Synergistic action, on the masculinization of female chick embryo gonads, of a graft of embryonic testis and administration of an anti-estrogenic substance (tamoxifen)

The capability of an embryonic testis graft to induce a male gonadal sex differentiation in genetically female chick embryos was significantly enhanced by the administration of a antiestrogenic drug (tamoxifen). This observation reinforces the view that the experimentally-induced testis differentiation depends chiefly on a inhibition of estrogen influence.     

Impaired blood clearance of bacteria and phagocytic activity in vitamin A-deficient rats

The effect of vitamin A deficiency on the functional integrity of the reticuloendothelial system and the phagocytic capacity of circulating polymorphonuclear leukocytes was evaluated in retinoate-cycled vitamin A-deficient rats under conditions such that secondary dietary imbalances were eliminated. Kinetics of blood clearance of 2 X 10(7) Escherichia coli injected intravenously was depressed within 8 days of the withdrawal of retinoic acid; all animals were profoundly affected by Day 12 of deficiency. In vitro, the phagocytic activity of polymorphonuclear leukocytes was similarly affected; by Day 12 of deficiency, phagocytic capacity in all deficient animals was less than 40% of the appropriate control values (P less than 0.01). Animals rendered vitamin A deficient by this procedure also displayed marked susceptibility to endogenous bacterial infection, as judged from the proportion of deficient rats that spontaneously developed bacteremia during the later stages of deficiency. These data together demonstrate unequivocally that reticuloendothelial and polymorphonuclear leukocytic functions are impaired in vitamin A deficiency in the absence of other dietary imbalances.     

Segmental homology between T-cell receptors and immunoglobulin variable regions: evidence that antisera to synthetic JH1 peptide react with murine and human T-cell products

To determine precisely the nature of serological determinants shared between T-cell surface molecules and immunoglobulin variable regions, the capacity of antisera directed against a synthetic peptide corresponding to the entire JH 1 region of classical immunoglobulin plus five residues of the D region were tested for their capacity to bind to T-cell membranes and isolated T-cell products. The anti-JH 1 antisera reacted with normal and monoclonal in vitro grown T-cell lines as judged by microhemagglutination and binding in enzyme-linked immunosorbent assays. Immunologically cross-reactive membrane components disclosed by immunoblot transfer analysis ("Western blots") consisted of major components in the molecular weight range 30-35,000 and minor components in the range 65-70,000. The major product of the human T-cell leukemia line MOLT-3 had an approximate mass of 34,000 Da, a value consistent with the predicted size of the molecule specified by the recently described putative T-cell receptor gene YT35. The 65 to 70,000-Da components are most probably tightly associated dimers of the 30 to 35,000-Da forms. It was possible to align the JH sequences of molecules reactive with the anti-JH 1 antisera and other characterized VH sequences of molecules known to be cross-reactive with T-cell products. This facilitated a comparison disclosing clear segmental homology between the protein sequence derived from the YT35 gene and immunoglobulin VH framework regions sharing approximately 50% of sequence identity. The identification of VH-related T-cell products (termed VT-bearing molecules) with products of putative T-cell receptor genes gained further support by N-terminal sequence of the 68,000-Da product of the 70-N2 T-cell line which showed homology to the predicted N-terminal region of the YT35 product. These serological and protein chemical data, coupled with the comparison to gene sequence, show that T-cell components that bear serological determinants cross-reactive with VH show segmental homology with products of putative T-cell receptor genes and immunoglobulin VH.     

Novel silylated steroids as aromatase inhibitors

Androst-4-en-3-one analogs incorporating a trimethylsilyl or a trimethylsilylmethyl group at C-1, C-2 or C-19 were prepared and evaluated as inhibitors of aromatase. Only 10-[1-hydroxy-2-(trimethylsilyl)ethyl]estr-4-ene-3,17-dione inhibited human placental aromatase. Enzyme kinetic analysis revealed competitive inhibition [apparent dissociation constant (Ki) of 562 +/- 12 nM] associated with marginal time-dependent inhibition.     

