Urinary dehydroepiandrosterone sulphate excretion in women with idiopathic hirsutism.

Urinary dehydroepiandrosterone sulphate (DHAS) concentration was measured as DHA in 100 non-obese females with idiopathic hirsutism, while, at the same time, a similar number of normal males and normal females, of comparable age group and with normal body weight, were also included in this study. The method applied involved hydrolysis of DHA-sulphate conjugate after boiling the urine samples, followed by ether extraction of the free steroid, evaporation of ether, Girard T separation of ketones from non-ketones, paper chromatographic separation of DHAS from 6beta-hydroxy-3,5-cycloandrostan-17-one and, finally DHAS was estimated spectrophotometrically after Zimmermann reaction. For comparison, 30 urine samples were estimated by a g.l.c. procedure and good agreement was found with the spectrophotometric method. It was necessary to purify DHAS by paper chromatography before Zimmerman reaction was carried out as, in the same 30 urine samples, DHAS was consistently being overestimated. Urinary DHAS (mg/24 h) in normal males gave a mean of: 2.5+/-0.94 (SD), range: 0.35+/-4.8; in normal females, mean: 1.3+/-0.57 (SD), range: 0.28-2.6; in hirsute females, mean: 2.1+/-1.5 (SD), range: 0.65-9.7. Of these hirsute females, 26 were above the range found in normal women and mean values were higher than those from the same number of normal females of comparable age group; this was found to be significant (P less than 0.0005). These results indicate that urinary DHAS (as DHA) estimation in women with idiopathic hirsutism is clinically useful and could be applied as an index of androgen status in these women.     

Humidity‐controlled cold storage of Neoseiulus californicus (Acari: Phytoseiidae): effects on male survival and reproductive ability

Humidification can suppress water loss from an organism and has great potential for improving the cold storage of short‐lived arthropods, such as predatory mites. The effectiveness of humidity‐controlled cold storage was recently verified for Neoseiulus californicus (McGregor) females but was not examined for males. Combining both males and females in one storage protocol might increase the predator population because it would enhance the opportunity for multiple mating, which is necessary for females to maximize their egg production. Newly emerged adult males were stored at an air temperature of 5°C and relative humidity (RH) of 100% or 80%. The median survival time (LT₅₀) was 32 days at 100% RH and 14 days at 80% RH; the survival curves differed significantly. Males stored at 100% RH for 0, 10, 20 and 30 days were introduced to virgin females for mating at 25°C to evaluate their reproductive ability. The pre‐oviposition period was significantly prolonged in the females mated with males stored for ≥20 days. No negative effects of storage were observed on the oviposition period, total number of eggs or net reproduction rate (R₀) in the females mated with males stored for ≤20 days or on the mean generation time (T) for those stored for 30 days. A slight decrease in the intrinsic rate of increase (r_m) was observed in the females mated with males stored for ≥20 days. Our storage method can preserve N. californicus males for 20 days with only a minor reduction in their survival and reproductive ability.     

Non-invasive repeated measurement of urinary progesterone, 17beta-estradiol, and testosterone in developing, cycling, pregnant, and postpartum female mice

Excretory samples from adult female mice were collected non-invasively during development, estrous cycling, pregnancy, and postpartum. In initial studies, urinary measures were statistically more dynamic over days than were fecal measures; thus subsequent studies focused on urine. Higher 17beta-estradiol levels were present in isolated females than in those exposed to males. In cycling females, urinary 17beta-estradiol was more variable than were measures of testosterone or progesterone, showing peaks with an approximate 5-day periodicity. When urinary estradiol and progesterone were monitored in conjunction with vaginal smear cell counts, patterns were idiosyncratic; most females showed distinct peaks in urinary steroids, not in clear synchrony with vaginal cell cornification. Levels of progesterone rose markedly during the first 10 days of pregnancy, then declined before birth. Estradiol showed a substantial peak on days 7-8 of gestation in all females measured. Urinary testosterone was not dynamic during pregnancy, but rose in immediate prenatal and postpartum measures. During post-weaning, pre-pubertal development, urinary levels of progesterone remained constant but levels of estradiol rose substantially over time.     

