Interactions of culture vessels, media volume, culture density, and carbon dioxide levels on lettuce and spearmint shoot growth in vitro.

The influence of culture chamber capacity, medium volume and culture density on the growth yields of lettuce (Lactuca sativa L.) and spearmint (Mentha spicata L.) shoots were determined in an environment containing either 350 or 10,000 µmol mol-1 CO2 after 8 weeks of incubation. High positive correlations occurred between the culture vessel capacity and spearmint fresh weight, leaf number, root number, and shoot number. Similarly, high positive correlations occurred between culture vessel capacity and lettuce fresh weight, leaf number, and root number. Higher fresh weights, leaf numbers, and root numbers were obtained from lettuce and spearmint shoots when cultured in 1-quart Mason jars containing 100- or 150-ml aliquots of medium compared to jars containing 25- or 50-ml aliquots of medium within an environment containing either 350 or 10,000 µmol mol-1 CO2. High culture density decreased growth yields, and this phenomenon could only be slightly off-set by the employment of an elevated CO2 environment or larger culture vessels.

The pineal and extra-pineal origins of 5-sulphatoxy N-acetyl-serotonin in humans.

In humans 6-sulphatoxy melatonin (SaMT) is the principal metabolite of endogenous and exogenous melatonin. 5-sulphatoxy N-acetyl-serotonin (SNAS) is a minor metabolite of exogenous melatonin, but it has not been established whether the levels of endogenous SNAS in plasma derives principally from endogenous melatonin. We have developed the first radioimmunoassay (RIA) for SNAS and used it (together with RIAs for melatonin and SaMT) to determine whether endogenous SNAS derives from endogenous melatonin or from platelet serotonin. Our results show a) the values of endogenous SNAS, unlike endogenous SaMT, increased with blood collection procedures that increased the values of serotonin, b) the values of endogenous SNAS in urine or in platelet-poor plasma were approximately the same as those of endogenous SaMT, but, unlike SaMT, did not show a diurnal rhythm, and c) we confirmed that SNAS was a minor metabolite of orally ingested melatonin. Thus, our conclusion is that SNAS is a minor metabolite of exogenous melatonin, but is not a significant metabolite of endogenous melatonin. In all probability, endogenous SNAS is principally the metabolite of platelet serotonin.

Elevated nocturnal melatonin is a consequence of gonadotropin-releasing hormone deficiency in women with hypothalamic amenorrhea.

Elevated nocturnal melatonin is found in women with idiopathic hypogonadotropic hypogonadism (IHH), but it is not known whether this is implicated in the etiology of their GnRH deficiency. It is unlikely that nocturnal melatonin can be implicated in the etiology of the GnRH deficiency of Kallmann's syndrome (KS), because this condition is caused by defective neuronal migration in embryonic life. We therefore measured nocturnal melatonin in women with IHH and KS to determine whether it was elevated in one or both conditions and thereby to determine whether it was implicated as cause or consequence of GnRH deficiency. Four women with IHH, 3 women with KS, and 7 individually matched (age and body size) controls were recruited. Frequent day- and nighttime samples were taken for LH pulsatility studies. All patients showed absent or diminished LH pulsatility, compared with their respective controls. Samples were also taken over 24 h for melatonin and 6-sulphatoxymelatonin (the principle metabolite of melatonin and an independent marker of its secretion). Melatonin and 6-sulphatoxymelatonin levels were elevated in 6 of 7 patients (compared with their matched controls) and were significantly elevated in the KS group (compared with their controls). The finding of elevated nocturnal melatonin (and its metabolite) in GnRH-deficient women with KS (as well as IHH) suggests that nocturnal melatonin is elevated as a consequence of GnRH deficiency, irrespective of its etiology.

First trimester maternal serum concentrations of fetal antigen 2 in normal pregnancies and those affected by trisomy 21.

Serum concentrations of fetal antigen 2 (FA-2), the amino-propeptide of the alpha1 chain of collagen type I, were measured in peripheral blood from women with normal (n = 234) and trisomy 21 affected (n = 14) pregnancies between 9 and 11 weeks gestation. Serum FA-2 concentrations were seen to be stable throughout this period, and though raised FA-2 concentrations were seen at the 10th week of gestation, a statistically significant difference between normal and trisomy 21 affected pregnancies was not found overall. Therefore it seems unlikely that FA-2 has a role in first trimester screening for trisomy 21, despite the fact that significantly higher FA-2 concentrations in trisomy 21 and significantly lower concentrations in trisomy 18 had been previously demonstrated in amniotic fluid in the second trimester.

