Adenovirus L1 52- and 55-kilodalton proteins are required for assembly of virions.

A variant of adenovirus type 5 that contained a mutation within the L1 52- and 55-kilodalton (52/55K) protein-coding region was isolated. The mutant, termed ts369, produced L1 52/55K proteins with a two-amino-acid substitution and was temperature sensitive. Temperature-shift experiments indicated that the ts369 defect was late in the viral growth cycle. DNA replication and synthesis of late proteins occurred normally in ts369-infected cells at the nonpermissive temperature, but mature virions were not produced. Rather, capsidlike particles associated with the left-terminal region of the viral chromosome accumulated. These incomplete particles could not be chased into mature virions when the infected cells were shifted to the permissive temperature. However, previously synthesized proteins could be assembled into virions in the presence of a protein synthesis inhibitor upon shiftdown from the nonpermissive temperature, suggesting that the inactivation of the L1 52/55K proteins was reversible. These results indicate that the adenovirus L1 52/55K proteins play a role in the assembly of infectious virus particles.     

Mutant Cells Selected during Persistent Reovirus Infection Do Not Express Mature Cathepsin L and Do Not Support Reovirus Disassembly

Persistent reovirus infections of murine L929 cells select cellular mutations that inhibit viral disassembly within the endocytic pathway. Mutant cells support reovirus growth when infection is initiated with infectious subvirion particles (ISVPs), which are intermediates in reovirus disassembly formed following proteolysis of viral outer-capsid proteins. However, mutant cells do not support growth of virions, indicating that these cells have a defect in virion-to-ISVP processing. To better understand mechanisms by which viruses use the endocytic pathway to enter cells, we defined steps in reovirus replication blocked in mutant cells selected during persistent infection. Subcellular localization of reovirus after adsorption to parental and mutant cells was assessed using confocal microscopy and virions conjugated to a fluorescent probe. Parental and mutant cells did not differ in the capacity to internalize virions or distribute them to perinuclear compartments. Using pH-sensitive probes, the intravesicular pH was determined and found to be equivalent in parental and mutant cells. In both cell types, virions localized to acidified intracellular organelles. The capacity of parental and mutant cells to support proteolysis of reovirus virions was assessed by monitoring the appearance of disassembly intermediates following adsorption of radiolabeled viral particles. Within 2 h after adsorption to parental cells, proteolysis of viral outer-capsid proteins was observed, consistent with formation of ISVPs. However, in mutant cells, no proteolysis of viral proteins was detected up to 8 h postadsorption. Since treatment of cells with E64, an inhibitor of cysteine-containing proteases, blocks reovirus disassembly, we used immunoblot analysis to assess the expression of cathepsin L, a lysosomal cysteine protease. In contrast to parental cells, mutant cells did not express the mature, proteolytically active form of the enzyme. The defect in cathepsin L maturation was not associated with mutations in procathepsin L mRNA, was not complemented by procathepsin L overexpression, and did not affect the maturation of cathepsin B, another lysosomal cysteine protease. These findings indicate that persistent reovirus infections select cellular mutations that affect the maturation of cathepsin L and suggest that alterations in the expression of lysosomal proteases can modulate viral cytopathicity.     

Adsorption of reovirus to clay minerals: effects of cation-exchange capacity, cation saturation, and surface area.

The adsorption of reovirus to clay minerals has been reported by several investigators, but the mechanisms defining this association have been studied only minimally. The purpose of this investigation was to elucidate the mechanisms involved with this interaction. More reovirus type 3 was adsorbed, in both distilled and synthetic estuarine water, by low concentrations of montmorillonite than by comparable concentrations of kaolinite containing a mixed complement of cations on the exchange complex. Adsorption to the clays was essentially immediate and was correlated with the cation-exchange capacity of the clays, indicating that adsorption was primarily to negatively charged sites on the clays. Adsorption was greater with low concentrations of clays in estuarine water than in distilled water, as the higher ionic strength of the estuarine water reduced the electrokinetic potential of both clay and virus particles. The addition of cations (as chloride salts) to distilled water enhanced adsorption, with divalent cations being more effective than monovalent cations and 10(-2) M resulting in more adsorption than 10(-3) M. Potassium ions suppressed reovirus adsorption to montmorillonite, probably by collapsing the clay lattices and preventing the expression of the interlayer-derived cation-exchange capacity. More virus was adsorbed by montmorillonite made homoionic to various mono-, di-, and trivalent cations (except by montmorillonite homoionic to potassium) than by comparable concentrations of kaolinite homoionic to the same cations. The sequence of the amount of adsorption to homoionic montmorillonite was Al greater than Ca greater than Mg greater than Na greater than K; the sequence of adsorption to kaolinite was Na greater than Al greater than Ca greater than Mg greater than K. The constant partition-type adsorption isotherms obtained when the clay concentration was maintained constant and the virus concentration was varied indicated that a fixed proportion of the added virus population was adsorbed, regardless of the concentration of infectious particles. A heterogeneity within the reovirus population was indicated.     

