HlyC, the internal protein acyltransferase that activates hemolysin toxin: the role of conserved tyrosine and arginine residues in enzymatic activity as probed by chemical modification and site-directed mutagenesis.

Internal fatty acylation of proteins is a recognized means of modifying biological behavior. Escherichia coli hemolysin A (HlyA), a toxic protein, is transcribed as a nontoxic protein and made toxic by internal acylation of two lysine residue epsilon-amino groups; HlyC catalyzes the acyl transfer from acyl-acyl carrier protein (ACP), the obligate acyl donor. Conserved residues among the respective homologous C proteins that activate 13 different RTX (repeats in toxin) toxins of which HlyA is the prototype likely include some residues that are important in catalysis. Possible roles of two conserved tyrosines and two conserved arginines were investigated by noting the effects of chemical modifiers and site-directed mutagenesis. TNM modification of HlyC at pH 8.0 led to extensive inhibition that was prevented by the presence of the substrate myristoyl-ACP but not by the product, ACPSH. NAI had no effect. Y70G and Y150G greatly diminished enzyme activity, whereas mutations Y70F and Y150F exhibited wild-type activity. Modification of arginine residues with PG markedly lowered acyltransferase activity with moderate protection by both myristoyl-ACP and ACPSH. Under optimum conditions, four separate mutations of the two conserved arginine residues (R24A, R24K, R87A, and R87K) had little effect on acyltransferase activity.     

Cytochrome P450 2E1-derived reactive oxygen species mediate paracrine stimulation of collagen I protein synthesis by hepatic stellate cells.

To evaluate possible fibrogenic effects of CYP2E1-dependent generation of reactive oxygen species, a model was developed using co-cultures of HepG2 cells, which do (E47 cells) or do not (C34 cells) express cytochrome P450 2E1 (CYP2E1) with stellate cells. There was an increase in intra- and extracellular H(2)O(2), lipid peroxidation, and collagen type I protein in stellate cells co-cultured with E47 cells compared with stellate cells alone or co-cultured with C34 cells. The increase in collagen was prevented by antioxidants and a CYP2E1 inhibitor. CYP3A4 did not mimic the stimulatory effects found with CYP2E1. Collagen mRNA levels remained unchanged, and pulse-chase analysis indicated similar half-lives of collagen I protein between both co-cultures. However, collagen protein synthesis was increased in E47 co-culture. Hepatocytes from pyrazole-treated rats (with high levels of CYP2E1) induced collagen protein in primary stellate cells, and antioxidants and CYP2E1 inhibitors blocked this effect. These results suggest that increased translation of collagen mRNA by CYP2E1-derived reactive oxygen species is responsible for the increase in collagen protein produced by the E47 co-culture. These co-culture models may be useful for understanding the impact of CYP2E1-derived ROS on stellate cell function and activation.     

Crystal structure of a novel mid-gut procarboxypeptidase from the cotton pest Helicoverpa armigera.

The cotton bollworm Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is one of the most serious insect pests in Australia, India and China. The larva causes substantial economical losses to legume, fibre, cereal oilseed and vegetable crops. This pest has proven to be difficult to control by conventional means, mainly due to the development of pesticide resistance. We present here the 2.5 A crystal structure from the novel procarboxypeptidase (PCPAHa) found in the gut extracts from H. armigera larvae, the first one reported for an insect. This metalloprotease is synthesized as a zymogen of 46.6 kDa which, upon in vitro activation with Lys-C endoproteinase, yields a pro-segment of 91 residues and an active carboxypeptidase moiety of 318 residues. Both regions show a three-dimensional structure quite similar to the corresponding structures in mammalian digestive carboxypeptidases, the most relevant structural differences being located in the loops between conserved secondary structure elements, including the primary activation site. This activation site contains the motif (Ala)(5)Lys at the C terminus of the helix connecting the pro- and the carboxypeptidase domains. A remarkable feature of PCPAHa is the occurrence of the same (Ala)(6)Lys near the C terminus of the active enzyme. The presence of Ser255 in PCPAHa instead of Ile and Asp found in the pancreatic A and B forms, respectively, enlarges the S1' specificity pocket and influences the substrate preferences of the enzyme. The C-terminal tail of the leech carboxypeptidase inhibitor has been modelled into the PCPAHa active site to explore the substrate preferences and the enzymatic mechanism of this enzyme.     

