Role of the membrane anchoring and cytoplasmic domains in intracellular transport and localization of viral glycoproteins.

The role of the transmembrane and the cytoplasmic regions of viral glycoproteins namely, the envelope glycoprotein gD of herpes simplex virus and the integral membrane glycoprotein E3-11.6 K of the nonenveloped adenovirus that are localized in the nuclear envelope has been studied. Chimeras of the cell surface glycoprotein G of vesicular stomatitis virus containing the transmembrane and (or) the cytoplasmic-tail domains of either herpes simplex virus gD or adenovirus E3-11.6 K protein were examined for their intracellular transport and localization. The results show that hybrids containing the membrane anchoring and (or) the cytoplasmic tail domains of either herpes simplex virus gD or adenovirus E3-11.6 K glycoprotein were localized in the nuclear envelope as well as in the endoplasmic reticulum and the Golgi complex. These results suggest that the membrane anchoring and the cytoplasmic domains of herpes simplex virus glycoproteins gD, as well as the adenovirus integral membrane protein E3-11.6 K, were necessary for localization in the nuclear envelope and could influence retention in the endoplasmic reticulum and the Golgi complex.     

Induction and intracellular localization of the 80-kilodalton heat-shock protein of Neurospora crassa.

The most abundant heat-shock protein of Neurospora crassa is a multimeric glycoprotein of 80-kilodaltons (i.e., HSP80), induced strongly by hyperthermia and at a lower level by sodium arsenite, ethanol, and carbon source depletion. Immunoelectron microscopy, using indirect immunogold labelling demonstrated that HSP80 was undetectable in mycelium cultured at the normal growth temperature of 28 degrees C, but it appeared rapidly following the commencement of heat-shock treatment at 48 degrees C. HSP80, visualized by the gold label, was observed almost exclusively in the cytoplasm, exhibiting a uniform distribution. Association of this protein with cellular membranes and (or) targeting to a particular subcellular compartment or organelle was not apparent.     

Requirements of cyclin a for mitosis are independent of its subcellular localization

Cyclin A (CycA), the only essential mitotic cyclin in Drosophila, is cytoplasmic during interphase and accumulates in the nucleus during prophase. We show that interphase localization is mediated by Leptomycin B (LMB)-sensitive nuclear export. This is a feature shared with human CyclinB1, and it is assumed that nuclear accumulation is necessary for mitotic entry. Here, we tested if the unique mitotic function of CycA requires nuclear accumulation. We fused subcellular localization signals to CycA and tested their mitotic capability. Surprisingly, nuclear accumulation was not required, and even a membrane-tethered form of CycA was able to induce mitosis. We noted that Cyclin B (CycB) protein disappears prematurely in CycA mutants, reminiscent of rca1 mutants. Rca1 is an inhibitor of Fizzy-related-APC/C activity, and in rca1 mutants, mitotic cyclins are degraded in G2 of the 16(th) embryonic cell cycle. Overexpression of Rca1 can restore mitosis in CycA mutants, indicating that the mitotic failure of CycA mutants is caused by premature activation of the APC/C. The essential mitotic function of CycA is therefore not the activation of numerous mitotic substrates by Cdk1-dependent phosphorylation. Rather, CycA-dependent kinase activity is required to inhibit one inhibitor of mitosis, the Fzr protein.     

Skin immune responses to peptide and protein antigen are TLR4 independent

Little is known about the innate immune mechanisms regulating adaptive immune responses elicited through the skin. Tissue injury is postulated to liberate Toll like receptor 4 (TLR4) ligands. In this study, we determined whether TLR4 signaling modulates the response to epidermal injury induced by tape stripping (TS) and whether it alters humoral and cellular immune responses generated through epicutaneous immunization with peptide+cholera toxin (CT). The combined use of cholera toxin and TS with antigen promoted optimal antigen-specific CD4(+) and CD8(+) T cell proliferation in Balb/c and C57BL/6 mice, respectively. TLR4 mutant mice had similar T cell responses to wild type mice. Further, OVA-protein specific IgG, IgG(1), IgG(2a), and IgE titers were similar in wild type and TLR4 mutant mice. Thus, TLR4 signaling was not required for the generation of epicutaneous T cell or antibody mediated immune responses and did not alter the quality of the immune responses elicited.     

