In vitro assembly properties of human immunodeficiency virus type 1 Gag protein lacking the p6 domain

Human immunodeficiency virus type 1 (HIV-1) normally assembles into particles of 100 to 120 nm in diameter by budding through the plasma membrane of the cell. The Gag polyprotein is the only viral protein that is required for the formation of these particles. We have used an in vitro assembly system to examine the assembly properties of purified, recombinant HIV-1 Gag protein and of Gag missing the C-terminal p6 domain (Gag Deltap6). This system was used previously to show that the CA-NC fragment of HIV-1 Gag assembled into cylindrical particles. We now report that both HIV-1 Gag and Gag Deltap6 assemble into small, 25- to 30-nm-diameter spherical particles in vitro. The multimerization of Gag Deltap6 into units larger than dimers and the formation of spherical particles required nucleic acid. Removal of the nucleic acid with NaCl or nucleases resulted in the disruption of the multimerized complexes. We conclude from these results that (i) N-terminal extension of HIV-1 CA-NC to include the MA domain results in the formation of spherical, rather than cylindrical, particles; (ii) nucleic acid is required for the assembly and maintenance of HIV-1 Gag Deltap6 virus-like particles in vitro and possibly in vivo; (iii) a wide variety of RNAs or even short DNA oligonucleotides will support assembly; (iv) protein-protein interactions within the particle must be relatively weak; and (v) recombinant HIV-1 Gag Deltap6 and nucleic acid are not sufficient for the formation of normal-sized particles.     

Increased susceptibility of thymocytes to apoptosis in mice lacking AIM, a novel murine macrophage-derived soluble factor belonging to the scavenger receptor cysteine-rich domain superfamily

Apoptosis of cells must be regulated both positively and negatively in response to a variety of stimuli in the body. Various environmental stresses are known to initiate apoptosis via differential signal transduction cascades. However, induction of signals that may inhibit apoptosis is poorly understood, although a number of intracellular molecules that mediate inhibition of apoptosis have been identified. Here we present a novel murine macrophage-specific 54-kD secreted protein which inhibits apoptosis (termed AIM, for apoptosis inhibitor expressed by macrophages). AIM belongs to the macrophage scavenger receptor cysteine-rich domain superfamily (SRCR-SF), members of which share a highly homologous conserved cysteine-rich domain. In AIM-deficient mice, the thymocyte numbers were diminished to half those in wild-type mice, and CD4/CD8 double-positive (DP) thymocytes were strikingly more susceptible to apoptosis induced by both dexamethasone and irradiation in vivo. Recombinant AIM protein significantly inhibited cell death of DP thymocytes in response to a variety of stimuli in vitro. These results indicate that in the thymus, AIM functions in trans to induce resistance to apoptosis within DP cells, and thus supports the viability of DP thymocytes before thymic selection.     

Plasminogen activator inhibitor type 1 in rat spermatozoa: localisation in the tail, in the acrosome and on the surface of the head

Isolated seminiferous tubules of rat testis contain considerable urokinase-inhibiting activity. An immunohistological analysis revealed the presence of plasminogen activator inhibitor type 1 (PAI-1) in the basement membrane as well as in the interior of the tubules. Distribution and intensity of the intratubular immunoreactivity depends on the stage of the seminiferous cycle. A relatively weak signal is present around elongated nuclei of spermatids at the beginning of chromatin condensation. The signal intensity increases in the course of differentiation until a maximum is reached at stages VII-VIII. In these stages PAI-1 immunoreactivity is localised around the nuclei of the late spermatids as well as along their tails. Spermatozoa in the ductus epididymis also strongly react with the PAI-1-specific antiserum, suggesting that the inhibitor remains associated with the germ cells after spermiation and during maturation in the epididymis. In intact mature spermatozoa isolated from epididymis cauda by "swimming-up' in non-capacitation medium, PAI-1 antigen is localised on the plasma membrane surrounding the head. In addition, in fixed and permeabilised cells the immunoreactivity is detectable in the acrosome and in the tail. Possible functions of PAI-1 in spermatogenesis, sperm motility and sperm-egg interaction are discussed.     

Change in the hexokinase activity of the rat myocardial and skeletal muscle mitochondria and cytoplasm in fasting and on a carbohydrate diet

Our experience using the lateral simplified thoracotomy incision for most chest work other than operations requiring median sternotomy is reported. The incision provides adequate exposure, yet preserves major muscle masses and decreases the postoperative morbidity. Return of normal ipsilateral arm function appears to be hastened in the postoperative period. In addition, the incision is easier to open and close than the standard incision.     