Radioimmunoassay of bound and free melatonin in plasma.

We describe a nonextraction procedure, and two extraction procedures, for RIA of melatonin in human plasma. All procedures showed a diurnal rhythm of melatonin in human subjects, with interindividual differences greater than interprocedure differences. However, further investigations demonstrated considerable variability of recovery in the nonextraction procedure, suggesting a variability of binding proteins between samples. Combining recovery and dialysis experiments in the extraction procedures, we demonstrated that chloroform was unable to extract albumin-bound melatonin from a human serum albumin solution but, paradoxically, was able to extract bound and free melatonin from a plasma sample. The methanol extraction procedure extracted free and bound melatonin from all sources. These results indicate that albumin binding can substantially affect the RIA procedures. We conclude that assays should be validated against free and bound melatonin and that the two forms should be independently investigated when assessing bioactivity.

An enzyme immunoassay for 6-sulphatoxy-melatonin in human urine.

We describe a newly developed enzyme immunoassay (EIA) for the determination of 6-sulphatoxy-melatonin (aMT6s) in human urine, using a aMT6s-bovine serum albumin-horseradish peroxidase (aMt6s-BSA-HRP) conjugate as the enzyme label. The assay incorporates a highly specific antibody raised in rabbits. The EIA has a sensitivity of 2 pg/well (40 pg/ml) with intraassay coefficients of variation of 2.3-6.1% in the range of the assay. The material with the highest level of cross-reactivity was N-acetyl serotonin sulphate, with a relative potency of 0.000078%. One hundred thirty-four urine samples from children and adults at different time points were assayed and the results compared with those from an established radioimmunoassay (RIA) and with a newly developed RIA using the same antibody as the EIA. The correlation coefficient, r, comparing the two RIA's was 0.9869, and the regression equation was log (kit) = 0.9340 log (new) + 0.1213. The correlation coefficient, r, comparing the EIA with the newly developed RIA, was 0.9686, and regression equation log (new) = 0.9674 log (EIA) + 0.0600. The EIA for the measurement of aMT6s in urine represents a new approach in the investigation of pineal function.

The purification and characterization of biological 6-sulphatoxymelatonin and comparison with synthetic 6-sulphatoxymelatonin.

We have purified the major metabolite of melatonin, 6-sulphatoxymelatonin, from urine and compared it to its synthetic counterpart. For preparation of the biological material, oral melatonin was administered to human volunteers and their urine extracted onto Amberlite XAD-2 resin to remove urea; the glucuronide metabolites of melatonin were removed by silica chromatography; and 6-sulphatoxymelatonin was separated from N-acetyl serotonin sulphate, the other sulphate metabolite of melatonin, by preparative thin-layer chromatography. Synthetic 6-sulphatoxymelatonin was produced by reacting 6-hydroxymelatonin with chlorosulphonic acid in dimethylformamide; the reaction mixture was purified on Florisil and preparative thin-layer chromatography was used to remove indolic by-products of the reaction. Elemental and X-ray microanalysis of the biological and synthetic products showed that classical methods used for their purification introduced inorganic impurities, such as silicon- and chlorine-containing compounds, which were not detectable by thin-layer chromatography, infrared spectroscopy, nuclear magnetic resonance spectroscopy, or gas chromatography-mass spectrometry. We introduced further purification steps to remove these inorganic impurities, monitoring the process using elemental and X-ray microanalysis. Extensive characterization of the resulting purified products showed that the biological and synthetic compounds were identical.

Evidence for the Bauman variant in Kallmann's syndrome.

