In vitro modification of Candida albicans invasiveness

Candida albicans produces germ-tubes (GT) when it is incubated in animal or human serum. This dimorphism is responsible for its invasive ability.The purpose of the present paper is (1) to evaluate the ability of rat peritoneal macrophages to inhibit GT production of ingested Candida albicans, obtained from immunized rats and then activated in vitro with Candida-induced lymphokines; (2) to determinate any possible alteration of phagocytic and candidacidal activities.The phagocytes were obtained from rats immunized with viable C. albicans. Some of them were exposed to Candida-induced lymphokines in order to activate the macrophages in vitro. The monolayers of activated, immune and normal macrophages were infected with a C. albicans suspension during 4 hr.Activated macrophages presented not only the highest phagocytic and candidacidal activities but a noticeable inhibition of GT formation and incremented candidacidal activity.     

Protein kinase from Mucor rouxii

Summary Cyclic AMP binding to Mucor rouxii protein kinase holoenzyme and free regulatory subunit was measured by the classical membrane filtration technique and by equilibrium dialysis. The results obtained demonstrate that the filtration method can be used without loss of any cyclic AMP binding site. Both methods unambiguously demonstrate that the number of molecules of cyclic AMP bound to the holoenzyme are half of those bound to the regulatory subunit. This result suggests that unshielding of new cyclic AMP binding sites occurs upon dissociation of the ternary complex holoenzyme-cyclic AMP.     

Response to osmotic medium and fusicoccin by seeds of radish (Raphanus sativus) in the early phase of germination

The effect of increasing osmotic values of the medium (mannitol) on the growth and the response mechanisms of seeds of radish ( Raphanus sativus L., cv. Ton do Rosso Quarantino) during the early phase of germination was investigated in the presence or absence of fusicoccin (FC). Decreasing the water potential in the medium inhibited the growth and the evolution of protein synthesis and enhanced H⁺ extrusion, net uptake of K⁺ and malic acid synthesis. FC, which stimulates these latter functions, counteracted the inhibitory effect of the decreasing water potential of the medium on growth and protein synthesis. Neither in the absence nor in the presence of FC did decreasing water potential of the medium enhance the synthesis of soluble sugars and amino acids to support the osmotic pressure of the seeds. The osmotic and water potentials of the seeds increased during germination. FC made the increase more rapid, while mannitol kept both potentials low. The pressure potentials of the seeds also decreased with time, and both FC and mannitol enhanced this change. If the seeds were without turgor, the development of protein synthesis was blocked. The seeds counteract the effect of decreasing water potentials in the medium by: a) enhancing H⁺ extrusion (and, as a consequence, wall loosening and transport mechanisms) and the synthesis of malic acid as apparent in the presence of FC; b) regulating the osmotic potentials of the cells (with a lower dilution of the osmotic compounds present in the seeds due to the diminished uptake of water); c) controlling the growth through the effects of a) and b) on the pressure potentials (internal hydrostatic pressure) of the seeds and on protein synthesis.     

The β4Integrin Subunit Is Expressed in Mouse Fibroblasts and Modulated by Transforming Growth Factor-β1

Integrin β₄subunit is present in association with α₆chain on both normal and transformed epithelial cells. Recently α₆β₄heterodimer was found on the endothelium of medium-sized blood vessels and on immature thymocytes. In this report we show, by Northern blotting, indirect immunofluorescence, immunoprecipitation, and Western blotting, that β₄subunit is expressed also on cells of mesenchymal origin such as fibroblasts, myoblasts, and myotubes. Increased expression of α₆β₄has been related to the aggressive metastatic phenotype of human and murine carcinomas. The transforming growth factor β₁(TGF-β₁) has been found to modulate the expression of several integrins and intracellular matrix proteins, as well as to stimulate cell invasion and metastatic potential. To evaluate whether α₆β₄expression is modulated by TGF-β₁, we transfected 3T3 fibroblasts with an expression vector carrying the human TGF-β₁cDNA driven by the SV40 early promoter. We observed by indirect immunofluorescence a modification in the subcellular distribution of β₄subunit, which acquires a perinuclear localization. This finding suggests this integrin subunit correlates with the cytoskeletal reorganization induced by TGF-β₁.     

Effect of Protease Inhibitors on Early Events of Apoptosis

Proteolysis is an early event of apoptosis which appears to be associated with activation of the endonuclease which is responsible for internucleosomal DNA cleavage. The present study was designed to reveal the possible role of proteolysis in other early events, such as chromatin condensation, nuclear breakdown, and destabilization ofin situDNA double-stranded structure. Apoptosis of human leukemic HL-60 cells and rat thymocytes was induced by different agents, including DNA topoisomerase inhibitors, an RNA antimetabolite, and the glucocorticosteroid, prednisolone. DNA degradation was evaluated by pulsed field and conventional gel electrophoresis and by the presence ofin situDNA strand breaks. DNA stability was estimated by the measure of its sensitivityin situto denaturation. Chromatin condensation, nuclear breakdown, and other morphological changes were monitored by interference contrast and UV microscopy following cell staining with the DNA-specific fluorochrome 4′,6-diamidino-2-phenylindole. Several irreversible or reversible serine protease inhibitors prevented internucleosomal DNA degradation, nuclear breakdown, and destabilization of DNA double-stranded structure. The effective inhibitors, however, did not prevent the onset of chromatin condensation, nor the loss of the fine structural framework, nor the initial step of DNA cleavage generating DNA fragments of ≥50 kb in size. The data indicate that in both cell systems the activity of proteases sensitive to the inhibitors tested is needed for internucleosomal DNA cleavage to occur. The data also suggest that these proteases may be involved in dissolution of the nuclear envelope. Because nuclear matrix proteins and histones stabilize DNAin situ,and the decrease in DNA stability which occurs during apoptosis is precluded by the inhibitors, it is likely that serine proteases may degrade DNA stabilizing proteins. The activity of these proteases, however, appears needed neither for DNA cleavage to ≥50-kb fragments nor for the onset of chromatin condensation which is associated with dissolution of the structural framework of the nucleus.     

