Premila Rathnam

Sustained release of microbicides by newly engineered vaginal rings.

Objective:An effective vaginal microbicide against sexual HIV transmission remains elusive, with requirements for adherence to appropriate application of effective, nontoxic products being a major deterrent. We explored methods to enable sustained release of combinations of antiretroviral microbicides, utilizing intravaginal rings composed of biosoluble Acacia gum or nonbiodegradable hydrogel of 2-hydroxyethyl methacrylate and sodium methacrylate, materials approved for use by the US Food and Drug Administration. Design and methods:The reverse transcriptase inhibitors TMC120, PMPA, 3′-azido-3′-deoxythymidine, and a newly characterized anti-HIV agent, Boc-lysinated betulonic acid, were incorporated into vaginal rings with different combinations. Daily and cumulative release rates of these inhibitors in ring eluates were determined by high-performance liquid chromatography, gas chromatography, or immunoassay. Anti-HIV effects were measured by assessment of p24 Gag antigen in T-cell cultures exposed to HIV-1 isolates. Results:Drug release rates were sustained at concentrations higher than the minimum effective dose for HIV inhibition. The release was maintained for no less than 15 and 28 days from the Acacia gum and 2-hydroxyethyl methacrylate and sodium methacrylate rings, respectively. Boc-lysinated betulonic acid showed more than 90% inhibition of HIV-1 infection in H9 cells, with little toxicity to normal cells. Conclusion:The intravaginal rings described here are capable of efficacious drug delivery. Incorporation of several antiretroviral agents, including betulinol derivatives, which act at multiple levels of the HIV life cycle, may provide a synergistic effect to achieve higher efficacy on the inhibition of HIV infection.

Studies on the disulfide bonds in human pituitary follicle-stimulating hormone

Human follicle-stimulating hormone (FSH) was digested with subtilisin, thermolysin, cyanogen gromide, pronase and trypsin to isolate the cystine-containing peptides. These peptides were purified by gel filtration through Sephadex G-50 column and by high-voltage paper electrophoresis at pH 6, 3.5 and/or 2. The location of the cystine-containing peptides in human FSH alpha- and beta-subunits was established by amino acid composition, end-group analysis and determination of the amino acid sequence by Edman degradation. The results indicate that the disulfide bonds are present between half-cystine residues located between positions 7 and 10, 28 and 87 and 82 and 84 in the alpha-subunit, and between positions 3 and 28, 17 and 51 and 32 and 104 in the beta-subunit of human FSH.

Clinical validation of enzymeimmunoassay of human luteinizing hormone (hLH) in the detection of the preovulatory luteinizing hormone (LH) surge in urine

The preovulatory luteinizing hormone (LH) surge (mean, 60.7 standard error +/- 4.7 mIU/ml) as determined by a solid-phase enzymeimmunoassay in urine has been correlated with clinical parameters in 24 women. In group A, of seven women, the preovulatory LH surge correlated with basal body temperature and cervical mucus. In one of the women in group A, serum levels of pituitary and gonadal hormones confirmed ovulation. In group B, of 17 women, the urinary estrone-3-glucuronide (E1-3-G) peak was either coincident with or preceded the LH surge. The LH surge in all cases occurred 12 to 24 hours prior to follicular rupture, as visualized by real-time sonography. The enzymeimmunoassay for the detection of the preovulatory LH surge is useful in patients for artificial insemination and for aspiration of mature oocytes for in vitro fertilization.

