Feasibility of the immunohistochemical detection of endogenous steroids in paraffin-embedded ovarian tumours

Summary Formalin-fixed, paraffin-embedded samples from 25 ovarian granulosa cell tumours and six poorly differentiated ovarian carcinomas examined immunohistochemically for the presence of estradiol, testosterone and progesterone. Twenty-four of the 25 granulosa cell tumours stained for estradiol predominantly in granulosa cells and also in the theca cells, but none of the carcinomas were positive. All the granulosa cell tumours and five out of six carcinomas were positive for progesterone, while 13 granulosa cell tumours and three carcinomas stained moderately positive for testosterone.No clear-cut relationship was observed between positivity for estradiol in granulosa cell tumours and occurrence of endometrial hyperplasia. In two cases of granulosa cell tumours, 1–15% cells showed ultrastructural features of steroid synthesis: i.e. moderate to abundant smooth endoplasmic reticulum and mitochondria with tubular cristae. This is in contrast with the diffuse immunohistochemical staining reaction of granulosa cell tumours with antisteroid antibodies.To evalutate the significance of immunohistochemical for estradiol, four estradiol-containing tumours were tested by radioimmunoassay for the presence of estradiol before and after dehydration. As expected, treatment with organic solvent leads to a drastic reduction in estradiol content below the detection level of the assay. The result indicate that immunohistochemical staining for steroids of paraffin-embedded granulosa cell tumours if of limited value.     

Culture conditions effecting plant regeneration from cotyledons of Vigna radiata (L.) Wilczek

Conditions for plant regeneration from excised cotyledons of Vigna radiata were studied. Complete plant developed from the uncallused proximal ends of cotyledons on Murashige & Skoog's (MS), Gamborg's (B₅) and C (MS salts + B₅ vitamins) basal media. The basal medium C was found to be best for plant regeneration. Regeneration frequency, however, varied with genotype, size, orientation and age of explant and the different plant growth regulators combination in the medium. Addition of cytokinins induced callusing at the proximal ends of cotyledons followed by multiple shoot formation. Out of 6-benzyl aminopurine (BAP), kinetin (KIN), N (^–2 isopentyl) adenine (2iP) and adenine sulphate (AS), only BAP and KIN were found to be more effective in enhancing the frequency of shoot regeneration. BAP at 1×10^-1M induced maximum (60%) shoot regeneration whereas maximum number of shoots (8 to 9 shoots) per explant was observed with 5×10^-6M BAP. Cotyledons excised from two-day old seedlings were most regenerative. The regenerative response of cotyledons decreased when sliced into two equal parts either longitudinally or transversely. Callusing and organogenic differentiation occured only if the petiolar end of cotyledons was in contact with medium. None of the tested treatments were effective in inducing shoot bud differentiation from subcultured callus. Well developed shoots rooted when incubated on half strength MS, MS and MS basal medium supplemented with IAA (5×10^-6M). The rooted plants were transferred to pots and later established in the field with 60% success.Abbreviations AS adenine sulphate - BAP 6-benzylaminopurine - B₅ medium after Gamborg et al. [6], - C Medium with MS salts + B₅ vitamins - 2iP N (^–2 isopentyl) adenine - IAA indole-3-acetic acid - KIN Kinetin - MS medium after Murashige & Skoog [21] - NAA 1-napthaleneacetic acid     

Water-soluble macromolecular fluorogenic substrates for assaying proteinases: determination of pancreatic elastase activity.

Water-soluble macromolecular fluorogenic substrates were synthesized in order to develop an easy specific proteinase assay. The validity of this method was tested with porcine pancreatic elastase by using its specific peptidic substrate Ala-Ala-Pro-Ala linked to a hydrosoluble polymer. The octapeptidic sequence FTC-epsilon Aca-Ala-Ala-Pro-Ala-Gly-Gly-Gly was linked to a water-soluble and neutral poly-L-lysine derivative. The aminocaproyl residue and the triglycyl sequence were added in order to improve the stability of the substrate, and the accessibility of the specific sequence Ala-Ala-Pro-Ala to elastase, respectively. The assay is based on the quantitative precipitation of the polymeric substrate in isopropanol while the released soluble fluorescent peptidic moiety is fluorometrically titrated in the supernatant.     

Maternal Magnesium Restriction Elevates Glucocorticoid Stress and Inflammation in the Placenta and Fetus of WNIN Rat Dams

Magnesium plays a major role in many vital functions in the body. We reported earlier that maternal magnesium restriction altered body composition, fat metabolism, and insulin resistance in WNIN rat offspring and was associated with increased glucocorticoid stress in the offspring in their later life. We hypothesize that increased glucocorticoid stress and inflammation which originate in Mg restricted rat dams is transmitted through placenta to the fetus and underlie the metabolic disturbances in the later life of the offspring. Female weanling WNIN rats received ad libitum, a control diet (MgC) or the same with 62% restriction of Mg (MgR) for 3 months, and their plasma magnesium, inflammatory cytokines, and corticosterone were determined (n = 6 per group) before mating. Following mating with control males, placentae, and fetuses were collected on gestational day 15 (GD 15) from MgC and MgR dams (eight dams from each group and three samples from each dam) and used to determine the levels of inflammatory cytokines, corticosterone, and expression of relevant genes. MgR placentae and fetuses had higher (than MgC) levels of corticosterone and proinflammatory cytokines. Expression of Hsd11b1 was increased (sixfold, p < 0.05), while that of Hsd11b2 was decreased (0.4-fold, p < 0.05) in MgR (than MgC) placenta, whereas expression of Hsd11b1was increased (3.4-fold, p < 0.05) in MgR fetus. Chronic dietary magnesium restriction in WNIN female rats increased their levels of corticosterone, leptin, and proinflammatory cytokines which appeared to be transmitted through placenta to the fetus and could thus be associated with increased stress, altered body composition, fat metabolism, and insulin resistance in the later life of the offspring.     

