Tetraphenylphosphonium is an indicator of negative membrane potential in Candida albicans

The characteristics of the uptake of lipophilic cations tetraphenylphosphonium (TPP+) into Candida albicans have been investigated to establish whether TPP+ can be used as a membrane potential probe for this yeast. A membrane potential (delta psi, negative inside) across the plasma membrane of C. albicans was indicated by the intracellular accumulation of TPP+. The steady-state distribution of TPP+ was reached within 60 min and varied according to the expected changes of delta psi. Agents known to depolarize membrane potential caused a rapid and complete efflux of accumulated TPP+. The initial influx of TPP+ was linear over a wide range of TPP+ concentrations (2.5-600 microM), indicating a non mediated uptake. Thus, TPP+ is a suitable delta psi probe for this yeast.     

The role of macrophages in B cell tolerance. I. Antigen-specific failure of Thy-1, Ly-2 negative adherent cells from tolerant mice to reconstitute immunocompetence in adherent cell deficient spleen cells

T-Cell-independent B-cell tolerance to the hapten derivatives of carboxymethyl cellulose (CMC) or methyl cellulose (MC) appears to be controlled by Thy-1-, Ly-2- adherent (A) cells contained in the spleen or peritoneal fluid. Immunocompetence in nonadherent (NA) normal spleen cells could be restored in vitro by irradiated A cells from normal mice. However, NA cells reconstituted with irradiated A cells derived from hapten specifically tolerant mice failed to respond to the same hapten, but responded normally to an immunogenic challenge with another unrelated antigen. A cells that had been preincubated at 4 degrees C with hapten derivatized MC also failed to restore immunocompetence. While preincubation of unfractionated spleen cells with the tolerogen under the same conditions resulted in B-cell unresponsiveness, such treatment of NA cells failed to render B cells tolerant. Treatment of A cells from tolerant mice with the reducing agent potassium iodide (KI) in vitro restored their capacity to render cultures of NA cells immunocompetent to the relevant hapten. Moreover, treatment with KI of spleen cells from mice injected with the tolerogen was shown to render them responsive. We suggest that B-cell tolerance induced by hapten derivatives of CMC and MC is mediated by suppressive macrophages contained among A cells. Certain subpopulations of macrophages are known to exert cytotoxic effects upon target cells by the release at close range of oxidating agents. We postulate that hapten derivatized CMC and MC, through unique properties of the carrier, bind to and possibly activate macrophages rendering them specifically suppressive for hapten binding B cells.     

An intermediate polymer in the assembly of clathrin baskets

Clathrin (8 S) is known to polymerize into two varieties of basket structures (150 S or 300 S) under the normal buffer conditions [100 mM 2-(N-morpholino)ethanesulfonic acid (Mes), pH 5.9-6.7] used for the isolation of coated vesicles. However, it is now observed that under very low salt conditions (2 mM Mes, pH 5.9), it forms a homogeneous species with a sedimentation coefficient of 27 S. Increasing the salt concentration to 50 mM Mes completely converts all the 27S species into 150S baskets. Sedimentation equilibrium data show that this 27S species has a molecular weight that is 6 times that of the clathrin protomer and is the result of highly cooperative reversible self-association of the 8S protomer. Light-scattering studies show that the stabilities of 27S species and baskets (150 S or 300 S) are comparable. Fluorescent labeling of sulfhydryl groups with N-(1-anilinonaphthalenyl)maleimide indicates that the conformation of clathrin in 27S species and baskets (150 S or 300 S) is similar. Trypsin digestion reveals that in the 27S species clathrin has a conformation differing from that in both the 8S species and baskets.     

Inactivation of D-(-)-beta-hydroxybutyrate dehydrogenase by modifiers of carboxyl and histidyl groups

Data presented in this paper suggest that D-(-)-beta-hydroxybutyrate dehydrogenase (BDH) purified from bovine heart mitochondria contains an essential carboxyl group and an essential histidyl residue at or near the active site. Lactate and malate dehydrogenases, which catalyze reactions analogous to that catalyzed by BDH, also contain an aspartyl and a histidyl residue at the active site [Birktoft, J.J., & Banaszak, L.J. (1983) J. Biol. Chem. 258, 472-482]. In addition, all three enzymes contain an essential arginyl residue, apparently concerned with electrostatic interaction with their respective carboxylic acid substrates, and promote ternary adduct formation involving the enzyme, NAD, and sulfite.     

Precise location of breakpoints in a frequent reciprocal translocation between chromosomes 11 and 22

One of the most frequent chromosomal translocations in human beings is 11q/22q, which results in the "partial trisomy of 22q syndrome." However, the breakpoint on the long arms of chromosomes 11 and 22 is still a matter of controversy. In the present study, we have used chromosomes from lymphocytes of a neonate who happened to have this classical abnormality, and by R-banding prometaphase chromosomes with acridine orange it has been possible to establish that the translocation between chromosomes 11 and 22 resulted from 3:1 meiotic maternal nondisjunction. A detailed analysis of the chromosome regions involved in this translocation revealed that the breakpoints on chromosomes 11 and 22 were at 11q23.3 and 22q11.1, respectively.     