Conjugation of a fetuin glycopeptide to human follicle-stimulating hormone and its subunits by photoactivation

Human follicle-stimulating hormone (human FSH), dansylated human FSH, human FSH-alpha and human FSH-beta subunits were individually conjugated by photoactivation to an azidobenzoyl derivative of a glycopeptide isolated from fetuin. The conjugates were purified on a column of Sephadex G-100. The carbohydrate content of the conjugated human FSH increased 2.7-fold with a concomitant 1.5- and 2-fold increase in the immunological and biological activity of the human FSH. The glucosamine content of the human FSH-alpha and human FSH-beta increased 6- and 1.4-fold, respectively, after conjugation. Human FSH-alpha conjugate, when recombined with untreated human FSH-beta showed up to 50% increase in the biological activity over the control. When the conjugated human FSH-beta was combined with untreated human FSH-alpha, there was little change in the biological activity. These experiments demonstrate that the photoactivation procedure, although random and site-nonspecific in nature, provides a potential means of attachment of glycopeptides to the protein moiety and enhancement of the hormonal activity of human FSH.

Elucidation of the disulfide bond positions of the β-subunit of human follicle-stimulating hormone

Abstract From a proteolytic digest of human follicle-stimulating hormone (FSH), three of the five disulfide bonds in the α-subunit of human FSH, namely, those between half-cystine residues at positions 7–10, 28–87 and 82–84, and three of the six disulfide bonds in the β-subunit of human FSH, namely, those between half-cystine residues at positions 3–28, 17–51 and 32–104, have been previously identified. The remaining fractions of the proteolytic digest containing the rest of the cystine peptides of human FSH were pooled, and fractionated by gel filtration on Sephadex G-50 (superfine) and by high-pressure liquid chromatography. One high molecular weight fragment containing half-cystine residues was recovered. The fragment was resistant to a sequential digestion with specific proteolytic enzymes, such as thermolysin and l -(1-tosylamido-2-phenyl)ethyl chloromethyl ketone (TPCK)-treated trypsin. This fragment, on the basis of amino acid analysis, N- and C-terminal and limited amino acid sequence analysis, was identified as a single peptide containing the remaining six half-cystine residues or three disulfide bonds of the human FSH β-subunit. Hence, the fragment was digested with a mixture of proteases such as pronase, papain, subtilisin, chymotrypsin, thermolysin and TPCK-treated trypsin, in order to achieve a random cleavage of the peptide. From this enzyme digest, two cystine-containing peptides were isolated by gel filtration on Sephadex G-50, and high-voltage paper electrophoresis at pH 2 and pH 3.5. The peptides were identified by amino acid, N- and C-terminal and sequence analyses as containing disulfide bonds between βCys-66-βCys-94 and βCys-20-βCys-82. The single remaining bond was assigned between βCys-84-βCys-87 by elimination, thus completing the identification of all six disulfide bonds of the β-subunit of human FSH.

Isolation of prolactin from human amniotic fluid

Amniotic fluid, obtained from women at term, was centrifuged and concentrated by ultrafiltration. The concentrated amniotic fluid was purified by gel filtration on Sephadex G-100 and ion-exchanged chromatography on DEAE-cellulose. The fractions obtained during purification procedures were assayed for prolactin and somatotropin activities by respective radioimmunoassays. The prolactin-rich fraction obtained from ion-exchange chromatography was further purified by isoelectric focusing. A yield of 0.21 mg of highly pruified prolactin containing 40 units/mg was obtained from 1 liter of the amniotic fluid. The prolactin was free of somatotropin and human placental lactogen.

Biological actions of monoclonal antibodies to bovine lutropin receptor

Monoclonal antibodies (Mabs) were produced against bovine Lutropin receptor (LH-R). Antibodies were detected by an enzyme linked immunosorbant assay (ELISA). Hybridomas were subcloned to achieve monoclonality. Ascites were developed in Balb/c mice. Hybridoma supernatants were purified by ammonium sulfate precipitation and chromatography on hydroxylapatite columns. LH-R antibodies showed upto 50% inhibition of 125I-labeled hCG binding to bovine luteal cell membranes and up to 80% inhibition of testosterone production by hCG stimulated mouse Leydig cells. LH-R antibodies were predominantly IgM isotype. Purified antibodies showed a 78-kDa band, in SDS-PAGE, as the heavy chain of the immunoglobulin. LH-R antibodies were localized specifically in the thecal and luteal cells of the rat ovaries as well as in the Leydig cell of mouse testes. Injections of the LH-R antibody caused a constant estrus in normal rats. One month after the cessation of the injections the animals returned to normal estrus cycle and fertility. Pregnant mice injected with LH-R antibodies produced only 3 viable pregnancies and 10 pups, as compared to 8 pregnancies and 45 pups born to normal controls. LH-R antibodies also caused, approximately, a 50% reduction in testosterone production in normal male rats. These observations indicate a high degree of specificity of the Mab to LH-R and their potential in studies on gonadal function.