Social suppression of ovarian cyclicity in captive and wild colonies of naked mole-rats, Heterocephalus glaber

To investigate the endocrine cause of reproductive suppression in nonbreeding female naked mole-rats, animals from 35 colonies were studied in captivity. Urinary and plasma progesterone concentrations were elevated in pregnant females (urine: 10.0-148.4 ng/mg Cr, 27 samples from 8 females; plasma: 3.6-30.0 ng/ml, 5 samples from 5 females; Days 21-40 of pregnancy) and cyclic breeding females (urine: 0.5-97.8 ng/mg Cr, 146 samples from 7 females; plasma: less than 1.0-35.4 ng/ml, 25 samples from 7 females). The latter group showed cyclic patterns of urinary progesterone, indicating a mean ovarian cycle length of 34.4 +/- 1.6 days (mean +/- s.e.m.) with a follicular phase of 6.0 +/- 0.6 days and a luteal phase of 27.5 +/- 1.3 days (19 cycles from 9 breeding females). In non-breeding females urinary and plasma progesterone values were undetectable (urine: less than 0.5 ng/mg Cr, 232 samples from 64 females; plasma: less than 1.0 ng/ml, 7 samples from 6 females). Breeding females had higher (P less than 0.001) plasma LH concentrations (3.0 +/- 0.2 mi.u./ml, 73 samples from 24 females) than did non-breeding females (1.6 +/- 0.1 mi.u./ml, 57 samples from 44 females). Urinary and plasma progesterone concentrations in non-breeding females from wild colonies situated near Mtito Andei, Kenya, were either below the assay sensitivity limit (urine: less than 0.5 ng/mg Cr, 11 females from 2 colonies; plasma: less than 1.0 ng/ml, 25 females from 4 colonies), or very low (plasma: 1.6 +/- 0.6 ng/ml, 15 females from 4 colonies). In captivity, non-breeding females removed from their colonies (i.e. the dominant breeding female) and either paired directly with a non-breeding male (N = 2), or removed and housed singly for 6 weeks before pairing with a non-breeding male (N = 5) may develop a perforate vagina for the first time in as little as 7 days. Urinary progesterone concentrations rose above 2.0 ng/mg Cr (indicative of a luteal phase) for the first time 8.0 +/- 1.9 days after being separated. These results suggest that ovulation is suppressed in subordinate non-breeding female naked mole-rats in captive and wild colonies, and show that plasma LH concentrations are significantly lower in these non-breeding females. This reproductive block in non-breeding females is readily reversible if the social factors suppressing reproduction are removed.     

Improvement in the long-term stability of freeze-dried mouse spermatozoa by adding of a chelating agent

This study demonstrates that a small amount of chelating agent in the freeze-drying solution is necessary to prevent the deterioration of spermatozoa during freeze-drying and subsequent preservation at 4 degrees C. We freeze-dried mouse epididymal spermatozoa in the solutions containing Tris-HCl and ethylenediaminetetraacetic acid (EDTA) as a chelating agent. Spermatozoa stored for various times up to 1 year at 4 degrees C were injected intracytoplasmically into individual oocytes, and the normality of chromosomes in fertilized oocytes was analyzed. In addition, embryos derived from freeze-dried spermatozoa were transferred into recipients to determine their developmental ability. Chromosomes were maintained well when spermatozoa were freeze-dried in a solution containing 10 mM Tris-HCl and 1mM EDTA (73%), and 57% of embryos developed to term. Of embryos derived from spermatozoa stored for 1 year, 65% developed into live offspring. On the other hand, when spermatozoa were freeze-dried in a solution containing 10 mM Tris-HCl and 0 or 50 mM EDTA, spermatozoa that maintained karyotypically normal chromosomes were 64% or 22%, and only 16% or 3% of embryos were developed to term, respectively. This finding suggested that mouse spermatozoa can be freeze-dried in a simple solution containing the same composition as that used to preserve extracted DNA.     