Isolation and characterization of the two major intracellular Glut4 storage compartments.

In rat adipose cells, intracellular Glut4 resides in two distinct vesicular populations one of which contains cellugyrin whereas another lacks this protein (Kupriyanova, T. A., and Kandror, K. V. (2000) J. Biol. Chem. 275, 36263--36268). Cell surface biotinylated MPR and (125)I-labeled transferrin are accumulated in cellugyrin-positive vesicles and to a lesser extent in cellugyrin-negative vesicles. An average cellugyrin-positive vesicle carries not more than one molecule of either Glut4, insulin-responsive aminopeptidase (IRAP), or transferrin receptor (TfR), whereas cellugyrin-negative vesicles contain five to six molecules of Glut4, more than 10 molecules of IRAP, and still one molecule of TfR per vesicle. Cellugyrin-negative vesicles are translocated to the cell surface after insulin stimulation, whereas cellugyrin-positive vesicles maintain intracellular localization both in the absence and in the presence of insulin and, therefore, may be involved in interendosomal protein transport. Both cellugyrin-positive and cellugyrin-negative vesicles are present in extracts of non-homogenized cells and therefore may represent the major form of Glut4 storage in vivo.     

Mutations in two independent genes lead to suppression of the shoot apical meristem in maize

The shoot apical meristem (SAM), initially formed during embryogenesis, gives rise to the aboveground portion of the maize (Zea mays) plant. The shootless phenotype (sml) described here is caused by disruption of SAM formation due to the synergistic interaction of mutations at two genetic loci. Seedlings must be homozygous for both sml (shootmeristemless), and the unlinked dgr (distorted growth) loci for a SAM-less phenotype to occur. Seedlings mutant only for sml are impaired in their morphogenesis to different extents, whereas the dgr mutation alone does not have a recognisable phenotype. Thus, dgr can be envisaged as being a dominant modifier of sml and the 12 (normal):3 (distorted growth):1 (shoot meristemless) segregation observed in the F(2) of the double heterozygote is the result of the interaction between the sml and dgr genes. Other segregation patterns were also observed in the F(2), suggesting instability of the dgr gene. Efforts to rescue mutant embryos by growth on media enriched with hormones have been unsuccessful so far. However, mutant roots grow normally on medium supplemented with kinetin at a concentration that suppresses wild-type root elongation, suggesting possible involvement of the mutant in the reception or transduction of the kinetin signal or transport of the hormone. The shootless mutant appears to be a valuable tool with which to investigate the organization of the shoot meristem in monocots as well as a means to assay the origins and relationships between organs such as the scutellum, the coleoptile, and leaves that are initiated during the embryogenic process.     

The chemokinetic and chemotactic behavior of Rhodobacter sphaeroides: two independent responses.

Rhodobacter sphaeroides exhibits two behavioral responses when exposed to some compounds: (i) a chemotactic response that results in accumulation and (ii) a sustained increase in swimming speed. This latter chemokinetic response occurs without any apparent long-term change in the size of the electrochemical proton gradient. The results presented here show that the chemokinetic response is separate from the chemotactic response, although some compounds can induce both responses. Compounds that caused only chemokinesis induced a sustained increase in the rate of flagellar rotation, but chemoeffectors which were also chemotactic caused an additional short-term change in both the stopping frequency and the duration of stops and runs. The response to a change in chemoattractant concentration was a transient increase in the stopping frequency when the concentration was reduced, with adaptation taking between 10 and 60 s. There was also a decrease in the stopping frequency when the concentration was increased, but adaptation took up to 60 min. The nature and duration of both the chemotactic and chemokinetic responses were concentration dependent. Weak organic acids elicited the strongest chemokinetic responses, and although many also caused chemotaxis, there were conditions under which chemokinesis occurred in the absence of chemotaxis. The transportable succinate analog malonate caused chemokinesis but not chemotaxis, as did acetate when added to a mutant able to transport but not grow on acetate. Chemokinesis also occurred after incubation with arsenate, conditions under which chemotaxis was lost, indicating that phosphorylation at some level may have a role in chemotaxis. Aspartate was the only chemoattractant amino acid to cause chemokinesis. Glutamate caused chemotaxis but not chemokinesis. These data suggest that (i) chemotaxis and chemokinesis are separate responses, (ii) metabolism is required for chemotaxis but not chemokinesis, (iii) a reduction in chemoattractant concentration may cause the major chemotactic signal, and (iv) a specific transport pathway(s) may be involved in chemokinetic signalling in R. sphaeroides.     

