Distinctive solution conformation of phosphatase inhibitor CPI-17 substituted with aspartate at the phosphorylation-site threonine residue

We present solution NMR structures for wild-type and mutated forms of CPI-17, a phosphoinhibitor for protein phosphatase 1. Phosphorylation of Thr38 of CPI-17 produces a >1000-fold increase in inhibitory potency for myosin phosphatase. We compared the 1H-15N heteronuclear single quantum coherence spectroscopy (HSQC) chemical shifts of wild-type CPI-17, partially phosphorylated CPI-17 and CPI-17 with Thr38 replaced with Asp to introduce a negative charge. There was a switch in the protein conformation due to either Asp substitution or phosphorylation, so we determined the solution NMR structure of the CPI-17 T38D mutant as a model for the active (phospho-) conformation. The structures reveal a molecular switch in conformation that involves the rotation of two of the four helices in the four helix bundle. Despite this conformational switch, there was little increase in the inhibitory potency with T38D. We propose that for this inhibitor, a negative charge at residue 38 is sufficient to trigger an active conformation, but a phosphoryl group is required for full inhibitory potency against protein phosphatase-1.     

Regioselective synthesis of kojic acid esters by Bacillus subtilis protease

The lipophilicity of kojic acid [5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one] was improved by esterifying kojic acid with either divinyl adipate, vinyl hexanoate, vinyl octanoate or vinyl decanoate using protease from Bacillus subtilis for 7 d. ¹H-NMR and ¹³C-NMR showed that the primary hydroxyl group at the C-7 position of kojic acid was regioselectively esterified to afford 7-O-vinyl adipoyl kojic acid, 7-O-hexanoyl kojic acid, 7-O-octanoyl kojic acid and 7-O-decanoyl kojic acid (13–27% yield). The kojic acid esters had radical scavenging activities, inhibited tyrosinase activity and was biodegradable.     

Isolation and characterization of an active mannanase-producing anaerobic bacterium, Clostridium tertiumKT-5A, from lotus soil

Of 10 strains of mannanase-producing anaerobicbacteria isolated from soils and methanogenic sludges, Clostridium tertium KT-5A,which was isolated from lotus soil, produced high amounts of extracellular β-1,4-mannanase. The isolate was an aerotolerant anaerobe without quinon systems; the cell growthcultivated with no addition of reducing agents was also stable. High yields of mannanasewere obtained by inducing enzyme production with galactomannan guar gum and beef extract/peptone as carbon and nitrogen sources, respectively. Fermentation endproducts on galactomannan fermentation were formate, acetate, lactate, butyrate, carbondioxide and hydrogen. The extracellular mannanase displayed high activity ongalactomannans of locust bean gum galactose/mannose (G/M) ratio 1:4 and spinogum (G/M 1:3), but weak activity on guar gum galactomannan (G/M 1:2) and konjac glucomannan. As far as is known, this is the first report on the isolation of an activemannanase-producing anaerobic bacterium from natural environments.     

Polyacrylamides containing sugar residues: synthesis,characterization and hepatocyte attachment studies

Homo polymers of acrylamide having glucose (PAAm-glucose) and galactose (PAAm-galactose) as pendent groups were synthesized. Tissue culture polystyrene (TCPS) plates coated with these polymers showed increased surface wettability. Coating of PAAm-glucose and PAArn-galactose on to TCPS plates was also confirmed by X-ray photoelectron spectroscopic (XPS) characterization. Rat hepatocytes in primary culture attached to the surfaces of PAArn-galactose homopolymer, but not to those of PAAm-glucose homopolymer. © Rapid Science Ltd. 1998     

Electrophysiology of trichobothria in orb-weaving spiders (Agelenidae,Araneae)

Summary Trichobothria of the house spidersTegenaria atrica (C.L. Koch) andTegenaria derhami (Scopoli) were stimulated by linear deflection of the hair from its resting position to one side. The pulse response of the receptor cell was analyzed. At angular deflection velocities of 10^–21 deg/ms the receptor begins to discharge at an angle of 3 degrees. While the mean pulse rate remains constant during deflections of 10^–210^–1deg/ms the pulse train may be interrupted by repeated breaks. Discharge continues when the hair is bent proximally beyond the bothrium edge. When the hair is bent distally and touches the bothrium edge, however, discharge ceases. Responsible for this phenomenon seems to be an asymmetry of hair suspension. — Repeated deflection leads to logarithmically ascending latency curves and logarithmically falling curves of the pulse numbers. The function coefficients depend on velocities and repetition rates of the deflections. The adaptational effect is heightened by preceding stationary deflection in the direction of the dynamical stimulus. The mean pulse rate as a function of hair deflection velocity increases logarithmically. The mean pulse rate as a function of hair movement direction obeys a cosine law, provided that a given velocity and a definite deflection angle are used.Supported by grants of the Deutsche Forschungsgemeinschaft given to Prof. Dr. P. Görner in the field of the Schwerpunktprogramm RezeptorphysiologieThe present paper is part of a doctoral thesis. My thanks are due to Prof. Dr. P. G6rner for the theme, many valuable discussions and his constant readiness to help.     

Isolation and characterization of diploid clones from adult and newborn rat liver cell lines.

A high frequency of diploid and near-diploid clones were developed from cell lines derived from adult and newborn rat liver using micropipettes. There were some differences in morphology, biochemical properties and growth rate between clones. Cloned cells had low levels of tyrosine transaminase activity, glucose-6-phosphatase activity and albumin content. A diploid clone and pseudodiploid clone derived from adult rat liver cell line were positive for alpha-fetoprotein.     

Effect of 3′-methyl-4-dimethylaminoazobenzene in the induction of malignant transformation and of 8-azaguanine-resistant mutations and chromosomal aberrations in a diploid clone derived from normal rat liver cells in culture

Summary The effect of 3′methyl-4-dimethylaminoazobenzene (3′-Me-DAB) in the induction of malignant transformation and of 8-azaguanine-resistant mutations and chromosomal aberrations was studied in a diploid strain derived from normal rat liver cells. The cells were malignantly transformed by treatment with 3′-Me-DAB 1.7 μg/ml for 130 to 221 d or 1.7 μg/ml for 53 d followed by 24.9 μg/ml for 27 to 77 d. The untreated control cells did not transform spontaneously until the 232nd d in culture. Some properties of the 3′-Me-DAB-treated cells were compared to those of untreated control cells but no reliable marker for predicting the tumorigenic potential of the cells was found. The single addition of 3′-Me-DAB caused little induction of 8-azaguanine-resistant mutations and chromosomal aberrations to the cells. However, mutations and chromosomal aberrations were significantly induced byN-acetoxy-4-methylaminoazobenzene, an active metabolite of 4-dimethylaminoazobenzene or 3′-Me-DAB in the presence of liver microsomes. This study was supported by a grant for cancer research from the Japanese Ministry of Education.     

Degradation of Polycarbonate by a Polyester-Degrading Strain, Amycolatopsis sp. Strain HT-6

Amycolatopsis sp. strain HT-6, a poly(tetramethylene succinate) (PTMS)-degrading actinomycete, was observed to degrade poly(tetramethylene carbonate) (PTMC). In a liquid culture with 150 mg of PTMC film, 59% degradation was achieved, but with a low yield of cell growth. On the other hand, PTMS copolymerized with a small amount of PTMC, forming a copolyester carbonate (PEC) that was completely and rapidly degraded with a high yield of cell growth.