In situ hybridization and immunocytochemical localization of SERCA2 encoded Ca2+ pump in rabbit heart and stomach

Heart tissue contains large amounts of the protein encoded by the Ca²⁺ pump gene SERCA2. The SERCA2 RNA can be spliced alternatively to produce mRNA encoding the proteins SERCA2a and SERCA2b which differ in their C-terminal sequences. In this study we report the tissue distribution of SERCA2a and SERCA2b isoforms byin situ hybridization to rabbit heart and stomach. The expression of SERCA2 mRNA was high in myocardial cells, being the highest in the atrial region. In contrast, there was more SERCA2 protein in Western blots in ventricles than in atria. Myocardial cells expressed predominantly the mRNA for the isoform SERCA2a. Whereas the stomach smooth muscle and the neuronal plexus expressed SERCA2 at levels much lower than myocardial cells, the expression was very high in the stomach mucosa. Mucosa contained mainly the mRNA for SERCA2b. From immunocytochemistry it was concluded that the anti-heart SR Ca²⁺ pump antibody IID8 reacted much better with heart and surface mucosal cells in the stomach than with the stomach smooth muscle, and that IID8 reactivity was intracellular. In contrast PM4A2B, an antibody against the plasma membrane Ca²⁺ pump, reacted well with heart and stomach smooth muscle, plexus and mucosa, and its localization appeared to be in the plasma membrane. Thus, stomach smooth muscle expressed SERCA2b mRNA and protein at low levels, mucosa expressed SERCA2b mRNA and protein at high levels, atria and ventricle expressed SERCA2a mRNA and protein at high levels, mRNA being more in atria, but protein being more in ventricles.Deceased August 14, 1992     

INFLUENCE OF CELL SHAPE AND SIZE ON ALGAL COMPETITIVE ABILITY1

Allometric relations between physiological processes and cell volume and surface area are combined with the variable-internal-stores model of growth to predict the ability of hypothetical phytoplankton to compete for phosphorus at equilibrium. The analysis shows that for spherical cells, smaller cells are better competitors than large ones. For cells that are very elongated in shape, however, large cells are often better competitors than small ones. The cells predicted to be the best competitors compare favorably in size and shape with the species observed to dominate in phosphorus-limited chemostats at equilibrium.     

Immunological evidence for accumulation of two high-molecular-weight (104 and 90 kDa) HSPs in response to different stresses in rice and in response to high temperature stress in diverse plant genera

Rice seedlings accumulate stainable amounts of the 104 and 90 kDa polypeptides in response to high temperature stress. We have purified and raised highly specific polyclonal antisera against both of these polypeptides. In western blotting experiments, we find that these proteins are accumulated to different extents in rice seedlings subjected to salinity (NaCl), water stress, low-temperature stress and exogenous abscisic acid application. These proteins also accumulated when rice seedlings grown in pots under natural conditions were subjected to water stress by withholding watering. Seedlings of Triticum aestivum, Sorghum bicolor, Pisum sativum, Zea mays, Brassica juncea and mycelium of Neurospora crassa showed accumulation of the immunological homologues of both the 104 and the 90 kDa polypeptides, in response to high-temperature stress. We have earlier shown that shoots of rice seedlings exposed to heat shock accumulate a 110 kDa polypeptide which is an immunological homologue of the yeast HSP 104 (Singla and Grover, Plant Mol Biol 22: 1177–1180, 1993). Employing anti-rice HSP 104 antibodies and anti-yeast HSP 104 antibodies together, we provide evidence that rice HSP 104 is different from the earlier characterized rice HSP 110.     

Mg-activated double-stranded DNA cleavage by [(bpy)2(OH2)Ru---O---Ru(H2O) (bpy)2]

The reaction of the title complex with DNA has been examined. Addition of [(bpy)₂(OH₂)RuORu(OH₂) (bpy)₂]⁴⁺ to DNA leads to the reduction of the complex to Ru(bpy)₂(OH₂)₂²⁺, as indicated by absorption spectroscopy and cyclic voltammetry. The reaction is accelerated by Mg²⁺. The combined evidence points to a mechanism where the oxo-bridged dimer is hydrolyzed to a monomeric Ru(III) complex that is capable of oxidizing DNA to effect strand scission. Gel electrophoresis demonstrates nicking of supercoiled /gfX174 DNA by [(bpy)₂(OH₂)RuORu(OH₂) (bpy)₂]⁴⁺, and double-stranded cleavage is observed in the presence of Mg²⁺. Linearization of the plasmid prior to treatment with the complex does not lead to further fragmentation, suggesting that supercoiling is required to realize double-stranded cleavage.     

Morphometrics of Infective Juveniles of Steinernema spp. and Heterorhabditis bacteriophora (Nemata: Rhabditida)

Selected morphometrics of Heterorhabditis bacteriophora and seven species of Steinernema from in vivo culture were compared in relation to time of harvest. In addition, five Steinernema species were reared in vitro and their morphometrics were compared with those from in vivo culture. With in vivo culture, there was generally a negative linear relationship between body length of infective juveniles (IJ) and time of harvest. The distance from the anterior end to the excretory pore (EP) and the tail length (T) of IJ also varied with time of harvest. The E percentage (= EP/T x 100) was the least variable. Body lengths of IJ reared in vitro were much less than those of IJ reared in vivo. The study suggests that IJ harvested from in vivo culture within 1 week of emergence from cadavers are best for species identification. Infective juveniles from in vitro culture should not be used for species identification.     

