Combined Genetic and Genealogic Studies Uncover a Large BAP1 Cancer Syndrome Kindred Tracing Back Nine Generations to a Common Ancestor from the 1700s

We recently discovered an inherited cancer syndrome caused by BRCA1-Associated Protein 1 (BAP1) germline mutations, with high incidence of mesothelioma, uveal melanoma and other cancers and very high penetrance by age 55. To identify families with the BAP1 cancer syndrome, we screened patients with family histories of multiple mesotheliomas and melanomas and/or multiple cancers. We identified four families that shared an identical BAP1 mutation: they lived across the US and did not appear to be related. By combining family histories, molecular genetics, and genealogical approaches, we uncovered a BAP1 cancer syndrome kindred of ~80,000 descendants with a core of 106 individuals, whose members descend from a couple born in Germany in the early 1700s who immigrated to North America. Their descendants spread throughout the country with mutation carriers affected by multiple malignancies. Our data show that, once a proband is identified, extended analyses of these kindreds, using genomic and genealogical studies to identify the most recent common ancestor, allow investigators to uncover additional branches of the family that may carry BAP1 mutations. Using this knowledge, we have identified new branches of this family carrying BAP1 mutations. We have also implemented early-detection strategies that help identify cancers at early-stage, when they can be cured (melanomas) or are more susceptible to therapy (MM and other malignancies).     

Complete mitochondrial DNA sequence of Aulopus japonicus (Teleostei: Aulopiformes), a basal Eurypterygii: longer DNA sequences and higher-level relationships

For the first step toward resolution of the higher-level relationships of the order Aulopiformes (Teleostei: Eurypterygii) using longer DNA sequences, we determined the complete mitochondrial DNA sequence for Aulopus japonicus (Aulopodidae). The entire genome was purified by gene amplification using a long PCR technique, and the products were subsequently used as templates for PCR with 63 fish-versatile and 3 species-specific primers that amplify contiguous, overlapping segments of the entire genome. Direct sequencing of the PCR products demonstrated that the genome (16 653 base pairs [bp]) contained the same 37 mitochondrial genes (2 ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes) as found in other vertebrates, with the gene order identical to that in typical vertebrates. Maximum-parsimony analysis using nucleotide sequences from the concatenated 12 protein-coding genes (no third codon positions and excluding the ND6 gene) plus 22 tRNA genes (stem regions only) from eight teleosts placed A. japonicus in a reasonable phylogenetic position; those from individual protein-coding genes and the concatenated 22 tRNA genes alone, however, did not reproduce the expected phylogeny with few exceptions, probably owing to insufficient phylogenetic information in these smaller data sets. This result suggests that further taxonomic sampling and sequencing efforts may clarify limits and intra- and interrelationships of this morphologically and ecologically diverse group of fishes using mitochondrial genomic (mitogenomic) data. Received: August 31, 2000 / Revised: December 20, 2000 / Accepted: January 23, 2001     

Detecting nonculturable bacteria in the active mycorrhizal zone of the pine mushroom <Emphasis Type="Italic">Tricholoma matsutake</Emphasis>

The fungus Tricholoma matsutake forms an ectomycorrhizal relationship with pine trees. Its sporocarps often develop in a circle, which is commonly known as a fairy ring. The fungus produces a solid, compact, white aggregate of mycelia and mycorrhizae beneath the fairy ring, which in Japanese is called a ’shiro’. In the present study, we used soil dilution plating and molecular techniques to analyze the bacterial communities within, beneath, and outside the T. matsutake fairy ring. Soil dilution plating confirmed previous reports that bacteria and actinomycetes are seldom present in the soil of the active mycorrhizal zone of the T. matsutake shiro. In addition, the results showed that the absence of bacteria was strongly correlated with the presence of T. matsutake mycorrhizae. The results demonstrate that bacteria, especially aerobic and heterotrophic forms, and actinomycetes, are strongly inhibited by T. matsutake. Indeed, neither bacteria nor actinomycetes were detected in 11.3% of 213 soil samples from the entire shiro area by culture-dependent methods. However, molecular techniques demonstrated that some bacteria, such as individual genera of Sphingomonas and Acidobacterium, were present in the active mycorrhizal zone, even though they were not detected in soil assays using the dilution plating technique.     

