Epitope tag mapping of the extracellular and cytoplasmic domains of the rat parathyroid hormone (PTH)/PTH-related peptide receptor

The PTH/PTH-related peptide (PTHrP) receptor is predicted to span the plasma membrane seven times with an amino-terminal extracellular extension and a cytoplasmic carboxyl-terminal tail. To assess this prediction, we inserted 10- or 9-amino acid epitope tags from c-myc or hemophilus influenza hemaglutinin (HA), which are recognized by the monoclonal antibodies 9E10 and 12Ca5, respectively, in different extracellular and cytoplasmic regions of the receptor and examined the immunoreactivity of the epitopes in intact and permeabilized cells. The data show that the epitopes were well tolerated when introduced into the E2 region of the extracellular amino-terminus (E2-myc and E2-HA), in the first extracellular loop (EL1), in the second and third cytoplasmic loops (CL2c and CL3), or in the carboxyl-terminal tail (T-myc). Receptors tagged at these locations were well expressed, bound PTH with high affinity, and increased cAMP accumulation with a good efficiency. Receptors tagged in the second and third extracellular loops (EL2c and EL3c) or the first cytoplasmic loop (CL1c) bound the PTH radioligand with a low affinity, stimulated cAMP accumulation with a low efficiency, and had low expression levels. The receptors tagged on presumed extracellular regions, E2-myc, E2-HA, EL1, EL2c, and EL3c, were readily detected on the surface of intact cells with the monoclonal antibody against the epitope tag. In contrast, receptors tagged with the c-myc epitope in the cytoplasmic loops (CL1c, CL2c, and CL3) or in the carboxyl-terminal tail (T-myc) did not show any 9E10 binding in intact cells. These receptors, however, were well expressed on the cell surface, as detected by the binding of the monoclonal antibody, 12Ca5, to the HA tag that was introduced into the E2 region of these constructs. The c-myc epitopes, however, became accessible after permeabilization of the cell membrane. In conclusion, these data provide experimental evidence for the sidedness of the extracellular and cytoplasmic domains of the PTH/PTHrP receptor.     

Two-year follow-up of directly-observed intermittent regimens for smear-positive pulmonary tuberculosis in China

SETTING: The tuberculosis component of the Infectious and Endemic Disease Control Project in the People's Republic of China is the largest single tuberculosis control project in the world using directly-observed therapy and standardized intermittent regimens. OBJECTIVE: To determine the two-year relapse and mortality rates following completion of treatment. DESIGN: A prospective cohort study of 649 cases cured in this project. The 306 new and 343 retreatment cases were treated under field conditions with 2H3R3Z3S3/4H3R3 and 2H3R3Z3E3S3/6H3R3E3, respectively. Following treatment completion, two sputum samples were collected every six months for two years and examined for acid-fast bacilli. Causes of death were identified. RESULTS: The two-year relapse rates for new and retreatment cases were 3.3% and 5.6%, respectively. Retreatment cases with delayed sputum conversion had a greater risk for subsequent relapse. The two-year mortality rate for new and retreatment cases was 3.3% and 8.5%, respectively. The higher mortality rate in retreatment cases was not attributable to relapse of disease, but rather to non-infectious sequelae of tuberculosis. CONCLUSION: The use of directly-observed intermittent regimens is effective in permanently removing infectious tuberculosis cases from the community.     

The influence of hypoxia on the cytotoxicity of concomitant KW-2149 and ionizing irradiation in Chinese hamster fibroblasts

7-N-((2-((2-(gamma-L-glutamylamino)ethyl)dithio)ethyl))-mitomycin C (KW-2149) is a newly synthesized water-soluble mitomycin C (MMC) analog. Preclinical testing showed an interesting activity profile and a superior hematological tolerance in murine models. The aim of this study was to investigate the interaction of this compound with ionizing radiation, both under normoxic and hypoxic conditions, in Chinese hamster fibroblasts (V79). V79 cells were irradiated both under normoxic conditions and after a 1 h period of hypoxia. Paired irradiation dose-response curves confirmed the significance of radioresistance under hypoxia with an oxygen enhancement ratio of approximately 3. In contrast to MMC, KW-2149 showed no increased cytotoxic effect on hypoxic V79 cells. The cytotoxic effect of KW-2149 increased with increasing concentration, irrespective of the ambient oxygen pressure. When KW-2149 was combined with irradiation under hypoxic conditions, cytotoxicity was significantly enhanced under these conditions. The difference in survival between normoxic and hypoxic conditions was statistically significant (p < 0.004). These data suggest a radiosensitizing effect of KW-2149, more pronounced under hypoxic conditions. This effect increases with radiation dose. It also corroborates earlier suggestions of a different mode of action of KW-2149 as compared to MMC.     

