Function of the central domain of streptokinase in substrate plasminogen docking and processing revealed by site-directed mutagenesis

The possible role of the central beta-domain (residues 151-287) of streptokinase (SK) was probed by site-specifically altering two charged residues at a time to alanines in a region (residues 230-290) previously identified by Peptide Walking to play a key role in plasminogen (PG) activation. These mutants were then screened for altered ability to activate equimolar "partner" human PG, or altered interaction with substrate PG resulting in an overall compromised capability for substrate PG processing. Of the eight initial alanine-linker mutants of SK, one mutant, viz. SK(KK256.257AA) (SK-D1), showed a roughly 20-fold reduction in PG activator activity in comparison to wild-type SK expressed in Escherichia coli (nSK). Five other mutants were as active as nSK, with two [SK(RE248.249AA) and SK(EK281.282AA), referred to as SK(C) and SK(H), respectively] showing specific activities approximately one-half and two-thirds, respectively, that of nSK. Unlike SK(C) and SK(H), however, SK(D1) showed an extended initial delay in the kinetics of PG activation. These features were drastically accentuated when the charges on the two Lys residues at positions 256 and 257 of nSK were reversed, to obtain SK(KK256.257EE) [SK(D2)]. This mutant showed a PG activator activity approximately 10-fold less than that of SK(D1). Remarkably, inclusion of small amounts of human plasmin (PN) in the PG activation reactions of SK(D2) resulted in a dramatic, PN dose-dependent rejuvenation of its PG activation capability, indicating that it required pre-existing PN to form a functional activator since it could not effect active site exposure in partner PG on its own, a conclusion further confirmed by its inability to show a "burst" of p-nitrophenol release in the presence of equimolar human PG and p-nitrophenyl guanidino benzoate. The steady-state kinetic parameters for HPG activation of its 1:1 complex with human PN revealed that although it could form a highly functional activator once "supplied" with a mature active site, the Km for PG was increased nearly eightfold in comparison to that of nSK-PN. SK mutants carrying simultaneous two- and three-site charge-cluster alterations, viz., SK(RE24249AA:EK281.282AA) [SK(CH)], SK(EK272.273AA;EK281.282AA) [SK(FH)], and SK(RE248.249AA;EK272.273AA:EK281.282AA+ ++) [SK(CFH)], showed additive/synergistic influence of multiple charge-cluster mutations on HPG activation when compared to the respective "single-site" mutants, with the "triple-site" mutant [SK(CFH)] showing absolutely no detectable HPG activation ability. Nevertheless, like the other constructs, the double- and triple-charge cluster mutants retained a native like affinity for complexation with partner PG. Their overall structure also, as judged by far-ultraviolet circular dichroism, was closely similar to that of nSK. These results provide the first experimental evidence for a direct assistance by the SK beta-domain in the docking and processing of substrate PG by the activator complex, a facet not readily evident probably because of the flexibility of this domain in the recent X-ray crystal structure of the SK-plasmin light chain complex.     

Demonstration of protein-protein interaction specificity by NMR chemical shift mapping.

Chemical shift mapping is becoming a popular method for studying protein-protein interactions in solution. The technique is used to identify putative sites of interaction on a protein surface by detecting chemical shift perturbations in simple (1H, 15N)-HSQC NMR spectra of a uniformly labeled protein as a function of added (unlabeled) target protein. The high concentrations required for these experiments raise questions concerning the possibility for non-specific interactions being detected, thereby compromising the information obtained. We demonstrate here that the simple chemical shift mapping approach faithfully reproduces the known functional specificities among pairs of closely related proteins from the phosphoenolpyruvate:sugar phosphotransferase systems of Escherichia coli and Bacillus subtilis.     

Response of brown mussel, Perna indica, to elevated temperatures in relation to power plant biofouling control

1. 1. Results of a study on lethal and sublethal responses of different size groups of the tropical brown mussel, Perna indica, when exposed to different temperatures are presented.

2. 2. Exposure to a temperature of 38°C showed 100% mortality of 9 mm size group mussels in 120 min.

3. 3. Mortality was dependent on age (size) of the mussels, young ones being more susceptible than older ones.

4. 4. All size groups showed a progressive reduction in physiological activities such as filtration rate, foot activity and byssus thread production when temperature was increased from 30°C.

5. 5. This study suggests that heat treatment is an attractive alternative to chlorination for mussel fouling control in tropical power stations.

