Effects of a foliar fertilizer containing boron on the development of Sclerotinia stem rot (Sclerotinia sclerotiorum) on canola (Brassica napus L.) leaves

Sclerotinia stem rot (Sclerotinia sclerotiorum Lib. De Bary) is one of the most destructive fungal diseases on canola (Brassica napus L.). The effect of a foliar fertilizer containing 3% boron (Active Flower™ [AF]) in reducing disease severity was evaluated. AF at 0.1, 0.3 and 0.5 ml/100 ml was first tested for growth inhibition of S. sclerotiorum in potato dextrose broth. Growth was reduced at 0.5 ml/100 ml by around 90%. Boric acid (BA), an important component of AF, was tested against fungal growth at 10 ml/L, and no significant effect (p = .05) was found. Foliar applications of AF and AF formulation that did not contain boron at 0.1, 0.3 and 0.5 ml/100 ml were made weekly to canola ‘Westar’ grown under greenhouse conditions. Treatments were also made with BA at 10 ml/L to canola plants. After four applications, AF at 0.5 ml/100 ml and BA at 10 ml/L enhanced boron levels in leaves by fivefold and threefold, respectively, compared with the control. Lesion size of S. sclerotiorum on detached leaves was significantly (p < .05) reduced by AF at 0.5 ml/100 ml, but lesion size was not reduced on AFWB-treated leaves. Experiments were repeated twice with the same results. Levels of phenolic compounds in leaves treated with 0.5 ml/100 ml AF were enhanced by twofold compared with the control. There were no significant differences in lignin, peroxidase (POD) or polyphenoloxidase (PPO) between the control and AF treatments. These results suggest that enhanced boron levels in canola leaves were associated with a suppressive effect on disease due to S. sclerotiorum.     

A Method for Chromosome Preparation of Sea Urchin Embryos

Chromosome preparations of the sea urchin Clypeaster japonicus were made from early embryos. To obtain well spread chromosomes in air dried preparations, fertilization membranes were removed, embryos treated with colcemid (0.1-2.0 μg/ml) were dissociated into their component cells by Ca-Mg-free sea water, and dissociated cells were swollen with hypotonic (0.5 M) KCl. Thanks to adequate spreading of the cytoplasm, only the chromosomes stained well with Giemsa.     

Thyroid stimulating hormone enhancement of bovine oocyte maturation in vitro.

Efforts to improve bovine embryonic development in vitro involved study of effects of thyroid stimulating hormone (TSH) alone or in combination with LH on bovine oocyte maturation (IVM). Putative effects were assessed by observing cumulus expansion (CE), fertilization (IVF), and development to morulae/blastocysts (M/B). Effects of prolactin (PRL) were also investigated. Variables for the 24-hr IVM interval were no hormone (control), TSH (0.1, 0.5, or 1.0 micrograms/ml) or PRL (10, 100, or 1000 micrograms/ml), luteinizing hormone (LH) (0, 10, or 100 micrograms/ml) + TSH (0.1 or 0.5 micrograms/ml), and serum (20%, v/v) + 0.5 micrograms TSH/ml; data were from 4-5 trials for each IVM treatment. Higher proportions of oocytes exhibited complete CE with hormones or serum than without (P less than 0.05). All oocytes (with and without CE) were inseminated with heparin-capacitated sperm. A higher proportion of inseminated oocytes cleaved after IVM with 0.5 micrograms TSH/ml (53.4%) than for other TSH treatments (P less than 0.05). The combination of TSH (0.1 and 0.5 micrograms/ml) with 10 micrograms LH/ml for IVM enabled higher proportions (P less than 0.05) of ova to fertilize (67.4 and 69.2%) than did medium alone (28.3%), LH (10 micrograms/ml) alone (54.1%) or serum + 0.5 micrograms TSH/ml (55.6%). No improvement in proportions undergoing fertilization was seen after addition of TSH to 100 micrograms LH/ml for IVM. Frequency of CE and cleavage did not differ among PRL treatments. More M/B developed from cleaved ova after IVM with LH or TSH than with PRL or no hormone (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma glucagon-immunoreactive components in early life in dogs

