Ultrahigh density microarrays of solid samples

We present a sectioning and bonding technology to make ultrahigh density microarrays of solid samples, cutting edge matrix assembly (CEMA). Maximized array density is achieved by a scaffold-free, self-supporting construction with rectangular array features that are incrementally scalable. This platform technology facilitates arrays of >10,000 tissue features on a standard glass slide, inclusion of unique sample identifiers for improved manual or automated tracking, and oriented arraying of stratified or polarized samples.     

Effects of egg yolk testosterone on growth and immunity in a precocial bird

In oviparous vertebrates, maternal steroid allocation to eggs can have important fitness consequences for the offspring. However, elevated testosterone levels are not only associated with beneficial postnatal effects, such as enhanced growth and high social status, but may also entail costs by suppressing the immune system. In this study, testosterone levels in eggs of Chinese painted quail (Coturnix chinensis) were experimentally manipulated to evaluate its effects on growth and immunocompetence. Testosterone did not affect embryonic development, body size or growth during the first 20 days. However, elevated testosterone levels during embryonic development were immunosuppressive for chicks with inherently higher growth rate. Adaptive scenarios where only beneficial effects of increased testosterone levels are considered may therefore need to be re-evaluated.     

High doses of simvastatin, pravastatin, and cholesterol reduce brain cholesterol synthesis in guinea pigs

Recent epidemiological studies suggest that inhibitors of 3-hydroxy-3-methyl-glutaryl CoA reductase, so-called statins, are effective in lowering the prevalence of Alzheimer's disease. Whether the effect of statins is due to a local inhibition of cholesterol synthesis in the brain or whether it is mediated by the reduced levels of cholesterol in the circulation is not known. In the present work, we tested the possibility that high doses of lipophilic and hydrophilic statins, simvastatin and pravastatin, respectively, or a diet high in cholesterol could affect cholesterol homeostasis in the brain of guinea pigs. The total brain cholesterol levels were not affected by high-dose simvastatin or pravastatin treatment. Significantly lower levels of the cholesterol precursor lathosterol and its ratio to cholesterol were found in the brains of simvastatin and pravastatin-treated animals. 24S-Hydroxycholesterol, the transportable form of cholesterol across the blood-brain barrier, was significantly lower in the brain of pravastatin-treated animals. Excessive cholesterol feeding resulted in higher serum cholesterol levels but did not affect total brain cholesterol level. However, de novo cholesterol synthesis in the brain seemed to be down-regulated, as indicated by lower absolute levels and cholesterol-related ratios of lathosterol compared with controls. The passage of deuterium-labeled cholesterol across the blood-brain barrier in one animal was found to be approximately 1%. Our results suggest that brain cholesterol synthesis in guinea pigs can be slightly, but significantly, influenced by high doses of lipophilic and hydrophilic statins as well as by high dietary cholesterol intake, while total brain cholesterol content and thus, cholesterol homeostasis is maintained.     

Expression profiling of the gamma-subunit isoforms of AMP-activated protein kinase suggests a major role for gamma3 in white skeletal muscle

Expression patterns of the three isoforms of the regulatory gamma-subunit of AMP-activated protein kinase (AMPK) were determined in various tissues from adult humans, mice, and rats, as well as in human primary muscle cells. Real-time PCR-based quantification of mRNA showed similar expression patterns in the three species and a good correlation with protein expression in mice and rats. The gamma3-isoform appeared highly specific to skeletal muscle, whereas gamma1 and gamma2 showed broad tissue distributions. Moreover, the proportion of white, type IIb fibers in the mouse and rat muscle samples, as indicated by real-time PCR quantification of Atp1b2 mRNA, showed a strong positive correlation with the expression of gamma3. In samples of white skeletal muscle, gamma3 clearly appeared to be the most abundant gamma-isoform. Differentiation of human primary muscle cells from myoblasts into multinucleated myotubes was accompanied by upregulation of gamma3 mRNA expression, whereas levels of gamma1 and gamma2 remained largely unchanged. However, even in these cultured myotubes, gamma2 was the most highly expressed isoform, indicating a considerable difference compared with adult skeletal muscle. Immunoblot analysis of mouse gastrocnemius and quadriceps muscle extracts precipitated with a gamma3-specific antibody showed that gamma3 was exclusively associated with the alpha2- and beta2-subunit isoforms. The observation that the AMPKgamma3 isoform is expressed primarily in white skeletal muscle, in which it is the predominant gamma-isoform, strongly suggests that gamma3 has a key role in this tissue.     

