Cholera toxin A subunit: functional sites correlated with regions of secondary structure

The A subunit of cholera toxin contains the ADP-ribosyltransferase activity in its major constituent polypeptide A1 (Mr 23,000) which is responsible for the elevation of cAMP typically observed with most mammalian cell types after exposure to the toxin. The primary structure of the A subunit, recently established by sequence analyses, is presented and used as the basis for the secondary structure prediction according to the method of Chou and Fasman. The results indicated the presence of 27% alpha-helix, 25% beta-structure, 12% beta-turn, and 36% random coil. The majority of the beta-structure consisted of six strands located in the NH2-terminal portion of the molecule (residues 33-106) covering one-half of the region corresponding to the A1 polypeptide portion. The beta-sheet domain led immediately into the active site region characterized by the alternating structures of beta-pleated sheet and alpha-helix (residues 95-140) similar to that reported for other NAD+ binding proteins. The presence of this structural feature in the region was confirmed by the use of another predictive method (J. Garnier et al., J. Mol. Biol. 1978, 120, 97-120). In addition, two regions (residues 14-18 and 200-214), previously identified to contain binding sites for the B subunit as evidenced by chemical modification and monoclonal antibody studies, were found to be in alpha-helix configuration.     

Biomass: Is it a useful tool in paleocommunity reconstruction?

In general, more of the biomass of the community is preserved than is its numerical abundance. Thus, the paleontologist, on the average, works with more of the community when biomass is used. Community characteristics such as taxon dominance and habitat proportions are at least as accurately derived from biomass as numerical abundance. The use of biomass is clearly more appropriate in describing energy flow and trophic proportions. Whenever possible, biomass should be used as a complement to numerical abundance in future paleoecologic reconstructions.     

Ca2+/calmodulin-dependent protein kinase II. Isozymic forms from rat forebrain and cerebellum

Ca2+/calmodulin-dependent protein kinase II, an abundant brain protein proposed to mediate a number of Ca2+-regulated processes in neuronal tissue, is composed of autophosphorylatable subunits of Mr 50,000 and 60,000/58,000. A recent study (McGuinness, T. L., Lai, Y., Greengard, P., Woodgett, J.R., and Cohen, P. (1983) FEBS Lett. 163, 329-334) suggested that this kinase exists as isozymes which vary in the relative ratio of these subunits in different tissues or species. Other studies (Walaas, S. I., Nairn, A. C., and Greengard, P. (1983) J. Neurosci. 3, 291-301, 302-311) provided evidence which suggested that the ratio of these phosphopeptides might vary in different brain regions. In the present investigation, we have tested this possibility by comparing Ca2+/calmodulin-dependent protein kinase II purified from rat forebrain and cerebellum. The two kinases had similar purification characteristics, subunit compositions, physical properties, and substrate specificities. Gel filtration and sucrose density gradient centrifugation provided an estimated molecular weight of 550,000 for the forebrain kinase and 615,000 for the cerebellar kinase. The kinases from the two regions clearly differed in the relative proportions of the Mr 50,000 and 60,000/58,000 subunits. Three independent methods indicated that the forebrain kinase contained the Mr 50,000/(60,000/58,000) subunits in approximately a 3:1 ratio, while the cerebellar kinase contained the Mr 50,000/(60,000/58,000) subunits in approximately a 1:4 ratio. The forebrain kinase subunits were shown to be identical to the corresponding subunits of the cerebellar kinase by several criteria. The data are consistent with the existence in various brain regions of isozymic forms of Ca2+/calmodulin-dependent protein kinase II which differ in their relative subunit ratios.     

Immunochemical Characterization of Pyruvate Dehydrogenase Complex in Rat Brain

Pyruvate dehydrogenase complex (PDHC) in rat brain was studied immunochemically, using antibodies against the bovine kidney PDHC, by immunoblotting, immunoprecipitation, inhibition of enzyme activity, and enzyme-linked immunoabsorbent assay (ELISA). The immunoblots showed that the antibodies bound strongly to the alpha peptide of the pyruvate dehydrogenase (E1) component, and to the dihydrolipoyl transacetylase (E2) and the dihydrolipoyl dehydrogenase (E3) components of PDHC. A similar immunoblotting pattern was observed in all eight brain regions examined. On immunoblotting of the subcellular fractions, these PDHC peptides were observed in mitochondria and synaptosomes but not in the postmitochondrial supernatants. This agrees with other evidence that brain PDHC is localized in the mitochondria. These results, together with those from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitin, also showed that the alpha E1, beta E1, and E3 peptides of rat brain PDHC are very similar in sizes to those of the bovine kidney PDHC, being 42, 36, and 58 kD, respectively. The size of the E2 peptide, 66 kD, is different from that of bovine kidney E2, 73 kD. The relative abundance of PDHC protein in nonsynaptic mitochondria was compared by enzyme activity titration and ELISA. Both methods demonstrated that the amount of PDHC antigen in the mitochondria from cerebral cortex is greater than that in the olfactory bulb mitochondria. This is consistent with the results of the activity measurement. The ELISA also showed that the PDHCs in both mitochondrial populations are antigenically similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific absorption rate in rats exposed to 2,450-MHz microwaves under seven exposure conditions

Both positive and negative biological effects of microwaves on drug actions in rats exposed to 1-mW/cm2, 2,450-MHz microwaves have been reported by several investigators. We conducted dosimetry studies for seven different exposure conditions to determine whether these different results could be due to the rats having been exposed differently. They included anterior and posterior exposures in a circular waveguide, near field, far field with E- or H-field parallel to the long axis of the body and dorsal exposure in a miniature anechoic chamber with E- or H-field parallel to the long axis of the body. The average specific absorption rates (SARs) in the head, tail, and body of the exposed rats were measured by means of a calorimetry system. The local SARs at eight locations in the brain were determined by temperature measurement with Vitek probes. Intensive coupling of energy to the tail when it was exposed parallel to the E-field was shown by thermography. For the same average incident power density, the average SARs in the heads of rats were about two times higher in the circular waveguide than for other exposures. The local SARs in the brain varied for different exposure conditions. Statistical comparisons of SARs under the different exposure conditions are presented.     