Adsorption of reovirus to clay minerals: effects of cation-exchange capacity, cation saturation, and surface area

The adsorption of reovirus to clay minerals has been reported by several investigators, but the mechanisms defining this association have been studied only minimally. The purpose of this investigation was to elucidate the mechanisms involved with this interaction. More reovirus type 3 was adsorbed, in both distilled and synthetic estuarine water, by low concentrations of montmorillonite than by comparable concentrations of kaolinite containing a mixed complement of cations on the exchange complex. Adsorption to the clays was essentially immediate and was correlated with the cation-exchange capacity of the clays, indicating that adsorption was primarily to negatively charged sites on the clays. Adsorption was greater with low concentrations of clays in estuarine water than in distilled water, as the higher ionic strength of the estuarine water reduced the electrokinetic potential of both clay and virus particles. The addition of cations (as chloride salts) to distilled water enhanced adsorption, with divalent cations being more effective than monovalent cations and 10(-2) M resulting in more adsorption than 10(-3) M. Potassium ions suppressed reovirus adsorption to montmorillonite, probably by collapsing the clay lattices and preventing the expression of the interlayer-derived cation-exchange capacity. More virus was adsorbed by montmorillonite made homoionic to various mono-, di-, and trivalent cations (except by montmorillonite homoionic to potassium) than by comparable concentrations of kaolinite homoionic to the same cations. The sequence of the amount of adsorption to homoionic montmorillonite was Al greater than Ca greater than Mg greater than Na greater than K; the sequence of adsorption to kaolinite was Na greater than Al greater than Ca greater than Mg greater than K. The constant partition-type adsorption isotherms obtained when the clay concentration was maintained constant and the virus concentration was varied indicated that a fixed proportion of the added virus population was adsorbed, regardless of the concentration of infectious particles. A heterogeneity within the reovirus population was indicated.     

Regulation of nerve terminal calcium channel selectivity by a weak acid site.

The effects of low pH, and of alkaline earth cations, were examined on calcium uptake by pinched-off nerve terminals (synaptosomes). This uptake appears to be mediated by voltage-sensitive Ca channels (J. Physiol. 247:617, 1975). Ca uptake was measured in low (5 mM) or high (77 mM) potassium media. The extra uptake promoted by depolarizing (K-rich) media was almost maximal at pH 7.5, and decreased as the pH was lowered. Data relating depolarization-induced 45Ca uptake to pH fit a titration curve with a pKa approximately 6. Experiments in which Ca concentration and pH were both varied indicated that Ca2+ and H+ compete for a common binding site. Inhibition of depolarization-induced 45Ca uptake by the alkaline earth cations was studied to determine the apparent binding sequence for these cations in the Ca channels: Ca greater than Sr greater than Ba greater than Mg. This sequence resembles that observed for block of Ca channels in other preparations. The apparent binding sequence of the alkaline earth cations and the apparent pKa (approximately 6) of the Ca-binding site indicate that the Ca channel is a "high field strength" system. Protonation of a Ca channel binding site could explain the inhibitory effect of low pH on Ca-dependent neurotransmitter release (cf. Del Castillo et al., J. Cell. Comp. Physiol. 59:35, 1962).     

