Electrophoretic patterns of protein synthesis and turnover in apical plasma membrane and outer bilayer of Schistosoma mansoni

Polypeptide fractions labelled with [¹⁴C]leucine and associated with fractioned inner plasma membrane and outer bilayer (envelope) from the apical double bilayer complex of the surface epithelium of the human blood fluke, Schistosoma mansoni, were analyzed by two-dimensional electrophoresis and fluorography. In contrast to the distribution of alkaline phosphatase, the polypeptide profiles of the two bilayer fractions were similar due to cross contamination between one membrane containing larger amounts of protein (inner) and the second bilayer having more heavily labelled proteins (outer bilayer). Convincing evidence for only two of 35 polypeptides could be provided for localization to the outer bilayer. These results suggest that the marker enzyme used for the inner bilayer, alkaline phosphatase, may not be homogeneously distributed in this membrane. In pulse-chase studies a correction factor for cross-contamination was derived. The rate to turnover of the polypeptide fractions was twice as fast for the outer compared to the inner membrane, this difference being consistent with the view that multilamellar bodies are the precursors of the apical double bilayer complex. Comparing the rates of surface renewal in adult and juvenile schistosomes leads to the suggestion that membrane turnover can be correlated with susceptibility to host immune effector mechanisms.     

Scanning electron microscopic observations of early stages of phagocytosis of E. coli by human neutrophils

Summary Changes in surface morphology, as observed by scanning electron microscopy, appear rapidly when human polymorphonuclear neutrophils (PMN) are challenged with bacteria. Monolayers of PMN adhering to glass were incubated with opsonized E. coli from 5 sec to 10 min, and then fixed and prepared for SEM. As early as 5 sec after phagocytic challenge, E. coli are found in contact with PMN and in the process of engulfment into open cavities formed by lamellipodia. The shape of the mouth of the forming phagocytic vacuole is related to the orientation of bacteria during entry. Bacteria engulfed into early forming phagosomes are surrounded by a large open space between the bacteria and the phagosome wall. As phagocytosis proceeds, the space is reduced and the loose fit around the entering bacteria becomes tight. By 30 sec, bacteria may be completely internalized and by 1 min phagocytized E. coli are packed into bulging PMN. The observations reveal the variability and rapidity of the phagocytic response and confirm the presence of sensitive mechanisms for host defense by PMN.This work was supported by research grants from the University of North Carolina Research Council and the National Institutes of Health (A1 02430)     

β-Hydroxy-α-ketobutyric acid in Drosophila melanogaster,with reference to biosynthesis of drosopterins

A substance designated as compound D, which reacts spontaneously with 7,8-dihydropterin to give drosopterins, is found in Drosophila melanogaster. The compound was partially purified from the extract of flies by column chromatography and identified as β-hydroxy-α-ketobutyric acid by analysis of its 2,4-dinitrophenylhydrazone, mass spectrometry and reactivity with 7,8-dihydropterin. A highly significant correlation (r = 0.969, p < 0.001) was found between the amounts of the compound and drosopterins in the eye-pigment mutants of Drosophila. Changes of the compound during development of flies were also closely related to those of drosopterins. Based on these observations, a role of the compound in biosynthesis of drosopterins has been discussed.     

Methylglyoxal: Enzyme distributions relative to its presence in Douglas-fir needles and absence in Douglas-fir needle callus

Methylglyoxal was isolated as its 2,4-dinitrophenylosazone from an insoluble fraction from Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] needles but was not observed in a similar Douglas-fir needle callus preparation. This result was consistent with the distribution of enzymes of methylglyoxal metabolism between needles and needle callus. Only catabolic glyoxalases and methylglyoxal reductase could be found in the needle callus, whereas extracts of needles of various ages contained methylglyoxal synthetase and methylglyoxal reductase in a manner suggestive of a function for methylglyoxal in needle development and maturation. While glyoxalases I and II were active in all callus clones tested, activities of these enzymes were not immediately evident in needle extracts. However, it was demonstrated that at least small amounts of glyoxalase I occurred in needle extracts in an inhibited state. From the viewpoint that mature needles and needle callus represent resting and proliferative cellular states, respectively, the data indicate that methylglyoxal may be operating in conifers as a cell division inhibitor as envisioned by Szent-Gyorgyi.     

