Phytoreovirus T = 1 core plays critical roles in organizing the outer capsid of T = 13 quasi-equivalence.

The structures of the double-shelled rice dwarf virus and of its single-shell core have been determined by cryoelectron microscopy and image reconstruction. The core carries a prominent density located at each of the icosahedral faces of its T = 1 lattice. These protrusions are formed by outer shell trimers, tightly inserted at the threefold positions of the core. Such configuration of the core may guide the assembly of the outer shell, aided by lateral interactions between its subunits, into a T = 13 lattice. The organization of the phytoreovirus capsid elucidates for the first time a general model for assembling two unique T numbers of quasi-equivalence.

Recombinant hepatitis E capsid protein self-assembles into a dual-domain T = 1 particle presenting native virus epitopes.

The three-dimensional structure of a self-assembled, recombinant hepatitis E virus particle has been solved to 22-A resolution by cryo-electron microscopy and three-dimensional image reconstruction. The single subunit of 50 kDa is derived from a truncated version of the open reading frame-2 gene of the virus expressed in a baculovirus system. This is the first structure of a T = 1 particle with protruding dimers at the icosahedral two-fold axes solved by cryo-electron microscopy. The protein shell of these hollow particles extends from a radius of 50 A outward to a radius of 135 A. In the reconstruction, the capsid is dominated by dimers that define the 30 morphological units. The outer domain of the homodimer forms a protrusion, which corresponds to the spike-like density seen in the cryo-electron micrograph. This particle retains native virus epitopes, suggesting its potential value as a vaccine.

Binding of Galanthus nivalis lectin to Chlamydia trachomatis and inhibition of in vitro infection.

A glycoprotein present in Chlamydia trachomatis, serotype L1, elementary bodies (EBs) was earlier found to bind the lectin from Galanthus nivalis (GNA). In the present paper we investigate the interaction of GNA with chlamydial EBs and its effect on in vitro infectivity. The binding affinity was studied with 125I-GNA lectin. Within 15 min about 80% maximal binding was obtained. The chlamydia-GNA interaction was inhibited by alpha-methylmannoside, causing a decrease of about 50% at 1 mM. Curve fit analyses indicated two types of binding sites for GNA on the EBs. The affinity to these differed by a factor of 15. The influence of the lectin on the ability of C. trachomatis to infect McCoy cells was also investigated. There was a GNA-dependent inhibition with a 50% reduction in the number of intracellular inclusions at 0.2 microM of the lectin. The findings indicate the presence of terminal mannose structures on the chlamydial surface at or in the proximity of the cell-binding domains. Mannose-binding proteins of eukaryotic cells could be important for the initial uptake of EBs.

Lectin-mediated effects on HIV type 1 infection in vitro.

Lectins with specificity for terminal mannose residues and anti-mannan antibodies neutralize HIV-1 infection in vitro. This is assumed to be caused by binding of the agents to the viral glycoproteins. In this study we show that one such agent, the Galanthus nivalis lectin (GNA), also blocks infection at the target cell level. To explore the effect of GNA on HIV infection we used the two HIV-1 isolates LAV and NDK, representing in the first case a prototype virus and in the latter case a highly cytopathic virus, which spreads preferentially via cell-to-cell contact. MT-4 cells were used as target cells and infection was determined from the occurrence of syncytia. Cell-to-cell infection was studied with CEM cells persistently infected with the two virus isolates. GNA, at concentrations in the nanogram per milliliter range, neutralized the HIV-1 isolates LAV, NDK, and MN as well as HIV-2ROD. Pretreatment of cells with the lectin, before addition of virus, or of infected cells, also blocked infection. This effect was more pronounced with HIV-1NDK than with HIV-1LAV. Mannosidase treatment of the target cells abolished the GNA effect on HIV-1NDK infection. It is concluded that GNA inhibits infection of several HIV isolates. It neutralizes infection by binding to the virion but also blocks infection at the target cell level. The latter effect may be different for different virus isolates. Mannosyl residuals at the cell surface are targets for GNA modulation of infection with the cytopathic HIV-1NDK. These do not represent essential virus receptors.

Purification of simian immunodeficiency virus, SIVMAC251, and of its external envelope glycoprotein, gp148.

Two-phase extraction in a system composed of dextran and polyethylene glycol was used to purify simian immunodeficiency virus, SIVMAC251 (32H isolate) from 25 l of culture supernatant. The virus partitioned to the interphase with 80% recovery of gag peptide p27 and reverse transcriptase and an about 25% recovery of the external env glycoprotein, gp148. The virus was treated with octylglycoside and its subcomponents separated. Two gag-p27 containing fractions were obtained; gag-1, which also contained reverse transcriptase and nucleopeptides, and gag-2, which contained the major portion of the p27. The env gp148 was purified by chromatography through a series of lectin columns. The prepared materials are characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immuno- and lectin blotting.