Vaccinia virus penetration requires cholesterol and results in specific viral envelope proteins associated with lipid rafts

Vaccinia virus infects a wide variety of mammalian cells from different hosts, but the mechanism of virus entry is not clearly defined. The mature intracellular vaccinia virus contains several envelope proteins mediating virion adsorption to cell surface glycosaminoglycans; however, it is not known how the bound virions initiate virion penetration into cells. For this study, we investigated the importance of plasma membrane lipid rafts in the mature intracellular vaccinia virus infection process by using biochemical and fluorescence imaging techniques. A raft-disrupting drug, methyl-beta-cyclodextrin, inhibited vaccinia virus uncoating without affecting virion attachment, indicating that cholesterol-containing lipid rafts are essential for virion penetration into mammalian cells. To provide direct evidence of a virus and lipid raft association, we isolated detergent-insoluble glycolipid-enriched membranes from cells immediately after virus infection and demonstrated that several viral envelope proteins, A14, A17L, and D8L, were present in the cell membrane lipid raft fractions, whereas the envelope H3L protein was not. Such an association did not occur after virions attached to cells at 4 degrees C and was only observed when virion penetration occurred at 37 degrees C. Immunofluorescence microscopy also revealed that cell surface staining of viral envelope proteins was colocalized with GM1, a lipid raft marker on the plasma membrane, consistent with biochemical analyses. Finally, mutant viruses lacking the H3L, D8L, or A27L protein remained associated with lipid rafts, indicating that the initial attachment of vaccinia virions through glycosaminoglycans is not required for lipid raft formation.     

Induction of thermal and chemical stability of O6-methylguanine-DNA methyltransferase by Ca2+

Divalent cations stabilized rat recombinant O6-methylguanine-DNA methyltransferase (rMGMT) protein against heat treatment. Activity of rMGMT was completely abolished by incubating at 45 degrees C for 30 min, however, addition of 1.0 mM Mg2+, Ca2+ or Mn2+ significantly protected heat-induced inactivation of MGMT activity (50-60% vs. 97% inactivation). Protective effect of Ca2+ on the MGMT activity was concentration-dependent up to 3 mM, and the thermal protection was effective up to 45 degrees C. In order to investigate Ca2+ binding site in rMGMT protein, truncated GST-rMGMT proteins containing N-terminal 39 amino acids (GST-rMGMT39), 70 amino acids (GST-rMGMT70) and full-length protein (GST-rMGMT) were prepared. Radiolabeled calcium ion [45Ca2+] was bound only to the GST-rMGMT70 and GST-rMGMT, but not to the GST-rMGMT39, indicating that divalent cations could bind the residues between 40th and 70th of the rMGMT protein. Calcium binding was not observed in the site-directed mutant rMGMT proteins (rMGMT(D42A) and rMGMT(E45A)), confirmed by autoradiography using [45Ca2+] after nondenaturing gel electrophoresis; however, the above two mutants had the same catalytic activity as well as proteolytic sensitivity as the wild MGMT protein. Analysis by equilibrium dialysis revealed stoichiometric binding of one molecule of Ca2+ to one molecule of the protein. Since circular dichroism (CD) spectra indicated no discernible difference before and after Ca2+ binding, the above results suggested that neutralization of two negative charges of Asp42 and Glu45 by Ca2+ resulted in thermal stabilization of the protein with minimum perturbation of its tertiary structure.     

The Role of Bivalent Cations on the Isolation of Allantoinase from the Soybean Seed Extracts

Allantoinases (ALNase) in the water extracts distilled from seeds and leaves of soybean (Glycine max L. cv. Keyu 10) were. remarkably heat stable. However the enzyme and non-enzyme protein in the seed extract, but not leaf extract, lost their activity and were denaturated at 75℃ for 5 min in the presence of Ca2+, Mg2+. or Mn2+ions respecitively. At room temperature over 40%～50% of the non-enzyme proteins in the seed extract could be removed by the bivalent cations without affecting the enzyme activity. This effect was weakend by the increase of concentration. Both extracts had different responses to all sorts of insoluble Ca2. salts. For the seed extract about 50 % of the non-enzyme proteins were removed by 5 % CaSO4 (W/V), without effecting the enzyme activity, while the leaf extract was sensible to Ca3 (PO4) 2. After treatment with 5 % Ca3 (PO4) 2 about 50 % of the enzyme activities and about 70% of proteins were lost. Mn2+ ions could enhance the enzyme activity in crude seed extract, but had no effect on partially purified enzyme from seeds and enzyme in crude extract from leaves. Further, EDTA had no effect on enzyme activity in both extracts.     

