An immunoenzymatic solid-phase assay for quantitative determination of HIV-1 protease activity

A novel immunoenzymatic procedure for the quantitative determination of HIV protease activity is provided. An N-terminal biotinylated peptide (DU1) that comprises an HIV-1 protease (HIV-PR) cleavage sequence was bound to streptavidin-coated microtiter plates. The bound peptide can be quantified by an immunoenzymatic procedure (enzyme-linked immunosorbent assay, ELISA) that includes a monoclonal antibody (Mab 332) against the peptide (DU1) C-terminal. The incubation of the bound peptide with HIV-PR in solution resulted in a signal decrement, as the peptide was hydrolyzed and the released C-terminal segment washed away. An equation that relates the amount of added enzyme to the kinetics of the reaction was written in order to describe this heterogeneous enzyme-quasi-saturable system. This equation allows quantitative determination of protease activity, a feature widely underrated in previous similar assays. The assay also allows evaluation of the inhibitory activity of HIV-PR inhibitors. Due to the intrinsic advantages of the ELISA format, this method could be used in high-throughput screening of HIV protease inhibitors. The assay can be extended to other proteolytic enzymes.     

Conditional loss of PTEN leads to testicular teratoma and enhances embryonic germ cell production

The tumor suppressor gene PTEN, which is frequently mutated in human cancers, encodes a lipid phosphatase for phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3] and antagonizes phosphatidylinositol 3 kinase. Primordial germ cells (PGCs), which are the embryonic precursors of gametes, are the source of testicular teratoma. To elucidate the intracellular signaling mechanisms that underlie germ cell differentiation and proliferation, we have generated mice with a PGC-specific deletion of the Pten gene. Male mice that lacked PTEN exhibited bilateral testicular teratoma, which resulted from impaired mitotic arrest and outgrowth of cells with immature characters. Experiments with PTEN-null PGCs in culture revealed that these cells had greater proliferative capacity and enhanced pluripotent embryonic germ (EG) cell colony formation. PTEN appears to be essential for germ cell differentiation and an important factor in testicular germ cell tumor formation.     

Chlamydomonas contains calcium stores that are mobilized when phospholipase C is activated

Mastoparan induces Ca²⁺-dependent deflagellation of the unicellular green alga Chlamydomonas moewusii Gerloff, as well as the activation of phospholipase C and the production of inositol 1,4,5-trisphosphate (InsP₃; T. Munnik et al., 1998, Planta 207: 133–145). Even in the absence of extracellular Ca²⁺, mastoparan still induces deflagellation (L.M. Quarmby and H.C. Hartzell, 1994, J Cell Biol 124: 807–815; J.A.J. van Himbergen et al., 1999, J Exp Bot, in press) suggesting that InsP₃ mediates Ca²⁺ release from intracellular stores. To test this hypothesis, cells were pre-loaded with ⁴⁵Ca²⁺ and their plasma membranes permeabilized by digitonin. Subsequent treatment of the cells with mastoparan (3.5 μM) induced release of intracellular ⁴⁵Ca²⁺. Mastoparan also activated phospholipase C in permeabilized cells, as demonstrated by the breakdown of ³²P-phosphatidylinositol 4,5-bisphosphate and the production of diacylglycerol. The mastoparan analogues mas7 and mas17 were also effective and their efficacy was correlated with their biological activity. X-ray microanalysis showed that electron-dense bodies (EDBs) are a major Ca²⁺ store in  C. moewusii. Analysis of digitonin-permeabilized cells showed that EDBs lost calcium at digitonin concentrations that released radioactivity from ⁴⁵Ca²⁺-labelled cells, suggesting that ⁴⁵Ca²⁺ monitored the content of EDBs. X-ray microanaysis of living cells treated with mastoparan also revealed that calcium was released from EDBs. Received: 30 December 1998 / Accepted: 25 June 1999     

Fractional phosphate composition in sediments from a tropical river (Catatumbo River, Venezuela)

The distribution of phosphate species in the Catatumbo River, Venezuela, was studied using a sequential extraction scheme. Extraction was performed using 1.0 M NH₄Cl, 0.1 M Na₂S₂O₄–NaHCO₃, 1.0 M NaOH and 0.5 M HCl. Total phosphate was in the range between 121 and 581 g g^–1 D.W. About 64% of the total phosphate is inorganic and present the following composition: 1% NH₄Cl-P, 10% BD-P, 20% NaOH-P, 33% HCl-P; while org-P is 36%. The main form of P in sediment from the Catatumbo River is calcium bound-P, like apatite. The metal concentrations present in sediment are in the following order: Fe> Al > Ca > Mg > Mn. Relationships between P and Fe, Al and Ca were found. Cluster analysis showed that speciation is not dependent on the inorganic matrix of the sediments. Analysing the influence of NaOH concentration and duration of the extraction invalidated the use of NaOH as extracting agent in P-speciation.     

