Fingerprinting human chromosomes by polymerase chain reaction-mediated DNA amplification.

We describe here a method for DNA fingerprinting of human chromosomes by Alu-polymerase chain reaction (PCR) amplification of DNA from monochromosomal hybrids, following digestion with restriction endonucleases. DNA digestion with restriction enzymes prior to PCR amplification reduces the total number of amplified fragments. The number and pattern of bands of PCR products observed in an electrophoretic medium are chromosome specific and provide a "fingerprint signature" for individual human chromosomes. Using this approach, we have produced fingerprints for human chromosomes 2, 5, 7, 9, and 12. The applicability of this approach to chromosome identification was assessed by comparing the fingerprints obtained for two different hybrids containing chromosome 7. DNA fragments specific for the long and the short arms of human chromosome 12 have also been identified. In addition, Alu-PCR-generated DNA fragments, specific for different chromosomes, were used to probe Southern blots of a hybrid cell panel to identify human chromosomes present in hybrid cell lines. The chromosomal specificity of these probes permits the identification of intact as well as rearranged chromosomes composed of segments arising from more than one chromosome.     

Sulfhydryl group(s) in the ligand binding site of the D-1 dopamine receptor: specific protection by agonist and antagonist

An iodinated compound, [125I]-8-iodo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin -7-ol, has been recently reported [Sidhu, A., & Kebabian, J.W. (1985) Eur. J. Pharmacol. 113, 437-440] to be a specific ligand for the D-1 dopamine receptor. Due to its high affinity and specific activity, this ligand was chosen for the biochemical characterization of the D-1 receptor. Alkylation of particulate fractions of rat caudate nucleus by N-ethylmaleimide (NEM) caused an inactivation of the D-1 receptor, as measured by diminished binding of the radioligand to the receptor. The inactivation of the receptor sites by NEM was rapid and irreversible, resulting in a 70% net loss of binding sites. On the basis of Scatchard analysis of binding to NEM-treated tissue, the loss in binding sites was due to a net decrease in the receptor number with a 2-fold decrease in the affinity of the receptor for the radioligand. Receptor occupancy by either a D-1 specific agonist or antagonist protected the ligand binding sites from NEM-mediated inactivation. NEM treatment of the receptor in the absence or presence of protective compound abolished the agonist high-affinity state of the receptor as well as membrane adenylate cyclase activity. The above-treated striatal membranes were fused with HeLa membranes and assayed for dopamine-stimulated adenylate cyclase activity. When the sources of D-1 receptors were from agonist-protected membranes, the receptors retained their ability to functionally couple to the HeLa adenylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uptake of γ-Aminobutyric Acid by Brain Tissue Preparations: A Reevaluation

The kinetic constants Km and Vmax for the uptake of gamma-aminobutyric acid (GABA) by various preparations from rat cerebral cortex were determined by means of Eadie-Hofstee plots and computer analysis. The Km values were much greater in 0.1-mm slices than in synaptosomal preparations, and the Km value increased further with the thickness of the slices. The apparent high Km values in slices were probably due to depletion of the GABA concentration in the extracellular fluid as the exogenous GABA ran the gauntlet of competing uptake sites on its way to sites deep within the slice, thereby bringing about a requirement for higher GABA concentrations in the incubation medium in order to maintain the internal GABA levels at the "Km level." Evidence was obtained for three GABA uptake systems with Km values (in synaptosomes) of 1.1 microM, 43 microM, and 3.9 mM, respectively. In contrast, only two uptake systems for D-aspartate were detected, with Km values of 1.8 microM and 1.8 mM, respectively. The implications of the findings in the study with respect to previous data in the literature are discussed.     

Reproductive life of some Gujar women of Punjab

Data was collected on current age, age at menarche, marriage age, maternal age at 1st birth, age at the birth of last child, age at menopause, total number of conceptions, live births, stillbirths, abortions, dead children and living children for a sample of 150 Gunjar women of Punjab, India, during September and October 1977 to study their reproductive life. The women ranged in age from 45-55 years. The mean age at menarche was 14.90 years for the sample. The median age at menopause was 46.20 years. The mean age at marriage of the present sample was 12.56+-2.50 years; the mean age of the mother at the birth of her 1st child was 16.85 years; and the mean age at the birth of the last child was 38.68 years. The average number of conceptions was 7.2; the average number of live births of these 150 women was 6.90. The fertility of this population was natural as they were not using any family planning method.     

Effect of uterine fluid on in vitro acrosome reaction of buffalo (Bubalus bubalis ) spermatozoa

The present study was undertaken to examine in vitro acrosome reaction in the uterine fluid of estrous buffalo. Successful acrosome reaction was achieved by incubating buffalo spermatozoa in 2% detoxified uterine fluid in Biggers Whitten Whittingham (BWW) medium, pH 7.4 at 37 degrees C and a sperm concentration of 36 x 10/ml. Neat uterine fluid has been found to be toxic to spermatozoa, hence we detoxified the uterine fluid at 56 degrees C for 30 min, which resulted in higher percentage of sperm motility, viability, and acrosome reaction. All the three stages of acrosome reaction i.e., acrosome swelling, vesiculation and shedding, were observed and they reached an apparent maximum at 4, 7 and 8 h of incubation, respectively. The significance of the findings in relation to the role of female reproductive tract in acrosome reaction is discussed.     