Investigation of phosphorylation status of OdhI protein during penicillin- and Tween 40-triggered glutamate overproduction by <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Glutamate overproduction by Corynebacterium glutamicum is triggered by treatment with penicillin or Tween 40 and is accompanied by a decrease in 2-oxoglutarate dehydrogenase complex (ODHC) activity. We have reported that de novo synthesis of OdhI, which inhibits ODHC activity by interacting specifically with the E1o subunit of ODHC (OdhA), is induced by penicillin, and that odhI overexpression induces glutamate overproduction in the absence of any triggers for glutamate overproduction. In this study, to determine the function of OdhI in glutamate overproduction by C. glutamicum, changes in OdhI levels and phosphorylation status during penicillin- and Tween 40-induced glutamate overproduction were examined by western blot. The synthesis of both unphosphorylated and phosphorylated OdhI was increased by addition of Tween 40 or penicillin and the levels of unphosphorylated OdhI, which can inhibit ODHC activity, was significantly higher than those of phosphorylated OdhI, which is unable to inhibit ODHC activity. Meanwhile, the OdhA levels were maintained throughout the culture. These results indicate that OdhI synthesis is induced by additions of penicillin and Tween 40 and most synthesized OdhI is unphosphorylated, resulting in the decrease in ODHC activity and glutamate overproduction. Similarly, in the odhI-overexpressing strain, both unphosphorylated and phosphorylated OdhI were synthesized, while the levels of OdhA were nearly constant throughout culture. Our results suggest that high level of unphosphorylated OdhI regulates glutamate overproduction by C. glutamicum.     

Molecular basis of guanine nucleotide dissociation inhibitor activity of human neuroglobin by chemical cross-linking and mass spectrometry

Oxidized human neuroglobin (Ngb), a heme protein expressed in the brain, has been proposed to act as a guanine nucleotide dissociation inhibitor (GDI) for the GDP-bound form of the heterotrimeric G protein alpha-subunit (Galpha(i)). Here, to elucidate the molecular mechanism underlying the GDI activity of Ngb, we used an glutathione-S-transferase pull-down assay to confirm that Ngb competes with G-protein betagamma-subunits (Gbetagamma) for binding to Galpha(i), and identified the Galpha(i)-binding site in Ngb by chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and sulfo-N-hydroxysuccinimide, coupled with mass spectrometry (MS). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis for tryptic peptides derived from the cross-linked Ngb-Galpha(i) complex revealed several binding regions in Ngb. Furthermore, MALDI-TOF/TOF MS analysis of the cross-linked Ngb and Galpha(i) peptides, together with the MS/MS scoring method, predicted cross-linking between Glu60 (Ngb) and Ser206 (Galpha(i)), and between Glu53 (Ngb) and Ser44 (Galpha(i)). Because Ser206 of Galpha(i) is located in the region that contacts Gbetagamma, binding of Ngb could facilitate the release of Gbetagamma from Galpha(i). Binding of Ngb to Galpha(i) would also inhibit the exchange of GDP for GTP, because Ser44 (Galpha(i)) is adjacent to the GDP-binding site and Glu53 (Ngb), which is cross-linked to Ser44 (Galpha(i)), could be located close to GDP. Thus, we have identified, for the first time, the sites of interaction between Ngb and Galpha(i), enabling us to discuss the functional significance of this binding on the GDI activity of Ngb.     

Proximity of reactive lysyl residue to the antigenic site in rabbit skeletal myosin against the monoclonal antibody (MF-18) generated to chicken skeletal myosin

MF-18, one of the monoclonal antibodies generated to chicken myosin, cross-reacted with rabbit skeletal myosin subfragment-1 (S1). Utilizing an improved procedure of immuno-blotting, a decrease in reactivity of MF-18 to S1 by trinitrophenylation was observed. This indicates that the reactive lysyl residue is very close to the hapten site. This is consistent with the evidence that the hapten site resides in the 26,000 dalton tryptic fragment of S1. Use of such antibodies as labels may open the way to determining the location of specific hapten sites in the three-dimensional image of actin-S1 complex reconstructed from the electron micrographs.     