C. elegans tubby regulates life span and fat storage by two independent mechanisms

In C. elegans, similar to in mammals, mutations in the tubby homolog, tub-1, promote increased fat deposition. Here, we show that mutation in tub-1 also leads to life span extension dependent on daf-16/FOXO. Interestingly, function of tub-1 in fat storage is independent of daf-16. A yeast two-hybrid screen identified a novel TUB-1 interaction partner (RBG-3); a RabGTPase-activating protein. Both TUB-1 and RBG-3 localize to overlapping neurons. Importantly, RNAi of rbg-3 decreases fat deposition in tub-1 mutants but does not affect life span. We demonstrate that TUB-1 is expressed in ciliated neurons and undergoes both dendritic and ciliary transport. Additionally, tub-1 mutants are chemotaxis defective. Thus, tub-1 may regulate fat storage either by modulating transport, sensing, or responding to signals in ciliated neurons. Taken together, we define a role for tub-1 in regulation of life span and show that tub-1 regulates life span and fat storage by two independent mechanisms.     

Skeletal muscle capillary responses to insulin are abnormal in late-stage diabetes and are restored by angiotensin-converting enzyme inhibition

Acute physiological hyperinsulinemia increases skeletal muscle capillary blood volume (CBV), presumably to augment glucose and insulin delivery. We hypothesized that insulin-mediated changes in CBV are impaired in type 2 diabetes mellitus (DM) and are improved by angiotensin-converting enzyme inhibition (ACE-I). Zucker obese diabetic rats (ZDF, n = 18) and control rats (n = 9) were studied at 20 wk of age. One-half of the ZDF rats were treated with quinapril (ZDF-Q) for 15 wk prior to study. CBV and capillary flow in hindlimb skeletal muscle were measured by contrast-enhanced ultrasound (CEU) at baseline and at 30 and 120 min after initiation of a euglycemic hyperinsulinemic clamp (3 mU.min(-1).kg(-1)). At baseline, ZDF and ZDF-Q rats were hyperglycemic and hyperinsulinemic vs. controls. Glucose utilization in ZDF rats was 60-70% lower (P < 0.05) than in controls after 30 and 120 min of hyperinsulinemia. In ZDF-Q rats, glucose utilization was impaired at 30 min but similar to controls at 120 min. Basal CBV was lower in ZDF and ZDF-Q rats compared with controls (13 +/- 4, 7 +/- 3, and 9 +/- 2 U, respectively). With hyperinsulinemia, CBV increased by about twofold in control animals at 30 and 120 min, did not change in ZDF animals, and increased in ZDF-Q animals only at 120 min to a level similar to controls. Anatomic capillary density on immunohistology was not different between groups. We conclude that insulin-mediated capillary recruitment in skeletal muscle, which participates in glucose utilization, is impaired in animals with DM and can be partially reversed by chronic ACE-I therapy.     

Identification of two intracellular mechanisms leading to reduced expression of oncoretrovirus envelope glycoproteins at the cell surface

All retrovirus glycoproteins have a cytoplasmic domain that plays several roles in virus replication. We have determined whether and how the cytoplasmic domains of oncoretrovirus glycoproteins modulate their intracellular trafficking, by using chimeric proteins that combined the alpha-chain of the interleukin-2 receptor with the glycoprotein cytoplasmic domains of five oncoretroviruses: human T-cell leukemia virus type 1 (HTLV-1), Rous sarcoma virus (RSV), bovine leukemia virus (BLV), murine leukemia virus (MuLV), and Mason-Pfizer monkey virus (MPMV). All of these proteins were synthesized and matured in the same way as a control protein with no retrovirus cytoplasmic domain. However, the amounts of all chimeric proteins at the cell surface were smaller than that of the control protein. The protein appearing at and leaving the cell surface and endocytosis were measured in stable transfectants expressing the chimera. We identified two groups of proteins which followed distinct intracellular pathways. Group 1 included chimeric proteins that reached the cell surface normally but were rapidly endocytosed afterwards. This group included the chimeric proteins with HTLV-1, RSV, and BLV cytoplasmic domains. Group 2 included chimeric proteins that were not detected at the cell surface, despite normal intracellular concentrations, and were accumulated in the Golgi complex. This group included the chimeric proteins with MuLV and MPMV cytoplasmic domains. Finally, we verified that the MuLV envelope glycoproteins behaved in the same way as the corresponding chimeras. These results indicate that retroviruses have evolved two distinct mechanisms to ensure a similar biological feature: low concentrations of their glycoproteins at the cell surface.     