Two distinct low-pH steps promote entry of vaccinia virus

Entry of vaccinia virus into cells occurs by an endosomal route as well as through the plasma membrane. Evidence for an endosomal pathway was based on findings that treatment at a pH of <6 of mature virions attached to the plasma membrane enhances entry, whereas inhibitors of endosomal acidification reduce entry. Inactivation of infectivity by low-pH treatment of virions prior to membrane attachment is characteristic of many viruses that use the endosomal route. Nevertheless, we show here that the exposure of unattached vaccinia virus virions to low pH at 37 degrees C did not alter their infectivity. Instead, such treatment stably activated virions as indicated by their accelerated entry upon subsequent addition to cells, as measured by reporter gene expression. Moreover, the rate of entry was not further enhanced by a second low-pH treatment following adsorption to the plasma membrane. However, the entry of virions activated prior to adsorption remained sensitive to inhibitors of endosomal acidification, whereas virions treated with low pH after adsorption were resistant. Activation of virions by low pH was closely mimicked by proteinase digestion, suggesting that the two treatments operate through a related mechanism. Although proteinase cleavage of the virion surface proteins D8 and A27 correlated with activation, mutant viruses constructed by individually deleting these genes did not exhibit an activated phenotype. We propose a two-step model of vaccinia virus entry through endosomes, in which activating or unmasking the fusion complex by low pH or by proteinase is rate limiting but does not eliminate a second low-pH step mediating membrane fusion.     

Biological characterization of the lytic cycle of actinophage φA7 in Streptomyces antibioticus

Some basic parameters of the lytic development of phage phi A7 in Streptomyces antibioticus are described. One-step growth experiments demonstrated that at 28 degrees C phi A7 has a latent period of about 60 min and an exponential growth period of about 35 min. The average burst size ranged from 70-100 plaque forming units per infected cell. At the same temperature 50% of the virions were adsorbed to germ tubes of S. antibioticus in about 10 min. This corresponds to an adsorption constant of 6.5 x 10(-10) ml/min. The phage was unable to adsorb the host at other stages of the life cycle (spores or mycelium). Divalent cations are not required for phi A7 stability but Ca2+ proved to be essential for adsorption and also for a later stage of the vegetative development of the phage.     

Adenovirus L1 52- and 55-kilodalton proteins are present within assembling virions and colocalize with nuclear structures distinct from replication centers.

Analysis of a temperature-sensitive mutant, Ad5ts369, had indicated that the adenovirus L1 52- and 55-kDa proteins (52/55-kDa proteins) are required for the assembly of infectious virions. By using monoclonal antibodies directed against bacterially produced L1 52-kDa protein, the L1 52/55-kDa proteins were found to be differentially phosphorylated forms of a single 48-kDa polypeptide. Both phosphoforms were shown to be present within all suspected virus assembly intermediates (empty capsids, 50 to 100 molecules; young virions, 1 to 2 molecules) but not within mature virions. The mobilities of these proteins in polyacrylamide gels were affected by reducing agents, indicating that the 52/55-kDa proteins may exist as homodimers within the cell and within assembling particles. Immunofluorescence analysis revealed that the 52/55-kDa proteins localize to regions within the infected nucleus that are distinct from viral DNA replication centers, indicating that replication and assembly of viral components likely occur in separate nuclear compartments. Immunoelectron microscopic studies determined that the 52/55-kDa proteins are found in close association with structures that appear to contain assembling virions. These results are consistent with an active but transient role for the L1 products in assembly of the adenovirus particle, perhaps as scaffolding proteins.     

Adsorption ofRhizobium meliloti to alfalfa roots: Dependence on divalent cations and pH

Adsorption ofRhizobium meliloti L5-30 in low numbers to alfalfa (Medicago sativa L.) roots was dependent on the presence of divalent cations, and required neutral pH. Adsorption was proportional to Ca and/or Mg concentrations up to 1.5 mM. Ca was not substituted by Sr, Ba or Mn. Adsorption was abolished and viability decreased at pH6. When lowering pH, higher Ca concentrations were required to attain similar adsorption levels, indicating a marked interactive effect between Ca and H ions. Pretreatment of the roots with Ca and low pH did not affect subsequent adsorption of the bacteria. However, Ca pretreatment ofR. meliloti sustained further adsorption at low Ca levels and low pH substantially affected their ability to adsorb. Low pH appears to affect the stability of binding causing desorption of the previously bound bacteria. The presence of saturating concentrations of heterologousR. leguminosarum bv.trifolii A118, did not prevent the expression of divalent cations and pH requirements, as well as their interaction. Our results suggest that rhizobial binding to the root surface already shows the Ca and pH dependence of alfalfa nodulation, which was generally associated to some event prior to rhizobial penetration of root hairs.     