Effects of nonselective cation channels and PI3K on endothelin-1-induced PYK2 tyrosine phosphorylation in C6 glioma cells

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca²⁺-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) in C6 glioma cells. In the present study, we investigated the effects of NSCCs on the ET-1-induced proline-rich tyrosine kinase 2 (PYK2) phosphorylation in C6 glioma cells. In addition, we examined the effects of phosphoinositide 3-kinase (PI3K) on the ET-1-induced NSCCs activation and PYK2 phosphorylation. The PI3K inhibitors wortmannin and LY-294002 inhibited ET-1-induced Ca²⁺ influx through NSCC-2 but not NSCC-1. On the other hand, addition of these inhibitors after stimulation with ET-1 failed to suppress Ca²⁺ influx through NSCC-2. PYK2 phosphorylation was abolished by blocking Ca²⁺ influx through NSCCs. The PI3K inhibitors blocked the NSCC-2-dependent part of ET-1-induced PYK2 phosphorylation. These results indicate that 1) NSCC-2 is stimulated by ET-1 via a PI3K-dependent cascade, whereas NSCC-1 is stimulated via a PI3K-independent cascade; 2) PI3K seems to be required for the activation of the Ca²⁺ entry, but not for its maintenance; 3) Ca²⁺ influx through NSCC-1 and NSCC-2 plays an essential role in ET-1-induced PYK2 phosphorylation; and 4) PI3K is involved in the ET-1-induced PYK2 phosphorylation that depends on the Ca²⁺ influx through NSCC-2. endothelin; phosphoinositide 3-kinase; nonselective cation channel; proline-rich tyrosine kinase 2; glioma cell     

Ranging behavior of Mahale chimpanzees: a 16 year study

We have analyzed the ranging patterns of the Mimikire group (M group) of chimpanzees in the Mahale Mountains National Park, Tanzania. During 16 years, the chimpanzees moved over a total area of 25.2 or 27.4 km², as estimated by the grid-cell or minimum convex polygon (MCP) methods, respectively. Annually, the M group used an average of 18.4 km², or approximately 70 %, of the total home-range area. The chimpanzees had used 80 % of their total home range after 5 years and 95 % after 11 years. M group chimpanzees were observed more than half of the time in areas that composed only 15 % of their total home range. Thus, they typically moved over limited areas, visiting other parts of their range only occasionally. On average, the chimpanzees used 7.6 km² (in MCP) per month. Mean monthly range size was smallest at the end of the rainy season and largest at the end of the dry season, but there was much variability from year to year. The chimpanzees used many of the same areas every year when Saba comorensis fruits were abundant between August and January. In contrast, the chimpanzees used several different areas of their range in June. Here range overlap between years was relatively small. Over the 16 years of the study we found that the M group reduced their use of the northern part of their range and increased their frequency of visits to the eastern mountainous side of their home range. Changes in home-range size correlated positively with the number of adult females but not with the number of adult males. This finding does not support a prediction of the male-defended territory model proposed for some East African chimpanzee unit-groups.     

Inhibition of chromosome separation in fertilized starfish eggs by kalihinol F, a topoisomerase I inhibitor obtained from a marine sponge

Kalihinol F, a naturally occurring diterpene from a marine sponge, Acanthella sp., inhibited chromosome separation in fertilized starfish (Asterina pectinifera) eggs but allows the first cleavage to occur, thereby forming unseparated metaphase chromosomes which were elongated between the two daughter cells. The chromosomes were eventually torn off in the embryonic cells. Most of the cells gradually lost the chromosomes during the cell cycle progression. The embryonic development halted at the morula stage just before the onset of blastulation. The mitotic failure occurred when kalihinol F was applied to a fertilized egg during the second meiotic process, but not after the completion of the second meiotic division. Kalihinol F inhibited topoisomerase I activity in vitro, but had no effects on activities of DNA polymerases alpha, beta, and gamma, and of topoisomerase II. These results suggest that the topoisomerase I plays an essential role in meiosis II in this species.