Comparison of developmental stages of inflorescence for high frequency plant regeneration in Triticum aestivum L. and T. durum Desf.

Summary Whole immature inflorescences at 4 different developmental stages (0.5, 1.0, 1.5, 2.0 cm in size) of different genotypes of Triticum aestivum and T. durum were cultured to see the morphogenetic responses on Murashige and Skoog's (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (2.5 mg/l). Very young inflorescences 0.5 and 1.0cm long formed embryogenic callus from their entire surface while 1.5 and 2.0 cm long inflorescences formed embryogenic callus from the basal spikelets and rachis only. This embryogenic callus was maintained by regular subcultures on MS medium with 2,4-D (2.5 mg/l) for more than a year. Plantlets were regenerated by transferring the embryogenic callus on hormone-free MS medium. Inflorescences (0.5 and 1.0 cm long) responded best in forming callus as well as plantlets at a very high frequency. Variation in response was observed amongst the genotypes but the qualitative response of formation of embryogenic callus and later regeneration of plantlets was observed from all the genotypes. Immature young inflorescence explants could provide a suitable material for particle gun mediated genetic transformation in wheat.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige and Skoog (1962)     

Actinomyces in cervical smears of women using intrauterine contraceptive devices

Cervical smears from 1,784 women who attended the family planning clinics of the Institute for Research in Reproduction were examined for the presence of Actinomyces-like organisms. Among 815 intrauterine contraceptive device (IUD) users, the repeat smears from 57 women were positive for Actinomyces-like organisms, giving a prevalence rate of 6.99%. Immunofluorescent staining using specific antisera confirmed the presence of A. israelii in all 57 women. Forty IUD users whose smears were positive for Actinomyces-like organisms underwent bacteriologic culture studies; A. israelii was isolated in 23 of these cases. The clinical findings at the time of smear collection in the 57 IUD users were within normal limits. The initial cervical smears of all IUD users and both the initial and repeat smears of all nonusers were negative for Actinomyces-like organisms. The data indicate that prolonged use (greater than 2 years) of an inert or copper intrauterine device promotes the overgrowth of Actinomyces in the vagina and that this can be detected by routine cervical cytology.     

Synthesis of prebiotic molecules — Role of some carbonyl compounds in prebiotic chemistry

Methyleneaminoacetonitrile(MAAN) resulting from the interaction of formaldehyde, ammonia and hydrogen cyanide on hydrolysis under mildly alkaline conditions gives a number of amino acids and peptides. Various aldehydes react with glycine to give corresponding hydroxyalkyl amino acids, which on reduction with formic acid are converted to reduced amino acids. Formaldehyde reacts with uracil to give 5-hydroxymethyl uracil which on reduction with formic acid yields thymine. Pyrrole formed by heating serine reacts with aldehydes to form porphyrins. Clays do not seem to influence most of these reactions, except the uracil-formaldehyde — formic acid reaction which results in enhanced yield of thymine.     

RGS2 inhibits β-adrenergic receptor-induced cardiomyocyte hypertrophy

The chronic stimulation of certain G protein-coupled receptors promotes cardiomyocyte hypertrophy and thus plays a pivotal role in the development of human heart failure. The beta-adrenergic receptors (β-AR) are unique among these in that they signal via Gs, whereas others, such as the alpha1-adrenergic (α1-AR) and endothelin-1 (ET-1) receptors, predominantly act through Gq. In this study, we investigated the potential role of regulator of G protein signalling 2 (RGS2) in modulating the hypertrophic effects of the β-AR agonist isoproterenol (ISO) in rat neonatal ventricular cardiomyocytes. We found that ISO-induced hypertrophy in rat neonatal ventricular myocytes was accompanied by the selective upregulation of RGS2 mRNA, with little or no change in RGS1, RGS3, RGS4 or RGS5. The adenylyl cyclase activator forskolin had a similar effect suggesting that it was mediated through cAMP production. To study the role of RGS2 upregulation in β-AR-dependent hypertrophy, cardiomyocytes were infected with adenovirus encoding RGS2 and assayed for cell growth, markers of hypertrophy, and β-AR signalling. ISO-induced increases in cell surface area were virtually eliminated by the overexpression of RGS2, as were increases in α-skeletal actin and atrial natriuretic peptide. RGS2 overexpression also significantly attenuated ISO-induced extracellular signal-regulated kinases 1 and 2 (ERK1/2) and Akt activation, which may account for, or contribute to, its observed antihypertrophic effects. In contrast, RGS2 overexpression significantly activated JNK MAP kinase, while decreasing the potency but not the maximal effect of ISO on cAMP accumulation. In conclusion, the present results suggest that RGS2 negatively regulates hypertrophy induced by β-AR activation and thus may play a protective role in cardiac hypertrophy.