Immunohistochemical localization of gamma-aminobutyric acid (GABA) in the rat pancreas

The localization of gamma-aminobutyric acid (GABA) in rat pancreas was investigated using antiserum raised against GABA conjugated to bovine serum albumin with glutaraldehyde. Immunoreactive cells were only found in the center of the pancreatic islets, and these cells were surrounded by nonimmunoreactive cells. When two serial sections of rat pancreas were consecutively stained with GABA antiserum and with antibodies against insulin, both antisera stained the same population of endocrine cells within the islets. In rats pretreated with streptozotocin, a B-cell toxin, we observed a marked decrease in the number of cells exhibiting GABA-like immunoreactivity. These observations indicate that GABA is present in the B cells of rat pancreatic islets.     

Identifying the fluorescent body (F-body) with quinacrine mustard staining of canine

The fluorescent body (F-body) was identified with quinacrine mustard (Q-M) staining in spermatozoon and lymphocyte of canine. Well washed sperm suspension was treated with protease (125 mg/ml) or dispase (2000p. u./ml) and staining with Q-M (final dilution 50 micrograms/ml) for 15 min to 24 hr at 37 degrees C. The lymphocyte cultures from whole blood were prepared as routine human investigation. The chromosomal preparation made by air dry method was stained with Q-M (final dilution 0.5 to 50 micrograms/ml) after pretreatment of enzyme digestion. The examination using a reflected fluorescent microscope revealed that the same F-body in human was present in both spermatozoon (20.1-39.7%) and interphase of lymphocyte (0.37.2%) of male origin.     

Stimulatory release of lipoprotein lipase activity from rat fat pads by vanadate

Vanadate stimulated the release of lipoprotein lipase (LPL) activity from rat fat pads into the medium in a time- and dose-dependent manner. It exerted the synergetic effect with heparin. The stimulatory effects of vanadate and heparin were decreased by incubation in Na+- or Ca2+-free media but were well preserved in K+-free medium. Amiloride inhibited the vanadate-stimulated release of LPL activity in a dose-dependent manner, but did not inhibit the heparin-stimulated release of LPL activity. Colchicine, antimycin A, and carbonyl cyanide m-chlorophenylhydrazone suppressed the stimulatory effect of vanadate, but cycloheximide did not. Preincubation of the fat pads with the tetrakis (acetoxymethyl) ester of quin 2 (quin 2-AM) inhibited the vanadate-stimulatory release of LPL activity without affecting basal activity. The concentration required for half-maximal inhibition of the action of vanadate by quin 2-AM was calculated to be 39 microM, suggesting that the action of vandate was dependent on intracellular Ca2+ concentration. The heparin-stimulated release, on the other hand, was not inhibited even at higher concentrations of quin 2-AM (up to 200 microM). These findings suggest that vanadate stimulates the release of LPL activity through mechanisms of action involving amiloride-sensitive and calcium-dependent pathways with a requirement of metabolic energy.     

Macrophage-mediated N-nitrosation of thioproline and proline

Thioproline (Thiazolidine-4-carboxylic acid) and proline were nitrosated by stimulated mouse macrophages in vitro. A macrophage cell line (J774.1, 1.0 x 10(6)/well, 1 ml) was incubated with Escherichia coli lipopolysaccharide, interferon-gamma and thioproline (5 mM) or proline (5 mM). After 72 hr incubation at 37 degrees C, 4 microM N-nitrosothioproline was produced. The amount of N-nitrosoproline was much lower than that of N-nitrosothioproline. Thioproline and proline inhibited the formation of carcinogenic N-nitrosomorpholine. N-nitrosothioproline and N-nitrosoproline are found as major N-nitroso compounds in human urine. Macrophage mediated N-nitrosation may contribute to the formation of these N-nitrosamino acids in the human body.     

Photoreceptor channel activation by nucleotide derivatives

Cyclic nucleotide activated sodium currents were recorded from photoreceptor outer segment membrane patches. The concentration of cGMP and structurally similar nucleotide derivatives was varied at the cytoplasmic membrane face; currents were generated at each concentration by the application of a voltage ramp. Nucleotide-activated currents were analyzed as a function of both concentration and membrane potential. For cGMP, the average K0.5 at 0 mV was 24 microM, and the activation was cooperative with an average Hill coefficient of 2.3. Of the nucleotide derivatives examined, only 8-[[(fluorescein-5-yl-carbamoyl)methyl]thio]-cGMP (8-Fl-cGMP) activated the channel at lower concentrations than cGMP with a K0.5 of 0.85 microM. The next most active derivative was 2-amino-6-mercaptopurine riboside 3',5'-monophosphate (6-SH-cGMP) which had a K0.5 of 81 microM. cIMP and cAMP had very high K0.5 values of approximately 1.2 mM and greater than 1.5 mM, respectively. All nucleotides displayed cooperativity in their response and were rapidly reversible. Maximal current for each derivative was compared to the current produced at 200 microM cGMP; only 8-Fl-cGMP produced an identical current. The partial agonists 6-SH-cGMP, cIMP, and cAMP activated currents which were approximately 90%, 80%, and 25% of the cGMP response, respectively. 5'-GMP, 2-aminopurine riboside 3',5'-monophosphate, and 2'-deoxy-cGMP produced no detectable current. The K0.5 values for cGMP activation, examined from -90 to +90 mV, displayed a weak voltage dependence of approximately 400 mV/e-fold; the index of cooperativity was independent of the applied field.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dependence on exercise intensity of changes in electrolyte secretion from the skin sampled by a simple method

