Purification of goat serum retinol-binding protein and preparation of its antibodies

The method for the purification of goat serum retinol-binding protein consists of DEAE-cellulose chromatography of the serum followed by preparative polyacrylamide disc gel electrophoresis. After electrophoresis, the retinol-binding protein containing zone is identified by the specific fluorescence of retinol. For raising the antibodies, the portion of the gel containing retinol binding protein is homogenized and injected intradermally and intramuscularly to rabbits. The availability of this simple method for the isolation of retinol-binding protein and production of its antibodies enables the development of a radioimmunoassay for this protein.

Proteomic Identification of Haptoglobin alpha 2 as a Glioblastoma Serum Biomarker: Implications in Cancer Cell Migration and Tumor Growth

Glioblastoma (GBM; grade IV astrocytoma) is the most malignant and common primary brain tumor in adults. Using combination of 2-DE and MALDI-TOF MS, we analyzed 14 GBM and 6 normal control sera and identified haptoglobin alpha 2 chain as an up-regulated serum protein in GBM patients. GBM-specific up-regulation was confirmed by ELISA based quantitation of haptoglobin (Hp) in the serum of 99 GBM patients as against lower grades (49 grade III/AA; 26 grade II/DA) and 26 normal individuals (p = 0.0001). Further validation using RT-qPCR on an independent set (n = 78) of tumor and normal brain (n = 4) samples and immunohistochemcial staining on a subset (n = 42) of above samples showed increasing levels of transcript and protein with tumor grade and were highest in GBM (p = < 0.0001 and < 0.0001, respectively). Overexpression of Hp either by stable integration of Hp cDNA or exogenous addition of purified Hp to immortalized astrocytes resulted in increased cell migration. RNAi-mediated silencing of Hp in glioma cells decreased cell migration. Further, we demonstrate that both human glioma and mouse melanoma cells overexpressing Hp showed increased tumor growth. Thus, we have identified haptoglobin as a GBM-specific serum marker with a role on glioma tumor growth and migration.

Ability of human chorionic gonadotropin beta-subunit to inhibit the steroidogenic response to lutropin.

Ability of the beta-subunit of human chorionic gonadotropin to inhibit the response to lutropin (luteinizing hormone, LH) was tested in the immature rat ovarian system and pregnant-mare-serum-gonadotropin-primed rat ovarian system with progesterone production being used as the response. Human chorionic gonadotropin beta-subunit was found to inhibit human and ovine lutropin-stimulated progesterone production. At a constant dose of lutropin, inhibition was dependent on the concentration of beta-subunit. When concentration of the beta-subunit was kept constant at 5.0 microgram/ml and the concentration of lutropin was varied, the inhibition was maximum at the saturating concentration of the native hormone. The alpha-subunit of the human chorionic gonadotropin did not inhibit the response to lutropin. The lutropin/beta-subunit ratio required to produce an inhibition of response was much lower than that required to bring about an observable inhibition of binding.

Common epitopes of serum retinol-binding proteins: a study with monoclonal antibodies

Monoclonal antibodies were raised against purified chicken retinol-binding protein. These were characterised extensively with respect to their ability to recognize retinol-binding proteins from different species. The monoclonal antibodies exhibited differential recognition characteristics. Though the majority presented restricted reactivities, one out of the four monoclonal antibodies studied cross-reacted with retinol-binding proteins from all species tested so far.

Antigenic determinants of human serum retinol binding protein as probed with monoclonal antibodies

Monoclonal antibodies raised against human serum retinol-binding protein (hRBP) were used as probes for the study of the antigenic determinants of hRBP and those shared with the same protein from other species. The antibodies could be classified into four distinct groups and react with the homologous proteins from the rat as well as the rabbit sera. Three of these antibodies recognize sequential or continuous epitopes while the remaining antibody is directed against a discontinuous or conformational epitope. By chemical cleavage with cyanogen bromide, the domains recognized by the monoclonal antibodies could be delineated. By solid-phase synthetic approach, the core sequences recognized by two of these monoclonal antibodies were identified to amino acid sequences 45–51 and 128–131 of the primary amino acid sequence of hRBP.

Development of an LH receptor assay capable of measuring serum LH/CG in a wide variety of species

The development of a radioreceptor assay (RRA) that can measure serum LH in a variety of species and CG in sera and urine of pregnant women and monkeys is reported. Using sheep luteal membrane as the receptor source and I-125-labelled hLH/hCG as the tracer, dose-response (displacement) curves were obtained using hLH or hCG as standard. The addition of LH-free serum (200 mul per tube) had no affect on the standard displacement curve. The assay is simple, requires less than 90 min to complete and provides reproducible results. The sensitivity of the assay was 0.6 ng hLH per tube and the intra- and interassay variations were 9.6 and 9.8, respectively. Sera obtained from male and female bonnet monkeys (Macaca radiata) and monkey pituitary extract showed parallelism to the standard curve. The concentrations of LH measured correlated with the physiological status of the animals. Sera of rats, rabbits, hamsters, guinea-pigs, sheep and humans showed parallelism to the hLH standard curve indicating the viability of the RRA to measure serum LH of different species. Since the receptors recognize LH and CG, detection of pregnancy in monkeys and women was possible using this assay. The sensitivity of the assay for hCG was 8.7 miu per tube. This RRA could be a convenient alternative to the Leydig cell bioassay for obtaining the LH bioactivity profile of sera and biological fluids.

