Sortase from Staphylococcus aureus does not contain a thiolate-imidazolium ion pair in its active site

Many surface proteins are anchored to the cell wall by the action of sortase enzymes, a recently discovered family of cysteine transpeptidases. As the surface proteins of human pathogens are frequently required for virulence, the sortase-mediated anchoring reaction represents a potential target for new anti-infective agents. It has been suggested that the sortase from Staphylococcus aureus (SrtA), may use a similar catalytic strategy as the papain cysteine proteases, holding its Cys184 side chain in an active configuration through a thiolate-imidazolium ion interaction with residue His120. To investigate the mechanism of transpeptidation, we have synthesized a peptidyl-vinyl sulfone substrate mimic that irreversibly inhibits SrtA. Through the study of the pH dependence of SrtA inhibition and NMR, we have estimated the pKas of the active site thiol (Cys184) and imidazole (His120) to be approximately 9.4 and 7.0, respectively. These measurements are inconsistent with the existence of a thiolate-imidazolium ion pair and suggest a general base catalysis mechanism during transpeptidation.     

The TIM gene family: emerging roles in immunity and disease

The search for cell-surface markers that can distinguish T helper 1 (T(H)1) cells from T(H)2 cells has led to the identification of a new gene family, encoding the T-cell immunoglobulin mucin (TIM) proteins, some of which are differentially expressed by T(H)1 and T(H)2 cells. The role of the TIM-family proteins in immune regulation is just beginning to emerge. Here, we describe the various TIM-family members in mice and humans, and discuss the genetic and functional evidence for their role in regulating autoimmune and allergic diseases.     

KLP-18, a Klp2 kinesin,is required for assembly of acentrosomal meiotic spindles in Caenorhabditis elegans

The proper segregation of chromosomes during meiosis or mitosis requires the assembly of well organized spindles. In many organisms, meiotic spindles lack centrosomes. The formation of such acentrosomal spindles seems to involve first assembly or capture of microtubules (MTs) in a random pattern around the meiotic chromosomes and then parallel bundling and bipolar organization by the action of MT motors and other proteins. Here, we describe the structure, distribution, and function of KLP-18, a Caenorhabditis elegans Klp2 kinesin. Previous reports of Klp2 kinesins agree that it concentrates in spindles, but do not provide a clear view of its function. During prometaphase, metaphase, and anaphase, KLP-18 concentrates toward the poles in both meiotic and mitotic spindles. Depletion of KLP-18 by RNA-mediated interference prevents parallel bundling/bipolar organization of the MTs that accumulate around female meiotic chromosomes. Hence, meiotic chromosome segregation fails, leading to haploid or aneuploid embryos. Subsequent assembly and function of centrosomal mitotic spindles is normal except when aberrant maternal chromatin is present. This suggests that although KLP-18 is critical for organizing chromosome-derived MTs into a parallel bipolar spindle, the order inherent in centrosome-derived astral MT arrays greatly reduces or eliminates the need for KLP-18 organizing activity in mitotic spindles.     