High plasma concentrations of C-terminal immunoreactive glucagon (IRG) have been found during early life in several mammalian species. We have analyzed the plasma IRG of 12 h to 60 day-old dogs in terms of the 4 peaks (IRG greater than 20,000, IRG9000, IRG3500 and IRG2000) obtained by gel filtration on Bio-Gel P-30. Significant changes with age and in response to administered agents were confined to IRG9000 and IRG3500. IRG9000 was 9-fold higher in 12-36 h old dogs than in adults (108 +/- 24 pg/ml pancreatic glucagon equivalents v. 12 +/- 3 pg/ml, mean +/- SEM) and showed a decline to 2-fold higher (27 +/- 5 pg/ml) at 31-60 days. IRG3500 was higher than in the adult only during the first 36 h of life (36 +/- 5 pg/ml v. 15 +/- 3 pg/ml). Arginine infusion (0.5 g/kg over 15 min) caused an increase in plasma levels of both IRG9000 and IRG3500 in the newborn, whereas in adult dogs only IRG3500 was increased. Insulin injection (0.2 U/kg intravenously) causing a marked hypoglycemia had no significant effect on the plasma level of any IRG component in newborn dogs. Dihydrosomatostatin infusion (10 micrograms/kg bolus +/- 90 micrograms/kg over 30 min) caused a decrease in both IRG9000 and IRG3500. The increased basal level and secretory response to arginine of IRG9000 in newborn dogs may reflect an immaturity of the A cells, whereby more of this component, which may represent a precursor of pancreatic glucagon, is secreted than in the adult. The immature A cells also appear to have an impaired secretory response to hypoglycemia.     

Plasma progesterone levels during the estrous cycle of Holstein and Brahman cows, Carora type and cross-bred heifers

Daily plasma progesterone (P(4)) was determined during one estrous cycle of 19 cows and 18 heifers of four different breeds: Holstein (H), Brahman (B), Carora-type (C) and crossbred (CB) females. Estrus detection was made by visual observation and using a teaser bull with a chin-ball marker. The P(4) profiles showed no differences among groups. In Group 1 (H), P(4) levels ranged from 0.5 ng/ml plasma on the day of estrus (Day 0) to 5.1 ng/ml at the luteal phase peak (Day 13). In Group 2 (B), P(4) levels ranged from 0.5 ng/ml on Day 0 to 9.2 ng/ml on Day 13. In Groups 3 (C) and 4 (CB), P(4) levels ranged from 0.5 ng/ml, on Day 0, to 13.7 ng/ml on Day 12 and 8.8 ng/ml on Day 13. These last two groups were moved to the same location and then compared. It was found that P(4) concentrations were significantly higher (P < 0.025) in Group 3 between Days 7 and 14 of the estrous cycle. In all groups, P(4) levels were lower than 1 ng/ml one day before the next estrus, and levels of 0.4, 0.5, 0.4 and 0.4 ng/ml were obtained the day of estrus in Groups 1 to 4, respectively. Results indicated that the pattern of P(4) for each one of the groups was similar to those reported by other investigators.     

天然药物对牙龈卟啉单胞菌的体外抗菌研究

目的 观察天然药物对牙龈卟啉单胞菌（Porphyromonas gingivalis,P.gingivalis）生长的影响,筛选抗菌作用最强的天然药物。方法 选用7种天然药物,采用液体稀释法,结合吸光度测定以确定7种天然药物对P.gingivalis的最小抑菌浓度（MIC）与最小杀菌浓度（MBC）。结果 黄芩、三七、白芷、大黄、五倍子、血藤和川芎对P.gingivalis的MIC值分别为1、1、0.5、0.0156、1、0.5、0.125 mg/ml;MBC值分别为1、1、0.5、0.0625、1、1、0.25mg/ml。结论 7种天然药物对P.gingivalis的生长均表现出明显的抑制作用,大黄的抑菌作用最强,川芎其次。     

An analytical method with a single extraction procedure and two separate high performance liquid chromatographic systems for the determination of artesunate, dihydroartemisinin and mefloquine in human plasma for application in clinical pharmacological studies of the drug combination

