Alpha-thrombin stimulates nuclear diglyceride levels and differential nuclear localization of protein kinase C isozymes in IIC9 cells.

The mechanism by which an agonist, binding to a cell surface receptor, exerts an effect on events in the nucleus is not known. We have previously shown (Leach, K. L., Ruff, V. A., Wright, T. M., Pessin, M. S., and Raben, D. M. (1991) J. Biol. Chem. 266, 3215-3221) that alpha-thrombin treatment of IIC9 cells results in increased levels of cellular 1,2-diacylglycerol (DAG) and activation of protein kinase C (PKC). Here, we have examined whether changes in nuclear PKC and nuclear DAG also are induced following alpha-thrombin treatment. IIC9 cells were treated with 500 ng/ml alpha-thrombin, and nuclei were then isolated. Western blot analysis using isozyme-specific antibodies demonstrated the presence of PKC alpha, but not PKC epsilon or zeta in the nuclei of cells treated with either phorbol 12-myristate 13-acetate or alpha-thrombin. The increase in nuclear PKC alpha levels was accompanied by a 10-fold increase in nuclear PKC specific activity and stimulated phosphorylation of at least six nuclear proteins. The rise in nuclear PKC levels occurred rapidly and reached a maximum at 30-60 s, which was followed by a decline back to the control level over the next 15 min. In addition, alpha-thrombin treatment resulted in an immediate rise in DAG mass levels in the nuclear fractions. Kinetic analysis indicated that a maximum increase in DAG levels occurred 2.5-5 min after the addition of alpha-thrombin and remained elevated for at least 30 min. In cells labeled with [3H]myristic acid, alpha-thrombin treatment induced an increase in radiolabeled nuclear diglycerides, suggesting that the stimulated nuclear DAGs are derived, at least in part, from phosphatidylcholine. Our results suggest that increases in both nuclear DAG levels and PKC activity following alpha-thrombin treatment may play a role in mediating thrombin-induced nuclear responses such as changes in gene expression and cellular proliferation.     

A phenylalanine in peptide substrates provides for selectivity between cGMP- and cAMP-dependent protein kinases.

Bovine lung cGMP-binding cGMP-specific phosphodiesterase (cG-BPDE) is a potent and relatively specific substrate for cGMP-dependent protein kinase (cGK) as compared to cAMP-dependent protein kinase (cAK) (Thomas, M. K., Francis, S. H., and Corbin, J. D. (1990) J. Biol. Chem. 265, 14971-14978). A synthetic peptide, RKISASEFDRPLR (BPDEtide), was synthesized corresponding to the sequence surrounding the phosphorylation site in cG-BPDE. BPDEtide retained the cGK/cAK kinase specificity demonstrated by native cG-BPDE: the apparent Km of BPDEtide for cGK was 5-fold lower than that for cAK (Km = 68 and 320 microM, respectively). Vmax values were 11 mumol/min/mg for cGK and 3.2 mumol/min/mg for cAK. The peptide was not phosphorylated to a measurable extent by protein kinase C or by calcium/calmodulin-dependent protein kinase II. Thus, the primary amino acid sequence of the peptide substrate was sufficient to confer kinase specificity. Studies in crude tissue extracts indicated that BPDEtide was the most selective peptide substrate documented for measuring cGK activity. Peptide analogs of BPDEtide were synthesized to determine the contribution of specific residues to cGK or cAK substrate specificity. Substitution of a Lys for the amino-terminal Arg did not reduce cGK/cAK specificity; neither did the exchange of an Ala for the non-phosphorylated Ser nor the removal of the 3 carboxyl-terminal residues. A truncated BPDEtide (RKISASE) served equally well as substrate (Km approximately 90 microM) for both kinases. However, restoration of the Phe, to yield RKISASEF, reproduced the original cGK/cAK specificity for BPDEtide (Km = 120 and 480 microM, respectively), primarily by decreasing the affinity of cAK. Addition of a carboxyl-terminal Phe to the peptide RKRSRAE (derived from the sequence of the cGK phosphorylation site in histone H2B) or to the peptide LRRASLG (derived from the sequence of the cAK phosphorylation site in pyruvate kinase) also improved the cGK/cAK specificity by decreasing the affinity of cAK. These data suggested that the Phe in each substrate tested is a negative determinant for cAK.     

