Early events in the cellular formation of proparathyroid hormone

Early events in the cellular synthesis and subsequent transfer into membrane-limited compartments of pre-proparathyroid hormone (pre-proPTH) and proparathyroid hormone (proPTH) were investigated by electrophoretic analyses of newly synthesized proteins in subcellular fractions of parthyroid gland slices pulse-labeled for 0.5-5 min with [(35)S] methionine. During these short times of incubation, both pre-proPTH and proPTH were confined to the microsomal fraction. Labeled pre-proPTH and proPTH were detected in a 30-s interval between 0.5 and 1.0 min of incubation. The radioactivity in proPTH became relatively constant between 3 and 5 min, whereas the radioactivity in ProPTH increased markedly over this period. When corrected for the known content of methionine in the prohormone and the prohormone, we found four times as much radiolabeled prohormone as prehormone between 0.5 and 1.0 min of synthesis. Sequestration of labeled prohomrone into endoplasmic reticulum compartments was shown by treatment of the microsomal fraction with chymotrypsin and trypsin, which resulted in the degradation of the prehormone but not of the prohormones. Approximately 50 percent of pre-prohormone and 25 percent of prohormone were released from the microsomes by their extraction with 1.0 M KCl, whereas 80-90 percent of both was released by treatment with Triton X-100. These results in intact cells support the signal hypothesis proposed by Blobel and his co-workers in studies utilizing cell-free systems, inasmuch as the results indicate transfer of prohormone into the cisternal space of the rough endoplasmic reticulum concomitant with the growth of the nascent polypeptide chain. Appearance of membrane-sequestered proPTH takes place without entry of pre-proPTH into the cisternal space, suggesting that proteolytic removal of the leader peptide occurs during transfer of the polypeptide through the lipid bilayer. Further evidence in support of this process is that pre-proPTH is only partly extracted from the microsomes by treatment with 1.0 M KCl, suggesting that a substantial fraction of the nascent pre-proPTH is integrally inserted into the membranes before it is cleaved to form proPTH.     

The Bacillus megaterium ribonucleotide reductase: Evidence for a B12 coenzyme requirement

Summary On the basis of the following evidence, it has been concluded that extracts ofBacillus megaterium KM contain a B₁₂ coenzyme-dependent ribonucleotide reductase. The reduction of cytidine nucleotides to deoxycytidine nucleotides is enhanced by the addition of B₁₂ coenzyme. In addition, the presence of hydroxyurea, a specific inhibitor of non-B₁₂ coenzymedependent reductases, has only a slight effect on this reduction. Finally,B. megaterium extracts catalyze a transfer of tritium from 5-deoxyadenosylcobalamin-5-³H₂ to water, a reaction specific for B₁₂ coenzyme-dependent ribonucleotide reductases.     

Evidence for Four Deoxynucleoside Kinase Activities in Extracts of Lactobacillus leichmannii

Extracts of Lactobacillus leichmannii (ATCC 7830) catalyze the phosphorylation of the four principal deoxynucleosides. Thymidine, deoxyguanosine, and deoxycytidine kinase activities were found to be optimal with deoxyadenosine triphosphate as the phosphoryl donor, whereas deoxycytidine triphosphate was the optimal donor for deoxyadenosine kinase activity. L. leichmannii catalyzes the conversion of deoxycytidine to deoxyuridylic acid, probably by a pathway involving deoxycytidylate deaminase.     

Ribonucleoside Triphosphate-Dependent Pyrophosphate Exchange of Reovirus Cores

Reovirus cores catalyze a ribonucleoside triphosphate (rNTP)-dependent pyrophosphate exchange reaction in the presence of all four rNTP species. When rNTP species are tested individually, only guanosine-5'-triphosphate supports pyrophosphate exchange.     

Methylated Bases of Bacillus megaterium KM Nucleic Acids: Comparison with Escherichia coli

Parallel studies were performed with methionineless derivatives of Escherichia coli 15 T(-) and Bacillus megaterium KM: T(-). Methylated bases are present in the total cell ribonucleic acid (RNA) of B. megaterium. The level of RNA methylation in E. coli is about 60% greater than that in B. megaterium. Although E. coli deoxyribonucleic acid (DNA) was found to contain 0.12% 5-methylcytosine (5-MC) and 0.24% 6-methylaminopurine (6-MA), methylated bases were not detected in the DNA of B. megaterium. Assuming a molecular weight of 7 x 10(9) daltons for B. megaterium DNA, it was calculated that this organism could not contain more than one molecule of 5-MC or 6-MA per genome, and that possibly no methylated bases were present. Methylated bases were also not detected in the DNA of thymine-starved B. megaterium. Crude extracts of this organism possess RNA methylase activity but no detectable DNA methylase activity.     

Effect of Selenomethionine on Growth of Escherichia coli and Bacillus megaterium

Studies with methionineless strains of Escherichia coli WWU and Bacillus megaterium KM have shown that selenomethionine only partially satisfies their methionine requirement.     

Erratum

Neurotransmitter receptor trafficking and the regulation of synaptic strength. Traffic 2001:2(7):437–448.     

Delineation of amplification,hybridization and location effects in microarray data yields better-quality normalization

Background  

Oligonucleotide arrays have become one of the most widely used high-throughput tools in biology. Due to their sensitivity to experimental conditions, normalization is a crucial step when comparing measurements from these arrays. Normalization is, however, far from a solved problem. Frequently, we encounter datasets with significant technical effects that currently available methods are not able to correct.