Inhibition of sarcoplasmic reticulum calcium pump by cytosolic protein(s) endogenous to heart and slow skeletal muscle but not fast skeletal muscle

Cytosol from rabbit heart and slow and fast skeletal muscles was fractionated using (NH₄)₂SO₄ to yield three cytosolic protein fractions, viz., CPF-I (protein precipitated at 30% saturation), CPF-II (protein precipitated between 30 and 60% saturation), and cytosol supernatant (protein soluble at 60% saturation). The protein fractions were dialysed and tested for their effects on ATP-dependent, oxalate-supported Ca²⁺ uptake by sarcoplasmic reticulum from heart and slow and fast skeletal muscles. CPF-I from heart and slow muscle, but not from fast muscle, caused marked inhibition (up to 95%) of Ca²⁺ uptake by sarcoplasmic reticulum from heart and from slow and fast muscles. Neither unfractionated cytosol nor CPF-II or cytosol supernatant from any of the muscles altered the Ca²⁺ uptake activity of sarcoplasmic reticulum. Studies on the characteristics of inhibition of sarcoplasmic reticulum Ca²⁺ uptake by CPF-I (from heart and slow muscle) revealed the following: (a) Inhibition was concentration- and temperature-dependent (50% inhibition with approx. 80 to 100 μg CPF-I; seen only at temperatures above 20°C). (b) The inhibitor reduced the velocity of Ca²⁺ uptake without appreciably influencing the apparent affinity of the transport system for Ca²⁺. (c) Inhibition was uncompetitive with respect to ATP. (d) Sarcoplasmic reticulum washed following exposure to CPF-I showed reduced rates of Ca²⁺ uptake, indicating that inhibition results from an interaction of the inhibitor with the sarcoplasmic reticulum membrane. (e) Concomitant with the inhibition of Ca²⁺ uptake, CPF-I also inhibited the Ca²⁺-ATPase activity of sarcoplasmic reticulum. (f) Heat-treatment of CPF-I led to loss of inhibitor activity, whereas exposure to trypsin appeared to enhance its inhibitory effect. (g) Addition of CPF-I to Ca²⁺-preloaded sarcoplasmic reticulum vesicles did not promote Ca²⁺ release from the vesicles. These results demonstrate the presence of a soluble protein inhibitor of sarcoplasmic reticulum Ca²⁺ pump in heart and slow skeletal muscle but not in fast skeletal muscle. The characteristics of the inhibitor and its apparently selective distribution suggest a potentially important role for it in the in vivo regulation of sarcoplasmic reticulum Ca²⁺ pump, and therefore in determining the duration of Ca²⁺ signal in slow-contracting muscle fibers.     

Further studies on phosphorylation and the thiol/disulphide ratio of histones in growth and development

1. The proportion of thiol groups in the total thiol+disulphide of histone extracts from fertilized eggs from Echinus and Psammechinus was increased during periods of structural alterations in the nucleus. 2. The probable start of DNA synthesis in the fertilized eggs coincided with periods of maximum thiol content. 3. Histone extracts from rat liver and regenerating liver were predominantly in the thiol form and no significant variations could be detected during the first 30hr. after partial hepatectomy. 4. An assay system was developed to follow the phosphorylation believed to be associated with the arginine-rich histone F3. Phosphorylation increased by 50% at 1 and 2hr. after partial hepatectomy. The phosphate content also increased during the period of DNA synthesis. 5. The increased phosphorylation found 1hr. after partial hepatectomy was not prevented by actinomycin or prior irradiation. 6. The phosphate content of histone F1 was very high in livers from foetal rats and declined in neonatal rats similarly to the decline in DNA synthesis.     

Metabolic and chemical properties of basic proteins isolated from nuclei of rat liver and thymus gland

1. The effects of alkylating agents and disulphides on the thiol-containing proteins of nuclei from rat thymus and liver were studied. Three protein fractions were examined: histones extracted with 50mm- and 250mm-hydrochloric acid and the residual protein. None of the reagents selectively reacted with any one of the protein fractions. 2. Amino acid uptake in vitro into the histones of nuclei from rat thymus was analysed by preparative electrophoresis of the proteins extracted with 50mm- and 250mm-hydrochloric acid. After 1hr. at 37° the greater incorporation was into the proteins extracted with 50mm-hydrochloric acid. 3. Preparative electrophoresis was used to study the relative thiol contents of the proteins of the 50mm-hydrochloric acid extract from thymus nuclei by labelling the histones in vitro with ¹⁴C-labelled N-ethylmaleimide. 4. The capacity of the proteins extracted from rat thymus with 50mm- and 250mm-hydrochloric acid, and of the components from these extracts separated by preparative electrophoresis, to combine with DNA and to depress DNA-dependent RNA synthesis was studied. The histones extracted with 50mm-hydrochloric acid were more lysine-rich than those extracted with 250mm-hydrochloric acid. Wide variations were found in the abilities of the separated components to depress RNA synthesis.     

Further Evaluation of a Rapid Immunofluorescence Technique for Detecting Salmonellae in Meat and Poultry

A rapid 18–24 h immunofluorescence technique detected 14 of 15 positive samples in tests on 706 routine samples, which included 656 home produced raw beef samples. The rapid technique also recorded 49 false positive results, i.e. samples which proved negative in subsequent cultural tests. The immunofluorescence technique could be used as a presumptive screening test aimed at the rapid detection of negative samples. In this way salmonella free raw materials should usually be cleared for production within 1 day of sampling.     

Enzyme induction in rat liver: The effects of Be2+ in vivo

Rats given an LD₅₀ dose of Be²⁺ showed reduced activities of ornithine decarboxytase and tyrosine aminotransferase in liver in response to dexamethasone induction. Control fed animals showed superinduction. Be²⁺ also inhibited the uptake of [³H]orotic acid into rapidly labelled RNA of ribonucleoprotein particles extracted from liver nuclei in isomolar solutions at pH 8.0. Consistent with inhibition of cytoplasmic protein kinase reported previously (Kaseret at., 1980), the uptake of [³²P]P_i into proteins in the ribonucleoprotein particles was also diminished.     

Mitochondrial-like DNA sequences flanked by direct and inverted repeats in the nuclear genome of Toxoplasma gondii.

In the course of our genetic studies on Toxoplasma gondii, it was discovered that one cosmid hybridized to a repetitive element. The hybridization pattern observed for the enzyme BglII indicated that this cosmid hybridized to a large number of discrete, but related elements. Four BglII fragments were subcloned from the cosmid, and each was shown to hybridize with all the others, as well as to numerous dispersed sequences in genomic DNA. Three subclones were sequenced in their entirety, and shown to contain fragments of the genes for cytochrome oxidase subunit I and apocytochrome b, complete and functional copies of which have been found in only mitochondrial genomes. All the subcloned fragments were bounded at both ends by a 91 base-pair sequence, which contains a site for BglII. This 91 base-pair sequence could be found as either a direct or inverted repeat. It was determined that the BglII elements are arrayed downstream from a single copy nuclear gene. Comparison of genomic and cosmid DNAs confirmed that the cosmid faithfully reflects the nuclear genome. Although the mitochondrial genome of Toxoplasma has not been characterized, these nuclear mitochondrial-like sequences appear to be internally rearranged with respect to known, functional mitochondrial genomes, and with respect to each other. The finding of short repeated sequences flanking these elements may be a clue to the mechanism of their dissemination.