Enhancer-site downstream binding protein activity is enriched in rat tissues that express the class I alcohol dehydrogenase gene.

The activity of the rat class I alcohol dehydrogenase (ADH) is enriched in certain tissues including the liver, intestine and testis. The tissue-specific expression of the gene encoding ADH in the rat was studied and found to closely correlate with tissue isozymic activity. A factor designated enhancer-site downstream binding protein (EDBP) was recently identified in the rat liver and found to interact with the proximal promoter of the class I ADH gene. The distribution of EDBP in nuclear extracts obtained from various tissues was examined based on its sequence-specific DNA binding property and found to correlate with tissue ADH expression. These findings suggest that EDBP is potentially a positive regulatory factor which is involved in controlling the tissue-specific expression of the ADH gene.     

Nonserine esterases from rat liver cytosol

An effort to identify the major general esterases of rat liver cytosol that are insensitive to the serine esterase inhibitor paraoxon (diethyl 4-nitrophenyl phosphate) has led to the isolation of a dozen enzymes. Four of these are electrophoretically homogeneous. Although purified on the basis of their hydrolytic activity toward 4-nitrophenyl acetate, each of the enzymes has a very broad and overlapping substrate specificity for aromatic esters. Thiol esters serve as substrates but, within the limits of the methods used, amides are not hydrolyzed.     

Molecular diffusion into horse spleen ferritin: a nitroxide radical spin probe study.

Electron paramagnetic resonance spectroscopy and gel permeation chromatography were employed to study the molecular diffusion of a number of small nitroxide spin probes (approximately 7-9 A diameter) into the central cavity of the iron-storage protein ferritin. Charge and polarity of these radicals play a critical role in the diffusion process. The negatively charged radical 4-carboxy-2,2,6,6-tetramethylpiperidine-N-oxyl (4-carboxy-TEMPO) does not penetrate the cavity whereas the positively charged 4-amino-TEMPO and 3-(aminomethyl)-proxyl radical and polar 4-hydroxy-TEMPO radical do. Unlike the others, the apolar TEMPO radical does not enter the cavity but instead binds to ferritin, presumably at a hydrophobic region of the protein. The kinetic data indicate that diffusion is not purely passive, the driving force coming not only from the concentration gradient between the inside and outside of the protein but also from charge interactions between the diffusant and the protein. A model for diffusion is derived that describes the observed kinetics. First-order half-lives for diffusion into the protein of 21-26 min are observed, suggesting that reductant molecules with diameters considerably larger than approximately 9 A would probably enter the protein cavity too slowly to mobilize iron efficiently by direct interaction with the mineral core.     

Inhibition of gap junctional intercellular communication in heptachlor- and heptachlor epoxide-treated normal human breast epithelial cells

Based on the concern of organochlorides in the environment and in human tissue, this study was designed to determine whether various noncytotoxic levels of heptachlor and heptachlor epoxide could inhibit, reversibly, gap junctional intercellular communication in human breast epithelial cells (HBEC). Cytotoxicity and gap junctional intercellular communication (GJIC) were evaluated by lactate dehydrogenase assay and fluorescence redistribution after photobleaching analysis, respectively. Both heptachlor and heptachlor epoxide were noncytotoxic up to 10 μg/ml. At this concentration, heptachlor and heptachlor epoxide inhibited GJIC of normal human breast epithelial cells after 1 h treatment. Within a 24 h treatment with heptachlor and heptachlor epoxide at 10 μg/ml, recovery of GJIC had not returned. GJIC completely recovered after a 12 h treatment of 1 μg/ml heptachlor epoxide, but it did not recover after a 24 h treatment of 1 μg/ml heptachlor. RT-PCR and Western blots were analyzed to determine whether the heptachlor or heptachlor epoxide might have altered the steady-state levels of gap junction mRNA and/or connexin protein levels or phosphorylation state. No significant difference in the level of connexin 43 (Cx43) message between control and heptachlor-treated cells was observed. Western blot analyses showed hypophosphorylation patterns in cells treated with 10 μg/ml heptachlor and heptachlor epoxide for 1 h with no recovery within 24 h. Immunostaining of Cx43 protein in normal HBEC indicated that heptachlor and heptachlor epoxide caused a loss of Cx43 from the cell membranes at noncytotoxic dose levels. Taken together, these results suggest that heptachlor and heptachlor epoxide can alter GJIC at the post-translational level, and that, under the conditions of exceeding a threshold concentration in the breast tissue containing ‘initiated’ cells for a long time and not being counteracted by anti-tumor-promoting chemicals, they could act as breast tumor promoters.     

