Demonstration and cytochemical analysis of anionic sites on ejaculated boar spermatozoa: a scanning electron microscopy study using cationised colloidal gold

The plasmalemma of spermatozoa bears negative charges as is the case for most mammalian cells. This has been concluded from the sperm cell's electrophoretic behaviour and from labelling experiments with various cationic probes followed by transmission electron microscopy of ultrathin sections. An overall view of the cell surface, however, is necessary in order to assess the distribution and density of the anionic sites adequately. We, therefore, used scanning electron microscopy in combination with cationised colloidal gold labelling to analyse the presence of anionic sites on ejaculated boar spermatozoa. Incubations were performed at pH 3.5, 2.5 and 1.0. Labelling was specific and bound gold particles were unequivocally identified using the backscattered electron signal. The chemical nature of the anionic sites involved was investigated by treating spermatozoa with pronase, phosphatase and neuraminidase as well as by methylation, acid hydrolysis and beta-elimination prior to cationised gold labelling. Our results suggest that besides phosphates, carboxyl groups are predominantly accountable for the binding of cationised colloidal gold. Presumptive macromolecules bearing these anionic sites are phospholipids and sialic acid residues. The combination of methods presented herewith should be of value in order to elucidate charge interactions which have been shown to play a role in cellular recognition events and adhesion.     

Natural patterns of neural activity: how physiological mechanisms are orchestrated to cope with real life

Physiological mechanisms of neuronal information processing have been shaped during evolution by a continual interplay between organisms and their sensory surroundings. Thus, when asking for the functional significance of such mechanisms, the natural conditions under which they operate must be considered. This has been done successfully in several studies that employ sensory stimulation under in vivo conditions. These studies address the question of how physiological mechanisms within neurons are properly adjusted to the characteristics of natural stimuli and to the demands imposed on the system being studied. Results from diverse animal models show how neurons exploit natural stimulus statistics efficiently by utilizing specific filtering capacities. Mechanisms that allow neurons to adapt to the currently relevant range from an often immense stimulus spectrum are outlined, and examples are provided that suggest that information transfer between neurons is shaped by the system-specific computational tasks in the behavioral context.     

Geranylgeranyltransferase I of Candida albicans: null mutants or enzyme inhibitors produce unexpected phenotypes

Geranylgeranyltransferase I (GGTase I) catalyzes the transfer of a prenyl group from geranylgeranyl diphosphate to the carboxy-terminal cysteine of proteins with a motif referred to as a CaaX box (C, cysteine; a, usually aliphatic amino acid; X, usually L). The alpha and beta subunits of GGTase I from Saccharomyces cerevisiae are encoded by RAM2 and CDC43, respectively, and each is essential for viability. We are evaluating GGTase I as a potential target for antimycotic therapy of the related yeast, Candida albicans, which is the major human pathogen for disseminated fungal infections. Recently we cloned CaCDC43, the C. albicans homolog of S. cerevisiae CDC43. To study its role in C. albicans, both alleles were sequentially disrupted in strain CAI4. Null Cacdc43 mutants were viable despite the lack of detectable GGTase I activity but were morphologically abnormal. The subcellular distribution of two GGTase I substrates, Rho1p and Cdc42p, was shifted from the membranous fraction to the cytosolic fraction in the cdc43 mutants, and levels of these two proteins were elevated compared to those in the parent strain. Two compounds that are potent GGTase I inhibitors in vitro but that have poor antifungal activity, J-109,390 and L-269,289, caused similar changes in the distribution and quantity of the substrate. The lethality of an S. cerevisiae cdc43 mutant can be suppressed by simultaneous overexpression of RHO1 and CDC42 on high-copy-number plasmids (Y. Ohya et al., Mol. Biol. Cell 4:1017, 1991; C. A. Trueblood, Y. Ohya, and J. Rine, Mol. Cell. Biol. 13:4260, 1993). Prenylation presumably occurs by farnesyltransferase (FTase). We hypothesize that Cdc42p and Rho1p of C. albicans can be prenylated by FTase when GGTase I is absent or limiting and that elevation of these two substrates enables them to compete with FTase substrates for prenylation and thus allows sustained growth.     

Membrane Transport Generated by the Osmotic and Hydrostatic Pressure. Correlation Relation for Parameters L p, σ, and ω

Standard approach to membrane transport generated by osmotic andhydrostatic pressures, developed by Kedem and Katchalsky, is based onprinciples of thermodynamics of irreversible processes. In this paper wepropose an alternative technique. We derive transport equations from fewfairly natural assumptions and a mechanistic interpretation of the flows.In particular we postulate that a sieve-type membrane permeability isdetermined by the pore sizes and these are random within certain range.Assuming that an individual pore is either permeable or impermeable tosolute molecules, the membrane reflection coefficient depends on the ratioof permeable and impermeable pores. Considering flows through permeableand impermeable pores separately, we derive equations for the total volumeflux, solute flux and the solvent flux across the membrane. Comparing themechanistic equations to the Kedem-Katchalsky equations we find the formereasier to interpret physically. Based on the mechanistic equations we alsoderive a correlation relation for the membrane transport parameters L _p,, and . This relation eliminates the need for experimentaldetermination of all three phenomenological parameters, which in somecases met with considerable difficulties.     

Allogeneic bone marrow transplantation with co-stimulatory blockade induces macrochimerism and tolerance without cytoreductive host treatment

Allogeneic bone marrow transplantation (in immunocompetent adults) has always required cytoreductive treatment of recipients with irradiation or cytotoxic drugs to achieve lasting engraftment at levels detectable by non-PCR-based techniques ('macrochimerism' or 'mixed chimerism'). Only syngeneic marrow engraftment at such levels has been achieved in unconditioned hosts. This requirement for potentially toxic myelosuppressive host pre-conditioning has precluded the clinical use of allogeneic bone marrow transplantation for many indications other than malignancies, including tolerance induction. We demonstrate here that treatment of naive mice with a high dose of fully major histocompatibility complex-mismatched allogeneic bone marrow, followed by one injection each of monoclonal antibody against CD154 and cytotoxic T-lymphocyte antigen 4 immunoglobulin, resulted in multi-lineage hematopoietic macrochimerism (of about 15%) that persisted for up to 34 weeks. Long-term chimeras developed donor-specific tolerance (donor skin graft survival of more than 145 days) and demonstrated ongoing intrathymic deletion of donor-reactive T cells. A protocol of high-dose bone marrow transplantation and co-stimulatory blockade can thus achieve allogeneic bone marrow engraftment without cytoreduction or T-cell depletion of the host, and eliminates a principal barrier to the more widespread use of allogeneic bone marrow transplantation. Although efforts have been made to minimize host pre-treatment for allogeneic bone marrow transplantation for tolerance induction, so far none have succeeded in eliminating pre-treatment completely. Our demonstration that this can be achieved provides the rationale for a safe approach for inducing robust transplantation tolerance in large animals and humans.