Localization of phospholamban in smooth muscle using immunogold electron microscopy

Phospholamban, the putative regulator of the Ca2+-ATPase in cardiac sarcoplasmic reticulum, was immunolocalized in canine visceral and vascular smooth muscle. Gently disrupted tissues were labeled with an affinity-purified phospholamban polyclonal antibody and indirect immunogold, using preembedding techniques. The sarcoplasmic reticulum of smooth muscle cells was specifically labeled with patches of immunogold distributed in a nonuniform fashion, while the sarcolemma did not appear to contain any phospholamban. The outer nuclear envelopes were also observed to be heavily labeled with the affinity-purified phospholamban polyclonal antibody. These findings suggest that phospholamban may play a role in the regulation of cytoplasmic and intranuclear calcium levels in smooth muscle cells.     

IL-1 is an autocrine growth factor for T cell clones

Activation of Th lymphocytes requires that Ag be presented on the surface of accessory cells displaying Ia Ag. A number of studies have concluded that the T cell also requires IL-1 from accessory cells. However, we recently reported that one murine T cell clone (D10.G4.1) produced its own IL-1-like activity after encountering APC (9). In this report, we demonstrate that 1) IL-1 production is a common property of murine T cell clones, 2) T cell IL-1 activity is blocked by anti-IL-1-alpha antiserum, 3) IL-1-alpha mRNA can be directly visualized in individual cloned T cells using in situ hybridization techniques, and 4) IL-1 appears to serve an autocrine role in the activation of T cell clones inasmuch as anti-IL-1-alpha antiserum blocks cell proliferation when the T cell is the only IL-1 source.     

Glucose transport into rat skeletal muscle: interaction between exercise and insulin

This study was done to evaluate the effect of insulin on sugar transport into skeletal muscle after exercise. The permeability of rat epitrochlearis muscle to 3-O-methylglucose (3-MG) was measured after exposure to a range of insulin concentrations 30, 60, and 180 min after a bout of exercise. Thirty and 60 min after exercise, the effects of exercise and insulin on 3-MG transport were additive over a wide range of insulin concentrations, with no increase in sensitivity or responsiveness to insulin. After 180 min, when approximately 66% of the exercise-induced increase in sugar transport had worn off, both the responsiveness and sensitivity of the glucose transport process to insulin were increased. These findings appear compatible with the hypothesis that the actions of exercise and insulin result in activation and/or translocation into the plasma membrane of two separate pools of glucose transporters in mammalian skeletal muscle.     

The ontogeny of the vascular cambium inGinkgo biloba roots

Observation was made on early ontogeny of vascular cambium in the developing root ofGinkgo biloba L. After completion of root elongation, the vascular meristem gradually acquires cambial characteristics. Strips of the periclinal division of cells in transverse section are observed on the inner side of phloem when the primary xylem and phloem in the stele have been established. The strips are united into a continuous layer between phloem and xylem. In tangenital section, the procambium shows a homogeneous structure, which is initially composed of short cells with transverse end walls and subsequently, of long cells with tapering ends. Then, the procambium is organized into two systems of cells; axial strands of short cells with transverse end walls resulting from the sporadic transverse divisions of long cells, and long cells with tapering ends. Still later, the short cells are divided frequently in a trasverse plane exhibiting one or a few cells in width and several decades of cells in height, while the long cells are elongated. The frequency of transverse divisions of the short cells decreases in subsequent stages. Eventually, the short cells in axial strands are vertically separated from one another by the elongation of neighboring long cells and by the decrease in the frequency of transverse divisions of short cells themselves. Cambial initials occur in two forms; ray initials a few cells in height and one cell in width derived from the short cells, and fusiform initials with tapering ends derived from the long cells.     

Gene amplification at a locus encoding a putative Na+/H+ antiporter confers sodium and lithium tolerance in fission yeast.

We have identified a new locus, sodium 2 (sod2) based on selection for increased LiCl tolerance in fission yeast, Schizosaccharomyces pombe. Tolerant strains have enhanced pH-dependent Na+ export capacity and sodium transport experiments suggest that the gene encodes an Na+/H+ antiport. The predicted sod2 gene product can be placed in the broad class of transporters which possess 12 hydrophobic transmembrane domains. The protein shows some sequence similarity to the human and bacterial Na+/H+ antiporters. Overexpression of sod2 increased Na+ export capacity and conferred sodium tolerance. Osmotolerance was not affected and sod2 cells were unaffected for growth in K+. In a sod2 disruption strain cells were incapable of exporting sodium. They were hypersensitive to Na+ or Li+ and could not grow under conditions that approximate pH7. The sod2 gene amplification could be selected stepwise and the degree of such amplification correlated with the level of Na+ or Li+ tolerance.     

