Electronic Library of Scientific Literature



ENDOCRINE REGULATIONS



Volume 31 / No. 4 / 1997




MAMMARY-SPECIFIC TRANSGENES AS TOOLS IN SCIENCE AND INDUSTRY

D. L. Hadsell

USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030

The ability to modify the animal genome by microinjection of DNA fragments into fertilized embryos was first achieved in 1980. The most abundant mammary-specific milk protein genes were recognized shortly thereafter as valuable tools with which to apply this technology to the mammary gland. The expression of the six major milk protein genes accounts for up to 80 % of the total mRNA in lactating mammary tissue. A few of these genes function efficiently as transgenes and can target the expression of heterologous gene product in a tissue-specific, developmentally regulated fashion. The use of regulatory DNA from these genes in a variety of transgene configurations has resulted in scientific advances in three areas. First, a better understanding of milk protein gene expression has enabled the development of helpful guidelines for the construction of efficiently expressed transgenes, including a requirement for regulatory elements within flanking DNA, the presence of introns, processing signals, and translation and protein processing signals. Second, the ability of the mammary gland to exhibit efficient production of heterologous gene products has been tested and the limitations identified. Third, the ability to produce defined, tissue-specific genetic modifications within the mammary gland has allowed researchers to drastically change its development. Such changes could lead to alterations in the physical or chemical properties of manufactured milk, alterations in milk yield or milk composition, alterations in the metabolism and disease resistance of the lactating female, and alterations in the growth and development of the suckling neonate. This review summarizes the current state of transgenic technology as it relates to mammary specific transgenes and illustrates the application of these transgenes to scientific endeavors and to areas of potential commercial interest.

Key words: Transgenic Mice - Chromatin Domains - Lactation - Milk Protein Genes - Biotechnology - Neonatal
pp. 175-185

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DISTRIBUTION OF SOMATOSTATIN RECEPTOR TYPE 3 IN THE RAT: IMMUNOHISTOCHEMICAL STUDY

T. Mitsuma, N. Rhue, Y. Hirooka, M. Kayama, Y. Mori, K. Adachi, T. Wago, J. Takagi, Jing Ping, T. Nogimori, J. Sakai

The Fourth Department of Internal Medicine;
Department of Laboratory Medicine;
The First Department of Physiology, Aichi Medical University, Nagakute Aichi, Aichi 480-11 Japan;
Department of Internal Medicine, Konanshowa Hospital, Konan Aichi, Japan

Somatostatin receptor type 3 (SSTR-3) was identified immunohistochemically in the rat tissues using specific anti-SSTR-3 serum which was raised in New Zealand white rabbits immunized with a conjugate of synthetic SSTR-3 peptide (28-41) with bovine serum albumin. Immunohistochemical analysis was performed by avidin-biotin complex method. SSTR-3 immunoreactivity was visualized in the central nervous system, anterior pituitary, gastric and duodenal mucosa, Auerbach's and Meissner's nervous branch of gastrointestinal tract, adrenal medulla, testis and pancreas. Significant staining was detected in neural perikarya, axons and dendrites. When using antiserum preincubated with synthetic SSTR-3 peptide (28-41) or rat anterior pituitary homogenate which contains SSTR-3 peptide, no significant stain of the anterior pituitary or neurons in the hypothalamus was detected. These findings suggest that SSTR-3 is widely distributed and that this method is valuable in studying the distribution of SSTR-3 in rats.

Key words: Somatostatin Receptor Type 3 - Immunohistochemistry - Distribution in Rats
pp. 187-192

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ASSESSMENT OF BETA-ENDORPHIN AND MELATONIN CIRCADIAN RHYTHMS IN NORMOTENSIVE OBESE WOMEN OF REPRODUCTIVE AGE

Z. Ostrowska, B. Buntner, K. Zwirska-Korczala, M. Pardela, M. Drozdz, B. Marek

1st Department of Pathophysiology and Clinical Biochemistry, 2nd Department of General and Vascular Surgery, Silesian Academy of Medicine, 41-800 Zabrze, Poland

We have evaluated the relationship between beta-endorphin (beta-EP) and melatonin (MEL) secretion as determined at 3 h intervals over a 24 h period in 27 normotensive obese women of different phenotypes (12 showing gynoid-type and 15 showing android-type of adipose tissue distribution) as well as in 12 healthy volunteers with normal body weight, aged 30 to 40 years. A considerable increase of mean 24 h beta-EP secretion (mainly due to elevated afternoon and evening levels) and total absence of beta-EP circadian rhythm were observed in all obese patients. Mean 24 h MEL concentrations were markedly higher (mainly due to increased daytime levels) in all obese patients while the disturbances of MEL secretion in the form of acrophase shift and (or) suppression of its rhythmicity were observed especially in obese women with android phenotype. Circadian beta-EP levels correlated positively with BMI and WHR values and negatively with circadian MEL concentrations. In contrast, no significant correlation was found between the values of BMI, WHR and MEL levels. Our data indicate that alterations of beta-EP secretion occurring in obese women could play a role in inducing disturbances of MEL secretory pattern.

