Purification and characterization of a calmodulin-dependent kinase from rat brain cytosol able to phosphorylate tubulin and microtubule-associated proteins

Tubulin is a major substrate for endogenous Ca2+-calmodulin-dependent phosphorylation in synaptic cytoplasm. The present study details the purification to apparent homogeneity and characterization of a brain cytosolic Ca2+-calmodulin-dependent kinase which phosphorylates tubulin and microtubule-associated proteins as major substrates. The cytosolic kinase system, purified by sequential chromatography on phosphocellulose resin, calmodulin-affinity resin, and Fractogel TSK HW-55, chromatographs as a homogeneous complex of approximately 600,000 Da on Sephacryl S-300. This calmodulin-dependent kinase possesses a group of properties which specifically characterize this enzyme system: 1) the enzyme contains two calmodulin-binding doublets, rho and sigma, of approximately 52,000 and 63,000 Da, respectively; 2) both the rho and the sigma subunits demonstrate isoelectric points between 6.7 and 7.2; 3) both the rho and sigma subunits demonstrate autophosphorylation; 4) both the rho and sigma subunits show significant homologies as assessed by tryptic peptide fingerprints; 5) in the absence of substrate, both the rho and sigma subunits manifest lower mobility autophosphorylated species; 6) the kinase phosphorylates beta-tubulin equally on threonine and serine residues. Substrate specificity, kinetic parameters, calmodulin-binding properties, subunit composition, and subunit isoelectric points clearly differentiate this enzyme from other previously reported calmodulin-dependent kinases.     

Properties of r Mutants of Bacteriophage T4 Photodynamically Induced in the Presence of Thiopyronin and Psoralen

About 4 x 10(-4)r mutants were induced per lethal hit, a frequency characteristic of weak mutagens. Collections of mutants produced in the presence of either dye were indistinguishable in most of their properties. The rII mutants differed sharply from spontaneous mutants in their mutational spectra (fine scale map distribution) and their reversion responses to specific mutagens. Few or none of the induced mutants were induced to revert with proflavine (sign mutants; reading frame shift mutants). A majority were induced to revert with base analogues (base pair substitution mutants), and about half of these also responded to the hydroxymethylcytosine-specific agent hydroxylamine. A large minority of the mutants reverted spontaneously but failed to respond either to proflavine or to base analogues. We believe these mutants, as well as some of the mutants which did respond to base analogues, to be transversions (base pair substitutions which reverse the purine-pyrimidine orientation).     

Severity of arthritis is predicted by antibody response to gp135 in chronic infection with caprine arthritis-encephalitis virus.

Antibody titers to caprine arthritis-encephalitis virus surface glycoprotein gp135 and core protein p28 in synovial fluid and serum from 35 goats infected for 3 years were compared with the histologic severity of arthritis in these animals. Anti-gp135 antibody titers in synovial fluid and serum directly reflect the severity of carpal arthritis in chronically infected goats.     

Syncro-mate B induces estrus in ovariectomized cows and heifers

Eleven ovariectomized Hereford x Simmental cows and 10 ovariectomized crossbred heifers (primarily Angus and Hereford) were given the Syncro-Mate B (SMB) estrous synchronization treatment. The SMB treatment consisted of a 2 ml i.m. injection containing 5 mg of estradiol valerate and 3 mg of norgestomet plus a hydron ear implant containing 6 mg of norgestomet. The ear implant was removed 9 d later. Cows and heifers were considered in estrus only if they stood for mounting by a herdmate or a bull. Observations for estrus were made four or six times each day for 3 d after implant removal. The 21 animals were used in eight trials. Each trial involved 9 or 11 cows or 5 or 10 heifers. Four days to three weeks elapsed between implant removal and implant insertion for the next trial. No ovariectomized cow or heifer was observed in estrus for 21 d before treatment with SMB. In the eight trials, 3 of 9, 7 of 9 and 6 of 11 cows exhibited estrus, whereas 5 of 10, 1 of 5, 3 of 5, 3 of 5 and 5 of 5 heifers exhibited estrus after treatment. When data were pooled, 16 of 29 (55.2%) cows and 17 of 30 (56.7%) heifers exhibited estrus after treatment. Our data indicate that the SMB treatment can induce estrus in cows and heifers, independently of the ovaries.     

Influences on the antimicrobial activity of surface-adsorbed nisin

The efficacy of the antimicrobial peptide nisin was examined after adsorption to silica surfaces. Three protocols were used to evaluate nisin's activity against adhered cells ofListeria monocytogenes: bioassay usingPediococcus pentosaceous FBB 61-2 as the sensitive indicator strain; visualization and enumeration of cells by microscopic image analysis; and viability of adhered cells as determined by lodonitrotetrazolium violet uptake and crystallization. The activity of adsorbed nisin was highly dependent upon conditions of adsorption. The highest antimicrobial activity of adsorbed nisin occurred with high concentrations of nisin (1.0 mg ml^–1) and brief contact times (1 h) on surfaces of low hydrophobicity. Sequential adsorption of a second protein (-lactoglobulin or bovine serum albumin) onto surfaces consistently resulted in decreased nisin activity. These data provide direction for the development of applications to limit microbial attachment on food contact surfaces through the use of adsorbed antimicrobial peptides.     

Rapid prenatal diagnosis of chromosomal aneuploidies by fluorescence in situ hybridization: clinical experience with 4,500 specimens.

Detection of chromosome aneuploidies in uncultured amniocytes is possible using fluorescence in situ hybridization (FISH). We herein describe the results of the first clinical program which utilized FISH for the rapid detection of chromosome aneuploidies in uncultured amniocytes. FISH was performed on physician request, as an adjunct to cytogenetics in 4,500 patients. Region-specific DNA probes to chromosomes 13, 18, 21, X, and Y were used to determine ploidy by analysis of signal number in hybridized nuclei. A sample was considered to be euploid when all autosomal probes generated two hybridization signals and when a normal sex chromosome pattern was observed in greater than or equal to 80% of hybridized nuclei. A sample was considered to be aneuploid when greater than or equal to 70% of hybridized nuclei displayed the same abnormal hybridization pattern for a specific probe. Of the attempted analyses, 90.2% met these criteria and were reported as informative to referring physicians within 2 d of receipt. Based on these reporting parameters, the overall detection rate for aneuploidies was 73.3% (107/146), with an accuracy of informative results for aneuploidies of 93.9% (107/114). Compared to cytogenetics, the accuracy of all informative FISH results, euploid and aneuploid, was 99.8%, and the specificity was 99.9%. In those pregnancies where fetal abnormalities had been observed by ultrasound, referring physicians requested FISH plus cytogenetics at a significantly higher rate than they requested cytogenetics alone. The current prenatal FISH protocol is not designed to detect all chromosome abnormalities and should only be utilized as an adjunctive test to cytogenetics. This experience demonstrates that FISH can provide a rapid and accurate clinical method for prenatal identification of chromosome aneuploidies.