Lectin effects on HIV-1 infectivity.

Lectins with specificity for terminal mannose residues and anti-mannan antibodies are assumed to neutralize HIV-1 by binding to the viral glycoproteins. In this report we demonstrate that one such agents, the lectin from Galanthus nivalis (GNA), blocks infection also at the target cell level.

Characterization of glycoproteins from Chlamydia trachomatis using lectins.

Glycoproteins in Chlamydia trachomatis, serotype L1, elementary bodies were studied by lectin blotting. A panel of 23 lectins representing a variety of sugar specificities was used. The pattern of lectin-binding specificities at a peptide band was studied in order to determine the type and structure of its glycoconjugate. To establish chlamydial origin of the glycopeptide bands in the blot, control samples from non-infected host cell membranes were run in parallel. Terminal mannosidic structures were demonstrated in a 72 kDa glycopeptide (gp72) by its selective binding of Galanthus nivalis lectin (GNA). Sialic acids were found in two chlamydial glycopeptides, gp40 and gp64, which appear to carry O-linked glycoconjugates as they bound the peanut agglutinin (PNA, both gp40 and gp64) and jackfruit lectin (Jac, only gp40). Such structures were also present in other chlamydial glycopeptides. Lectins with specificities for fucose in different links, galactose and N-acetyl glucosamine bound to several chlamydial peptides. On the basis of our results we suggest an alternative mechanism for uptake of chlamydial elementary bodies into host cells, namely phagocytosis mediated by eukaryotic cell surface lectins.

Extraction of HIV-1 in aqueous two-phase systems to obtain a high yield of gp120.

The conventionally applied centrifugation protocols for the concentration and purification of human immunodeficiency virus type 1 (HIV-1) result in a low recovery of the external glycoprotein, gp120. This is consistent with what has been found with other retroviruses. In the search for a method allowing the copurification of the gp120 with the virion we have applied two-phase extraction based on water-soluble polymers. Several polymer systems were tested for their capacity to enrich HIV-1 from HTLV-IIIB-infected H9 cell culture medium. With a dextran-polyethylene glycol system the gp120 and the gag protein p24, used as marker of the virion, were recovered to about 60 and 70%, respectively, in 1% of the initial volume. The two proteins were both about 30-fold purified and reverse transcriptase activity and infectious titer were retained to a high degree. The calculated molar ratio of gp120 to p24 was twofold higher in the phase-extracted fraction than in material pelleted by ultracentrifugation. It is concluded that extraction in aqueous two-phase systems is a method well suited for the concentration and initial purification of HIV-1. The technique is adaptable to almost any scale and may replace ultracentrifugation. Qualitatively, its main advantage over the latter method is the enhanced recovery of the gp120 in the virion fraction.

Autoradiography of 3H-alpha-fluoromethyl histidine in mice: correlation with the kidney histidine decarboxylase activity.

Tritium-alpha-fluoromethyl histidine (3H-alpha-FMH), designed as a kcat-inhibitor of mammalian histidine decarboxylase (EC 4.1.1.22), was administered intravenously in male and pregnant female mice of the NMRI strain and the distribution of tritium in the body recorded by whole-body and microautoradiography. The results showed penetration of radioactivity into most tissues within 5 min. after the injection. After 4 hrs the highest levels of radioactivity were present in the intestinal content and in the kidneys. In the pregnant animal there was also a high labelling of the foetal tissues. When whole-body sections were washed in TCA prior to the autoradiographic exposure to retain only protein-bound radioactivity, a distinct labelling pattern was seen in the kidneys of the pregnant female mice but not in those of the male mice. Microautoradiography of the kidneys showed that the cells involved were located within the proximal convoluted tubuli. In several mouse strains, including the NMRI, the activity of kidney histidine decarboxylase is low in the males but high in females during a transient period of pregnancy. Incorporation of tritium into kidney protein after treatment with 3H-alpha-FMH, was correlated to a loss in histidine decarboxylase activity. The isotopic labelling was confined mainly to a component which cofractionated with histidine decarboxylase in polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions. Our data indicate that the cells described above represent the location of kidney histidine decarboxylase.

Human immunodeficiency virus glycoproteins: lectin binding properties.

Recombinant glycoproteins derived from the HIV env gene are available and are under evaluation as antigens in vaccines against AIDS. The importance of the glycoconjugate structure for eliciting a protective immune response by these proteins is incompletely known. In this report we devise a method for the characterization of the glycoconjugate and demonstrate gross differences in the composition of the carbohydrate moiety in glycoproteins derived from the HIV env gene when expressed in different cell lines.

