alpha-Amylase from developing embryos of the camel tick Hyalomma dromedarii

alpha-Amylase activity in the camel tick Hyalomma dromedarii was followed throughout embryogenesis. During purification of alpha-amylase III to homogeneity, ion exchange chromatography lead to four separate forms (termed I, II, III and IV). alpha-Amylase III with the highest specific activity was pure after chromatography on Sephacryl S-300. The molecular mass of alpha-amylase III was 106 kDa for the native enzyme, composed of two subunits of 43 and 66 kDa, respectively. alpha-Amylase had a value of 10 mg starch/ml. Varying alpha-amylase activity was detected when supplied with various substrates. alpha-Amylase III had a temperature optimum at 40 degrees C with heat stability up to 50 degrees C, and a pH optimum of 7.0. The enzyme activity was activated by CaCl2, MgCl2 and NaNO3, but not activated by NaCl, p-CMB, N-ethylmaleimide and iodoacetamide. EDTA and beta-mercaptoethanol strongly inhibited activity.     

Hypoxia induces differential expression of the integrin receptors alpha(vbeta3) and alpha(vbeta5) in cultured human endothelial cells

The integrins alpha(vbeta3) and alpha(vbeta5) have been implicated in playing a key role in the process of angiogenesis. In this study, we examined the effects of hypoxia, an important stimulus of angiogenesis, on the differential expression of the integrin subunits beta(3) and beta(5). beta(3) and beta(5) messenger RNA (mRNA), protein levels, and alpha(v)beta(3) function were measured in human umbilical vein endothelial cells (HUVECs) cultured under normoxic and hypoxic (1% O(2)) conditions. Cells exposed to hypoxic conditions for up to 72 h showed gradually increased mRNA levels of alpha(V) and beta(3), peaking at 24 h, in comparison with cells cultured under normoxic conditions. However, beta(5) mRNA levels, under the same hypoxic conditions, remained at a constant level. Results from Western blot analysis of HUVECs, cultured under hypoxic conditions, paralleled those of the Northern analysis with an increased expression in alpha(v)beta(3) protein levels, measured by blotting with LM609, evident by 24 h. alpha(v)beta(5) protein levels, measured by blotting with P1F6, did not change for up to 72 h. HUVECs cultured under hypoxic conditions for 72 h showed increased attachment to fibrinogen, an alpha(v)beta(3) mediated process. These results indicate that hypoxia can increase expression of alpha(v)beta(3) in HUVECs, and that hypoxic regulation of alpha(v)beta(3) may be an important regulator of angiogenesis.     

Influenza virus assembly: effect of influenza virus glycoproteins on the membrane association of M1 protein

Influenza virus matrix protein (M1), a critical protein required for virus assembly and budding, is presumed to interact with viral glycoproteins on the outer side and viral ribonucleoprotein on the inner side. However, because of the inherent membrane-binding ability of M1 protein, it has been difficult to demonstrate the specific interaction of M1 protein with hemagglutinin (HA) or neuraminidase (NA), the influenza virus envelope glycoproteins. Using Triton X-100 (TX-100) detergent treatment of membrane fractions and floatation in sucrose gradients, we observed that the membrane-bound M1 protein expressed alone or coexpressed with heterologous Sendai virus F was totally TX-100 soluble but the membrane-bound M1 protein expressed in the presence of HA and NA was predominantly detergent resistant and floated to the top of the density gradient. Furthermore, both the cytoplasmic tail and the transmembrane domain of HA facilitated binding of M1 to detergent-resistant membranes. Analysis of the membrane association of M1 in the early and late phases of the influenza virus infectious cycle revealed that the interaction of M1 with mature glycoproteins which associated with the detergent-resistant lipid rafts was responsible for the detergent resistance of membrane-bound M1. Immunofluorescence analysis by confocal microscopy also demonstrated that, in influenza virus-infected cells, a fraction of M1 protein colocalized with HA and associated with the HA in transit to the plasma membrane via the exocytic pathway. Similar results for colocalization were obtained when M1 and HA were coexpressed and HA transport was blocked by monensin treatment. These studies indicate that both HA and NA interact with influenza virus M1 and that HA associates with M1 via its cytoplasmic tail and transmembrane domain.     