Ventilatory and cardiac responses to hypoxia at submaximal exercise are independent of altitude and exercise intensity

The hypoxic exercise test combining a 4,800-m simulated altitude and a cycloergometer exercise at 30% of normoxic maximal aerobic power (MAP) is used to evaluate the individual chemosensitivity to hypoxia in submaximal exercise conditions. This test allows the calculation of three main parameters: the decrease in arterial oxygen saturation induced by hypoxia at exercise (ΔSa(e)) and the ventilatory (HVR(e)) and cardiac (HCR(e)) responses to hypoxia at exercise. The aim of this study was to determine the influence of altitude and exercise intensity on the values of ΔSa(e), HVR(e), and HCR(e). Nine subjects performed hypoxic tests at three simulated altitudes (3,000 m, 4,000 m, and 4,800 m) and three exercise intensities (20%, 30%, and 40% MAP). ΔSa(e) increased with altitude and was higher for 40% MAP than for 20% or 30% (P < 0.05). For a constant heart rate, the loss in power output induced by hypoxia, relative to ΔSa(e), was independent of altitude (4,000-4,800 m) and of exercise intensity. HVR(e) and HCR(e) were independent of altitude (3,000-4,800 m) and exercise intensity (20%-40% MAP). Moreover, the intraindividual variability of responses to hypoxia was lower during moderate exercise than at rest (P < 0.05 to P < 0.001). Therefore, we suggest that HVR(e) and HCR(e) are invariant parameters that can be considered as intrinsic physiological characteristics of chemosensitivity to hypoxia.     

Accumulation, subcellular localization and ecophysiological responses to copper stress in two Daucus carota L. populations

Copper accumulation, subcellular localization and ecophysiological responses to excess copper were investigated using pot culture experiments with two Daucus carota L. populations, from a copper mine and an uncontaminated field site, respectively. Significant differences of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) concentrations and antioxidant enzyme [superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX)] activities of leaves under Cu treatment were observed between the two populations. At high Cu concentrations (400 and 800 mg kg⁻¹), a significant increase in contents of MDA and H₂O₂ but a significant decrease in activities of SOD, CAT and APX were observed in uncontaminated population. Contrarily, the population from copper mine maintained a lower level of MDA and H₂O₂ but higher activities of SOD, CAT and APX. Copper accumulation in roots and shoots increased significantly with the increase of copper concentrations in soils in the two populations. No significant difference of the total Cu in roots and shoots was found between the two populations at same copper treatment. There were also no striking differences of cell wall-bound Cu and protoplasts Cu of leaves between the two populations. The difference was that Cu concentration in vacuoles of leaves was 1.5-fold higher in contaminated site (CS) population than in uncontaminated site population. Hence, more efficient vacuolar sequestration for Cu and maintaining high activities of SOD, CAT and APX in the CS population played an important role in maintaining high Cu tolerance.     