Affinity purification of DNA and RNA from environmental samples with peptide nucleic acid clamps

Bispeptide nucleic acids (bis-PNAs; PNA clamps), PNA oligomers, and DNA oligonucleotides were evaluated as affinity purification reagents for subfemtomolar 16S ribosomal DNA (rDNA) and rRNA targets in soil, sediment, and industrial air filter nucleic acid extracts. Under low-salt hybridization conditions (10 mM NaPO(4), 5 mM disodium EDTA, and 0.025% sodium dodecyl sulfate [SDS]) a PNA clamp recovered significantly more target DNA than either PNA or DNA oligomers. The efficacy of PNA clamps and oligomers was generally enhanced in the presence of excess nontarget DNA and in a low-salt extraction-hybridization buffer. Under high-salt conditions (200 mM NaPO(4), 100 mM disodium EDTA, and 0.5% SDS), however, capture efficiencies with the DNA oligomer were significantly greater than with the PNA clamp and PNA oligomer. Recovery and detection efficiencies for target DNA concentrations of > or =100 pg were generally >20% but depended upon the specific probe, solution background, and salt condition. The DNA probe had a lower absolute detection limit of 100 fg of target (830 zM [1 zM = 10(-21) M]) in high-salt buffer. In the absence of exogenous DNA (e.g., soil background), neither the bis-PNA nor the PNA oligomer achieved the same absolute detection limit even under a more favorable low-salt hybridization condition. In the presence of a soil background, however, both PNA probes provided more sensitive absolute purification and detection (830 zM) than the DNA oligomer. In varied environmental samples, the rank order for capture probe performance in high-salt buffer was DNA > PNA > clamp. Recovery of 16S rRNA from environmental samples mirrored quantitative results for DNA target recovery, with the DNA oligomer generating more positive results than either the bis-PNA or PNA oligomer, but PNA probes provided a greater incidence of detection from environmental samples that also contained a higher concentration of nontarget DNA and RNA. Significant interactions between probe type and environmental sample indicate that the most efficacious capture system depends upon the particular sample type (and background nucleic acid concentration), target (DNA or RNA), and detection objective.     

Effects of urinary proteins from certain leukemics upon macromolecular synthesis and enzyme levels in bone marrow cultures.

Urinary proteins from human leukemic patients have been found to alter quantitatively macromolecular synthesis in primary mouse bone marrow cultures. Urinary protein-stimulated incorporation of [3H]uridine into RNA was found after 1 day of culture. Increased levels of adenine phosphoribosyltransferase and lysozyme were demonstrable at 3 and 5 days, respectively, with urinary protein-supplemented cultures. The incorporation of 3H-labeled deoxynucleosides into DNA was higher in the presence of urinary proteins after 2 days of culture. The rate of incorporation of [3H]deoxyuridine into DNA was strongly inhibited by 10(-5) M Methotrexate and 10(-6) M 5-fluorodeoxyuridine, however, the effect of urinary proteins on incorporation of [3H]uridine into RNA and lysozyme accumulation were not inhibited. Urinary proteins also stimulated the formation of "colonies" (groups of at least 30 cells) in media containing methylcellulose. This latter phenomenon was also not inhibited by 10(-5) M Methotrexate or 10(-6) M 5-fluorodeoxyuridine. The results of these studies are consistent with the postulate that in the presence of human urinary proteins, mouse bone marrow cells in culture proceed to a phenotype characteristic of circulating peripheral white cells.     

Effects of testosterone on ontogeny of urinary behavior in male and female dogs

Development of urinary behavior from birth to adulthood was observed in six groups of beagles including normal males (NM), normal females (NF), males castrated soon after birth (CM), males castrated soon after birth and treated with testosterone (T) for the next 90 days (CMT), females exposed to T in utero (FTU), and females exposed to T in utero and during infancy, i.e., the first 30-40 days postpartum (FTUI). Prenatal treatment with T had masculinizing effects on juvenile urinary behavior in FTU and FTUI. On the other hand normal development of fully adult masculine urinary patterns in males and females necessitated both prenatal and postnatal androgenic stimulation. It was not necessary that T be present at the time the overt behavior developed. For example, adult male behavior appeared in FTUI at the same time as in NM, i.e., 6-10 months, although the supply of exogenous androgen in FTUI had been exhausted within 30-40 days after birth. CMT showed precocious development of all components of the adult male pattern. Development of adult responses was markedly retarded in most CM, and their performance did not equal that of NM at 23 months. They were then injected with TP which promptly evoked completely normal male urinary behavior. It is tentatively concluded that T acting before birth and during the juvenile period "prepares" critical CNS mechanisms so that when general maturation reaches the appropriate point adult male behavior develops. Although the preparatory role of T is essential, the behavior is not dependent on T after it has developed.     

