Isolation, partial chemical and biological characterization of thymone B

A new approach to the study of the molecular arrangements of proteins in membranes is described. Irradiation with visible light of native erythrocytes or washed erythrocyte membranes suspended in buffers containing a) riboflavin, fluorescein or fluorescein coupled to dextran and b) ³H-labelled tryptophan resulted in incorporation of radioactivity into the membrane proteins. Polyacrylamide gel electrophoresis of solubilized membranes followed by radioactivity measurements of the separated membrane proteins revealed that in native erythrocytes the protein components known to be located at the exterior cell surface, Band 3 and the major sialoglycoproteins became specifically labelled, whereas in washed lysed cells all of the major membrane proteins were labelled.     

Rapid production of a chimeric antibody-antigen fusion protein based on 2A-peptide cleavage and green fluorescent protein expression in CHO cells

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.     

5-Hydroxytryptamine-Stimulated Inositol Phospholipid Hydrolysis in the Mouse Cortex Has Pharmacological Characteristics Compatible with Mediation via 5-HT2 Receptors but This Response Does Not Reflect Altered 5-HT2 Function After 5,7-Dihydroxytryptamine Lesioning or Repeated Antidepressant Treatments

5-Hydroxytryptamine (5-HT; 3 x 10(-8)-1 x 10(-5)M) produced a dose-dependent increase in phosphatidylinositol/polyphosphoinositide (PI) turnover in mouse cortical slices, as measured by following production of 3H-labelled inositol phosphates (IPs) in the presence of 10 mM LiCl. Analysis of individual IPs, in slices stimulated for 45 min, indicated substantial increases in inositol monophosphate (IP1; 140%) and inositol bisphosphate (IP2; 95%) contents with smaller increases in inositol trisphosphate (IP3; 51%) and inositol tetrakisphosphate (IP4; 48%) contents. The increase in IP3 level was solely in the 1,3,4-isomer. This response was inhibited by the nonselective 5-HT antagonists methysergide, metergoline, and spiperone. It was also inhibited by the selective 5-HT2 antagonists ketanserin and ritanserin but not by the 5-HT1 antagonists isapirone, (-)-propranolol, or pindolol. 5-HT-stimulated IP formation was also unaltered by atropine, prazosin, and mepyramine. Lesioning brain 5-HT neurones using 5,7-dihydroxytryptamine (5,7-DHT; 50 micrograms i.c.v.) produced a 210% (p less than 0.01) increase in the number of 5-HT2-mediated head-twitches induced by 5-methoxy-N,N-dimethyltryptamine (2 mg/kg). However, 5,7-DHT lesioning had no effect on 5-HT-stimulated PI turnover in these mice. Similarly, an electroconvulsive shock (90 V, 1 s) given five times over a 10-day period caused an 85% (p less than 0.01) increase in head-twitch responses but no change in 5-HT-stimulated PI turnover. Decreasing 5-HT2 function by twice-a-day injection of 5 mg/kg of zimeldine or desipramine (DMI) produced 50% (p less than 0.01) and 56% (p less than 0.01), respectively, reductions in head-twitch behaviour.(ABSTRACT TRUNCATED AT 250 WORDS)     

Location of crosslinks in chemically stabilized horseradish peroxidase: implications for design of crosslinks.

The bifunctional compound, ethylene-glycol bis(N-hydroxysuccinimidylsuccinate) (EGNHS), stabilizes horseradish peroxidase C (HRP) by reaction with the enzyme's lysine residues. In this study we compare native and modified HRP by proteolytic fragmentation, peptide sequencing, and mass spectroscopy, and identify the sites of modification. Most significantly, EGNHS is shown to form a crosslink between Lys232 and Lys241 of HRP and modifies Lys174 without formation of a crosslink. These findings are in agreement with the lysine side-chain reactivities predicted from the surface accessibility of the amino groups, and the maximal span of 16 A of the EGNHS crosslinker.     

Peptides and multiple antigen peptides from Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase: preparation, immunogenicity and immunoprotective capacity in C57BL/6 mice.

Four monoepitopic MAPs (MAP A, B, C and E) and one bis-diepitopic MAP B-E derived fromthe primary sequence of Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase, previously tested in BALB/c mice, were examined for their immunogenicity and protective capacity in C57BL/6 mice. Despite multimerization into MAPs, MAP Aand MAP C were poorly immunogenic. In contrast toBALB/c mice, MAP E was non-immunogenic in C57BL/6 mice. Peptide B in the form of MAP B orbis-diepitopic MAPB-E elicited immune responses in C57BL/6 mice that were associated with a significant decrease in worm burden. The MAPs were prepared by the stepwise solid-phase peptide synthesis using Boc/Bzl chemistry, successfully purified on the RP-HPLC column and characterized by RP-HPLC, HPCE and MALDI-TOF MS techniques. A general strategy for MAPs purification is discussed here and the purification of MAP Band MAP E is documented in detail.     

