Identification of the gene encoding the mitochondrial elongation factor G in mammals.

Protein synthesis in cytosolic and rough endoplasmic reticulum associated ribosomes is directed by factors, many of which have been well characterized. Although these factors have been the subject of intense study, most of the corresponding factors regulating protein synthesis in the mitochondrial ribosomes remain unknown. In this report we present the cloning and initial characterization of the gene encoding the rat mitochondrial elongation factor-G (rEF-Gmt). The rat gene encoding EF-Gmt (rMef-g) maps to rat chromosome 2 and it is expressed in all tissues with highest levels in liver, thymus and brain. Its DNA sequence predicts a 752 amino acid protein exhibiting 72% homology to the yeast Saccharomyces cerevisiae mitochondrial elongation factor-G (YMEF-G), 62% and 61% homology to the Thermus thermophilus and E. coli elongation factor-G (EF-G) respectively and 52% homology to the rat elongation factor-2 (EF-2). The deduced amino acid sequence of EF-G contains characteristic motifs shared by all GTP binding proteins. Therefore, similarly to other elongation factors, the enzymatic function of EF-Gmt is predicted to depend on GTP binding and hydrolysis. EF-Gmt differs from its cytoplasmic homolog, EF-2, in that it contains an aspartic acid residue at amino acid position 621 which corresponds to the EF-2 histidine residue at position 715. Since this histidine residue, following posttranslational modification into diphthamide, appears to be the sole cellular target of diphtheria toxin and Pseudomonas aeruginosa endotoxin A, we conclude that EF-Gmt will not be inactivated by these toxins. The severe effects of these toxins on protein elongation in tissues expressing EF-Gmt suggest that EF-Gmt and EF-2 exhibit nonoverlapping functions. The cloning and characterization of the mammalian mitochondrial elongation factor G will permit us to address its role in the regulation of normal mitochondrial function and in disease states attributed to mitochondrial dysfunction.     

Transformation-associated cytokine 9E3/CEF4 is chemotactic for chicken peripheral blood mononuclear cells.

9E3/CEF4, which is released from transformed chicken embryo fibroblasts (CEF), is a member of the platelet factor 4 family of inflammatory proteins and may be the avian homolog of interleukin-8. Since the function of 9E3/CEF4 is unknown, we examined the effect of the protein on mitogenicity and chemotaxis, as well as its expression, in fibroblasts and peripheral blood cells. 9E3/CEF4 mRNA was expressed in chicken peripheral blood monocytes, and its expression was stimulated by incubation of the monocytes with lipopolysaccharide or phorbol myristic acetate. Boyden double-membrane analysis of chemotaxis showed that 9E3/CEF4 was chemotactic for chicken peripheral blood mononuclear cells, as well as for heterophils. Untransformed CEF and CEF transformed with Rous sarcoma virus also migrated to 9E3/CEF4 protein, as measured by Boyden single-membrane analysis. 9E3/CEF4 was slightly mitogenic for CEF, causing a doubling of [3H]thymidine uptake when added to serum-starved CEF.9E3/CEF4 was found associated not only with the cell and in the culture medium of Rous sarcoma virus-transformed CEF but also with the extracellular matrix. The in vivo role of 9E3/CEF4 may be involved with chemotaxis and metastasis, rather than with direct stimulation of mitogenicity.     

Preptetos cannoni n. sp. (Digenea: Lepocreadiidae) from Siganus lineatus (Teleostei: Siganidae) from the southern Great Barrier Reef,Australia

Preptetos cannoni n. sp. is described from Siganus lineatus from Heron Island on the southern Great Barrier Reef, Australia. Morphological features that are diagnostic of this species include: a genital pore that is sinistral to the ventral sucker and vitelline follicles that reach anteriorly to at least the bifurcation of the intestine. This is the first report of a species of the genus Preptetos from Australian waters and from fishes of the genus Siganus.     

