Proteolysis in the gut of mosquito larvae results in further activation of the Bacillus sphaericus toxin

Gut proteases from the larvae of the mosquito Culex pipiens convert the 43-kilodalton (kDa) toxin from Bacillus sphaericus 2362 to a 40-kDa peptide. The 50% lethal concentration of this peptide for tissue culture-grown cells of Culex quinquefasciatus was 1.0 microgram/ml (as determined by the intracellular ATP assay), 54-fold less than that of the 43-kDa peptide. Gut proteases from Anopheles gambiae and Aedes aegypti, as well as bovine pancreatic trypsin, also converted the 43-kDa protein to a 40-kDa peptide which was indistinguishable from the peptide formed by the proteases from C. pipiens with respect to its toxicity to tissue culture-grown cells of C. quinquefasciatus. Evidence for the in vivo conversion of the 43-kDa protein to the 40-kDa peptide was also obtained from experiments in which larvae of C. pipiens, Anopheles gambiae, and Aedes aegypti were fed crystals from B. sphaericus 2362. By using the exclusion of trypan blue as an indication of cell viability, it was shown that chitobiose, chitotriose, N-acetylmuramic acid, and N-acetylneuraminic acid decreased the toxicity of the 40-kDa peptide (from 100 to 50% mortality at about 10 mM concentrations of these sugars). Muramic acid, N-acetylgalactosamine, and N-acetylglucosamine were less effective, while several sugars had no effect, suggesting that the 40-kDa toxin binds to specific receptors on the cell membrane. The 40-kDa protein was less toxic to tissue culture-grown cells of Anopheles gambiae and Aedes dorsalis, and the same sugars which reduced the toxicity for cells of C. quinquefasciatus were also effective in reduction of toxicity for these cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lectin-labeled membrane is transferred to the Golgi complex in mouse pituitary cells in vivo

Labeling of the Golgi complex with the lectin conjugate wheat germ agglutinin-horseradish peroxidase (WGA-HRP), which binds to cell surface membrane and enters cells by adsorptive endocytosis, was analyzed in secretory cells of the anterior, intermediate, and posterior lobes of mouse pituitary gland in vivo. WGA-HRP was administered intravenously or by ventriculo-cisternal perfusion to control and salt-stressed mice; post-injection survival times were 30 min-24 hr. Peroxidase reaction product was identified within the extracellular clefts of anterior and posterior pituitary lobes through 24 hr but was absent in intermediate lobe. Endocytic vesicles, spherical endosomes, tubules, dense and multivesicular bodies, the trans-most saccule of the Golgi complex, and dense-core secretory granules attached or unattached to the trans Golgi saccule were peroxidase-positive in the different types of anterior pituitary cells and in perikarya of supraoptico-neurohypophyseal neurons; endoplasmic reticulum and the cis and intermediate Golgi saccules in the same cell types were consistently devoid of peroxidase reaction product. Dense-core secretory granules derived from cis and intermediate Golgi saccules in salt-stressed supraoptic perikarya likewise failed to exhibit peroxidase reaction product. The results suggest that in secretory cells of anterior and posterior pituitary lobes, WGA-HRP, initially internalized with cell surface membrane, is eventually conveyed to the trans-most Golgi saccule, in which the lectin conjugate and associated membrane are packaged in dense-core secretory granules for export and potential exocytosis of the tracer. Endoplasmic reticulum and the cis and intermediate Golgi saccules appear not to be involved in the endocytic/exocytic pathways of pituitary cells exposed to WGA-HRP.     

Bacillus sphaericus as a mosquito pathogen: properties of the organism and its toxins.

In the course of sporulation, Bacillus sphaericus produces an inclusion body which is toxic to a variety of mosquito larvae. In this review we discuss the general biology of this species and concentrate on the genetics and physiology of toxin production and its processing in the midgut of the larval host. The larvicide of B. sphaericus is unique in that it consists of two proteins of 51 and 42 kDa, both of which are required for toxicity to mosquito larvae. There is a low level of sequence similarity between these two proteins, which differ in their sequences from all the other known insecticidal proteins of Bacillus thuringiensis. Within the midgut the 51- and 42-kDa proteins are processed to proteins of 43 and 39 kDa, respectively. The conversion of the 42-kDa protein to a 39-kDa protein results in a major increase in toxicity; the significance of the processing of the 51-kDa protein is not known. In contrast to the results with mosquito larvae, the 39-kDa protein is alone toxic for mosquito-derived tissue culture-grown cells, and this toxicity is not affected by the 51-kDa protein or its derivative, the 43-kDa protein. Comparisons of larvae from species which differ in their susceptibility to the B. sphaericus toxin indicate that the probable difference resides in the nature of the target sites of the epithelial midgut cells and not in uptake or processing of the toxin. A similar conclusion is derived from experiments involving tissue culture-grown cells from mosquito species which differ in their susceptibility to the B. sphaericus toxin.     

Sporulation-associated activation of Bacillus sphaericus larvicide.