Specific CSF-1 binding on murine placental trophoblasts and macrophages serves as a link to placental growth

Previous studies have shown that the colony-stimulating factor-1 (CSF-1), stimulates the in vitro proliferation of a fetally-derived adherent, phagocytic and non-specific esterase positive placental cell population which stains positively for cytokeratin and Mac-1. Binding experiments were designed to test whether this is a direct effect of the factor on these cells. Binding/elution as well as autoradiography experiments, show that adherent placental cells specifically bind CSF-1. Based on the expression of the endothelial markers cytokeratin and vimentin three subpopulations of cells were isolated from the murine placenta: labyrinthine-derived trophoblasts (cytokeratin positive, vimentin negative), spongiotrophoblast-derived trophoblasts (cytokeratin positive, vimentin negative) and placental macrophages (cytokeratin negative, vimentin positive). 3H-Thymidine incorporation assays as well as binding experiments, showed that these cells simultaneously respond to and bind the macrophage-specific factor CSF-1. Furthermore, the results indicate that isolated trophoblasts have a low rate of growth and they are very sensitive to mitogenic stimulation, whereas placental macrophages alone have a high rate of growth and therefore are less sensitive to the mitogenic stimulus. These findings are in favour of the existence of an important cytokine regulatory network in the murine placenta, where two major cell populations may collaborate possibly via soluble factors to stimulate placental growth and thus fetal development.     

Identification of a new mutation of the myosin VII head region in Usher syndrome type 1

We characterized apneas by a quantitative method (esophageal pressure measurements) and by a qualitative method (strain gauges) at the same time in 22 patients with sleep-related breathing disorders. Detection of respiratory effort by strain gauges significantly overestimated the total number of central apneas in each patient. Despite this overestimation, none of the patients was wrongly diagnosed as having pure central sleep apnea syndrome. Strain gauges are sufficiently reliable for the characterization of apneas in most patients. When strain gauges reveal that most apneas are central in origin, verification by esophageal pressure measurements is recommended.     

Characterization of human type X procollagen and its NC-1 domain expressed as recombinant proteins in HEK293 cells

Type X collagen is a short-chain, network-forming collagen found in hypertrophic cartilage in the growth zones of long bones, vertebrae, and ribs. To obtain information about the structure and assembly of mammalian type X collagen, we generated recombinant human type collagen X by stable expression of full-length human alpha1(X) cDNA in the human embryonal kidney cell line HEK293 and the fibrosarcoma cell line HT1080. Stable clones were obtained secreting recombinant human type X collagen (hrColX) in amounts of 50 microg/ml with alpha1(X)-chains of apparent molecular mass of 75 kDa. Pepsin digestion converted the native protein to a molecule migrating as one band at 65 kDa, while bands of 55 and 43 kDa were generated by trypsin digestion. Polyclonal antibodies prepared against purified hrColX reacted specifically with type X collagen in sections of human fetal growth cartilage. Circular dichroism spectra and trypsin/chymotrypsin digestion experiments of hrColX at increasing temperatures indicated triple helical molecules with a reduced melting temperature of 31 degrees C as a result of partial underhydroxylation. Ultrastructural analysis of hrColX by rotary shadowing demonstrated rodlike molecules with a length of 130 nm, assembling into aggregates via the globular noncollagenous (NC)-1 domains as reported for chick type X collagen. NC-1 domains generated by collagenase digestion of hrColX migrated as multimers of apparent mass of 40 kDa on SDS-polyacrylamide gel electrophoresis, even after reduction and heat denaturation, and gave rise to monomers of 18-20 kDa after treatment with trichloroacetic acid. The NC-1 domains prepared by collagenase digestion comigrated with NC-1 domains prepared as recombinant protein in HEK293 cells, both in the multimeric and monomeric form. These studies demonstrate the potential of the pCMVsis expression system to produce recombinant triple helical type X collagens in amounts sufficient for further studies on its structural and functional domains.     

Replication and neutralization of human immunodeficiency virus type 1 lacking the V1 and V2 variable loops of the gp120 envelope glycoprotein

A human immunodeficiency virus type 1 (HIV-1) mutant lacking the V1 and V2 variable loops in the gp120 exterior envelope glycoprotein replicated in Jurkat lymphocytes with only modest delays compared with the wild-type virus. Revertants that replicated with wild-type efficiency rapidly emerged and contained only a few amino acid changes in the envelope glycoproteins compared with the parent virus. Both the parent and revertant viruses exhibited increased sensitivity to neutralization by antibodies directed against the V3 loop or a CD4-induced epitope on gp120 but not by soluble CD4 or an antibody against the CD4 binding site. This result demonstrates the role of the gp120 V1 and V2 loops in protecting HIV-1 from some subsets of neutralizing antibodies.     