Fot 1 Insertions in the Fusarium oxysporum f. sp. albedinis Genome Provide Diagnostic PCR Targets for Detection of the Date Palm Pathogen

Populations of Fusarium oxysporum f. sp. albedinis, the causal agent of Bayoud disease of date palm, are derivatives of a single clonal lineage and exhibit very similar Fot 1 hybridization patterns. In order to develop a sensitive diagnostic tool for F. oxysporum f. sp. albedinis detection, we isolated several DNA clones containing a copy of the transposable element Fot 1 from a genomic library of the date palm pathogen. Regions flanking the insertion sites were sequenced, and these sequences were used to design PCR primers that amplify the DNA regions at several Fot 1 insertion sites. When tested on a large sample of Fusarium isolates, including 286 F. oxysporum f. sp. albedinis isolates, 17 other special forms, nonpathogenic F. oxysporum isolates from palm grove soils, and 8 other Fusarium species, the primer pair TL3-FOA28 allowed amplification of a 400-bp fragment found only in F. oxysporum f. sp. albedinis. Sequence analysis showed that one of the Fot 1 copies was truncated, lacking 182 bp at its 3′ terminus. The primer pair BI03-FOA1 amplified a 204-bp fragment which overlapped the Fot 1 truncated copy and its 3′ site of insertion in the F. oxysporum f. sp. albedinis genome and identified 95% of the isolates. The primer pairs BIO3-FOA1 and TL3-FOA28 used in PCR assays thus provide a useful diagnostic tool for F. oxysporum f. sp. albedinis isolates.     

Acyl chain-length asymmetry alters the interfacial elastic interactions of phosphatidylcholines.

Phosphatidylcholines (PCs) with stearoyl (18:0) sn-1 chains and variable-length, saturated sn-2 acyl chains were synthesized and investigated using a Langmuir-type film balance. Surface pressure was monitored as a function of lipid molecular area at various constant temperatures between 10 degrees C and 30 degrees C. Over this temperature range, 18:0-10:0 PC displayed only liquid-expanded behavior. In contrast, di-14:0 PC displayed liquid-expanded behavior at 24 degrees C and 30 degrees C, but two-dimensional phase transitions were evident at 20 degrees C, 15 degrees C, and 10 degrees C. The average molecular area of 18:0-10:0 PC was larger than that of liquid-expanded di-14:0 PC at equivalent surface pressures, and the shapes of their liquid expanded isotherms were somewhat dissimilar. Analysis of the elastic moduli of area compressibility (Cs(-1)) as a function of molecular area revealed shallower slopes in the semilog plots of 18:0-10:0 PC compared to di-14:0 PC. At membrane-like surface pressures (e.g., 30 mN/m), 18:0-10:0 PC was 20-25% more elastic (in an in-plane sense) than di-14:0 PC. Other PCs with varying degrees of chain-length asymmetry (18:0-8:0 PC, 18:0-12:0 PC, 18:0-14:0 PC, 18:0-16:0 PC) were also investigated to determine whether the higher in-plane elasticity of fluid-phase 18:0-10:0 PC is a common feature of PCs with asymmetrical chain lengths. Two-dimensional phase transitions in 18:0-14:0 PC and 18:0-16:0 PC prevented meaningful comparison with other fluid-phase PCs at 30 mN/m. However, the Cs(-1) values for fluid-phase 18:0-8:0 PC and 18:0-12:0 PC were similar to that of 18:0-10:0 PC (85-90 mN/m). These values showed chain-length asymmetrical PCs to have 20-25% greater in-plane elasticity than fluid-phase PCs with mono- or diunsaturated acyl chains.     

Do random fluctuations in daily ration affect the growth rate of juvenile three-spined sticklebacks?