Distinct regulatory effects of the Na,K-ATPase gamma subunit

The two variants of the gamma subunit of the rat renal sodium pump, gamma(a) and gamma(b), have similar effects on the Na,K-ATPase. Both increase the affinity for ATP due to a shift in the enzyme's E(1) <--> E(2) conformational equilibrium toward E(1). In addition, both increase K(+) antagonism of cytoplasmic Na(+) activation. To gain insight into the structural basis for these distinct effects, extramembranous N-terminal and C-terminal mutants of gamma were expressed in rat alpha1-transfected HeLa cells. At the N terminus, the variant-distinct region was deleted (gammaNDelta7) or replaced by alanine residues (gammaN7A). At the C terminus, four (gamma(a)CDelta4) or ten (gamma(a)CDelta10) residues were deleted. None of these mutations abrogates the K(+)/Na(+) antagonism as evidenced in a similar increase in K'(Na) seen at high (100 mm) K(+) concentration. In contrast, the C-terminal as well as N-terminal deletions (gammaNDelta7, gamma(a)CDelta4, and gamma(a)CDelta10) abolished the decrease in K'(ATP) seen with wild-type gamma(a) or gamma(b). It is concluded that different regions of the gamma chain mediate the distinct functional effects of gamma, and the effects can be long-range. In the transmembrane region, the impact of G41R replacement was analyzed since this mutation is associated with autosomal dominant renal Mg(2+)-wasting in man (Meij, I. C., Koenderink, J. B., van Bokhoven, H., Assink, K. F. H., Groenestege, W. T., de Pont, J. J. H. H. M., Bindels, R. J. M., Monnens, L. A. H., Van den Heuvel, L. P. W. J., and Knoers, N. V. A. M. (2000) Nat. Genet. 26, 265-266). The results show that Gly-41 --> Arg prevents trafficking of gamma but not alphabeta pumps to the cell surface and abrogates functional effects of gamma on alphabeta pumps. These findings underscore a potentially important role of gamma in affecting solute transport, in this instance Mg(2+) reabsorption, consequent to its primary effect on the sodium pump.     

Motivations and concerns of women considering genetic testing for breast cancer: a comparison between affected and at-risk probands

Since the discovery of the BRCA1 and BRCA2 genes, there has been an increasing demand for breast cancer risk assessment programs. In an effort to understand and serve the population such programs target better, several studies have identified factors influencing high-risk women to pursue breast cancer risk assessment and genetic testing services; none, however, has focused on how the motivations and concerns of at-risk women may differ from their previously affected counterparts, who are typically the initial members of their families to undergo genetic testing. The majority of both previously affected and unaffected women felt that preventative surgery decisions, surveillance practices, the assessment of children's risks, and increased breast cancer anxiety were "more important" or "very important" issues regarding their thoughts about genetic testing. Significantly more affected women deemed family members' opinions "more" or "very important" (p < 0.01). Opinions concerning insurance and employment discrimination did not vary significantly between groups; however, a larger percentage of affected women felt this issue was of importance. Although all issues above should be addressed with women seeking cancer risk assessment and genetic testing, this research may help health care providers to gain a greater understanding of how the motivators and concerns of high-risk women can differ with personal cancer status so that referral, counseling, and education can be executed optimally.     

Effect of the rearing diet on gene expression of antimicrobial peptides in Hermetia illucens (Diptera: Stratiomyidae)

Insect proteins have been proposed for human and animal food production. Safeguarding the health status of insects in mass rearing allows to obtain high-quality products and to avoid severe economic losses due to entomopathogens. Therefore, new strategies for preserving insect health must be implemented. Modulation of the insect immune system through the diet is one such strategy. We evaluated gene expression of two antimicrobial peptides (one defensin and one cecropin) in Hermetia illucens (L.) (Diptera: Stratiomyidae) reared on different diets. Analyses were performed on prepupae and 10-day-old larvae reared on cereal- and municipal organic waste-based diets and on only prepupae reared on a cereal-based diet supplemented with sunflower, corn, or soybean oil. The inclusion of sunflower oil at different points in the cereal-based diet was also evaluated. Moreover, diet-driven differences in the inhibitory activity of the hemolymph were tested against Escherichia coli DH5α and Micrococcus yunnanensis HI55 using diffusion assays in solid media. Results showed that a municipal organic waste-based diet produced a significant overexpression of antimicrobial peptides only in prepupae. Inclusion of vegetable oils caused an upregulation of at least one peptide, except for the corn oil. Higher expression of both genes was observed when sunflower oil was added 5 days before pupation. All hemolymph samples showed an inhibitory activity against bacteria colonies. Our results suggest that municipal organic waste-based diet and vegetable oil-added diet may successfully impact the immune system of H. illucens. Such alternatives may also exist for other species of economic interest.     

