O-(4-Diazo-3,5-di[125I]iodobenzoyl)sucrose, a novel radioactive label for determining organ sites of catabolism of plasma proteins.

A method is described for radiolabelling proteins with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose (DD125IBS). When proteins so labelled were degraded within lysosomes, the radioactive fragments were largely retained within the organelle. High specific radioactivities were obtained without changing the properties of the protein. The validity of the method was demonstrated in vivo in rats using the short-lived protein lactate dehydrogenase, isoenzyme M4, and the long-lived protein bovine serum albumin. Derivatization with DD125IBS did not alter the clearance of either protein. Uptake of DD125IBS-labelled lactate dehydrogenase, isoenzyme M4, by liver and spleen of rats was determined. Radioactivity in these tissues increased up to about 2 h after injection (at this time the protein has been almost completely cleared from the blood) and subsequently declined with a half-life of approx. 20 h. After differential fractionation of liver, radioactivity was largely found in the mitochondrial and lysosomal fraction. The results of these studies establish that DD125IBS covalently coupled to plasma proteins should be a useful radioactive tracer for identifying the tissue and cellular sites of catabolism of relatively long-lived circulating proteins.     

Genetic recombination in Micromonospora rosaria by protoplast fusion

Auxotrophic strains of Micromonospora rosaria were isolated by N-methyl-N'-nitro-N'-nitrosoguanidine mutagenesis and used in intraspecific recombination by protoplast fusion. High-frequency fusion of protoplasts of M. rosaria strains was induced by polyethylene glycol (molecular weight, 1,000) (PEG 1,000). The optimum concentration of PEG 1,000 for fusion of M. rosaria was 50% (wt/vol). PEG 4,000 was slightly better than PEG 1,000 at concentrations lower than 50% (wt/vol). The recombinant frequency did not increase after treatment with PEG 1,000 (50% [wt/vol]) for longer than 20 min. Under these conditions, fusion with many auxotrophic strains of M. rosaria resulted in a high frequency of formation of true recombinants (sometimes more than 10%). Additionally, when ros (rosamicin nonproducing) strains were crossed by protoplast fusion; about 5% of the resultant prototrophic recombinants were shown to have the ros+ (rosamicin producing) characteristic restored. Rosamicin production by M. rosaria colonies was clearly distinguished by the broth overlay method. The results of fusion experiments between ros and ros+ strains indicated that either the chromosomal mutation or pleiotrophic effect of some auxotrophic markers is involved.     

A region in domain 1 of CD4 distinct from the primary gp120 binding site is involved in HIV infection and virus-mediated fusion.

The high affinity binding site for human immunodeficiency virus (HIV) envelope glycoprotein gp120 resides within the amino-terminal domain (D1) of CD4. Mutational and antibody epitope analyses have implicated the region encompassing residues 40-60 in D1 as the primary binding site for gp120. Outside of this region, a single residue substitution at position 87 abrogates syncytium formation without affecting gp120 binding. We describe two groups of CD4 monoclonal antibodies (mAbs) which recognize distinct epitopes associated with these regions in D1. These mAbs distinguish between the gp120 binding event and virus infection and virus-induced cell fusion. One cluster of mAbs, which bind at or near the high affinity gp120 binding site, blocked gp120 binding to CD4 and, as expected, also blocked HIV infection of CD4+ cells and virus-induced syncytium formation. A second cluster of mAbs, which recognize the CDR-3 like loop, did not block gp120 binding as demonstrated by their ability to form ternary complexes with CD4 and gp120. Yet, these mAbs strongly inhibited HIV infection of CD4+ cells and HIV-envelope/CD4-mediated syncytium formation. The structure of D1 has recently been solved at atomic resolution and in its general features resembles IgVk regions as predicted from sequence homology and mAb epitopes. In the D1 structure, the regions recognized by these two groups of antibodies correspond to the C'C" (Ig CDR2) and FG (Ig CDR3) hairpin loops, respectively, which are solvent-exposed beta turns protruding in two different directions on a face of D1 distal to the D2 domain. This face is straddled by the longer BC (Ig CDR1) loop which bisects the plain formed by C'C' and FG. This structure is consistent with C'C' and FG forming two distinct epitope clusters within D1. We conclude that the initial interaction between gp120 and CD4 is not sufficient for HIV infection and syncytium formation and that CD4 plays a critical role in the subsequent virus-cell and cell-cell membrane fusion events. We propose that the initial binding of CD4 to gp120 induces conformational changes in gp120 leading to subsequent interactions of the FG loop with other regions in gp120 or with the fusogenic gp41 potion of the envelope gp160 glycoprotein.     