BACKGROUND AND OBJECTIVE: Idiopathic hypogonadotrophic hypogonadism (IHH) is a condition of gonadotrophin releasing hormone (GnRH) deficiency. IHH associated with anosmia is Kallmann's syndrome. A variant has been described by Bauman where a patient with Kallmann's syndrome apparently regained normal hypothalamo-pituitary function 2 years after the initial diagnosis. GnRH secretory activity can be assessed by measuring LH pulsatility. Our objective was to define the pattern of LH pulsatility in men with IHH and Kallmann's syndrome compared with those of normal controls, and to determine whether there is evidence for a Bauman variant of Kallmann's syndrome. DESIGN: Patients with IHH and Kallmann's syndrome were recruited from the endocrine clinic. Long-term hormone replacement therapy was discontinued. LH pulsatility was determined. PATIENTS: Three men with IHH, 3 men with classical Kallmann's syndrome and 5 normal male volunteers. MEASUREMENTS: Baseline serum FSH, LH and testosterone. Intensive blood sampling every 10 minutes for serum LH from 1000 to 1600 h during the day and 2200 to 0400 h during the night to measure LH pulsatility. RESULTS: The volunteer group showed normal LH pulsatility. In the patient group, LH secretion was apulsatile in one, showed significantly diminished amplitude in four, and there was normal pulsatility in one patient which remained normal 5 months later. CONCLUSION: Three patients with idiopathic hypogonadotrophic hypogonadism and 2 with Kallmann's syndrome had variable degrees of GnRH deficiency. One patient with Kallmann's syndrome had apparently normal GnRH activity, which remained normal 5 months later. This patient appears to have the Bauman variant of Kallmann's syndrome.

Abnormal amniotic fetal antigen 2 concentrations in trisomy 18 and trisomy 21.

Fetal antigen 2 (FA-2) is a human protein, first identified in amniotic fluid, and shown to be identical to the aminopropeptide of the alpha 1 chain of collagen type I. It exists in several different size and charge forms. In the present study, FA-2 was measured in amniotic fluid using two different assays: a rocket line immunoelectrophoretic assay which measured total FA-2, and a radioimmunoassay which was specific for the high molecular mass forms of FA-2. Both assays gave similar results. FA-2 concentrations were measured in amniotic fluid samples collected from normal pregnancies at 10-23 weeks gestation; they were shown to rise steeply from 10-14 weeks, peak at 17 weeks and then fall slightly by 23 weeks. Comparison between amniotic fluid from normal pregnancies and pregnancies affected by trisomy, showed significantly higher FA-2 concentrations in trisomy 21 and significantly lower concentrations in trisomy 18.

The typing of fetal antigen 2 in human amniotic fluid.

Fetal antigen 2 (FA-2) has been identified in amniotic fluid and shown to be of fetal origin. In this study we have extended previous observations on FA-2 heterogeneity with respect to both size and charge using gel filtration, ion-exchange chromatography, non-dissociating polyacrylamide gel electrophoresis and sodium dodecyl sulphate polyacrylamide gel electrophoresis. From the diversity of forms we have been able to define two principal FA-2 types, type A and type B. Type A has a high molecular mass (140 kDa), has subunits of 33 kDa and 29 kDa, and elutes at approximately 0.27 mol/l sodium chloride from diethylaminoethyl (DEAE)-Sephacel. Type B has the same mass and subunits as type A, but elutes at approximately 0.24 mol/l sodium chloride from DEAE-Sephacel. Other low molecular mass forms of FA-2 have also been identified. All FA-2 forms described were shown to be common to all amniotic fluid samples studied and were not attributable to artefacts of collection or storage. It was also demonstrated that the recently described FA-2 RIA is specific for FA-2 types A and B and the conversion of arbitrary units FA-2 into micrograms applies to type A. The typing is discussed with respect to (i) the aminopropeptide of the alpha 1 chain of human procollagen type I, (ii) the 24 kDa phosphoprotein in developing bone and (iii) fetal calf ligament protein 1 (FCL-1), suggesting that they are the same protein.

HPLC assay of melatonin in plasma with fluorescence detection.

We report an HPLC assay for melatonin that incorporates automated injection, methanol/water mobile phase, and fluorescence detection. Plasma samples were extracted by solid and liquid phases. Recovery was > 70% for 1-10 mL of plasma extracted, approximately 40 pg-250 ng of melatonin. Samples were dried and reconstituted in 100 mL/L methanol. Injections were 25 microL or 150 microL, depending on sample concentration, and the melatonin peak was eluted in 380 mL/L methanol. The detection limit of the assay was 6 pg on the column, allowing a practical sensitivity in plasma of 11 pmol/L for 8-mL samples and 34 pmol/L for 2-mL samples. More than 100 plasma samples from volunteers and patients were assayed and the results compared with an established RIA. The mean daytime concentration of melatonin was 20.7 pmol/L (SEM = 1.2) and 18.5 pmol/L (SEM = 1.6) for HPLC and RIA, respectively, and the mean nighttime concentration was 82.4 pmol/L (SEM = 6.5) and 82.2 (SEM = 7.3), respectively.