     

Effect of Cassia auriculata Linn. Root extract on cisplatin and gentamicin-induced renal injury

The ethanol extract of the roots of Cassia auriculata was studied for its nephroprotective activity in cisplatin- and gentamicin-induced renal injury in male albino rats. In the cisplatin model, the extract at doses of 300 and 600 mg/kg body wt. reduced elevated blood urea and serum creatinine and normalized the histopathological changes in the curative regimen. In the gentamicin model, the ethanol extract at a dose of 600 mg/kg body wt. reduced blood urea and serum creatinine effectively in both the curative and the preventive regimen. The extract had a marked nitric oxide free-radical-scavenging effect. The findings suggest that the probable mechanism of nephroprotection by C. auriculata against cisplatin- and gentamicin-induced renal injury could be due to its antioxidant and free-radical-scavenging property.     

Mechanistic and kinetic analysis of the DcpS scavenger decapping enzyme

Decapping is an important process in the control of eukaryotic mRNA degradation. The scavenger decapping enzyme DcpS functions to clear the cell of cap structure following decay of the RNA body by catalyzing the hydrolysis of m(7)GpppN to m(7)Gp and ppN. Structural analysis has revealed that DcpS is a dimeric protein with a domain-swapped amino terminus. The protein dimer contains two cap binding/hydrolysis sites and displays a symmetric structure with both binding sites in the open conformation in the ligand-free state and an asymmetric conformation with one site open and one site closed in the ligand-bound state. The structural data are suggestive of a dynamic decapping mechanism where each monomer could alternate between an open and closed state. Using transient state kinetic studies, we show that both the rate-limiting step and rate of decapping are regulated by cap substrate. A regulatory mechanism is established by the intrinsic domain-swapped structure of the DcpS dimer such that the decapping reaction is very efficient at low cap substrate concentrations yet regulated with excess cap substrate. These data provide biochemical evidence to verify experimentally a dynamic and mutually exclusive cap hydrolysis activity of the two cap binding sites of DcpS and provide key insights into its regulation.     

Differentiated structure and function of primary cultures of monkey oviductal epithelium

Summary We have established well-differentiated, polarized cultures of monkey oviductal epithelium. Oviductal epithelial cells were isolated by protease digestion and plated on collagen-coated, porous cell culture inserts. About 5 d after plating, cells developed detectable transepithelial electrical resistance of up to 2000 Ω.cm² (an index of tight junction formation) and transepithelial voltages of up to 20 mV (an index of vectorial transepithelial ion transport). Measurements of short-circuit current in Ussing chambers indicated that active secretion of Cl was the major transepithelial active ion transport process, and that this was stimulated by elevation of either cAMP or Ca. Furthermore, estimates of the volume of mucosal liquid were consistent with Cl secretion mediating fluid secretion. Various microscopical methods showed that the cultures were densely ciliated and contained mature secretory cells. Transport across the oviductal epithelium determines the composition of the oviductal fluid, and the study of the relevant transport processes will be greatly enhanced by well-differentiated cultures of oviductal epithelium of the kind established here.     

Life table and predatory efficiency of Stethorus gilvifrons (Coleoptera: Coccinellidae), an important predator of the red spider mite, Oligonychus coffeae (Acari: Tetranychidae), infesting tea

The ladybird beetle, Stethorus gilvifrons, is a major predator of the red spider mite, Oligonychus coffeae, infesting tea. Biology, life table and predatory efficiency of S. gilvifrons were studied under laboratory conditions. Its average developmental period from egg to adult emergence was 19.2 days. After a mean pre-oviposition period of 5.3 days, each female laid an average of 149.3 eggs. Adult females lived for 117.3 days and males for 41.5 days. The life table of the beetle was characterized by an intrinsic rate of increase (r) of 0.066 day⁻¹, net reproductive rate (R ₀) of 72.2 eggs/female, gross reproduction rate (Σm _x ) of 82.3 eggs/female, generation time (T) of 64.9 days, doubling time of 10.5 days and finite rate of increase (λ) of 1.07 day⁻¹. Population dynamics of S. gilvifrons and its prey, O. coffeae, was monitored by sampling 25 tea leaves from each experimental block grown under the prevailing field conditions. Populations of S. gilvifrons reached a peak during January to March and had low incidence during June to November. Peaks in the populations of S. gilvifrons coincided with the abundance of O. coffeae in tea fields. Weather factors such as low temperature, high humidity and heavy rainfall adversely affected the populations of S. gilvifrons. The predatory efficiency of S. gilvifrons increased during the growth of larval instars. An adult female consumed 205.0 eggs, 92.2 larvae, 81.8 nymphs and 52.4 adult mites per day.     

