The infectivity and purification of cultured Plasmodium berghei ookinetes

Plasmodium berghei ookinetes were cultured from hamster blood as described previously (Kurtti and Munderloh, 1986). An average of 7.3 X 10(6) ookinetes was harvested from each ml of blood. Ookinetes were purified by centrifugation on first a 40% and then a 36% Percoll gradient. The final preparation comprised 32.8% of the ookinetes initially obtained, and contained 3.3 other parasite stages or blood cells per ookinete. Unpurified and purified ookinetes were resuspended in hamster blood and fed to Anopheles stephensi. There was a strong linear correlation between the concentration of purified or unpurified ookinetes and the number of oocysts formed. With unpurified ookinetes, a maximum was reached when preparations containing 1 X 10(7) ookinetes/ml were fed, and feeding preparations containing a higher concentration did not produce more oocysts. Sporozoites were found in the salivary glands of mosquitoes fed ookinetes by days 14 (unpurified) or 15 (purified) PI. Approximately 5 times as many purified as unpurified ookinetes were required to produce each oocyst.     

The effect of repeated administration of insulin on pain threshold and gamma-aminobutyric acid level in mouse brain

In experiments on male Albino-Swiss mice weighing 18-22 g insulin given in doses of 2 i.u./kg caused no change in the time of reaction to pain, while the same dose administered daily for 7 days potentiated the analgesic action of morphine (3 mg/kg s.c.). Glucose caused no change in this effect of insulin. After 14 days of insulin treatment the time of reaction to pain in the animals subjected to the action of morphine returned to its initial value. Twenty-four hours after the last administration of morphine the level of gamma-aminobutyric acid (GABA) was found to be decreased in the animals receiving insulin with glucose. These results suggest that the central action of insulin is dependent not only on hypoglycaemia produced by it, but may be due also to its direct action on the central structures and an indirect action mediated by its effect on other neurotransmitter systems.     

Subcellular location of enzymes involved in the N-glycosylation and processing of asparagine-linked oligosaccharides in Saccharomyces cerevisiae

A particulate translation system isolated from the yeast Saccharomyces cerevisiae was shown to translate faithfully in-vitro-transcribed mRNA coding for a mating hormone precursor (prepro-alpha-factor mRNA) and to N-glycosylate the primary translation product after its translocation into the lumen of the microsomal vesicles. Glycosylation of its three potential sugar attachment sites was found to be competitively inhibited by acceptor peptides containing the consensus sequence Asn-Xaa-Thr, supporting the view that the glycan chains are N-glycosidically attached to the prepro-alpha-factor polypeptide. The accumulation in the presence of acceptor peptides of a membrane-specific, unglycosylated translation product (pp-alpha-F0) differing in molecular mass from a cytosolically located, protease-K-sensitive alpha-factor polypeptide (pp-alpha-Fcyt) by about 1.3 kDa, suggests that, in contrast to previous reports, a signal sequence is cleaved from the mating hormone precursor on/after translocation. This conclusion is supported by the observation that the multiply glycosylated alpha-factor precursor is cleaved by endoglucosaminidase H to a product with a molecular mass smaller than the primary translation product pp-alpha-Fcyt but larger than the membrane-specific pp-alpha-F0. Translation and glycosylation experiments carried out in the presence of various glycosidase inhibitors (e.g. 1-deoxynojirimycin, N-methyl-1-deoxynojirimyin and 1-deoxymannojirimycin) indicate that the N-linked oligosaccharide chains of the glycosylated prepro-alpha-factor species are extensively processed under the in vitro conditions of translation. From the specificity of the glycosidase inhibitors applied and the differences in the molecular mass of the glycosylated translation products generated in their presence, we conclude that the glycosylation-competent microsomes contain trimming enzymes, most likely glucosidase I, glucosidase II and a trimming mannosidase, which process the prepro-alpha-factor glycans down to the (Man)8(GlcNAc)2 stage. Furthermore, several arguments strongly suggest that these three enzymes, which apparently represent the full array of trimming activities in yeast, are exclusively located in the lumen of microsomal vesicles derived from endoplasmic reticulum membranes.     

Macromolecules in the receptor lymph of campaniform sensilla

Summary The receptor lymph of campaniform sensilla on the halteres of the blowfly, Calliphora vicina, was analyzed histochemically. Acid mucopolysaccharides were demonstrated by a test for iron-binding capacity (Hale-reaction). Further characterization by enzyme treatment showed that the receptor lymph contains hyaluronic acid and/or chondroitin sulfate. Ultrahistochemical studies gave evidence for glycoproteins in the inner and outer receptor lymph space. The significance of acid mucopolysaccharides for arthropod sensilla is discussed.     