Exposure to developing females induces polyuria, polydipsia, and altered urinary levels of creatinine, 17β-estradiol, and testosterone in adult male mice (Mus musculus)

Novel male mice can accelerate reproductive maturation in proximal developing females, an effect mediated by the chemistry of the males' urine. Exogenous estrogens can similarly accelerate female sexual development. In Experiment 1, adult male mice were housed across wire grid from either empty compartments or those containing post-weanling females. Proximity of females caused males to urinate more, progressively over days of exposure, with most urination directed towards females' compartments. Male urine collected after 5 days in these conditions was analyzed by enzyme immunoassay for 17β-estradiol, testosterone, and creatinine. Urinary creatinine of isolated males significantly exceeded that of female-exposed males. Unadjusted urinary steroids also trended toward higher levels in isolates, but creatinine-adjusted estradiol and testosterone of female-exposed males significantly exceeded that of isolated males. In Experiment 2, measurement of water consumption indicated significantly greater drinking by female-exposed as opposed to isolated males. In Experiment 3, males were housed in isolation or beside post-weanling intact (sham-operated) females, ovariectomized females, or intact (sham-operated) males. Male water consumption was elevated in all conditions involving social contact. Urinary creatinine was significantly lower in female-exposed males compared to isolated controls, while unadjusted testosterone was significantly lower in males in all social conditions. Again, creatinine-adjusted estradiol in female-exposed males significantly exceeded that of isolates. These data indicate that adult males drink and urinate more, have more dilute urine, and have a higher ratio of estradiol to creatinine when they are near developing females. These dynamics increase females' exposure to urinary steroids and other urinary constituents that can hasten sexual maturity.     

Affinity Purification of DNA and RNA from Environmental Samples with Peptide Nucleic Acid Clamps

Bispeptide nucleic acids (bis-PNAs; PNA clamps), PNA oligomers, and DNA oligonucleotides were evaluated as affinity purification reagents for subfemtomolar 16S ribosomal DNA (rDNA) and rRNA targets in soil, sediment, and industrial air filter nucleic acid extracts. Under low-salt hybridization conditions (10 mM NaPO₄, 5 mM disodium EDTA, and 0.025% sodium dodecyl sulfate [SDS]) a PNA clamp recovered significantly more target DNA than either PNA or DNA oligomers. The efficacy of PNA clamps and oligomers was generally enhanced in the presence of excess nontarget DNA and in a low-salt extraction-hybridization buffer. Under high-salt conditions (200 mM NaPO₄, 100 mM disodium EDTA, and 0.5% SDS), however, capture efficiencies with the DNA oligomer were significantly greater than with the PNA clamp and PNA oligomer. Recovery and detection efficiencies for target DNA concentrations of ≥100 pg were generally >20% but depended upon the specific probe, solution background, and salt condition. The DNA probe had a lower absolute detection limit of 100 fg of target (830 zM [1 zM = 10⁻²¹ M]) in high-salt buffer. In the absence of exogenous DNA (e.g., soil background), neither the bis-PNA nor the PNA oligomer achieved the same absolute detection limit even under a more favorable low-salt hybridization condition. In the presence of a soil background, however, both PNA probes provided more sensitive absolute purification and detection (830 zM) than the DNA oligomer. In varied environmental samples, the rank order for capture probe performance in high-salt buffer was DNA > PNA > clamp. Recovery of 16S rRNA from environmental samples mirrored quantitative results for DNA target recovery, with the DNA oligomer generating more positive results than either the bis-PNA or PNA oligomer, but PNA probes provided a greater incidence of detection from environmental samples that also contained a higher concentration of nontarget DNA and RNA. Significant interactions between probe type and environmental sample indicate that the most efficacious capture system depends upon the particular sample type (and background nucleic acid concentration), target (DNA or RNA), and detection objective.     

