Profiling the specificity of neutralizing antibodies in a large panel of plasmas from patients chronically infected with human immunodeficiency virus type 1 subtypes B and C

Identifying the viral epitopes targeted by broad neutralizing antibodies (NAbs) that sometimes develop in human immunodeficiency virus type 1 (HIV-1)-infected subjects should assist in the design of vaccines to elicit similar responses. Here, we investigated the activities of a panel of 24 broadly neutralizing plasmas from subtype B- and C-infected donors using a series of complementary mapping methods, focusing mostly on JR-FL as a prototype subtype B primary isolate. Adsorption with gp120 immobilized on beads revealed that an often large but variable fraction of plasma neutralization was directed to gp120 and that in some cases, neutralization was largely mediated by CD4 binding site (CD4bs) Abs. The results of a native polyacrylamide gel electrophoresis assay using JR-FL trimers further suggested that half of the subtype B and a smaller fraction of subtype C plasmas contained a significant proportion of NAbs directed to the CD4bs. Anti-gp41 neutralizing activity was detected in several plasmas of both subtypes, but in all but one case, constituted only a minor fraction of the overall neutralization activity. Assessment of the activities of the subtype B plasmas against chimeric HIV-2 viruses bearing various fragments of the membrane proximal external region (MPER) of HIV-1 gp41 revealed mixed patterns, implying that MPER neutralization was not dominated by any single specificity akin to known MPER-specific monoclonal Abs. V3 and 2G12-like NAbs appeared to make little or no contribution to JR-FL neutralization titers. Overall, we observed significant titers of anti-CD4bs NAbs in several plasmas, but approximately two-thirds of the neutralizing activity remained undefined, suggesting the existence of NAbs with specificities unlike any characterized to date.     

The molecular basis of an avian plumage polymorphism in the wild: a melanocortin-1-receptor point mutation is perfectly associated with the melanic plumage morph of the bananaquit, Coereba flaveola

BACKGROUND: Evolution depends on natural selection acting on phenotypic variation, but the genes responsible for phenotypic variation in natural populations of vertebrates are rarely known. The molecular genetic basis for plumage color variation has not been described in any wild bird. Bananaquits (Coereba flaveola) are small passerine birds that occur as two main plumage variants, a widespread yellow morph with dark back and yellow breast and a virtually all black melanic morph. A candidate gene for this color difference is the melanocortin-1 receptor (MC1R), a key regulator of melanin synthesis in feather melanocytes. RESULTS: We sequenced the MC1R gene from four Caribbean populations of the bananaquit; two populations of the yellow morph and two populations containing both the yellow morph and the melanic morph. A point mutation resulting in the replacement of glutamate with lysine was present in at least one allele of the MC1R gene in all melanic birds and was absent in all yellow morph birds. This substitution probably causes the color variation, as the same substitution is responsible for melanism in domestic chickens and mice. The evolutionary relationships among the MC1R haplotypes show that the melanic alleles on Grenada and St. Vincent had a single origin. The low prevalence of nonsynonymous substitutions among yellow haplotypes suggests that they have been under stabilizing selection, whereas strong selective constraint on melanic haplotypes is absent. CONCLUSIONS: We conclude that a mutation in the MC1R is responsible for the plumage polymorphism in a wild bird population and that the melanic MC1R alleles in Grenada and St. Vincent bananaquit populations have a single evolutionary origin from a yellow allele.     

Steroid receptors in the human prostate. 1. Estradiol - 17beta binding in benign prostatic hypertrophy.

Agar gel electrophoresis and ultracentrifugation on continuous sucrose gradients revealed the presence of a 4S estradiol 'receptor' in cytosols of samples of human benign hyperplastic prostate tissue. High affinity (charcoal stability) and saturability (disappearance with excess competitor) binding characteristics of the molecular species concerned facilitated its clear distinction from similarly migrating serum albumin-steroid complexes. Our data, obtained with human serum, purified human albumin and albumin-enriched cytosol strongly suggest that agar gel electrophoresis, when used alone, may lack specificity for the quantification of estrogen 'receptors'. Radioligand binding to these molecules may be obscured by similarly migrating albumin-steroid complexes which fail to dissociate during electrophoresis. We advocate the inclusion of competitor experiments to improve the specificity of the method.     