Cross-neutralization potential of native human papillomavirus N-terminal L2 epitopes

Background

Human papillomavirus (HPV) capsids are composed of 72 pentamers of the major capsid protein L1, and an unknown number of L2 minor capsid proteins. An N-terminal “external loop” of L2 contains cross-neutralizing epitopes, and native HPV16 virions extracted from 20-day-old organotypic tissues are neutralized by anti-HPV16 L2 antibodies but virus from 10-day-old cultures are not, suggesting that L2 epitopes are more exposed in mature, 20-day virions. This current study was undertaken to determine whether cross-neutralization of other HPV types is similarly dependent on time of harvest and to screen for the most effective cross-neutralizing epitope in native virions.

Methodology and Principal Findings

Neutralization assays support that although HPV16 L2 epitopes were only exposed in 20-day virions, HPV31 or HPV18 epitopes behaved differently. Instead, HPV31 and HPV18 L2 epitopes were exposed in 10-day virions and remained so in 20-day virions. In contrast, presumably due to sequence divergence, HPV45 was not cross-neutralized by any of the anti-HPV16 L2 antibodies. We found that the most effective cross-neutralizing antibody was a polyclonal antibody named anti-P56/75 #1, which was raised against a peptide consisting of highly conserved HPV16 L2 amino acids 56 to 75.

Conclusions and Significance

This is the first study to determine the susceptibility of multiple, native high-risk HPV types to neutralization by L2 antibodies. Multiple anti-L2 antibodies were able to cross-neutralize HPV16, HPV31, and HPV18. Only neutralization of HPV16 depended on the time of tissue harvest. These data should inform attempts to produce a second-generation, L2-based vaccine.     

Influence of cations on the blue to purple transition of bacteriorhodopsin. Comparison of Ca2+ and Hg2+ binding and their effect on the surface potential

We have investigated the effect of Ca2+ and Hg2+ binding on various properties of the blue membrane prepared by deionization of the Halobacterium halobium purple membrane. Binding of radioactive 45Ca2+ and 203Hg2+ was monitored by a filtration technique. Five high and medium affinity sites for Ca2+ and seven low affinity sites for Hg2+ were found per bacteriorhodopsin. Competitive binding was observed only for three Ca2+ and three Hg2+. Visible absorption studies indicated that Ca2+ binding could restore the purple color of bacteriorhodopsin while Hg2+ was inefficient. Hg2- could partially reverse to blue the Ca2+-regenerated purple membrane in parallel with the displacement of three Ca2+. Effects of cation binding on the surface potential of the membrane were measured by Electron Spin Resonance spectroscopy using a cationic spin-labeled amphiphile. Cations such as La3+, Ca2+, Mg2+, or Na+ strongly increased (i.e. rendered less negative) the surface potential. An univocal correlation was found between the cation-induced variation of surface potential and the extent of regeneration of the purple color. Hg2+ induced a smaller increase in surface potential than that corresponding to the effective divalent cations. This lower effect appears to be due to binding to sites not related to those of other cations.     

重金属离子对猪红细胞膜Ca~(2+)-ATP酶的作用

猪红细胞膜Ca~(2+)-ATP酶是一种钙调蛋白(CaM)依赖酶,其活力又依赖巯基的完整性。实验应用Ca~(2+)-ATP酶这一模型体系观察到重金属离子,Pb~(2+)、Cd~(2+)和Hg~(2+)都能替代Ca~(2+),激活CaM,从而激活Ca~(2+)-ATP酶;其最大刺激活力分别为85％、80％和30％,半刺激浓度分别为32、27和0.7μmol/L。当三种重金属离子的浓度增加时,则与Ca~(2+)-ATP酶的巯基结合,抑制酶的活力,Pb2~(2+)、Cd~(2+)和Hg~(2+)的半抑制浓度分别为370、440和2μmol/L。抑制作用为渐进性过程,而刺激作用为即时效应。抑制作用可为巯基化物,特别是二巯基化物所逆转。研究结果提示,CaM可能是重金属中毒最初作用的靶分子,而重金属中毒不仅使CaM“开关”失灵,还可能导致细胞内Ca~(2+)的调节全面失控。     

Dissociation of polyoma virus by the chelation of calcium ions found associated with purified virions.