Effect of treatment with the MER tubercle bacillus fraction on syngeneic plasma cell tumors of BALB/c mice

Summary MER administered prophylactically prolonged the survival of BALB/c mice challenged with transplants of syngeneic plasmacytomas to a moderate but significant extent. In contrast, MER exerted little therapeutic action when given alone at or after tumor implantation.Combined treatment, with MER introduced prophylactically and cyclophopshamide (CY) after tumor implantation, decreased the incidence of recurrence of one of the tumors tested (MPC-11 NP, a non-myeloma protein producer) and prolonged host survival significantly as compared with animals subjected to CY therapy alone. When, instead, MER was introduced at the time of tumor challenge or thereafter to animals also treated with CY, the therapeutic response was not appreciably different from that of mice under therapy with CY only. With regard to the second plasmacytoma (MPC-11 P, a myeloma protein producer), mice treated with CY and given MER prior to or after challenge showed similar responses to animals given chemotherapy only; when MER was injected at the time of challenge and CY thereafter, the chemoimmunotherapy was somewhat inferior to chemotherapy alone.This work was supported by US Public Health Service Contract NO1-CM-12127, and by grants from Mrs. J. H. Hazen, Ruth Estrin Goldberg Memorial for Cancer Research, Leukemia Research Foundation, Inc., Concern Foundation, Inc., and Mr. and Mrs. M. Gordon     

METABOLIC SEGREGATION OF INTRACELLULAR FREE AMINO ACIDS IN PLATYMONAS (CHLOROPHYTA)1

¹⁴C-amino acids were supplied to Platymonas subcordiformis (Wille) Hazen and the incorporation of radioactivity into protein and other compounds was followed. Alanine was rapidly metabolized by both N-limited and N-sufficient cells. Arginine and lysine were metabolized rapidly by N-limited cells, but were sequestered from metabolism in N-sufficient cells. This suggests the existence of two functionally distinct pools; a “metabolic” pool that is rapidly metabolized and preferentially used for incorporation into protein, and a “storage” pool rich in basic amino acids that is sequestered from metabolism.     

Hydroxyl radical production in a purified NADPH--cytochrome c (P-450) reductase system.

Using the spin trap, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) we have demonstrated that hydroxyl radicals are generated indirectly from purified preparations of rat liver microsomal NADPH-cytochrome c (P-450) reductase during NADPH oxidation. Hydroxyl radical formation is completely inhibited by p-chloromercuribenzoate, but not by metyrapone. In addition, hydroxyl radical DMPO adduct formation is blocked by added linolenic acid which, in turn, is peroxidatively degraded into malondialdehyde, suggesting that hydroxyl radicals formed from purified NADPH-cytochrome c (P-450) reductase are capable of initiating lipid peroxidation. A mechanism for the indirect production of hydroxyl radicals from NADPH-cytochrome P-450 reductase is discussed.     

Effect of DNA repair on the cytotoxicity and mutagenicity of polycyclic hydrocarbon derivatives in normal and xeroderma pigmentosum human fibroblasts

The cytotoxicity of the “K-region” epoxides as well as several other reactive metabolites or chemical derivatives of polycyclic hydrocarbons was compared in normally-repairing human diploid skin fibroblasts and in fibroblasts from a classical xeroderma pigmentosum (XP) patient (XP2BE) whose cells have been shown to carry out excision repair of damage induced in DNA by ultraviolet (UV) radiation at a rate approx. 20% that of normal cells. Each compound tested exhibited a 2- to 3-fold greater cytotoxicity in this XP strain than in the normal strain. To determine whether this difference in survival reflected a difference in the capacity of the strains to repair DNA damage caused by such hydrocarbon derivatives, we compared the cytotoxic effect of several “K-region” epoxides in two additional XP strains, each with a different capacity for repair of UV damage. The ration of the slopes of the survival curves for each of the XP strains to that of the normal strain, following exposure to each epoxide, was very similar to that which we had previously determined for their respective UV curves, suggesting that human cells repair damage induced in DNA by exposure to hydrocarbon derivatives with the same system used for UV-induced lesions.To determine whether the deficiency in rate of excision repair in this classical XP strain (XP2BE) causes such cells to be abnormally susceptible to mutations induced by “K-region” epoxides of polycyclic hydrocarbons, we compared them with normal cells for the frequency of induced mutations to 8-azaguanine resistance. The XP cells were two to three times more susceptible to mutations induced by the “K-region” epoxide of benzo(a)pyrene (BP), 7,12-dimethylbenz(a)anthracene (DMBA), and dibenz(a,h)anthracene (DBA). Evidence also was obtained that cells from an XP variant patient are abnormally susceptible to mutations induced by hydrocarbon epoxides and, as is the case following exposure to UV, are abnormally slow in converting low molecular weight DNA, synthesized from a template following exposure to hydrocarbon epoxides, into large-size DNA.