Secreta from the palm and forearm was sampled for 1-min periods by a new technique, using a glass cylinder. Subjects exercised for 10-min periods at successive intensities of 40%, 50% and 65% VO2max with a leg ergometer operated in the supine position. Changes in the concentrations (values) of Na+, K+ and Cl- in their secreta during exercise were investigated. Significant positive correlations were found between the values of any two electrolytes in samples from the palm or the forearm, but the correlations between values for any one of the three electrolytes from the two sites were not significant. Values for concentrations of the electrolytes were significantly higher in samples from the palm than in those from the forearm at rest, 10 min after the beginning of exercise and at the end of exercise. No significant correlation was found between values for electrolytes in samples from the palm and the exercise intensity, but values for Na+ in samples from the forearm increased stepwise with increase in exercise intensity, and similar tendencies were observed for values of K+ and Cl-. The values for the three electrolytes in samples from the forearm, but not the palm, were significantly correlated with values for blood lactate, the percentage of VO2max and the heart rate. These results suggest that the present technique is suitable for successive samplings of secreta from the forearm, and that values for the electrolytes in samples are useful indices of exercise intensity.     

Studies on the structure and stabilizing factor of the CUUCGG hairpin RNA using chemically synthesized oligonucleotides.

A tridecaribonucleotide, r-UGAGCUUCGGCUC, and two analogues r(UGAGC)d(UUCG)r(GCUC) and r-UGAGCUUCIGCUC, which form a hairpin structure with a four-base-paired stem and a UUCG loop, were synthesized by the solid-phase phosphoramidite method. Properties of these three oligomers and d-TGAGCTTCGGCTC, the DNA analogue, were studied by UV, CD and NMR spectroscopy. The melting temperature (Tm) data suggest that the 2'-hydroxy1 groups and the 2-amino group of guanosine in the loop (9G) stabilize the CUUCGG hairpin which is known to have an unusually high Tm. NMR studies show that this 9G takes a syn conformation and the phosphodiester backbone has a turn at 9G-10G which is a junction of the stem and loop.     

Multiple expression of keratins, vimentin, and S-100 protein in pleomorphic salivary adenomas.

Immunohistochemical staining for S-100 protein and the intermediate filaments keratin and vimentin, was made in 41 salivary adenomas. In pleomorphic adenomas, great heterogeneity in the staining, as well as multiple and co-expressions of these proteins were found in the outer tumor cells of tubulo-ductal structures and modified myoepithelial cells, but not in the luminal tumor cells. All the outer tumor cells stained for S-100 protein, 97% for K8.12 keratin and 85% for vimentin. Of these cells, 29% showed multiple expression of K8.12 keratin, vimentin, and S-100 protein, and 17% showed co-expression of K8.12 and S-100 protein. Modified and neoplastic myoepithelial cells showed similar expressions of these proteins to those of outer tumor cells; myoepithelioma cells displayed the most complicated pattern, being positive for KL1, PKK1, and K8.12 keratins, vimentin and S-100 protein. In luminal tumor cells there was a heterogeneous expression of KL1 and PKK1 in 82%, and of KL1, PKK1, and K8.12 in only 14.7%. Based on the immunohistochemical findings obtained with different monoclonal antibodies in pleomorphic salivary adenomas, outer tumor cells may be derived from ductal basal cells and luminal tumor cells from intercalated duct cells.     

Lobeline production by hairy root culture of Lobelia inflata L.

Hairy roots were obtained following inoculation of the stems of Lobelia inflata L. with Agrobacterium rhizogenes strain ATCC 15834. These hairy roots contained agropine and mannopine. In addition, lobeline was detected by HPLC and confirmed by mass spectrometry. Various media were tested for the growth of hairy roots as well as for the content of lobeline in hairy roots. The growth rate of hairy roots cultured in Nitsch and Nitsch's medium was approximately one third of those cultured in other media. The lobeline content of hairy roots (18–54 g/g dry weight) cultured in these media was the same order of magnitude compared with that of roots of L. inflata (24 g/g dry weight) cultivated in pots. The hairy roots cultured in Nitsch and Nitsch's medium were morphologically different from those cultured in other media.Abbreviations MS medium Murashige and Skoog's medium - 1/2 MS medium one-half strength of the standard Murashige and Skoog's medium - B5 medium Gamborg's B5 medium - NN medium Nitsch and Nitsch's medium - FW fresh weight - DW dry weight