The use of aqueous two-phase systems to concentrate and purify bovine leukemia virus outer envelope protein gp51.

Enzootic bovine leucosis is a chronic lymphoproliferative disease of cattle. The causative agent, bovine leukemia virus (BLV), is related to the human retroviruses HTLV-I and -II. The external env-protein of BLV, a glycoprotein of 51 kDa, carries neutralizing epitopes and should be an essential component in a vaccine against the virus. Problems have been encountered with the concentration and purification of intact virions of BLV and other retroviruses. During centrifugation procedures the external env-proteins are to a great extent detached and consequently poorly recovered with the virion particles. Therefore, other methods are sought to obtain a high yield of the external glycoproteins. The use of two-phase systems based on water soluble polymers is described for the extraction of BLV-gp51 from culture medium. Several polymer systems were tested and the results showed that some were attractive for large scale application. The classical combination dextran-polyethylene glycol gave promising results; a partition coefficient of about 0.02 was obtained for the distribution of the gp51 between the top and combined inter- and bottom phases. In a single extraction step it was possible to obtain 45% of the glycoprotein in a small volume bottom phase and at the same time about 15-fold purified. That should be compared with a recovery of less than 20% with the conventional centrifugation procedures. It is concluded that extraction in phase systems based on water soluble polymers is a methodology well suited for the concentration and purification of BLV-gp51.

Concentration and purification of feline leukaemia virus (FeLV) and its outer envelope protein gp70 by aqueous two-phase systems.

The major protective antigens of retroviruses are considered to be their glycosylated envelope proteins. However, the methods commonly employed to enrich and purify virus from culture media such as pelleting and density-gradient centrifugation result in a low recovery of the viral external glycoproteins. This is an obvious drawback when the virus is intended for use in a vaccine. In search for alternative methods to concentrate and purify FeLV, we have attempted extraction in two-phase systems based on water-soluble polymers (Albertsson PA., Biochem Biophys Acta 1958; 27: 378-395). A variety of polymer systems was tested. Some of them seem attractive for a large-scale concentration of the virus and/or its glycoprotein. The distribution between the phases of two FeLV proteins, the outer envelope protein, gp70, and the gag protein, p27, was determined. With a system composed of dextran sulfate and polyvinyl alcohol both the glycoprotein and the gag protein were almost completely recovered in the lower phase which constitutes about 3% of the total system in weight. The two proteins were more than 40-fold purified as calculated on protein basis. The proteins can be extracted readily.

Monoclonal antibody directed detection of cytochrome P-450 (PCN) in human fetal liver.

Monoclonal antibodies (MAbs) raised to rat liver cytochrome P-450s induced by phenobarbital, 3-methylcholanthrene, and pregnenolone-16 alpha-carbonitrile were used to detect these epitope specific P-450s in human abortion fetuses 14-24 weeks of age. This was performed using a Western blot technique. In parallel, ECOD was determined in the same tissue specimens. Of seven different MAbs used MAb PCN 2-13-1/C2 was the only one that immunodetected a cytochrome P-450 band with Western blot analyses of human fetal liver microsomes. This band was consistently detected in all fetal liver specimens studied although the intensity varied among samples. No bands were detected in microsomal preparations from adrenal and renal tissues obtained from the same fetuses. The human adult liver microsomal specimens also contained a MAb PCN 2-13-1/C2 identified cytochrome P-450 band. ECOD activity was detected in all but one of the human fetal livers and varied between 0.22 and 47.5 pmol min-1 mg protein-1, as compared to 113 to 489 pmol min-1 mg protein-1 in human adult livers. In all of the fetuses except one the adrenal ECOD activity (0.63-37.0 pmol min-1 mg protein-1) exceeded that in the liver. The renal ECOD activities were, however, low. The hepatic and adrenal ECOD activities correlated with each other (r = 0.95). Although the ECOD activity is a function of several different P-450s there was also a correlation (r = 0.78) between the ECOD activity and the MAb immunodetected protein band intensity in Western blots of human fetal liver microsomes. The presence of a MAb PCN 2-13-1/C2 identified band in fetal liver microsomes may be indicative of a steroid-dependent effect in fetal life.

Human liver morphine UDP-glucuronyl transferase enantioselectivity and inhibition by opioid congeners and oxazepam.

