Biomedical subjects

U Schubert

Publications and source records attributed to U Schubert.

At least 37 records · Page 2Linked to original sources

PubMed1998

Safety and toxicity of amphotericin B in glucose 5% or intralipid 20% in neutropenic patients with pneumonia or fever of unknown origin: randomised study.

OBJECTIVE: To compare the feasibility of treatment, safety, and toxicity of intravenous amphotericin B deoxycholate prepared in either glucose or intralipid for empirical antimycotic treatment of neutropenic cancer patients. DESIGN: Single centre stratified, randomised non-blinded phase II study. SETTING: University hospital providing tertiary clinical care. SUBJECTS: 51 neutropenic patients (leukaemia (35), lymphoma (11), solid tumours (5)) with refractory fever of unknown origin (24) or pneumonia (27). INTERVENTIONS: Amphotericin B 0.75 mg/kg/day in 250 ml glucose 5% solution or mixed with 250 ml intralipid 20%, given on eight consecutive days then alternate days, as a 1-4 hour infusion. MAIN OUTCOME MEASURES: Feasibility of treatment, subjective tolerance (questionnaire), and objective toxicity (common toxicity criteria of the National Cancer Institute). RESULTS: Study arms were balanced for age, sex, underlying malignancy, renal and liver function, and pre- and concomitant treatment with antibiotics and nephrotoxic agents. No statistically significant or clinically relevant differences were found between the treatment groups for: daily or cumulative dose and duration of treatment with amphotericin B; incidence and time of dose modifications or infusion duration changes related to toxicity; dose or duration of symptomatic support with opiates, antipyretics, or antihistamines; renal function; subjective tolerance; most common toxicity scores; course of infection; and incidence of treatment failures. Patients treated with amphotericin B in intralipid were given fewer diuretics (P<0.05) and therefore had more peripheral oedema (P<0.01) and needed less potassium supplementation (P<0.05) than patients given amphotericin in glucose. Acute respiratory events were more common in the intralipid arm (P<0.05). CONCLUSIONS: Amphotericin B 0.75 mg/kg/day in intralipid given on eight consecutive days then alternate days provides no benefit and is associated with potential pulmonary side effects possibly because of fat overload or an incompatibility of the two drugs.

Adolescent↗

PubMed1998

Solution structure of the cytoplasmic domain of the human CD4 glycoprotein by CD and 1H NMR spectroscopy: implications for biological functions.

The human T cell receptor CD4 is a type I integral membrane glycoprotein that is involved in T cell activation and also acts as the primary coreceptor for human immunodeficiency viruses (HIV). Here the structure of a synthetic 38 amino acid peptide corresponding to the complete cytoplasmic domain of CD4 (CD4CYTO) has been investigated under a variety of solution conditions using a combination of circular dichroism and homonuclear two-dimensional 1H nuclear magnetic resonance spectroscopy. In the presence of the membrane mimetic 2,2,2-trifluoroethanol (TFE), a conformational change of CD4CYTO from a random coil to an alpha-helical structure was observed. In keeping with this, CD4CYTO has the potential to associate with membranes as demonstrated by binding studies of in vitro phosphorylated CD4CYTO with microsomal membranes. Both chemical shift and nuclear Overhauser enhancement data in 50% 2,2, 2-trifluoroethanol solution provide direct experimental evidence for the predominance of a short amphiphatic alpha-helix that is approximately 4 turns in length and extends from positions Arg-402 to Lys-417. The present data provide, for the first time, compelling experimental evidence that only a fraction of CD4CYTO has a propensity for adopting secondary structure under conditions that are assumed to exist at or near to the membrane surface and that this alpha-helical structure is located in the membrane-proximal region of CD4CYTO. The N-terminal residues, that link the alpha-helix to the transmembrane anchor of CD4, and a substantial C-terminal portion (14-18 residues) of CD4CYTO are unstructured under the solution conditions investigated. Correlation of our structural data with recent studies on the biological activity of CD4CYTO indicates that the alpha-helix is of crucial importance for the interaction of CD4 with Nef and Vpu in the process of HIV-mediated CD4 down-regulation.

Amino Acid Sequence↗

PubMed1998

CD4 glycoprotein degradation induced by human immunodeficiency virus type 1 Vpu protein requires the function of proteasomes and the ubiquitin-conjugating pathway.

