Rajan Dighe - Academia.edu (original) (raw)

Papers by Rajan Dighe

Journal of Biosciences, 1988

The mechanism of 'down regulation' of luteinizing hormone receptors was investigated in pseudopre... more The mechanism of 'down regulation' of luteinizing hormone receptors was investigated in pseudopregnant rats using a modified radioimmunoassay capable of measuring endogenous tissue-bound hormone. Treatment of pseudopregnant animals with a desensitizing dose (desensitization treatment) of human chorionic gonadotropin resulted in a decrease in receptor concentration. This decrease was prevented if the animals were treated prior to the desensitization treatment with indomethacin, an inhibitor of prostaglandin biosynthesis, suggesting a role for prostaglandins in down regulation. The desensitization treatment resulted in a time-dependent decrease in subsequent responsiveness of the tissue to luteinizing hormone. Basal progesterone production rate was also decreased following desensitization. Total tissue cholesterol was found to be decreased following desensitization treatment, without any change in the ratio of free to esterified cholesterol. Mitochondrial cholesterol was significantly reduced and pregnenolone production by the mitochondria of desensitized corpora lutea was also markedly reduced. However, when cholesterol was added to the mitochondria of desensitized corpora lutea, pregnenolone production was increased, reaching values almost equal to that shown by the control mitochondria. These results show that decrease in the responsiveness following desensitization treatment is due to, besides receptor loss, decrease in tissue cholesterol, in particular mitochondrial cholesterol. The cholesterol side chain cleavage activity, although low, appears to be functionally intact; the low activity could be attributed to low levels of mitochondrial cholesterol.

The gonadotropins, luteinizing hormone (lutropin, LH) and follicle-stimulating hormone (follitrop... more The gonadotropins, luteinizing hormone (lutropin, LH) and follicle-stimulating hormone (follitropin, FSH) through their ability to regulate the synthesis of the sex steroids (androgens and estrogens), are indispensable for proper gonadal development and function (1). The synthesis and secretion of gonadotropins are regulated by the hormones produced by the pituitary itself (such as follistatin and activin), as well as the hormones from the hypothalamus and the gonads (2,3,4,5,6) . This tripartite regulatory network is known as the hypothalamuspituitary-gonadal (HPG) axis (Fig. 1).

Spermatogenesis is characterized by sequential gene-expression at precise stages in progression o... more Spermatogenesis is characterized by sequential gene-expression at precise stages in progression of differentiation of the germ cells. Any alteration in expression of the critical genes is responsible for arrest of spermatogenesis associated with infertility. Inspite of advances the differential gene expression accompanying spermatogenesis, the corresponding regulatory mechanisms and their correlation to human infertility have not been clearly established. This study aims to identify the gene expression pattern of the human testicular germ cells from the patients either with obstructive azoospermia with complete intra-testicular spermatogenesis or non-obstructive azoospermia with spermatogenesis arrested at different stages and correlate the same to infertility. The testicular transcriptomes of 3 OA and 8 NOA patients and pooled testicular RNA (commercial source) were analyzed for their differential gene expression to identify potential regulators of spermatogenesis and the results w...

The association between the upregulated Notch and FSH signaling and ovarian cancer is well docume... more The association between the upregulated Notch and FSH signaling and ovarian cancer is well documented. However, their signaling has been investigated independently and only in the primary tumor tissues. The aim of this study was to investigate the interactive effects of FSH and Notch signaling on the ovarian cancer proliferation, formation and maintenance of the disseminated ovarian cancer cells. Roles of Notch and FSH in the ovarian cancer pathogenesis was investigated using ovarian cancer cell lines and specific antibodies against Notch and FSH receptor (FSHR). FSH upregulated Notch signaling and proliferation in the ovarian cancer cells. High levels of FSH were detected in the ascites of patients with serous ovarian adenocarcinoma. The spheroids from the ascites of the patients, as well as, the spheroids from the ovarian cancer cell lines under low attachment culture conditions, expressed FSH beta subunit mRNAs and secreted the hormone into the medium. In contrast, the primary ov...

