Genetic variation of the East Balkan Swine (Sus scrofa) in Bulgaria, revealed by mitochondrial DNA and Y chromosomal DNA.

East Balkan Swine (EBS) Sus scrofa is the only aboriginal domesticated pig breed in Bulgaria and is distributed on the western coast of the Black Sea in Bulgaria. To reveal the breed's genetic characteristics, we analysed mitochondrial DNA (mtDNA) and Y chromosomal DNA sequences of EBS in Bulgaria. Nucleotide diversity (πn ) of the mtDNA control region, including two newly found haplotypes, in 54 EBS was higher (0.014 ± 0.007) compared with that of European (0.005 ± 0.003) and Asian (0.006 ± 0.003) domestic pigs and wild boar. The median-joining network based on the mtDNA control region showed that the EBS and wild boar in Bulgaria comprised mainly two major mtDNA clades, European clade E1 (61.3%) and Asian clade A (38.7%). The coexistence of two mtDNA clades in EBS in Bulgaria may be the relict of historical pig translocation. Among the Bulgarian EBS colonies, the geographical differences in distribution of two mtDNA clades (E1 and A) could be attributed to the source pig populations and/or historical crossbreeding with imported pigs. In addition, analysis of the Y chromosomal DNA sequences for the EBS revealed that all of the EBS had haplotype HY1, which is dominant in European domestic pigs.

Characterization of the 4C8 antigen involved in transendothelial migration of CD26(hi) T cells after tight adhesion to human umbilical vein endothelial cell monolayers.

In extravasation of T cells, little is known about the mechanisms of transendothelial migration subsequent to the T cells' tight adhesion to endothelium. To investigate these mechanisms, we developed a monoclonal antibody (mAb), termed anti-4C8, that blocks transmigration but not adhesion in a culture system in which high CD26-expressing (CD26(hi)) T cells preferentially migrate through human umbilical vein endothelial cell (HUVEC) monolayers cultured on collagen gels. Anti-4C8 reacted with all CD3(+) T cells and monocytes but not neutrophils or HUVECs. The structure defined by this antibody was an 80-kD molecule. The mAb at 1 mug/ml inhibited 80-90% of migration of CD3(+) T cells through unstimulated and interferon gamma-stimulated HUVEC monolayers without interfering with adhesion and cell motility. When added to the cultures after the adhesion, anti-4C8 completely blocked subsequent transmigration of adherent T cells. Phase-contrast and electron microscopy revealed that T cells are arrested at the intercellular junctions of HUVECs in the presence of anti-4C8. Anti-4C8 exhibited agonistic effects on resting T cells without other stimuli under culture conditions in which anti-4C8 can stimulate T cells. First, in the checkerboard assay using collagen gels, the antibody promoted chemokinetic migration of the cells in a dose-dependent manner from 0.1 to 10 mug/ml. The predominant population of T cells that migrated into collagen gels with impregnated anti-4C8 were CD26(hi). Second, solid-phase-immobilized anti-4C8 induced adhesion of T cells to the substrate, often with polarizations in cell shape and large pseudopods rich in filamentous (F-) actin. Third, soluble anti-4C8 augmented F-actin content preferentially in CD26(hi) T cells when added to T cells at a high dose of 10 mug/ml. Finally, both anti-4C8-induced chemokinetic migration and transendothelial migration were inhibited by pretreatment of T cells with pertussis toxin. These findings suggest that stimulation via the 4C8 antigen increases cell motility of CD26(hi) cells with profound cytoskeletal changes through signaling pathways including G proteins. The 4C8 antigen may be involved in preferential transmigration of CD26(hi) cells adherent to HUVECs.

Fission yeast alpha-glucan synthase Mok1 requires the actin cytoskeleton to localize the sites of growth and plays an essential role in cell morphogenesis downstream of protein kinase C function.

