Suppression of pacemaker activity by rapid repetitive phase delay

Spontaneous activity of pacemaker cells or structures may be suppressed by rapid repetitive stimulation. Conditions are that the oscillator's phase reset curve, characterizing the phase resetting effect of single stimuli, has a phase delay part and that the interval between the stimuli falls within a range of values, determined by the form oo the phase reset curve. Under these conditions, which appeared the same as those for stable underdrive pacing, the pacemaker becomes stably entrained to the stimuli without firing, i.e. it is kept within a certian part of its limit cycle because the pulses repeatedly delay the next coming action potential. This rapid stimulation suppression of pacemaker activity is demonstrated experimentally on a simple electronic pacemaker cell model for two types of phase reset curves, a biphasic one for depolarizing and a monophasic one for hyperpolarizing pulses. Computer simulations of coupled pacemaker cells, interacting by phase reset curves, illustrate how this type of pacemaker suppression may protect a population of pacemaker cells like the sinus node in the heart against arrhythmias.Supported by the Netherlands Foundation for Medical Research FUNGOSupported by the Netherlands Organization for the Advancement of Pure Research ZWO     

Modulation of protein levels in chromosomal dosage series of maize: the biochemical basis of aneuploid syndromes

Genetically defined dosage series of chromosome arms 1L, 3L, 4S, 5L, 7L, 9S, 10L and combinations of 1L-3L, collectively spanning approximately one-third of the maize genome, were examined for alterations in the expression of total protein profiles in scutellar tissue. The major effects found were negative correlations of specific proteins with the dosage of particular regions in a manner similar to that previously described for enzyme activity levels (Birchler 1979). Chromosome arms 1L, 4S and 5L produced the most severe negative effects, with 3L and 7L exhibiting this phenomenon to a lesser degree. Positive correlations of certain proteins were observed with the dosage of the 1L, 3L, 5L and 7L regions. The structural locus of one of the major scutellar proteins (PRO) is present in the long arm of chromosome 1 (Schwartz 1979), but exhibits compensation in a dosage series involving whole-arm comparisons. Multiple factors in 1L affect the level of the protein. The compound TB-1La-3L4759-3 (1L 0.20-0.39) has a slight negative effect on PRO, while TB-1La-3Le (1L 0.20-0.58) and TB-1La-3L5267 (1L 0.20-0.72) have a more pronounced negative influence. The level of this protein is not altered by the dosage of 3L. These observations suggest that compensation is brought about by the cancellation of a positive structural gene dosage effect by the negative inverse effect. Other regions of the genome that contribute to the control of PRO levels are 4S and 5L. Total protein profiles were also compared in haploid, diploid and tetraploid maize as a comparison to the aneuploid series. Most proteins exhibit structural-gene-dosage effects through the ploidy series, but others show a positive effect greater than expected from varying the structural genes. Still others are negatively affected by ploidy changes. In general, the ploidy alterations are not as great as predicted from the cumulative action of the aneuploid effects. The bearing of these observations on the biochemical basis of aneuploid syndromes is discussed.     

Development of bacterial spoilage at adipose tissue surfaces of fresh meat.

Adipose tissue contains low-molecular-weight soluble substances which are utilized in preference to lipid for bacterial growth. These components are present in low concentration at the surface of adipose tissue, and the pH of the surface is high (greater than 7.0). Bacteria growing on a thin layer of agar over an adipose tissue surface utilized glucose preferentially, but this was soon exhausted in the vicinity of colonies. Amino acids were then attacked, producing malodorous substances which were detectable as spoilage odors when the cell density was about 10(6)/cm2. Growth ceased at a cell density approaching 10(8)/cm2 because of substrate limitation. Bacterial lipolytic activity is not necessary for the development of bacterial spoilage of adipose tissue.     

Experimental confirmation of ecosystem model predictions comparing transient and equilibrium plant responses to elevated atmospheric CO2

Ecosystem models predict that short-term responses to elevated atmospheric CO₂ may differ substantially from the "real" long-term responses expected at equilibrium. Experimental validation of these model predictions is difficult as the data available are from short-term studies that do not include biogeochemical feedbacks typical of long-term exposure. Using a reciprocal transplant design at a natural CO₂ spring, we generated combinations of atmospheric and soil conditions that represented both short- and long-term elevated CO₂ conditions. Plant responses were significantly different between these treatments, confirming model predictions that there is not a simple relationship between transient and equilibrium responses to elevated CO₂.     

Genetic identification of multiple loci that control breast cancer susceptibility in the rat.