Journal of Steroid Biochemistry, 1984

Glycoprotein hormones, Thyrotropin(TSH), Follitropin(FSH), Lutropin(LH) and Chorionic Gonadotropi... more Glycoprotein hormones, Thyrotropin(TSH), Follitropin(FSH), Lutropin(LH) and Chorionic Gonadotropin(CG) are heterodimers composed of a common \alpha subunit, non covalently associated with hormone specific \beta subunit. They are interesting models to study protein folding, protein-protein interaction and role of carbohydrates in protein structure and function. The studies using site directed mutagenesis are often hampered by the loss of heterodimerization. This limitation can be overcome by translationally fusing the subunits to obtain a single chain hormone. Single chain hCG , thus produced in the laboratory, has been shown to be structurally and functionally similar to native hCG. This fusion protein approach was used to study a mutation that disrupts heterodimerization, where a Proline38(P38) residue in \alpha was substituted by Alanine. The mutant was expressed using Pichia expression system. It was observed that though the overall conformation of the mutant was altered, several epitopes in the subunits were maintained as tested by RIA. The mutant retained its receptor binding ability but was biologically inactive. It can be inferred that the fusion protein strategy has successfully overcome the constraint of heterodimerization imposed by the mutation. P38 residue appears to play an important role in attaining a conformation that is optimal for bioactivity.

Molecular and Cellular Endocrinology, 2016

The Luteinizing hormone receptor (LHR) has a large extracellular domain (amino acid residues, a.a... more The Luteinizing hormone receptor (LHR) has a large extracellular domain (amino acid residues, a.a.1-355) and a transmembrane domain (TMD; a.a. 356-699), essential for hormone binding and signaling, respectively. The LHR hinge region (a.a. 256-355) connects the two domains and acts as an activating switch for the receptor by an unknown mechanism. LHR hinge-specific Single chain fragment variables (ScFv) stimulated cAMP production by the stable and transiently transfected cell lines expressing LHR in a hormone-independent manner and the C-terminal region of LHR hinge (a.a. 313-349) was identified as the probable epitope for one agonistic ScFv. This epitope attained a helical conformation upon agonistic ScFv binding and the activity of the ScFv was dependent on Y331 sulfation. ScFv was also able to activate TMD mutants, D578Y and A593P, reemphasizing the role of TM helix VI in LHR activation.

PloS one, 2012

The mechanism by which the hinge regions of glycoprotein hormone receptors couple hormone binding... more The mechanism by which the hinge regions of glycoprotein hormone receptors couple hormone binding to activation of downstream effecters is not clearly understood. In the present study, agonistic (311.62) and antagonistic (311.87) monoclonal antibodies (MAbs) directed against the TSH receptor extracellular domain were used to elucidate role of the hinge region in receptor activation. MAb 311.62 which identifies the LRR/Cb-2 junction (aa 265-275), increased the affinity of TSHR for the hormone while concomitantly decreasing its efficacy, whereas MAb 311.87 recognizing LRR 7-9 (aa 201-259) acted as a non-competitive inhibitor of Thyroid stimulating hormone (TSH) binding. Binding of MAbs was sensitive to the conformational changes caused by the activating and inactivating mutations and exhibited differential effects on hormone binding and response of these mutants. By studying the effects of these MAbs on truncation and chimeric mutants of thyroid stimulating hormone receptor (TSHR), th...