Effect of altering endogenous gonadotrophin concentrations on the kinetics of testicular germ cell turnover in the bonnet monkey (Macaca radiata)

The role of FSH and diurnal testosterone rhythms in specific germ cell transformations during spermatogenesis were investigated using DNA flow cytometry and morphometry of the seminiferous epithelium of the adult male bonnet monkey (Macaca radiata), the endogenous hormone levels of which were altered by two different protocols. (1) Active immunization of five monkeys for 290 days using ovine FSH adsorbed on Alhydrogel resulted in the neutralization of endogenous FSH, leaving the LH and diurnal testosterone rhythms normal. (2) Desensitization of the pituitary gonadotrophs of ten monkeys by chronically infusing gonadotrophin-releasing hormone analogue, buserelin (50 micrograms/day release rate), via an Alzet pump implant (s.c.) led to a 60-80% reduction in LH and FSH as well as total abolition of testosterone rhythms. The basal testosterone level (3.3 +/- 2.0 micrograms/l), however, was maintained in this group by way of an s.c. testosterone silicone elastomer implant. Both of the treatments caused significant (P < 0.01) nearly identical reduction in testicular biopsy scores, mitotic indices and daily sperm production rates compared with respective controls. The germ cell DNA flow cytometric profiles of the two treatment groups, however, were fundamentally different from each other. The pituitary-desensitized group exhibited a significant (P < 0.001) increase in 2C (spermatogonial) and decrease in 1C (round spermatid) populations while S-phase (preleptotene spermatocytes) and 4C (primary spermatocytes) populations were normal, indicating an arrest in meiosis caused presumably by the lack of increment in nocturnal serum testosterone. In contrast, in the FSH-immunized group, at day 80 when the FSH deprivation was total, the primary block appeared to be at the conversion of spermatogonia (2C) to cells in S-phase and primary spermatocytes (4C reduced by > 90%). In addition, at this time, although the round spermatid (1C) population was reduced by 65% (P < 0.01) the elongate spermatid (HC) population showed an increase of 52% (P < 0.05). This, taken together with the fact that sperm output in the ejaculate is reduced by 80%, suggests a blockade in spermiogenesis and spermiation. Administration of booster injections of oFSH at time-points at which the antibody titre was markedly low (at days 84 and 180) resulted in a transient resurgence in spermatogenesis (at day 180 and 228), and this again was blocked by day 290 when the FSH antibody titre increased.

In vivo and in vitro studies on the differential role of luteinizing hormone and follicle-stimulating hormone in regulating follicular function in the bonnet monkey (Macaca radiata) using specific gonadotropin antibodies

Although a distinct need for FSH in the regulation of follicular maturation in the primate is well recognized, it is not clear how FSH controls the functionality of different cellular compartments of the follicle. It is also not evident whether there is a requirement for LH in follicular maturation in the primate. In the first part of the present study, female bonnet monkeys were administered a well-characterized ovine (o) LH antiserum to neutralize endogenous monkey LH for different periods during the follicular phase, and the effect on the overall follicular maturation process was assessed by analyzing serum estrogen (E) and progesterone (P) profiles. Neither continuous LH deprivation from Day 8 of the cycle nor deprivation of LH on any one day between Days 6 and 10 had a significant effect on serum E and P profiles and the follicular maturation process. The period for which the antiserum was effective was dependent upon the dose injected; 1 ml of the antiserum given on Day 8 blocked ovulation but not follicular maturation. To assess the effect of deprivation of LH/FSH at the cellular level, animals were deprived in vivo of LH (on Days 8 and 9 of the cycle) or of FSH (on Day 6 of the cycle) by injection of highly characterized hCG and ovine (o) FSH antisera, respectively; the in vitro responsiveness of granulosa and thecal cells isolated on Day 10 from these animals was then determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of beta-lactamase-catalyzed hydrolysis of cephalosporins on peroxynitrite-mediated nitration of serum albumin and cytochrome c

The hydrolysis of beta-lactam antibiotics by beta-lactamases is one of the major bacterial defense systems. These enzymes generally hydrolyze a variety of antibiotics including the latest generation of cephalosporins, cephamycins and imipenem. In this paper, the effect of cephalosporins-based antibiotics on the peroxynitrite-mediated nitration of protein tyrosine is described. Although some of the antibiotics have weak inhibitory effect on the nitration reactions in the absence of beta-lactamase, they exhibit very strong inhibition in the presence of beta-lactamase. This is due to the elimination of heterocyclic thiol/thione moieties from cephalosporins by beta-lactamase-mediated hydrolysis. After the elimination, the thiols/thiones effectively scavenge peroxynitrite, leading to the inhibition of the nitration reactions.