The effect of random fluctuations in daily ration on the growth performance of individual juvenile three-spined sticklebacks Gasterosteus aculeatus was studied in experiments lasting 21 days at 14°C and a photoperiod of 10L:14D. Two mean ration levels were used: a maintenance ration of 2%, and a high ration of 6% of initial body weight per day. For experimental fish, the daily ration varied randomly about the required mean value with a coefficient of variation of 33%. The controls received a constant daily ration. The experiment was replicated in winter (Dec.-Jan.) and spring (Mar.-Apr.). At a given ration, there was no significant difference between the specific growth rates of fish receiving constant or varying ration. Neither the final dry weight, final body water content nor final lipid content (% dry weight) differed significantly. As expected, ration had a significant effect on growth rate, final dry weight and body lipid and water content. There was a significant difference in mean growth rate between the winter and spring replicates. The growth rates observed in these experiments were compared with those predicted from a previously described empirical growth model developed for sticklebacks fed constant rations. The model underestimated mean growth rates.     

Phosphorus Limitation of Heterotrophic Biofilms from the Fraser River, British Columbia, and the Effect of Pulp Mill Effluent

Abstract Experiments were conducted to determine the limiting nutrient and the effect of pulp mill effluent (PME) on heterotrophic biofilms in the Fraser River, a northern, temperate river in British Columbia, Canada. Biofilms were cultured by irrigating flow cells with Fraser River water alone (control), or river water amended with glucose; ammonium; phosphate; glucose, ammonium, and phosphate combined; or 3% PME. Phosphorus increased bacterial growth in the biofilm, which was rapidly transferred to protists, and reduced the amount of extracellular polymeric substances accumulated by biofilm bacteria. This work demonstrates phosphorus limitation of biofilm bacteria in a major river system and emphasizes the importance of nutrients in industrial discharges. Received: 7 October 1997; Accepted: 8 January 1998     

Human Immunodeficiency Virus (HIV) Envelope Binds to CXCR4 Independently of CD4, and Binding Can Be Enhanced by Interaction with Soluble CD4 or by HIV Envelope Deglycosylation

Chemokine receptor CXCR4 (also known as LESTR and fusin) has been shown to function as a coreceptor for T-cell-tropic strains of human immunodeficiency virus type 1 (HIV-1). We have developed a binding assay to show that HIV envelope (Env) can interact with CXCR4 independently of CD4 but that this binding is markedly enhanced by the previous interaction of Env with soluble CD4. We also show that nonglycosylated HIV-1_SF-2 gp120 or sodium metaperiodate-treated oligomeric gp160 from HIV-1₄₅₁ bound much more readily to CXCR4 than their counterparts with intact carbohydrate residues did.In the recent past, several members of the family of chemokine receptors have been identified as cofactors for human immunodeficiency virus type 1 (HIV-1) entry (1, 6, 8, 10). Specifically, CCR5 (as well as CCR3 and CCR2b in some instances) has been shown to mediate entry of viruses characterized as macrophage tropic or dual tropic (1, 5–8), while CXCR4 has been shown to mediate entry of T-cell-tropic or dual-tropic strains (7, 10). While several ligands have been found for CCR5, CXC chemokine stromal derivative factor (SDF1) remains the only known ligand for CXCR4 (4, 24). Coimmunoprecipitation studies have shown that HIV-1 Env from T-cell-tropic strains forms a complex with CD4 and CXCR4 (18), but the nature of the binding events leading to the formation of this complex and the possibility of a direct interaction between HIV Env and CXCR4 remained speculative. Data from Hesselgesser et al. (15) have more recently shown that gp120 from the T-cell-tropic strains IIIB or BRU was able to compete with SDF1 for binding to CXCR4 in hNT cells (a neuronal CD4-negative cell line), indicating the possibility of a direct interaction between CXCR4 and gp120, but no information was presented on the relevance of the interaction with CD4. Other data have shown that gp120 from macrophage-tropic strains of HIV might be able to bind directly to CCR5 and that the affinity for binding between the two molecules can be increased significantly by the presence of soluble CD4 (sCD4) (34), although this effect could not be reproduced by a different group (32).We have performed the following studies to determine if HIV Env binds to CXCR4 independently of CD4 and, if so, what would be the effect of previous binding of HIV Env to sCD4.

CD4-independent binding of HIV Env to CXCR4.