Heterotrophic bacteria, activity and bacterial diversity in two coastal lagoons as detected by culture and 16S rRNA genes PCR amplification and partial sequencing

Activity and numbers of heterotrophic bacteria have indicated that, as expected, Prevost Lagoon is more eutrophic than Arcachon Bay. Amplification and sequence analysis of the 16S rRNA genes from DNA samples extracted directly from the environment allow the determination of phylogenetic relationships among members of microbial communities in natural ecosystems without the need for cultivation. Analysis of partial 16S rRNA gene sequences obtained from Stations A and 11 revealed that, in both environments, a relatively large number of clones related to Cytophaga/FlexibacterBacteroides as well as to -Proteobacteria were found. One hundred percent similarity with the sequences of the data bases were not found for any of the more than a hundred clones studied. In fact for most clones maximum similarity was below 95% for the nucleotide series sequenced. Similarity was not higher with any of the sequences found for the 14 isolates (pure cultures) obtained from the same samples. Redundancy, i.e. number of identical sequences, was higher in the samples from Arcachon. In addition, sequences related to representatives of ten major phylogenetic branches of Eubacteria were obtained from Prevost Lagoon, however only five branches were represented by the data from Arcachon. These findings indicate a higher bacterial diversity in Prévost Lagoon.     

Nuclear matrix involvement in sperm head structural organization

Summary— In the sperm nuclei the DNA is packaged into a highly condensed form and is not organized into nucleosome and solenoid but is bound and stabilized mainly by the protamines that arrange the DNA in an almost crystalline state. As demonstrated for somatic cells, the sperm DNA has been reported to be organized in loop domains attached to the nuclear matrix structures. However, the possible role of the sperm head matrix in maintaining the loop organization in absence of a typical nucleosomal structures has not been fully elucidated. By using in situ nick translation at confocal and electron microscope level, we analyzed the organization of the DNAprotamine complex and its association with the sperm nuclear matrix. The data obtained indicate that the chromatin organization in sperm nuclei is maintained during the sperm condensation by means of interactions with the nuclear matrix at fixed sites. The fine stucture of sperm nucleus and of sperm nuclear matrix, investigated on sections and replicas of freeze-fractured specimens, suggests that the lamellar array, observed by freeze-fracturing in the sperm nuclei, could depend on the inner matrix which presents a regular organization of globular structures possibly involved in the maintenance of chromatin domains in highly condensed sperm nuclei also.     

Genetic variation in North Africa and Eurasia: Neolithic demic diffusion vs. paleolithic colonisation

The hypothesis that both genetic and linguistic similarities among Eurasian and North African populations are due to demic diffusion of neolithic farmers is tested against a wide database of allele frequencies. Demic diffusion of farming and languages from the Near East should have determined clines in areas defined by linguistic criteria; the alternative hypothesis of cultural transmission does not predict clines. Spatial autocorrelation analysis shows significant gradients in three of the four linguistic families supposedly affected by neolithic demic diffusion; the Afroasiatic family is the exception. Many such gradients are not observed when populations are jointly analyzed, regardless of linguistic classification. This is incompatible with the hypothesis that major cultural transformations in Eurasia (diffusion of related languages and spread of agriculture) took place without major demographic changes. The model of demic diffusion seems therefore to provide a mechanism explaining coevolution of linguistic and biological traits in much of the Old World. Archaeological, linguistic, and genetic evidence agree in suggesting a multidirectional process of gene flow from the Near East in the neolithic. However, the possibility should be envisaged that some allele frequency patterns can predate the neolithic and depend on the initial spread of Homo sapiens sapiens from Africa into Eurasia. © 1994 Wiley-Liss, Inc.     

Physiologic target of the Air-1 trans-activator revealed by stable transfection assay

RJ 2.2.5 is a human B cell mutant, derived from Raji cells, which has lost expression of major histocompatibility complex (MHC) class II genes because of a defect in the AIR1 locus function. The MHC class II-positive phenotype can be restored by introducing an active AIR1 locus or its mouse equivalent, Air-1. An example of the latter is the H4 cell hybrid, derived by somatic cell fusion between RJ 2.2.5 and mouse class II-positive spleen cells. H4 contains a single mouse chromosome, autosome 16, in which the Air-1 locus maps, and an entire RJ 2.2.5-derived genome. In the present study we show that the physiologic target of the Air-1 locus product is contained within a limited HLA-DRA promoter sequence of 300 base pairs, encompassing the crucial Y, X, and W cis-acting elements. A plasmid construct, pDRA300neo, containing the HLA-DRA promoter sequence which drives the expression of the neomycin resistance gene, has been stably integrated in the genome of the H4 hybrid. Transfectants selected in the presence of G418 retain mouse chromosome 16 and express the DR genes. On the other hand, transfectants grown in a non-selective medium segregate mouse chromosome 16; this is accompanied by a loss of DRA gene expression and G418 resistance, although pDRA300neo is still integrated in the genome. These results offer scope for using this experimental model to clone the Air-1 gene in a straightforward way. Correspondence to: R. S. Accolla.