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.     

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.     

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.     

In vivo EMG biofeedback in violin and viola pedagogy

In vivo EMG biofeedback was found to be an effective pedagogical tool for removing unwanted left-hand tension in nine violin and viola players. Improvement occurred rapidly and persisted throughout a 5-month follow-up period. Further studies will be necessary to assess the effect of biofeedback independent of placebo effects. The brevity of the method and the magnitude of improvement warrant further investigation.     

Unlikelihood that minimal phylogenies for a realistic biological study can be constructed in reasonable computational time

The problem of determining a phylogeny of maximum parsimony from a given set of protein sequences is defined. It is shown that this problem is what is called, in computer science, NP-complete. The implication of this result is that it is equivalent in difficulty to a host of other problems in combinatorial optimization which are notorious for their intractability. This implies that it is more fruitful to attempt to develop heuristic techniques (which do not guarantee maximum parsimony but which do run in reasonable computer time) than to try to develop exact algorithms for phylogeny construction     

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.     

Absorption of light in photoreceptors: Effect of waveguiding property

The effect of waveguiding property (i.e., the intensity distribution) of the photoreceptor on the number of photons absorbed in a photoreceptor has been studied. It has been found that the effect is significant only for large values of the exposure and the maximum effect is less than 11% in the case of human rod photoreceptor. In the analysis, the funnelling effect, which follows from the coupling between the interior and exterior fields, has not been considered.Work partially supported by the Department of Science and Technology (India)B. D. Gupta is associated with the School of Bioscience Studies