Facultative polyploidy in endocrine tissues.

Adrenal tissue from 44 autopsies and 30 biopsies of thyroid gland were analyzed by flow--and scanning-cytophotometry. Most nuclei were diploid and the 4C fraction ranged from 1.9 to 6.1% according to the technique, but with no significant difference between controls and adenomas, hyperplasias, adenomatous goiters, or two carcinomas of the thyroid gland. The only significant increase of 4C fraction was found in normal adrenals of patients above 50 years of age (P less than 0,025). Similarly constant was the proportion of 8C nuclei which ranged from 0.5 to 1.3 per thousand. However, in one of 23 adenomatous goiters a total polyploidization was was observed. Comparable results in the literature show that the thyroid gland like the epithelium of seminal vesicles can facultatively become polyploidized, in contrast to the obligatory polyploid orgnas namely the liver and heart. This is not related to malignancy.     

Lithium accumulation in some endocrine tissues.

Lithium levels were measured in several tissues of rats after oral lithium administration for various periods of time. The lithium levels in the brain approached those of the serum while there was a marked accumulation of lithium in the pituitary and thyroid glands. When lithium was stopped there was no noticeable lag in clearance for any of the tissues examined, with the pituitary maintaining a significant elevation of lithium over the serum levels. Animals receiving lithium had higher levels of adrenal corticosterone under quiescent conditions and higher levels of plasma corticosterone thirty minutes after a brief electric footshock.     

Turnover of carnitine by rat tissues.

A small release of Pi from a diphenylamine-formic acid digest of DNA was detected after elimination of interpurine phosphodiester bonds was complete. Minor components in the DNA digest were identified as pyrimidine oligonucleotides which had lost one terminal phosphate. Isolated pyrimidine tracts released Pi on redigestion with the formic acid-diphenylamine reagent in amounts that increased with the number of nucleotides in the oligonucleotide taken. The oligonucleotides were also partially degraded by the formic acid-diphenylamine reagent and the degradation (2-3% of phosphodiester bonds between consecutive nucleotides) was almost independent of chain length. The cleavage was random with no preference for a phosphodiester bond flanked by particular nucleosides. This minor lack of specificity in the formic acid-diphenylamine-catalysed degradation of DNA can, however, account for the low recoveries of long pyrimidine tracts previously reported. Any analysis of pyrimidine tracts in a DNA molecule should make some correction for this small degree of degradation if exact assignments of the numbers of pyrimidine tracts are to be made.     

Species-common antigen of connective tissues.

Collagen-free extracts were prepared from bovine, porcine and canine hyaline, elastic and fibrous cartilages, articular capsule, tendon, aorta, cortical bone and regenerating articular surfaces. The extracts were investigated with antisera to bovine nasal septal cartilage, dog articular cartilage and non-collagenous protein fraction of bovine cortical bone. Immunodiffusion, immunoelectrophoresis, and immunohistochemical methods were used. In the different supporting tissues of the three animal species a common antigen, probably of proteoglycan origin, was demonstrated. The finer differences in antigenicity between the different tissues are probably due to the variations in proteoglycan composition of the given supporting tissues. Owing to the wide-spread occurrence of the antigen, the authors suggest the term "species-common connective tissue antigen" instead of the "species-common cartilage antigen" used so far.     

Thromboxane receptors in human kidney tissues.

Thromboxane (TX) A2 effects in the kidneys include contraction of glomerular mesangial cells and intrarenal vascular tissue. A kidney cDNA encoding a TX receptor expressed in rat renal glomeruli and rat renal arterial smooth muscle cells has been reported. However, TXA2 receptors in human kidneys have not been documented. The purpose of this study was to identify and characterize TXA2 receptors in glomeruli and intrarenal arteries isolated from human kidneys. Normal kidneys, not used for transplant because of technical reasons, were kept at -70 degrees C and used for research purposes. The glomeruli and intrarenal arteries were isolated from renal cortical tissue by a mechanical sieving technique. The equilibrium dissociation constant and receptor number were determined by nonlinear analysis of binding inhibition data. The data were generated in radioreceptor assays using [125I]-BOP, a stable analog of TXA2. The dissociation constants (mean +/- SEM) for binding of I-BOP to human glomeruli and intrarenal arterial membranes were 6.6 +/- 1.1 nM (n = 7) and 20 +/- 6 nM (n = 7), respectively (p < 0.05). The receptor number was 311 +/- 91 fmol/mg protein (n = 7) in glomeruli and 74 +/- 16 fmol/mg protein (n = 7) in intrarenal arterial membranes (p < 0.04). The order of specificity of TXA2 analogs for [125I]-BOP binding sites was similar in glomeruli and in arterial membranes and was I-BOP > or = U46619 > or = pinane TXA2 > or = carbocyclic TXA2 > or = PGH2. These findings provide direct evidence for the presence of specific, high-affinity [125I]-BOP binding sites in human renal glomeruli and extraglomerular vascular tissue. These data also indicate that the human binding sites have higher affinity for the TXA2 agonist I-BOP than for PGH2.     