Trauma, especially of the submandibular glands, causes release of epidermal growth factor into bloodstream in mice

Submandibular glands in mice were traumatized by handling and then removed. Immunoreactive epidermal growth factor (EGF) in serum increased after 5 min and continued to increase, reaching at 1 h a peak of 50-fold normal in males and twice normal in females. If after traumatization the glands were repositioned with their blood supply intact, maximal increase of serum EGF at 1 h was 190-fold control values in males and 2-fold in females. In male mice, incision of abdominal wall skin led to a 15-fold increase of EGF in the serum; this rise was absent 3 days after sialoadenoectomy. After traumatization, repositioned submandibular glands lost 80% of their EGF; after the abdominal wall incision, only 30%. Following removal of submandibular glands, decrease of EGF level in serum was very slow: to 60% of the initial value after 3 days and to 40% after 10 days. By the HPLC characteristics, immunoreactive EGF in control serum and at its peak were indistinguishable. Urinary excretion of EGF was significantly elevated only when its serum level was 190-fold normal. We conclude that traumatized submandibular glands discharge into circulation a large part of their stored EGF. A similar but much less pronounced process takes place after abdominal skin incision. The presence of EGF in serum after its slow decline in sialoadenoectomized mice shows that a fraction of circulating EGF may recirculate prolonging its apparent half-life.     

Urinary endocrine monitoring of the ovarian cycle and pregnancy in Goeldi's monkey (Callimico goeldii)

A non-invasive study of urinary hormones in 6 captive female Goeldi's monkeys provided accurate information on reproductive function. Conjugated oestrone accounted for 80-85% of the urinary oestrone and oestradiol measured. Radioimmunoassay measurements of conjugated oestrone provided a reliable indicator of cyclic ovarian function (mean cycle length: 24.1 +/- 0.9 days; n = 9) and pregnancy (gestation: 145, 155 days; n = 2). Measurements of urinary progesterone and pregnanediol glucuronide were only reliable as indicators of ovarian cyclicity. Elevations in urinary conjugated oestrone coincided with luteal-phase elevations of urinary progesterone and pregnanediol glucuronide. Urinary LH concentrations provided no indication of pituitary activity. However, the frequencies of female sexual solicitations of males were maximal when oestrone conjugate concentrations rose, suggesting a peri-ovulatory period. Ovulation was suppressed in 1 of 3 subordinate females housed in male-female-female trios.     

Long-term preservation of freeze-dried mouse spermatozoa

Many genetically engineered mice strains have been generated worldwide and sperm preservation is a valuable method for storing these strains as genetic resources. Freeze-drying is a useful sperm preservation method because it requires neither liquid nitrogen nor dry ice for preservation and transportation. We report here successful long-term preservation at 4 °C of mouse spermatozoa freeze-dried using a simple buffer solution (10mM Tris, 1mM EDTA, pH 8.0). Offspring with fertility were obtained from oocytes fertilized with freeze-dried spermatozoa from C57BL/6 and B6D2F1 mouse strains stored at 4 °C for 3 years. This freeze-drying method is a safe and economical tool for the biobanking of valuable mouse strains.     

Development of the metanephric kidney. Protein and nucleic acid synthesis

The metanephric kidney was studied in fetal and older mice beginning at 16 days after mating of the parents. Polyribosomes from fetal kidneys labeled in vitro with 14C-labeled amino acids had 10-20 times more acid-precipitable radioactivity associated with them than polysomes from adult kidneys similarly labeled. Between 3 and 6 days after birth the rate incorporation of labeled amino acids by polyribosomes from neonatal kidneys declined sharply to only twice the value found for adult kidneys. There was no change in the shape of the polyribosome profile with increasing age, but before birth few, if any, ribosomes were bound to membranes compared with 20% 2 days after birth and between 20 and 30% in the adult. Total protein represented less than 10% of the wet weight in the fetal kidney but increased to 17% of the wet weight in the adult kidney. There was a steady decline in the concentration of RNA and DNA with respect to dry weight throughout kidney development. DNA concentration declined more rapidly than RNA concentration, so that the milligram to milligram ratio of RNA to DNA increased. In males the RNA/DNA ratio was stable at 1.3 at 40 days after birth; but in females the decline in DNA concentration was more protracted, and at 200 days after birth the RNA/DNA ratio was only 0.99. Thus, total nucleic acids show only gradual changes in concentration throughout development of the kidney, but a sharp change in the synthetic activity of the ribosomes and in their binding to membranes occurs in kidneys soon after birth.     