The combination of two sensitive, selective and reproducible reversed phase liquid chromatographic (RP-HPLC) methods was developed for the determination of artesunate (AS), its active metabolite dihydroartemisinin (DHA) and mefloquine (MQ) in human plasma. Solid phase extraction (SPE) of the plasma samples was carried out on Supelclean LC-18 extraction cartridges. Chromatographic separation of AS, DHA and the internal standard, artemisinin (QHS) was obtained on a Hypersil C4 column with mobile phase consisting of acetonitrile-0.05 M acetic acid adjusted to pH 5.2 with 1.0M NaOH (42:58, v/v) at the flow rate of 1.50 ml/min. The analytes were detected using an electrochemical detector operating in the reductive mode. Chromatography of MQ and the internal standard, chlorpromazine hydrochloride (CPM) was carried out on an Inertsil C8-3 column using methanol-acetonitrile-0.05 M potassium dihydrogen phosphate adjusted to pH 3.9 with 0.5% orthophosphoric acid (50:8:42, v/v/v) at a flow rate of 1.00 ml/min with ultraviolet detection at 284 nm. The mean recoveries of AS and DHA over a concentration range of 30-750 ng/0.5 ml plasma and MQ over a concentration of 75-1500 ng/0.5 ml plasma were above 80% and the accuracy ranged from 91.1 to 103.5%. The within-day coefficients of variation were 1.0-1.4% for AS, 0.4-3.4% for DHA and 0.7-1.5% for MQ. The day-to-day coefficients of variation were 1.3-7.6%, 1.8-7.8% and 2.0-3.4%, respectively. Both the lower limit of quantifications for AS and DHA were at 10 ng/0.5 ml and the lower limit of quantification for MQ was at 25 ng/0.5 ml. The limit of detections were 4 ng/0.5 ml for AS and DHA and 15 ng/0.5 ml for MQ. The method was found to be suitable for use in clinical pharmacological studies.     

Characterization of epithelial cell cultures derived from human tracheal glands

Summary Cultures of normal human tracheal gland epithelial cells that exhibit functional differentiation have been propagated in serum-free medium supplemented with insulin (5 μg/ml), epidermal growth factor (10 ng/ml), hydrocortisone (0.5 μg/ml), and bovine pituitary extract (25 μg/ml). The cells retain many characteristics of epithelial cells including microvilli on cell surfaces, desmosomes between cells, and tonofilaments in the cytoplasm. In addition, they exhibit keratin-positive titers and react positively with Peanut agglutinin, which is specific for the disaccharide β-d-galactose-(l→ 3)N-acetyld-galactosamine, a major component of mucin glycoprotein. The cells also exhibit normal Cl⁻ channel activity which was enhanced by the cAMP agonist Forskolin. The major component of the cellular secretion was hyaluronic acid; approximately 10% of the void volume material was resistant to hyaluronidase and may contain material similar to mucin glycoprotein. Some of the cell cultures have been maintained in serum-free conditions for 6 to 7 passages. This model will be important to study regulation of ion-channel activities and mucous glycoprotein secretion and to compare such regulations with the tracheal mucosal epithelial cells already established. This research was supported by USPHS grants HL 41979 and HL 33142 from the National Heart, Lung and Blood Institutes.     

Effects of inhibitors of integrin binding on cellular outgrowth from bovine inner cell masses in vitro

Summary Bovine inner cell masses (ICM) cultured on fibronectin give rise to extensive cellular outgrowths containing endoderm. Peptides with the Glu-Ile-Leu-Asp-Val (EILDV) and Arg-Gly-Asp (RGD) sequences inhibit cell migration on fibronectin by binding to the fibronectin-recognition site in several integrins. To identify integrins involved in endodermal cell outgrowth on fibronectin and vitronectin, the effects of the EILDV and RGD peptides were evaluated in vitro. In experiment 1, ICM were cultured on fibronectin in medium containing 0.5 or 1.0 mg/ml EILDV or RGD (or both). Compared with 0 mg/ml, 0.5 mg/ml EILDV suppressed (P<0.10) outgrowth area overall, and 1.0 mg/ml EILDV reduced (P<0.05) outgrowth area after 72 h of culture. Compared with 0 mg/ml, 0.5 and 1.0 mg/ml RGD reduced (P<0.05) outgrowth area after 72 h of culture. Plasminogen activator activity in conditioned medium increased (P<0.05) in 0.5 mg/ml RGD but decreased (P<0.10) in 1.0 mg/ml RGD compared with 0 mg/ml RGD. In experiment 2, bovine ICM were cultured on vitronectin in medium containing 0.5 or 1.0 mg/ml RGD. Neither concentration of RGD (P>0.10) affected the extent of cellular outgrowth on vitronectin. Bovine endodermal cell migration on fibronectin can be modulated by the RGD and EILDV peptides. Despite inhibition, neither peptide completely prevented outgrowth on fibronectin. In contrast, cellular outgrowth on vitronectin was unaffected by RGD. The persistence of cellular outgrowth on fibronectin and the absence of inhibition by RGD for ICM cultured on vitronectin suggests that bovine endodermal cells can use alternative cellular adhesion systems, such as nonintegrin receptors, during outgrowth.     