Approximate methods for estimating the pattern of nucleotide substitution and the variation of substitution rates among sites

We propose two approximate methods (one based on parsimony and one on pairwise sequence comparison) for estimating the pattern of nucleotide substitution and a parsimony-based method for estimating the gamma parameter for variable substitution rates among sites. The matrix of substitution rates that represents the substitution pattern can be recovered through its relationship with the observable matrix of site pattern frequences in pairwise sequence comparisons. In the parsimony approach, the ancestral sequences reconstructed by the parsimony algorithm were used, and the two sequences compared are those at the ends of a branch in the phylogenetic tree. The method for estimating the gamma parameter was based on a reinterpretation of the numbers of changes at sites inferred by parsimony. Three data sets were analyzed to examine the utility of the approximate methods compared with the more reliable likelihood methods. The new methods for estimating the substitution pattern were found to produce estimates quite similar to those obtained from the likelihood analyses. The new method for estimating the gamma parameter was effective in reducing the bias in conventional parsimony estimates, although it also overestimated the parameter. The approximate methods are computationally very fast and appear useful for analyzing large data sets, for which use of the likelihood method requires excessive computation.      

A protein that is highly related to GTPase-activating protein-associated p62 complexes with phospholipase C gamma.

p62 is a highly tyrosyl phosphorylated protein that was first identified in immunoprecipitates of the GTPase-activating protein (GAP) of p21ras from cells transformed by oncogenic nonreceptor tyrosine kinases or stimulated through tyrosine kinase receptors (C. Ellis, M. Moran, F. McCormick, and T. Pawson, Nature 343:377-381, 1991). In this article we describe a highly related 62-kDa protein that becomes tyrosyl phosphorylated and associated with phospholipase C gamma (PLC gamma) in C3H10T1/2 cells stimulated with epidermal growth factor (EGF) or transformed by v-src. GAP-associated and PLC gamma-associated p62 comigrated in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited nearly identical phosphotryptic peptide patterns. That the association of p62 with PLC gamma was direct and not mediated through binding of GAP-p62 to PLC gamma or to the EGF receptor (and coprecipitation of the receptor with PLC gamma) was demonstrated by (i) the inability to detect GAP in PLC gamma immunocomplexes or PLC gamma in GAP immunocomplexes, (ii) the association of p62 with PLC gamma in v-src-transformed cells in the absence of EGF stimulation, and (iii) in vitro solution binding and direct blotting of p62 with a glutathione S-transferase fusion protein containing the Src homology 2 (SH2) domains of PLC gamma. Unlike GAP, whose N-terminal SH2 mediates the interaction between GAP and p62, PLC gamma was found to require both its N- and C-terminal SH2 regions for p62 binding. These studies demonstrate that a protein identical to or highly related to GAP-associated p62 binds PLC gamma and suggest a means by which "cross-talk" between PLC gamma- and GAP-mediated signalling may occur.     

Stochastic versus deterministic variability in simple neuronal circuits: II. Hippocampal slice.

Long time series of Schaffer collateral to CA1 pyramidal cell presynaptic volleys (stratum radiatum) and population spikes (stratum pyramidale) were evoked (driven) in rat hippocampal slices. From the driven CA1 region in normal [K+] perfusate, both population spike amplitude and an input-output function consisting of population spike amplitude divided by the presynaptic volley amplitude were analyzed. Raising [K+] in the perfusion medium to 8.5 mM, slices were induced to spontaneously burst fire in CA3 and long time series of inter-burst intervals were recorded. Three tests for determinism were applied to these series: a discrete adaptation of a local flow approach, a local dispersion approach, and nonlinear prediction. Surrogate data were generated to serve as mathematical and statistical controls. All of the population spike (6/6) and input-output (6/6) time series from the normal [K+] driven circuitry were stochastic by all three methods. Although most of the time series (5/6) from the autonomously bursting high [K+] state failed to demonstrate evidence of determinism, one (1/6) of these time series did demonstrate significant determinism. This single instance of predictability could not be accounted for by the linear correlation in these data.     

聚焦色谱法测定肌型肌酸激酶亚型

报道了测定CK-MM亚型的聚焦色谱法,此法简单,快速,结果可靠,线性范围宽,最低检测限（8U／L）较正常参考值低,比国外报道的类似方法高6倍以上,分离度亦有改进.测定了20例健康人血清亚型分布,与文献报道结果相近.该法自动化程度高,已在急性心梗的诊断中实际应用.