nanos1: a mouse nanos gene expressed in the central nervous system is dispensable for normal development

A mouse nanos (nanos1) gene was cloned and its function was examined by generating a gene-knockout mouse. The nanos1 gene encodes an RNA-binding protein, which contains a putative zinc-finger motif that exhibits similarity with other nanos-class genes in vertebrates and invertebrates. Although nanos1 is not detected in primordial germ cells, it is observed in seminiferous tubules of mature testis. Interestingly, maternally expressed nanos1 is observed in substantial amounts in oocytes, but the amount of maternal RNA is rapidly reduced after fertilization, and the transient zygotic nanos1 expression is observed in eight-cell embryos. At 12.5 days postcoitum, nanos1 is re-expressed in the central nervous system and the expression continues in the adult brain, in which the hippocampal formation is the predominant region. The nanos1 -deficient mice develop to term without any detectable abnormality and they are fertile. No significant neural defect is observed in terms of their behavior to date.     

Efflux of sphingomyelin, cholesterol, and phosphatidylcholine by ABCG1

Cholesterol and phospholipids are essential to the body, but an excess of cholesterol or lipids is toxic and a risk factor for arteriosclerosis. ABCG1, one of the half-type ABC proteins, is thought to be involved in cholesterol homeostasis. To explore the role of ABCG1 in cholesterol homeostasis, we examined its subcellular localization and function. ABCG1 and ABCG1-K120M, a WalkerA lysine mutant, were localized to the plasma membrane in HEK293 cells stably expressing ABCG1 and formed a homodimer. A stable transformant expressing ABCG1 exhibited efflux of cholesterol and choline phospholipids in the presence of BSA, and the cholesterol efflux was enhanced by the presence of HDL, whereas cells expressing ABCG1-K120M did not, suggesting that ATP binding and/or hydrolysis is required for the efflux. Mass and TLC analyses revealed that ABCG1 and ABCA1 secrete several species of sphingomyelin (SM) and phosphatidylcholine (PC), and SMs were preferentially secreted by ABCG1, whereas PCs were preferentially secreted by ABCA1. These results suggest that ABCA1 and ABCG1 mediate the lipid efflux in different mechanisms, in which different species of phospholipids are secreted, and function coordinately in the removal of cholesterol and phospholipids from peripheral cells.     

Inhibition of poly(ADP-ribose) polymerase activity by Bcl-2 in association with the ribosomal protein S3a.

We screened a human lymphocyte cDNA library using the yeast two-hybrid system and an automodification domain of PARP as a probe. The DNA sequence of an isolated clone (clone 3-9) was identical to the partial cDNA sequence of the human ribosomal protein S3a. We confirmed that PARP interacts with clone 3-9 by performing binding studies using a GST-3-9 fusion protein as bait. We also demonstrated that native S3a in nuclear extracts of HL-60 cells interacts with the automodification domain of PARP and that PARP from nuclear extracts is coprecipitated with the GST-3-9 fusion protein. Furthermore, we demonstrated that Bcl-2 interacts with PARP in association with S3a and that the interaction of S3a and Bcl-2 with PARP causes a significant decrease in PARP activity. Since Bcl-2 failed to inhibit PARP activity in the absence of S3a, we suggest that Bcl-2 together with S3a prevents apoptosis probably by inhibiting PARP activity.     

Correlation between the leaf turnover rate and anti-herbivore defence strategy (balance between ant and non-ant defences) amongst ten species of <Emphasis Type=Italic>Macaranga</Emphasis> (Euphorbiaceae)

We measured variation in the intensities of ant and non-ant anti-herbivore defences amongst ten Macaranga species in Sarawak, Malaysia. Intensities of non-ant defences were estimated by measuring effects of fresh leaves (provided as food) of these Macaranga species on survival of common cutworm larvae [Spodoptera litura (Fabricius), Lepidoptera: Noctuidae]. Intensities of ant defences were estimated by measuring ant aggressiveness in the presence of artificial damage inflicted on plants. As part of our examination of non-ant defences, we measured leaf toughness (punch strength, by penetrometry), and the contents of total phenols and condensed tannin. We demonstrated interspecific variation in intensities of both ant and non-ant defences amongst ten Macaranga species and showed that the rank order of ant defence intensity was negatively correlated with the intensity of non-ant defence. We also found that the balance between ant and non-ant defence intensity was correlated with the rates of leaf turnover and shoot growth. Species investing more in ant defence tended to have higher leaf turnover rates. Macaranga species that occur preferentially in shadier microhabitats had lower leaf turnover rates, suggesting that non-ant defences are more cost-effective in more shade-tolerant species. Our results also suggest that the total intensity of non-ant defences is positively correlated with both leaf toughness and total phenol content.     

Alterations in cardiac function and subcellular membrane activities after hypervitaminosis D3

The present study was designed to induce massive accumulation of calcium in the myocardium and to evaluate the effect of calcium overload on myocardial contractile function and biochemical activity of cardiac subcellular membranes. Rats were treated with an oral administration of 500,000 units/kg of vitamin D₃ for 3 consecutive days, and their hearts were sampled on the 5th day for biochemical analysis. On the 4th and 5th days, heart rate, mean aortic pressure, left ventricular systolic pressure and left ventricular dP/dt were significantly lowered in vitamin D₃-treated rats, demonstrating the existence of appreciable myocardial contractile dysfunction. Marked increases in the myocardial calcium (67-fold increase) and mitochondrial calcium contents (24-fold increase) were observed by hypervitaminosis D3. Mitochondrial oxidative phosphorylation and ATPase activity were significantly reduced by this treatment. A decline in sarcolemmal Na⁺, K⁺-ATPase activity was also observed, while relatively minor or insignificant changes in calcium uptake and ATPase activities of sarcoplasmic reticulum were detectable. Electron microscopic examination revealed calcium deposits in the mitochondria after vitamin D₃ treatment. The results suggest that hypervitaminosis D₃ produces massive accumulation of calcium in the myocardium, particularly in the cardiac mitochondrial membrane, which may induce an impairment in the mitochondrial function and eventually may lead to a failure in the cardiac contractile function.     