Immunohistochemical localization of follistatin in rat tissues.

We have used immunohistochemistry to localize follistatin/activin-binding protein in adult male and female rats. A polyclonal antibody directed against a follistatin peptide (residues 123-134) was used as a specific immunologic probe. Intense and specific follistatin immunoreactivity was evident in spermatogenic cells of seminiferous tubules in the testis. The predominant staining was in nuclei of spermatocytes and spermatids, but no immune reaction was observed in spermatogonia or spermatozoa. Moderate immunoreactivity was detected in Leydig cells. Sertoli cells were follistatin-negative. Significant immunoreactivity was evident in ovarian granulosa cells. The intensity of the staining changed with follicle development: no immunoreactivity was observed in granulosa cells of primordial to primary follicles, but the cells of secondary to Graafian follicles displayed moderate to strong staining and finally luteal cells of the corpus luteum became negative. The epithelial lining of the oviduct and the smooth muscle of the myometrium of the uterus were intensely immunoreactive. Immunoreactive follistatin staining was present in the pituitary: a group of round-shaped cells were specifically stained. Immunostainable follistatin was visible in the epithelial layers of renal tubules with moderate to strong staining reactivity. Hepatic cells in the liver demonstrated homogeneous immunoreactivity from moderate to strong. The cortex of the adrenal gland, white pulp of the spleen and the brain cortex were also stained weakly but distinctly with the antiserum. In conclusion, immunoreactive follistatin is widespread in rat tissues, suggesting that follistatin/activin-binding protein is a ubiquitous protein, regulating a wide variety of activin actions.     

Lysine-ketoglutarate reductase in human tissues.

Lysine-ketoglutarate reductase (saccharopine dehydrogenase (NADP+, lysine-forming) EC 1.5.1.8) from human liver has been partially purified and characterized. A spectrophotometric assay is described. The Michaelis constants have been determined for lysine (1.5-10-3 M), alpha-ketoglutarate (1-10-3 M) and NADPH (8-10-5 M). The pH optimum is 7.8. The enzyme is product inhibited. The specificity of the enzyme, response to inhibitors, pH and thermal stability are reported. Lysine-ketoglutarate reductase is present in high concentration in liver and heart, to a lesser degree in kidney and skin and in trace amounts in several other tissues. Saccharopine dehydrogenase (saccharopine dehydrogenase (NAD+, L-glutamate-forming) EC 1.5.1.9) was demonstrable only in liver and kidney. Lysine-ketoglutarate reductase reacts effectively with delta-hydroxylysine.     

Distribution of fibronectin in foetal tissues.

The distribution of fibronectin in tissues of four human foetuses (7-14 gestation weeks/GW) and twenty seven pig foetuses (25-114 days of gestation) was investigated using immunofluorescence and avidin/biotin methods. Fibronectin was abundant in the circulatory and gastrointestinal system and its derivates, in reticular stroma of immune organs, and in connective tissues and chorionic villi at all developmental stages.     

Histochemical localization of glutathione in tissues.