Temperature Sensitivity of Agonist High-Affinity Binding Sites of Solubilized and Reconstituted D1 Dopamine Receptors

Abstract: Solubilization of rat striatal membranes with sodium cholate, followed by reconstitution into phospholipid vesicles, leads to a 6.5-fold increase in the agonist high-affinity binding sites of the D₁ dopamine receptor. These high-affinity binding sites display differential sensitivity toward temperature. When reconstituted receptors were preincubated for 1 h at 0–4°C (on ice) or at 22°C (room temperature) followed by radioligand binding assays with dopamine, neither the high-affinity values of the receptor for dopamine nor the percent receptors in the high-affinity state (31–39%) were changed from control reconstituted receptors, which were not subject to any preincubations. At 30°C, there was a partial loss in the number of high-affinity D₁ receptors with only 25% of the total receptor population in the high-affinity state; there was no change in the affinity values of the high-affinity binding sites. At 37°C, there was a 40% loss in total number of D₁ receptor binding sites. All the high-affinity binding sites were lost and the remaining 60% of binding activity represented the low-affinity binding state of the receptor. These results indicate that the high-affinity binding sites of the reconstituted D₁ dopamine receptors are uniquely sensitive to higher temperatures.     

IMPRINTING EFFECTS OF THREE AMINO ACIDS (ALANINE,LYSINE AND GLYCINE) AND THEIR OLIGOPEPTIDES INTETRAHYMENA PYRIFORMIS. DATA FROM THE HORMONE AND HORMONE RECEPTOR EVOLUTION

Hormonal imprinting takes place at the first encounter of the hormone and receptor, and results in a changed binding capacity and reaction of the cell and its progeny generations. The imprinting effect of three amino acids and their oligopeptides is studied using fluorescent-labelled peptides. Glycine and lysine could provoke positive imprinting (increased binding in the progeny generations) for their own peptides, but alanine could not. Mostly positive imprinting was provoked by glycine and lysine peptides for their own peptides of different chain length. The optimal chain length provoking self-imprinting was four for glycine, two for lysine and three for alanine. Except in this case, alanine was neutral or provoked mostly negative imprinting. After reaching the optimal chain length, there is a decline in binding. Evolutionary conclusions are discussed.     

Induction of buffalo (Bubalus bubalis ) sperm capacitation and acrosome reaction in the excised reproductive tract of hamsters

A study was conducted on the induction of buffalo sperm capacitation and acrosome reaction in the excised reproductive tract of hamsters at the estrogen- and progesterone-dominated stages of estrus. The percentages of the maximum capacitation and acrosome reaction were significatly (P < 0.01) higher for spermatozoa incubated in the uterus with oviducts of estrogen dominated hamsters compared with those incubated in BWW medium in a test tube (64.6%, 60.2%; 16.2%, 14.7%). Buffalo spermatozoa incubated in the uterus and oviducts of progesterone-dominated hamsters showed significantly (P < 0.01) lower capacitation and acrosome reaction rates than those incubated in the uterus and oviducts of estrogen-dominated hamsters (34.8%, 34.3%: 64.6%, 60.2%). The percentage of capacitation and acrosome reaction in spermatozoa were significantly (P < 0.01) more when incubated in the uterus plus oviducts than without the oviduct irrespective of whether the reproduct tract of hamster was estrogen- or progesterone-dominated. The time for the onset of maximum capacitation and acrosome reaction was reduced from 12 to 10 h when the spermatozoa were incubated in the hamster reproductive tract rather than in BWW medium in test tubes. The significance of the results in relation to hormonal regulation of sperm capaciation and acrosome reaction are also discussed.     

A Simple Staining Procedure for Detecting the true Acrosome Reaction in Buffalo (Bubalus bubalis) Spermatozoa

A simple dual staining procedure for detecting the true acrosome reaction in dried smears of buffalo spermatozoa is described. Trypan blue is used first to differentiate live from dead spermatozoa and the dried smears which have been prepared are stained with Giemsa for acrosome evaluation. Four categories of spermatozoa were recognized: A) live, intact acrosome (acrosome pink, postnuclear cap clear); B) dead, intact acrosome (acrosome pink, postnuclear cap blue); C) live, detached acrosome (acrosome clear, postnuclear cap clear); and D) dead, detached acrosome (acrosome clear, postnuclear cap blue). The procedure is simple, rapid and convenient for assessing true acrosome reaction in buffalo spermatozoa. Simultaneous assessment of sperm viability and its acrosomal status in dried smears makes this procedure attractive because the true acrosome reaction can be studied thoroughly at a later state after the incubation period.