Aging impairs the essential contributions of non‐glial progenitors to neurorepair in the dorsal telencephalon of the Killifish Nothobranchius furzeri

The aging central nervous system (CNS) of mammals displays progressive limited regenerative abilities. Recovery after loss of neurons is extremely restricted in the aged brain. Many research models fall short in recapitulating mammalian aging hallmarks or have an impractically long lifespan. We established a traumatic brain injury model in the African turquoise killifish (Nothobranchius furzeri), a regeneration‐competent vertebrate that evolved to naturally age extremely fast. Stab‐wound injury of the aged killifish dorsal telencephalon unveils an impaired and incomplete regeneration response when compared to young individuals. In the young adult killifish, brain regeneration is mainly supported by atypical non‐glial progenitors, yet their proliferation capacity clearly declines with age. We identified a high inflammatory response and glial scarring to also underlie the hampered generation of new neurons in aged fish. These primary results will pave the way to unravel the factor age in relation to neurorepair, and to improve therapeutic strategies to restore the injured and/or diseased aged mammalian CNS.     

Influence of zoo visitor presence on the behavior of captive Indian gaur (Bos gaurus gaurus) in a zoological park

Visitors to zoos can be a source of potential disturbance and stress to some captive, nonhuman animals in the wild. To determine the influence of visitor presence on captive bison (Bos gaurus gaurus), the study analyzed the behavior of 4 individuals at the Arignar Anna Zoological Park, India. The study often observed the behavior of the animals on visitor-present days and on days when visitors were absent. In the presence of zoo visitors, the bison showed a higher level of intragroup aggression and moving behavior. In contrast, the bison rested more when no visitors were present. The results revealed that the presence of zoo visitors significantly influenced the behavior of captive bison and thereby may have affected their welfare.     

Three-dimensional models of NB-ARC domains of disease resistance proteins in tomato, Arabidopsis, and flax

Three dimensional models of NB-ARC domains in five different proteins were constructed based on the recently published crystal structure of the apoptotic protease activating factor 1, of which two are for tomato species, one each for flax, Arabidopsis, and nematode. Standard multiple sequence alignment was performed for chosen members of the NB-ARC domains, very divergent from each other in protein sequence, followed by homology model building and structure refinement. In this alignment, amino acid insertions and deletions between members generally fall in loop regions or at ends of alpha helices. Despite the presence of sequence divergence between the species, it is argued that the NB-ARC domains carry out the similar biological functions in the various species, highlighting the ATP binding and ATPase activity. By our comparative study of these models, it is predicted that NB-ARC domains should bind ADP/ATP rather than GDP/GTP. Both natural and induced mutants of Arabidopsis within the RPS2 locus and their phenotypes for disease reaction against Pseudomonas syringae are rationalized from the protein model. Apaf-1 Thr263 and Arg265 positions conserved totally within the NB-ARC domains are predicted to take active part in the catalytic activity of kinase-3 motif, the arginine known as the sensor I motif in AAA+ proteins. This was later verified for the Ced-4 crystal structure in complex with Ced-9. Our model of Ced-4 based on Apaf-1 was also compared with its crystal structure in the Ced-4-Ced-9 complex; the 3 layered alpha/beta domain superposes quite well, helical domain I is shifted by about 5 A but the winged helix domain is rotated away to a new position. Since Apaf-1 was crystallized with ADP and Ced-4-Ced9 with magnesium-ATP, this rotation signifies a change in structure of these NB-ARC domains between the two forms. Further, we hypothesize that certain mutants in the plant R proteins called 'constitutive gain-of-function' or 'autocatalytic' dispose their winged helix domains permanently like the magnesium-ATP form as observed for Ced-4, avoiding the closed ADP conformation. The models are also validated with mutagenesis data for a related tomato protein I-2, tomato prf and flax, including loss of function, wild type and autocatalytic phenotypes, and compared with similar data for potato and tobacco proteins, for which models were not built. These three dimensional models would help us to understand the spatial arrangement, function of R proteins and their conserved motifs.