hSK4, a member of a novel subfamily of calcium-activated potassium channels

The gene for hSK4, a novel human small conductance calcium-activated potassium channel, or SK channel, has been identified and expressed in Chinese hamster ovary cells. In physiological saline hSK4 generates a conductance of approximately 12 pS, a value in close agreement with that of other cloned SK channels. Like other members of this family, the polypeptide encoded by hSK4 contains a previously unnoted leucine zipper-like domain in its C terminus of unknown function. hSK4 appears unique, however, in its very high affinity for Ca2+ (EC50 of 95 nM) and its predominant expression in nonexcitable tissues of adult animals. Together with the relatively low homology of hSK4 to other SK channel polypeptides (approximately 40% identical), these data suggest that hSK4 belongs to a novel subfamily of SK channels.     

Treatment of sepsis by extracorporeal elimination of endotoxin using fiber-immobilized polymyxin B

To remove endotoxin directly from the blood, an affinity adsorbent (PMX) has been developed. PMX is composed of a ligand of polymyxin B and a carrier of polystyrene fibers. We treated 16 patients, suffering from septic shock or multiple organ failure by PMX-F hemoperfusion. The pretreatment level of endotoxin decreased significantly from 80pg/ml to 21pg/ml on average 2 hours after the hemoperfusion. The hyperdynamic status in the cardiac index and the decreased systemic vascular resistance, which are characteristic to endotoxin shock, were normalized after the treatment. In septic shock patients with systolic pressure under 10mmHg, the systolic pressure increased significantly from pretreatment level. Nine of the sixteen patients remained alive for two weeks after the therapy, and seven out of the nine patients discharged alive. Hemoperfusion with PMX is likely to become an effective treatment for sepsis and septic shock.     

Reinterpretation of luminiscence properties of neurotoxins from the venom of Middle-Asian corba Naja oxiana eichw

A new interpretation of previous work (Bukolova-Orlova, T. G., Burstein, E.A. and Yukelson, L. Ya (1974) Biochim. Biophys. Acta 342, 272-280) and some new data on the luminescence of neurotoxins I and II from Naja oxiana venom is given, based on the newer data on their complete amino acid sequences. Very effective excitation energy exchange exists between Trp-27 and Trp-33 in neurotoxin I and between Trp-27 and Trp-28 in neurotoxin II, Which results in the tryptophanyl fluorescence spectra of each of the proteins seeming to be monocomponent ones. The lowered fluorescence quantium yield value, the shortened phosphorescence lifetime (80% of the emission has tau p less than 0.5 s, 20% has tau p = 4.8 s, comparing with usual tau p = 5.5-5.9 s) and decreased phosphorescence to fluorescence ratio (0.042, as compared to the usual 0.4-0.7) for neurotoxin I suggest that the indole chromophore of Trp-27 and/or Trp-33 are in contact with heavy sulfur atoms of disulfide, most probably of Cys(28)-Cys(32). Tryptophanyls in neurotoxin II are exposed to the solvent, however their accessibility in relation to that of the free tryptophan to the negatively charged quencher I- (0.455) is much higher than that for the positively charged Cs+ (0.08), which is probably due to the proximity of cationic Lys-25, Lys-26 and His-31. The difference of accessibility to the negative and positive quenchers is even more pronounced in the case of the neurotoxin I (1.04 and 0 +/- 0.02, respectively), though in its chromophore vicinity along the primary structure there is only one cationic group, Lys-25. This fact together with the analysis of the amino acid sequence, suggest that the space folding of this polypeptide results in the close proximity of Trp-27 and/or Trp-33 with the C-terminal peptide segment 67-73, which contains four cationic groups (His-67, Lys-69, Lys-71 and Arg-72).     

Lead Oxide Coatings on Sol–Gel-Derived Lead Lanthanum Zirconium Titanate Thin Layers for Enhanced Crystallization into the Perovskite Structure

An improved method is described for the sol–gel preparation of PLZT thin layers in the perovskite structure. The method uses a PbO cover coat. Details are reported for the sol-gel processing route and heat-treatment conditions. Through use of this method it is possible to prepare singlephase perovskite material with improved properties. The deleterious effect of additional phases—which are not present when a PbO cover coat is used—is attributed to Pb loss from the surface during thermal processing. Examples are given for PLZT thin layers integrated on Si with and without a PbO cover coat. The dielectric and ferroelectric properties were always found to be superior for coated structures.