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.     

The conversion of tubulin carboxyl groups to amides has a stabilizing effect on microtubules.

In a recent study, we demonstrated that the conversion of carboxyl residues in the C-termini of tubulin to neutral amides with glycine ethyl ester enhanced the ability of the protein to assemble into microtubules and decreased its interaction with microtubule-associated proteins (MAPs). In this work, we investigated the effects of carboxyl modification on the dynamic behavior of microtubules at polymer mass steady state. After steady state, microtubules assembled from unmodified tubulin were sheared, and the mean polymer lengths decreased to 5 microns and then increased to 29 microns within 130 min. In contrast, lengths of sheared microtubules polymerized from tubulin containing 23 modified carboxyl groups increased by only 2-fold. Stabilization of polymer lengths was also observed directly by video-enhanced light microscopy of microtubules grown off of axonemes. Rapid shortening was seen in microtubules composed of unmodified but not modified tubulin. Further evidence for the less dynamic behavior of microtubules as a result of carboxyl modification was obtained from kinetic studies of the elongation phase during assembly which showed a 3-fold lower off-rate constant, k-, for modified microtubules. Another effect of the modification was a 12-fold reduction in the steady-state rate constant for GTP hydrolysis (165 s-1 for unmodified and 14 s-1 for modified). These results suggest that reduction of the negative charges in the C-termini by modification of the acidic residues stabilizes microtubules against depolymerization. MAPs may stabilize microtubules in an analogous manner.     

Effect of alkyl side chain variation on the electron-transfer activity of ubiquinone derivatives

The effect of the alkyl side chain of the ubiquinone molecule on the electron-transfer activity of ubiquinone in mitochondrial succinate-cytochrome c reductase is studied by using synthetic ubiquinone derivatives that possess the basic ubiquinone structure of 2,3-dimethoxy-5-methyl-1,4-benzoquinone with different alkyl side chains at the 6-position. The alkyl side chains vary in chain length, degree of saturation, and location of double bonds. When a ubiquinone derivative is used as an electron acceptor for succinate-ubiquinone reductase, an alkyl side chain of six carbons is needed to obtain the maximum activity. However, when it serves as an electron donor for ubiquinol-cytochrome c reductase or as a mediator in succinate-cytochrome c reductase, an alkyl side chain of 10 carbons gives maximal efficiency. Introduction of one or two isolated double bonds into the alkyl side chain of the ubiquinone molecule has little effect on electron-transfer activity. However, a conjugated double bond system in the alkyl side chain drastically reduces electron-transfer efficiency. The effect of the conjugated double bond system on the electron-transferring efficiency of ubiquinone depends on its location in the alkyl side chain. When location is far from the benzoquinone ring, the effect is minimal. These observations together with the results obtained from photoaffinity-labeling studies lead us to conclude that flexibility in the portion of the alkyl side chain immediately adjacent to the benzoquinone ring is required for the electron-transfer activity of ubiquinone.     

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.     

Hyperoxia causes increased albumin permeability of cultured endothelial monolayers

The effect of phasic eye movement activity on ventilation during rapid-eye-movement (REM) sleep was studied in seven healthy young adults by use of the respiratory inductive plethysmograph. Mean ventilation (VE) and ventilatory components during REM sleep were not significantly different from that seen in either stages 1-2 or 3-4 sleep. The percent of rib cage contribution to ventilation in REM sleep, 29.3 +/- 5.1%, was reduced compared with 54.4 +/- 5.8% in stage 1-2 and 52.2 +/- 4.3% in stage 3-4 sleep (P less than 0.005). When one separated breaths by the degree of associated phasic eye movement activity, it became apparent that breathing during REM sleep is very heterogeneous. Increasing eye movement activity was associated with inhibition of ventilation with a reduction in VE from 5.1 +/- 0.3 to 3.8 +/- 0.3 l/min. Tidal volume and frequency both fell, whereas inspiratory duration was unchanged. Compartmental ventilation was also affected, with the fall in the rib cage contribution from 37.8 +/- 6.4 to 15.3 +/- 5.6%. Chest wall and abdominal movement became more asynchronous as phasic-eye-movement activity increased and frank paradoxical breathing was seen.     

A numerical taxonomic study of Actinobacillus, Pasteurella and Yersinia

A numerical taxonomic study of strains of Actinobacillus, Pasteurella and Yersinia, with some allied bacteria, showed 23 reasonably distinct groups. These fell into three major areas. Area A contained species of Actinobacillus and Pasteurella: A. suis, A. equuli, A. lignieresii, P. haemolytica biovar A, P. haemolytica biovar T, P. multocida, A. actinomycetemcomitans, 'P. bettii', 'A. seminis', P. ureae and P. aerogenes. Also included in A was a composite group of Pasteurella pneumotropica and P. gallinarum, together with unnamed groups referred to as 'BLG', 'Mair', 'Ross' and 'aer-2'. Area B contained species of Yersinia: Y. enterocolitica, Y. pseudotuberculosis, Y. pestis and a group 'ent-b' similar to Y. enterocolitica. Area C contained non-fermenting strains: Y. philomiragia, Moraxella anatipestifer and a miscellaneous group 'past-b'. There were also a small number of unnamed single strains.     

Electrophysiological and behavioral correlates of sleep in the desert iguana, Dipsosaurus dorsalis Hallowell

The circadian behavior of the desert iguana, Dipsosaurus dorsalis, was investigated on the basis of behavioral observation and electrophysiological recording. D. dorsalis adequately complies with accepted criteria for both behavioral sleep and paradoxical sleep. At 20 degrees C, 12% of the photophase is spent in sleep, 95% of the scotophase is spent in sleep. Paradoxical sleep occurs at all times of the year, but only at temperatures of 20 and 30 degrees C. Amounts of behavioral sleep are affected by both temperature and time of year. Total sleep increases with decreased day length and decreased temperature. Daytime sleep increases with decreased temperature.     

An isocitrate lyase of higher plants: analysis and comparison of some molecular properties

A new purification procedure for isocitrate lyase from Pinus pinea is reported. The final preparation shows charge homogeneity and a purity degree higher than 95%. It is possible to remove catalase completely by exploiting the high hydrophobicity of isocitrate lyase. The enzyme has a Mr of 264,000 and is likely composed of four subunits, each with a Mr of 66,000. The binding of radioactively labeled oxalate revealed four catalytic sites per oligomer. These data suggest that isocitrate lyase subunits are similar, if not identical. The Michaelis constant for isocitrate is equal to 33 microM; molecular activity is about 2670 mol X min-1 X mol of enzyme-1. The amino acid composition of the enzyme was also determined. Isocitrate lyase appears resistant to proteolysis by carboxypeptidase A. Hydrazinolysis, Edman degradation, and dansyl chloride treatment indicate that both carboxy and amino terminals are probably inaccessible or blocked.