Cloning and characterization of a testis-specific, developmentally regulated A-kinase-anchoring protein (TAKAP-80) present on the fibrous sheath of rat sperm

(-)-beta-D-2-Aminopurine dioxolane (APD), (-)-beta-D-2-amino-6-chloropurine dioxolane (ACPD) and dioxolane guanine (DXG) are nucleoside analogues possessing potent activity against human immunodeficiency virus (HIV) and hepatitis B virus (HBV) in vitro. APD and ACPD are metabolized in vivo to yield DXG. Reversed-phase HPLC analytical methodologies were developed for the simultaneous determination of APD and DXG, and for ACPD and DXG in monkey serum, urine and cerebrospinal fluid (CSF). 2-Fluoro-2',3'-dideoxyinosine (FDDI) served as the internal standard. The extraction recoveries of the nucleoside analogues from serum samples were similar, averaging approximately 90%. The limit of quantitation of the analytical method for serum samples was 0.1 microg/ml for DXG, and 0.25 microg/ml for APD and ACPD. The intra- and inter-day relative standard deviations for each compound at low, medium and high nucleoside concentrations were less than 9.0%. The accuracy of the assay methods was greater than 90% for prodrugs and parent compound. Similar results were observed with urine and CSF samples. Thus, these methods provide sensitive, accurate and reproducible determination of the prodrugs and parent nucleoside in biological samples.     

Linkage mapping of Lims1l, the murine homolog of the human LIM domain gene PINCH, to mouse chromosome 10

In order to compare the efficacy of immediate intravenous oxytocin administration and intracervical prostaglandin E2 gel application in premature rupture of membranes with unfavorable cervices at term, 45 term pregnant patients with premature rupture of membranes were randomized into two groups. Twenty women received immediate intravenous oxytocin after cleansing enema while the rest were treated with intracervical prostaglandin E2 gel. Means of maternal age, gestational age, Bishop score at admission and the rates of nulliparity did not show any significant differences between the two groups (p > 0.05). The mean rupture to delivery time was 12.6 +/- 4.4 hours in the oxytocin group and 16.5 +/- 4.5 hours in the prostaglandin group (p < 0.01). Mean birth weights and Apgar scores were insignificant. Cesarean section rates were 24% in the oxytocin group and 5% in the other (p < 0.05). No infectious morbidity was seen in any case. In conclusion, although delivery is delayed with the intracervical prostaglandin approach, cesarean section rate is lowered without an increase in infectious morbidity.     

Targeting of NH2-terminal-processed microsomal protein to mitochondria: a novel pathway for the biogenesis of hepatic mitochondrial P450MT2

Cytochrome P4501A1 is a hepatic, microsomal membrane-bound enzyme that is highly induced by various xenobiotic agents. Two NH2-terminal truncated forms of this P450, termed P450MT2a and MT2b, are also found localized in mitochondria from beta-naphthoflavone-induced livers. In this paper, we demonstrate that P4501A1 has a chimeric NH2-terminal signal that facilitates the targeting of the protein to both the ER and mitochondria. The NH2-terminal 30-amino acid stretch of P4501A1 is thought to provide signals for ER membrane insertion and also stop transfer. The present study provides evidence that a sequence motif immediately COOH-terminal (residues 33-44) to the transmembrane domain functions as a mitochondrial targeting signal under both in vivo and in vitro conditions, and that the positively charged residues at positions 34 and 39 are critical for mitochondrial targeting. Results suggest that 25% of P4501A1 nascent chains, which escape ER membrane insertion, are processed by a liver cytosolic endoprotease. We postulate that the NH2-terminal proteolytic cleavage activates a cryptic mitochondrial targeting signal. Immunofluorescence microscopy showed that a portion of transiently expressed P4501A1 is colocalized with the mitochondrial-specific marker protein cytochrome oxidase subunit I. The mitochondrial-associated MT2a and MT2b are localized within the inner membrane compartment, as tested by resistance to limited proteolysis in both intact mitochondria and mitoplasts. Our results therefore describe a novel mechanism whereby proteins with chimeric signal sequence are targeted to the ER as well as to the mitochondria.     