In fission yeast protein kinase C homologues (Pck1 and Pck2) are essential for cell morphogenesis. We have isolated mok1(+) in a genetic screen to identify downstream effectors for Pck1/2. mok1(+) is essential for viability and encodes a protein that has several membrane-spanning domains and regions homologous to glucan metabolic enzymes. mok1 mutant shows abnormal cell shape, randomization of F-actin and weak cell wall. Biochemical analysis shows that Mok1 appears to have alpha-glucan synthase activity. Mok1 localization undergoes dramatic alteration during the cell cycle. It localizes to the growing tips in interphase, the medial ring upon mitosis, a double ring before and dense dot during cytokinesis. Double immunofluorescence staining shows that Mok1 exists in close proximity to actin. The subcellular localization of Mok1 is dependent upon the integrity of the F-actin cytoskeleton. Conversely, overexpression of mok1(+) blocks the translocation of cortical actin from one end of the cell to the other. pck2 mutant is synthetically lethal with mok1 mutant, delocalizes Mok1 and shows a lower level of alpha-glucan. These results indicate that Mok1 plays a crucial role in cell morphogenesis interdependently of the actin cytoskeleton and works as one of downstream effectors for Pck1/2.

Functional dissection and hierarchy of tubulin-folding cofactor homologues in fission yeast.

We describe the isolation of fission yeast homologues of tubulin-folding cofactors B (Alp11) and E (Alp21), which are essential for cell viability and the maintenance of microtubules. Alp11(B) contains the glycine-rich motif (the CLIP-170 domain) involved in microtubular functions, whereas, unlike mammalian cofactor E, Alp21(E) does not. Both mammalian and yeast cofactor E, however, do contain leucine-rich repeats. Immunoprecipitation analysis shows that Alp11(B) interacts with both alpha-tubulin and Alp21(E), but not with the cofactor D homologue Alp1, whereas Alp21(E) also interacts with Alp1(D). The cellular amount of alpha-tubulin is decreased in both alp1 and alp11 mutants. Overproduction of Alp11(B) results in cell lethality and the disappearance of microtubules, which is rescued by co-overproduction of alpha-tubulin. Both full-length Alp11(B) and the C-terminal third containing the CLIP-170 domain localize in the cytoplasm, and this domain is required for efficient binding to alpha-tubulin. Deletion of alp11 is suppressed by multicopy plasmids containing either alp21(+) or alp1(+), whereas alp21 deletion is rescued by overexpression of alp1(+) but not alp11(+). Finally, the alp1 mutant is not complemented by either alp11(+) or alp21(+). The results suggest that cofactors operate in a linear pathway (Alp11(B)-Alp21(E)-Alp1(D)), each with distinct roles.

Identification of novel temperature-sensitive lethal alleles in essential beta-tubulin and nonessential alpha 2-tubulin genes as fission yeast polarity mutants.

We have screened for temperature-sensitive (ts) fission yeast mutants with altered polarity (alp1-15). Genetic analysis indicates that alp2 is allelic to atb2 (one of two alpha-tubulin genes) and alp12 to nda3 (the single beta-tubulin gene). atb2(+) is nonessential, and the ts atb2 mutations we have isolated are dominant as expected. We sequenced two alleles of ts atb2 and one allele of ts nda3. In the ts atb2 mutants, the mutated residues (G246D and C356Y) are found at the longitudinal interface between alpha/beta-heterodimers, whereas in ts nda3 the mutated residue (Y422H) is situated in the domain located on the outer surface of the microtubule. The ts nda3 mutant is highly sensitive to altered gene dosage of atb2(+); overexpression of atb2(+) lowers the restrictive temperature, and, conversely, deletion rescues ts. Phenotypic analysis shows that contrary to undergoing mitotic arrest with high viability via the spindle assembly checkpoint as expected, ts nda3 mutants execute cytokinesis and septation and lose viability. Therefore, it appears that the ts nda3 mutant becomes temperature lethal because of irreversible progression through the cell cycle in the absence of activating the spindle assembly checkpoint pathway.

Isolation and characterization of SSE1 and SSE2, new members of the yeast HSP70 multigene family.