A histochemical method has been developed for the localization of glutathione (GSH) in frozen sections from various tissues including liver, lung, kidney, testis and eye. The reliability and specificity of the method has been investigated by comparing the rates of reaction in tissue and gelatin sections and after depletion of GSH in liver by diethyl maleate. In principle, the method is based on the formation of an irreversible complex of mercury orange with the --SH group of GSH. A 5-min staining period was found to be optimal for staining the --SH group of GSH. In brief, frozen sections 8 mu thick are stained with a 50 muM solution of mercury orange dissolved in toluene, counterstained in 0.05 per cent methylene blue and mounted in Histoclad. Pretreatment of the sections with fixatives or drying them in air completely prevented the staining. In hepatic lobules the brick red granules of the GSH mercury orange complex were distributed uniformly, whereas in other tissues they were not uniform. The GSH staining was localized in the proximal convoluted tubules in the cortex of the kidney, the interalveolar epithelial cells of lungs, the epididymis and the capsule of testis, epithelial cells of vas deferens and the periphery of the lens.     

Guanosine triphosphate cyclohydrolase activity in rat tissues.

The GTP cyclohydrolase activity of rat tissues has been studied by means of the measurement of formic acid release and neopterin synthesis from GTP. After gel filtration of a 45%-satd.-(NH4)2SO4 fraction of liver homogenates, three enzyme fractions were separated and named A1, A2 and A3 according to the order of their elution. Fractions A1 and A3 displayed an 8-formyl-GTP deformylase activity; no proof of cyclized product has yet been established. This activity was heat-labile and required Mg2+ for maximal activity. Fraction A2 displayed a 'neopterin-synthetase' activity, with dihydroneopterin triphosphate and formic acid formed in stochiometric amounts. Fraction A1 isolated from heat-treated homogenates also produced dihydroneopterin triphosphate. Neopterin synthetase activity in fractions A1 and A2 was heat-resistant and inhibited by Mg2+. In liver the A2 fraction represented 70-75% of the neopterin synthetase capacity and was inhibited by reduced pterines (sepiapterin, dihydrobiopterin and tetrahydrobiopterin) and to a lesser extent by reduced forms of folic acid. In kidney and brain, fraction A1 and A3 GTP 8-formylhydrolase activities were found in significant amounts, in contrast with the neopterin synthetase activity, which was low and appeared to be confined to the A1 fraction.     

Presence of a protein methylesterase in mammalian tissues.

Using enzymatically methylated protein-³H methyl esters as substrates, a protein methylesterase activity was observed in mammalian tissues. The product of hydrolysis was identified as radioactive methanol by microdistillation under reduced pressure and formation of a 3,5-dinitrobenzoate derivative. The enzyme was active over a broad range of pH and appeared in all tissues investigated. Since protein carboxyl-methylation is involved in chemotaxis of leukocytes and exocytotic secretion, this protein methylesterase probably participates in the regulation of these processes.     

Elastomechanical characterization of brain tissues.

The fluid-induced changes in the intracranial pressure which have important clinical implications are believed to be largely determined by the elastomechanical properties of the brain tissues. To define and evaluate the elastomechanical characteristics of the brain tissues a nonlinear hyperelastic hollow spherical shell has been employed to model the craniospinal complex for its fluid-induced intracranial pressure volume changes. The strain energy function proposed by Hart-Smith has been used to derive the constitutive equations. In 10 dogs, fluid has been infused in the lateral ventricle of the brain. The resulting changes in the ventricular fluid pressure (VFP) and the epidural pressure (EDP) have been recorded. The plot of pressure as a function of volume increases first, reaches a maximum, decreases, reaches a minimum and increases monotonously. The values of maximum and minimum pressures (pv max and pv min) due to fluid infusion are found to be, respectively, 42.4 +/- 15.4 mmHg and 33.1 +/- 12.2 mmHg. The pressure achieved the maximum and minimum values with infusion of 0.19 +/- 0.09 ml and 0.51 +/- 0.15 ml of fluid, respectively. The elastomechanical parameters of the Hart-Smith function that characterize the brain tissues have been evaluated by matching the experimentally obtained pressure-volume curves with the corresponding model generated curves. It is found that the agreement between the experimentally obtained pressure-volume curves and the corresponding Hart-Smith profile is satisfactory at a high inflation level but less so at the lower inflation level.     

Glutamate signaling in peripheral tissues.

