Long-term storage and safe retrieval of DNA from microorganisms for molecular analysis using FTA matrix cards.

We assessed the potential use of Whatman FTA paper as a device for archiving and long-term storage of bacterial cell suspensions of over 400 bacterial strains representing 61 genera, the molecular applications of immobilised DNA on FTA paper, and tested its microbial inactivation properties. The FTA paper extracted bacterial DNA is of sufficiently high quality to successfully carryout the molecular detection of several key genes including 16S rRNA, esp (Enterococcus surface protein), Bft (Bacteroides fragilis enterotoxin) and por (porin protein) by PCR and for DNA fingerprinting by random amplified polymorphic DNA-PCR (RAPD-PCR). To test the long-term stability of the FTA immobilised DNA, 100 of the 400 archived bacterial samples were randomly selected following 3 years of storage at ambient temperature and PCR amplification was used to monitor its success. All of the 100 samples were successfully amplified using the 16S rDNA gene as a target and confirmed by DNA sequencing. Furthermore, the DNA was eluted into solution from the FTA cards using a new alkaline elution procedure for evaluation by real-time PCR-based assays. The viability of cells retained on the FTA cards varied among broad groups of bacteria. For the more fragile gram-negative species, no viable cells were retained even at high cell densities of between 10(7) and 10(8) colony forming units (cfu) ml(-1), and for the most robust species such as spore-formers and acid-fast bacteria, complete inactivation was achieved at cell densities ranging between 10(1) and 10(4) cfu ml(-1). The inactivation of bacterial cells on FTA cards suggest that this is a safe medium for the storage and transport of bacterial nucleic acids.

Microbiological studies of an anaerobic baffled reactor: microbial community characterisation and deactivation of health-related indicator bacteria.

This WRC funded project has studied the appropriateness of the ABR (anaerobic baffled reactor) for on-site primary sanitation in low-income communities. A 3,000 L pilot reactor was located at the Kingsburgh wastewater treatment plant south of Durban, South Africa. Feed to the reactor was raw domestic wastewater containing a significant proportion of particulate organic matter. The compartments of the ABR were routinely monitored for pH, COD, and gas production, among other physical-chemical determinants. The microbial population in each compartment was analysed by fluorescent in situ hybridisation, using general oligonucleotide probes for eubacteria and archeae and a suite of 10 genera or family specific probes. Scanning electron microscopy was conducted on the sludge fraction of each compartment. Mixed fractions from each compartment were also analysed for health-related indicator bacteria (total coliforms and E. coli). Results indicated that methanogenesis was not occurring to the expected extent in the latter compartments, and that this was probably due to a hydraulic load limitation. This contrasted with earlier studies on industrial effluent, for which the organic load was exclusively in soluble form. Inactivation of health-related indicator bacteria was less than 1 log, indicating the need for an additional post-treatment of the effluent to protect community health.

Isolation of genomic DNA from blood using a novel filter-based DNA purification technology.

A novel DNA purification technology is described that enables the purification of pure dsDNA from blood. When compared to existing DNA purification methods, the Whatman BioScience Purification System (WBPS) offers a fast and convenient way to recover high yields of DNA. WBPS is based on a unique filter system that entraps DNA within a matrix. This allows the process to be performed in a single unidirectional reaction vessel, reducing user interaction and multiple centrifugation steps.

Postembryonic hematopoiesis in Drosophila.