Two new members of the Saccharomyces cerevisiae heat-shock protein 70 multigene (HSP70) family were isolated from a yeast expression library using antisera made against a yeast calmodulin-binding fraction. They are designated as SSE1 and SSE2, because their predicted amino acid (aa) sequences are highly homologous to each other (76% identical), and share homology with known members of the yeast HSP70 multigene family, but their homologies (13 to 28% identity) are not high enough to place them in known subfamilies. SSE1 and SSE2 are thought to encode polypeptides of 693 aa with calculated M(r)'s of 77,408 and 77,619, respectively. The SSE1 mRNAs were moderately abundant during steady-state growth at 23 degrees C, and increased a few-fold upon upshift to 37 degrees C. SSE2 mRNAs were present at low level during steady-state growth at 23 degrees C, and greatly increased upon upshift to 37 degrees C. Disruption of SSE1 results in slow-growing cells at any temperature. No phenotypic effects of the mutation in SSE2 were detected, and the growth property of the sse1sse2 double mutant was the same as that of the sse1 single mutant.

Cloning and characterization of the Saccharomyces cerevisiae SVS1 gene which encodes a serine- and threonine-rich protein required for vanadate resistance.

A novel Saccharomyces cerevisiae (Sc) SVS1 gene was cloned as a multicopy suppressor of vanadate (Vn) sensitivity (VnS) due to a calcineurin (CaN) null mutation. SVS1 encoded a 260-amino-acid protein abundant in Ser and Thr residues, with a putative signal sequence at the N terminus. Deletion of SVS1 resulted in increased sensitivity to Vn, but not to other metallic ions or drugs. Northern analysis of the SVS1 mRNA indicated that the induction of the gene occurred specifically in the response to Vn. These results suggested that Sc has a mechanism to enhance the tolerance to Vn by increasing the expression of SVS1. The results of genetic experiments suggested that CaN and the Svs1 proteins act in separate pathways to enhance the tolerance to Vn.

Mutational analysis of Yap1 protein, an AP-1-like transcriptional activator of Saccharomyces cerevisiae.

To define the essential amino acid residues of Yap1 in stress response, we generated yap1 mutations by in vitro mutagenesis, which cause defects in mediating resistance to the stress of H2O2, but not of CdCl2. Sequence analysis of the mutant yap1 genes revealed three point mutations and two truncation mutations near the carboxy-terminus. The truncation mutations resulted in hyperresistance to cadmium. Northern blot analysis of stress-induced levels of TRX2 and GSH1 mRNAs indicated that the ability of the mutant Yap1 protein to induce transcriptional activation of target genes correlates well with its ability to confer stress resistance. The carboxy-terminal domain of Yap1 appears to act negatively in cadmium resistance.

Regulation of Wee1 kinase in response to protein synthesis inhibition.

To investigate the mechanism coupling growth (protein synthesis) with cell division, we examined the relationship between the tyrosine kinase Wee1 that inhibits Cdc2-Cdc13 mitosis-inducing kinase by phosphorylating it, and protein synthesis inhibition in fission yeast. The wee1-50 mutant showed supersensitivity to protein synthesis inhibitor, cycloheximide. Wee1 was essential for the G(2) delay upon a partial inhibition of protein synthesis. Indeed, the protein synthesis inhibition caused an increase in the Wee1 protein by the Sty1/Spc1 MAPK-dependent transcriptional and the Sty1/Spc1 MAPK-independent post-transcriptional regulations. Further, the results indicated that the post-transcriptional regulation is important for the G(2) delay.

The involvement of the Saccharomyces cerevisiae multidrug resistance transporters Pdr5p and Snq2p in cation resistance.

The ATP-binding cassette superfamily proteins Pdr5p and Snq2p of Saccharomyces cerevisiae are implicated in multidrug resistance. Here, we show that these transporters are also involved in cation resistance. Null mutants of PDR5 and SNQ2 genes exhibit increased sensitivity to NaCl, LiCl and MnCl2. The mutant cells grown in the presence of high concentrations of these metal salts contain higher levels of the metals than wild-type cells. The expression of PDR5 and SNQ2 is induced by the metal salts. These results provide evidence that the yeast drug transporters contribute to cation resistance by regulating cellular cation homeostasis under ionic stress conditions.

Antineutrophil cytoplasmic autoantibody specific for proteinase 3 in a patient with shunt nephritis induced by Gemella morbillorum.