Studies on the unique presence of an N-acetylgalactosamine residue in the carbohydrate moieties of human follicle-stimulating hormone

Abstract Glycopeptides located at asparagine residues at position 52 in the α-subunit and at position 7 in the β-subunit of human follicle-stimulating hormone (FSH) were obtained by pronase digestion of the intact human FSH molecule. The glycopeptide fractions were isolated by gel filtration and by high-voltage electrophoresis on paper at pH 6 and/or pH 3.5. Alkaline degradation studies performed on the α- and β-subunits of human follicle-stimulating hormone confirmed that carbohydrate moieties linked O -glycosidically to threonine or to serine are not present in either subunit. Carbohydrate composition of both α-52 and β-7 glycopeptides was similar. Sialic acid, mannose, galactose, N -acetylglucosamine, N -acetylgalactosamine and fucose were present in a ratio of 3:4:3:4:1:1 in both glycopeptides. The results of sequential enzymatic digestion revealed that the peripheral monosaccharide sequence of both the α-52 and β-7 glycopeptides consisted of three chains each containing the following sequence: sialic acid galactose β - N -acetylglucosamine mannose. The composition of the undigested ‘inner core’ of both carbohydrate moieties was found to be similar and consisted of one residue/mol glycopeptide each of mannose, N -acetylglucosamine, N -acetylgalactosamine, and fucose. The unique presence of GalNAc attached to an N -glycosidically linked GlcNAc was confirmed by hexosamine analysis on an amino acid analyzer, by thin-layer chromatography, by digestion with enzymes such as exo- α - N -acetylgalactosaminidase and endoglycosidases D and H, and by ion-exchange chromatography on DEAE-Sephacel. The monosaccharide sequence of the α-52 and the β-7 glycopeptides of human follicle-stimulating hormone is shown below and is identical to the one at the α-78 position.

Human Chorionic Gonadotropin in Early Pregnancy

Publisher Summary Because of the structural similarities between hLH and hCG, the two hormones are similar in their primary biological and immunological activities. Since the 30 amino acid carboxy-terminal sequence is not present in the hLH-β subunit, it is reasonable to assume that portion of the hCG molecule is not necessary for biologic activity. hCG and both of its subunits are highly antigenic. Maintenance of corpus luteum gestation is essential for the maintenance of pregnancy during periimplantation. The primary biological role of chorionic gonadotropin extends the functional life span of the corpus luteum in the fertile menstrual cycle. As human trophoblast cells in tissue culture can produce ample amounts of hCG without involvement of any maternal factor, it would seem more likely that the 99 trophoblast cells present in the 107 cells of human blastocyst could already produce sufficient hCG to provide the necessary signal to the corpus luteum to persist.

Removal of residual luteinizing hormone by the use of a specific receptor and antibody coupled to glass beads.

We describe here, a novel and effective use of a specific receptor for Luteinizing Hormone (LH) coupled to controlled pore-glass beads (CPG) and LH antibodies to glass beads (GB), in selective removal of residual LH from purified Follicle Stimulating Hormone (FSH) and Thyroid Stimulating Hormone (TSH). The LH receptor was prepared from bovine corpora lutea. LH antisera were raised in rabbits and purified. FSH and TSH preparations were purified by treatment with the LH receptor coupled to CPG-beads; and/or LH-antibody coupled to glass beads. This procedure avoided dilution of the FSH or TSH during purification.