We have investigated the blood cell types present in Drosophila at postembryonic stages and have analysed their modifications during development and under immune conditions. The anterior lobes of the larval hematopoietic organ or lymph gland contain numerous active secretory cells, plasmatocytes, few crystal cells, and a number of undifferentiated prohemocytes. The posterior lobes contain essentially prohemocytes. The blood cell population in larval hemolymph differs and consists mainly of plasmatocytes which are phagocytes, and of a low percentage of crystal cells which reportedly play a role in humoral melanisation. We show that the cells in the lymph gland can differentiate into a given blood cell lineage when solicited. Under normal nonimmune conditions, we observe a massive differentiation into active macrophages at the onset of metamorphosis in all lobes. Simultaneously, circulating plasmatocytes modify their adhesion and phagocytic properties to become pupal macrophages. All phagocytic cells participate in metamorphosis by ingesting doomed larval tissues. The most dramatic effect on larval hematopoiesis was observed following infestation by a parasitoid wasp. Cells within all lymph gland lobes, including prohemocytes from posterior lobes, massively differentiate into a new cell type specifically devoted to encapsulation, the lamellocyte.

Novel inducible antibacterial peptides from a hemipteran insect, the sap-sucking bug Pyrrhocoris apterus.

Insects belonging to the recent orders of the endopterygote clade (Lepidoptera, Diptera, Hymenoptera and Coleoptera) respond to bacterial challenge by the rapid and transient synthesis of a battery of potent antibacterial peptides which are secreted into their haemolymph. Here we present the first report on inducible antibacterial molecules in the sap-sucking bug Pyrrhocoris apterus, a representative species of the Hemiptera, which predated the Endoptergotes by at least 50 million years in evolution. We have isolated and characterized from immune blood of this species three novel peptides or polypeptides: (i) a 43-residue cysteine-rich anti-(Gram-positive bacteria) peptide which is a new member of the family of insect defensins; (ii) a 20-residue proline-rich peptide carrying an O-glycosylated substitution (N-acetylgalactosamine), active against Gram-negative bacteria; (iii) a 133-residue glycine-rich polypeptide also active against Gram-negative bacteria. The proline-rich peptide shows high sequence similarities with drosocin, an O-glycosylated antibacterial peptide from Drosophila, and also with the N-terminal domain of diptericin, an inducible 9 kDa antibacterial peptide from members of the order Diptera, whereas the glycine-rich peptide has similarities with the glycine-rich domain of diptericin. We discuss the evolutionary aspects of these findings.

Immunocytochemical localization of Bombyx-PTTH-like molecules in neurosecretory cells of the brain of the migratory locust, Locusta migratoria. A comparison with neuroparsin and insulin-related peptide.

Using a monoclonal antibody directed against a synthetic pentadecapeptide corresponding to the N-terminus of the prothoracicotropic hormone (PTTH) of Bombyx mori, we report the presence of immunoreactive molecules in a large number of median neurosecretory cells of the pars intercerebralis of the migratory locust, Locusta migratoria. These cells correspond to the A1 cell type which we show to contain also neuroparsins, a family of predominant neurohormones of the migratory locust. In contrast, PTTH-like molecules are absent from A2 cells of the pars intercerebralis which contain Locusta insulin-related peptide (LIRP). Developmental studies show the presence of PTTH-related substances in neurosecretory cells of Locusta migratoria from late embryogenesis to adult development, including ageing vitellogenic female adults.

Cortisol receptor resistance: the variability of its clinical presentation and response to treatment.

Primary (partial) cortisol receptor resistance was previously reported in a total of 7 patients and 14 asymptomatic family members. Its occurrence is considered to be extremely rare. In the present study we report on 6 patients (2 males and 4 females) with the syndrome. The first male patient presented with mild hypertension. Hydrochlorothiazide therapy resulted in life-threatening hypokalemia. The second male patient had slight hypertension without hypokalemia. All four female patients presented between the age of 20-30 yr with acne, hirsutism, and irregular menstruations. Low dose dexamethasone therapy (1-1.5 mg/day) was of clinical benefit in these patients. All patients showed insufficient suppression of serum cortisol concentrations in the overnight 1-mg dexamethasone test. The diurnal rhythm of ACTH and cortisol was intact, albeit at an elevated level. There was a normal increase in ACTH, cortisol, and GH (except in one obese patient) in response to insulin-induced hypoglycemia, while cortisol production was elevated in three patients. Circulating adrenal androgen levels were increased in all patients. Glucocorticoid receptors were investigated in a whole cell dexamethasone binding assay in mononuclear leukocytes. In the first male patient, the number of receptors was very low, while the affinity was lower than that in controls. A lowered affinity to dexamethasone was found in one female patient, while a lowered number of receptors was found in three patients. In the second male patient, no abnormalities were found. As a bioassay for glucocorticoid action we also measured dexamethasone suppressibility of mitogen-stimulated incorporation of [3H]thymidine in mononuclear leukocytes. In the male patient with normal receptor status, dexamethasone suppressibility of [3H]thymidine incorporation was significantly lower than that in healthy controls with respect to both maximal suppression and IC50. Partial cortisol receptor resistance might be less rare than previously thought. In the six patients presented, at least three different forms can be recognized. Therapy with dexamethasone was successful in female patients with acne and hirsutism, as the secondary increase in the production of adrenal androgens was effectively controlled.