Synthesis and localization of two sulphated glycoproteins associated with basement membranes and the extracellular matrix

Two sulphated glycoproteins (sgps) of apparent molecular weight (Mr) 180,000 and 150,000, are synthesized by murine PYS and PF HR9 parietal endoderm and Swiss 3T3 cells. The Mr 150,000 sgp has a similar chemical structure to the sulphated glycoprotein, C, synthesized and laid down in Reichert's membrane by mouse embryo parietal endoderm cells (Hogan, B. L.M., A. Taylor, and A.R. Cooper, 1982, Dev. Biol., 90:210-214). Both the Mr 180,000 and 150,000 sgps are deposited in the detergent- insoluble matrix of cultured cells, but they do not apparently undergo any disulphide-dependent intermolecular interactions and are not precursors or products of each other. They contain asparagine-linked oligosaccharides, but these are not the exclusive sites of sulphate labeling. Antiserum raised against the Mr 150,000 sgp C of Reichert's membranes has been used in an immunohistochemical analysis of rat skin. In early foetal and adult skin the antigen is present only in basement membranes, but transiently before and after birth it is also found throughout the upper part of the dermis. This suggests that 150,000 sgp C is at times synthesized by nonepithelial cells and contributes to the extracellular matrix of mesenchymal tissues.     

Intrinsic mitochondrial membrane potential and associated tumor phenotype are independent of MUC1 over-expression

We have established previously that minor subpopulations of cells with stable differences in their intrinsic mitochondrial membrane potential (Δψm) exist within populations of mammary and colonic carcinoma cells and that these differences in Δψm are linked to tumorigenic phenotypes consistent with increased probability of participating in tumor progression. However, the mechanism(s) involved in generating and maintaining stable differences in intrinsic Δψm and how they are linked to phenotype are unclear. Because the mucin 1 (MUC1) oncoprotein is over-expressed in many cancers, with the cytoplasmic C-terminal fragment (MUC1 C-ter) and its integration into the outer mitochondrial membrane linked to tumorigenic phenotypes similar to those of cells with elevated intrinsic Δψm, we investigated whether endogenous differences in MUC1 levels were linked to stable differences in intrinsic Δψm and/or to the tumor phenotypes associated with the intrinsic Δψm. We report that levels of MUC1 are significantly higher in subpopulations of cells with elevated intrinsic Δψm derived from both mammary and colonic carcinoma cell lines. However, using siRNA we found that down-regulation of MUC1 failed to significantly affect either the intrinsic Δψm or the tumor phenotypes associated with increased intrinsic Δψm. Moreover, whereas pharmacologically mediated disruption of the Δψm was accompanied by attenuation of tumor phenotype, it had no impact on MUC1 levels. Therefore, while MUC1 over-expression is associated with subpopulations of cells with elevated intrinsic Δψm, it is not directly linked to the generation or maintenance of stable alterations in intrinsic Δψm, or to intrinsic Δψm associated tumor phenotypes. Since the Δψm is the focus of chemotherapeutic strategies, these data have important clinical implications in regard to effectively targeting those cells within a tumor cell population that exhibit stable elevations in intrinsic Δψm and are most likely to contribute to tumor progression.     

The localization of proteoglycans and glycoproteins in the hyaline cartilage.

Antibodies to proteoglycan (PG) and glycoprotein of bovine nasal cartilage were conjugated with fluorescein isothiocyanate and with horseradish peroxidase. Hyaluronidase digestion of cartilage tissue-specimens increased the intensity of immune reactions; pronase digestion or extraction with 4 M guanidinium chloride abolished the staining. In the intercellular matrix fine filaments beaded with small granules were seen forming an irregular network. The interstices of the network are filled with collagen fibers linked together by the filaments and granules. In view of the linear conformation of core proteins of PGs and the globular conformation of glycoproteins (link proteins), it may be supposed that the granules and filaments represent these two protein components of PG-aggregates. In chondrocytes a homogeneous staining was recorded in the endoplasmic reticulum, in the juxtanuclear areas and in several smooth-walled vesicles and elongated areas situating subjacent to the cell membrane. In contrast to the extracellular immune reactions, this homogeneous intracellular staining was never enhanced by hyaluronidase digestion. This is interpreted in the sense that conformation changes of molecules secreted, and the aggregation of PGs, occur extracellularly.     

Monoclonal antibodies to two glycoproteins of herpes simplex virus type 2.