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a type I anchored integral membrane phosphoprotein with two independent functions. First, it regulates virus release from a post-endoplasmic reticulum (ER) compartment by an ion channel activity mediated by its transmembrane anchor. Second, it induces the selective down regulation of host cell receptor proteins (CD4 and major histocompatibility complex class I molecules) in a process involving its phosphorylated cytoplasmic tail. In the present work, we show that the Vpu-induced proteolysis of nascent CD4 can be completely blocked by peptide aldehydes that act as competitive inhibitors of proteasome function and also by lactacystin, which blocks proteasome activity by covalently binding to the catalytic beta subunits of proteasomes. The sensitivity of Vpu-induced CD4 degradation to proteasome inhibitors paralleled the inhibition of proteasome degradation of a model ubiquitinated substrate. Characterization of CD4-associated oligosaccharides indicated that CD4 rescued from Vpu-induced degradation by proteasome inhibitors is exported from the ER to the Golgi complex. This finding suggests that retranslocation of CD4 from the ER to the cytosol may be coupled to its proteasomal degradation. CD4 degradation mediated by Vpu does not require the ER chaperone calnexin and is dependent on an intact ubiquitin-conjugating system. This was demonstrated by inhibition of CD4 degradation (i) in cells expressing a thermally inactivated form of the ubiquitin-activating enzyme E1 or (ii) following expression of a mutant form of ubiquitin (Lys48 mutated to Arg48) known to compromise ubiquitin targeting by interfering with the formation of polyubiquitin complexes. CD4 degradation was also prevented by altering the four Lys residues in its cytosolic domain to Arg, suggesting a role for ubiquitination of one or more of these residues in the process of degradation. The results clearly demonstrate a role for the cytosolic ubiquitin-proteasome pathway in the process of Vpu-induced CD4 degradation. In contrast to other viral proteins (human cytomegalovirus US2 and US11), however, whose translocation of host ER molecules into the cytosol occurs in the presence of proteasome inhibitors, Vpu-targeted CD4 remains in the ER in a transport-competent form when proteasome activity is blocked.

Acetylcysteine↗

PubMed1998

Effective biomodulation by leucovorin of high-dose infusion fluorouracil given as a weekly 24-hour infusion: results of a randomized trial in patients with advanced colorectal cancer.

PURPOSE: To determine whether high-dose infusional fluorouracil (FU) is effectively modulated by leucovorin (LV), interferon (IFN) alpha-2b, or both when given to patients with metastatic colorectal cancer. PATIENTS AND METHODS: Patients (n = 236) with progressive, measurable disease were randomized to three groups and received FU 2,600 mg/m2 as a 24-hour continuous infusion (CI) weekly for 6 weeks with 2 weeks rest (FU24h) and LV 500 mg/m2 as a 2-hour infusion before FU or IFN 3 x 10(6) U subcutaneously 3 times weekly or both. Treatment continued until progressive disease or unacceptable toxicity was observed. Pairs of treatment arms were analyzed sequentially to detect equivalence or a 25% difference in response rates. RESULTS: The rate of objective remission in patients who received FU24h/LV (44%; 40 of 91) was significantly higher than in patients who received FU24h/IFN (18%; 16 of 90; P < .05). The response rates of patients who received FU24h/LV versus FU24h/LV/IFN (27%; 13 of 49) were statistically equivalent. Significant differences were observed for time to tumor progression (TTP) (FU24h/LV, 7.1 months; FU24h/IFN, 3.9 months; FU24h/LV/IFN, 6.3 months; global P value < .009) and survival (16.6 months, 12.7 months, 19.6 months, respectively; global P value < .04). Unpredictable and life-threatening toxicity in the FU24h/LV/IFN arm required dose reduction of FU to 2,000 mg/m2/day and early stoppage of this arm. Toxicity was manageable in patients who received both FU24h/LV (grade 3 to 4 diarrhea, 21%) and FU24h/IFN (grade 3 to 4 diarrhea, 15%). CONCLUSION: Response rate, TTP, and overall survival were superior for LV-containing regimens compared with IFN modulation alone. The addition of IFN to high-dose infusional FU plus LV offers no advantage and may increase toxicity. The regimen of high-dose infusional FU24h/LV warrants further evaluation in patients with metastatic colorectal cancer.