Desensitization and resensitization of rat pituitary tumor cells in long-term culture to the effects of the somatostatin analogue SMS 201-995 on cell growth and prolactin secretion.

Cultured cells, prepared from the transplantable rat prolactin (rPRL)-secreting rat pituitary tumor 7315b were found to be inhibited in a dose-dependent way in their cell growth and hormone secretion by the somatostatin analogue SMS 201-995 (Sandostatin). In short-term (1 week) experiments these effects were not time dependent and of similar magnitude (an inhibition of approximately 50% at 100 nM SMA 201-995) both for the rate of rPRL secretion and for the rate of incorporation of tritiated thymidine into the tumor cells. When freshly isolated 7315b cells were used for long-term experiments (38 days), continuous exposure to SMS 201-995 at all concentrations tested (0.1 nM, 10 nM, and 1 microM) resulted in desensitization of the cells to the peptide with respect to rPRL secretion. Using a stable cell line derived from the long-term experiment and designated 7315c, we show that (a) long-term exposure of 7315c cells to SMS 201-995 leads to loss of sensitivity with respect to both rPRL secretion and cell growth, (b) this loss of sensitivity is accompanied by complete disappearance of the somatostatin receptors from the cells, (c) withdrawal of treatment from desensitized cells leads to reappearance of receptors and of sensitivity to SMS 201-995, showing that selection for a non-receptor-bearing population was not the cause of desensitization, and (d) since these experiments were carried out with a pure population of 7315c cells the effects of SMS 201-995 are direct effects on these cells and not effects mediated by other cell or organ systems.

Effects of suramin on hormone release by cultured rat anterior pituitary cells.

Suramin is a polyanionic compound which has been used in the treatment of trypanosomiasis and acquired immunodeficiency syndrome (AIDS), while preliminary success has been reported in the treatment of cancer. However, suramin also causes adrenal insufficiency. We have previously reported that suramin selectively inhibited corticotropin (ACTH)-stimulated corticosterone release by dispersed adrenal cells in a dose-dependent manner via a direct interaction with the ACTH molecule. The present study was undertaken in order to investigate the effect of suramin on hormone release by dispersed rat anterior pituitary cells. Suramin at a concentration of 100 microM inhibited both basal and secretagogue-stimulated ACTH release by cells cultured in minimal essential medium (MEM) only, while it had no effect on ACTH release by cells cultured in MEM + 10% fetal calf serum (FCS) or MEM + 0.1% bovine serum albumin (BSA). In addition, suramin also caused a parallel decrease of prolactin (PRL) and growth hormone (GH) release by cells cultured in MEM only, suggesting a toxic, rather than a selective effect of suramin on anterior pituitary cells cultured in MEM only. In addition, suramin potentiated the effect of thyrotropin-releasing hormone (TRH) on PRL release by cells cultured in MEM + 10% FCS and suppressed the inhibitory effect of dopamine (DA) on PRL release by cells cultured in MEM + 10% FCS and in MEM + 0.1% BSA. Comparable suppressive effects of suramin on growth hormone-releasing hormone (GHRH)-stimulated and somatostatin (SRIH)-inhibited GH release were found in cells cultured in MEM + 0.1% BSA but not in cells cultured in MEM + 10% FCS.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the conditions determining the inhibitory effect of somatostatin on adrenocorticotropin, prolactin and thyrotropin release by cultured rat pituitary cells.