Analysis of polyoma virions by X-ray fluorometry demonstrated that calcium (Ca2+) was associated with the purified virion. Treatment of purified virions with ethyleneglycol-bis-N,N'-tetraacetic acid (EGTA), which chelates Ca2+, and the reducing agent dithiothreitol caused the virions to dissociate. Electron microscopy revealed that the virions were dissociated to the capsomere level. Incubation of polyoma virions with 150 mM NaCl, 10 mM EGTA, and 3 mM dithiothreitol was optimum for the dissociation reaction. The pH for the dissociation reaction ranged from 7.5 to 10.5. Cesium chloride density gradient centrifugation indicated that both EGTA and dithiothreitol were necessary for dissociation to occur; neither reagent alone dissociated the virus. The major protein product of the dissociated viral particles sedimented at 12S. Relationships between these experiments and the alkaline carbonate-bicarbonate dissociation of polyoma are discussed.     

Structural Proteins of Vesicular Stomatitis Viruses

Three major and three minor structural proteins were identified by polyacrylamide gel electrophoresis of purified infectious virions of the Indiana serotype of vesicular stomatitis (VS) virus disrupted with acetic acid, 0.5 m urea, sodium dodecyl sulfate (SDS), and 2-mercaptoethanol. Molecular weights of the six virion proteins were estimated by comparative electrophoretic migration of known marker proteins in the presence of SDS. The following values were obtained: major proteins P6 congruent with 34,500, P5 congruent with 59,500, and P4 congruent with 81,500; minor proteins P3 congruent with 140,000, P2 congruent with 186,000, and P1 congruent with 275,000. P1 did not disaggregate in 8 m urea, but P2 and P3 did. The possibility that P1 is an uncleaved large polypeptide chain could not be ruled out. Six identical protein components were dissociated from Indiana VS virions grown in chick and mouse cells; no cellular proteins could be detected in purified virions. Of six proteins identified in virions of the New Jersey serotype, only the smallest protein (P6) could be distinguished from any of the six proteins of the Indiana serotype on the basis of migration in SDS gels. The defective T particles of Indiana VS virus contained the same six proteins in essentially the same proportions as those of the infectious B virions. Only P6 and P5 could be cleanly separated by preparative gel electrophoresis.     

Early Interactions of Rhizobium leguminosarum bv. phaseoli and Bean Roots: Specificity in the Process of Adsorption and Its Requirement of Ca(sup2+) and Mg(sup2+) Ions

Roots of Phaseolus vulgaris L. were incubated with dilute suspensions (1 x 10(sup3) to 3 x 10(sup3) bacteria ml(sup-1)) of an antibiotic-resistant indicator strain of Rhizobium leguminosarum bv. phaseoli in mineral medium and washed four times by a standardized procedure prior to quantitation of adsorption (G. Caetano-Anolles and G. Favelukes, Appl. Environ. Microbiol. 52:371-376, 1986). The population of rhizobia remaining adsorbed on roots after washing was homogeneous, as indicated by the first-order course of its desorption by hydrodynamic shear. Rhizobia were maximally active for adsorption in the early stationary phase of growth. The process leading to adsorption was rapid, without an initial lag, and slowed down after 1 h. Adsorption of the indicator strain at 10(sup3) bacteria ml(sup-1) was inhibited to different extents in the presence of 10(sup3) to 10(sup8) antibiotic-sensitive competitor rhizobia ml(sup-1). After a steep rise above 10(sup4) bacteria ml(sup-1), inhibition by heterologous competitors in the concentration range of 10(sup5) to 10(sup7) bacteria ml(sup-1) was markedly less than by homologous strains, while at 10(sup8) bacteria ml(sup-1) it approached the high level of inhibition by the latter. At 10(sup7) bacteria ml(sup-1), all of the heterologous strains tested were consistently less inhibitory than homologous competitors (P < 0.001). These differences in competitive behavior indicate that in the process of adsorption of R. leguminosarum bv. phaseoli to its host bean roots, different modes of adsorption occur and that some of these modes are specific for the microsymbiont (as previously reported for the alfalfa system [G. Caetano-Anolles and G. Favelukes, Appl. Environ. Microbiol. 52:377-381, 1986]). Moreover, whereas the nonspecific process occurred either in the absence or in the presence of Ca(sup2+) and Mg(sup2+) ions, expression of specificity was totally dependent on the presence of those cations. R. leguminosarum bv. phaseoli bacteria adsorbed in the presence of Ca(sup2+) and Mg(sup2+) were more resistant to desorption by shear forces than were rhizobia adsorbed in their absence. These results indicate that (i) symbiotic specificity in the P. vulgaris-R. leguminosarum bv. phaseoli system is expressed already during the early process of rhizobial adsorption to roots, (ii) Ca(sup2+) and Mg(sup2+) ions are required by R. leguminosarum bv. phaseoli for that specificity, and (iii) those cations cause tighter binding of rhizobia to roots.     