Micronuclei in blood lymphocytes and genetic polymorphism for GSTM1, GSTT1 and NAT2 in pesticide-exposed greenhouse workers.

The frequency of micronuclei (MN) in cultured peripheral lymphocytes was used as a biomarker of genotoxic effects in 34 Italian pesticide-exposed greenhouse workers and 33 unexposed referents matched with the exposed workers for age and smoking habits. The possible influence of the genetic polymorphisms of xenobiotic metabolizing enzymes glutathione S-transferase M1 (GSTM1), T1 (GSTT1), and N-acetyltransferase 2 (NAT2) was also evaluated. To restrict the analysis primarily to cells that have divided once in vitro, MN were scored only in cells showing label after a 42-h incubation with bromodeoxyuridine (BrdU), as detected by immunofluorescence (anti-BrdU technique). Two different concentrations of BrdU (0.5 and 1 microg/ml) were compared. Individual frequencies of micronucleated cells (MNCs) obtained with the two concentrations of BrdU significantly correlated with each other (r=0.55, P<0.001). Higher mean MNCs frequencies (per 1000 cells) were detected among exposed smokers (9.0 at 0.5 microg/ml BrdU and 7.8 at 1 microg/ml BrdU) than in smoking referents (6.3 and 5.9, respectively). In multiple regression analysis controlling for age, sex, smoking and genotypes, a significant elevation of MNC frequency (P=0.004 at 1 microg/ml BrdU; P=0.052 at 0.5 microg/ml BrdU) was observed in greenhouse workers with a work history of extensive pesticide spraying (n=17). Increased MNC frequencies were also associated with ageing at 0.5 microg/ml BrdU, with the GSTM1-positive genotype at both 1 (P=0.028) and 0.5 (P=0.056) microg/ml BrdU in all subjects, and with the NAT2 fast acetylator genotype in smokers at 0.5 microg/ml BrdU (P=0.043). The results indicate that MN rates are increased in greenhouse workers, especially in those involved in pesticide spraying. The GSTM1 positive and NAT2 fast genotypes appear to be associated with elevated MNC frequencies, which contradicts with earlier results on elevated chromosomal aberration rates in GSTM1 null smokers and NAT2 slow subjects.     

Analysis of chromosome aberrations and sister-chromatid exchanges in human lymphocytes exposed in vitro to Hydergine.

Hydergine (dihydroergotoxine mesylate, Sandoz) was examined for its capability to induce chromosome damage and sister-chromatid exchanges (SCEs) in human lymphocyte in vitro. For the chromosome-aberration study, cultures set up from 6 individuals were divided into 5 groups: negative control, positive control (caffeine, 0.5 mg/ml), and Hydergine (0.1, 0.25 and 0.5 micrograms/ml). For the SCE examination, which used 8 individuals, 4 cultures were made per person in the following way: negative control, positive control (mitomycin C, 0.1 microgram/ml), and Hydergine (0.1 and 0.5 micrograms/ml). Lymphocytes were cultivated for 72 h, being exposed to the respective treatments during the final 24 h. The results showed that Hydergine induced no chromosome damage in human lymphocytes in vitro.     

