Plasma catecholamine levels during water immersion in man

In ten normal subjects thermoneutral neck-out water immersion produced a highly significant natriuresis and diuresis mediated via an induced central hypervolaemia. During immersion suppression of plasma noradrenaline and adrenaline was observed but no change occurred in plasma dopamine levels. No correlation was found between the suppression of noradrenaline and the diuresis and natriuresis. The reduction in plasma noradrenaline observed may reflect a widespread diminution of sympatho-adrenal activity during water immersion. This reduction could be a consequence of the cardiovascular changes of immersion and may not be directly involved in the mechanism of the renal response.     

Intracellular location of unoccupied 1,25-dihydroxyvitamin D receptors: a nuclear-cytoplasmic equilibrium

In order to investigate the subcellular distribution of unoccupied 1,25-dihydroxyvitamin D3 receptors, highly purified cytoplasts and nucleoplasts were prepared from two kidney cell lines (PK1 and MDBK). This was accomplished utilizing the technique of enucleation by cytochalasin B and density gradient centrifugation. Unoccupied 1,25-dihydroxyvitamin D3 receptors were found in both the nuclear and cytosolic compartments, with approximately 70% of the receptors localized in the cytoplasm. When cells were pretreated with 1,25-[3H]dihydroxyvitamin D, prior to enucleation, it was found that 90% of the receptor-hormone complex was associated with nucleoplasts, thus demonstrating that cytochalasin B treatment does not alter the high-affinity association of the receptor-hormone complex with the nucleus. The ratio of unoccupied receptor/protein was found to be the same in whole cells, cytoplasts, and nucleoplasts for both cell types. The ratio of unoccupied receptor/DNA was highest in cytoplasts and lowest in nucleoplasts. Taken together, these data indicate that the unoccupied 1,25-dihydroxyvitamin D receptor is generally associated with cell proteins and not specifically associated with cell DNA. We therefore propose, at least for these cells, that the unoccupied 1,25-dihydroxyvitamin D receptor exists in equilibrium between the nuclear and cytosolic compartments of the whole cell, and receptor-hormone binding shifts this equilibrium to favor nuclear localization.     

Membrane vesicles of cellular dimensions fit in two geometric series

Summary Preparations of basal-lateral plasma membranes from rat intestinal epithelial cells were analyzed with the analytical centrifuge. In these preparations a number of well-defined membrane fractions were observed. The particle weights of these fractions appear to fit in two geometric series. Until now only relatively small vesicles up to a diameter of about 1 m were observed. In our preparation we have observed vesicles up to a diameter of 7.5 m. Therefore, even vesicles with the same size as the plasma membranes of intact cells fit in the two geometric series.     

ICV-CRH potently affects behavior without altering antinociceptive responding

To explore the hypothesized integrative function of corticotropin releasing hormone (CRH) in the stress response, stress-related behaviors including antinociception were studied in rats after either intracerebroventricular (ICV) or peripheral administration of CRH. The effects of low-dose (0.3 microgram) and high-dose (3.0 micrograms) ICV-CRH were compared to those of vehicle, employing a within-S design. The two doses yielded comparable behavioral changes suggestive of increased arousal and stress. These changes were characterized by significant increases in grooming, walking, burrowing, self-gnawing, and pica, and decreases in rearing and sleeping. None of these effects of ICV-CRH were obtained with peripheral administration of the same doses. The hot-plate test of analgesia failed to show a significant effect of ICV-CRH or peripherally administered CRH. A between-S experiment incorporating both the tail-flick and the hot-plate tests of analgesia compared ICV-CRH (3.0 micrograms) with vehicle. ICV-CRH did not affect antinociceptive responding in either of these tests. In contrast, ICV morphine (10 micrograms) yielded potent analgesia in both tests. Thus, with doses of ICV-CRH yielding clear evidence of stress-related behavior, no evidence of analgesia was obtained. These findings question the possible role of central CRH systems in antinociceptive processes.     

Amino acid replacements in yeast iso-1-cytochrome c. Comparison with the phylogenetic series and the tertiary structure of related cytochromes c

The structural and folding requirements of eukaryotic cytochromes c have been investigated by determining the appropriate DNA sequences of a collection of 46 independent cyc 1 missense mutations obtained in the yeast Saccharomyces cerevisiae and by deducing the corresponding amino acid replacements that abolish function of iso-1-cytochrome c. A total of 33 different replacements at 19 amino acid positions were uncovered in this and previous studies. Because all of these nonfunctional iso-1-cytochromes c are produced at far below the normal level and because a representative number are labile in vitro, most of the replacements appear to be affecting stability of the protein or heme attachment. By considering the tertiary structure of related cytochromes c, the loss of function of most of the mutant iso-1-cytochromes c could be attributed to either replacements of critical residues that directly interact with the heme group or to replacements that disrupt the proper folding of the protein. The replacements of residues interacting with the heme group include those required for covalent attachment (Cys-19 and Cys-22), ligand formation (His-23 and Met-85), and formation of the immediate heme environment (Leu-37, Tyr-53, Trp-64, and Leu-73). Proper folding of the protein is prevented by replacements of glycine residues at sites that cannot accommodate side chains (Gly-11 and Gly-34); by replacements of residues with proline, which limit the torsion angle (Leu-14 and His-38); and by replacements apparently unable to direct the local folding of the backbone into the proper conformation (Pro-35, Tyr-72, Asn-75, Pro-76, Lys-84, Leu-99, and Leu-103). Even though most of the missense mutations occurred at sites corresponding to evolutionarily invariant or conserved residues, a consideration of the replacements in functional revertants indicates that the requirement for residues evolutionarily preserved is less stringent than commonly assumed.     