UNLABELLED: Somatostatin (SRIH) is a physiological inhibitor of growth hormone (GH) secretion, but its role in the regulation of adrenocorticotropic hormone (ACTH), prolactin (PRL) and thyroid-stimulating hormone (TSH) release is unclear. SRIH (1 pM to 1 microM) did not affect basal and corticotropin-releasing hormone (CRH)-stimulated ACTH release by normal rat pituitary cells cultured in medium with 10% fetal calf serum (FCS). In cells deprived of serum for 48 h, or preincubated with the glucocorticoid-receptor-blocking agent, RU 38486, CRH-stimulated ACTH release was significantly suppressed by 1 pM to 0.10 nM SRIH. Preincubation with 5 nM dexamethasone completely abolished this inhibitory effect of SRIH on ACTH release. PRL release by pituitary cells cultured in phenol red-free culture medium with 10% estrogen-stripped FCS showed a very low sensitivity to SRIH. Increasing concentrations of 10 and 50 pM and 1 nM estradiol made PRL release by these cells significantly less sensitive to 50 nM dopamine, whereas the sensitivity to SRIH increased to a similar extent. In all instances dopamine and SRIH together exerted additive inhibitory effects, the extent of which remained similar under all conditions. After a 2-hour incubation, thyrotropin-releasing hormone-stimulated TSH secretion was significantly suppressed by 100 nM and 1 microM SRIH only in cells cultured in medium with 10% hypothyroid serum, and not in cells cultured in medium with 10% FCS. Such a difference in the sensitivity of thyrotrophs to SRIH disappeared during longer incubation. CONCLUSIONS: (1) ACTH release by normal corticotrophs is only sensitive to SRIH in the absence of the physiological peripheral feedback regulation by glucocorticoids.(ABSTRACT TRUNCATED AT 250 WORDS)

Familial and iatrogenic cortisol receptor resistance.

A family is described in which the father, a daughter, and two sons had cortisol receptor resistance. Hirsutism, skull baldness, and menstrual irregularities existed only in the female patient, while all three males were asymptomatic. The syndrome was transmitted via a dominant autosomal trait. A lowered dexamethasone affinity and a lowered number of glucocorticoid receptors were detected on the peripheral mononuclear leukocytes of the female patient. Chronic administration of the glucocorticoid receptor-blocking agent mifepristone (RU 38486) to normal individuals resulted in resetting of the hypothalamo-pituitary-adrenal axis at a higher level, while the diurnal rhythm of cortisol and the responsiveness to corticotropin releasing factor remained present. In four postmenopausal women this resulted in a considerable increase in circulating androstenedione levels (and eventually of estradiol levels). This iatrogenic RU 38486-induced biochemical syndrome is similar and difficult to differentiate from familial cortisol receptor resistance. Among a group of patients with so-called "idiopathic hirsutism," four patients were recognized with elevated androstenedione and (slightly) elevated cortisol levels which showed abnormalities in the affinity and/or number of glucocorticoid receptors on their peripheral mononuclear leukocytes. Therefore it is hypothesized that the syndrome of (partial) glucocorticoid receptor resistance is far more common than currently thought, especially among the group of patients with so-called "idiopathic hirsutism."

The interrelationship between the effects of insulin-like growth factor I and somatostatin on growth hormone secretion by normal rat pituitary cells: the role of glucocorticoids.