Preparations of the larvicidal crystal from 46-h cultures of Bacillus sphaericus 2362 contain 125-, 110-, 63-, and 43-kilodalton (kDa) proteins (P. Baumann, B. M. Unterman, L. Baumann, A.H. Broadwell, S.J. Abbene, and R.D. Bowditch, J. Bacteriol. 163:738-747, 1985). The 63- and 43-kDa proteins, which have been purified, are not immunologically cross-reactive, and only the 43-kDa protein is toxic to mosquito larvae. Since antigenic determinants of the two smaller proteins have been detected in the higher-molecular-weight proteins (125 and 110 kDa), it has been suggested that the latter are precursors of the 43- and 63-kDa peptides. In the present study, purified 110-kDa protein was found to be toxic to the larvae of Culex pipiens (50% lethal concentration = 115 ng/ml). A luciferase-luciferin assay for intracellular ATP as well as an assay based on the exclusion of Trypan Blue by live cells indicated that the 110-kDa protein had no effect on tissue-culture-grown cells of C. quinquefasciatus, while cells exposed to the 43-kDa protein rapidly lost viability (50% lethal concentration = 54 microgram(s)/ml by the intracellular ATP assay). These findings suggested that the 110-kDa protein and, by extension, the 125-kDa protein are protoxins which are activated during sporulation by cleavage to a 43-kDa toxin. To further investigate the origins and relationships of the crystal proteins of B. sphaericus, we analyzed samples during the growth and sporulation of the culture. Synthesis of crystal proteins was initiated at the end of exponential growth and was completed after about 7 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

血红细胞超氧化物歧化酶(SOD)制备工艺的研究初报

本文报告了本实验室设计的由血红细胞自溶液60℃热变性, 乙醇——氯仿法除血红蛋白,旋转蒸发法减压浓缩抽去氯仿、乙醇,硫酸铵分级盐析法沉降SOD,Sepbadex G-75层析提纯SOD等步骤构成的一条成本低、设计合理、简便实用的分离纯化SOD的工艺路线。     

Construction by site-directed mutagenesis of a 39-kilodalton mosquitocidal protein similar to the larva-processed toxin of Bacillus sphaericus 2362.

After ingestion of the parasporal crystals of Bacillus sphaericus, mosquito larvae process the 42-kilodalton (kDa) toxin to a protein of 39 kDa, which has an increased toxicity (A. H. Broadwell and P. Baumann, Appl. Environ. Microbiol. 53:1333-1337, 1987). A similar activation is performed by trypsin and chymotrypsin. Using site-directed mutagenesis, we have constructed derivatives of the 42-kDa toxin with a deletion of 10 amino acids at the N terminus and deletions of 7, 17, or 20 amino acids at the C terminus. Toxicity for mosquito larvae was retained upon deletion of 7 or 17 amino acids but was lost upon deletion of 20 amino acids. Evidence is presented indicating that the protein containing deletions of 10 amino acids at the N terminus and 17 amino acids at the C terminus (corresponding to potential chymotrypsin cleavage sites) is similar to the 39-kDa protein produced in mosquito larvae or by digestion with chymotrypsin. Digestion with trypsin appears to generate a protein lacking 16 or 19 amino acids from the N terminus and 7 amino acids from the C terminus. As is the case with the recombinant-made 42-kDa protein, toxicity of its derivatives is dependent on the presence of a 51-kDa protein which is a component of the parasporal crystal of B. sphaericus 2362.     

Affinity of Ionic Species of Nucleoside Transport Protein Inhibitors

Abstract

A class of very potent nucleoside transport inhibitors is present in two molecular forms around physiological pH. We investigated whether the monoprotonated or the unionized species of these molecules binds to this camer protein with higher affinity.     

THE EFFECTS OF INJURY PREVENTION WARM-UP PROGRAMMES ON KNEE STRENGTH IN MALE SOCCER PLAYERS

The study investigates the effects of the 11+ and HarmoKnee injury prevention programmes on knee strength in male soccer players. Under-21-year-old players (n=36) were divided equally into: the 11+, HarmoKnee and control groups. The programmes were performed for 24 sessions (20-25 min each). The hamstrings and quadriceps strength were measured bilaterally at 60°·s^-1, 180°·s^-1 and 300°·s^-1. The concentric quadriceps peak torque (PT) of the 11+ increased by 27.7% at 300°·s^-1 in the dominant leg (p<0.05). The concentric quadriceps PT of HarmoKnee increased by 36.6%, 36.2% and 28% in the dominant leg, and by 31.3%, 31.7% and 20.05% at 60°·s^-1, 180°·s^-1 and 300°·s^-1 in the non-dominant leg respectively. In the 11+ group the concentric hamstring PT increased by 22%, 21.4% and 22.1% at 60°·s^-1, 180°·s^-1 and 300°·s^-1, respectively in the dominant leg, and by 22.3%, and 15.7% at 60°·s^-1 and 180°·s^-1, in the non-dominant leg. In the HarmoKnee group the hamstrings in the dominant leg showed an increase in PT by 32.5%, 31.3% and 14.3% at 60°·s^-1, 180°·s^-1 and 300°·s^-1, and in the non-dominant leg hamstrings PT increased by 21.1% and 19.3% at 60°·s^-1 and 180°·s^-1 respectively. The concentric hamstrings strength was significantly different between the 11+ and control groups in the dominant (p=0.01) and non-dominant legs (p=0.02). The HarmoKnee programme enhanced the concentric strength of quadriceps. The 11+ and HarmoKnee programmes are useful warm-up protocols for improving concentric hamstring strength in young professional male soccer players. The 11+ programme is more advantageous for its greater concentric hamstring strength improvement compared to the HarmoKnee programme.