Equol: a contributor to enigmatic immunoassay measurements of estrogen

The efficacy of radioimmunoassay (RIA) for the measurement of estradiol-17 beta (E2) in murine plasma was investigated. When Sephadex LH-20 or celite column chromatography was used to separate E2 from estrone (E1) and other cross-reacting compounds, the results were erratic if small volumes of mouse plasma were resolved. Assay of a diethyl ether extract of plasma (500 microL) was the most practical method for estimating the concentration of estradiol-17 beta in mice. This method was used to determine the pattern of estrogen secretion during the estrous cycle, on the day of implantation and during pregnancy. No convincing change in estrogen secretion was observed in the diestrous/proestrous mouse. By comparison, estrogen levels were elevated during pregnancy. Taken together, these results implied that cross-reactive components in plasma masked low levels of endogenous estrogen. Further evaluation of mouse plasma and urine using a co-chromatography technique to examine estrogen elution from a reverse-phase HPLC system followed by GC/MS analysis indicated the presence of equol [7-hydroxy-3-(4-hydroxyphenyl)chroman], a phytoestrogen metabolite with a ring structure similar to estradiol-17 beta. Equol and possibly other cross-reactive components of plasma may account for the apparent lack of increased estrogen secretion during the mouse estrous cycle and on the day of implantation as determined by the radioimmunoassay of ether extracts of plasma.     

Quantization of membrane potential generation by cytochrome c oxidase in small vesicles

Proteoliposomes incorporating cytochrome c oxidase have been prepared by the cholate dialysis method and by sonication. Sonication produces multilamellar vesicles heterogeneous in size in contrast to a more uniform preparation of unilamellar vesicles produced by the dialysis procedure. Respiratory control in both preparations ranges between 4 and 8. From an electron microscopic analysis of proteoliposome size, the average electrical capacitance/vesicle for the dialyzed and sonicated preparations is calculated as 15 X 10(-18) F and 130 X 10(-18) F, respectively. These capacitance values would lead to a quantization of membrane potential generation by the enzyme at 77 mV/turnover for the dialyzed preparation and 9 mV/turnover for sonicated vesicles. It is argued that these differences can explain the dependence of H+ translocation on the number of turnovers of cytochrome c oxidase in dialyzed preparations in contrast to the lack of dependence on number of turnovers in sonicated preparations.     

NADPH-specific and NADH-specific xylose reduction is catalyzed by two separate enzymes in Pachysolen tannophilus

Summary Pachysolen tannophilus contains — in addition to an NADPH-linked xylose reductase — a separate NADH-linked one, in this respect differing from the yeast Pichia stipitis. Both enzyme proteins can conveniently be separated from each other by either ion exchange chromatography or chromatofocusing.     

Rationalization of some genetic anticodonic assignments

The genetic code appears to be a logic matrix in which, generally speaking, there is a correlation between the hydrophobicities of amino acids and their anticodonic nucleotides. There are several exceptions to this generality, however, and using previous data on hydrophobicity and binding constants, coupled with new data on reaction rates, we rationalize several of the anticodonic assignments.     

Characterization of monoclonal antibodies to bromodeoxyuridine

The characteristics of three mouse monoclonal antibodies to halogenated uridine derivatives are presented. Two, IU-1 and IU-2, are produced by hybridomas derived in our laboratory, and the third is the B-44 hybridoma described by Gratzner (7) and obtained commercially from Becton-Dickinson Monoclonal Center. Hybridomas IU-1 and IU-2 were derived from the fusion of spleen cells from a Biozzi High Responder mouse immunized with iododeoxyuridine (IdUrd) conjugated to bovine serum albumin and SP2/0 mouse myeloma cells. This paper presents methods and results for enzyme-linked immunosorbent assays (ELISA) against whole cells labeled with bromodeoxyuridine (BrdUrd), ELISA against BrdUrd-labeled DNA, and a competition ELISA for free BrdUrd. All three antibodies show similar binding affinities and specificities. The IU antibodies react with BrdUrd and IdUrd when the nucleosides are either free in solution or incorporated into single-stranded DNA (ss-DNA). The antibodies do not recognize either halogenated base in double-stranded DNA (ds-DNA), nor do they react with uracil or bromocytidine. Weak binding to thymidine, 5-fluorodeoxyuridine, and unsubstituted ss-DNA occurs.