Oocyte generation in adult mammalian ovaries by putative germ cells in bone marrow and peripheral blood

It has been suggested that germline stem cells maintain oogenesis in postnatal mouse ovaries. Here we show that adult mouse ovaries rapidly generate hundreds of oocytes, despite a small premeiotic germ cell pool. In considering the possibility of an extragonadal source of germ cells, we show expression of germline markers in bone marrow (BM). Further, BM transplantation restores oocyte production in wild-type mice sterilized by chemotherapy, as well as in ataxia telangiectasia-mutated gene-deficient mice, which are otherwise incapable of making oocytes. Donor-derived oocytes are also observed in female mice following peripheral blood transplantation. Although the fertilizability and developmental competency of the BM and peripheral blood-derived oocytes remain to be established, their morphology, enclosure within follicles, and expression of germ-cell- and oocyte-specific markers collectively support that these cells are bona fide oocytes. These results identify BM as a potential source of germ cells that could sustain oocyte production in adulthood.     

Homologous chromosomes characteristics by sequential banding procedures in rainbow trout Oncorhynchus mykiss

The development of high resolution methods of chromosome banding helped the finding of homologous chromosomes, detecting chromosomal abnormalities, and assigning the gene loci to particular chromosomes in mammals. Unfortunately, small and numerous fish chromosomes do not show GC rich and GC poor compartments, this preventing the establishment of G banding pattern. The combination of techniques enabling the identification of constitutive heterochromatin (C-banding), heterochromatin resistant to restriction endonucleas, NOR bearing chromosomes (AgNO3 banding), or AT rich regions on chromosomes (DAPI banding) in sequential staining provides a better characteristic of fish chromosomes. In this work sequentially DAPI, DdeI, AgNO3 stained chromosomes of rainbow trout resulted in the characteristic banding pattern of some homologous chromosomes. Procedure of FISH with telomere probe and DAPI as a counterstaining fluorochrome visualized simultaneous hybridization signals and DAPI banding. Possibility of detection both FISH and DAPI signals can help in procedures of gene mapping on chromosomes.     

The Staphostatin-staphopain complex: a forward binding inhibitor in complex with its target cysteine protease

Staphostatins are the endogenous inhibitors of the major secreted cysteine proteases of Staphylococcus aureus, the staphopains. Our recent crystal structure of staphostatin B has shown that this inhibitor forms a mixed, eight-stranded beta-barrel with statistically significant similarity to lipocalins, but not to cystatins. We now present the 1.8-A crystal structure of staphostatin B in complex with an inactive mutant of its target protease. The complex is held together through extensive interactions and buries a total surface area of 2300 A2. Unexpectedly for a cysteine protease inhibitor, staphostatin B binds to staphopain B in an almost substrate-like manner. The inhibitor polypeptide chain runs through the protease active site cleft in the forward direction, with residues IG-TS in P2 to P2' positions. Both in the free and complexed forms, the P1 glycine residue of the inhibitor is in a main chain conformation only accessible to glycines. Mutations in this residue lead to a loss of affinity of the inhibitor for protease and convert the inhibitor into a substrate.     

Enhanced longevity in tau mutant Syrian hamsters,Mesocricetus auratus

The single-gene mutation tau in the Syrian hamster shortens the circadian period by about 20% in the homozygous mutant and simultaneously increases the mass-specific metabolic rate by about 20%. Both effects might be expected to lead to a change in longevity. To test such expectations, the life span of male and female hamsters from three genotypes (wild-type, heterozygous, and homozygous tau mutants, all derived from heterozygote crosses to randomize the genetic background) was recorded in constant darkness. Male hamsters lived significantly longer than females: the overall average life span was 96.9 weeks (SE = 2.5, n = 118) for males and 82.0 weeks (SE = 2.1, n = 99) for females. To our surprise, male and female homozygous mutant hamsters lived significantly longer rather than shorter compared to wild-types. For males, the difference between the two genotypes was on average 14%; for females, the difference was 16%. The mortality rate of wild-type males was significantly different from that of homozygous tau males but not different from that of heterozygotes. Overall, survival of wild-type females was statistically distinguishable from both heterozygous and homozygous mutant females. Male and female wild-type hamsters were heavier than homozygote mutants throughout the entire life span, and heterozygous mutants had intermediate weights. There was no correlation between body mass and life span, and the causes of the extended life span in tau mutant hamsters remain unresolved.     

