Immunological and biochemical analysis of a null mutant of α-glycerolphosphate dehydrogenase from Drosophila melanogaster

Summary A Drosophila null mutant(BO-1-4) of -glycerolphosphate dehydrogenase induced by ethylmethane sulfonate(EMS) was analyzed by double immunodiffusion, enzyme immuno-inactivation, immunoelectrophoresis and two-dimensional electrophoresis. Based on all the immunological evidence, this mutant appears to express no protein that can cross-react with the antiserum specific to -glycerolphosphate dehydrogenase. A protein spot corresponding to -glycerolphosphate dehydrogenase was identified on two-dimensional gels of the soluble fly homogenates. The absence of this protein spot on two-dimensional gels of this null mutant further supported the immunological data. The activities of seven other enzymes in the related metabolic pathways were determined for the mutant and the control Drosophila. The null mutant does not show significant alterations in activities of these enzymes. The relationship between the deficiency of this enzyme and the inability for the sustained flight of the null mutant was discussed in terms of cellular metabolic regulations.Abbreviations used -GPD -glycerolphosphate dehydrogenase (EC 1.1.1.8) - EMS ethylmethane sulfonate - TEMED N,N,N,N-tetramethylene diamine - pI isolectric point - CRM immunological cross-reacting material     

Microbiological quality of frozen cauliflower, corn, and peas obtained at retail markets.

The microbiological quality of blanched frozen cauliflower, cut corn, and peas at the retail level was determined. At 35 degrees C, mean aerobic plate count (APC) values for cauliflower, corn, and peas, respectively, were 30,000, 6,100, and 4,700 per g; at 30 degrees C, the mean APC values were 45,000, 8,500, and 6,800 per g, respectively. Geometric means for coliform, Escherichia coli, and Staphylococcus aureus counts for all three vegetables were less than 10 per g.     

Microbiological quality of some spices and herbs in retail markets.

The microbiological quality of 10 spices or herbs was determined by a national survey at the retail level. Aerobic plate count values for the 10 products ranged from less than 100 to 3.1 X 10(8) per g; mean values of the individual spices or herbs ranged from 1,400 to 820,000 per g. Coliform counts ranged from less than 3 to 1.1 X 10(6) per g; however, mean values were less than 20 per g for all products. Escherichia coli counts ranged from less than 3 to 2,300 per g. Except for celery seed, which had a mean value of 7 per g, all mean values were less than 3 per g. Yeast and mold counts were made for 5 of the 10 products. Mean values were generally low; the highest mean (290 per g) was obtained for cinnamon.     

The cDNA for rat selenoprotein P contains 10 TGA codons in the open reading frame

Selenoprotein P is a plasma protein recently purified and characterized as containing 7.5 +/- 1.0 selenium atoms/molecule as selenocysteine. In rats maintained on a defined diet containing nutritionally adequate amounts of selenate as the sole selenium source, over half the selenium in plasma is accounted for by selenoprotein P. Its cDNA has been cloned from a rat liver library and sequenced. The sequence is highly unusual, containing 10 TGA codons in its open reading frame prior to the TAA termination codon. TGA designates selenocysteine in other selenoproteins, and limited peptide sequencing that included the amino acids encoded by two of the TGA codons verified that they correspond to selenocysteine. The deduced 366-amino acid sequence is histidine- and cysteine-rich and contains 9 of its selenocysteines in the terminal 122 amino acids. Comparison of the deduced amino acid sequence of selenoprotein P with those of other selenoprotein reveals no significant similarities. Selenoprotein P represents a new class of selenoproteins and is the first protein described with more than 1 selenocysteine in a single polypeptide chain. The primary structure of selenoprotein P suggests that it might be responsible for some of the antioxidant properties of selenium.     

Point mutations in the DNA polymerase gene of human cytomegalovirus that result in resistance to antiviral agents.

