Wild chimpanzee infant urine and saliva sampled noninvasively usable for DNA analyses

In many genetic studies on the great apes, fecal or hair samples have been used as sources of DNA. However, feces and hairs are difficult to collect from chimpanzee infants under 3 years of age. As alternative DNA sources, we investigated the efficiency of collecting urine samples from infants compared with fecal samples, as well as the validity of the DNA extracted from urine and saliva samples of well-habituated M group chimpanzees (Pan troglodytes schweinfurthii) in the Mahale Mountains National Park, Tanzania. We collected 40 urine and 3 fecal samples from 10 infants under 3 years. Compared with feces, the urine samples were relatively easy to collect. The saliva of infants, which remained on the twigs sucked by them, was collected using cotton swabs. The average amounts of DNA extracted from the 40 urine and 6 saliva samples were 3,920 and 458 pg/μl, respectively. The rate of positive PCR was low and the allelic dropout rate was high when using less than 25 pg of template DNA in the PCR mixtures. Based on the amounts of DNA, 50% of the urine samples and 100% of the saliva samples were judged usable for accurate microsatellite genotyping. For infant chimpanzees in particular, collecting urine and saliva as an alternative to fecal and hair samples can reduce the effort invested in collection in the field.     

Comparative analysis of estrogen receptor gene polymorphisms in apes

Polymorphic microsatellite repeats in the promoter region of estrogen receptor α gene (ESRα and the intron 6 region of estrogen receptor β gene (ESRβ) have been reported in human populations. To examine the evolutional state of both repeats, we surveyed the corresponding regions in DNA sequences from the following great apes and gibbons: 56 chimpanzees, 3 bonobos, 16 gorillas, 20 orangutans and 60 gibbons (four species: 17 of Hylobates agilis, 11 of H. lar, 15 of H. muelleri, and 17 of H. syndactylus). In the corresponding region of the TA repeat of human ESRα, chimpanzees and bonobos had two motifs in the repeat tract, (TA)_7–9 and (CA)_4–6. Gorillas had the (TA)_9–10 repeat tracts and orangutans had monomorphic (TA)₇ repeats. Although all great apes maintained the TA expansion, all gibbon sequences contained (TA)₂, implying that the CA dinucleotide expansion arose in the ancestor of chimpanzees and bonobos. The nucleotide sequences of ESRβ showed a very complex repeat pattern in apes. The human sequences had a non-variable preceding sequence at (CA) _n , (GA)₂(TA)₈(CA)₄(TA). In apes that region included {(TA) _n (CA) _n } _n . Gibbon sequences included (TATG) _n and (TATC) _n and no regular construction was observed. A deletion event in the reverse primer site seems to have occurred in the orangutan lineage. In addition, a great diversity of allele length was detected in each gibbon species.     

Crystal structure of monofunctional histidinol phosphate phosphatase from Thermus thermophilus HB8

Monofunctional histidinol phosphate phosphatase from Thermus thermophilus HB8, which catalyzes the dephosphorylation of l-histidinol phosphate, belongs to the PHP family, together with the PHP domain of bacterial DNA polymerase III and family X DNA polymerase. We have determined the structures of the complex with a sulfate ion, the complex with a phosphate ion, and the unliganded form at 1.6, 2.1, and 1.8 A resolution, respectively. The enzyme exists as a tetramer, and the subunit consists of a distorted (betaalpha)7 barrel with one linker and one C-terminal tail. Three metal sites located on the C-terminal side of the barrel are occupied by Fe1, Fe2, and Zn ions, respectively, forming a trinuclear metal center liganded by seven histidines, one aspartate, one glutamate, and one hydroxide with two Fe ions bridged by the hydroxide. In the complexes, the sulfate or phosphate ion is coordinated to three metal ions, resulting in octahedral, trigonal bipyramidal, and tetrahedral geometries around the Fe1, Fe2, and Zn ions, respectively. The ligand residues are derived from the four motifs that characterize the PHP family and from two motifs conserved in histidinol phosphate phosphatases. The (betaalpha)7 barrel and the metal cluster are closely related in nature and architecture to the (betaalpha)8 barrel and the mononuclear or dinuclear metal center in the amidohydrolase superfamily, respectively. The coordination behavior of the phosphate ion toward the metal center supports the mechanism in which the bridging hydroxide makes a direct attack on the substrate phosphate tridentately bound to the two Fe ions and Zn ion to hydrolyze the phosphoester bond.     

Involvement of KC, MIP-2, and MCP-1 in leukocyte infiltration following injection of necrotic cells into the peritoneal cavity

Although necrotic cells are known to induce inflammation in vivo, the underlying mechanism remains largely unexplored. In order to examine the mechanism, we used an inflammation model induced by injection of necrotic leukemic P388 cells into the peritoneal cavity in this study. The injection of necrotic cells induced the infiltration of neutrophils and subsequently that of monocytes/macrophages. In agreement with this, the injection also induced the production of KC and MIP-2, and subsequently that of MCP-1. Although the level of KC was higher than that of MIP-2, both anti-KC Ab and anti-MIP-2 Ab significantly inhibited the infiltration of neutrophils. Antibodies against CXCR2, a sole receptor for KC and MIP-2, almost completely inhibited the infiltration of neutrophils and monocytes/macrophages. Anti-MCP-1 Ab, on the other hand, inhibited the infiltration of monocytes/macrophages but not neutrophils. These results indicate that KC and MIP-2 play important roles in the infiltration of neutrophils into the site of injection of necrotic cells and that neutrophils may regulate monocyte/macrophage infiltration in our model.     