We have used a rat model of induced mammary carcinomas in an effort to identify breast cancer susceptibility genes. Using genetic crosses between the carcinoma-resistant Copenhagen (COP) and carcinoma-sensitive Wistar-Furth rats, we have confirmed the identification of the Mcs1 locus that modulates tumor number. We have now also identified two additional loci, Mcs2 and Mcs3. These three loci map to chromosomes 2, 7, and 1, respectively, and interact additively to suppress mammary carcinoma development in the COP strain. They are responsible for a major portion of the tumor-resistant phenotype of the COP rat. No loss of heterozygosity was observed surrounding the three loci. A fourth COP locus, Mcs4, has also been identified on chromosome 8 and acts in contrast to increase the number of carcinomas. These results show that mammary carcinoma susceptibility in the COP rat is a polygenic trait. Interestingly, a polymorphism in the human genomic region homologous to the rat Mcs4 region is associated with an increased breast cancer risk in African-American women. The isolation of the Mcs genes may help elucidate novel mechanisms of carcinogenesis, provide information important for human breast cancer risk estimation, and also provide unique drug discovery targets for breast cancer prevention.     

Assignment of Biochemical Functions to Glycosyl Transferase Genes Which Are Essential for Biosynthesis of Exopolysaccharides in Sphingomonas Strain S88 and Rhizobium leguminosarum

Glycosyl transferases which recognize identical substrates (nucleotide-sugars and lipid-linked carbohydrates) can substitute for one another in bacterial polysaccharide biosynthesis, even if the enzymes originate in different genera of bacteria. This substitution can be used to identify the substrate specificities of uncharacterized transferase genes. The spsK gene of Sphingomonas strain S88 and the pssDE genes of Rhizobium leguminosarum were identified as encoding glucuronosyl-(β1→4)-glucosyl transferases based on reciprocal genetic complementation of mutations in the spsK gene and the pssDE genes by segments of cloned DNA and by the SpsK-dependent incorporation of radioactive glucose (Glc) and glucuronic acid (GlcA) into lipid-linked disaccharides in EDTA-permeabilized cells. By contrast, glycosyl transferases which form alternative sugar linkages to the same substrate caused inhibition of polysaccharide synthesis or were deleterious or lethal in a foreign host. The negative effects also suggested specific substrate requirements: we propose that spsL codes for a glucosyl-(β1→4)-glucuronosyl transferase in Sphingomonas and that pssC codes for a glucuronosyl-(β1→4)-glucuronosyl transferase in R. leguminosarum. Finally, the complementation results indicate the order of attachment of sphingan main-chain sugars to the C₅₅-isoprenylphosphate carrier as -Glc-GlcA-Glc-isoprenylpyrophosphate.     

Embryonic growth rate and birth weight of the offspring of apterous and alate aphids: a cost of dispersal

Apterous and alate Sitobion avenae (Fab.) were dissected from the third instar onwards and the size and degree of development of their largest embryos recorded. From at least the third instar apterae had larger and more well developed embryos than alatae. Embryos in both morphs showed an exponential increase in volume with time during their mothers' nymphal development, but those in apterous mothers grew faster. Although alatae had a longer pre-reproductive development than apterae, it was not long enough to compensate for the lower embryo growth rates in alatae and as a consequence they initially produced smaller and fewer offspring than apterae. After 4 days of reproduction apterous and alate mothers produced similar sized offspring and at the same rate. The total fecundity of apterae was greater than that of alatae.

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Résumé La taille et l'état de développement des plus gros embryons de Sitobion avenae Fab ont été examinés par dissection d'ailés et aptères à partir du troisième stade. La taille des embryons des 2 morphes augmente pendant le développement larvaire des mères, mais ceux des mères aptères grossissent plus vite. Bien que la période précédant la reproduction des alates soit plus longue chez les ailés que chez les aptères, les embryons des ailés sont néanmoins incapables de l'emporter en taille sur les aptères, et les ailés produisent initialement moins de pucerons et plus petits que les aptères. Après 4 jours de reproduction, les mères ailées et aptères produisent des pucerons de même taille au même rythme. La fécondité totale des aptères est supérieure à celle des ailés; la différence est liée à la biologie des 2 morphes.

     

Rational immunotherapy with ribonuclease chimeras. An approach toward humanizing immunotoxins.

