In vitro antigen challenge of human antibody libraries for vaccine evaluation: the human immunodeficiency virus type 1 envelope.

Human antibody responses, or versions thereof, can be cloned as phage display libraries. In vaccine evaluation, the possibility therefore exists of challenging the human response in vitro, rather than in vivo, in order to assist in establishing the most promising vaccine leads. The characteristics of the antibodies retrieved directly indicate the strengths and weaknesses of the vaccine at the molecular level. We applied this approach to compare recombinant and native human immunodeficiency virus type 1 envelope preparations. We conclude that recombinant gp160, gp140, and, to a lesser extent, gp120 present epitopes around the CD4 binding site in a conformation different from that of the native multimer and contrary to expected vaccine requirements. Antibodies to the potently neutralizing b12 epitope were selected preferentially from an immune library by purified human immunodeficiency virus type 1 virions. This suggests that b12 is a major epitope on the virions, in contrast to recombinant envelope preparations, in which related, weakly neutralizing epitopes predominate. Although the majority of virions in the preparation used are expected to be noninfective, it appears that they predominantly express a native envelope configuration and would be able to elicit potent neutralizing antibodies.     

Epilithic bacterial and algal colonization in a stream run, riffle, and pool: a test of biomass covariation

Epilithic bacterial and algal biomass were compared among a run, riffle, and pool along an open-canopy section of a third-order, temperate stream. Epilithic biofilms were sampled after 3, 7, 14, 21, 28, and 35 days colonization on unglazed ceramic tiles that were attached to plastic trays (n = 3) placed across each of the three habitats (i.e., run, riffle, pool). The diverse habitats and sampling regime were selected to provide a range in algal biomass so that potential covariation between epilithic bacterial and algal biomass could be assessed. There were significant differences among habitats and among trays within each habitat for both chlorophyll a and AFDM. Chlorophyll a and AFDM increased in the run and pool throughout the colonization period. In the riffle, chlorophyll a and AFDM increased rapidly early in colonization, then decreased. Epilithic bacterial biomass increased rapidly with no significant differences among the three habitats throughout colonization. Further, bacterial biomass did not correlate with either chlorophyll a or AFDM in any of the three habitats or on any of the sampling days. These results suggest that epilithic algal and bacterial biomass may be regulated by independent controls in some stream environments.     

Neutrophil priming mechanisms of sulfolipid-I and n-formyl-methionyl-leucyl-phenylalanine

The principal sulfatide of virulentMycobacterium tuberculosis, sulfolipid-I (SL-I), both directly stimulates neutrophil superoxide (O ₂ ^– ) release and, at substimulatory concentrations, primes these cells for markedly enhanced oxidative responsiveness to other stimuli. The present study was undertaken to clarify the priming mechanisms by comparing cellular events following priming doses of SL-I with those following priming with N-formyl-methionyl-leucyl-phenylalanine (FMLP). We compared the involvement of the calcium cation (Ca²⁺), as well as membrane protein kinase C (PKC) activity and the translocation of NADPH oxidase-cytosolic cofactor effected by priming levels of the two agonists. The investigation led to two important conclusions. First, we clearly demonstrate that priming by both SL-I and FMLP results from activation of cellular processes that are not involved in direct oxidative activation. For example, whereas direct induction of O ₂ ^– generation by FMLP and SL-I required increases in intracellular Ca²⁺, an increase in intracellular calcium concentration ([Ca²⁺]_i) above basal levels was not required for priming. Second, we identified key differences in the cellular responses to priming doses of SL-I and FMLP. Whereas increased membrane PKC activity caused by priming doses of FMLP was only partially blocked by chelation of intracellular Ca²⁺, Ca²⁺ chelation completely inhibited the increase in membrane PKC activity caused by SL-I. NADPH oxidase-cytosolic factor translocation to plasma membranes was completely blocked by pertussis toxin when priming doses of SL-I were used. This guanine-nucleotide-binding protein inhibitor had no effect on FMLP-dependent translocation of the oxidase cofactors. The comparative approach introduced in this report provides a valuable and novel method to discern the complex interactions of various cellular processes that regulate the state of activation of stimulated cells.     

Activity of mushroom polyphenol oxidase in organic medium

A kinetic study of the activity of mushroom polyphenol oxidase in an organic system was carried out to obtain detailed enzyme kinetic data in relation to optimization of reaction conditions and substrate specificity. A simple method for consistent measurement of reaction rates in the heterogeneous enzyme/organic solvent system (consisting of immobilized polyphenol oxidase and a hydrated solution of the substrate in chloroform) was designed. The aqueous content of the system was optimized using p-cresol as the substrate. With this system, a crude extract of Agaricus bisporus was used to hydroxylate and oxidize a range of selected p-substituted phenolic substrates, yielding o-quinone products. Michaelis-Menten kinetics were used to obtain apparent K(M) and V(max) values with respect to each of these substrates. Results from this analysis indicated a correlation between the enzymic kinetic parameters obtained and the steric requirements of the substrates, which could be rationalized in terms of the restricted flexibility of the enzyme when it is in chloroform and also in terms of substrate and solvent hydrophobicity. In the course of the investigation UV molar absorption coefficients of several o-quinones were measured by a novel method: (1)H nuclear magnetic resonance (NMR) spectroscopy was employed to determine component concentrations in reaction mixtures resulting from the transformation of phenols by polyphenol oxidase in chloroform. Thus the UV molar absorption coefficients could be obtained directly, avoiding the necessity to isolate the water-sensitive, unstable o-quinones. (c) 1993 John Wiley & Sons, Inc.     