During the initiation of fermentation overexpression of hexokinase PII in yeast transiently causes a similar deregulation of glycolysis as deletion of Tps1

In the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 (= GGS1 = FDP1 = BYP1 = CIF1 = GLC6 = TSS1)-encoded trehalose-6-phosphate synthase, is essential for restriction of glucose influx into glycolysis apparently by inhibiting hexokinase activity in vivo. We show that up to 50-fold overexpression of hexokinase does not noticeably affect growth on glucose or fructose in wild-type cells. However, it causes higher levels of glucose-6-phosphate, fructose-6-phosphate and also faster accumulation of fructose-1,6-bisphosphate during the initiation of fermentation. The levels of ATP and Pi correlated inversely with the higher sugar phosphate levels. In the first minutes after glucose addition, the metabolite pattern observed was intermediate between those of the tps1 delta mutant and the wild-type strain. Apparently, during the start-up of fermentation hexokinase is more rate-limiting in the first section of glycolysis than phosphofructokinase. We have developed a method to measure the free intracellular glucose level which is based on the simultaneous addition of D-glucose and an equal concentration of radiolabelled L-glucose. Since the latter is not transported, the free intracellular glucose level can be calculated as the difference between the total D-glucose measured (intracellular + periplasmic/extracellular) and the total L-glucose measured (periplasmic/extracellular). The intracellular glucose level rose in 5 min after addition of 100 mM-glucose to 0.5-2 mM in the wild-type strain, +/- 10 mM in a hxk1 delta hxk2 delta glk1 delta and 2-3 mM in a tps1 delta strain. In the strains overexpressing hexokinase PII the level of free intracellular glucose was not reduced. Overexpression of hexokinase PII never produced a strong effect on the rate of ethanol production and glucose consumption. Our results show that overexpression of hexokinase does not cause the same phenotype as deletion of Tps1. However, it mimics it transiently during the initiation of fermentation. Afterwards, the Tps1-dependent control system is apparently able to restrict properly up to 50-fold higher hexokinase activity.     

Comparative characterization of a wild type and transmembrane domain-deleted fatty acid amide hydrolase: identification of the transmembrane domain as a site for oligomerization

Fatty acid amide hydrolase (FAAH) is an integral membrane protein responsible for the hydrolysis of a number of primary and secondary fatty acid amides, including the neuromodulatory compounds anandamide and oleamide. Analysis of FAAH's primary sequence reveals the presence of a single predicted transmembrane domain at the extreme N-terminus of the enzyme. A mutant form of the rat FAAH protein lacking this N-terminal transmembrane domain (DeltaTM-FAAH) was generated and, like wild type FAAH (WT-FAAH), was found to be tightly associated with membranes when expressed in COS-7 cells. Recombinant forms of WT- and DeltaTM-FAAH expressed and purified from Escherichia coli exhibited essentially identical enzymatic properties which were also similar to those of the native enzyme from rat liver. Analysis of the oligomerization states of WT- and DeltaTM-FAAH by chemical cross-linking, sedimentation velocity analytical ultracentrifugation, and size exclusion chromatography indicated that both enzymes were oligomeric when membrane-bound and after solubilization. However, WT-FAAH consistently behaved as a larger oligomer than DeltaTM-FAAH. Additionally, SDS-PAGE analysis of the recombinant proteins identified the presence of SDS-resistant oligomers for WT-FAAH, but not for DeltaTM-FAAH. Self-association through FAAH's transmembrane domain was further demonstrated by a FAAH transmembrane domain-GST fusion protein which formed SDS-resistant dimers and large oligomeric assemblies in solution.     

Sequences present in the cytoplasmic domain of CD4 are necessary and sufficient to confer sensitivity to the human immunodeficiency virus type 1 Vpu protein

CD4 is an integral membrane glycoprotein which functions as the human immunodeficiency virus receptor for infection of human host cells. We have recently demonstrated that Vpu, a human immunodeficiency virus type 1-encoded integral membrane phosphoprotein, induces rapid degradation of CD4 in the endoplasmic reticulum. Using an in vitro model system, we demonstrated that Vpu targets specific sequences in the cytoplasmic domain of CD4 to promote its degradation. In this report, we have further delineated regions within CD4 which are required for susceptibility to Vpu. Transfer of the CD4 cytoplasmic region into a heterologous protein, CD8, rendered the chimeric protein sensitive to Vpu-dependent degradation. In contrast, substitution of the CD8 transmembrane domain with the analogous region from CD4 did not confer sensitivity to Vpu. Finally, mutant forms of the CD4 protein containing the extracellular region alone or the extracellular and transmembrane regions linked to a heterologous cytoplasmic domain were not targeted by Vpu. Thus, sequences present in the cytoplasmic domain of CD4 are necessary and sufficient to confer sensitivity to Vpu.