尖核和杏仁核在脑啡肽和β—内啡肽的释放中的相互作用

The functional relations between nucleus accumbens and amygdala were investigated with intracranial microinjection, push-pull perfusion and radioimmunoassay in the rabbit. Microinjection of morphine 20 micrograms into nucleus accumbens increased the immunoreactive (ir) enkephalin content in amygdala perfusate from a control level of 0.43 +/- 0.43 fmol/0.5 ml (normal saline group) to 61.6 +/- 16.3 fmol/0.5 ml (P less than 0.01); and ir-beta-endorphin content from 1.88 +/- 0.98 fmol/0.5 ml to 4.80 +/- 1.12 fmol/0.5 ml (P less than 0.05). On the other hand, microinjection of morphine into amygdala increased the release of ir-enkephalins (2.41 +/- 1.41 vs 34.6 +/- 8.4, P less than 0.01) and ir-beta-endorphin (1.79 +/- 0.64 vs 5.58 +/- 1.39 P less than 0.05) in the perfusates of N. accumbens. The results indicate the existence of reciprocal reinforcement of opioid release between the two nuclei, which may take part in a putative positive feedback mechanism in the cerebral analgesic system.     

Effect of Gynostemma Pentaphyllum Polysaccharide on boar spermatozoa quality following freezing–thawing

Gynostemma Pentaphyllum Polysaccharide (GPP) was added at concentrations of 0.25, 0.5, 1.0, 1.5 and 2.0 mg/ml to the extenders used to freeze boar semen and its effects on the quality of frozen–thawed sperm were assessed. The sperm motility was significantly higher in the extenders containing 0.25 and 0.5 mg/ml GPP, as compared to other groups (P < 0.05). The extender supplemented with 0.5 mg/ml GPP favored the highest intact membrane and intact acrosome percentages in comparison with other groups (P < 0.05), respectively. The mitochondrial activity was significantly higher at the concentrations of 0.25, 0.5 and 1.0 mg/ml GPP than that of other treatments, and the control group (P < 0.05). In biochemical assays, the extender supplemented with 0.25 and 0.5 mg/ml GPP significantly improved SOD levels, compared to other groups (P > 0.05). However, the extenders supplemented with GPP did not cause significant differences in levels of CAT and GSH-Px, compared to the control (P > 0.05). In summary, GPP exhibited a dose-related response and the lower concentration produced greater protective effect. According to the standard semen quality parameters and antioxidant activities measured in this study, the concentration of 0.5 mg/ml GPP caused a beneficial cryoprotective effects on the quality of frozen–thawed boar semen. It is proposed that an extender containing 0.5 mg/ml GPP could be used as cryoprotective medium of better efficiency.     

Binding proteins for sweet compounds from gustatory papillae of the cow, pig and rat

The intensely sweet proteins thaumatin and monellin were covalently attached to affinity column supports. Lingual tissue extracts were incubated with the affinity columns which were then eluted with glycine-HCl pH 3.4, the sweet peptide aspartame, or gymnemic acid, which is a sweet taste modifier. SDS-PAGE analysis of eluates from the columns showed that 156 kDa and 47 kDa proteins were the main components from cow fungiform papillae which were specifically bound to thaumatin and monellin. These proteins could be displaced from the column with 0.5 mM aspartame or 0.5 mg/ml gymnemic acid. With circumvallate papillae small amounts of 47 kDa protein were also found. The 47 kDa protein was also the major component bound to a gymnemic acid affinity column and could be displaced from the column with 0.5 mg/ml gymnemic acid. Control experiments with other lingual tissue components indicated that these proteins are localised in the gustatory papillae. Similar protein patterns were also found in extracts of pig fungiform papillae and rat lingual preparations.     

Stage of ovarian follicular development associated with the initiation of steroidogenic competence in avian granulosa cells.

