A 41 kDa protein isolated from maize mesocotyl cell walls immunolocalizes to plasmodesmata

Summary Plasmodesmata, dynamic pore structures that traverse plant cell walls, function in cytoplasmic transport between contiguous cells. Cell walls containing embedded plasmodesmata were isolated from mesocotyls of etiolated maize seedlings. Proteins associated with the isolated walls were separated by SDS-PAGE and antibodies were generated against a 41 kDa protein, one of several associated with this wall fraction. Immunoblot analysis showed that the 41 kDa polypeptide was also associated with other subcellular fractions obtained following tissue homogenization and differential centrifugation. The wall associated 41 kDa protein is apparently a peripheral membrane protein since it could be extracted by high salt and high pH. Silver-enhanced immunogold light microscopy showed that the 41 kDa protein was associated with the cell walls of cells both in the stele and cortex. The immunolabeling pattern was transwall and punctate. Electron microscopic immuno-gold labeling localized the polypeptide to plasmodesmata and to electron dense cytoplasmic structures that are apparently Golgi membranes. The significance of the presence of this protein in the Golgi is discussed.Abbreviations ER endoplasmic reticulum     

Analysis of a Candida albicans gene that encodes a novel mechanism for resistance to benomyl and methotrexate

Summary The pathogenic yeast, Candida albicans, is insensitive to the anti-mitotic drug, benomyl, and to the dihydrofolate reductase inhibitor, methotrexate. Genes responsible for the intrinsic drug resistance were sought by transforming Saccharomyces cerevisiae, a yeast sensitive to both drugs, with genomic C. albicans libraries and screening on benomyl or methotrexate. Restriction analysis of plasmids isolated from benomyl- and methotrexate-resistant colonies indicated that both phenotypes were encoded by the same DNA fragment. Sequence analysis showed that the fragments were nearly identical and contained a long open reading frame of 1694 bp (ORF1) and a small ORF of 446 bp (ORF2) within ORF1 on the opposite strand. By site-directed mutagenesis, it was shown that ORF1 encoded both phenotypes. The protein had no sequence similarity to any known proteins, including -tubulin, dihydrofolate reductase, and the P-glycoprotein of the multi-drug resistance family. The resistance gene was detected in several C. albicans strains and in C. stellatoidea by DNA hybridization and by the polymerase chain reaction.     

Growth Cycle of Predacious Bdellovibrios in a Host-Free Extract System and Some Properties of the Host Extract

Host-free growth and reproduction of a host-dependent strain of Bdellovibrio bacteriovorus incubated with an extract from host cells were studied. The morphological changes occurring in the cells were correlated with deoxyribonucleic acid (DNA) synthesis as measured by labeled nucleotide or orthophosphate incorporation. The host-free developmental cycle of Bdellovibrio is similar to that of the two-membered system; the early loss of flagella, the elongation into filaments, and multiple fission into flagellated progeny are typical for both host-free and intraperiplasmic development of bdellovibrios. Filament length and time of division appear to depend on the concentration of the host extract. Host extract was found to be heat stable and DNase stable, and Pronase sensitive and RNase sensitive. Addition of ribonucleic acid to the extract medium at various times during the Bdellovibrio growth cycle demonstrated that host extract is required continuously during the cycle for growth. The observations reported give a unified picture of Bdellovibrio development and allow for the suggestion that wild-type bdellovibrios depend upon the presence of some host factor for induction of DNA synthesis, whereas depletion of host factor triggers division. The ecological implications of such host dependence are discussed.     

Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche

Satellite cells are situated beneath the basal lamina that surrounds each myofiber and function as myogenic precursors for muscle growth and repair. The source of satellite cell renewal is controversial and has been suggested to be a separate circulating or interstitial stem cell population. Here, we transplant single intact myofibers into radiation-ablated muscles and demonstrate that satellite cells are self-sufficient as a source of regeneration. As few as seven satellite cells associated with one transplanted myofiber can generate over 100 new myofibers containing thousands of myonuclei. Moreover, the transplanted satellite cells vigorously self-renew, expanding in number and repopulating the host muscle with new satellite cells. Following experimental injury, these cells proliferate extensively and regenerate large compact clusters of myofibers. Thus, within a normally stable tissue, the satellite cell exhibits archetypal stem cell properties and is competent to form the basal origin of adult muscle regeneration.     

