Phospholipid-Sensitive Ca2+-Dependent Protein Kinase Preferentially Phosphorylates Serine-115 of Bovine Myelin Basic Protein

Phospholipid-sensitive Ca2+ -dependent protein kinase (PL-Ca-PK) and cyclic AMP-dependent protein kinase (A-PK) both preferentially phosphorylated serine residues of bovine myelin basic protein (MBP). Tryptic peptide maps of MBP phosphorylated by PL-Ca-PK or A-PK, however, revealed different phosphopeptides, suggesting a difference in the intramolecular substrate specificity for the two enzymes. Serine-115 of MBP, in the sequence (-Arg-Phe-Ser(115)-Trp-), was found to be a preferred and probably major phosphorylation site for PL-Ca-PK. Because serine-115 of bovine MBP corresponds to serine-113 of rabbit MBP, an in vivo phosphorylation site reported by Martenson et al. (1983), and PL-Ca-PK is present at a very high level in brain and myelin, it is suggested that the enzyme may be responsible for the in vivo phosphorylation of this and other sites in MBP.     

A novel method for the rapid cloning in Escherichia coli of Bacillus subtilis chromosomal DNA adjacent to Tn917 insertions

Summary A rapid and general procedure has been devised for the pBR322-mediated cloning in Escherichia coli of Bacillus subtilis chromosomal DNA extending in a specified direction from any Tn917 insertion. Derivatives of Tn917 have been constructed that contain a pBR322-derived replicon, together with a chloramphenicol-resistance (Cm^r) gene of Gram-positive origin (selectable in B. subtilis), inserted by ligation in two orientations into a SalI restriction site located near the center of the transposon. When linearized plasmid DNA carrying such derivatives was used to transform to Cm^r B. subtilis bacteria already containing a chromosomal insertion of Tn917, the pBR322 sequences efficiently became integrated into the chromosomal copy of the transposon by homologous recombination. It was then possible to clone chromosomal sequences adjacent to either transposon insertion junction into E. coli, using a selection for ampicillin-resistance, by transforming CaCl₂-treated cells with small amounts of insert-containing DNA that had been digested with various restriction enzymes and then ligated at a dilute concentration. Because pBR322 sequences may be inserted by recombination in either orientation with respect to the transposon arms, a single restriction enzyme (such as EcoRi or SphI) that has a unique recognition site in pBR322 DNA may be used to separately clone chromosomal DNA extending in either direction from the site of any transposon insertion. A family of clones generated from the region of an insertional spo mutation (spoIIH::Tn917) was used in Southern hybridization experiments to verify that cloned material isolated with this procedure accurately reflected the arrangement of sequences present in the chromosome. Strategies are discussed for taking advantage of certain properties inherent in the structure of clones generated in this way to facilitate the identification and study of promoters of insertionally mutated genes.     

Physical mechanisms by which low-frequency magnetic fields can affect the distribution of counterions on cylindrical biological cell surfaces

Effects of dc and low-frequency ac magnetic fields on the motion and distribution of counterions on surfaces of cylindrical biological cells are examined. Magnetic fields along the cell axis as well as perpendicular to it are considered. When a dc magnetic field of any physically realizable magnitude is parallel to the cell axis it has no effect on ion motion, since the resulting Lorentz force is much smaller than the counterion-to-ion attractive force. However a dc magnetic field perpendicular to the cell surface will distort any preexisting ion motion and the resulting current (i) perpendicular to the original motion will be much larger than any current induced by a low-frequency ac magnetic field of the same magnitude as the dc field and parallel to it. Nevertheless i will still be much smaller than the current i_o constituting the original ion motion since (i/i_o)=/, where is the ion cyclotron frequency and the effective counterion collision frequency. With no preexisting coherent ion motion (i_o=0) the circulating current induced by a sinusoidally time-varying magnetic field parallel to the cell axis will be well below thermal fluctuation noise as long as only a single cell is considered; however when even an infinitesimal exchange of ions between adjacent cells is assumed the magnetic field will cause a redistribution of counterions on the cell surface. The resulting steady-state distribution becomes independent of the frequency of the applied magnetic field () when , where is 1/2 of the relaxation frequency for counterion diffusion. On the basis of these results it is suggested that whenever modification of cell behavior in response to application of a low-frequency magnetic field is established, measurements of dielectric permittivity versus frequency of the cell preparation be performed. Redistribution of counterions on the cell surface would be a likely cause if the noted effect becomes independent of the frequency of the applied magnetic field above the counterion dielectric relaxation frequency. It is also suggested that in magnetic field exposure of cell preparations the size of the sample (e.g. diameter of Petri dish) and direction of the magnetic field relative to average cell orientation can critically affect experimental results.     