Monoclonal antibodies to herpes simplex virus type 2 were found to precipitate different numbers of radiolabeled polypeptides from lysates of virus-infected cells. Antibodies directed against two viral glycoproteins were characterized. Antibodies from hybridoma 17 alpha A2 precipitated a 60,000-molecular-weight polypeptide which chased into a 66,000- and 79,000-molecular-weight polypeptide. All three polypeptides labeled in the presence of [3H]glucosamine and had similar tryptic digest maps. The 60,000-molecular-weight polypeptide also chased into a 31,000-molecular-weight species which did not label with [3H]glucosamine. Antibodies from hybridoma 17 beta C2 precipitated a 50,000-molecular-weight polypeptide which chased into a 56,000- and 80,000-molecular weight polypeptide. These polypeptides also shared a similar tryptic digest map and labeled with [3H]glucosamine. Both monoclonal antibodies were herpes simplex virus type 2 specific. The viral proteins precipitated by 17 alpha A2 antibodies had characteristics similar to those reported for glycoprotein E, whereas the proteins precipitated by 17 beta C2 antibodies appeared to represent a glycoprotein not previously described. This glycoprotein should be tentatively designated glycoprotein F.     

Muskelin, a novel intracellular mediator of cell adhesive and cytoskeletal responses to thrombospondin-1.

We have used an expression cloning strategy based on a cell-attachment assay screen to seek identification of molecules required in cellular responses to thrombospondin-1, a regulated macromolecular component of extracellular matrix. We report the identification and functional characterization of a novel, widely expressed, intracellular protein, named muskelin, which contains dispersed motifs with homology to the tandem repeats first identified in the Drosophila kelch ORF1 protein. In adherent C2C12 cells, muskelin localizes in the cytoplasm and at cell margins. Over-expression of muskelin in C2C12 cells promotes cell attachment to the thrombospondin-1 C-terminal domain, alters the mechanisms of attachment to intact thrombospondin-1 and correlates with decreased formation of fascin microspikes and increased assembly of focal contacts by cells adherent on thrombospondin-1. Reciprocally, cell attachment, spreading and cytoskeletal organization are specifically reduced in TSP-1-adherent cells after antisense depletion of muskelin. These results establish a requirement for muskelin in cell responses to thrombospondin-1 and demonstrate that such responses involve a novel process which is integrated into the regulation of cell-adhesive behaviour and cytoskeletal organization.     

Induction and activity of NO synthase in bone-marrow-derived macrophages are independent of Ca2+.

The aim of the present study was to analyse whether an increase in the intracellular free Ca2+ concentration ([Ca2+]i) plays a role as a signal mediating synthesis of nitric oxide (NO) in bone-marrow-derived macrophages, either by stimulating induction of NO synthase or by regulating the activity of the enzyme. Therefore we compared the effects of various synthetic analogues of bacterial lipopeptide and of lipopolysaccharide (LPS) on NO production (assessed as nitrite formation during an incubation for 24 h) and on [Ca2+]i [measured with the fluorescent probe indo-1 (1-[2-amino-5-(6-carboxyindol-2-yl)phenoxy]-2- 2-(2'-amino-5'-methylphenoxy)ethane-NNN'N'-tetra-acetic acid)]. Strongly dissociating effects were evoked on nitrite formation and on [Ca2+]i by the stimuli. LPS was preferentially effective on nitrite formation, whereas the Ca2+ ionophore ionomycin and AlF3 induced increases only in [Ca2+]i. The lipopeptides N-palmitoyl-(S)-[2,3-bis(palmitoyloxy)-(2RS)- propyl]-(R)-cysteinylalanylglycine, N-palmitoyl-(S)-[2,3-bis(palmitoyloxy)- (2RS)-propyl]-(R)-cysteinylseryl-lysyl-lysyl-lysine and (S)-(1,2- dicarboxyhexadecyl)ethyl-N-palmitoylcysteinylseryl-lysyl-lys yl-lysine stimulated both parameters, but the maximal effects on nitrite formation and the shape of the dose-response curves did not parallel the effects on [Ca2+]i. Reduction of extracellular Ca2+ with EGTA significantly inhibited increases in [Ca2+]i, but did not change nitrite formation. Furthermore, NO synthesis in the cytosolic fraction of stimulated macrophages was not affected by Ca2+ over the concentration range 10 nM-2 microM. We conclude that increases in [Ca2+]i are not required for NO production in bone-marrow-derived macrophages. Thus the cellular regulation of NO production strikingly differs from that in the vascular endothelium, brain and adrenal gland.