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.     

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.     

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.     

Biocompatibility of radiolucent breast implants

Current implants for breast augmentation containing silicone gel, saline, or both are radiopaque on mammographic examination and can totally obscure microcalcifications and soft-tissue masses. The effect of these implants on the detection of early breast cancers in patients who have undergone augmentation mammaplasty remains unproven and controversial. Implants filled with medium-chain triglycerides (peanut oil) are radiolucent on mammographic examination and allow visualization of both soft-tissue masses and microcalcifications. To investigate the biocompatibility of radiolucent implants, 10 cc of sterile, nonpyrogenic peanut oil was injected subcutaneously into rats using silicone gel as a control. Twenty-one rabbits had two 125-cc silicone shell implants inserted on either side of the chest wall. The right-sided shell was filled with 125 cc of sterile saline, and the left-sided shell was filled with 125 cc of sterile, nonpyrogenic peanut oil. Results were determined by both histologic and radiographic examination. Rats injected with peanut oil equivalent to 7 percent of their body weight rapidly absorbed the freely injected oil without detriment. Histologic examination of the lungs, liver, kidneys, and tissues adjacent to the injection sites demonstrated no abnormalities. There was no evidence of allergic, toxic, inflammatory, or neoplastic response. Eighteen of 21 rabbits survived more than 3 months. Radiographs showed the oil-filled implants to be radiolucent, whereas the saline-filled controls obscured the surrounding soft and bony tissues. Histologic examination demonstrated a fibrous capsule surrounding both types of implants. Histologic examination of the lungs, liver, and kidneys showed no significant abnormalities. These and previous studies have shown peanut oil to be biocompatible when freely injected either intramuscularly or subcutaneously. This study demonstrates that a radiolucent, peanut oil-filled implant is biocompatible in animals and that further long-term studies for its use in humans are merited.     

Oxygen transport and peripheral microcirculation in long-term diabetes

The purpose of this investigation was to evaluate the impact of long-term diabetes on muscle blood flow (MBF) and oxygen transport (vO2) during exercise. Twelve male patients (58 +/- 8 years, mean +/- SD), with at least a 10-year history of diabetes controlled by insulin, and seven age-matched controls (56 +/- 5 years, mean +/- SD) participated in this study. No patient had been clinically diagnosed as having peripheral vascular disease, and on the average resting ankle/arm systolic blood pressure ratios were normal. Following a baseline period, 5 min of cycle ergometer exercises at 75 W were performed in the upright position and, after 1-hr recovery, in the supine position. Continuous vO2 was determined via breath-by-breath analysis. MBF was measured in the vastus lateralis (VL) and tibialis anterior (TA) by 133Xe clearance. In the erect position, the diabetic group (compared with the control group, respectively) exhibited significantly (P less than 0.05) lower exercise MBF [ml. (100 g.min)-1] in both VL (19 +/- 2.5 vs 30.9 +/- 2) and TA (13.7 +/- 2 vs 22.0 +/- 4), a lower steady-state VO2 (1.3 +/- 0.3 vs 1.7 +/- 0.2 liters.min-1) during exercise including the values in the last 15 sec of exercise, and greater accumulation of blood lactate (35 +/- 2 vs 22.0 +/- 2 mg/100 ml). The same trends in the data were observed during supine exercise; however, the blood pressure of the diabetics was significantly elevated during exercise when compared with that of controls. The reduced exercise MBF in the TA and VL demonstrated that impaired microvascular flow, without clinically overt peripheral vascular disease, in long-term diabetics leads to reduced oxygen delivery and exercise tolerance.     

Degradation of toluene and m-xylene and transformation of o-xylene by denitrifying enrichment cultures

Seven different sources of inocula that included sediments, contaminated soils, groundwater, process effluent, and sludge were used to establish enrichment cultures of denitrifying bacteria on benzene, toluene, and xylenes in the absence of molecular oxygen. All of the enrichment cultures demonstrated complete depletion of toluene and partial depletion of o-xylene within 3 months of incubation. The depletion of o-xylene was correlated to and dependent on the metabolism of toluene. No losses of benzene, p-xylene, or m-xylene were observed in these initial enrichment cultures. However, m-xylene was degraded by a subculture that was incubated on m-xylene alone. Complete carbon, nitrogen, and electron balances were determined for the degradation of toluene and m-xylene. These balances showed that these compounds were mineralized with greater than 50% conversion to CO2 and significant assimilation into biomass. Additionally, the oxidation of these compounds was shown to be dependent on nitrate reduction and denitrification. These microbial degradative capabilities appear to be widespread, since the widely varied inoculum sources all yielded similar results.     

Co-localization of nitric oxide synthase immunoreactivity and NADPH diaphorase staining in neurons of the guinea-pig intestine

Summary Neuronal nitric oxide synthase (NOS), an enzyme capable of synthesizing nitric oxide, appears to be identical to neuronal NADPH diaphorase. The correlation was examined between NOS immunoreactivity and NADPH diaphorase staining in neurons of the ileum and colon of the guinea-pig. There was a one-to-one correlation between NOS immunoreactivity and NADPH diaphorase staining in all neurons examined; even the relative staining intensities obtained were similar with each technique. To determine whether pharmacological methods could be employed to demonstrate that NADPH diaphorase staining was due to the presence of NOS, tissue was pre-treated with N^G-nitro-l-arginine, a NOS inhibitor, or l-arginine, a natural substrate of NOS. In these experiments on unfixed tissue, it was necessary to use dimethyl thiazolyl tetrazolium instead of nitroblue tetrazolium as the substrate for the NADPH diaphorase histochemical reaction. Neither treatment caused a significant decrease in the level of NADPH diaphorase staining, implying that arginine and NADPH interact at different sites on the enzyme.     

Purification and properties of intracellular fructosyl transferase fromAureobasidium pullulans

Intracellular frustosyl transferase was purified fromAureobasidium pullulans C-23 by ethanol fractionation, CM-Sephadex chromatography and preparative disc gel electrophoresis. It was shown to be homogeneous on disc polyacrylamide gel electrophoresis, with a molecular size of 190kDa. The pI value of the enzyme was about 3.7. The enzyme has aK _m value of 0.43 mM for sucrose and was optimally active at pH 5.0 and 60°C. The enzyme was stable from pH 2.5 to 12. It was almost completely inhibited by 5mM Hg²⁺ but was not significantly affected by other cations. The transferase was inactivated by treatment with the tryptophan-specific reagentN-bromosuccinimide and the tyrosine-specific reagent, I₂, suggesting that tryptophan and tyrosine residues are probably located at or near the active site of the enzyme.