Adult↗

PubMed1997

The human immunodeficiency virus type 1 (HIV-1) Vpu protein interferes with an early step in the biosynthesis of major histocompatibility complex (MHC) class I molecules.

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a small integral membrane phosphoprotein with two established functions: degradation of the viral coreceptor CD4 in the endoplasmic reticulum (ER) and augmentation of virus particle release from the plasma membrane of HIV-1-infected cells. We show here that Vpu is also largely responsible for the previously observed decrease in the expression of major histocompatibility complex (MHC) class I molecules on the surface of HIV-1-infected cells. Cells infected with HIV-1 isolates that fail to express Vpu, or that express genetically modified forms of Vpu that no longer induce CD4 degradation, exhibit little downregulation of MHC class I molecules. The effect of Vpu on class I biogenesis was analyzed in more detail using a Vpu-expressing recombinant vaccinia virus (VV). VV-expressed Vpu induces the rapid loss of newly synthesized endogenous or VV-expressed class I heavy chains in the ER, detectable either biochemically or by reduced cell surface expression. This effect is of similar rapidity and magnitude as the VV-expressed Vpu-induced degradation of CD4. Vpu had no discernible effects on cell surface expression of VV-expressed mouse CD54, demonstrating the selectivity of its effects on CD4 and class I heavy chains. VV-expressed Vpu does not detectably affect class I molecules that have been exported from the ER. The detrimental effects of Vpu on class I molecules could be distinguished from those caused by VV-expressed herpes virus protein ICP47, which acts by decreasing the supply of cytosolic peptides to class I molecules, indicating that Vpu functions in a distinct manner from ICP47. Based on these findings, we propose that Vpu-induced downregulation of class I molecules may be an important factor in the evolutionary selection of the HIV-1-specific vpu gene by contributing to the inability of CD8+ T cells to eradicate HIV-1 from infected individuals.

CD8-Positive T-Lymphocytes↗

PubMed1996

Identification of an ion channel activity of the Vpu transmembrane domain and its involvement in the regulation of virus release from HIV-1-infected cells.

HIV-1 Vpu catalyzes two independent functions, degradation of the virus receptor CD4 in the endoplasmic reticulum and enhancement of virus release from the cell surface. These activities are confined to distinct structural domains of Vpu, the cytoplasmic tail and the transmembrane (TM) anchor, respectively. It was recently reported that Vpu forms cation-selective ion channels in lipid bilayers. Here we report that this property of Vpu is a characteristic of its TM anchor. Expression of full-length Vpu in Xenopus oocytes increases membrane conductance. The Vpu-induced conductance is selective to monovalent cations over anions, does not discriminate Na+ over K+ and shows marginal permeability to divalent cations. Notably, introduction of the scrambled TM sequence into full-length Vpu abrogates its capacity to increase membrane conductance in oocytes and to promote virus release from infected cells. Reconstitution of synthetic Vpu fragments in lipid bilayers identified an ion channel activity for a sequence corresponding to the TM domain of Vpu. In contrast, a peptide with the same amino acid composition but with a scrambled sequence does not form ion channels. Our findings therefore suggest that the ability of Vpu to increase virus release from infected cells may be correlated with an ion channel activity of the TM domain, thereby providing a potential target for drug intervention based on the development of Vpu-specific channel blockers.

Amino Acid Sequence↗

PubMed1996

Solution structure of the cytoplasmic domain of the human immunodeficiency virus type 1 encoded virus protein U (Vpu).