Mammalian thioredoxin reductase alters cytolytic activity of an antibacterial peptide

Granulysin is a disulfide rich 9 kDa human tumoricidal protein produced by cytolytic cells. Here we show that thioredoxin reductase (TrxR) reduced a 23-residue peptide from granulysin (GranF2), and this markedly enhanced the killing of small cell lung cancer cells (SCLC) by GranF2. Cells treated with reduced GranF2 showed rapid ATP deletion within 90 min and strong annexin V staining after 4 h incubation. SCLC with elevated TrxR levels was more sensitive to oxidized GranF2 than normal cells. The levels of TrxR are enhanced in many cancer cells, including SCLC, and it is possible that cytolytic activity of cytolytic cells on SCLC may in part be mediated by granulysin and modulated by TrxR.     

Mitochondrial toxicity detected in a health product with a boar spermatozoan bioassay

Seaweed and organic alfalfa capsules sold as "health promoting" products had repeatedly caused emesis in a consumer. Using the boar spermatozoan bioassay, the capsule contents were found to contain a toxic substance that inhibited boar sperm motility and depolarised mitochondria at low exposure concentrations of 10 microg/ml. The capsule also contained high amounts (10(5)-10(7) cfu/g), of endospore-forming bacteria and Streptomyces-like bacteria. Bacteria from the capsule produced toxic substances when cultured in the laboratory. Three different toxic responses were provoked in the spermatozoa exposed to extracts from the Streptomyces-like isolates: a) hyperpolarisation of the plasma membrane and depolarisation of the mitochondria; b) depolarisation of mitochondria similar to that caused by the capsule content extract; and c) motility inhibition, with no observed change of any cytosolic transmembrane potential. Membrane potential changes in the sperm cells exposed to the bacterial extracts were similar to those provoked by exposure to valinomycin and bafilomycin A1, to nigericin, and to oligomycin and ionomycin, respectively. Extracts prepared from Bacillus isolated from the capsule non-specifically depolarised all the cellular transmembrane potentials. The results demonstrate the potential value of a cell toxicity assay with boar spermatozoa for detecting hazardous substances in products intended for human consumption, without whole-animal exposure or using fetal calf serum for cell cultures.     

Effect of growth rate on mean microfibril angle and cross-sectional shape of tracheids of Norway spruce

The variation of the mean microfibril angle (MFA) and the shape of the cross-section of lumen with the distance from the pith in fast grown Norway spruce were studied by X-ray scattering and optical microscopy. The samples were from stems of a clone of Norway spruce [ Picea abies (L.) Karst.] grown in a fertile site at Nurmijärvi, southern Finland Both the mean MFA and the circularity index of the lumen of the fast-grown trees decreased more gradually as the distance from the pith increased than those in reference trees grown in a medium fertility site. However, in mature wood the mean MFA reached the same level in fast-grown trees as in reference trees (5°–10°) but the cross-sections of the cells remained more circular in fast-grown trees than in reference trees. The dependence of the mean MFA on the distance from the pith was similar for earlywood and latewood, but the values of the mean MFA of latewood were systematically smaller than those of earlywood. Two different X-ray diffraction geometries were compared from the points of view of biology and data analysis.     

ALK7, a receptor for nodal, is dispensable for embryogenesis and left-right patterning in the mouse

Mesendoderm formation and left-right patterning during vertebrate development depend upon selected members of the transforming growth factor beta superfamily, particularly Nodal and Nodal-related ligands. Two type I serine/threonine kinase receptors have been identified for Nodal, ALK4 and ALK7. Mouse embryos lacking ALK4 fail to produce mesendoderm and die shortly after gastrulation, resembling the phenotype of Nodal knockout mice. Whether ALK4 contributes to left-right patterning is still unknown. Here we report the generation and initial characterization of mice lacking ALK7. Homozygous mutant mice were born at the expected frequency and remained viable and fertile. Viability at weaning was not different from that of the wild type in ALK7(-/-); Nodal(+/-) and ALK7(-/-); ALK4(+/-) compound mutants. ALK7 and ALK4 were highly expressed in interdigital regions of the developing limb bud. However, ALK7 mutant mice displayed no skeletal abnormalities or limb malformations. None of the left-right patterning abnormalities and organogenesis defects identified in mice carrying mutations in Nodal or in genes encoding ActRIIA and ActRIIB coreceptors, including heart malformations, pulmonary isomerism, right-sided gut, and spleen hypoplasia, were observed in mice lacking ALK7. Finally, the histological organization of the cerebellum, cortex, and hippocampus, all sites of significant ALK7 expression in the rodent brain, appeared normal in ALK7 mutant mice. We conclude that ALK7 is not an essential mediator of Nodal signaling during mesendoderm formation and left-right patterning in the mouse but may instead mediate other activities of Nodal and related ligands in the development or function of particular tissues and organs.