Activity of long chain acyl-CoA synthetase in isolated alveolar type II cells

Fatty-acyl-CoA synthetase activity was determined in rat alveolar type II cells. Compared to whole-lung homogenate, the enzyme specific activity with palmitic acid was 3.6-fold higher in isolated type II alveolar cells. The enzyme in rat alveolar type II cells did not discriminate among various fatty acids, suggesting that supply of fatty acids rather than specificity might be an important factor for their activation in these cells.     

Glutamic acid-dihydrogen phosphate hydrogen-bonded networks: their proton polarizability as a function of cations present. Infrared investigations.

Glutamic acid [(L-glu)n] + dihydrogen phosphate systems are studied by infrared (IR) spectroscopy dried and hydrated at 75% relative humidity, as a function of both the phosphate-glutamic acid residue (Pi/glu) ratio and the type of cations present. It is shown that the glutamic acid groups form hydrogen-bonded chains with the phosphates. In these chains the positive charge fluctuates, and they show very large proton polarizability which increases in the series Li+,Na+,K+ systems. These chains are cross-linked via phosphate-phosphate hydrogen bonds, in which the proton is almost localized at one Pi. The comparison of the (L-glu)n + dihydrogen phosphate systems with the results obtained earlier in the case of (L-glu)n + hydrogen phosphate systems shows that the behavior of (L-glu)n + Pi systems strongly depends on the pH. Only with decreasing pH the conducting chains are formed. Finally, a hypothesis is discussed with regard to the charge conduction in the F0 subunit of the H+-ATPase in mitochondria.     

Stimulation of pancreatic secretory process in the rat by low-molecular weight proteinase inhibitor

Oral application of a single dose of a new synthetic proteinase inhibitor Camostate (Foy-305) in male Wistar rats was carried out together with studies of in vitro amino acid incorporation followed by separation of proteins by two-dimensional gel electrophoresis. The aim of this experiment was to analyze changes produced by the inhibitor in total protein and individual enzyme biosynthesis. Administration of 100 mg/kg Foy-305 resulted in significant inhibition of total pancreatic protein synthesis, without changes in fractional rates for individual enzymes. 50 mg/kg Foy-305 induced a 10-fold elevation of cholecystokinin (CCK) levels in serum; this persisted for 3 h and led to a significant increase in the total rate of protein synthesis with peak values at 6 and 9 h (78% and 84% above control levels, respectively), returning to control by 15 h. Changes in fractional rates of synthesis occurred with a latency of 6 h and were restricted to amylase and the anionic form of trypsinogen and chymotrypsinogen. Amylase biosynthesis decreased by about 40% from control levels at 9 h to return to control levels by 15 h. Increased synthesis of trypsinogen and chymotrypsinogen was observed; this was also phasic. The results show similar enzyme-specific regulation as previously described for exogenous CCK stimulation and for the adaptation of the pancreas to diets enriched in protein. They demonstrate the effectiveness of pulsatory endogenous hormone release in the regulation of protein synthesis.     

Mammalian DNA polymerase alpha: a replication competent holoenzyme form from calf thymus.

A complex "replication competent" holoenzyme form of DNA polymerase alpha (RC-alpha) was purified 10,000 fold from calf thymus through the use of an assay employing primed single stranded circular DNA template. The RC-alpha form could partially replicate a double-stranded oligo(dT)-tailed linear DNA and could completely convert primed single-stranded circular DNA to its double stranded form. The RC-alpha was resolved by denaturing gel electrophoresis into at least 10 discrete polypeptide species ranging in apparent molecular mass from 200 to 47 kilodaltons; three of the bands (apparent Mr of 200, 118 and 63 kilodaltons) displayed DNA polymerase activity in denaturing gel activity assay. The isolation of RC-alpha required the use of absolutely fresh calf thymus, the inclusion of ATP and protease inhibitors throughout the purification procedure. Treatment of the RC-alpha with the neutralizing anti-DNA polymerase alpha monoclonal antibody SJK 132-20 (Tanaka et al. (1982), J. Biol. Chem. 257, 8386-8390) in nondenaturing conditions selected the complete set of 10 polypeptides, whereas treatment in denaturing conditions selected the 200 kilodalton catalytic DNA polymerase active polypeptide. The properties and the behaviour of the RC-alpha preparation following removal of specific polypeptides strongly suggested that the capacity of RC-alpha to extend and replicate long template requires the function of nonproteolysed form of the 200 kilodaltons catalytic DNA polymerase core and at least 6 other auxiliary polypeptides of, respectively, 98, 87, 63, 54, 49 and 47 kilodaltons.