The HIV-1-specific Vpu protein is an 81 amino acid class I integral membrane phosphoprotein that induces degradation of the virus receptor CD4 in the endoplasmic reticulum and enhances the release of virus particles from infected cells. Vpu is of amphipathic nature and consists of a hydrophobic N-terminal membrane anchor proximal to a polar C-terminal cytoplasmic domain. In our recent work, focussed on the structural analysis of the cytoplasmic tail, we established an alpha-helix-flexible-alpha-helix-turn model. Now we present the experimental solution structure of the Vpu cytoplasmic domain which has been elucidated in aqueous 50% trifluoroethanol solution by 2D 1H NMR spectroscopy, and restrained molecular dynamics and energy minimization calculations. Under these conditions the peptide, Vpu32-81, is predominantly monomeric and adopts a well defined helix-interconnection-helix-turn conformation, in which the four regions are bounded by residues 37-51, 52-56, 57-72 and 73-78. The presence of the cis isomer of Pro-75 manifests itself as a doubling of cross peaks of neighbouring residues in the 2D spectra. A related variant peptide, Vpum32-81, in which the Vpu-phosphoacceptor sites Ser52 and Ser56 were exchanged for Asn, adopts a very similar structure and, taken together, provides evidence that the second helix and the turn form a comparatively rigid region. Both helices are amphipathic in character, but show different charge distributions. In general the cytoplasmic region is N-terminally positively charged, passes through a region of alternating charges in helix 1 and then becomes negatively charged. The flexibility of the interconnection permits orientational freedom of the two helices. The motif found here is the first experimentally refined solution structure of the cytoplasmic domain of Vpu, and it is conceivable that these alpha-helices are important for a previously defined physical interaction with an alpha-helical Vpu-responsive element located within the cytoplasmic tail of CD4.

Amino Acid Sequence↗

PubMed1996

The two biological activities of human immunodeficiency virus type 1 Vpu protein involve two separable structural domains.

The human immunodeficiency virus type 1 (HIV-1) Vpu protein is an integral membrane phosphoprotein that induces CD4 degradation in the endoplasmic reticulum and enhances virus release from the cell surface. CD4 degradation is specific, requires phosphorylation of Vpu, and involves the interaction between Vpu and the CD4 cytoplasmic domain. In contrast, regulation of virus release is less specific and not restricted to HIV-1 and may be mechanistically-distinct from CD4 degradation. We show here that a mutant of Vpu, Vpu35, lacking most of its cytoplasmic domain has residual biological activity for virus release but is unable to induce CD4 degradation. This finding suggests that the N terminus of Vpu encoding the transmembrane (TM) anchor represents an active domain important for the regulation of virus release but not CD4 degradation. To better define the functions of Vpu's TM anchor and cytoplasmic domain, we designed a mutant, VpuRD, containing a scrambled TM sequence with a conserved amino acid composition and alpha-helical structure. The resulting protein was integrated normally into membranes, was able to form homo-oligomers, and exhibited expression levels, protein stability, and subcellular localization similar to those of wild-type Vpu. Moreover, VpuRD was capable of binding to CD4 and to induce CD4 degradation with wild-type efficiency, confirming proper membrane topology and indicating that the alteration of the Vpu TM domain did not interfere with this function of Vpu. However, VpuRD was unable to enhance the release of virus particles from infected or transfected cells, and virus encoding VpuRD had replication characteristics in T cells indistinguishable from those of a Vpu-deficient HIV-1 isolate. Mutation of the phosphorylation sites in VpuRD resulted in a protein which was unable to perform either function of Vpu. The results of our experiments suggest that the two biological activities of Vpu operate via two distinct molecular mechanisms and involve two different structural domains of the Vpu protein.

Amino Acid Sequence↗

PubMed1996

The envelope glycoprotein of human immunodeficiency virus type 2 enhances viral particle release: a Vpu-like factor?

The Vpu protein is a human immunodeficiency virus type 1 (HIV-1)-specific accessory protein that is required for the efficient release of viral particles from infected cells. Even though HIV-2 does not encode Vpu, we found that this virus is nevertheless capable of efficiently releasing virus particles. In fact, the rate of virus release from HeLa cells transfected with a full-length molecular clone of HIV-2, ROD10, was comparable to that observed for the vpu+ HIV-1 NL4-3 isolate and was not further enhanced by expression of Vpu in trans. However, consistent with previous observations showing that HIV-2 particle release is Vpu responsive in the context of HIV-1/HIV-2 chimeric constructs; exchanging the gag-pol region of NL4-3 with the corresponding region from pROD10 rendered the resulting chimeric virus Vpu responsive. Our finding that the responsiveness of HIV-2 particle release to Vpu is context dependent suggested the presence of a Vpu-like factor(s) encoded by HIV-2. Using chimeric proviruses encoding HIV-2 gag and pol in the context of the HIV-1 provirus that were coexpressed with subgenomic HIV-2 constructs, we found that the HIV-2 envelope glycoprotein had the ability to enhance HIV-2 particle release with an efficiency comparable to that of the HIV-1 Vpu protein. Conversely, inactivation of the HIV-2 env gene in the original ROD10 clone resulted in a decrease in the rate of viral particle release to a level that was comparable to that of Vpu-deficient HIV-1 isolates. Providing the wild-type envelope in trans rescued the particle release defect of the ROD10 envelope mutant. Thus, unlike HIV-1, which encodes two separate proteins to regulate virus release or to mediate viral entry, the HIV-2 Env protein has evolved to perform both functions.