Three independently isolated mutants of human cytomegalovirus strain AD169 were found to be resistant to ganciclovir at a 50% effective dose of 200 microM. Phosphorylation of ganciclovir was reduced 10-fold in mutant-infected cells compared with AD169-infected cells. All three mutants were also determined to be resistant to the nucleotide analogs (S)-1-[(3-hydroxy-2- phosphonylmethoxy)propyl]adenine (HPMPA) and (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine (HPMPC) and hypersensitive to thymine-1-D-arabinofuranoside (AraT). Single base changes resulting in amino acid substitutions were demonstrated in the nucleotide sequence of the DNA polymerase gene of each mutant. The polymerase mutation contained in one of the mutants was transferred to the wild-type AD169 background. Ganciclovir phosphorylation in cells infected with the recombinant virus produced by this transfer was found to be equivalent to that of AD169-infected cells. The ganciclovir resistance of the recombinant was reduced fourfold compared with that of the parental mutant; however, the recombinant remained resistant to HPMPA and HPMPC and hypersensitive to AraT. The ganciclovir resistance of the mutants therefore appears to result from mutations in two genes: (i) a kinase which phosphorylates ganciclovir and (ii) the viral DNA polymerase.     

Achlorhydria induced changes in gastrin, somatostatin, H+/K(+)-ATPase and carbonic anhydrase in the sheep.

Gastrin, somatostatin, H+/K(+)-ATPase and carbonic anhydrase are principal elements of acid secretion. We investigated in the conscious sheep the effect of 24 h omeprazole (an H+/K(+)-ATPase inhibitor) infusion on these elements at the level of synthesis, storage and secretion. Omeprazole inhibited acid secretion-pH increased from 3.0 to 7.1 at 24 h. Plasma amidated and glycine extended gastrin increased 3-fold while the ratio of amidated to glycine extended gastrins (4:1) remained unchanged. Despite the increase in circulating gastrin, antral gastrin concentration and mRNA did not change significantly. Gastrin-17 (amidated and glycine extended) was the predominant form in the circulation and antrum, although there were preferential increases in larger forms following omeprazole treatment. Omeprazole had no effect on somatostatin mRNA or peptide levels in the fundus. Similarly, plasma somatostatin remained unchanged. However, antral somatostatin increased significantly (63%) following omeprazole treatment accompanied by a 4-fold increase in its mRNA. Fundic H+/K(+)-ATPase mRNA was unchanged but a significant increase (87%) in carbonic anhydrase II mRNA was observed. Omeprazole induced hypergastrinaemia occurred without a measurable reduction in storage or increased synthesis of gastrin at 24 h. Increased antral somatostatin synthesis and storage may result from stimulation by plasma gastrin on antral D cells, independent of acid. The rise in carbonic anhydrase II mRNA in the absence of any change in H+/K(+)-ATPase mRNA may reflect the differential sensitivity of the genes encoding these two enzymes to the stimulatory action of gastrin.     

Microbiological quality of frozen breaded fish and shellfish products.

A survey was made of the microbiological quality of seven frozen, breaded, precooked fish and shellfish products and of frozen, breaded, uncooked shrimp at the retail level. Geometric mean aerobic plate counts per gram (and number of units examined) were as follows: fish sticks, 8,300 (1,539); fish cakes, 5,600 (1,378); crab cakes, 4,900 (1,226); scallops, 1,700 (1,392); clams, 450 (1,384); haddock, 15,000 (1,306); fish in fish and chips dinner, 7,200 (1,485); and uncooked shrimp, 220,000 (1,462). Geometric mean coliform, Escherichia coli, and Staphylococcus aureus counts for all eight products ranged from 1 to 10/g.     

Kinetics of cell fusion induced by a syncytia-producing mutant of herpes simplex virus type I.

We have isolated a number of plaque-morphology mutants from a strain of herpes simplex virus type I which, unlike the wild type, cause extensive cell fusion during a productive viral infection. After the onset of fusion, there is an exponential decrease in the number of single cells as a function of time after infection. At a multiplicity of infection (MOI) of 3.8 plaque-forming units per cell, fusion begins 5.3 h after infection with the number of single cells decreasing to 10% of the original number 10.2 h after infection. As the MOI is gradually increased from 0.4 to 8, the onset of fusion occurs earlier during infection. However, when the MOI is increased from 8 to 86, the onset of fusion does not occur any earlier. The rate of fusion is independent of the MOI for an MOI greater than 1. The rate of fusion varies linearly with initial cell density up to 3.5 X 10(4) cells/cm2 and is independent of initial cell density at higher cell concentrations. To assay cell fusion we have developed a smiple quantitative assay using a Coulter counter to measure the number of single cells as a function of time after infection. Data obtained using a Coulter counter are similar to those obtained with a microscope assay.