Biochemical acclimation of metabolic enzymes in response to lowered temperature in tadpoles of Limnodynastes peronii

We measured the rate at which the metabolic enzymes lactate dehydrogenase (LDH), citrate synthase (CS), and cytochrome c oxidase (CCO) acclimate in the response to lowered temperature in the axial muscle of tadpoles of Limnodynastes peronii (Anura: Myobatrachidae) over 6 weeks. In addition, we measured growth rates of the tadpoles kept at both temperatures and examined the activities of these enzymes in the liver tissue of the control group and cold-acclimated group at the end of the experiment. We found that LDH acclimates in axial muscle; the differences between the control and cold-acclimated group became apparent after 21 days. After 42 days, the activity of LDH in axial muscle in the cold-acclimated group was 30% greater than the control group. Growth rates were maintained at 0.7 mm/week within both treatments despite the 10 degrees C difference in temperature between experimental groups. Both LDH and CS were increased in activity in the liver (5 and 1.3 times greater, respectively, in the cold-acclimated group). The thermal sensitivity (Q(10)) of LDH was between 20 and 30 degrees C in the cold-acclimated group (1.2+/-0.01) when compared to the control group (1.6+/-0.15). The rate at which acclimation in this species occurs is appropriate for seasonal changes in temperature, and these animals may not be able to respond to a rapid drop in temperature.     

Exploiting dendritic cells for cancer immunotherapy: genetic modification of dendritic cells

Dendritic cells (DCs) are pivotal regulators of immune reactivity and immune tolerance. The observation that DCs can recruit naive T cells has invigorated cancer immunology and led to the proposal of DCs as the basis for vaccines designed for the treatment of cancer. Designing effective strategies to load DCs with antigens is a challenging field of research. The successful realization of gene transfer to DCs will be highly dependent on the employed vector system. Here, we review various viral and non-viral gene transfer systems, and discuss their distinct characteristics and possible advantages and disadvantages in respect to their use in DC-based immunotherapy.     

Chronic prenatal exposure to cocaine alters cerebrovascular responses in newborn pigs

Maternal cocaine abuse may increase the incidence of perinatal asphyxia. In nonexposed asphyxiated neonates, decreased cerebrospinal fluid (CSF) cAMP concentrations are associated with poor neurological outcome. On the other hand, cocaine increases central nervous system (CNS) cAMP. Therefore, we hypothesized that in utero cocaine exposure may increase brain cAMP and thereby preserve cerebrovascular responses to cAMP-dependent stimuli following asphyxia. Pregnant pigs received either cocaine (1 mg/kg, i.v.) twice weekly during the last trimester or normal saline vehicle (sham-control) and were allowed to deliver vaginally at term. Cranial windows were implanted in the newborn pigs within the first week of life and used to collect CSF for cAMP determinations and to assess changes in pial arteriolar diameters (PAD). In the first part of the study, pial arteriolar responses to different vasodilator and vasoconstrictor stimuli were evaluated in piglets prior to asphyxia (n = 20). In newborn pigs exposed to cocaine, cerebrovascular responses to hypercapnia and norepinephrine were significantly exaggerated compared to controls. Then, piglets were randomly selected for the second part of the study that involved prolonged asphyxia (n = 12). In cocaine-exposed but not sham-control piglets, CSF cAMP increased markedly during asphyxia. In the sham piglets, but not the cocaine-exposed piglets, CSF cAMP fell progressively below the baseline during recovery. Cerebrovascular reactivity to cAMP-dependent stimuli (hypercapnia and isoproterenol) was preserved during recovery from asphyxia in the cocaine-exposed piglets but significantly attenuated in the sham controls. We conclude that piglets with chronic prenatal exposure to cocaine show exaggerated cerebrovascular responses to vasogenic stimuli and preserved cAMP-dependent cerebral vasoreactivity following asphyxia.     

Rsp1p, a J domain protein required for disassembly and assembly of microtubule organizing centers during the fission yeast cell cycle

Regulation of microtubule organizing centers (MTOCs) orchestrates the reorganization of the microtubule (MT) cytoskeleton. In the fission yeast Schizosaccharomyces pombe, an equatorial MTOC (eMTOC) at the cell division site disassembles after cytokinesis, and multiple interphase MTOCs (iMTOCs) appear on the nucleus. Here, we show that, upon eMTOC disassembly, small satellites carrying MTOC components such as the gamma-tubulin complex travel in both directions along interphase MTs. We identify rsp1p, an MTOC protein required for eMTOC disassembly. In rsp1 loss-of-function mutants, the eMTOC persists and organizes an abnormal microtubule aster, while iMTOCs and satellites are greatly reduced. Conversely, rsp1p overexpression inhibits eMTOC formation. Rsp1p is a J domain protein that interacts with an hsp70. Thus, our findings suggest a model in which rsp1p is part of a chaperone-based mechanism that disassembles the eMTOC into satellites, contributing to the dynamic redistribution of MTOC components for organization of interphase microtubules.     