DNA isolation by a rapid method from human blood samples: Effects of MgCl2, EDTA,storage time,and temperature on DNA yield and quality

The isolation of DNA from whole blood by a modified rapid method (RM) was tested using various detergents and buffer conditions. Extraction of DNA with either NP-40 or Triton X-100 gave a high yield of undegraded DNA in less than an hour. The concentration of magnesium ion in the buffers was critical to obtaining intact, high molecular weight (HMW) DNA. Greater than 10 mM MgCl₂ led to degradation. Addition of EDTA to the buffer inhibits this degradation. Preparation of DNA from blood stored at room temperature or incubated at 37°C for 24 hr resulted in the same amount and quality of DNA as from samples frozen at −70°C. DNA from blood samples that had undergone more than four freeze-thaw cycles was found to be partially degraded. The modified RM can be applied to extract DNA from as little as 10 μl of blood (340 ng of DNA) and from dried blood samples. DNA samples remained intact and undegraded for longer times when DNA was dissolved in higher concentrations of EDTA. This work was supported by grants from the Indiana Department of Mental Health and PHS RO1 AG10297.     

Biodegradation of Naphthalene by Pseudomonas stutzeri in Marine Environments: Testing Cells Entrapment in Calcium Alginate for Use in Water Detoxification

The biodegradation of naphthalene in sea water by freely suspended and alginate-entrapped cells of Pseudomonas stutzeri 19SMN4 has been investigated in batch cultures. The results showed that immobilized cells can be stored at 4°C for 1 month without loss of viability. The biodegradation was highly affected by the availability of nitrogen and phosphorous, so at 30°C a naphthalene concentration of 25 mM was almost completely degraded (93%) by free cells in 6 days in samples supplemented with these nutrients, whereas only 42% naphthalene was consumed in the nonsupplemented samples. Biodegradation was much slower at 16°C than at 30°C; after 6 days of culture at 30°C, almost all naphthalene was degradated by free and immobilized cells, whereas only 22% and 34% at 16°C, respectively. The degradation rate remained unaffected when the naphthalene concentration was reduced from 25 to 10 mM. Alginate of three different viscosities was used for immobilization of cells. After 7 days of culture, beads formed with 31.4 cP alginate were fragmented, whereas beads formed with 240 and 3600 cP did not display structural changes and afforded the same degradation rate. Beads formed with high-viscosity alginate retained cells more efficiently.     

An optimized method for extraction and detection of Coconut cadang-cadang viroid(CCCVd) from oil palm

Coconut cadong-cadong viroid (CCCVd) causes the Lethal cadang-cadang disease of coconut palms in the Philippines and it is recently reported to be associated with the orange spotting disease on oil palm in Malaysia. The low concentration of the viroid RNA in oil palm as well as the high content of polyphenols and polysaccharides in this plant which interfere with the purification steps makes it difficult to extract and detect this viroid from oil palm. A previously described method was modified and optimized for extraction and detection of CCCVd from infected oil palms. Briefly, 7 g of leaf material was homogenized in a mortar or a blender using liquid nitrogen. 10 ml of extraction buffer (100 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA) along with 100 mM 2-mercaptoethanol and 10 ml water saturated phenol was added to the frozen powder. After centrifuging at 4 degrees C, 4000 g for 30 min, the aqueous phase was extracted once more with phenol then once with chloroform-isoamyl alcohol (24:1). After adding sodium acetate, pH 5.6 to 200 mM, the mixture was precipitated with 2.5 vol ethanol overnight in -20 freezer and then the pellet was washed with 70% ethanol and air-dried. One milliliter of 8 M LiCl was added to the dried pellet and after shaking overnight at 4 degrees C and another centrifugation step the supernatant was collected and precipitated again with ethanol and then the resulting pellet was washed and air-dried. To carry out northern blotting, samples equivalent to 40 g of plant tissue were mixed with formamide buffer and loaded onto a 12% polyacrylamide gel containing 7 M urea and after separation by electrophoresis, were electroblotted onto membrane and fixed by UV cross-linking. Pre-hybridization and hybridization using hybridization buffer (50% formamide, 25%SSPE, 0.1% Ficol and PVP, 0.1 % SDS, 0.02 % DNA (5mg/ml)) was carried out at 45 degrees C for 90 min and 16 h, respectively followed by two low stringency washes (0.5 X SSC, 0.1% SDS, at room temperature for 5 min) and one high stringency wash (0.1X SSC, 0.1% SDS at 60 degrees C for 1 hour). In vitro synthesized DIG-labeled full-length CCCVd(-) RNA probe was used in hybridization step. DIG Nucleic Acid Detection Kit (Roche) instructions were followed for detection procedure and as a result the blue bands corresponding to the position of the viroid were appeared on the membrane. The result of this study showed the ability of DIG labeled probe in detection of the viroid and also provided a suitable extraction and hybridization method for the detection of CCCVd from oil palm.     