Enhancement of water status by calcium pretreatment in groundnut and cowpea plants subjected to moisture stress

Summary The effect of calcium in the water relations and tolerance to moisture deficits was tested in groundnut and cowpea. In both species, enrichment of tissue with calcium resulted in maintenance of a higher water status under stress associated with low proline accumulation. The extent of membrane damage (as reflected by the absorbance at 273 nm) was lesser in leaves of plants fed with higher levels of Ca⁺⁺ when subjected to simulated stress. The rate of water loss from the leaves of Ca⁺⁺-enriched plants was also lower. The possible role of Ca⁺⁺ in inducing membrane stability and maintenance of higher water status is discussed.     

Changes in lipoprotein composition during larval-pupal metamorphosis of an insect, Manduca sexta

During the transition from the last feeding larval stage to the pupal stage of the tobacco hornworm, Manduca sexta, significant changes occur in the properties of lipophorin, the major hemolymph lipoprotein. Within the first 24 h after cessation of feeding, the larval lipophorin (HDLp-L) is first converted to a higher density form (HDLp-W2) and then HDLp-W2 is converted to a lower density form (HDLp-W1). HDLp-W1 remains in the hemolymph until pupation, when another form, HDLp-P, with a density between HDLp-W1 and HDLp-L, is present. Although all the lipophorins contain identical apoproteins, they differ in lipid content and composition; the differences in density being primarily related to diacylglycerol content. The conversion of HDLp-L to HDLp-W1 is accompanied by a loss of hydrocarbon and uptake of carotenes. These latter changes in lipophorin composition reflect alterations in cuticular lipid composition. HDLp-L was radiolabeled in the apoproteins by injecting animals with 3H-amino acids early in the last larval stage. Subsequently HDLp-L was isolated at the end of the larval stage, HDLp-W2 and HDLp-W1 were isolated during the wandering stage, and HDLp-P was isolated after pupation. The specific activity of the apoproteins in the four lipophorins was not significantly different, suggesting that the observed alterations in lipophorin properties do not require synthesis of new apoproteins but result from retailoring the lipid composition of preexisting molecules. Examination of the hemolymph of individual animals during these transitions showed that only one species of lipoprotein was present, never a mixture of two or more species. These observations suggest that the lipoprotein conversions are precisely timed and that lipoprotein metabolism during larval development and pupation cannot be considered a static process. The unique finding of these studies was that synthesis of lipophorin apoproteins proceeds actively during the first part of the fifth instar but then ceases and does not recommence during the wandering or early pupal stages.     

Characterization of Bacteria by Particle Beam Mass Spectrometry

A technique is described for detecting and characterizing bacteria on a single-particle basis by mass spectrometry. The method involves generation of a particle beam of single whole cells which are rapidly volatilized and ionized in vacuum in the ion source of a quadrupole mass spectrometer. The particle beam can be generated, with minimal sample handling, from a naturally occurring aerosol or from a solution of bacteria that can be dispersed as an aerosol. The mass spectrum is generated by successively measuring the average intensities of different mass peaks. The average intensity is obtained by measuring the ion intensity distribution at the particular mass (m/e) for ion pulses from more than 1,000 bacteria particles. Bacillus cereus, Bacillus subtilis, and Pseudomonas putida samples were analyzed to test the capability of the instrument for differentiating among species of bacteria. Significant ion-intensity information was produced over the m/e range of 50 to 300, an improvement over previous pyrolysis-mass spectrometry results. The complex mass spectra contained a few unique peaks which could be used for the differentiation of the bacteria. A statistical analysis of the variations in peak intensities among the three bacteria provided a quantitative measure of the reproducibility of the instrument and its ability to differentiate among bacteria. The technique could lead to a new rapid method for the analysis of microorganisms and could be used for the detection of airborne pathogens on a continuous, real-time basis.     

Regulation of toxin biosynthesis by plasmids in Vibrio cholerae

Vibrio cholerae strain 569B Inaba harbouring P plasmid produced less toxin than the parent strain. To examine the effect of plasmid loss on toxin production, temperature-sensitive (ts) mutants of P, unable to replicate at 42 degrees C, were isolated. One ts plasmid was unstable at 42 degrees C and its loss yielded a cured strain that resumed a normal level of toxin biosynthesis characteristic of the plasmid-free parent strain. Toxin production was again suppressed in the cured strain after reacquisition of P plasmid. This suggested a role for plasmid-borne genes in the regulation of toxin biosynthesis. A mutant of strain 569B Inaba that produced mutant toxin was isolated by transfer of P and V plasmids. The mutant toxin was similar to choleragenoid because it did not give rise to symptoms of cholera but induced antitoxin immunity in rabbits.