Characterization and preliminary crystallographic data on the VL-related fragment of the human kI Bence Jones protein Wat

A “naturally occurring” human κI V_L dimer, designated Wat, has been isolated and crystallized. Protein Wat consists of two non-covalently bound monomers, each having a molecular weight of ~ 11,500. The monomer subunit is composed of an entire variable region light chain (V_L) domain closely homologous to that of the κI Bence Jones protein Roy (Hilschmann &; Craig, 1965) as evidenced from amino acid composition, tryptic peptide map, and sequence analysis. Immunochemical studies substantiated that protein Wat is of the κ chain subgroup κI and lacks the isotypic and allotypic antigenic determinants associated with the κ constant region light chain domain. Two types of crystals of V_L dimer Wat were obtained from ammonium sulfate or polyethylene glycol solutions. The type I crystals have unit cell dimensions of $\text{a = b = 82.6}\text{A}\text{?}\text{, c = 60.3}\text{A}\text{?}$, and the space group is hexagonal P6₂ or P6₄. The asymmetric unit consists of one V_L dimer; the fractional volume of unit cell occupied by solvent is 0.51. The unit cell dimensions of the type II crystals are $\text{a = b = 1,08.3}\text{A}\text{?}\text{, c = 108.8}\text{A}\text{?}$; the space group is hexagonal P6₁22 or P6₅22. Three variable domains constitute the asymmetric unit of the type II crystals; the fractional value of the solvent (0.52) is compatible with the value obtained for the type I crystals.     

Characterization of experimental phenylketonuria augmentation of hyperphenylalaninemia with α-methylphenylalanine and p-chlorophenylalanine

Phenylalanine in conjuction with p-chlorophenylalanine or α-methylphenylalanine was administered to suckling rats to induce hyperphenylalaninemia reminiscent of untreated phenylketonuria, and developmental parameters were monitored. The experimental model utilizing p-chlorophenylalanine was found to be unsatisfactory, in that the drug had general deleterous effects on growth, numerous side effects including increased mortality, and affected brain levels of biogenic monoamine neurotransmitters. The model utilizing α-methylphenylalalanine was relatively free from nonspecific effects and thus, changes observed in the animals were attributable to expereimental phenylketonuria. The latter animals had slightly decreased body and brain weights, and exhibited grossly elevated serum phenylalanine and urinary excretion of phenylketone metabolites. Hyperphenylalaninemia produced greatly disrupted brain amino acids at 10 days of age (prior to the formalization of the blood-brain barrier and specific transport systems) which was limited by 30 days of age to changes in glycine, γ-aminobutyric acid and the aliphatic and aromatic amino acids which compete for uptake in t he brain by a common carrier. These animals also exhibited a myelin deficit and changes in proteins from isolated nerve cell preparations. Mature animals which had daily treatment up to 60 days of age results obtained with animal models and the clinical findings in untreated phenylketonuric patients.     

Species novae generisEremostachys (Labiatae)

Five new species are described:Eremostachys codonocalyx from NE. Iran (related toE. subspicata),E. stenocalycina from NE. Afghanistan (related toE. alberti andE. hissarica),E. salangensis from NE. Afghanistan (close toE. bamianica),E. freitagii from E. Afghanistan (similar but not closely related toE. vulnerans), andE. andersii from E. Afghanistan (similar but not closely related toE. vulnerans), andE. andersii from NE. Afghanistan of isolated systematic position.

Florae Iranicae praecursores 5–9.     

Tritium labeling of thermolysin, elastase, and ribonuclease by exposure to tritium gas at low pressure

The bacterial metalloendoprotease thermolysin, bovine pancreatic ribonuclease, and porcine pancreatic elastase have been tritiated by exposure to subcurie amounts of tritium gas at pressures below 50 mTorr for periods of 1 to 6 h. Thermolysin, ribonuclease, and elastase have been purified to specific radioactivities of 15, 5, and 1 Ci/mol, respectively. Amino acid analyses of the tritiated enzymes revealed higher relative specific radioactivities for His, Pro, and Phe in all three proteins while Val and Ile were among the residues with the lowest relative specific radioactivities. The recovery of enzyme activity was always greater than 95% and the formation of tritiated decomposition products was not observed. This lowpressure gas exposure process requires less tritium gas and less time than the original method of Wilzbach to achieve equal or higher levels of tritium incorporation. In addition, the enzymes were completely active and did not show the presence of highly radioactive byproducts which have been observed in earlier studies of the Wilzbach labeling of proteins.