Brefeldin A↗

PubMed1995

Solution structure of the hydrophilic region of HIV-1 encoded virus protein U (Vpu) by CD and 1H NMR spectroscopy.

The HIV-1 specific Vpu is a class I oligomeric membrane phosphoprotein of unknown structure and mechanism. The first experimental evidence for the position of secondary structural elements present in the hydrophilic C-terminal region of Vpu under various solution regimes is reported. CD data for nine overlapping 15 amino-acid fragments and 3 longer fragments indicate the presence of only transitory amounts of stable structure in aqueous solution alone, while with increasing trifluoroethanol content limiting structures were found indicating two helical segments in the hydrophilic region of Vpu. These limiting structures were more precisely defined from a detailed study of Vpu41-58, Vpu52-74 and Vpu63-81, by a combination of 2D 1H NMR spectroscopy, distance geometry, and restrained molecular dynamics and energy minimization calculations. Sets of low-energy conformations compatible with the quantitative NOE data indicate that Vpu41-58 has an alpha-helix from residues 42 to 50 while a second helix is found for Vpu52-74 from residues 57 to 69. Vpu63-81 shows only the presence of a single reverse turn at residues 74 to 77, without any evidence of helix, under the same conditions. From CD measurements the first helix extends back to residue 30 and is connected to the N-terminal anchor of Vpu. Thus the hydrophilic region of Vpu consists of two alpha-helices joined by a flexible region of 6 or 7 residues, which contains the phosphoacceptor sites of Vpu at positions 52 and 56. The second helix is followed by a single reverse turn and a flexible C-terminus.

Amino Acid Sequence↗

PubMed1995

Augmentation of virus secretion by the human immunodeficiency virus type 1 Vpu protein is cell type independent and occurs in cultured human primary macrophages and lymphocytes.

The human immunodeficiency virus type 1-specific Vpu protein is a small integral membrane phosphoprotein that induces degradation of the virus receptor CD4 in the endoplasmic reticulum and, independently, increases the release of progeny virions from infected cells. To address the importance of Vpu for virus replication in primary human cells such as peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM), we used three different sets of monocyte-tropic molecular clones of human immunodeficiency virus type 1: a primary isolate, AD8+, and two chimeric variants of the T-cell-tropic isolate NL4-3 carrying the env determinants of either AD8+ or SF162 monocyte-tropic primary isolates. Isogenic variants of these chimeric viruses were constructed to express either wild-type Vpu or various mutants of Vpu. The effects of these mutations in the vpu gene on virus particle secretion from infected MDM or PBMC were assessed by determination of the release of virion-associated reverse transcriptase into culture supernatants, Western blot (immunoblot) analysis of pelleted virions, and steady-state or pulse-chase metabolic labeling. Wild-type Vpu increased virus release four- to sixfold in MDM and two- to threefold in PBMC, while nonphosphorylated Vpu and a C-terminal truncation mutant of Vpu were partially active on virus release in primary cells. These results demonstrate that Vpu regulates virus release in primary lymphocyte and macrophage cultures in a similar manner and to a similar extent to those previously observed in HeLa cells or CD4+ T-cell lines. Thus, our findings provide evidence that Vpu functions in a variety of human cells, both primary cells and continuous cell lines, and mutations in Vpu affect its biological activity independent of the cell type and virus isolate used.

Cells, Cultured↗

PubMed1995

The human immunodeficiency virus type 1 Vpu protein specifically binds to the cytoplasmic domain of CD4: implications for the mechanism of degradation.

