effect of AB and CS-AB on concomitant O2 deprivation/reoxygenation (30 min/15 min) PTS injury was also assessed. AB and hypoxia/reoxygenation caused additive, not synergistic, LDH release whereas CS-AB had no adverse effect.(ABSTRACT TRUNCATED AT 250 WORDS)


Hydrated clearance of gadolinium-DTPA as a measurement of glomerular filtration rate.

Technetium (99mTc)-diethylene triamine pentaacetic acid (DTPA) hydrated clearance studies are accurate for determining GFR but require special facilities for handling and measuring samples. We investigated the potential of a non-radioactive paramagnetic analog, Gadolinium (Gd)-diethylene triamine pentaacetic acid (DTPA), an approved NMR contrast agent, as a glomerular filtration marker. Instead of relying on the radioactivity of technetium, this test is based on the fact that gadolinium induces alterations in the NMR T1 relaxation times in blood and urine samples. Ninety patients underwent simultaneous determinations of GFR using 1 mCi of Tc-DTPA and 0.05 mmol/kg Gd-DTPA (Berlex Labs) IV. The patients were hydrated with oral and intravenous fluid. Following a one hour equilibrium period, three or four consecutive urine collections were obtained; plasma samples were acquired at the beginning and end of each approximately 20-minute interval. 99mTc-DTPA radioactivity was determined with a scintillation counter. T1 relaxation times were measured on a 10 MHz NMR spectrometer. These were converted to Gd-DTPA concentration by comparison with standard solutions. The Gd-DTPA derived GFR closely approximated the 99mTc-DTPA derived GFR which ranged from 15 to 147 ml/min. The equation and correlation coefficient of the regression line is y = 1.04 x -2.2, r = 0.94. Thus, Gd-DTPA is a safe, non-radioactive indicator of GFR that may provide an alternative renal clearance method for clinical studies of progressive renal disease and nephrotoxicity.


The sodium retaining effects of cyclosporine.

The effect of chronic cyclosporine administration on volume regulation was studied in mongrel dogs. Dogs received either cyclosporine (20 mg/kg/day p.o.; N = 7) or vehicle (N = 6) while being maintained on a constant sodium diet. Dogs had measurement of baseline vasoactive hormones. Daily sodium excretion was determined. Following eight days of drug administration, dogs were anesthetized, pre-volume expansion data was collected, and dogs underwent a one hour, 10% body weight 0.9% saline volume expansion. Daily sodium balance was +8.6 +/- 2.2 mEq in the cyclosporine group versus 0.4 +/- 1.8 mEq (P less than 0.05) in the control group after 8 days. Prior to acute volume expansion, aldosterone was 22.5 +/- 7.1 ng% in the cyclosporine group versus 4.7 +/- 0.7 ng% in controls (P less than 0.05). ANF was suppressed in the animals receiving cyclosporine. In response to volume expansion, the cyclosporine group demonstrated an attenuation of maximum urine flow by 56%, fractional excretion of sodium by 52%, and electrolyte free water clearance by 75% when compared to controls (P less than 0.05). We demonstrate that chronic cyclosporine administration activates the renin-angiotensin-aldosterone system, suppresses circulating ANF, and results in chronic sodium retention. Additionally, cyclosporine attenuates the natriuretic and diuretic response to acute volume expansion.


Childhood membranoproliferative glomerulonephritis type I: limited steroid therapy.

Nineteen patients with biopsy proven membranoproliferative glomerulonephritis type I (MPGN I) and a minimum of three years of follow-up (mean 6.5 +/- 0.7 years) have been treated with an uncontrolled regimen of limited corticosteroids. Initial therapy ranged from 20 mg per os (po) every other day to 30 mg/kg/day i.v. for three consecutive days, depending on clinical disease severity. Therapy was then decreased based on each patient's improving clinical status. At diagnosis creatinine clearance (CCr) was less than 80 ml/min/1.73 m2 in 12 patients and less than 50 in 2. All patients had hematuria and proteinuria, with 15 in the nephrotic range. Hypertension, present at diagnosis in 13, developed in five others following institution of prednisone, and was controlled medically. Renal biopsy was repeated after two years of therapy prior to cessation of treatment (mean total treatment duration 38 +/- 3 months). Follow-up biopsy revealed decreased glomerular inflammatory activity in 88% of patients. All patients have now been off prednisone for 40 +/- 9 months. The mean CCr is 126 +/- 5 ml/min/1.73 m2. Eight patients have normal urinalyses. These data suggest that early therapy with a limited course of corticosteroids, and control of associated hypertension, may forestall progressive renal insufficiency in children with MPGN type I.


Carnitine metabolism during exercise in patients on chronic hemodialysis.

Patients on hemodialysis (HD) have impaired exercise performance. Carnitine homeostasis is also abnormal in this population. As carnitine is an important cofactor for muscle energy metabolism, exercise performance and skeletal muscle carnitine metabolism were characterized in eight HD patients, and in five age-matched controls. Each patient underwent graded bicycle exercise testing to define maximal performance, and prolonged exercise at 70% of their peak work capacity. Muscle (vastus lateralis) total carnitine content (carnitine plus all acylcarnitines) at rest was lower in HD patients than in controls (2320 +/- 1190 vs. 3800 +/- 940 nmol/g, P less than 0.05). In patients on HD, muscle carnitine content was inversely correlated to time on HD (r = -0.74, P less than 0.05), and positively correlated to peak exercise performance (r = 0.77, P less than 0.05). In patients on HD, 8 +/- 7% of the muscle carnitine pool at rest was short-chain acylcarnitines (similar to the distribution in controls), but 32 +/- 5% of the plasma carnitine pool consisted of short-chain acylcarnitines. With high-intensity exercise in patients on HD, muscle short-chain acylcarnitine content increased from 130 +/- 130 to 1380 +/- 820 nmol/g (P less than 0.01). The change in muscle short-chain acylcarnitine content with exercise was correlated with the increase in muscle lactate content (r = 0.88, P less than 0.01). In summary, patients on HD had a lower muscle total carnitine content than control subjects which was correlated to exercise performance.(ABSTRACT TRUNCATED AT 250 WORDS)