Differentiation of a hepatic phenotype after heterotropic transplantation of heart, kidney, brain, and skin tissues into liver in F344 rats

While organ-specific stem cells with roles in tissue injury repair have been documented, their pathogenic significance in diseases and the factors potentially responsible for their activation remain largely unclear. In the present study, heart, kidney, brain, and skin samples from F344 transgenic rats carrying the GFP gene were transplanted into normal F344 rat liver one day after an intraperitoneal injection (i.p.) of carbon tetrachloride (CCl(4)) to test their differentiation capacity. The transplantation was carried out by female donors to male recipients, and vice versa. One week after transplantation, GFP antigen-positive cells with phenotypic characteristics of hepatocytes were noted. After two weeks, their extent increased, and at 4 weeks, large areas of strongly GFP-stained cells developed. All recipient livers had GFP antigen-positive hepatocyte cells. PCR analysis coupled with laser capture micro-dissection (LCM) revealed those cells to contain GFP DNA. Thus, our results indicate that tissue stem cells have multipotential ability, differentiating into hepatocytes when transplanted into an injured liver.     

Phospho-dependent functional modulation of GABA(B) receptors by the metabolic sensor AMP-dependent protein kinase

GABA(B) receptors are heterodimeric G protein-coupled receptors composed of R1 and R2 subunits that mediate slow synaptic inhibition in the brain by activating inwardly rectifying K(+) channels (GIRKs) and inhibiting Ca(2+) channels. We demonstrate here that GABA(B) receptors are intimately associated with 5'AMP-dependent protein kinase (AMPK). AMPK acts as a metabolic sensor that is potently activated by increases in 5'AMP concentration that are caused by enhanced metabolic activity, anoxia, or ischemia. AMPK binds the R1 subunit and directly phosphorylates S783 in the R2 subunit to enhance GABA(B) receptor activation of GIRKs. Phosphorylation of S783 is evident in many brain regions, and is increased dramatically after ischemic injury. Finally, we also reveal that S783 plays a critical role in enhancing neuronal survival after ischemia. Together our results provide evidence of a neuroprotective mechanism, which, under conditions of metabolic stress or after ischemia, increases GABA(B) receptor function to reduce excitotoxicity and thereby promotes neuronal survival.     

In vitro susceptibility of dermatomycoses agents to six antifungal drugs and evaluation by fractional inhibitory concentration index of combined effects of amorolfine and itraconazole in dermatophytes

To investigate the antifungal drug susceptibility of fungi responsible for dermatomycoses, minimum inhibition concentration (MIC) tests were performed in 44 strains of dermatophytes, including Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton tonsurans, Microsporum canis, Microsporum gypseum and Epidermophyton floccosum, with six antifungal drugs (amorolfine, terbinafine, butenafine, ketoconazole, itraconazole and bifonazole) by broth microdilution assay according to Clinical Laboratory Standard Institute protocols. Six possible dermatomycosis‐causing non‐dermatophytic fungi were also tested. The two major causes of tinea, T. rubrum and T. mentagrophytes, showed significantly different sensitivities to ketoconazole and bifonazole. Clinically derived dermatophytes were sensitive to the six antifungal drugs tested. However, non‐dermatophytes, especially Fusarium spp., tended to be resistant to these antifungal drugs. In Trichophyton spp., the MICs of non‐azole drugs had narrower distributions than those of azoles. To evaluate the effects of antifungal drug combinations, the fractional inhibitory concentration index was calculated for the combination of amorolfine and itraconazole as representative external and internal drugs for dermatophytes. It was found that this combination had synergistic or additive effects on most dermatophytes, and had no antagonistic effects. The variation in susceptibility of clinically derived fungal isolates indicates that identification of causative fungi is indispensable for appropriately choosing effective antifungal drugs in the early stages of infection. The results of combination assay suggest that multiple drugs with different antifungal mechanisms against growth of dermatophytes should be used to treat refractory dermatomycoses, especially onychomycosis.     

gone early,a Novel Germline Factor,Ensures the Proper Size of the Stem Cell Precursor Pool in the Drosophila Ovary

In order to sustain lifelong production of gametes, many animals have evolved a stem cell–based gametogenic program. In the Drosophila ovary, germline stem cells (GSCs) arise from a pool of primordial germ cells (PGCs) that remain undifferentiated even after gametogenesis has initiated. The decision of PGCs to differentiate or remain undifferentiated is regulated by somatic stromal cells: specifically, epidermal growth factor receptor (EGFR) signaling activated in the stromal cells determines the fraction of germ cells that remain undifferentiated by shaping a Decapentaplegic (Dpp) gradient that represses PGC differentiation. However, little is known about the contribution of germ cells to this process. Here we show that a novel germline factor, Gone early (Goe), limits the fraction of PGCs that initiate gametogenesis. goe encodes a non-peptidase homologue of the Neprilysin family metalloendopeptidases. At the onset of gametogenesis, Goe was localized on the germ cell membrane in the ovary, suggesting that it functions in a peptidase-independent manner in cell–cell communication at the cell surface. Overexpression of Goe in the germline decreased the number of PGCs that enter the gametogenic pathway, thereby increasing the proportion of undifferentiated PGCs. Inversely, depletion of Goe increased the number of PGCs initiating differentiation. Excess PGC differentiation in the goe mutant was augmented by halving the dose of argos, a somatically expressed inhibitor of EGFR signaling. This increase in PGC differentiation resulted in a massive decrease in the number of undifferentiated PGCs, and ultimately led to insufficient formation of GSCs. Thus, acting cooperatively with a somatic regulator of EGFR signaling, the germline factor goe plays a critical role in securing the proper size of the GSC precursor pool. Because goe can suppress EGFR signaling activity and is expressed in EGF-producing cells in various tissues, goe may function by attenuating EGFR signaling, and thereby affecting the stromal environment.