Molecular cancer therapeutics, 2014

Aberrant activation of Notch and Ras pathways has been detected in breast cancers. A synergy betw... more Aberrant activation of Notch and Ras pathways has been detected in breast cancers. A synergy between these two pathways has also been shown in breast cell transformation in culture. Yet, the clinical relevance of Notch-Ras cooperation in breast cancer progression remains unexplored. In this study, we show that coordinate hyperactivation of Notch1 and Ras/MAPK pathways in breast cancer patient specimens, as assessed by IHC for cleaved Notch1 and pErk1/2, respectively, correlated with early relapse to vital organs and poor overall survival. Interestingly, majority of such Notch1(high)Erk(high) cases encompassed the highly aggressive triple-negative breast cancers (TNBC), and were enriched in stem cell markers. We further show that combinatorial inhibition of Notch1 and Ras/MAPK pathways, using a novel mAb against Notch1 and a MEK inhibitor, respectively, led to a significant reduction in proliferation and survival of breast cancer cells compared with individual inhibition. Combined in...

Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences, 2013

Iron(III) complexes [FeL(B)] ( 1 – 4 ) of a tetradentate phenolate-based ligand (H 3 L) and bioti... more Iron(III) complexes [FeL(B)] ( 1 – 4 ) of a tetradentate phenolate-based ligand (H 3 L) and biotin-conjugated dipyridophenazine bases (B), viz. 7-aminodipyrido [3,2- a :2′,3′- c ]-phenazine (dppza in 1 ), ( N -dipyrido[3,2- a :2′,3′- c ]-phenazino)amidobiotin (dppzNB in 2 ), dipyrido [3,2- a :2′,3′- c ]-phenazine-11-carboxylic acid (dppzc in 3 ) and 2-((2-biotinamido)ethyl) amido-dipyrido[3,2- a :2′,3′- c ]-phenazine (dppzCB in 4 ) are prepared, characterized and their interaction with streptavidin and DNA and their photocytotoxicity and cellular uptake in various cells studied. The high-spin iron(III) complexes display Fe(III)/Fe(II) redox couple near −0.7 V versus saturated calomel electrode in dimethyl sulfoxide–0.1 M tetrabutylammonium perchlorate. The complexes show non-specific interaction with DNA as determined from the binding studies. Complexes with appended biotin moiety show similar binding to streptavidin as that of free biotin, suggesting biotin conjugation to dppz does...

Molecular Cancer Therapeutics, 2011

Journal of Molecular Endocrinology, 1999

Human chorionic gonadotropin (hCG), a heterodimeric glycoprotein hormone, is composed of an alpha... more Human chorionic gonadotropin (hCG), a heterodimeric glycoprotein hormone, is composed of an alpha subunit noncovalently associated with the hormone-specific beta subunit. The objective of the present study was recombinant expression of properly folded, biologically active hCG and its subunits using an expression system that could be used for structure-function studies while providing adequate quantities of the hormone for immunocontraceptive studies. We report here expression of biologically active hCG and its subunits using a yeast expression system, Pichia pastoris. The recombinant hCGalpha and hCGbeta subunits were secreted into the medium and the levels of expression achieved at shake culture level were 24 and 2.7-3 mg/l secretory medium respectively. Co-expression of both subunits in the same cell resulted in secretion of heterodimeric hCG into the medium. The pichia-expressed hCG was immunologically similar to the native hormone, capable of binding to the LH receptors and stim...

Journal of Molecular Endocrinology, 2000

The strategy of translationally fusing the two subunits of human chorionic gonadotropin (hCG) has... more The strategy of translationally fusing the two subunits of human chorionic gonadotropin (hCG) has been used to produce recombinant single chain hCG in which the C-terminus of the alpha subunit is fused to the N-terminus beta without any linker using Pichia pastoris expression system. The Pichia-expressed hCGalphabeta (phCGalphabeta) attained an overall conformation similar to that of hCG, and could bind to the receptor and elicit biological response, suggesting that receptor binding and signal transduction can take place even with a molecule having blocked the C-terminus of the alpha subunit. The carboxyl terminal of the alpha subunit has been shown to be involved in hormone binding and signal transduction of all the heterodimeric glycoprotein hormones. However, deletion of five amino acids from the C-terminus of the alpha subunit in the single chain hCG did not alter the overall conformation of the fusion molecule and its receptor binding ability, but led to a significant reduction...