DMPO-Alkyl radical spin trapping: an in situ radiolysis steady-state ESR study

Short-lived free radicals formed in the reaction of 11 substrates and radiolytically produced hydroxyl radicals were trapped successfully with 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) in dilute aqueous solution. The in situ radiolysis steady-state ESR spectra of the spin adducts were analyzed to determine accurate ESR parameters for these spin adducts in a uniform environment. Parent alkyl radicals include methyl, ethyl, 1-propyl and 2-propyl (1-methylethyl). Hydroxyalkyl parent radicals were hydroxymethyl, hydroxyethyl, 2-hydroxy-2-propyl (1-methyl-1-hydroxyethyl), 1-hydroxypropyl and 2-hydroxy-2-methylpropyl. Carboxyl radical (carbon dioxide anion, formate radical) and sulfite anion radical were the sigma radicals studied. The DMPO spin adduct of 1-propyl was identified for the first time. For most spin adducts, g factors were also determined for the first time. In DMPO spin adducts of hydroxyalkyl radicals, nitrogen and C(2)-proton hyperfine coupling constants are smaller than those of alkyl radical adducts; the hydroxyalkyl spin adducts possess larger g values than their unsubstituted counterparts. These changes are ascribed to the spread of pi conjugation to include the hydroxyl group. Strong evidence of spin addend-aminoxyl group interaction can be seen in the asymmetrical line shapes in the hydroxyethyl and the hydroxypropyl spin adducts.     

Immunocytochemical studies on the pancreatic endocrine cells in the Japanese newt (Cynopus pyrrhogaster)]

Pancreatic endocrine cells were examined by light and electron microscopic immunocytochemistry to discuss the co-localization of peptides in one cell type. A cells were irregular in shape with an occasional long cytoplasmic process, and contained glucagon-immunoreactive granules with various contours. These granules were 160-300nm in diameter with various density, and also immunoreactive to anti-human pancreatic polypeptide (PP) serum. A part of them were further immunoreactive to anti-somatostatin serum. B cells were round to elliptical in shape, and often aggregated around the capillaries. Granules of B cells were round to irregular in shape, 270-410 nm in diameter, and immunoreactive to anti-insulin serum. D cells were irregular in shape with meager cytoplasm, and contained somatostatin-immunoreactive granules. These granules were ovoid or teardrop in shape, 140-250nm in longitudinal diameter, and immunoreactive to both anti-somatostatin and anti-human PP sera. PP cells were round to spindle-shaped, and contained human PP-immunoreactive round granules 150-35nm in diameter. These findings reveal the existence of at least 4 types of endocrine cells secreting glucagon, insulin, somatostatin, and PP, respectively, in the newt pancreas, and suggest the co-localization of some of these peptides in one cell type.     

Cloning and sequencing of the human homeobox gene HOX4A.

The HOX4A gene, one of the homeobox-containing genes on human chromosome 2, has been isolated by screening a genomic cosmid library with a HOX4B cDNA probe. The HOX4A gene consists of at least two exons separated by a long intron of 1860 bp. According to conceptual translation, the HOX4A protein is predicted to be composed of 416 amino acid residues. Interestingly, the HOX4A protein has a sequence, Pro-Ala-Ser-Gln-Ser-Pro-Glu-Arg-Ser, eight amino acids downstream from the homeodomain, which is similar to that containing a phosphorylation site in pp60c-src, Pro-Ala-Ser-Gln-Thr-Pro-Asn-Lys-Thr. However, the HOX2G protein, which exhibits a paralogous relationship with the HOX4A protein, does not possess the sequence which is similar to that in pp60c-src. A comparison of the predicted HOX4A protein with the HOX2G protein revealed four regions of amino acid sequence similarities: an N-terminal tetrapeptide, a pentapeptide (pre-box) upstream of the homeodomain, the homeodomain and a C-terminal octapeptide.     

Three-dimensional common-feature hypotheses for octopamine agonist 2-(arylimino)imidazolidines.

Three-dimensional pharmacophore hypotheses were built from a set of 10 octopamine (OA) agonist 2-(Arylimino)imidazolidines (AIIs), 2-(Arylimino)thiazolidines (AITs) and 2-(Arylimino)oxazolidines (AIOs). Among the 10 common-featured models generated by program Catalyst/HipHop, a hypothesis including a ring aromatic (RA), a positive ionizable (PI) and three hydrophobic aliphatic (HpAl) features was considered to be important in evaluating the OA-agonist activity. Active OA agonist 2,6-Et2 AII mapped well onto all the RA, PI and HpAl features of the hypothesis. On the other hand, less active compounds were shown to be difficult to achieve the energetically favorable conformation which is found in the active molecules in order to fit the 3-D common-feature pharmacophore models. Taken together, 2,6-Et2-Ph and foramidine structures are important as OA agonists. The present studies on OA agonists demonstrate that a RA, a PI and three HpAl sites located on the molecule seem to be essential for OA-agonist activity.