A 17-year-old girl had been placed with ventriculoperitoneal, then ventriculoatrial shunts for congenital hydrocephalus since birth. The patient originally was diagnosed as having a lupus-like disease, but later turned out to have shunt nephritis, presenting with fever, proteinuria, pancytopenia, and hypocomplementemia. Antineutrophil cytoplasmic autoantibody specific for proteinase 3 (PR3-ANCA) was detected in her serum. The patient received oral prednisolone and repeated methylprednisolone pulses, with essentially no beneficial effects. A gram-positive coccus, Gemella morbillorum, was recovered from her blood as well as cerebrospinal fluid, and the culture of the shunt catheter established the diagnosis of shunt nephritis. Removal of the shunt catheter improved symptoms dramatically and decreased PR3-ANCA in serum to an undetectable level. Because steroids had no effects and the control of bacterial infection lowered PR3-ANCA levels, the antibody would have been induced by continuous infection with G morbillorum.

Autoantibodies against chaperonin CCT in human sera with rheumatic autoimmune diseases: comparison with antibodies against other Hsp60 family proteins.

Chaperonin CCT containing t-complex polypeptide 1 is a cytosolic molecular chaperone that assists in the folding of actin, tubulin, and other proteins and is a member of the 60-kDa heat shock protein (Hsp60) family. We examined antibody titers against human CCT and other Hsp60 family members in the sera of patients with rheumatic autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematodes, Sjögren syndrome, and mixed connective tissue disease. Autoantibody titers against not only human mitochondrial Hsp60 but also CCT were significantly higher in the sera of patients with rheumatic autoimmune diseases than in healthy control sera. Although immunoglobulin G (IgG) titers against Escherichia coli GroEL were high in all the groups of sera tested, no significant differences in anti-GroEL responses were detected between patients and healthy controls. IgG titers against mycobacterial Hsp65 showed a similar pattern to titers of autoantibodies recognizing GroEL. Immunoabsorption experiments demonstrated that most of the autoantibodies recognizing CCT were cross-reactive with mitochondrial Hsp60, E coli GroEL, and mycobacterial Hsp65. Although most of the anti-Hsp60 IgG recognized CCT, anti-GroEL (or antimycobacterial Hsp65) IgG contained antibodies specific for GroEL (or mycobacterial Hsp65) in addition to antibodies cross-reactive with CCT and Hsp60. Results from immunoblot analyses, together with weak (15% to 20%) amino acid sequence identities between CCT and the other Hsp60 family members, suggested that CCT-reactive autoantibodies recognize conformational epitopes that are conserved among CCT and other Hsp60 family members.

Nuclear structure relevant to neutrinoless double beta decay: 76Ge and 76Se.

The possibility of observing neutrinoless double beta decay offers the opportunity of determining the effective neutrino mass if the nuclear matrix element were known. Theoretical calculations are uncertain, and measurements of the occupations of valence orbits by nucleons active in the decay can be important. The occupation of valence neutron orbits in the ground states of 76Ge (a candidate for such decay) and 76Se (the daughter nucleus) were determined by precisely measuring cross sections for both neutron-adding and removing transfer reactions. Our results indicate that the Fermi surface is much more diffuse than in theoretical calculations. We find that the populations of at least three orbits change significantly between these two ground states while in the calculations, the changes are confined primarily to one orbit.

Functional analysis of the promoter of the Saccharomyces cerevisiae multidrug resistance gene YDR1, which encodes a member of the ATP binding cassette (ABC) superfamily.

The Saccharomyces cerevisiae gene YDR1/PDR5/STS1, which encodes a member of the ATP binding cassette (ABC) superfamily, is important for cross-resistance to apparently unrelated drugs. The expression of YDR1 is induced by various drugs and heat shock [Hirata et al., Curr. Genet., 26, 285-294 (1994)]. By deletion analysis of the 5'-noncoding region of the YDR1 gene, two drug responsive regions (-604 to -485 and -335 to -275) were identified with fluphenazine and cycloheximide. Three conserved palindrome sequences were found in these regions.