Journal of Molecular Endocrinology, 2005

Human chorionic gonadotrophin (hCG) is secreted during early pregnancy and is required for implan... more Human chorionic gonadotrophin (hCG) is secreted during early pregnancy and is required for implantation and maintenance of the pregnancy. Active or passive immunoneutralization of hCG results in termination of pregnancy and this forms the basis of the hCG-based female contraceptive vaccine. However, the β subunit of hCG possesses 85% sequence homology with the first 114 amino acids of the β subunit of pituitary human LH (hLH), which is required for ovulation and maintenance of the corpus luteum function during the menstrual cycle. Immunization against hCG or its β subunit leads to generation of antibodies that can neutralize hLH due to many shared epitopes and hence may cause abnormal menstrual cycles. Therefore, it is essential to identify epitopes that are different in the two hormones. In the present study, we report a monoclonal antibody (MAb) specific for hCG that shows no binding to the isolated subunits. Interestingly, the MAb also does not bind hLH at all. The epitope mappin...

Journal of Clinical Investigation, 1984

Journal of Biosciences, 1996

The relative regulatory roles of the pituitary gonadotropins, luteinizing hormone and follicle st... more The relative regulatory roles of the pituitary gonadotropins, luteinizing hormone and follicle stimulating hormone in the spermatogonial proliferation has been studied using specific antibodies against these hormones in the immature rats. Immunoneutralization of luteinizing hormone for 7 days resulted in significant reduction in tetraploid cells and total absence of haploid cells, while there was a relative increase in the diploid population. This was also accomopanied by a decrease in spermatogonial proliferation as indicated by a decrease in [ 3 H] thymidine incorporation into DNA by purified spermatogonia. Administration of follicle stimulating hormone a/s for 7 days also caused a significant decrease in the rate of spermatogonial proliferation. Withdrawal of follicle stimulating hormone led to a significant reduction in tetraploid and haploid cells. However interestingly, it failed to totally abolish the appearance of these cells. Administration of testosterone (3 mg/day/rat) for 2 days along with the gonadotropin a/s could partially reverse the effect on spermatogonial proliferation. It is concluded that (i) both luteinizing hormone and follicle stimulating hormone are involved in spermatogonial proliferation, (ii) lack of testosterone consequent of the neutralization of luteinizing hormone prevented the entry of spermatogonial cells into meiosis, (iii) testosterone may be involved in spermatogonia] proliferation providing a mitotic signal and (v) both follicle stimulating hormone and testosterone act synergistically and lack of any one of the hormones results in impairment of spermatogonial proliferation.

Journal of Biosciences, 1989

The relative ability of ovine follicle stimulating hormone and its β-subunit, two potential candi... more The relative ability of ovine follicle stimulating hormone and its β-subunit, two potential candidates for male contraceptive vaccine, to generate antibodies in monkeys capable of bioneutralizing follicle stimulating hormone was assessed using in vitro model systems. Antiserum against native ovine follicle stimulating hormone was found to be highly specific to the intact form with no cross-reactivity with either of the two subunits while the antiserum against β-subunit of follicle stimulating hormone could bind to the βsubunit in its free form as well as when it is combined with α-subunit to form the intact hormone. Both antisera could block the binding of the hormone to the receptor if the hormone was preincubated with the antibody. However, the follicle stimulating hormone β-antisera could only inhibit the binding of the hormone partially (33% inhibition) if the antibody and receptor were mixed prior to the addition of the hormone, while antisera to the native follicle stimulating hormone could block the binding completely (100% inhibition) in the same experiment. Similarly antisera to the native follicle stimulating hormone was significantly effective in blocking (100%) response to follicle stimulating hormone but not the β-subunit antisera (0%) as checked using an in vitro granulosa cell system. Thus the probability of obtaining antibodies of greater bioneutralization potential is much higher if intact hormone is used as an antigen rather than its β-subunit as a vaccine.