Effects of saponin extracts from Solanum elaeagnifotium fruits on ion uptake by clover seedlings

Solanum elaeagnifolium Cav. fruits contain high concentrations of steroidal saponins. Treatment of 3-day-old clover seedlings with aqueous fruit extracts modified Ca²⁺ uptake without significantly altering K⁺ and H₂PO₄⁻ uptake. The extracts increased Ca²⁺ uptake in the concentration range of 0.2 to 20 m M Ca²⁺. Uptake curves could be represented by two phases. In the lower phase (0.2-1.0 m M Ca²⁺), this change could be related to an increase in V_max. Pretreatment of seedlings with saponin extracts significantly reduced ATP-dependent Ca²⁺ uptake and Ca²⁺-dependent ATPase activity in a fraction isolated from root homogenates by centrifugation at 1500 g for 15 min. Saponins purified from S. eleagnifolium extracts by thin-layer chromatography modified in vitro the Ca²⁺-ATPase activity of this fraction, indicating that the steroid may act directly on Ca²⁺ transport across membranes.     

Imino-proton resonances of yeast tRNAPhe studied by two-dimensional nuclear Overhauser enhancement spectroscopy

Application of two-dimensional nuclear Overhauser enhancement (NOE) spectroscopy to yeast tRNAPhe in H2O solution demonstrates that all imino-proton resonances, related to the secondary structure, and nearly all imino proton resonances, originating from the tertiary structure, can be assigned efficiently by this method. The results corroborate the assignments of the imino-proton resonances of this tRNA as established previously by one-dimensional NOE experiments (only the assignment of base pairs G1 X C72 and C2 X G71 should be reversed). The advantages of two-dimensional NOE spectroscopy over one-dimensional NOE spectroscopy for the assignments of imino-proton resonances and the structure elucidation of tRNA are illustrated and discussed. Furthermore, the use of non-exchangeable proton resonances as probes of the molecular structure is explored.     

Evolution and expression of the transplantation antigen gene family

We have cloned 26 different class I genes that are located in the major histocompatibility complex of the C57BL/10 mouse. Two of the three class I genes found in the H-2 complex encode the H-2Kb and H-2Db antigens; the other 23 class I genes map to the adjacent Tla complex. We have grouped the cosmids containing these genes into three clusters: one cluster links the H-2K and I-A regions, one cluster links the H-2D and Qa-2 regions, and the final cluster maps to the TL region. The class I gene organizations in the Qa-2 and TL regions of the C57BL/10 and BALB/c mice are generally similar, but there are several polymorphic segments. The Qa-2 region of both mice seems to have evolved by the duplication of gene pairs; furthermore, the H-2K region may have been generated by the translocation of a gene pair from the Qa-2 region. We have evidence that several of the genes in the Qa-2 region are expressed.     

Effects of Systemically Administered Lithium on Phosphoinositide Metabolism in Rat Brain, Kidney, and Testis

A single subcutaneous dose of 10 mEq/kg LiCl gives rise to an increase in the cerebral cortex level of myo-inositol-1-P (I1P) that closely follows cortical lithium levels and, at maximum, is 40-fold above the control value. Kidney and testis show smaller increases in I1P level following LiCl administration. The I1P level is still sixfold greater than that of untreated rat cortex 72 h later. In cortex, parallel increases also occur in myo-inositol-4-P (I4P) and myo-inositol 1,2-cyclic-P (cI1,2P), whereas myo-inositol-5-P (I5P) remains unchanged. The cortical increases in I1P and I4P levels are partially reversed by administering 150 mg/kg of atropine 22 h after the LiCl, treatment that does not affect cI1,2P. When doses of LiCl from 2 to 17 mEq/kg are given, the cerebral cortex levels of I1P and myo-inositol, measured 24 h later, are found to reach a plateau at about 9 mEq/kg of LiCl, whereas cortical lithium levels continued to increase with greater LiCl doses. Levels of all three of the brain phosphoinositides are unchanged by the 10 mEq/kg LiCl dose, as is the uptake of 32Pi into these lipids. Chronic dietary administration of LiCl for 22 days showed that the effects of lithium on I1P and myo-inositol levels persist for that period. Over the course of the chronic administration of the lithium, levels of I1P, myo-inositol, and of lithium in cortex remained significantly correlated. We believe that these increases in inositol phosphates result from endogenous phosphoinositide metabolism in cerebral cortex and that lithium is capable of modulating that metabolism by reducing cellular myo-inositol levels. The size of the effect is a function of both lithium dose and the degree of stimulation of receptor-linked phosphoinositide metabolism. This property of lithium may explain part of its ability to moderate the symptoms of mania. Our chronic study suggests that prolonged administration of LiCl does not result in compensatory changes in myo-inositol-1-P synthase or myo-inositol-1-phosphatase.     

New method for visualization of vascular networks in nonperfused fixed tissues

A new method of visualizing the angioarchitecture of tissues has been developed that uses blood components in nonperfused materials. Tissue blocks are fixed in 4% paraformaldehyde and cut with a vibratome into 50-60 micron sections. Endogenous peroxidase in red blood cells is then reduced in the presence of hydrogen peroxide with the resultant oxidation of the chromogen 3,3'-diaminobenzidine (DAB). This generates a dark, highly insoluble reaction product throughout the vascular system. The visualization of vascular components can be further enhanced by exposing the sections to peroxidase-conjugated IgG to increase the background staining of the blood plasma. The technique minimizes preparation artifact and permits the application of morphometric analytical methods, thus allowing parameters such as the volume density of the vascular bed to be quantified.