Determination of kinetic parameters related to the piasmid instability: for the recombinant fermentation under repressed condition

The plasmid stability under the repressed state of cloned gene was studied theoretically as well as experimentally using recombinant E. coli K12DeltaH1Deltatrp/pPLc23trpA1 as a "host-vector" model system. The important kinetic parameters studied were the plasmid loss rate (theta) describing the rate at which the plasrnid-harboring cells lose plas-mids and the plasmid-free cells are generated per unit time and the difference in growth rates (Delta) between the two genotypes. These parameters were carefully defined, studied, and compared with other key kinetic parameters involved in the recombinant fermentation to further our understanding of metabolism of recombinants. The ratio of the concentration of plasmid-free cells to plasmid-harboring cells (Omega) was introduced, and the mathematical model was derived and used for the determination of the kinetic parameters associated with plasmid instability. These methods developed based on the theoretical considerations were tested experimentally. The results of these methods were compared, and the best method was selected and recommended. The effect of temperature and dilution rate on kinetic parameters theta and Delta were also studied in continuous culture, in order to provide some practical information related to the operation and control of recombinant fermentation processes.     

Oculocerebral syndrome with hypopigmentation (Cross syndrome): the mixed pattern of hair pigmentation as an important diagnostic sign.

The present report describes two female siblings of mixed ancestry (Cape Coloured) with consanguineous parents as further examples of Cross oculocerebral hypopigmentation syndrome. The mixed pattern of hair pigmentation as an important diagnostic sign is emphasized.     

Evolution of the V3 envelope domain in proviral sequences and isolates of human immunodeficiency virus type 1 during transition of the viral biological phenotype.

The third variable domain (V3) of the envelope gene of human immunodeficiency virus type 1 contains a major neutralization epitope and determinants of syncytium-inducing (SI) capacity and replication rate (reviewed by J. P. Moore and P. L. Nara, AIDS Suppl. 2:S21-S33, 1991). Sequences were generated from DNA of samples taken 3 months apart over a period of 24 and 30 months from peripheral blood mononuclear cells (PBMC) of two individuals, both before and after cocultivation with uninfected donor PBMC. The isolated virus shifted from the non-syncytium-inducing (NSI) phenotype to the SI phenotype during the study period. This shift was associated with distinct changes in the V3 domain in both patients. The association of the phenotype shift with the V3 sequence changes was confirmed by construction of viruses with chimeric V3 loops. The shift from NSI- to SI-associated V3 variants was also seen in the uncultured PBMC of both patients, but not until 3 and 9 months after the detection of SI virus in culture. In the samples of uncultured PBMC DNA, several subgroups of sequences were found, indicating that the process of evolution may not be gradual and that several distinct populations can coexist. The paucity of intermediate sequences indicated that strong selection pressure was exerted on this part of the envelope. The early emergence of disease-associated SI variants in cultured material indicates that virus culture may have relevance for the in vivo situation.     

Plant size,geitonogamy and seed set in Ipomopsis aggregata

Summary We used powdered fluorescent dyes to estimate receipt of self vs. outcross pollen in the self-incompatible species Ipomopsis aggregata (Polemoniaceae). Flowers on small and large plants received equal amounts of outcross pollen, whereas flowers on large plants received more self pollen, so the proportion of self pollen delivered through geitonogamy increased with plant size. In natural populations emasculation of all flowers on a plant raised average seed set per flower from 5.19 to 6.99 and also raised fruit set, though not significantly. From these results one expects a negative correlation between plant size and seeds per flower. The opposite trend was observed in a sample of plants in the field, suggesting that deleterious effects of geitonogamy on female fecundity in large plants can be overruled by other factors such as size-related fruit or seed abortion. Results are discussed in relation to the evolution of gynodioecy.     

Binding of matrix attachment regions to lamin B1.

Eukaryotic chromatin is organized into topologically constrained loops that are attached to the nuclear matrix. The regions of DNA that interact with the matrix are called matrix attachment regions (MARs). We studied the spatial distribution of MAR-binding sites in the nuclear matrix from rat liver cells, following a combined biochemical and ultrastructural approach. We found that MAR-binding sites are distributed equally over the internal fibrogranular network and the peripheral nuclear lamina. Internal and peripheral binding sites have similar binding characteristics: both sets of binding sites show specific and saturable binding of MARs from different organisms. By means of a DNA-binding protein blot assay and in vitro binding studies, we identified lamin B1 as a MAR-binding protein, which provides evidence for a specific interaction of DNA with the nuclear lamina.     

Kinetic properties of a L-cysteine desulfhydrase-deficient mutant in the enzymatic formation of L-cysteine from D,L-ATC

Summary A mutant strain lacking in activity of L-cysteine desulfhydrase, a L-cysteine-decomposing enzyme, was screened after UV-treatment ofPseudomonas sp. CU6. The properties of the two strains, original and mutant, were compared on the basis of parameter values estimated from kinetic simulations of the enzymatic formation of L-cysteine from D,L-ATC. Both strains suffered from product inhibition, though inhibition was less for the mutant strain.