Detection of Brucella abortus in mammalian tissue, using biotinylated, whole genomic DNA as a molecular probe.

A method has been developed for the detection of Brucella abortus in complex tissue homogenates. The technique uses tissue homogenization in the presence of sucrose and Triton X-100 and subsequent filtration through a 5-microns pore size filter to remove mammalian nuclei and cellular debris. The DNA from the bacteria is then extracted, dot blotted onto nitrocellulose, and hybridized with a biotinylated probe of B abortus strain 19 DNA. In the present study, BALB/C mice were inoculated intraperitoneally with either 10(9) or 10(11) B abortus strain 2308S organisms. After 6 days, the mice were euthanatized by cervical dislocation and the livers were removed, weighed, and the appearance of each was noted. The tissues were homogenized, and a viable cell count was performed to determine the number of bacteria in each organ. The DNA was extracted, blotted onto nitrocellulose, and hybridized with the Brucella probe. The biotin label was detected by use of a commercially available streptavidin/alkaline phosphatase system. In control experiments, the technique detected 10(5) organisms in a mixture of bacteria and 1 g of rat liver. The technique also detected 10(7) B abortus organisms/g of tissue from experimentally inoculated mice. The probe was specific for Brucella and had no affinity for contaminating bovine or bacterial DNA.

Evidence for an influence of the ovary on circulating dehydroepiandrosterone sulfate levels.

Circulating dehydroepiandrosterone sulfate (DS) declines in old age, falling to almost undetectable levels after the seventh decade. Since an accelerated decrease occurs after the menopause, we sought to determine whether ovarian factors may influence adrenal DS secretion independent of chronological age. DS, cortisol, and estradiol levels were compared in subjects grouped according to age, ovarian function, and estrogen replacement. Our data show that premature ovarian failure and ovariectomy in young as well as in postmenopausal subjects precipitate an earlier decline in DS levels. There were no accompanying changes in cortisol levels and no correlations among levels of DS, cortisol, and estradiol. Long term estrogen replacement (over 10 yr) in postmenopausal women over age 65 had no beneficial effect on the age-related DS decline. We suggest that ovarian factors separate from estrogen-mediated effects significantly influence the reduction of DS levels independent of age.

Fetal lung maturation: amniotic fluid catecholamines, phospholipids, and cortisol.

The identification of beta-adrenergic receptors in the fetal lung led us to investigate amniotic fluid catecholamine levels in relation to other indices of fetal pulmonary maturity in late pregnancies (n = 62). Significant correlations were found between the percentage of phosphatidylglycerol and concentrations of norepinephrine (r = 0.44, p less than 0.001), its intraneuronal deaminated metabolite 3,4-dihydroxyphenylglycerol (DOPEG; r = 0.74, p less than 0.0001), epinephrine (4 = 0.43, p less than 0.001), and cortisol (r = 0.78, p less than 0.001). Highly significant elevations of these substances were noted with accelerated pulmonary maturation. Lecithin/sphingomyelin (L/S) ratios showed a significant correlation with cortisol levels (r = 0.50, p less than 0.001); however, significant associations between L/S ratios and catecholamine levels were found only when complicated pregnancies were excluded. These findings support the contention that fetal contention that fetal adrenergic activity participates in the process of pulmonary maturation.

Acute steroidogenic responsiveness to human luteinizing hormone in hypogonadotropic hypogonadism.

The responses of circulating levels of androgens, estrogens, and their C-21 biosynthetic precursors to a 6-h constant infusion of human Lh (hLH; 2000 IU) were studied in four males with hypogonadotropic hypogonadism (HH) and compared with those in normal male controls. Although similar levels of circulating LH were achieved, the initial and secondary increases in testosterone were significantly greater in the hypogonadotropic subjects than in the normal controls. In contrast, the responses of estradiol, estrone, 17 alpha-hydroxyprogesterone, and 17 alpha-hydroxypregnenolone to exogenous hLH were significantly lower in HH than in normal controls. The data demonstrate a different pattern of testicular steroidogenic responsiveness after pharmacological doses of hLH, with increased concentrations of circulating testosterone in subjects with HH compared to a disproportionate increase in estrogen and progestin levels in normal men.

Altered neuroendocrine status of middle-aged rats prior to the onset of senescent anovulation.

The incidence of senescent anovulation (constant estrus) in female rats increases sharply in the age interval 10--14 months. We have compared the neuroendocrine status of 12-month-old rats, which were still cycling, with that of 6-month-old rats in the reproductive prime. Norepinephrine content in the median eminence of the hypothalamus and circulating levels of FSh and androstenedione were significnatly higher in middle-aged rats (12 months old) than in young controls (6 months old). These increases were selective, in that ten other neuroendocrine parameters measured were unchanged. These results indicate that changes occur at multiple levels of the neuroendocrine system during the transitional phase prior to the onset of senescent anovulation.

A simple method for the assay of eight steroids in small volumes of plasma.

A simple method is described for the simultaneous radioligand assay of four delta5-3beta-hydroxysteroids adjacent to one another on the biosynthetic pathway (pregnenolone [1], 17alpha-hydroxypregnenolone, dehydroepiandrosterone and 5-androsterone-3beta, 17beta-diol), and their four delta4-3keto products (progesterone, 17alpha-hydroxyprogesterone, 4-androstene-3, 17-dione and testosterone). Two plasma aliquots are extracted and fractionated each for four steroids and individual corrections are made for losses. For fractionation, maximum use is made of the high resolution and reproducibility of celite minicolumns, using propylene glycol as stationary phase, and a discontinuous gradient of ethyl acetate in iso-octane as mobile phase. The fractions are then assayed in the appropriate radioligand end-assay system. Each assay was finally validated by demonstrating coincidence of peaks of immuno- and radioactive steroid in extracts of female plasma. Results in pre-pubertal girls and women in the follicular phase of the menstrual cycle suggest that the major change in adrenal steroid production at puberty may be an increase in 17, 20-desmolase activity. There appears to be little reversal of this change in adrenal function after ovariectomy.

Circulating concentrations of dehydroepiandrosterone and dehydroepiandrosterone sulfate during puberty.

In order to quantitate the chronological change in circulating dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DS) levels during the period of sexual maturation, serum DHEA and DS concentration (3-5 PM) in 76 boys and 65 girls (ages 8 to 15) as well as in adult male and female subjects were measured by a specific and sensitive radioimmunoassay technique. Our data show a progressive and parallel increase in serum DHEA and DS concentrations in boys, and adult male levels were reached earlier for DHEA (age 13) than for DS (age 14). From age 8 to adult male, there was a 2.6-fold increase in DHEA (1.52 plus or minus 0.16 ng/ml to 3.91 lus or minus 0.34 ng/ml) and a 7.7-fold increase in DS (0.40 plus or minus 0.08 mug/ml to 3.09 plus or minus 0.36 mug/ml). The rise of DHEA and DS was not in a parallel fashion in girls; while DS rose progressively, DHEA showed an abrupt increase between 11 and 12 yr of age. Adult female range was reached by age 12 for DHEA and by age 15 for DS. From age 8 to adultfemale there was a 2.3-fold increase in DHEA (1.93 plus or minus 0.19 ng/ml to 4.49 plus or minus 0.76 ng/ml) and a 7.5-fold increase in DS (0.29 PLUS OR MINUS 0.05 MUg/ml to 2.17 plus or minus 0.34 mug/ml). The role of increased adrenal androgens inthe sexual development during early stages of puberty is discussed.