Key words: beta-endorphin - Melatonin - Circadian Variations - Obese Women - Gynoid Phenotype - Android Phenotype
pp. 193-200

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INFLUENCE OF EGF ON THE RELATIVE VOLUMES OF COLLOID, EPITHELIUM AND STROMA AND ON THYROCYTE NUCLEI SIZE IN RAT THYROID LOBES IN VITRO

J. Brzezinski, D. Slowinska-Klencka, A. Lewinski, M. Klencki, A. Gesing, M. Stasiolek, L. Sowinska-Neuman

Department of Endocrine Surgery,
Department of Experimental Endocrinology and Hormone Diagnostics and
Department of Thyroidology, Institute of Endocrinology, Medical University of Lodz , Poland

The epidermal growth factor (EGF) is believed to be a potent growth factor for the thyroid gland. In the present study, we have examined the relative volumes of the main histological compartments (colloid, epithelium and stroma) and the size of thyrocyte nuclei (the mean volume, the mean intersection area and the mean perimeter) in the rat thyroid lobes incubated in vitro for 18 hrs with EGF, applied in 5 different concentrations: 0.1, 1.0, 10, 100 and 1000 ng/ml. Morphometric evaluation was performed, using a computer image analysis system, developed by us. We found that EGF - in concentration of 100 ng/ml - increased the relative volume of stroma when compared to controls, as well as to all the other groups incubated in exposure to that growth factor (used in different concentrations); at the same time, EGF decreased the relative volume of epithelium in the thyroid gland (statistical significance has been recorded only vs. EGF concentrations of 10 ng/ml and 1000 ng/ml). On the other hand, we observed that EGF - in concentration of 100 ng/ml - significantly increased the mean nuclear volume and the mean intersection area of thyrocyte nuclei when compared to the controls, as well as to EGF in concentrations of 1 ng/ml and 1000 ng/ml. With regards to the mean perimeter, a significant increase of its length was noted in the EGF(100 ng/ml)-exposed group vs. the group incubated with an addition of EGF (1 ng/ml).

Key words: EGF - Thyroid morphometry - Experiment in vitro - Computer-aided analysis
pp. 201-205

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CAFFEINE INJECTION IN THE DARK PHASE PROLONGS THE NOCTURNAL RISE IN SEROTONIN N-ACETYLTRANSFERASE ACTIVITY AND MELATONIN CONTENT IN THE PINEAL GLAND OF MALE RATS

I. Sabry

Zoology Department, Faculty of Science, Kuwait University, P.O. Box 5969, SAFAT - 13060 Kuwait

Caffeine, an important member of methylxanthines, induced a prolonged nocturnal rise in pineal melatonin content and an increase in its rate-limiting enzyme serotonin N-acetyltransferase (NAT) activity. The highest levels were reached five hours after subcutaneous caffeine injection to male rats in the dark phase, where the NAT activity increased from 920±70 pM/pineal/h in the control group to 1190±120 pM/pineal/h (P<0.001) in the treated group. The pineal melatonin content, as well, was elevated from 520±40 pg/pineal in the control group to 1120±80 pg/pineal (P<0.001) in caffeine treated group. These changes could be attributed to the depressive effect of caffeine on the activity of phosphodiesterase (PDE), the enzyme responsible for the hydrolysis of the intracellular second messenger cyclic adenosine monophosphate (cAMP).

Key words: Caffeine - Pineal Gland - Melatonin - N-Acetyltransferase
pp. 207-210

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MINIREVIEW
7-HYDROXYLATED DERIVATIVES OF DEHYDROEPIANDROSTERONE: WHAT ARE THEY GOOD FOR?

R. Hampl, R. Morfin, L. Starka

Institute of Endocrinology, 116 94 Praha, Czech Republic;
Bio-industries, Laboratoire de Biologie, Conservatoire National des Arts et Métiers, 75141 Paris, France

In the late fifties and early sixties our group has found that 3beta,7alpha-dihydroxy-5-androsten-17-one (7alpha-hydroxy-dehydroepiandrosterone) is a natural constituent of human body fluids. Later, the enzyme activity responsible for its formation has been demonstrated in many animal and human tissues including the foetal ones. The physiological role of this and related steroids has not been understood well for decades. As late as in 1994 Morfin and his group have shown that 7alpha-hydroxy-dehydroepiandrosterone may be a locally active metabolite, responsible for recently discovered immunostimulatory or immunomodulatory effects of dehydroepiandrosterone.

Key words: 7alpha-Hydroxy-dehydroepiandrosterone - Dehydroepiandrosterone - Antiglucocorticoid effects - Review
pp. 211-218

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EFFECT OF PROTEIN-MODIFYING REAGENTS ON STRUCTURAL ALTERATION OF RAT OVARIAN LH/hCG RECEPTORS

J. Kolena, S. Scsukova, M. Jezova, J.Vranova

Institute of Experimental Endocrinology, Slovak Academy of Sciences, 833 06 Bratislava, Slovak Republic

Treatment of rat ovarian membrane LH/hCG receptor with tyrosine-specific reagents 2,4-dinitrofluorobenzene (2,4-DNFB) and N-acetylimidazole (N-AcIA), as well as acetic anhydride (AcA), resulted in a concentration-dependent decrease in hCG binding activity. No effect on LH/hCG receptors was observed with ethylacetimidate, a reagent specific for á- and å-amino groups. The fluorescence quenching experiments indicated that 2,4-DNFB and AcA decreased the accessibility of fluorophore for acrylamide. Alterations of quenching rate generally suggest exposure of tryptophanyl residues. Modification of amino acid residues was connected with alteration of membrane lipid rigidity. Thermal inactivation of hCG-binding sites demonstrated that there was a significant destabilization of the receptor structure when ovarian membranes were treated with tyrosine-modifying reagent. Thermal destabilization produced by 10 and 20 mmol l-1 2,4-DNFB caused a decrease in T50 values by about 3 and 8 °C, respectively. These results suggest that tyrosyl residues are essential for hCG binding to LH/hCG receptor.

Key words: LH/hCG Receptor - Tyrosine Residues - Fluorescence Quenching - Lipid Rigidity - Thermal Inactivation
pp. 219-224

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