The phenotypes of the T-cell lines CEM-SS and Jurkat 25 (J25) were evaluated with respect to surface expression of both CD4 and CXCR4. J25 clone 22F6 cells (3, 21) were grown in complete medium (RPMI 1640, 2% penicillin-streptomycin, 2% l-glutamine; BioWhittaker, Walkersville, Md.) containing heat-inactivated 10% fetal calf serum at 37°C in a 5% CO₂ atmosphere. CEM-SS is a T-cell line that was obtained from the AIDS Research and Reference Reagent Program and maintained in complete medium. CEM-SS cells were derived from a human lymphoblastoid tumor (22, 23). Commercial monoclonal antibody (MAb) to CD4 (mouse immunoglobulin G2a [IgG2a], clone S3.5), fluorescein isothiocyanate (FITC) labeled, and the necessary isotypic controls were obtained from Caltag Laboratories (San Francisco, Calif.). Mouse MAb 12G5 against CXCR4 was raised in BALB/c mice and has been described previously (9). Goat anti-mouse IgG–FITC was purchased from Becton Dickinson (San Jose, Calif.). Flow cytometric analysis was performed on a Becton Dickinson FACScan cytometer equipped with a 15-mW argon laser emitting at 488 nm. Dead cells were detected on the basis of their scatter and eliminated from the analysis. Live cells (10,000) were analyzed for each marker. CXCR4 surface expression was determined by washing the cells taken in logarithmic growth phase with phosphate-buffered saline (PBS) containing 1% horse serum and incubating them with 10 μl of 12G5 antibody/100 μl (0.16 mg/ml) at 4°C for 30 min. The cells were then washed again in PBS, and a secondary goat anti-mouse IgG–FITC (Becton Dickinson) was incubated with the cells for another 30 min at 4°C. Finally, the cells were washed with PBS and fixed with 2% paraformaldehyde. As a control, equal amounts of mouse IgG2a (the same isotype as 12G5) were used. Both cell lines expressed significant levels of CXCR4 on their surfaces (Fig. ​(Fig.1),1), but only CEM-SS had measurable levels of surface CD4. This characteristic of the phenotype of J25 cells, with respect to CD4 expression, has been reported before (3). To assess binding of HIV Env to CXCR4, the following binding assay was developed. Oligomeric gp160 (ogp160) was purified from cell cultures (obtained from T. C. Van Cott (Henry M. Jackson Foundation, Rockville, Md.) infected with HIV₄₅₁ (17). The cells were washed once with PBS and then incubated with ogp160 for 1 h at 37°C in RPMI medium. The cells were washed again in PBS and incubated with 10 μg of human MAb 1331A [IgG3(λ)]/ml, which is specific for the C terminus of gp120 (i.e., amino acids 510 to 516 of HIV_LAI), or with a human MAb against p24 (MAb 71-31) as a control (12) for 30 min at 4°C. The secondary antibody was a goat anti-human IgG phycoerythrin labeled (Caltag). The cells were fixed in 2% paraformaldehyde, and the fluorescence intensity was determined by flow cytometry. Background was obtained by adding MAb 1331 and goat anti-human IgG, phycoerythrin labeled, to the cells in the absence of ogp160. The results of the binding assay with ogp160 from HIV₄₅₁ and both cell lines are shown in Fig. ​Fig.2A.2A. By using the high-affinity human MAb 1331A against the C-terminal region of gp120, our assay was able to detect significant binding of the ogp160 molecule to the surfaces of both cell lines even at concentrations of only 88 nM. The very high relative affinity of MAb 1331A for the gp120 molecule