Systemic vaccination with anti‐oligomeric monoclonal antibodies improves cognitive function by reducing Aβ deposition and tau pathology in 3xTg‐AD mice

Alzheimer's disease (AD) is a devastating disorder that is clinically characterized by a comprehensive cognitive decline. Accumulation of the amyloid‐beta (Aβ) peptide plays a pivotal role in the pathogenesis of AD. In AD, the conversion of Aβ from a physiological soluble monomeric form into insoluble fibrillar conformation is an important event. The most toxic form of Aβ is oligomers, which is the intermediate step during the conversion of monomeric form to fibrillar form. There are at least two types of oligomers: oligomers that are immunologically related to fibrils and those that are not. In transgenic AD animal models, both active and passive anti‐Aβ immunotherapies improve cognitive function and clear the parenchymal accumulation of amyloid plaques in the brain. In this report we studied effect of immunotherapy of two sequence‐independent non‐fibrillar oligomer specific monoclonal antibodies on the cognitive function, amyloid load and tau pathology in 3xTg‐AD mice. Anti‐oligomeric monoclonal antibodies significantly reduce the amyloid load and improve the cognition. The clearance of amyloid load was significantly correlated with reduced tau hyperphosphorylation and improvement in cognition. These results demonstrate that systemic immunotherapy using oligomer‐specific monoclonal antibodies effectively attenuates behavioral and pathological impairments in 3xTg‐AD mice. These findings demonstrate the potential of using oligomer specific monoclonal antibodies as a therapeutic approach to prevent and treat Alzheimer's disease.     

Life table and predation of Neoseiulus longispinosus (Acari: Phytoseiidae) on Oligonychus coffeae (Acari: Tetranychidae) infesting tea

Life table and predation of the predatory mite Neoseiulus longispinosus (Evans) on the red spider mite (RSM), Oligonychus coffeae (Nietner), a major pest of tea in India, were studied in the laboratory. Developmental time from egg to adult varied from 4 to 14 days at 30 to 15 °C, respectively; at 35 °C no larva survived. Survival of immature stages was more than 94 % at all temperatures. Threshold temperature for development of immature stages of females and males was 10 and 9.9 °C, respectively, and thermal constant was 84.03 degree-days for females and 80 for males. Sex ratio was female biased and temperature (20–30 °C) had no clear effect on sex determination. Egg hatchability was 73 % at 35 °C and >97 % at lower temperatures. Average number of eggs laid per female/day was higher at 30 °C than at 20 or 25 °C. The highest net reproductive rate (R ₀) was 40.7, at 20 °C. Mean generation time (T) decreased from 28 to 13 days with temperature increasing from 20 to 30 °C. Weekly multiplication (6.5) and intrinsic rate of natural increase (r _m ) (0.268) were highest at 30 °C. Males lived longer than females at every temperature tested. Longevity was highest at 20 °C (50 days for females and 55 for males). Survival and longevity were adversely affected by temperature above 30 °C. Daily consumption of prey increased with the advancement of predator’s life stages; adult females consumed the highest numbers of prey items, preferably larvae and nymphs.     

Efficacy,prey stage preference and optimum predator–prey ratio of the predatory mite,Neoseiulus longispinosus Evans (Acari: Phytoseiidae) to control the red spider mite,Oligonychus coffeae Nietner (Acari: Tetranychidae) infesting tea

The predatory mite, N. longispinosus preys up on red spider mite, O. coffeae infesting tea in south India. An attempt has been made to determine the predatory potential, prey stage preference and optimum predator–prey ratio of N. longispinosus under laboratory and green house conditions. When 50 adult female O. coffeae were given, the number of adults reduced by eight days along with an increase in the number of predators. The larvae hatched from the eggs laid by O. coffeae were fed by predatory mite. N. longispinosus preyed up on all life stages with a preference to larvae and nymphs of red spider mite. Predator–prey ratios of 1:33 and 1:50 were effective in lab, and 1:25 was found to be effective in green house. These results revealed that N. longispinosus could be used as a successful biocontrol candidate of O. coffeae in tea through augmentation or mass rearing and field release.     

An Analysis of Interactions between Fluorescently-Tagged Mutant and Wild-Type SOD1 in Intracellular Inclusions

Background

By mechanisms yet to be discerned, the co-expression of high levels of wild-type human superoxide dismutase 1 (hSOD1) with variants of hSOD1 encoding mutations linked familial amyotrophic lateral sclerosis (fALS) hastens the onset of motor neuron degeneration in transgenic mice. Although it is known that spinal cords of paralyzed mice accumulate detergent insoluble forms of WT hSOD1 along with mutant hSOD1, it has been difficult to determine whether there is co-deposition of the proteins in inclusion structures.

Methodology/Principal Findings

In the present study, we use cell culture models of mutant SOD1 aggregation, focusing on the A4V, G37R, and G85R variants, to examine interactions between WT-hSOD1 and misfolded mutant SOD1. In these studies, we fuse WT and mutant proteins to either yellow or red fluorescent protein so that the two proteins can be distinguished within inclusions structures.

Conclusions/Significance

Although the interpretation of the data is not entirely straightforward because we have strong evidence that the nature of the fused fluorophores affects the organization of the inclusions that form, our data are most consistent with the idea that normal dimeric WT-hSOD1 does not readily interact with misfolded forms of mutant hSOD1. We also demonstrate the monomerization of WT-hSOD1 by experimental mutation does induce the protein to aggregate, although such monomerization may enable interactions with misfolded mutant SOD1. Our data suggest that WT-hSOD1 is not prone to become intimately associated with misfolded mutant hSOD1 within intracellular inclusions that can be generated in cultured cells.