Expression of lactate dehydrogenase isozyme 5 (LDH-5) in cultured mouse blastocysts in the absence of implantation and outgrowth

Extensive extraction studies with Triton X-100 revealed only LDH-1 (B₄) but no trace of LDH-5 (A₄) in one-cell and two-cell mouse and rat embryos. The LDH isozyme pattern of preimplantation mouse embryos changes from the maternally inherited B subunit isozyme (LDH-1) to a pattern dominated by LDH-5 when mouse blastocysts are cultured under conditions that prevent hatching but allow trophoblast giant cell transformation. During differentiation of mouse blastocysts in vitro, implantation is therefore not essential for the appearance of the A subunit form of LDH (LDH-5) coded for by the embryonic genome. Mechanisms controlling the expression of LDH-5 in mouse blastocysts during in vivo development are discussed.This work was supported by grants of the Deutsche Forschungsgemeinschaft awarded to the Sonderforschungsbereich 29—Embryonale Entwicklung and Differenzierung.     

The influence of externally applied organic substances on phloem translocation in detached maize leaves

Summary Solutions of organic substances show differing influences on the direction of phloem transport of ¹⁴C-labeled assimilates in predarkened maize leaf strips, when externally applied to one end of the strip. One group of substances pushes the assimilates away from the site of application. Examples of this group are 75 mM solutions of sucrose, trehalose, maltose, D-glucose, D-fructose, glucose-6-phosphate, raffinose and galactose. There is strong evidence that pushing substances are taken up from the apoplast and loaded into the phloem. Another group of substances attracts the assimilates, it seems to pull the assimilates in direction to the site of application. Examples of this second group are 75 mM solutions of arabinose, melibiose, myo-inositol, D-mannitol, polyethylene glycol 2000, and Na₂-EDTA (ethylene-diaminetetraacetate). The pulling substances obviously are not taken up into living cells. It is assumed that they accumulate in the apoplast and build up a water stress (water potential), which is counteracted by an increase of solute concentration in the parenchyma, thus creating a sink for assimilates. A third group of substances shows inert behaviour, having no perceptible influence on phloem transport, at least not, when applied as 75 mM solutions. At concentrations of more than 300 mM, inert substances tend to attract assimilates like those of the second group. Inert substances are xylose, sorbose, 2-deoxy-D-glucose, mannose and sorbitol.abbreviation EDTA ethylenediaminetetraacetate Supported by Deutsche Forschungsgemeinschaft     

Repetitive firing deficits and reduced sodium current density in retinal ganglion cells developing in the absence of BDNF.

Previous work by Cellerino et al. has shown that chronic absence of brain-derived neurotrophic factor (BDNF) resulted in hypomyelination of the optic nerve. Since myelination is influenced by neuronal activity, it is possible that a deficiency in BDNF during early development could alter the firing properties of retinal neurons. To test this hypothesis, patch-clamp recordings were performed in retinal whole mounts from BDNF-deficient (bdnf-/-), heterozygote (bdnf+/-) or wild-type control mice (bdnf+/+). Ganglion cell layer neurons (RGNs) were tested at different age [postnatal day (P)1-11] for their ability to encode graded depolarization with variable action potential frequency. At all developmental ages examined, RGNs exhibiting frequency coding were less frequently encountered in bdnf-/- than in bdnf+/+ mice. At P1, none of the RGNs from bdnf-/- mice displayed repetitive firing compared to 50% in bdnf+/+ mice, and by P7-11, only 50% of bdnf-/- RGNs exhibited repetitive firing compared to 100% in bdnf+/+ mice. Moreover, in bdnf-/- RGNs repetitive discharge was characterized by a reduced frequency increment per current change. Acquisition of repetitive firing was paralleled by a decrease in input resistance and a steep increase of sodium current density. In bdnf-/- mice, the onset of this increase occurred at later stages of development than in wild-type controls (bdnf-/-: P6-11; bdnf+/+: P2-6). The discharge pattern of P7-11 bdnf-/- RGNs resembled that of RGNs in neonatal wild-type mice and was mimicked by acute application of a Ca(2+) channel blocker. We conclude that BDNF plays an important role in the ontogeny of repetitive firing of RGNs.