appears to be critical to demonstrate this interaction, as other antibodies with lower relative affinities for gp120 were incapable of detecting this low-level binding (data not shown). The binding of ogp160 to the CD4-expressing CEM-SS cells was several orders of magnitude higher than that to the J25 cells. To prove the specificity of the binding assay for CXCR4, a synthetic form of SDF1 was produced and tested for its ability to block infection by the HIV-1 strain NL_4-3 in HeLa CD4-positive long terminal repeat (LTR)-LacZ cells. These data have been published elsewhere (2). SDF1 synthesis and composition have been described previously (24). Exposure of J25 cells to SDF1 was shown to produce a dose-dependent blockage of the binding of ogp160 to the surfaces of the J25 cells (Fig. ​(Fig.2B),2B), indicating the specific nature of the assay. Open in a separate windowFIG. 1Phenotype analysis of CEM-SS and J25 cell lines. Thin solid line, background; thick solid line, CD4; dashed line, CXCR4.Open in a separate windowFIG. 2(A) Binding of ogp160 from HIV₄₅₁ to the surfaces of CEM-SS or J25 cells. Fluorescence intensity is expressed on a logarithmic scale on the x axis, with each line representing one-half log. Concentrations of ogp160 are shown at the right of each graph. The experiments were done in duplicate to ensure consistency of results. (B) Effect of RANTES (250 nM) or increasing amounts of SDF1 (up to 250 nM) on binding of ogp160 (355 nM) to J25 cells. The results are expressed as mean channel fluorescence. Experiments were repeated twice to ensure consistency of results.To further test the fact that HIV Env binding to CXCR4 could occur independently of CD4, and to evaluate the effect of prior binding of Env to sCD4, the following experiments were performed. We preexposed CEM-SS as well as J25 cells to either the anti-CD4 antibody Leu3a (Becton Dickinson), which blocks the CD4 binding domain of HIV Env, or OKT4 (Ortho Diagnostics, Costa Mesa, Calif.), which does not block binding of HIV Env to CD4. The cells were then tested for their ability to bind ogp160 to their surfaces. As shown in Fig. ​Fig.3,3, OKT4 had no significant effect on the binding of ogp160 to either CEM-SS or J25 cells while Leu3a readily inhibited binding of ogp160 to CEM-SS cells but had no such effect on J25 cells. Furthermore, when ogp160 was allowed to react in advance with recombinant sCD4 produced in CHO cells (Intracel, Issaquah, Wash.) for 30 min at 4°C at a concentration of 1 μg/ml, we were able to show a clear decrease in the surface binding of ogp160 to CEM-SS cells while the opposite, an obvious enhancement in surface binding, was demonstrated for J25 cells (Fig. ​(Fig.3).3). Open in a separate windowFIG. 3Binding of ogp160 to CEM-SS or J25 cells after exposure of the cells to the anti-CD4 antibodies Leu3a (thin solid lines), OKT4 (dotted lines), or a combination of ogp160 with sCD4 (dashed lines). The shaded areas represent background. The thick solid lines represent binding in the absence of antibodies or sCD4. The experiments were performed in quadruplicate with similar results. Mean channel fluorescence is represented on the x axis.Taken together, these data indicate that HIV Env can bind to CXCR4 independently of CD4. On the other hand, prior interaction of HIV Env with CD4 results in a clear increase in the binding of HIV Env to CXCR4.