1. Morphine uridine diphosphate glucuronyl transferase (UDP-GT) was studied in human liver microsomes. The (-)- and (+)-morphine enantiomers were used as substrates and inhibitors, such as oxazepam and various opioid congeners were employed to characterize the different glucuronidation pathways. The kinetics of the oxazepam inhibition were studied in the rat liver. 2. The overall glucuronidation of (+)-morphine was higher than that of (-)-morphine. The morphine congeners tested, potently inhibited the formation of (-)-morphine-3-glucuronide ((-)-M3G), except for normorphine and codeine. The formation of (+)-morphine-6-glucuronide [+)-M6G) was potently inhibited by only dextromethorphan and (+)-naloxone. All drugs except normorphine inhibited the formation of (+)-M3G by 18-50%. 3. The metabolism of (-)-morphine to (-)-M3G was more sensitive to oxazepam inhibition than the formation of (+)-M3G from (+)-morphine in the rat liver. 4. The glucuronidation of natural morphine is subject to in vitro interaction with oxazepam and several opiate drugs. Our study supports the theory of more than one type of UDP-GT being involved in morphine glucuronidation.

Morphine metabolism in mouse brain.

Morphine UDP-glucuronyltransferase activity was demonstrated in the brain of mice from recombinant inbred strains of the BXD series. The formation rate of morphine-3-glucuronide was about 4 fold higher in the progenitor DBA as compared to the C57BL strain.

Mammalian histidine decarboxylase: effect by protein kinase on mouse mastocytoma histidine decarboxylase.

Mouse mastocytoma histidine decarboxylase is decreased in activity when, in crude preparations, incubated with a cAMP-dependent protein kinase. This effect was not seen with purified preparations of the histidine decarboxylase (specific activity 7-13 mumol X mg-1 X h-1). Further, no incorporation of 32P from AT32P during incubation of this enzyme with the protein kinase could be demonstrated. Therefore, if protein phosphorylation operates in the regulation of the histidine decarboxylase, factors removed during the purification must be involved.

Effect of His-Phe, a competitive inhibitor of histidine decarboxylase, on gastric acid secretion in chronic gastric fistula rats: delay in acid secretion response to pentagastrin.

The dipeptide His-Phe, earlier shown to inhibit mammalian histidine decarboxylase, was analysed concerning its effect in vivo on pentagastrin-induced gastric acid secretion. Chronic gastric fistula rats were used and the effectors in saline were given as continuous i.v. infusions while acid was collected from the fistula. Addition of pentagastrin to the infusion solution resulted in an immediate increase in the acid output of the control runs. In the His-Phe experiments the dipeptide was introduced one hour before pentagastrin. A significant decrease in the acid output was obtained. This effect was optimal at a dose of about 6 mg/h and during the first few hours of the experiments. In spite of the continuous His-Phe infusion the acid secretion increased with time to the control values. These results are discussed in relation to preliminary observations on effects of alpha-fluoromethyl histidine on gastric acid secretion and the effect of this and His-Phe on gastric histamine content and histidine decarboxylase activity.

Changes in cadaverine and putrescine metabolism in the mouse kidney induced to growth by an anabolic steroid.

The formation of cadaverine and putrescine was studied in the kidneys of gonadectomized male mice stimulated to growth by nandrolone, an anabolic steroid with low androgenic activity. Administration of nandrolone resulted in an increased kidney weight and elevated activities of lysine and ornithine decarboxylase (assayed by measurement of the formation of 14CO2 from the 1-14C-labelled amino acids). The responses were dose and time dependent. The elevated enzyme activities were reflected by an increased endogenous kidney content of cadaverine and putrescine as well as in an increased urinary excretion of the diamines. Further, the kidney content and the urinary excretion of the polyamines spermidine and spermine were elevated on nandrolone treatment. Fractionation of kidney extracts on pore gradient electrophoresis revealed an apparent molecular weight of about 95 000 Daltons of the lysine decarboxylase as well as of the ornithine decarboxylase. On electrofocusing it was evident that both enzymes were present as more than one isoelectric form. However, the main form in both cases focused at a pH of about 5.0.

Lewis phenotype of erythrocytes and Leb-active glycolipid in serum of pregnant women.

In an attempt to provide an explanation for the previously reported effect of pregnancy on the Lewis phenotype of erythrocytes, the level of Leb-active glycolipid in serum was compared with the reactions of erythrocytes, using samples obtained from 73 nonpregnant women, 74 women at the time of delivery, and 2 women at weekly intervals during their pregnancy. In this Swedish population, the frequency of the Le(a--b-) blood group increased from 11% in nonpregnant women to 36% in women at the time of delivery. Among Le(a--b+) women of all ABO groups, those who were A1 most often became (Leb-) during pregnancy. The change in phenotype occurred as early as the 24th week of gestation; the Leb antigen was again detectable within 6 weeks after delivery. The concentration of Leb glycolipid in serum, as measured by radioimmunoassay, decreased only slightly during pregnancy. The repartition of glycolipids, secondary to the increased ratio of lipoprotein to red cell mass that occurs during pregnancy, may account for the relative lack of Lewis glycolipid on erythrocytes.