Previously described models for avian ovarian steroidogenesis, using mature, 25-40-mm preovulatory follicles as the source of tissues, were based on the assumption that interaction of the granulosa layer, as the predominant source of progesterone, with adjacent theca cells is required for maximal production of C21, C19, and C18 steroids. In the present study, we evaluated the steroidogenic capacity of ovarian cells isolated from less mature, 6-8-mm and 9-12-mm follicles in the chicken ovary (representative of a stage of development 2-3 wk prior to ovulation) to determine at which stage of follicular development granulosa and/or theca cells become steroidogenically competent. Granulosa cells collected from 6-8-mm follicles were found to be virtually incompetent to produce steroids, containing extremely low basal levels of progesterone (12 pg/5 x 10(5) cells) and failing to respond with increased steroid output following a 3-h exposure to ovine LH (oLH; 0.1 and 100 ng/0.5 ml), ovine FSH (oFSH; 100, 500, and 1,000 ng/0.5 ml), 8-bromo-cyclic adenosine monophosphate (8-bromo-cAMP; 0.33 and 3.33 mM) or 25-hydroxycholesterol (250 and 2,500 ng/0.5 ml). However, addition of pregnenolone (20 and 200 ng/0.5 ml) to granulosa incubations resulted in significantly increased progesterone levels. Granulosa cells of 6-8-mm follicles also failed to increase cAMP formation in the presence of oLH (10, 100, and 1,000 ng/0.5 ml) and 3-isobutyl-1-methylxanthine (IBMX; 10 microM), but responded to stimulation with 1,000 ng oFSH (4.4-fold increase over basal) or 10 microM forskolin (32-fold increase over basal) in the presence of IBMX. In contrast, granulosa cells isolated from 9-12-mm follicles and incubated for 3 h in vitro were found to contain basal progesterone levels 200-fold higher than those found in granulosa cells of 6-8-mm follicles. Furthermore, granulosa cells of 9-12-mm follicles markedly increased progesterone production following incubation in the presence of oFSH (100-1,000 ng/0.5 ml), 8-bromo-cAMP (0.33 and 3.33 mM), or 25-hydroxycholesterol (250 and 2,500 ng/0.5 ml). However, these granulosa cells remained unresponsive to oLH (0.1, 10, and 100 ng/0.5 ml), failing to increase cAMP accumulation (in the presence of IBMX) and progesterone output. Theca cells of small yellow follicles were found to produce measurable basal levels of progesterone, androstenedione, and estradiol, and levels of each steroid were significantly increased following a 3-h challenge with oLH, 8-bromo-cAMP, 25-hydroxycholesterol, and pregnenolone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Opioid action on luteinizing hormone secretion in newborn pigs: paradoxical effect of naloxone

German Landrace piglets, 6-7 days of age, received either saline (9 males, 8 females), 0.5 mg naloxone/kg body weight (7 males, 7 females), 2.0 mg naloxone/kg (7 males, 8 females) or 0.5 mg DADLE (potent leu-enkephalin analog)/kg (7 males, 7 females) through a catheter inserted into the jugular vein 2-4 days previously. Male or female piglets were allocated randomly, within litter, to the different experimental groups. Blood samples were withdrawn for a period of 240 min at 10-min intervals for the first 60 min following injection and at 20-min intervals for the rest of the test period. Piglets were separated from their mother via a detachable wall and were allowed to suckle every 50 min. DADLE failed to alter plasma levels of LH in both males and females. Naloxone induced a significant (P less than 0.01) decrease in LH concentrations in females 10 to 60 min after injection (saline: 2.3 +/- 0.2 ng/ml plasma (SEM); 0.5 mg naloxone/kg: 1.0 +/- 0.2 ng/ml plasma and 2 mg naloxone/kg 1.2 +/- 0.4 ng/ml plasma). In males low doses of naloxone reduced plasma LH levels 10 to 40 min after injection (saline: 2.0 +/- 0.3 ng/ml plasma and 0.5 ng naloxone/kg: 1.1 +/- 0.3 ng/ml), whereas a decrease in plasma LH levels occurred 80 to 140 min after injection of high doses of naloxone (saline: 2.1 +/- 0.2 ng/ml and 2 mg naloxone/kg: 1.0 +/- 0.2 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of calcium on somatostatin inhibition of insulin release and cyclic AMP production in mouse pancreatic islets.

The effect of somatostatin on glucose-induced insulin secretion and cyclic AMP accumulation in isolated islets from obese, hyperglycemic ob/ob mice was studied in a microperifusion system. The normal biphasic pattern of insulin release as well as the inhibitory pattern of insulin release produced by somatostatin (0.5--1 microgram/ml) was matched by similar changes in the intracellular concentration of cyclic AMP. When islets were stimulated by glucose (3 mg/ml) plus 3-isobutyl-1-methylxanthine (0.1 mM), somatostatin (0.5 microgram/ml) failed to inhibit insulin secretion or cyclic AMP formation in the second phase whereas in the first phase both parameters were significantly reduced by somatostatin (0.5 microgram/ml). In batch-type incubations it was shown that addition of excess calcium (to 6 mM) reversed this inhibition. In the second phase calcium potentiated the (glucose + 3-isobutyl-1-methylxanthine)-stimulated insulin secretion without affecting the cyclic AMP production. This potentiation was inhibited by somatostatin (0.1 microgram/ml). Somatostatin (1 microgram/ml) inhibited adenylate cyclase activity in islet homogenates. No effect of somatostatin on islet glucose utilization could be demonstrated. The results indicate a dual action of somatostatin in the inhibition of insulin release, one involving the islet adenylate cyclase and one affecting the islet uptake of calcium.     

