Na+-K+-Cl- cotransport in human fibroblasts is inhibited by cytomegalovirus infection

We examined the effects of human cytomegalovirus (HCMV)infection on theNa⁺-K⁺-Clcotransporter (NKCC) in a human fibroblast cell line. Using the Cl-sensitive dye MQAE, weshowed that the mock-infected MRC-5 cells express a functional NKCC.1) IntracellularCl concentration([Cl]_i)was significantly reduced from 53.4 ± 3.4 mM to 35.1 ± 3.6 mMfollowing bumetanide treatment. 2)Net Cl efflux caused byreplacement of external Clwith gluconate was bumetanide sensitive.3) InCl-depleted mock-infectedcells, the Cl reuptake rate(in HCO₃-free media) was reduced inthe absence of external Na⁺ and bytreatment with bumetanide. After HCMV infection, we found that although[Cl]_iincreased progressively [24 h postexposure (PE), 65.2 ± 4.5 mM; 72 h PE, 80.4 ± 5.0 mM], the bumetanide andNa⁺ sensitivities of[Cl]_iand net Cl uptake and losswere reduced by 24 h PE and abolished by 72 h PE. Western blots usingthe NKCC-specific monoclonal antibody T4 showed an approximatelyninefold decrease in the amount of NKCC protein after 72 h ofinfection. Thus HCMV infection resulted in the abolition of NKCCfunction coincident with the severe reduction in the amount of NKCCprotein expressed.

     

Altered Phase Behavior in Membranes of Aging Dry Pollen May Cause Imbibitional Leakage

Aging of dry pollen has been shown to coincide with increases of free fatty acids and lysophospholipids. These compounds reduce the integrity of hydrated liposomes made from isolated pollen phospholipids but do not lead to their total destruction. However, a massive, instantaneous leakage occurs upon imbibition of dry cattail pollen (Typha latifolia) that has aged to the point of complete loss of viability. To resolve the apparent discrepancy in stability between hydrated and dry membranes, the lyotropic phase behavior of two liposome systems containing lysophospholipid (12 mol%) was studied with differential scanning calorimetry and Fourier transform infrared spectroscopy. In both systems dehydration caused phase separation of the lipids. Fourier transform infrared data concerning phase behavior of isolated membranes from aging pollen and of membranes in situ did not show phase separations, probably because the assay technique was not sufficiently sensitive to detect them. However, aging of the pollen resulted in a permanent increase in the gel-to-liquid crystalline phase transition temperature (Tm) of isolated membranes and in a broadening of the transition in situ. We conclude that the increase in Tm of hydrated membranes may be more closely related to the leakage.     

Ribosomal RNA gene number and sequence divergence in the diploid-tetraploid species pair of north American hylid tree frogs

Hyla chrysoscelis (2n = 24) and H. versicolor (2n = 48) are a diploid-tetraploid species pair of tree frogs. Hybridization saturation of isolated ¹²⁵I-labeled ribosomal RNAs (rRNAs) with filter-immobilized DNA shows that there are twice as many rRNA genes in the tetraploid as in the diploid. For the diploid, saturation occurs at 0.037%, from which it is calculated that there are about 618 copies of the (18 S + 28 S) rRNA genes per haploid genome. Analysis of the extent of hybridization and also the thermal stability of homologous and heterologous hybrids shows that considerably more base substitutions have occurred in the tetraploid rDNA genes than in the diploid since their divergence. This is interpreted to reflect either a relaxation of the gene regulatory correction mechanism hypothesized to be responsible for the maintenance of identical tandem rRNA genes in the tetraploid or a release of one gene set from the normal selective constraints.This research supported by NSF Grants CDP-8002341 and PRM-8106947 to J.C.V. and by research grants from Miami University to L.A.T., D.T.C., R.J.D., and S.W.K.     

Germination and ion leakage are linked with phase transitions: of membrane lipids during imbibition of Typha latifolia pollen

In previous studies on the causes of imbibitional leakage in dry polien we have presented data which suggest that the leakage is due to a gel to liquid crystalline phase transition in membrane phospholipids during the rehydration event. In the present study we greatly extend and confirm those results. A supplemented phase diagram for the hydration dependent transition temperature of membrane phospholipids in pollen is presented. In pollen containing > 0.05 g H₂O g⁻¹ dry weight at the time of imbibition, this phase diagram for the phospholipids precisely predicts the conditions for rehydration under which germination is maximal and leakage is minimal. However, in extremely dry pollen, containing < 0.05 g H₂O g⁻¹ dry weight the predictive value of the phase diagram for phospholipids in the pollen is not in agreement with data for germination and leakage. Thus, an alternative explanation must be sought for leakage in these circumstances. We examined the available evidence and suggest here that a modified form of the non-bilayer phase hypothesis proposed by Simon (1974) may apply in the specialized case of extremely dry cells.     

Small intensely fluorescent (SIF) cells and sympathetic nerves in the adult rabbit portal vein and during perinatal development

Summary The ontogenesis of small intensely fluorescent (SIF) cells and the adrenergic nerve plexus is described in stretch preparations of the rabbit portal vein. On the 25 to 26th days of gestation there was a predominance of SIF cells (8 to 30 m in diameter), but a few nerve fibres in bundles were also present. Each portal vein preparation contained 6 to 9 groups of cells. The distribution and number of SIF cells and nerve bundles remained constant until the 31st day of gestation at which stage the number of SIF cells had decreased, while the density of the nerve plexus had increased approximately 4-fold. The adult portal vein exhibited a dense adrenergic plexus, but SIF cells were absent from nine out of ten preparations.     