Studies on the compaction of isolated nucleoids from Escherichia coli

The genomic DNA of Escherichia coli is contained in one or two compact bodies known as nucleoids. Isolation of typically shaped nucleoids requires control of DNA expansion, accomplished here by a modification of the polylysine-spermidine procedure. The ability to control expansion of in vitro nucleoids has application in nucleoid purification and in preparation of samples for high-resolution imaging, and may allow an increased resolution in gene localization studies. Polylysine of relatively low average molecular weight (approximately 3 kDa) is used to produce lysates containing nucleoids that are several-fold expanded relative to the sizes of in vivo nucleoids. These expanded forms can be converted to compact forms similar in dimensions to the cellular nucleoids by either a further addition of polylysine or by incubation of diluted lysates at 37 degrees C. The incubation at 37 degrees C is accompanied by autolytic degradation of most ribosomal RNA. Hyperchromism and circular dichroism spectra indicate that polylysine-DNA complexes are modified during the incubation. Compact forms of the nucleoid can be progressively reexpanded by exposure to salt solutions. Nucleoid compaction was similar in lysates made from rapidly or slowly growing cells or from cells that had been briefly treated with chloramphenicol to reduce linkages between DNA and cell envelope.     

SMP-1, a member of a new family of small myristoylated proteins in kinetoplastid parasites, is targeted to the flagellum membrane in Leishmania

The mechanisms by which proteins are targeted to the membrane of eukaryotic flagella and cilia are largely uncharacterized. We have identified a new family of small myristoylated proteins (SMPs) that are present in Leishmania spp and related trypanosomatid parasites. One of these proteins, termed SMP-1, is targeted to the Leishmania flagellum. SMP-1 is myristoylated and palmitoylated in vivo, and mutation of Gly-2 and Cys-3 residues showed that both fatty acids are required for flagellar localization. SMP-1 is associated with detergent-resistant membranes based on its recovery in the buoyant fraction after Triton X-100 extraction and sucrose density centrifugation and coextraction with the major surface glycolipids in Triton X-114. However, the flagellar localization of SMP-1 was not affected when sterol biosynthesis and the properties of detergent-resistant membranes were perturbed with ketoconazole. Remarkably, treatment of Leishmania with ketoconazole and myriocin (an inhibitor of sphingolipid biosynthesis) also had no affect on SMP-1 localization, despite causing the massive distension of the flagellum membrane and the partial or complete loss of internal axoneme and paraflagellar rod structures, respectively. These data suggest that flagellar membrane targeting of SMP-1 is not dependent on axonemal structures and that alterations in flagellar membrane lipid composition disrupt axoneme extension.     

Mitosis-specific anchoring of gamma tubulin complexes by pericentrin controls spindle organization and mitotic entry

Microtubule nucleation is the best known function of centrosomes. Centrosomal microtubule nucleation is mediated primarily by gamma tubulin ring complexes (gamma TuRCs). However, little is known about the molecules that anchor these complexes to centrosomes. In this study, we show that the centrosomal coiled-coil protein pericentrin anchors gamma TuRCs at spindle poles through an interaction with gamma tubulin complex proteins 2 and 3 (GCP2/3). Pericentrin silencing by small interfering RNAs in somatic cells disrupted gamma tubulin localization and spindle organization in mitosis but had no effect on gamma tubulin localization or microtubule organization in interphase cells. Similarly, overexpression of the GCP2/3 binding domain of pericentrin disrupted the endogenous pericentrin-gamma TuRC interaction and perturbed astral microtubules and spindle bipolarity. When added to Xenopus mitotic extracts, this domain uncoupled gamma TuRCs from centrosomes, inhibited microtubule aster assembly, and induced rapid disassembly of preassembled asters. All phenotypes were significantly reduced in a pericentrin mutant with diminished GCP2/3 binding and were specific for mitotic centrosomal asters as we observed little effect on interphase asters or on asters assembled by the Ran-mediated centrosome-independent pathway. Additionally, pericentrin silencing or overexpression induced G2/antephase arrest followed by apoptosis in many but not all cell types. We conclude that pericentrin anchoring of gamma tubulin complexes at centrosomes in mitotic cells is required for proper spindle organization and that loss of this anchoring mechanism elicits a checkpoint response that prevents mitotic entry and triggers apoptotic cell death.     

Comparison of physical and performance characteristics of NCAA Division I football players: 1987 and 2000

The purpose of this study was to compare normative data from present Division I National Collegiate Athletic Association football teams to those from 1987. Players were divided into 8 positions for comparisons: quarterbacks (QB), running backs (RB), receivers (WR), tight ends (TE), offensive linemen (OL), defensive linemen (DL), linebackers (LB), and defensive backs (DB). Comparisons included height, body mass, bench press and squat strength, vertical jump, vertical jump power, 40-yd-dash speed, and body composition. Independent t-tests were used to analyze the data with level of significance set at p < 0.01. Significant differences (p < 0.01) were found in 50 of 88 comparisons. From 1987 until 2000, Division I college football players in general have become bigger, stronger, faster, and more powerful. Further research is warranted to investigate if these trends will continue.