Relevance of the glycosylation state of HIV Env in binding to CXCR4.

The binding of HIV Env to CD4 is dependent on the appropriate conformation of the Env molecule (27), which can be significantly altered by changes in its carbohydrate content. We next tested the hypothesis that alterations in the carbohydrate moieties of Env would affect its binding to CXCR4. To do so, we used the gp120 molecule from HIV_SF2, produced in CHO cells, and its counterpart, nonglycosylated HIV_SF2 Env 2-3, produced in yeast strain 2150, and tested both in the binding assay with CEM-SS or J25 cells. HIV_SF-2 gp120 and its nonglycosylated counterpart, Env 2-3, were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, from Kathelyn Steimer, Chiron Corp. (13, 14, 19, 26, 29–31). The results are shown in Fig. ​Fig.4.4. As expected, nonglycosylated HIV_SF2 Env 2-3 bound to the surfaces of the CEM-SS cells to a lesser extent than did HIV_SF2 gp120. On the other hand, and unexpectedly, nonglycosylated HIV_SF2 Env 2-3 bound much more readily to the surfaces of the J25 cells than its glycosylated counterpart, HIV_SF-2 gp120, even when used at equal molar concentrations. To determine whether these findings could be generalized to other Env molecules that lacked intact carbohydrate molecules, we treated ogp160 with sodium metaperiodate. ogp160 from HIV₄₅₁ at 1.25 μg/ml was treated with sodium metaperiodate (Sigma, St. Louis, Mo.) in acetate buffer for 2 h at 4°C in the dark (33). The cells to be tested had been treated previously with 1% glycine (Sigma) for 30 min at 37°C. Such treatment results in the oxidation and cleavage of the carbohydrate hydroxyl groups without affecting the structure of the polypeptide chains (33). Nonspecific binding by the resulting aldehyde groups was prevented by blocking the target cells beforehand with 1% glycine. The results are shown in Fig. ​Fig.4.4. Sodium metaperiodate treatment of ogp160 resulted in a marked inhibition of the binding of ogp160 to the surfaces of the CEM-SS cells. In contrast, sodium metaperiodate treatment of ogp160 resulted in a very clear increase in the binding of HIV Env to the surfaces of the J25 cells. The preexposure of CEM-SS cells to SDF1 did not significantly affect the binding of ogp160 or sodium metaperiodate-treated ogp160. On the other hand, preexposure of J25 cells to 250 nM SDF1 resulted in a marked decrease in binding of both ogp160 and sodium metaperiodate-treated ogp160. These data indicate the specificity of the interaction of the deglycosylated form of ogp160 with CXCR4. The results of these experiments suggest that the alteration in the carbohydrate content of the HIV Env molecules resulted in a better exposure of the epitopes involved in gp120 binding to CXCR4. Open in a separate windowFIG. 4Binding of HIV_SF-2 gp120 or the nonglycosylated form, HIV_SF-2 Env 2-3 (Non-glyc SF-2 gp120), to CEM-SS or J25 cells. The concentration was 355 nM for both. The binding of ogp160 and sodium metaperiodate-treated ogp160 (De-glyc ogp160), each at a concentration of 355 nM, to CEM-SS or J25 cells is also shown. The two right-hand bars in each graph show results for cells preexposed to SDF1 at 150 nM. The results are expressed as mean channel fluorescence. The experiments were performed in duplicate with similar results.The understanding of the underlying mechanisms by which HIV Env, CD4, and the newly discovered HIV coreceptors interact to mediate viral entry remains a very significant issue. The way that HIV Env and CD4 interact is well established (28), and some information exists about the interaction between HIV Env, CCR5, and CD4 (34). In this paper we have shown that HIV Env is able to interact in a CD4-independent manner with CXCR4. Still, the extent of such interaction was clearly lower than that of the sCD4-HIV Env complex and CXCR4. This effect of sCD4 seems to be consistent with the observation that the complexing of this molecule with HIV Env from the strains JRFL or BAL resulted in a significant increase in the affinity of HIV Env for CCR5 (34). We speculate that this interaction between sCD4 and HIV Env results in a conformational change that exposes the binding epitopes in HIV Env relevant for binding to CXCR4, as it does with other gp120 epitopes (16). A different scenario would involve a change in both molecules, resulting in a newly formed common binding epitope. This second alternative seems less likely given our data showing CD4-independent binding of HIV Env to CXCR4, as well as previous data showing the existence of HIV strains capable of CD4-independent entry into target cells (9, 15).The gp120 molecule from HIV contains 20 potential N-linked glycosylation sites, with N-linked glycans representing at least 50% of the molecular mass. Their role in CD4 binding has been studied extensively, although some of the results remain somewhat controversial. Most of the available data seem to indicate that complete lack of glycosylation completely (20), or at least partially (25), inhibits HIV Env binding to CD4. Also, enzymatic manipulation of the carbohydrate residues results in a significant decrease but not in complete abrogation of the binding of HIV Env to CD4 (11, 20, 25). It was therefore somewhat unexpected to find that the nonglycosylated form, as well as the sodium metaperiodate-treated form, of HIV Env was able to bind in such an enhanced way to CXCR4. This would appear to reinforce the concept of the existence of a binding epitope for CXCR4 within HIV Env which is different from the one for CD4. It also suggests that the changes occurring as a consequence of the manipulation of the carbohydrate residues likely result in a better exposure of the CXCR4 binding epitope(s) within the HIV Env molecule.In summary, we have shown that HIV Env can interact with CXCR4 in a CD4-independent manner. We have also shown how the interaction of CD4 with HIV Env results in a significant increase in the binding of the latter to CXCR4 and how the alterations in the carbohydrate composition of the HIV Env molecule affect its binding to CXCR4. The complete definition of these interactions may result in novel approaches to protect against cell infection by HIV.