An HLA-C-specific DNA probe

Analysis of available nucleotide sequence data for class I HLA genes has established that the seventh intron is one of the gene regions which expresses the highest degree of locus specificity (the percentage sequence divergence between nonallelic genes minus the percentage sequence divergence between allelic genes). We have subcloned short DNA sequences including this region from the HLA-Cw3 gene. Two clones, pC250 and pC800, were tested by hybridizing them at high stringency to a panel of clones containing class I HLA genes. Under conditions permitting a strong hybridization signal with a C-locus gene, pC800 also expressed a weak but significant hybridization to other class I genes, while pC250 appeared to hybridize exclusively to the C-locus gene. Hybridization of the pC250 probe at high stringency to Hind III-digested genomic DNA from a panel of unrelated individuals and homozygous typing cell lines revealed a single band in all cases. However, equivalent hybridization against Eco RI-digested DNA revealed two hybridization bands, one at 7.9 kb which correlated with the serologically defined Cw5 and Cw8 alleles, and one at 7.6 kb which correlated with the Cw1, Cw2, Cw3, Cw4, Cw6, and Cw7 alleles.     

Effects of CuDIPS and tween 80 on SJL/J tumorigenesis

Plasma copper and zinc levels were measured in SJL/J mice, an inbred strain characterized by a high, spontaneous incidence of reticulum cell sarcoma (RCS). The changes with age in mean concentrations of these metals were consistent with a physiological response that is required for remission of neoplasia. Treatment of SJL/J mice with a copper complex, Cu(II)(3,4-diisopropylsalicylate)₂ (Cu 3,5-DIPS), dissolved in a 10% Tween 80-saline solution revealed a decrease in survival and decline in the incidence of RCS at 52 wk of age. The toxic effects of Cu 3,5-DIPS therapy appeared to be related to the intraperitoneal route of administration and to extracellular deposition of collagen. The inhibitory effect on tumor development was not related to Cu 3,5-DIPS. Rather, Tween 80 was found to be the factor of importance.     

The influence of androgen administration on the structure and function of the brain-pituitary-gonad axis of sexually immature platyfish,Xiphophorus maculatus

Summary This report demonstrates that the administration of testosterone (T) or 11-ketotestosterone (11-KT) to sexually immature (8 wks old) male platyfish (Xiphophorus maculatus) of early-and late-maturing genotypes affects the synthesis and/or release of luteinizing hormone-releasing hormone (LHRH), as assessed by immunocytochemical evaluation, increases the number and activity of pituitary gonadotropes, stimulates the production of sperm and, thus, advances the age of sexual maturation over that dictated by the genome. We also show that 11-KT and T affect different LHRH-containing centers in the brain and have differential effects on rate and degree of sexual maturation, regardless of whether the hormones are administered to early or late-maturing genotypes.     

The daily timing of lamprey attacks

Synopsis Laboratory and field data suggest that several species of parasitic lampreys forage primarily at night, but data forPetromyzon marinus are equivocal. Nocturnal foraging may minimize predation risk. This may be particularly important to those species of lampreys that inhabit shallow streams. Nocturnal foraging also may enhance foraging efficiency if potential hosts at night are quiescent and more easily approached. Future experiments should be designed to avoid several potential sources of bias.     

Partial purification,subunit structure and thermal stability of the photochemical reaction center of the thermophilic green bacterium Chloroflexus aurantiacus

Spectrally pure reaction center preparations from Chloroflexus aurantiacus have been obtained in a stable form; however, the product contained several contaminating polypeptides. The reaction center pigment molecules (probably three bacteriochlorophyll a and three bacteriopheophytin a molecules) are associated with two polypeptides (M_r = 30000 and 28000) in a reaction center complex of M_r = 52000. No carotenoid is present in the complex. These data together with previous spectral data suggest that the Chloroflexus reaction center represents a more primitive evolutionary form of the purple bacterial reaction center, and that it has little if any relationship to the green bacterial component. A reaction center preparation from Rhodopseudomonas sphaeroides R26 was fully denatured at 50°C while the Chloroflexus reaction center required higher temperatures (70–75°C) for complete denaturation. Thus, an intrinsic membrane protein of a photosynthetic thermophile has been demonstrated to have greater thermal stability than the equivalent component of a mesophile.     

Effect of methylation of histidine-48 on some enzymatic and pharmacological activities of snake venom phospholipases A2

The effects on some pharmacological and enzymatic properties were determined following methylation of histidine at the enzymatic active site of the basic relatively toxic $\text{Naja}$$\text{nigricollis}$ and the acidic relatively non-toxic $\text{Naja}$$\text{naja}$$\text{atra}$ phospholipases A₂. Following methylation a very low residual enzymatic activity (0.4 -- 1% of control) was accompanied by a parallel loss in intraventricular lethality, anticoagulant potency, direct hemolytic action and ability to block directly and indirectly evoked contractions of the mouse phrenic nerve-diaphragm preparation. Since methylation does not impair the enzyme's ability to bind monomeric of micellar substrates or Ca²⁺, the results suggest that the pharmacologicallly active region of the molecule is different from the micellular substrate binding site but strongly influenced by the invariant histidine-48 located at the enzymatic active site.     

An age dependent stochastic model of periodic screening: Basic properties

An age dependent stochastic model for the periodic screening of a progressive chronic disease is developed in this paper by combining results from previous modeling efforts. The basic concepts used are the random variables describing the disease free state and preclinical state sojourn times and the age at screening or observation, as well as generations of individuals defined according to time of entry into the preclinical state. At discrete time points, the model characterizes the density functions for individuals who are healthy, have preclinical disease, or have clinical disease. The joint density functions of age and sojourn times for cases detected by a periodic screening program and for cases which surface clinically between screens are derived as functions of screening interval, false negative rate, and disease natural history.