Increased Activity of a Cationic Peroxidase Associated with an Incompatible Interaction Between Xanthomonas oryzae pv oryzae and Rice (Oryza sativa)

Rice (Oryza sativa L.) cultivar Cas 209 carries the gene Xa-10 for resistance to race 2 of Xanthomonas oryzae pv oryzae, the bacterial blight pathogen. When seedling leaves of Cas 209 plants were infiltrated with bacterial cell suspensions of strain PXO86^Rif (race 2, incompatible), total peroxidase activity in extracts from extracellular spaces increased almost threefold between 16 and 24 hours after inoculation. The increase in total peroxidase activity in extracellular extracts was correlated with the appearance of a 43-kilodalton peroxidase isoenzyme with an isoelectric point of 8.6. Increases in the activities of two anionic peroxidase isoenzymes also were associated with the incompatible interaction. Later during the interactions, total peroxidase activities increased in both compatible (cv Cas 209 infiltrated with race 1, PXO61^Sm) and control (Cas 209 infiltrated with water) treatments, but final activity levels were less than that observed in the incompatible combination. Similarly, the cationic peroxidase was detected in all three treatments by 48 hours after infiltration, but at reduced levels in compatible and water-infiltrated control treatments relative to the incompatible combination. Accumulation of this peroxidase in extracellular spaces thus may play a role in the defense response in cultivar Cas 209.     

Assessment of genetic diversity and population structure of Xanthomonas oryzae pv. oryzae with a repetitive DNA element.

A repetitive DNA element cloned from Xanthomonas oryzae pv. oryzae was used to assess the population structure and genetic diversity of 98 strains of X. oryzae pv. oryzae collected between 1972 and 1988 from the Philippine Islands. Genomic DNA from X. oryzae pv. oryzae was digested with EcoRI and analyzed for restriction fragment length polymorphisms (RFLPs) with repetitive DNA element as a probe. Twenty-seven RFLP types were identified; there was no overlap of RFLP types among the six races from the Philippines. Most variability (20 RFLP types) was found in strains of races 1, 2, and 3, which were isolated from tropical lowland areas. Four RFLP types (all race 5) were found among strains isolated from cultivars grown in the temperate highlands. The genetic diversity of the total population of X. oryzae pv. oryzae was 0.93, of which 42% was due to genetic differentiation between races. The genetic diversities of strains collected in 1972 to 1976, 1977 to 1981, and 1982 to 1986, were 0.89, 0.90, and 0.92, respectively, suggesting a consistently high level of variability in the pathogen population over the past 15 years. Cluster analysis based on RFLP banding patterns showed five groupings at 85% similarity. The majority of strains from a given race were contained within one cluster, except for race 3 strains, which were distributed in three of the five clusters.     

Comparison of properties of commercially available crystalline native and recombinant firefly luciferases

Commercially available crystalline native and recombinant firefly luciferases were compared. The two types of luciferase had indistinguishable responses to variation in ATP and luciferin concentrations and to omission of reaction components. The time courses of light production, the responses to nucleotide analogues, and the stability of the enzymes under several storage conditions were identical. The native enzyme had a slightly greater specific activity and was more sensitive to trypsin degradation. These differeces are probably attributable to differences in conformation.     

A comparison of the inhibitory effects of melatonin and indomethacin on platelet aggregation and thromboxane release

Collagen-induced platelet aggregation and thromboxane release is inhibited, in a concentration response relationship, by preincubation of gel-filtered platelets with melatonin in the concentration range 430 nM – 4.3 mM. Inhibition of platelet aggregation and thromboxane release also occurs in the presence of indomethacin (4.3 nM – 4.3 mM), a known potent inhibitor of prostaglandin synthesis. Arachidonic acid-induced platelet aggregation and thromboxane release was inhibited in the presence of 4.0 mM melatonin. We therefore propose that inhibition of prostaglandin synthesis maybe the mechanism by which melatonin expresses its activity. Its antigonadotropic activity may result from inhibition of PGE₂ synthesis in the hypothalamus and median eminence.     

Anaerobic axillary abscess.