Immunologic and nonimmunologic responsiveness in ragweed-sensitized dogs

The relationship between airway responsiveness to inhaled antigen and histamine, immunologic release of lung histamine, immunologic responsiveness of skin, and specific immunoglobulin E (IgE) antibodies were examined in 11 inbred allergic dogs immunized with extracts of ragweed and grass and 5 nonimmunized control dogs from the same colony. Airway responsiveness to antigen and histamine was characterized by the doses that increased the airflow resistance of the total respiratory system to twice the control values (ED200). Highly significant correlations were found between airway responsiveness and cutaneous responsiveness to antigen and other immunologic characteristics (e.g., IgE and histamine released from lung by inhaled antigen) in all dogs. In ragweed-sensitized dogs, there was an inverse correlation between immunologic responsiveness (reflected by the cutaneous response to antigen and histamine released from lung by inhaled antigen) and nonimmunologic responsiveness of airways (histamine ED200: r = 0.73, P less than 0.05 and r = 0.75, P less than 0.01, respectively). Antigen ED200 was also correlated with histamine release from lung after antigen inhalation (r = 0.74; P less than 0.01). We conclude that airway reactions to inhaled antigen in allergic dogs are dependent not only on immunologic factors but also on the degree of nonimmunologic airway responsiveness to histamine and that these factors are correlated inversely.     

Changes in the levels of pituitary and steroid hormones in ovine and human ovarian follicular fluid

Changes in the protein and steroid hormones of follicular fluid, aspirated from different follicles of sheep and human ovaries, have been measured and correlated with the size of the follicles. As the fluid contains a number of proteins, steroids have been measured directly and after ether extraction. The follicular fluid concentrations of progesterone and 17 beta-oestradiol measured directly in the fluid increased with the size of the follicles. The levels of free testosterone remained constant in all sizes of follicles, while those of bound hormone showed a 10- to 15-fold increase over the free testosterone concentrations in both the sheep and human follicular fluid. A decrease in the levels of bound testosterone in the fluid of large follicles (LFFL) coincided with the increase in bound 17 beta-oestradiol, suggesting the possible conversion of bound testosterone to oestrogen as the follicle attained maturity. The ratio of follicle-stimulating hormone (FSH) to luteinizing hormone (LH) varied in the fluid obtained from different size follicles, being 1:7 in small (SFFL), 1.3.5 in medium (MFFL) and 1:2.3 in large (LFFL) follicles of sheep ovaries. The LH content of follicular fluid of different size follicles appeared to be the same, with LFFL showing a minor increase over SFFL. In the human, the fluid from medium follicles contained very little LH compared to LFFL. These differences in the pattern of LH levels present in the fluid from different size follicles between human and sheep ovaries presumably reflect species variations in the entry of LH into the follicles.     

CDC17: an essential gene that prevents telomere elongation in yeast

The CDC17 gene product performs an essential stage-specific function during the Saccharomyces cerevisiae cell cycle. When cdc17-1 strains are grown at the maximum permissive temperature, recombination is induced preferentially in the genetic interval of the chromosome closest to the telomere. Telomeres are longer in cdc17 strains than in CDC17 strains at the permissive temperature because of addition of sequence near or in the poly (C1-3A) telomeric DNA and become even longer when cells are propagated at elevated temperatures. The mitotic recombination events require RAD52 function, but telomere growth does not. Long telomeres are maintained for many generations when crossed into a CDC17+ background, suggesting that telomere length is largely conserved during replication. The altered telomere length phenotype of cdc17 mutations is recessive and coreverts and cosegregates with the temperature-sensitive lethal phenotype.     

The effect of carbohydrate depletion on procoagulant activity and in vivo survival of highly purified human factor VIII

Human factor VIII procoagulant protein (factor VIII) was purified using a modification of our previously described method, in which Sephacryl S-400 elution, rather than QAE-cellulose chromatography, served as the final purification step. The protein had a specific activity of more than 2500 U/mg and consisted of a single polypeptide (Mr 100 000) when analyzed by SDS-polyacrylamide gel electrophoresis. Factor VIII was shown to be a glycoprotein by staining with periodic acid-Schiff's reagent following electrophoresis. Treatment of factor VIII with a mixture of exo- and endoglycosidases caused a reduction by about 50% in the intensity of periodic acid-Schiff staining, as determined by scanning densitometry, and an increase in electrophoretic mobility (equivalent to a new Mr 95 000). Removal of this portion of the total carbohydrate had no significant effect on factor VIII clotting activity or on thrombin potentiation of clotting activity. The in vivo survival curves of a native and sugar-depleted 125I-labeled factor VIII both showed similar patterns of initial rapid decay to 60 and 40% activity, respectively, followed by a one-half decay time of 4 h for both. These results suggest that the carbohydrate portion of human factor VIII does not contribute significantly to either clotting function in vitro or to biological turnover in vivo.