Differing responses of Nijmegen breakage syndrome and ataxia telangiectasia cells to ionizing radiation

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder. Originally thought to be a variant of ataxia telangiectasia (AT), the cellular phenotype of NBS has been described as almost indistinguishable from that of AT. Since the gene involved in NBS has been cloned and its functions studied, we sought to further characterize its cellular phenotype by examining the response of density-inhibited, confluent cultures of human diploid fibroblasts to irradiation in the G(0)/G(1) phase of the cell cycle. Both NBS and AT cells were markedly sensitive to the cytotoxic effects of radiation. NBS cells, however, were proficient in recovery from potentially lethal damage and exhibited a pronounced radiation-induced G(1)-phase arrest. Irradiated AT cells showed no potentially lethal damage and no G(1)-phase arrest. Both cell types were hypersensitive to the induction of chromosomal aberrations, whereas the distribution of aberrations in irradiated NBS cells was similar to that of normal controls, AT cells showed a high frequency of chromatid-type aberrations. TP53 and CDKN1A (also known as p21(Waf1)) expression was attenuated in irradiated NBS cells, but maximal induction occurred 2 h postirradiation, as was observed in normal controls. The similarities and differences in cellular phenotype between irradiated NBS and AT cells are discussed in terms of the functional properties of the signaling pathways downstream of AT involving the NBS1 and TP53 proteins.     

Asymmetry of frameshift mutagenesis during leading and lagging-strand replication in Escherichia coli

Mutations in DNA, including frameshifts, may arise during DNA replication as a result of mistakes made by the DNA polymerase in copying the DNA template strands. In our efforts to better understand the factors that contribute to the accuracy of DNA replication, we have investigated whether frameshift mutations on the Escherichia coli chromosome occur differentially within the leading and lagging-strands of replication. The experimental system involves measurement of the reversion frequency for several defined lac frameshift alleles in pairs of strains in which the lac target is oriented in the two possible directions relative to the origin of chromosomal replication. Within these pairs any defined lac sequence will be subject to leading-strand replication in one orientation and to lagging-strand replication in the other. Fidelity differences between the two modes of replication can be observed as a differential lac reversion between the two strains. Our results, obtained with a series of lac alleles in a mismatch-repair-defective background, indicate that for at least some of the alleles there is indeed a difference in the fidelity of replication between the two modes of replication.     

A study on the nickel II-famotidine complexes

Potentiometric studies have shown that Ni(II) forms three pH-dependent complexes with famotidine (L), namely: [NiHL](3+), [NiL](2+) and [NiH(-2)L]. Two of them have been isolated from solution with a Ni/famotidine ratio of 1:1. At pH 6.0, a paramagnetic complex [NiL](2+) with octahedral geometry is formed in which, most likely thiazole N(9) and guanidine N(3) nitrogens are involved in the metal binding. Additionally, two water molecules and two perchlorate anions, ClO(4)(-), fulfil the coordination sphere. The second complex, [NiH(-2)L], that precipitates at pH 8 is diamagnetic and takes square-planar geometry in which four nitrogen donors: N(3), N(9), N(16) and N(20) coordinate to Ni(II). Potentiometric studies, mass spectrometry, FT-IR and Raman spectroscopy are employed to determine and discuss the structure of both complexes. Additionally, 1H, 13C and 15N NMR spectroscopy is used to confirm the binding site in a square-planar complex. The assignment of vibrational bands are made using ab initio HF/CEP-31G method.     

(-)6-n-Propylnicotine antagonizes the antinociceptive effects of (-)nicotine

Several 6-alkyl analogues of nicotine were examined in radioligand binding and in vivo functional assays. Although (-)6-ethylnicotine (3) binds with high affinity at nACh receptors (Ki=5.6 nM) and produces nicotine-like actions, its n-propyl homologue (-)4 (Ki=22 nM) failed to produce such effects. In fact, (-)4 antagonized the antinociceptive effects of (-)nicotine in the tail-flick assay in mice, but not the spontaneous activity or discriminative stimulus effects of (-)nicotine. Compound (-)4 appears to selectively antagonize only one of the three effects examined and is an interesting cholinergic agent for subsequent investigation.     

Archaeobotanical samples from non-specific urban contexts as a tool for reconstructing environmental conditions (examples from Elbląg and Kołobrzeg,northern Poland)

The botanical composition of samples from culture layers, explored in two medieval towns in northern Poland, is discussed with respect to their potential as a source of environmental data. The frequency of selected taxa and the proportion of their diaspores in the actualistic groups of weed and grassland species, as well as the distribution of indices for edaphic factors were used as indicators of the natural environment around and inside the towns, and of some aspects of agriculture. The comparison of the results from both towns affords new evidence for a better understanding of archaeobotanical data from culture layers of non-specific, complex origin.