Evidence for follicle-stimulating hormone mediation in the hemiorchidectomy-induced compensatory increase in the function of the remaining testis of the adult male bonnet monkey (Macaca radiata)

Hemiorchidectomy (HO) in the adult male bonnet monkey results in a selective increase in circulating concentrations of FSH and testosterone, and this is accompanied by compensatory increase in sperm production by the remaining testis. We investigated the possible role of increased FSH concentration that occurs after HO in the compensatory increase in the activity of the remaining testis. Of eight adult male bonnet monkeys that underwent HO, four received i.v. injections every other day for 30 days of a well-characterized ovine FSH antiserum (a/s) that cross-reacts with monkey FSH. The remaining four males received normal monkey serum (NMS) as control treatment in a protocol similar to that employed for ais-treated males. Blood samples were collected between 2100 and 2200 h before and 1/2, 1, 3, 5, 7, 14, 22, and 29 days after HO. Testicular weight, number of 3 beta-hydroxy steroid dehydrogenase-positive (3 beta-HSD+) cells, and DNA flow cytometric analysis of germ cell populations were obtained for testes collected before and at the termination of NMS or ais treatment. In NMS-treated males, circulating serum FSH concentrations progressively increased to reach a maximal level by Day 7 after HO (1.95 +/- 0.3 vs. 5.6 +/- 0.7 ng/ml on Days -1 and 7, respectively). Within 30 min of ais injection, FSH antibodies were detected in circulation, and the antibody level was maintained at a constant level between Day 7 and end of treatment (exhibiting 50-60% binding to I-125-hFSH). Although circulating mean nocturnal serum testosterone concentration showed an initial decrease, it rose gradually to pre-HO concentrations by Day 7 in NMS-treated males. In contrast, nocturnal mat serum testosterone concentrations in a/s-treated males remained lower than in NMS-treated controls (p < 0.05) up to Day 22 and thereafter only marginally increased. Testicular weights increased (p < 0.05) over the pre-HO weight in NMS- but not in ais-treated males. After HO, the number of 3 beta-HSD+ cells (Leydig cells) was markedly increased but was significantly (p < 0.05) higher in NMS-treated males compared to a/s-treated males. A significant (p < 0.05) reduction in the primary spermatocyte population of germ cells was observed in ais-treated compared to NMS-treated males. These results suggest that the increased FSH occurring after HO could be intimately involved in increasing the compensatory functional activity of the remaining testis in the male bonnet monkey.

Intranasally delivered microdoses of bromocriptine (BCR) effectively reduced serum prolactin levels in hyperprolactinemic patients

It is well known that hyperprolactinaemia in the human leads to infertility. The therapy of choice in India has been the administration of bromocriptine (BCR) as tablets, This mode of administration is generally accompanied by undesirable side-effects such as giddiness, nausea, vomiting and postural hypotension, We demonstrate here the efficacy of microdoses of BCR administered intranasally (IN) to hyperprolactinaemic patients (n = 6) in reducing significantly the elevated serum prolactin levels and maintain them within the normal range, The IN mode of BCR administration, in addition to reducing the effective dose of the drug by 4-20-fold, results in little or no side-effects otherwise associated with oral therapy.

CNT loading into cationic cholesterol suspensions show improved DNA binding and serum stability and ability to internalize into cancer cells

Methods which disperse single-walled carbon nanotubes (SWNTs) in water as `debundled', while maintaining their unique physical properties are highly useful. We present here a family of cationic cholesterol compounds (Chol(+)) {Cholest-5en-3 beta-oxyethyl pyridinium bromide (Chol-PB+), Cholest-5en-3 beta-oxyethyl N-methyl pyrrolidinium bromide (Chol-MPB+), Cholest-5en-3 beta-oxyethyl N-methyl morpholinium bromide (Chol-MMB+) and Cholest-5en-3 beta-oxyethyl diazabicyclo octanium bromide (Chol-DOB+)}. Each of these could be easily dispersed in water. The resulting cationic cholesterol (Chol(+)) suspensions solubilized single-walled carbon nanotubes (SWCNTs) by the non-specific physical adsorption of Chol(+) to form stable, transparent, dark aqueous suspensions at room temperature. Electron microscopy reveals the existence of highly segregated CNTs in these samples. Zeta potential measurements showed an increase in potential of cationic cholesterol aggregates on addition of CNTs. The CNT-Chol(+) suspensions were capable of forming stable complexes with genes (DNA) efficiently. The release of double-helical DNA from such CNT-Chol(+) complexes could be induced upon the addition of anionic micellar solution of SDS. Furthermore, the CNT-based DNA complexes containing cationic cholesterol aggregates showed higher stability in fetal bovine serum media at physiological conditions. Confocal studies confirm that CNT-Chol(+) formulations adhere to HeLa cell surfaces and get internalized more efficiently than the cationic cholesterol suspensions alone (devoid of any CNTs). These cationic cholesterol-CNT suspensions therefore appear to be a promising system for further use in biological applications.