Ca2+ and Mg2+ as modulators of mitochondrial L-glycerol-3-phosphate dehydrogenase

1. Physiological concentrations of either Ca2+ or Mg2+ stimulated L-glycerol 3-phosphate oxidation by intact mitochondria isolated from various mammalian tissues (hamster brown adipose tissue, rat brain, liver of normal and hyperthyroid rats). A higher cation concentration was required for stimulation by Mg2+ than by Ca2+. L-glycerol-3-phosphate dehydrogenase was the target of the stimulation by both cations as revealed by measurements with intact mitochondria as well as with the solubilized enzyme. With different electron acceptors Ca2+ and Mg2+ stimulation occurred at significantly different cation concentrations. 2. Substrate activation of mitochondrial L-glycerol-3-phosphate dehydrogenase was observed in intact mitochondria and with the solubilized enzyme isolated from hyperthyroid rats in the absence of Ca2+ and Mg2+. According to kinetic analysis two independent binding sites, functioning with different turnovers and with different affinities for the substrate, could account for the phenomenon. In the presence of Ca2+ or Mg2+ substrate activation could not be detected; the kinetic parameters apparently correspond to the tight substrate-binding site functioning with high turnover. 3. Thiol group(s), which in the absence of Ca2+ and Mg2+ did not participate in the functioning of the enzyme, played an essential role in the binding of these cations to the enzyme, as shown by chemical modification studies. 4. From the solubilized mitochondrial proteins L-glycerol-3-phosphate dehydrogenase was bound selectively to the hydrophobic phenyl-Sepharose 4B matrix in the presence Ca2+, and the bound enzyme could be eluted with EDTA. This suggests that Ca2+ caused an alteration in the conformation of the enzyme.     

Action of polyvalent cations on sodium transport across skin of larval and adult Rana catesbeiana

The actions of alkaline earth (AE) and transition element (TE) cations on Na+ transport across skin of larval and adult Rana catesbeiana were compared. Bathed on the outside by Ca+2-free Ringer's, both larval and adult skins maintained a stable short-circuit current (3-4 mu Amps cm-2 for larval skin and 20-30 mu Amps cm-2 for adult skin). Addition of Ca+2 to the external bath reduced the SCC; maximal inhibition was about 36% for larval skin and 22% for adult skin. Other AE divalent cations were also inhibitory. The order of effectiveness was: Ba+2 = Ca+2 greater than Sr+2 greater than Mg+2 for larval skin and Ba+2 greater than Ca+2 = Mg+2 for adult skin. Sodium influx was markedly elevated when Ca+2 was removed from the external medium. Current-voltage analysis indicated that Ca+2 increases the resistance of the active pathway without affecting the shunt resistance or the electromotive force of Na+ transport (ENa) in larval and adult skins. The SCC across adult skin was stimulated by TE cations (Co+2, Cd+2, La+3). These ions were inhibitory on larval skin. The transition in the response occurred at stage XXI. The inhibitory effect of TE on larvel skin resembles that seen in response to AE cations and we postulate a common mechanism. Since larval skin lacks the selective Na+ channels found in apical membranes of adult skin, we infer that the mechanism of inhibition by AE cations is not on these channels. A more general phenomenon such as change in surface charge at the apical membrane seems more reasonable.(ABSTRACT TRUNCATED AT 250 WORDS)