Endocrine changes during pregnancy, parturition and the early post-partum period in the llama (Lama glama)

Mean (+/- s.d.) pregnancy length for the 14 llamas in this study was 350 +/- 4.5 days. Plasma progesterone concentrations increased by 5 days after mating and remained elevated (greater than 2.0 ng/ml) throughout most of pregnancy. At about 2 weeks before parturition, plasma progesterone concentrations began to decline, dropped markedly during the final 24 h before parturition, and returned to basal concentrations (less than 0.5 ng/ml) by the day of parturition. The combined oestrone + oestradiol-17 beta and oestradiol-17 beta concentrations varied between 6 and 274 pg/ml and 4 and 114 pg/ml, respectively, during the first 9 months of pregnancy. Concentrations increased between 9 months after mating and the end of pregnancy with peak mean concentrations of 827 +/- 58 (s.e.m.) pg oestrone + oestradiol-17 beta/ml (range: 64-1658) and 196 +/- 10 pg oestradiol-17 beta/ml (31-294) during the last week of pregnancy. Concentrations then declined to 87 +/- 14 pg oestrone + oestradiol-17 beta/ml (7-488) and 25 +/- 5 pg oestradiol-17 beta/ml (2.5-142) during the first week post partum. Plasma cortisol concentrations varied between 2.6 and 51.9 ng/ml (14.0 +/- 0.5) from mating until 2 weeks before parturition when the concentrations began to decline. Only a slight increase in plasma cortisol concentrations was observed in association with parturition. Plasma triiodothyronine concentrations varied between 0.5 and 4.5 ng/ml (1.9 +/- 0.1) throughout pregnancy and the periparturient period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of testosterone in saliva and blow of bottlenose dolphins (Tursiops truncatus) using liquid chromatography-mass spectrometry

A rapid, accurate and reproducible assay utilising high performance liquid chromatography-mass spectrometry (LC-MS) has been developed and validated for determining testosterone concentrations in saliva and blow of bottlenose dolphins. Sample preparation used solid phase extraction with specific preconditioning of cartridges. Analytes were eluted with 100% acetonitrile, dried under nitrogen and stored at -80 degrees C. Samples were reconstituted in 60% acetonitrile for LC-MS analysis. Chromatographic separation was achieved with an Alltech Macrosphere C8 stainless steel analytical column (2.1 mm x 150 mm i.d., 5 microm particle size, 300 angstroms pore size) using a 55% mobile phase B isocratic method (mobile phase A = 0.5% acetic acid; mobile phase B = 0.5% acetic acid, 90% acetonitrile). Samples were analysed in SIM at m/z 289.20 (testosterone mw 288.40) and a positive ion ESI. The limit of quantification was 0.5 ng/ml with a limit of detection of 0.2 ng/ml. The concentration curve was linear from 0.5 to 50 ng/ml (y = 0.01x + 0.0045, r(2) = 0.959, r = 0.979, p < 0.001). The R.S.D.s of intra- and inter-batch precision were less than 15% for saliva and 11% blow. Recovery of the assay for saliva was 93.0 +/- 7.9% (50 ng/ml) and 91.5 +/- 3.72% (1 ng/ml), and for blow was 83.3 +/- 6.8% (50 ng/ml) and 85.8 +/- 4.6% (1 ng/ml). Recovery of the internal standard in saliva was 73.0 +/- 14.2% and in blow was 78.63 +/- 4.29. The described assay was used to determine the presence of endogenous testosterone in saliva (9.73-23 ng/ml, n = 10) and blow (14.71-86.20 ng/ml, n = 11) samples of captive bottlenose dolphins.