B cell overexpression of FCRL5 and PD-1 is associated with low antibody titers in HCV infection

Antibodies targeting the hepatitis C virus (HCV) envelope glycoprotein E2 are associated with delayed disease progression, and these antibodies can also facilitate spontaneous clearance of infection in some individuals. However, many infected people demonstrate low titer and delayed anti-E2 antibody responses. Since a goal of HCV vaccine development is induction of high titers of anti-E2 antibodies, it is important to define the mechanisms underlying these suboptimal antibody responses. By staining lymphocytes with a cocktail of soluble E2 (sE2) glycoproteins, we detected HCV E2-specific (sE2+) B cells directly ex vivo at multiple acute infection timepoints in 29 HCV-infected subjects with a wide range of anti-E2 IgG titers, including 17 persistently infected subjects and 12 subjects with spontaneous clearance of infection. We performed multi-dimensional flow cytometric analysis of sE2+ and E2-nonspecific (sE2-) class-switched B cells (csBC). In sE2+ csBC from both persistence and clearance subjects, frequencies of resting memory B cells (rMBC) were reduced, frequencies of activated MBC (actMBC) and tissue-like MBC (tlMBC) were increased, and expression of FCRL5, an IgG receptor, was significantly upregulated. Across all subjects, plasma anti-E2 IgG levels were positively correlated with frequencies of sE2+ rMBC and sE2+ actMBC, while anti-E2 IgG levels were negatively correlated with levels of FCRL5 expression on sE2+ rMBC and PD-1 expression on sE2+ actMBC. Upregulation of FCRL5 on sE2+ rMBC and upregulation of PD-1 on sE2+ actMBC may limit anti-E2 antibody production in vivo. Strategies that limit upregulation of these molecules could potentially generate higher titers of protective antibodies against HCV or other pathogens.     

RhoA Interacts with the Fusion Glycoprotein of Respiratory Syncytial Virus and Facilitates Virus-Induced Syncytium Formation

The fusion glycoprotein (F) of respiratory syncytial virus (RSV), which mediates membrane fusion and virus entry, was shown to bind RhoA, a small GTPase, in yeast two-hybrid interaction studies. The interaction was confirmed in vivo by mammalian two-hybrid assay and in RSV-infected HEp-2 cells by coimmunoprecipitation. Furthermore, the interaction of F with RhoA was confirmed in vitro by enzyme-linked immunosorbent assay and biomolecular interaction analysis. Yeast two-hybrid interaction studies with various deletion mutants of F and with RhoA indicate that the key binding domains of these proteins are contained within, or overlap, amino acids 146 to 155 and 67 to 110, respectively. The biological significance of this interaction was studied in RSV-infected HEp-2 cells that were stably transfected to overexpress RhoA. There was a positive correlation between RhoA expression and RSV syncytium formation, indicating that RhoA can facilitate RSV-induced syncytium formation.     

Mechanochemical coupling in spin-labeled, active, isometric muscle

Observed effects of inorganic phosphate (P(i)) on active isometric muscle may provide the answer to one of the fundamental questions in muscle biophysics: how are the free energies of the chemical species in the myosin-catalyzed ATP hydrolysis (ATPase) reaction coupled to muscle force?. Pflugers Arch. 414:73-81) showed that active, isometric muscle force varies logarithmically with [P(i)]. Here, by simultaneously measuring electron paramagnetic resonance and the force of spin-labeled muscle fibers, we show that, in active, isometric muscle, the fraction of myosin heads in any given biochemical state is independent of both [P(i)] and force. These direct observations of mechanochemical coupling in muscle are immediately described by a muscle equation of state containing muscle force as a state variable. These results challenge the conventional assumption mechanochemical coupling is localized to individual myosin heads in muscle.     

Real-time imaging of the dynamics of secretory granules in growth cones

Secretory granules containing a hybrid protein consisting of the regulated secretory protein tissue plasminogen activator and an enhanced form of green fluorescent protein were tracked at high spatial resolution in growth cones of differentiated PC12 cells. Tracking shows that granules, unlike synaptic vesicles, generally are mobile in growth cones. Quantitative analysis of trajectories generated by granules revealed two dominant modes of motion: diffusive and directed. Diffusive motion was observed primarily in central and peripheral parts of growth cones, where most granules diffused two to four orders of magnitude more slowly than comparably sized spheres in dilute solution. Directed motion was observed primarily in proximal parts of growth cones, where a subset of granules underwent rapid, directed motion at average speeds comparable to those observed for granules in neurites. This high-resolution view of the dynamics of secretory granules in growth cones provides insight into granule organization and release at nerve terminals. In particular, the mobility of granules suggests that granules, unlike synaptic vesicles, are not tethered stably to cytoskeletal structures in nerve terminals. Moreover, the slow diffusive nature of this mobility suggests that secretory responses involving centrally distributed granules in growth cones will occur slowly, on a time scale of minutes or longer.