Members of the pancreatic ribonuclease (RNase) family have diverse activities toward RNA that could cause them to function during host defense and physiological cell death pathways. This activity could be harnessed by coupling RNases to cell binding ligands for the purpose of engineering them into cell-type specific cytotoxins. Therefore, the cytotoxic potential of RNase was explored by linking bovine pancreatic ribonuclease A via a disulfide bond to human transferrin or antibodies to the transferrin receptor. The RNase hybrid proteins were cytotoxic to K562 human erythroleukemia cells in vitro with an IC50 around 10(-7) M, whereas > 10(-4) M of native RNase was required to inhibit protein synthesis. Cytotoxicity required both components of the conjugate since excess transferrin or ribonuclease inhibitors added to the medium protected the cells from the transferrin-RNase toxicity. Importantly, the RNase conjugates were found to have potent antitumor effects in vivo. Chimeric RNase fusion proteins were also developed. F(ab')2-like antibody-enzyme fusions were prepared by linking the gene for human RNase to a chimeric antitransferrin receptor heavy chain gene. The antibody enzyme fusion gene was introduced into a transfectoma that secreted the chimeric light chain of the same antibody, and cell lines were cloned that synthesized and secreted the antibody-enzyme fusion protein of the expected size at a concentration of 1-5 ng/mL. Culture supernatants from clones secreting the fusion protein caused inhibition of growth and protein synthesis toward K562 cells that express the human transferrin receptor but not toward a nonhuman derived cell line. Since human ribonucleases coupled to antibodies also exhibited receptor mediated toxicities, a new approach to selective cell killing is provided. This may allow the development of new therapeutics for cancer treatment that exhibit less systemic toxicity and, importantly, less immunogenicity than the currently employed ligand-toxin conjugates.     

Effects of age, photoperiod and follicle-stimulating hormone on lactate production by cultured Sertoli cells from prepubertal Siberian hamsters (Phodopus sungorus).

To define a functional difference in Sertoli cells of animals exposed to different photoperiodic conditions, we isolated Sertoli cells from the testes of juvenile Siberian hamsters and cultured them in serum-free medium. In all age groups studied, Sertoli cells isolated from hamsters with delayed and normal puberty responded to follicle-stimulating hormone (FSH) with an increase in lactate production. The increase in lactate production induced by 1000 ng FSH ml-1 was significantly greater in Sertoli cells isolated from hamsters with delayed puberty than in those with normal puberty. These results suggest that Sertoli cells of Siberian hamsters exposed to short photoperiod in vivo may respond to increases in plasma FSH concentrations associated with photostimulation or spontaneous sexual maturation by an increase in secretory activity that may be critical for the initiation of spermatogenesis.     

Cytotoxic ribonuclease chimeras. Targeted tumoricidal activity in vitro and in vivo.

Monoclonal antibodies to the transferrin receptor or to the T cell antigen, CD5, were chemically linked to mammalian RNase A and found to specifically inhibit protein synthesis in antigen-positive cells. Antibody-mediated specificity of these cytotoxic ribonuclease chimeras (CRCs) was demonstrated in three ways. 1) Toxicity was due to the chemical linkage of RNase to antibody, as the individual components added separately or in combination did not inhibit protein synthesis; 2) the anti-transferrin receptor CRCs inhibited protein synthesis in those cells expressing the human transferrin receptor (K562, U251, Jurkat cells) but had no detectable toxicity to cells lacking the human transferrin receptor (Vero or NIH 3T3 cells); 3) free antibody to either the human transferrin receptor (454A12 or 5E-9) or to the T cell antigen, CD5 (T101), blocked the cytotoxicity of the respective CRC. Two CRC species, designated P1 and P2, that differed in size and stoichiometry of RNase A to antibody, were purified by size-exclusion high performance liquid chromatography. The higher molecular weight P1 conjugate had an IC50 of 20-30 nM, whereas the P2 conjugate had a higher IC50 of 300-500 nM. Bioactivity could be reversibly increased more than 10-fold by freezing. The cytotoxicity of the CRCs was examined in vivo in a solid tumor animal model. Intratumoral injections of an anti-transferrin receptor CRC into established U251 human glioblastoma tumors grown in the flanks of nude mice prevented tumor growth, whereas RNase A mixed with antibody was ineffective. CRCs, therefore, express cytotoxicity in vitro and in vivo. Mammalian nucleases coupled to antibodies may be utilized as cell type-selective cytotoxins and have potential as pharmacologic reagents. The systemic toxicity and immunogenicity observed with mammalian derived cytotoxins may be significantly less than that of the currently employed plant- and bacterial-derived immunotoxins.