A mutation outside the two zinc fingers of ADR1 can suppress defects in either finger.

A second-site mutation that restored DNA binding to ADR1 mutants altered at different positions in the two zinc fingers was identified. This mutation (called IS1) was a conservative change of arginine 91 to lysine in a region amino terminal to the two zinc fingers and known from previous experiments to be necessary for DNA binding. IS1 increased binding to the UAS1 sequence two- to sevenfold for various ADR1 mutants and twofold for wild-type ADR1. The change of arginine 91 to glycine decreased binding twofold, suggesting that this arginine is involved in DNA binding in the wild-type protein. The increase in binding by IS1 did not involve protein-protein interactions between the two ADR1 monomers, nor did it require the presence of the sequences flanking UAS1. However, the effect of IS1 was influenced by the sequence of the first finger, suggesting that interactions between the region amino terminal to the fingers and the fingers themselves could exist. A model for the role of the amino-terminal region based on these results and sequence homologies with other DNA-binding motifs is proposed.     

Genotype of Yupik Eskimos with congenital adrenal hyperplasia due to 21-hydroxylase deficiency

Summary An A-to-G transition in the second intron was the sole mutation detected in four Yupik Eskimo patients with salt-wasting congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency. Allele-specific hybridization should be an efficient means of performing prenatal diagnosis of the disease in this highly inbred population.     

In vitro shoot regeneration from leaf mesophyll protoplasts of hybrid poplar (Populus nigra x P. maximowiczii)

Summary Protoplasts were isolated from leaf mesophyll of hybrid poplar (Populus nigra X P. maximowiczii) with a mean yield of 10.4 x 10⁶ protoplasts per g fresh weight using 2.0% Cellulase Onozuka R-10, 0.8% Macerozyme R-10, 1.2% Hemicellulase, 2.0% Driselase, and 0.05% Pectolyase Y-23 with CPW salts solution containing 0.6 M mannitol, 0.002 M DTT, 3 mM MES at pH 5.6. A liquid plating method produced the highest frequency of dividing protoplasts (48.6%) using an MS medium without NH₄NO₃. The highest percent of colony formation was 22.8%, produced with fabric supported semi-solid (0.5% w/v) agar plating method using the same culture medium. Growing cell colonies and/or micro-calli were transferred to a fresh semisolid agar medium containing 0.44 M BAP and 9.0 M 2,4-D. Multiple shoots were produced from protoplast-derived callus after culture on MS medium containing 6.8 M zeatin. After root induction on half-strength MS medium that lacked growth regulators, shoots were transferred to pots containing artificial soil mix.Abbreviations CPW Cell and Potoplast Wash solution - LPM Liquid Plating Method - LDM Liquid Drop Method - HDM Hanging Drop Method - FSPM Fabric supported Semi-solid agar Plating Method - DTT Dithiothreitol - MES 2-(N-morpholino) ethane sulfonic acid - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy acetic acid - NAA -naphthalene acetic acid - MS Murashige and Skoog (1962)     

Characterization of D2 receptors and dopamine levels in the thalamus of the rat.

We kinetically characterized D2 receptors in thalami pooled from a group of Sprague-Dawley rats and then determined thalamic levels of dopamine (DA), homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC), and norepinephrine (NE) in relation to a measure of thalamic DA D2 receptor densities in another group of rats. The equilibrium dissociation constant (kd) was estimated as 0.1 nM by three independent methods, while the Bmax for thalamic D2 receptors was found to be 6.4 fmol/mg p using 3H-spiperone as ligand and ketanserin to occlude 5HT2 binding. Kinetic constants were in agreement with previously reported kinetic data from rodent caudate-putamen. This suggests that thalamic D2 receptors are similar to D2 receptors from other brain areas. Mean thalamic levels of DA (22.6 ng/mg p), DOPAC (1.19 ng/mg p) and HVA (0.31 ng/mg p) concur with previous reports of a sparse distribution of thalamic DA neurons. D2 receptor densities were positively correlated with DA metabolites DOPAC (P less than .05; r = 0.423) and HVA (P less than .05; r = 0.368), but not DA or NE. These results establish fundamental characteristics of thalamic DA neurotransmission to assist in the investigation of behavioral pharmacology of this area.