Influence of oral dehydroepiandrosterone (DHEA) on urinary steroid metabolites in males and females

Oral dehydroepiandrosterone (DHEA) replacement therapy may have a multitude of potential beneficial effects and exerts its action mainly via peripheral bioconversion to androgens (and estrogens). A daily dose of 50-mg DHEA has been shown by us and others to restore low endogenous serum DHEA concentrations to normal youthful levels followed by an increase in circulating androgens and estrogens. As the hepatic first-pass effect may lead to a non physiological metabolism of DHEA after oral ingestion we studied the influence of two single DHEA doses (50 and 100 mg) on the excretion of steroid metabolites in 14 elderly males [age 58.8+/-5.1 years (mean +/- SEM)] with endogenous DHEAS levels <1500 ng/ml and in 9 healthy females (age 23.3+/-4.1 years) with transient suppression of endogenous DHEA secretion induced by dexamethasone (dex) pretreatment (4x0.5 mg/day/4 days). Urinary steroid profiles in the elderly males were compared to the steroid patterns found in 15 healthy young men (age 28.9+/-5.1 years). In the females the results were compared to their individual baseline excretion without dex pretreatment. Urinary steroid determinations were carried out by semiautomatic capillary gas-liquid chromatography. In both genders DHEA administration induced significant increases in urinary DHEA (females: baseline vs. 50 mg vs. 100 mg: 361+/-131 vs. 510+/-264 vs. 1541+/-587 microg/day; males: placebo vs. 50 mg vs. 100 mg: 434+/-154 vs. 1174+/-309 vs. 4751+/-1059 microg/day) as well as in the major DHEA metabolites androsterone (A) and etiocholanolone (Et). Fifty mg DHEA led to an excretion of DHEA and its metabolites only slightly above baseline levels found in young females and in young men, respectively, whereas 100 mg induced clearly supraphysiological values. After 50 mg DHEA the ratios of urinary DHEA metabolites (A/DHEA, Et/DHEA) were not significantly different between elderly males vs. young male volunteers and young healthy females versus their individual baseline levels. In conclusion, an oral dose of 30 to 50 mg DHEA restores a physiological urinary steroid profile in subjects with DHEA deficiency without evidence for a relevant hepatic first-pass effect on urinary metabolites.     

Improved electrophoretic separation of supercoiled and relaxed DNA in the presence of ethidium bromide.

Supercoiled and relaxed DNA were resolved electrophoretically in the presence of 0.5 micrograms/ml ethidium bromide. Under these conditions the Gaussian distributions of topological isomers of both supercoiled and relaxed DNA migrated as discrete bands. The separation of these DNAs was optimized by varying the concentration of electrode buffer. Electrophoresis in the presence of 160 mM Tris-acetate, pH 8.3, 4 mM EDTA resulted in a 20-fold increase in the separation of relaxed and supercoiled DNA relative to electrophoresis in 40 mM Tris-acetate, pH 8.3, 1 mM EDTA.     

Preservation of resuspended red cell concentrates. Rate of vesiculation and of spontaneous hemolysis

In resuspended red cell concentrates addition of sucrose, mannitol and sorbitol (30 mM final concentration each) to the SAG medium (150 mM NaCl, 50 mM glucose, 1.25 mM adenine) results in a significant reduction of the spontaneous hemolysis of the cells to about 25% after 3 weeks and to about 40% after 6 weeks preservation. Furthermore, in comparison to the SAG medium the vesiculation rate is reduced to about 40% after 3 weeks preservation. Clear cut differences in the effects between the three additives could not be found. The addition of guanosine (1.25 mM final concentration) to the SAG-sucrose or SAG-sorbitol medium has no significant effects on hemolysis and vesiculation.