The hypothesis that l-glutamate (Glu) is an excitatory amino acid neurotransmitter in the mammalian central nervous system is now gaining more support after the successful cloning of a number of genes coding for the signaling machinery required for this neurocrine at synapses in the brain. These include Glu receptors (signal detection), Glu transporters (signal termination) and vesicular Glu transporters (signal output through exocytotic release). Relatively little attention has been paid to the functional expression of these molecules required for Glu signaling in peripheral neuronal and non-neuronal tissues; however, recent molecular biological analyses show a novel function for Glu as an extracellular signal mediator in the autocrine and/or paracrine system. Emerging evidence suggests that Glu could play a dual role in mechanisms underlying the maintenance of cellular homeostasis - as an excitatory neurotransmitter in the central neurocrine system and an extracellular signal mediator in peripheral autocrine and/or paracrine tissues. In this review, the possible Glu signaling methods are outlined in specific peripheral tissues including bone, testis, pancreas, and the adrenal, pituitary and pineal glands.     

Aquaporins in complex tissues. II. Subcellular distribution in respiratory and glandular tissues of rat

The molecular pathways for fluid transport in pulmonary, oral,and nasal tissues are still unresolved. Here we use immunocytochemistry and immunoelectron microscopy to define the sites of expression of fouraquaporins in the respiratory tract and glandular epithelia, where theyreside in distinct, nonoverlapping sites. Aquaporin-1 (AQP1) is presentin apical and basolateral membranes of bronchial, tracheal, andnasopharyngeal vascular endothelium and fibroblasts. AQP5 is localizedto the apical plasma membrane of type I pneumocytes and the apicalplasma membranes of secretory epithelium in upper airway and salivaryglands. In contrast, AQP3 is present in basal cells of tracheal andnasopharyngeal epithelium and is abundant in basolateral membranes ofsurface epithelial cells of nasal conchus. AQP4 resides in basolateralmembranes of columnar cells of bronchial, tracheal, and nasopharyngealepithelium; in nasal conchus AQP4 is restricted to basolateralmembranes of a subset of intra- and subepithelial glands. These sitesof expression suggest that transalveolar water movement, modulation ofairway surface liquid, air humidification, and generation ofnasopharyngeal secretions involve a coordinated network of aquaporinwater channels.

     

Mechanisms of unidirectional block in cardiac tissues.

We used numerical solutions for cable equations representing nonuniform cardiac strands to investigate possible mechanisms of unidirectional block (UB) of action potential propagation. Because the presence of UB implies spatial asymmetry in some property along the strand, we varied membrane properties (gNa or leakage conductance), cell diameter, or intercellular resistance as functions of distance such that a propagating action potential encountered the parameter changes either gradually or abruptly. For changes in membrane properties there was very little difference in the effects on propagation for the gradual or abrupt encounter; but, for changes in cell diameter or in intercellular resistance, there were large differences leading to the production of UB over a wide range of parameter values.     

Nucleotide excision repair rates in rat tissues.

We have determined and compared nucleotide excision repair capability of several rat tissues by a method based on restoration of the transformation activity of UV-irradiated pBlueScript by incubation in repair-competent protein extracts. After 3 h of incubation, plasmid DNA was isolated and used to transform competent Escherichia coli cells. Damaged plasmids showed low transformation efficiency prior to incubation in repair-competent extracts. After incubation the transformation efficiency was restored to different extents permitting calculation of the repair capacity of the extracts. Our results showed that rapidly proliferating tissues such as liver, kidney and testis showed higher nucleotide excision repair capacity than slowly proliferating tissues such as heart, muscle, lung and spleen. When liver and splenocytes were stimulated to proliferation by partial hepatectomy and mitogen stimulation, their repair capability increased in parallel with the respective proliferative rates.     

Ice crystals formed in tissues during cryosurgery. I. Light microscopy

The size and distribution of ice crystals formed during cryosurgical procedures in intact animals are not clear. In the present experiment oral mucosa was frozen in situ by means of a surface applied cold probe and was excised and freeze substituted while in the frozen state. It was shown that the form of the frozen tissue was preserved during this procedure and the area frozen was divisible into a zone representing the central part of the lesion and a peripheral zone separating this from normal tissue. Ice crystals within the body of the lesion were intracellular in location but varied somewhat in size. Ice crystals in the boundary zone appeared to be intracellular in the epithelium and both intra- and extracellular in the muscle fibres.It is suggested that the intracellular crystals in the body of the frozen area result in cell death while the extracellular ice in the boundary zone results in a less predictable response.