Nitric oxide-induced cell death of cerebrocortical murine astrocytes is mediated through p53- and Bax-dependent pathways

We have investigated the mechanism by which nitric oxide (NO) induces the death of mouse astrocytes. We show that NO (from donor diethylenetriamine-NO adduct) induces death with several features of apoptosis, including chromatin condensation, phosphatidylserine exposure on the outer leaflet of the plasma membrane, Bax translocation to the mitochondria and cytochrome c release, but no caspase activation or nuclear fragmentation is observed. Nitric oxide also elevates p53 expression, causing a concomitant increase in p53 serine 18 phosphorylation and p53 translocation from the cytoplasm to the nucleus. Activation of Bax and p53 is important for NO-induced apoptosis-like cell death because Bax- or p53-deficient astrocytes are much more resistant than wild-type cells to the same NO treatment. We further demonstrate that LY294002-sensitive kinases are responsible for controlling serine 18 phosphorylation of p53, thereby regulating the pro-apoptotic activity of p53 in astrocytes. While apoptosis is suppressed in the presence of LY294002, however, death by necrosis is increased, suggesting that LY294002-sensitive kinases additionally suppress a latent necrotic response to NO. We conclude that NO-induced death in astrocytes is mediated by p53- and Bax-dependent mechanisms, although full manifestation of apoptosis is aborted by concomitant inhibition of caspase activation. More generally, our data suggest that apoptotic mediators should be evaluated as the cause of cell death even in cases where a full apoptotic phenotype is lacking.     

Does menarche mark a period of elevated resting metabolic rate?

Resting metabolic rate (RMR) and body composition were measured in 44 initially nonoverweight girls at three time points relative to menarche: premenarche (Tanner stage 1 or 2), menarche (+/-6 mo), and 4 yr after menarche. Mean absolute RMR was 1,167, 1,418, and 1,347 kcal/day, respectively. Absolute RMR was statistically significantly higher at menarche than at 4 yr after menarche despite statistically significantly less fat-free mass (FFM) and fat mass (FM), suggesting an elevation in RMR around the time of menarche. The pattern of change in RMR, adjusted for FFM, log transformed FM, age, race, parental overweight, and two interactions (visit by parental overweight, parental overweight by FFM), was also considered. Adjusted RMR did not differ statistically between the visits for girls with two normal-weight parents. For girls with at least one overweight parent, adjusted RMR was statistically significantly lower 4 yr after menarche than at premenarche or menarche. Thus parental overweight may influence changes that occur in RMR during adolescence in girls.     

Venom effects on monoaminergic systems

The monoamines, dopamine, epinephrine, histamine, norepinephrine, octopamine, serotonin and tyramine serve many functions in animals. Many different venoms have evolved to manipulate monoaminergic systems via a variety of cellular mechanisms, for both offensive and defensive purposes. One common function of monoamines present in venoms is to produce pain. Some monoamines in venoms cause immobilizing hyperexcitation which precedes venom-induced paralysis or hypokinesia. A common function of venom components that affect monoaminergic systems is to facilitate distribution of other venom components by causing vasodilation at the site of injection or by increasing heart rate. Venoms of some scorpions, spiders, fish and jellyfish contain adrenergic agonists or cause massive release of catecholamines with serious effects on the cardiovascular system, including increased heart rate. Other venom components act as agonists, antagonists or modulators at monoaminergic receptors, or affect release, reuptake or synthesis of monoamines. Most arthropod venoms have insect targets, yet, little attention has been paid to possible effects of these venoms on monoaminergic systems in insects. Further research into this area may reveal novel effects of venom components on monoaminergic systems at the cellular, systems and behavioral levels.     

Diploid males and their triploid offspring in the paper wasp Polistes dominulus

Although the hymenopteran sex-determining mechanism generally results in haploid males and diploid females, diploid males can be produced via homozygosity at the sex-determining locus. Diploid males have low fitness because they are effectively sterile or produce presumably sterile triploid offspring. Previously, triploid females were observed in three species of North American Polistes paper wasps, and this was interpreted as indirect evidence of diploid males. Here we report what is, to our knowledge, the first direct evidence: four of five early male-producing Polistes dominulus nests from three populations contained diploid males. Because haploid males were also found, however, the adaptive value of early males cannot be ignored. Using genetic and morphological data from triploid females, we also present evidence that both diploid males and triploid females remain undetected throughout the colony cycle. Consequently, diploid male production may result in a delayed fitness cost for two generations. This phenomenon is particularly relevant for introduced populations with few alleles at the sex-determining locus, but cannot be ignored in native populations without supporting genetic data. Future research using paper wasp populations to test theories of social evolution should explicitly consider the potential impacts of diploid males.