We have recently demonstrated that coexpression of Vpu and CD4 in HeLa cells results in the degradation of CD4 in the endoplasmic reticulum. The sensitivity of CD4 to Vpu-mediated degradation is conferred by the presence of specific sequences located between amino acids 402 and 420 in the CD4 cytoplasmic domain. Using an in vitro translation system, we also showed that degradation of CD4 by Vpu requires the two proteins to be present in the same membrane compartment. Although these results suggest that spatial proximity between CD4 and Vpu may be critical in triggering degradation, it remains unknown whether the two molecules have the ability to interact with each other. In order to better define the mechanisms involved in CD4 degradation, we investigated the existence and functional relevance of direct interactions between CD4 and Vpu. Coimmunoprecipitation experiments showed that Vpu specifically binds to the cytoplasmic tail of CD4. This phenomenon is relevant to the mechanism of CD4 degradation since the ability of CD8/CD4 chimeric molecules and various CD4 mutants to form complexes with Vpu correlates with their sensitivity to degradation. Accordingly, we found that amino acid residues in the CD4 cytoplasmic tail previously shown to be important for degradation are necessary for Vpu binding. We further demonstrate that a deletion mutant of Vpu as well as a phosphorylation mutant, both biologically inactive with regard to CD4 degradation, retained the capacity to interact with the CD4 cytoplasmic domain. Taken together, these results indicate that Vpu binding is necessary to trigger CD4 degradation. However, the binding to target molecules is not sufficient per se to cause degradation. Interaction between CD4 and Vpu is thus likely to be an early event critical in triggering a multistep process leading to CD4 degradation.

Amino Acid Sequence↗

PubMed1995

[Schwannoma of the gastric wall].

Clinical and ultrasound examination showed a tumor adjacent to the posterior gastric wall in two patients with noncharacteristic upper abdominal pain. The structure of the at least fist-size masses was partly solid, partly cystic and on palpation appeared soft and spongy with a smooth surface. Colour ultrasound showed a high vascularity. All these factors were confirmed by computerized tomography. History as well as clinical and ultrasound examination suggested the diagnosis of schwannoma and this was confirmed by histopathology following laparotomy.

Aged↗

PubMed1994

[Toxic diphtheria caused by Corynebacterium ulcerans].

A 42-year-old woman with bronchial asthma since childhood was admitted to hospital because of severe dyspnoea. Emergency bronchoscopic intubation had to be performed for life-threatening inspiratory and expiratory stridor. This demonstrated that the larynx was covered by a dirty-grey membrane and the vocal-cord gap was narrowed to a mere slit. As laryngeal diphtheria was suspected 2000 IU/kg diphtheria antitoxin was administered together with 1 mega U penicillin G four times daily intravenously. On the same day, Corynebacterium ulcerans, a very rare cause of diphtheria, was isolated from a coughed-up piece of the membrane. Toxic, massive swelling of the lymph nodes and soft tissues of the neck necessitated maintenance of an open upper airway by intubation and (later) tracheostomy for 41 days. A week later the patient was discharged without any permanent defect.

Adult↗

PubMed1994

The human immunodeficiency virus type 1 encoded Vpu protein is phosphorylated by casein kinase-2 (CK-2) at positions Ser52 and Ser56 within a predicted alpha-helix-turn-alpha-helix-motif.