Fifty-two patients with axillary abscesses were seen during two years. Staphylococcus aureus was isolated from 34, anaerobic bacteria from 12, and skin flora from five; in one case the pus was sterile. Seven patients with hidradenitis suppurativa had recurrent infection with abscess formation, which was bilateral in three. Anaerobes were isolated in five of these cases and skin flora alone in two. Anaerobes are secondary invaders in this condition, and histological examination shows that the primary abnormality is obstruction of pilosebaceous follicles and apocrine glands, associated with keratin plugging of the follicles. Chemotherapy offers little hope of cure, although metronidazole removes the offensive smell of the discharge. Radical surgery is usually indicated.     

NEUROTRANSMITTER UPTAKE INTO SLICES OF RAT CEREBRAL CORTEX IN VITRO: EFFECT OF SLICE SIZE

Abstract— The uptake of [¹⁴C]GABA, [¹⁴C]taurine, [³H] β -alanine and [¹⁴C]dopamine was compared in slices of rat cerebral cortex of three different sizes (0.1 × 0.1 × 2 mm, 0.2 × 0.2 × 2 mm and 0.4 × 0.4 × 2 mm prepared with a mechanical tissue chopper). [¹⁴C]Taurine and [³H] β -alanine uptake increased whereas [¹⁴C]GABA uptake decreased with increasing slice size. [¹⁴C]Dopamine uptake was optimal in 0.2 × 0.2 × 2 mm slices. Increasing slice size was shown to decrease inhibition of [³H] β -alanine and [¹⁴C]GABA uptake by l -2,4-diaminobutyric acid. Lactate dehydrogenase activity increased with increasing slice size indicating decreased tissue damage or increased cellular integrity. The possibility that varying slice size can be used to distinguish between neuronal and glial uptake is discussed. It is suggested that taurine uptake in the cerebral cortex is predominantly glial.     

Circular dichroism studies of myoglobin and leghemoglobin.

The circular dichroism spectra of leghemoglobin a from the root nodules of soybean have been compared with those for sperm whale myoglobin in the fat- and near-ultraviolet and the Soret and visible regions of the spectrum. Circular dichroism spectra in the far-ultraviolet show that the leghemoglobins all have a high alpha-helix content (soybean leghemoglobin a, 55%) regardless of the nature of bound ligands and oxidation or spin state of the heme iron. The known sequence homologies with mammalian hemoglobins may therefore be reflected in conformational homologies as suggested by the x-ray studies of Vainshtein et al. ((1975) Nature (London) 254, 163-164) on lupin leghemoglobin. Removal of the heme moiety decreases helicity by only 9% for leghemoglobins, compared with 23% for myoglobin. This, the much smaller heme contribution to the near-ultraviolet circular dichroism than in myoglobin, and the greater accessibility of the heme moiety to aqueous solvent (Nicola et al. (1974), Proc. Aust. Biochem. Soc. 7, 21) suggest that the association between heme and protein is much weaker in leghemoglobins than in myoglobin. The aromatic Soret and visible circular dichroism spectra for all derivatives of leghemoglobin are opposite in sense to those for myoglobin, showing that the patterns of protein side chain contacts with the heme are different in the two classes of heme proteins. There is strong evidence that one of the two tryptophans whose identity and structural role in myoglobin is known, is present also in plant leghemoglobins, hydrogen-bonded and in a similar nonpolar environment whether heme is present or not. The above findings help to explain the remarkably high oxygen affinity and some other ligand-binding properties of leghemoglobins which differ from those of myoglobin.     

The initial attachment of transforming DNA to competent Bacillus subtilis.

Summary The initial attachment of transforming DNA to competent Bacillus subtilis is temperature independent between 25° and 45°. However, below 15° there is a significant reduction in the amount of DNA attached to competent cells. The DNA that is attached at 4° can lead to transformation or interfere effectively with the subsequent attachment of a distinctive DNA when the cells are shifted to a permissive temperature (37°). These data suggest that the attachment of DNA at 4° is to sites normally involved in the transformation process. The amount of DNA that is initially attached to the bacteria at 4° or 37° after perturbation of the cells by ionic strength changes, repetitive washings, or periodate oxidation varies with the temperature at which the treatment occurs. These results are consistent with a reorientation of the DNA attachment sites upon lowering the temperature to 4°, such that their affinity for DNA and susceptibility inhibitory treatments are reduced.National Institutes of Health Research Career Program Awardee, CA-K3-6487 during a portion of this investigation.