Requirement of Skp1-Bub1 interaction for kinetochore-mediated activation of the spindle checkpoint

The spindle checkpoint transiently prevents cell cycle progression of cells that have incurred errors or failed to complete steps during mitosis, including those involving kinetochore function. The molecular nature of the primary signal transmitted from defective kinetochores and how it is detected by the spindle checkpoint are unknown. We report biochemical evidence that Bub1, a component of the spindle checkpoint, associates with centromere (CEN) DNA via Skp1, a core kinetochore component in budding yeast. The Skp1's interaction with Bub1 is required for the mitotic delay induced by kinetochore tension defects, but not for the arrest induced by spindle depolymerization, kinetochore assembly defects, or Mps1 overexpression. We propose that the Skp1-Bub1 interaction is important for transmitting a signal to the spindle checkpoint pathway when insufficient tension is present at kinetochores.     

Effects of UV dose on formation of spontaneously developing pocks in Streptomyces azureus ATCC14921

Spontaneously developing pocks (S pocks) of Streptomyces azureus ATCC14921 were formed by the both functions of conjugative plasmid pSA1 and lysogenic phage SAt2. The formation was affected by the dose of UV irradiation. The mean pock diameter in cultures treated with UV light at 0, 7.1, 14.2 and 21.3 x 10(2) microW. erg/cm, respectively, were 1.3, 0.4, 2.2, and 0.5 mm. The dose affected conjugative plasmid pSA1 related to pock formation. There was UV damage of autonomous pSA1 replicon and UV induction of the chromosomal integrated sequence. Increases and decreases in the amount of autonomous pSA1 replicon corresponded to increases and decreases, respectively, in the diameter of the pocks. Both pSA1 and SAt2 syntheses were developed in the large pocks (1.3 and 2.2 mm), but only SAt2 synthesis was developed in the pinhole pocks (0.4 and 0.5 mm).     

Immunochemical detection of precursor proteins of yolk and vitelline envelope, and their annual changes in the blood of Diodon holocanthus

Two distinct groups of female-specific proteins, vitellogenin (VTG) and vitelline envelope proteins (VEP), were detected in the blood of the porcupine fish Diodon holocanthus , and annual changes in concentration were measured immunochemically. Using antisera against yolk proteins (ab.a-E) and VEP (ab. a-VEP), VTG and VEP could be detected in the blood of maturing female fish and oestradiol-17β (E₂)-treated fish. Neither protein was detected in the blood of male fish. Immunohistochemistry showed that yolk globules and the vitelline envelope enclosing developing oocytes stained with ab.a-E. The vitelline envelope was stained specifically with ab.a-VEP. Hepatocytes from the E2-treated fish had immunoreactivity with both antisera. Thus, VTG and VEP appear to be synthesized in the liver by direct stimulation of E₂, released into the circulation, and incorporated into respective target sites. VTG and VEP in female serum maintained high levels from April until June, suggesting that yolk accumulation, as well as vitelline envelope formation, are occurring actively during these months. Unlike VTG, small amounts of VEP were detected between December and March, suggesting that vitelline envelope formation precedes yolk accumulation and that a slightly different hormonal regulation exists in the synthesis of both proteins in the liver during the early phase of oogenesis.     

Synchronized spawning ofanguilla japonica inferred from daily otolith increments in leptocephali

The exact timing ofAnguilla japonica spawning was determined from analyses of daily otolith increments in leptocephali collected near a spawning area west of the Mariana Islands on July 1–18, 1991. The birth dates of the 54 leptocephali examined (10.2 to 30.5 mm in total length) ranged from May 22 to June 24, 1991, the individuals clearly comprising two age groups, May-born fish (mode May 28) and June-born fish (mode June 21). The data showed thatA. japonica spawns intermittently during the spawning season, with fixed synchronized timing. Each group of leptocephali collected along three different north-south transects (131°, 134° and 137°E between 10° and 22°N) comprised both May-born and June-born fish. The latter were dominant along the easternmost and middle transects, whereas the May-born fish were more abundant along the westernmost transect. The modal ages of the June-born and May-born fish collected along 137°E on July 1–3 were 13 d and 35 d, respectively, while those of the two age groups collected along 134°E on July 17 and 18 were 28 d and 50 d, respectively. These data show that the interval between the sampling dates for the two transects (ca. 15 d) corresponded closely to the differences in modal ages of specimens from the two transects (15 d) for both the May- and June-born fish, and further, that the difference in modal age between the two age groups (22 d) was the same at both transects. A similar correspondence in total length was also observed between the two age groups at the above two transects. The findings clearly demonstrated parallel westward transport by the North Equatorial Current for both the May- and June-born eel leptocephali, which originated from a spawning area estimated as being between 141° and 143°E.     

Serotriflin, a CRISP family protein with binding affinity for small serum protein-2 in snake serum

Habu (Trimeresurus flavoviridis) serum contains 3 small serum proteins (SSP-1, SSP-2, and SSP-3) with molecular masses of 6.5 to 10 kDa. Gel filtration analysis showed that all the SSPs exist in high molecular mass forms of approximately 60 kDa in the serum. Ultrafiltration of Habu serum showed that SSPs dissociated from the complex below a pH of 4. An SSP-binding protein was purified from Habu serum by gel filtration, ion exchange, and reverse-phase HPLC. N-terminal sequencing yielded a 39-amino acid sequence, similar to the N-terminal region of triflin, which is a snake venom-derived Ca2+ channel blocker that suppresses smooth muscle contraction. The amino acid sequence of this protein, termed serotriflin, was established by peptide analysis and cDNA cloning. Serotriflin is a glycosylated protein and consists of 221 amino acids. Among the 3 SSPs, only SSP-2 formed a noncovalent complex with serotriflin. It was bound to triflin and serotriflin with high affinity, as evidenced by surface plasmon resonance. SSP-2 is considered to be a protein that prevents self injury by accidental leaking of venom into the blood.     

Carboxyl Proteinase from the Wood Deteriorating Basidiomycete Pycnoporus coccineus :Substrate Specificity with Oxidized Insulin Peptide B1 ~ B16 and B15 ~ B24, Angiotensin and Proangiotensin

The specificity of highly purified carboxyl proteinase from Pycnoporus coccineus (formerly designated Trametes sanguined) was investigated with oligopeptides at pH 2.7. Hydrolysis of oxidized insulin peptide Bl ~ B16 was observed at two peptide bonds (His¹⁰-Leu¹¹ and Ala¹⁴-Leu¹⁵) during 3-hr incubation. The enzyme did not hydrolyze oxidized insulin peptide B15 ~ B24. Hydrolysis of angiotensin (formerly designated angiotensin II) was observed at the Tyr⁴-Ile⁵ bond. Hydrolysis of proangiotensin (formerly designated angiotensin I) was also at the Tyr⁴-Ile⁵ bond. In conclusion, peptide bonds which have a hydrophobic amino acid in the P′₁ position (as defined by Schechter and Berger, Biochem. Biophys. Res. Commun., 27, 157 (1967)) are preferentially cleaved by the trypsinogen activating carboxyl proteinase of Pycnoporus coccineus.     

Use of an activated carbon column for the synthesis of disaccharides by use of a reversed hydrolysis activity of β-galactosidase

Summary An activated carbon column was utilized for the synthesis of disaccharides by use of a reversed hydrolysis activity of an immobilized -galactosidase column in order to shift the equilibrium to the direction of condensation. The yields of lactulose and allo-lactulose from galactose and fructose, and N-acetyl lactosamine and N-acetyl allolactosamine from galactose and N-acetyl glucosamine, were 11.3% and 10.0%, respectively.