Adult-derived stem cells and their potential for use in tissue repair and molecular medicine

This report reviews three categories of precursor cells present within adults. The first category of precursor cell, the epiblast-like stem cell, has the potential of forming cells from all three embryonic germ layer lineages, e.g., ectoderm, mesoderm, and endoderm. The second category of precursor cell, the germ layer lineage stem cell, consists of three separate cells. Each of the three cells is committed to form cells limited to a specific embryonic germ layer lineage. Thus the second category consists of germ layer lineage ectodermal stem cells, germ layer lineage mesodermal stem cells, and germ layer lineage endodermal stem cells. The third category of precursor cells, progenitor cells, contains a multitude of cells. These cells are committed to form specific cell and tissue types and are the immediate precursors to the differentiated cells and tissues of the adult. The three categories of precursor cells can be readily isolated from adult tissues. They can be distinguished from each other based on their size, growth in cell culture, expressed genes, cell surface markers, and potential for differentiation. This report also discusses new findings. These findings include the karyotypic analysis of germ layer lineage stem cells; the appearance of dopaminergic neurons after implantation of naive adult pluripotent stem cells into a 6-hydroxydopamine-lesioned Parkinson's model; and the use of adult stem cells as transport mechanisms for exogenous genetic material. We conclude by discussing the potential roles of adult-derived precursor cells as building blocks for tissue repair and as delivery vehicles for molecular medicine.     

The interaction of the steroidal antagonist faslodex and methotrexate.

Faslodex (FAS, ICI 182, 780), a novel steroidal estrogen antagonist decreased high-dose methotrexate (MTX) cytotoxicity in MCF-7 breast cancer cells. When FAS is given at least 24 hr prior to MTX, the resultant interaction is antagonistic. However, when breast cancer cells are exposed to FAS 24 hr after MTX, the interaction between FAS and MTX is not antagonistic. The proliferation of cells exposed to 0.1 microM FAS and 10 microM MTX alone or in combination with FAS 24 hr prior to MTX was in the following order: FAS>FAS 24 hr prior to MTX>MTX. MTX administration 24 hr prior to FAS had the following inhibitory effects on the growth of cells: MTX 24 hr prior to FAS >MTX>FAS 24 hr prior to MTX>FAS>control (no drug exposure). To determine if the antagonistic interaction between FAS and MTX was a function of sequence and time, cells were exposed to FAS 24 hr and 36 hr prior to MTX exposure. The percentages of control rates were 42.70 +/- 4.60% and 57.89 +/- 0.55%, respectively, from a 24 hr and 36 hr exposure of FAS prior to MTX. The growth rates after 24 and 36 hr exposures to MTX alone were 30.30 +/- 0.61% and 33.11 +/- 2.57% of control rates, respectively. These studies suggest that: a) the interactions between FAS and MTX are sequence-dependent; b) FAS antagonizes the effect of MTX when FAS administration precedes MTX, and c) FAS antagonism to MTX is a function of time.     

The interaction of α-chymotrypsin with isosteric substrates of different charge type

1. The synthesis of three substrates of alpha-chymotrypsin of closely similar steric requirements but different charge type is reported. 2. The interaction of these compounds [SS-dimethyl-(l-3-carboxymethyl-3-acetamido)propyl sulphonium iodide, l-2-acetamido-5-methylhexanoic acid methyl ester and N-acetyl-l-glutamic acid alpha-methyl ester] with alpha-chymotrypsin has been studied. 3. For the charged substrates, values of k(0) are two orders of magnitude smaller than, and values of K(m) two orders of magnitude larger than, the corresponding values for the uncharged isostere. 4. The results are interpreted in terms of the known specificity of the enzyme, and the relationship between binding and kinetic specificities is discussed.     

Genetic variation in mitochondrial DNA from some aboriginal Australians

Mitochondrial (mt) DNAs from 17 aboriginal Australians, predominantly from the coastal region of the Northern Territory were isolated and digested with four four-base restriction endonucleases, two of which revealed variation between samples. The observed fragment patterns were used directly in parsimony analyses of phylogenetic relationships between the samples, and were also converted to estimates of the number of substitutions per nucleotide position between samples (delta), which estimates were then used in distance analyses of phylogeny. The inferred fragment patterns of the completely sequenced 'Cambridge' human mtDNA were also included in these analyses. No strong evidence of geographic variation was found, consistent with previous findings of Australian aborigines and other humans generally, although the most divergent sample was one of two from Sydney, indicating that further work is desirable. The estimate of mean difference between samples (diversity), 0.0017 +/- 0.0003 (mean +/- 95% confidence interval), is significantly lower than that reported previously for humans generally.