On the Trail of Atmospheric Mutagens and Carcinogens: A Combined Chemical/Microbiological Approach

Major ecological problems of our polluted troposphere includeairborne toxic chemicals, acid rain and photochemical smog,all three of which are now recognized as being closely relatedchemical phenomena. We also recognize that inorder to developcost-effective strategies for their control, which protect publichealth and the environment, there must be close scientific interactionsbetween chemists and biological scientists. For example, ofrapidly emerging importance is the development of risk assessmentevaluations for specific aspects of each of these problem areas.In preparing such assessments, chemists must define the "exposure,"and biological scientists the "effects." In this paper, I discuss an example of how such close interactionsproved indispensible in our search for atmospheric mutagensand carcinogens. Thus, an integrated chemical/ microbiologicalprocedure for the isolation and identificationof particulatechemical mutagens in respirable diesel soot and ambient particlesis described. Emphasis is placed on our use of the short-term,Ames Salmonella typhimurium bacterial mutagenicity test as arapid, and relatively inexpensive, means of following the biologicalactivities of these environmental mutagens through the chemicalsteps of their separation, isolation and identification fromhighly complex environmental samples. Possible mechanisms offormation of these particulate mutagens are discussed. Theyinclude the reactions of polycyclic aromatic hydrocarbons presenton the surfaces of combustion-generated particles with gaseousco-pollutants such as nitrogen dioxide plus nitric acid, andozone. In discussing this research on a societally "relevant" problem,we illustrate the importance of "Science as a Way of Knowing."We further suggest that this integrated approach to scientificproblem solving by chemical and biological scientists mightserve as an example of a discussion topic on human ecology forundergraduate courses in the natural sciences.     

The nucleotide sequence of a rat liver glutathione S-transferase subunit cDNA clone

We have determined the nucleotide sequence of a cloned cDNA derived from liver poly(A) RNA of pentobarbital-treated rats encoding a glutathione S-transferase subunit. This cDNA clone pGTR261 contains one open reading frame of 222 amino acids, a complete 3' noncoding region, and 63 nucleotides in the 5' noncoding region. The cloned DNA hybridizes to rat poly(A) RNA in a tissue-specific fashion, with strong signals to liver and kidney poly(A) RNA(s) of approximately 1100 and approximately 1400 nucleotides in size but little or no hybridization to poly(A) RNAs from heart, lung, seminal vesicles, spleen, or testis under stringent conditions. Our sequence covers the cDNA sequence of pGST94 which contains a partial coding sequence for a liver glutathione S-transferase subunit of Ya size. Comparison of sequences with our earlier clone pGTR112 suggests that there are at least two mRNA species coding for two different subunits of the Ya (Mr = 25,600) subunit family with very limited amino acid substitutions mainly of conserved polarity. The divergent 3' noncoding sequences should be useful molecular probes in differentiating these two different but otherwise very similar subunits in induction and genomic structure analyses. Our results suggest that tissue-specific expression of the glutathione S-transferase subunits represented by the sequences of pGTR261 and pGTR112 may occur at or prior to the level of RNA processing.     

Interaction between 2-chloroadenosine and alpha-adrenoceptors in rat vas deferens

The effect of 2-chloroadenosine (2CA) on the binding of alpha 1- and alpha 2-adrenoceptor ligands in the rat vas deferens was investigated. In homogenates of vas deferens, 2CA (10(5)M) increased 3H-clonidine maximal binding sites from an undetectable level to 0.71 +/- 0.08 pmol/g. wet weight or 10.1 +/- 1.1 fmol/mg protein (N=12). This effect lasted for at least 5 hours after removal of 2CA. Concurrent addition of 1.25 mM theophylline completely abolished the effect of 2CA. A similar effect of 2CA on 3H-clonidine binding was observed following incubation of intact tissues with 2CA prior to homogenization. The effect of 2CA were similar in potency in the homogenate to that in the intact organ, suggesting that 2CA-sensitive sites are located on the outer surface of the plasma membrane. The binding of 3H-prazosin was not influenced by the presence of 10(-5)M 2CA. Contractions of isolated vasa deferentia induced by norepinephrine and phenylephrine were not changed by 10(-5)M 2CA, but the inhibition by clonidine of contractions induced by electric stimulation was enhanced by preincubation for 30 min with 10(-5)M 2CA. The results suggest that 2CA increases the number of available alpha 2-adrenoceptors and this interactions occurs, at least in part, presynaptically.     

Condensed tannins: co-occurrence of procyanidins,prodelphinidins and profisetinidins in the heartwood of Acacia baileyana

The flavonoids and condensed tannins of the heartwood of Acacia baileyana var. purpurea are described. In conformity with other Acacia species, the hydroxylation pattern of the flavonoids is of the resorcinol type but, in sharp contrast, the tannins are heterogeneous consisting of a mixture of the resorcinol and phtoroglucinol series. Dimeric proanthocyanidins of the phloroglucinol type were absent and this exception to the general observation that they invariably co-occur with the polymers may be explained by the relative nucleophilicity of the aromatic A-rings.