Melatonin: a contraceptive for the nineties.

The hypothalamic GnRH pulse generator activates the pituitary-gonadal reproductive axis, and contraceptive techniques have advanced to the point where GnRH analogues can block this effect. However, nature has an even finer form of contraception, whereby the GnRH pulse generator is activated or inactivated at different seasons of the year. Darkness affects the retino-pineal nervous pathway to cause the synthesis and release of melatonin from the pineal gland at night. The duration of the night time release of melatonin is longer in winter than in summer; and it is the prolongation in the duration of the night time release of melatonin, with the change of season from summer to winter, which acts as the endocrine signal for inactivating the hypothalamic GnRH pulse generator. Humans are not seasonal breeders, and evidence is presented to indicate that this is due to an impairment of the retino-pineal pathway rather than an impairment of melatonin hypothalamic function. Thus the way is open for utilising melatonin as a human contraceptive, and a melatonin-based contraceptive is at present undergoing phase III clinical trials. The challenge is to develop more refined methods for administering (or releasing) melatonin, so that it has a night time amplitude and duration which mimics that seen in long day breeders.

PIP: No contraceptive method which tricks nature by using nature's own method has yet been introduced. A prolonged release of melatonin from the pineal gland via the retina-pineal nervous pathway during the longer nighttime in winter operates as a signal to inactivate the hypothalamic gonadotropin releasing hormone (GnRH) pulse generator. Specifically, darkness activates the pathway. while light suppresses it. This process occurs in most mammals that are seasonal breeders. Yet, humans are not seasonal breeders. Scientists propose 2 possible reasons for the considerable variation in the nighttime release of melatonin in humans, which tends to be below the threshold needed to signal the GnRH pulse generator: an evolutionary phenomenon, or that humans are surrounded by artificial light. They have studied melatonin release in children before puberty and in women with functional hypothalamic amenorrhea to gain more insight. Increasing body mass and constant pineal production causes a reduced circulating level of melatonin to the point (during puberty) at which melatonin falls below a critical threshold and activates the GnRH pulse generator. Melatonin levels are significantly higher in functional hypothalamic amenorrheic patients than in normal controls. Phase III clinical trials of a melatonin-based contraceptive are occurring. At a dose of 75 mg melatonin, the maximum level of circulating melatonin is above 400,000 pmol/l, which maintains a nighttime level above the critical threshold for more than 12 hours. The strategies for developing more refined methods to mimic a nighttime amplitude and duration of long day breeders are likely a controlled release preparation or a method to activate N-acetyl transferase in the pineal body to synthesize and release melatonin at a sufficient level and duration.

Production of Colletotrichum truncatum for use as a mycoherbicide: effects of culture, drying and storage on recovery and efficacy.

Colletotrichum truncatum (Schwein.) Andrus and Moore NRRL 13737 (= NRRL 18434) is a fungal plant pathogen which shows promise as a bioherbicide against the troublesome weed Sesbania exaltata (Raf.) Rydb. ex A. W. Hill. Previous studies showed similar amounts of spores were produced/ml of medium in liquid and solid-state fermentations. In this study, Colletotrichum truncatum spores were produced in liquid (LC), solid/vermiculite (SV), and solid/perlite-cornmeal-agar (SP). After drying at room temperature with flowing air, SV and SP retained the most viability. Each product was then stored at 4 degrees, 15 degrees, and 25 degrees C. All three products stored at 4 degrees C and SP stored at 15 degrees C retained highest viability. Efficacy based upon assays utilizing equal numbers of viable spores showed SV and SP spores incited more severe disease symptoms than LC spores.

Cellular automata simulations of fungal growth on solid substrates.

Growth of filamentous fungi on the surface of cereal grains is a critical aspect of solid substrate fermentation (SSF). Numerous mathematical models have been developed to describe various aspects of fungal growth in SSF. These models consider hyphal geometry and nutrient availability as determinants of colony morphology and fungal physiological state. This work describes the use of cellular automata (CA) as an alternative method of modeling fungal growth. CA models reliant on a very limited set of rules or "knowledge base" display a rich array of behaviors that mimic fungal growth. By incorporating probablistic growth rules into CA models, colony characteristics such as biomass accumulation rate, colony radial growth rate, mycelial density and fungal differentiation are readily generated.