Both insulin-like growth factor I (IGF-I) and somatostatin (SRIH) have been shown to directly inhibit GH release and the total GH content of cultured pituitary cells. In the present study we evaluated the interrelationship between the effects of a recombinant human IGF-I analog ([Thr59]IGF-I) and SRIH on GH release by cultured normal rat pituitary cells together with the effects of glucocorticoids. In all experiments anterior pituitary cells were preincubated for 24 h without or with IGF-I, SRIH, and/or dexamethasone. Thereafter, 24-h incubations without or with IGF-I, dexamethasone, SRIH, and GHRH were performed. Both IGF-I and SRIH inhibited basal and GHRH-stimulated GH release in a dose-dependent manner; the maximal inhibitory concentrations were 5 nM IGF-I and 10 nM SRIH. These concentrations inhibited basal and GHRH-stimulated GH release by 23% and 40% (IGF-I) and 80% and 85% (SRIH), respectively. The combination of IGF-I and low concentrations of SRIH exerted an additive inhibitory effect on GHRH-stimulated GH release; IGF-I (1 nM) and SRIH (10 pM) together inhibited GH release by 50%, while the maximal inhibitory concentrations of 5 nM IGF-I and 10 nM SRIH virtually completely inhibited GH release (by 93%). Preincubation with 5 and 100 nM dexamethasone attenuated the sensitivity of somatotrophs to SRIH and completely abolished the inhibitory effects of IGF-I. This effect of dexamethasone could be reversed by coincubation with the glucocorticoid receptor antagonist RU 38486. High concentrations of 5-10 nM of the recombinant human IGF-I analog stimulated PRL cell content (5 and 10 nM) and release (10 nM), while a purified IGF-I preparation extracted from human blood exerted a parallel inhibitory effect on GH and PRL release. We conclude that 1) IGF-I and SRIH exert an additive direct inhibitory effect on basal and GHRH-stimulated GH secretion by normal cultured pituitary cells; 2) glucocorticoids directly attenuate the sensitivity of somatotrophs to SRIH, but completely prevent the inhibitory effects of IGF-I on GH secretion; and 3) in contrast to a purified IGF-I preparation extracted from human blood (which inhibits GH and PRL release) high concentrations of the recombinant IGF-I preparation (which inhibit GH release) stimulate PRL production.

[Vascular perfusion of an intestinal segment in the catfish (Ictalurus melas, R): demonstration of the vasoactive effects of mammalian VIP and of gastrointestinal extracts from teleost fish]. / Perfusion vasculaire d'un segment intestinal chez le poissonchat (Ictalurus mélas, R.): démonstration des effets vasoactifs du VIP de mammifère et d'extraits de tractus gastro-intestinal de Poisson téléostéen.

Through the middle mesenteric artery and the hepatic portal vein it is possible to perfuse specifically a small part of the catfish midgut. Using a tested VIP extraction procedure it is shown that trout and catfish extracts of oesophagus, stomach, midgut and hindgut induce a vasodilation similar to that of porcine VIP. The importance of the vasodilatory response depends on the level of perfused VIP and extracted tissue.

The vasotocin-like biological activity present in the bovine pineal is due to a compound different from vasotocin.

The vasotocin-like biological activity detected in an extract (E5 fraction) of bovine pineal gland was found not to be due to the presence of vasotocin, vasopressin or oxytocin. The data obtained by means of bio- and radioimmunoassays suggest that the peptide responsible for this biological activity, however, possess the same Pro-Arg-Gly(NH2) tripeptidic carboxy-terminal end as vasotocin.

[Radio-immunologic determination of hypophyseal vasotocin by Phoxinus laevis during experimental modification of osmotic equilibrium]. / Dosage radio-immunologique de la vasotocine hypophysaire chez Phoxinus laevis au cours de modifications expérimentales de l'équilibre osmotique.

Hypophysial vasotocin of Phoxinus laevis is determined by radio-immunoassay. With different sea water dilutions it is shown that hypophysial vasotocin concentration varies with ambient salinity. This fact agrees with histological pictures. Moreover, in comparison with the results of other authors, it is established that the hypophysial morphological modifications appeared only after the hormonal changes.