The human immunodeficiency virus type 1 (HIV-1) encoded Vpu is a small integral membrane phosphoprotein that functions in the enhancement of viral particle release and has more recently been shown to cause degradation of CD4 at the endoplasmic reticulum. We have demonstrated earlier that Vpu is phosphorylated by the ubiquitous casein kinase-2 (CK-2) in HIV-1 infected cells. The phosphoacceptor sites targeted by CK-2 in Vpu, however, have not been demonstrated and it was unclear whether Vpu was phosphorylated at one or more of its four serine residues. In this study we characterized the CK-2 phosphoacceptor sites in Vpu using recombinant CK-2 for in vitro phosphorylation of recombinant Vpu protein as well as synthetic peptides of Vpu. Phosphorylation of both Ser52 and Ser56 was demonstrated by in vitro phosphorylation using three 54-residue peptides comprising the entire hydrophilic part of Vpu and containing single serine to asparagine transitions in either position 52 or 56. The Km values of CK-2 to these peptides were established, revealing a preferential phosphorylation of Ser56. The Km values are: Ser56 = 31 microM; Ser 52 = 156 microM; wild type = 27 microM. In addition, we studied phosphorylation of Vpu by endogenous CK-2 following in vitro translation in rabbit reticulocyte lysate of wild-type Vpu or a mutant, Vpum2/6, carrying serine to asparagine changes at amino acid positions 52 and 56. The in vivo phosphorylation of Vpu was studied in transiently transfected human embryonic kidney (293) cells. In this system, the mutant Vpum2/6 was not phosphorylated, indicating that the seryl residues of Vpu at amino acid positions 52 and 56, but not those at positions 23 and 61, are phosphorylated by CK-2. The two CK-2 phosphorylation sites are conserved in all known Vpu sequences and represent the consensus Ser52GlyAsn(Glu/Asp)Ser(Glu/Asp)Gly(Glu/Asp)59. Prediction of the secondary structure revealed a conserved alpha-helix-turn-alpha-helix motif for the hydrophilic C-terminal part of Vpu. A structural model for Vpu is proposed in which the membrane anchor precedes a region comprising two amphipathic alpha-helices of opposed polarity, joined by a strongly acidic turn that protrudes into the cytoplasm and contains the CK-2 phosphorylation sites. Possible functional and structural homologies of Vpu to the membrane channel-forming M2 protein of influenza A viruses are discussed.

Amino Acid Sequence↗

PubMed1994

Detection of mycoplasma contaminations by the polymerase chain reaction.

The polymerase chain reaction (PCR) has been used for the general detection of Mollicutes. 25 Mycoplasma and Acholeplasma species were detected including important contaminants of cell cultures such as M. orale, M. arginini, M. hyorhinis, M. fermentans, A. laidlawii and additional human and animal mycoplasmas. PCR reactions were performed using a set of nested primers defined from conserved regions of the 16S rRNA gene. The detection limit was determined to be 1 fg mycoplasma DNA, which is equivalent to 1-2 genome copies of the 16S rRNA coding region. The identity of the amplification products was confirmed by agarose gel electrophoresis and restriction enzyme analysis. DNA from closely and distantly related micro-organisms did not give rise to specific amplification products. The method presented here offers a much more sensitive, specific and rapid assay for the detection of mycoplasmas than the existing ones.

Base Sequence↗

PubMed1994

Differential activities of the human immunodeficiency virus type 1-encoded Vpu protein are regulated by phosphorylation and occur in different cellular compartments.

The human immunodeficiency virus type 1 (HIV-1)-specific Vpu is an 81-amino-acid amphipathic integral membrane protein with at least two different biological functions: (i) enhancement of virus particle release from the plasma membrane of HIV-1-infected cells and (ii) degradation of the virus receptor CD4 in the endoplasmic reticulum (ER). We have previously found that Vpu is phosphorylated in infected cells at two seryl residues in positions 52 and 56 by the ubiquitous casein kinase 2. To study the role of Vpu phosphorylation on its biological activity, a mutant of the vpu gene lacking both phosphoacceptor sites was introduced into the infectious molecular clone of HIV-1, pNL4-3, as well as subgenomic Vpu expression vectors. This mutation did not affect the expression level or the stability of Vpu but had a significant effect on its biological activity in infected T cells as well as transfected HeLa cells. Despite the presence of comparable amounts of wild-type and nonphosphorylated Vpu, decay of CD4 was observed only in the presence of phosphorylated wild-type Vpu. Nonphosphorylated Vpu was unable to induce degradation of CD4 even if the proteins were artificially retained in the ER. In contrast, Vpu-mediated enhancement of virus secretion was only partially dependent on Vpu phosphorylation. Enhancement of particle release by wild-type Vpu was efficiently blocked when Vpu was artificially retained in the ER, suggesting that the two biological functions of Vpu are independent, occur at different sites within a cell, and exhibit different sensitivity to phosphorylation.

Base Sequence↗

PubMed1993

[Gas in the portal vein system of the liver. Value of ultrasound].

We report on 9 patients presenting with different diseases of varying severity, who were found to have hepatic portal venous gas (HPVG) identified by diagnostic ultrasonography. Intrahepatic gas was a shortlasting phenomenon, which could not be seen radiologically in most cases. Earlier beliefs that HPVG is a poor prognostic sign associated with high mortality, should be revised.

Abscess↗