COMPARISON OF THE STOMACH CONTENTS OF SOUTHERN ELEPHANT SEALS, MIROUNGA LEONINA, AT MACQUARIE AND HEARD ISLANDS

Abstract: There are three major breeding populations of southern elephant seals centered on Macquarie Island, Kerguelen-Heard Islands and South Georgia-Antarctic Peninsula. The composition of the diet differs between these populations based on published data from Signy Island and data presented here from Macquarie and Heard Islands. These differences in diet appear to be linked to the location at which seals were sampled ranging from the least Antarctic (Macquarie Island) to the most Antarctic (Signy Island). The major food remains consisted of cephalopod beaks and fish eye lenses. More benthic material was found at Heard Island than at Macquarie Island. The diet at Macquarie Island differed between summer and winter and between young animals and adults. The difficulty in collecting dietary samples of southern elephant seals near their main foraging areas makes the study of the feeding ecology of this species extremely difficult in comparison with other Southern Ocean species.     

FLOW-CYTOMETRIC ANALYSES OF NUCLEAR DNA CONTENT IN FOUR FAMILIES OF NEOTROPICAL BATS

Flow-cytometric analyses of 29 species of microchiropteran bats representing four families and 20 genera revealed that bats possess only 79% (5.43 pg) of the DNA content of a “typical” mammal (e.g., Mus musculus strain C57BL; 7 pg). Chiroptera, the second largest order of mammals, is thus an exception to the prevailing view that mammals possess a minimum nuclear DNA content of 7 pg. Limitations on cell size resulting from a high metabolic rate may have constrained evolution of DNA content and could explain why the extensive heterochromatic additions that are common in some groups of mammals are absent in bats. Chromosomes of bats have been well studied; detailed chromosomal banding data are available for nearly all the species used in this investigation. However, no significant correlations were found between DNA content and karyotypic characteristics such as 2n, fundamental number, and rate or pattern of chromosomal evolution.     

Primate terminal complement inhibitor homologues of human CD59

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers L22862 (African green monkey CD59) and L22863 (baboon CD59)     

Effects of defoliation on seed protein concentration in normal and high protein lines of soybean

Two high (NC106, NC111) and two normal (NC103, NC107) seed protein concentration lines, derived from two different recurrent selection populations of soybean (Glycine max L. Merr.) were subjected to partial defoliation at beginning seed fill (R5) under outdoor pot culture and field conditions. The aim of this study was to test the hypothesis that capacity to store N in vegetative organs and/or to mobilize that N to reproductive organs is associated with the high seed protein concentration trait. Symbiotic N₂ fixation was the sole source of N in the pot experiment and the major source of N (met > 50% of the N requirement) in the low N soil used in the field experiment. Seed protein concentration and seed yield at maturity in both experiments and N accumulation and mobilization between R5 and maturity in the pot experiment were measured. The four genotypes did not differ significantly with respect to the amount of N accumulated before beginning seed fill (R5). Removal of up to two leaflets per trifoliolate leaf at R5 significantly decreased the seed protein concentration of NC107/111 but had no effect on this trait in NC103/106. Defoliation treatments significantly decreased seed yield, whole plant N accumulation (N₂-fixation) during reproductive growth and vegetative N mobilization of all genotypes. Differences in harvest indices between the high and low protein lines accounted for approximately 35% of the differences in protein concentration. The two normal protein lines mobilized more vegetative N to the seed (average. 5.26 g plant^–1) than the two high protein lines (average. 4.28 g plant^–1). The two high seed protein lines (NC106, NC111) exhibited significantly different relative dependencies of reproductive N accumulation on vegetative N mobilization, 45% vs. 29%, in the control treatment. Whereas, NC103 with normal and NC106 with high seed protein concentration exhibited similar relative dependencies of reproductive N accumulation on vegetative N mobilization, (47% vs. 45%). Collectively, these results indicate that N stored in shoot organs before R5 and greater absolute and relative contribution of vegetative N mobilization to the reproductive N requirement are not responsible for the high seed protein concentration trait.Abbreviations DAT days after transplanting - R5 fifth reproductive stage according to Fehr and Caviness, 1977 Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

Elemental signatures of human diets from the Georgia Bight

Multielement analysis was performed on bone samples extracted from the femora of 39 adults from three mortuary sites (Johns Mound, Santa Catalina de Guale, and Santa Catalina de Guale de Santa Meria) and time periods (late preagricultural, early contact, and late contact) in the Georgia Bight. This study was used to investigate whether elemental analysis would support or contradict other lines of data regarding diets and dietary change previously generated for the region. The data are in agreement with an earlier interpretation, based on stable isotopes, that dietary maize increases through time but fails to support the idea that marine resources decreased in importance. Rather, it appears that the wild plant food component of the diets decreases as maize increases in importance; throughout the sequence, marine resources comprise a significant portion of the diets. © 1995 Wiley-Liss, Inc.     

Refinement by linkage analysis in two large families of the candidate region of the third locus (SCA3) for autosomal dominant cerebellar ataxia type I

The autosomal dominant cerebellar ataxias (ADCA) are clinically and genetically heterogeneous. To date, several loci (SCAI-V) have been identified for ADCA type I. We have studied two large families from the northern part of The Netherlands with ADCA type I with a broad intra-familial variation of symptoms. In both families significant linkage is shown of the disease to the markers of the SCA3 locus on chromosome 14. Through recombinations, the candidate region for SCA3 could be refined to a 13-cM range between D14S256 and D14S81. No recombinations were detected with the markers D14S291 and D14S280, which suggests that the SCA3 gene lies close to these loci. This finding will benefit the individuals at risk in these two families who are seeking predictive testing or prenatal diagnosis.