Uptake and assimilation of nitrogen from solutions containing multiple N sources

We assessed the extent to which plants can acquire amino acids when supplied as single N-sources or when plants have access to a mixture of amino- and inorganic N sources. Because the uptake of different N-sources is temperature-dependent, the effects of temperature on amino-N uptake were also tested. Lolium perenne (perennial rye-grass) was grown hydroponically at 11 °C or 21 °C. Uptake of N was determined using ¹⁵N tracers at the growth temperature from solutions containing either nitrate, ammonium or glycine as single N sources and from a mixture containing all three N-forms. Estimates of the relative importance of amino acids such as glycine to the total N budget of plants will have been underestimated in studies where uptake was determined in single source solutions compared with those from solutions containing a mixture of N-forms. The proportion of total N acquired from the mixed N source as ammonium increased as temperature was reduced. Regarding the uptake and initial metabolism of glycine, uptake was probably the rate limiting step at 11 °C whilst it was the metabolism of glycine to serine at 21 °C. Although ¹⁵N incorporation into the plant amino-N pool was generally in proportion to the abundance of individual amino acids, its incorporation into the glycine pool was sometimes significantly less than predicted.     

Bilayer stabilizing peptides and the inhibition of viral infection: antimeasles activity of carbobenzoxy-Ser-Leu-amide

A number of carbobenzoxy-dipeptide-amides raise the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine (stabilizes the bilayer). The potency of the peptides in stabilizing the bilayer phase is Z-Tyr-Leu-NH₂= Z-Gly-Phe-NH₂>Z-Ser-Leu-NH₂>Z-Gly-Leu-NH₂>Z-Gly-Gly-NH₂. A linear correlation was found between the respective HPLC retention time parameterk for the peptide and the slope of the bilayer stabilization curve determined with model membranes by differential scanning calorimetry. One dipeptide, Z-Ser-Leu-NH₂, reduces measles virus cytopathic effect (CPE) in Vero cells. The mechanism by which this peptide reduces the CPE is not known, although some peptides which raise the bilayer to hexagonal phase transition temperature of phospholipids inhibit membrane fusion.Abbreviations Z carbobenzoxy - DEPE dielaidoylphosphatidylethanolamine - DSC differential scanning calorimetry - HPLC high pressure liquid chromatography - CPE cytopathic effect To whom correspondence should be addressed.     

Problems of body-weight estimation in fossil primates

Body-weight estimates of fossil primates are commonly used to infer many important aspects of primate paleobiology, including diet, ecology, and relative encephalization. It is important to examine carefully the methodologies and problems associated with such estimates and the degree to which one can have confidence in them. New regression equations for predicting body weight in fossil primates are given which provide body-weight estimates for most nonhominid primate species in the fossil record. The consequences of using different subgroups (evolutionary “grades”) of primate species to estimate fossil-primate body weights are explored and the implications of these results for interpreting the primate fossil record are discussed. All species (fossil and extant) were separated into the following “grades”: prosimian grade, monkey grade, ape grade, anthropoid grade, and all-primates grade. Regression equations relating lower molar size to body weight for each of these grades were then calculated. In addition, a female-anthropoid grade regression was also calculated for predicting body weight infernales of extinct, sexually dimorphic anthropoid species. These equations were then used to generate the fossil-primate body weights. In many instances, the predicted fossil-primate body weights differ substantially from previous estimates.     

Reproductive strategies,body size,and encephalization in primate evolution

In order to understand fully the generally high level of encephalization observed in living primates, we must determine why early primates exhibited moderately large relative brain sizes compared to their early Tertiary contemporaries. The relatively high degree of encephalization in early primates may be related at least in part to the fact that they were highly unusualamong mammals in combining a small body size with a strongly precocial reporductive strategy. Other small, precocial mammals also exhibit moderately high levels of encephalization; but primates appear to have been truly uniquein being the only such small-sized and highly precocial group to give rise to extensive radiations of larger descendants. This is a key element in understanding primate brain evolution, because the initial “head start” of the early primates was translated up to larger sizes in descendants. The possible relationships among encephalization, precociality, small size, and arboreality are discussed, particularly in light of recent debates concerning the validity of the superorder Archonta. This work emphasizes that we need to consider relative brain size as but one element in a complex synergistic network of morphological and life-history features.