Use of Nematodes as Biomonitors of Nonfumigant Nematicide Movement through Field Soil

Three field experiments were established in a loamy sand soil in the Coastal Plain of North Carolina to determine downward movement of aldicarb and fenamiphos with a nematode bioassay. Penetration of bioassay plant roots by Meloidogyne incognita was measured at 1, 3, 7, 14, 21, and 28 days after treatment in the greenhouse as a means of determining nematicide effectiveness. Chemical movement was similar in planted and fallow soil. Nematicidal activity was greater in soil collected from the 0 to 10 cm depth than from the 10 to 20 cm depth. Fenamiphos suppressed host penetration by the nematode more than aldicarb under the high rainfall (19 cm) and low soil temperatures that occurred soon after application in the spring. During the summer, which had 13 cm precipitation and warmer soil temperatures, both chemicals performed equally well at the 0 to 10 cm depth. At the lower soil level (10 to 20 cm), aldicarb limited nematode penetration of host roots more quickly than fenamiphos. Both of these chemicals moved readily in the sandy soil in concentrations sufficient to control M. incognita. Although some variability was encountered in similar experiments, nematodes such as M. incognita have considerable potential as biomonitors of nematicide movement in soil.     

Weight in infancy and prevalence of coronary heart disease in adult life.

OBJECTIVE--To determine whether low birth weight and low weight at 1 year are followed by an increased prevalence of coronary heart disease in adult life. DESIGN--A follow up study of men born during 1920-30 whose birth weights and weights at 1 year were recorded. SETTING--Hertfordshire, England. SUBJECTS--290 men born and still living in East Hertfordshire. MAIN OUTCOME MEASURE--The prevalence of coronary heart disease, defined by the Rose/WHO chest pain questionnaire, standard electrocardiographic criteria, or history of coronary artery angioplasty or graft surgery. RESULTS--42 (14%) men had coronary heart disease. Their mean birth weight, 7.9 lb (3600 g), was the same as that of the other men. Their mean weight at 1 year, 21.8 lb (9.9 kg), was 1 lb (454 g) lower (95% confidence interval 0.1 to 1.8, P = 0.02). Percentages of men with coronary heart disease fell from 27% in those who weighed 18 lb (8.2 kg) or less at 1 year to 9% in those who weighed more than 26 lb (11.8 kg) (P value for trend = 0.03). This trend occurred in both smokers and non-smokers and within each social class. CONCLUSION--These findings add to the evidence that coronary heart disease is "programmed" during early growth.     

Impact of Soil Texture on the Reproductive and Damage Potentials of Rotylenchulus reniformis and Meloidogyne incognita on Cotton

The effects of soil type and initial inoculum density (Pi) on the reproductive and damage potentials of Meloidogyne incognita and Rotylenchulus reniformis on cotton were evaluated in microplot experiments from 1991 to 1993. The equilibrium nematode population density for R. reniformis on cotton was much greater than that of M. incognita, indicating that cotton is a better host for R. reniformis than M. incognita. Reproduction of M. incognita was greater in coarse-textured soils than in fine-textured soils, whereas R. reniformis reproduction was greatest in a Portsmouth loamy sand with intermediate percentages of clay plus silt. Population densities of M. incognita were inversely related to the percentage of silt and clay, but R. reniformis was favored by moderate levels of clay plus silt (ca. 28%). Both M. incognita races 3 and 4 and R. reniformis effected suppression of seed-cotton yield in all soil types evaluated. Cotton-yield suppression was greatest in response to R. reniformis at high Pi. Cotton maturity, measured as percentage of open bolls at different dates, was affected by the presence of nematodes in all 3 years.     

Influence of Clavicipitaceous Endophyte Infection in Ryegrass on Development of the ParasitoidMicroctonus hyperodaeLoan (Hymenoptera: Braconidae) inListronotus bonariensis(Kuschel) (Coleoptera: Curculionidae)

Laboratory experiments were conducted to examine the effect of ryegrass infection by the endophytic fungusAcremonium loliiLatch, Christensen and Samuels onMicroctonus hyperodaeLoan, a parasitoid ofListronotus bonariensis(Kuschel). Progression of parasitoids through the larval instar stages was shown to depend on adequate nutrition of the weevil host. Compared to confinement on endophyte-free ryegrass, parasitized weevils held on nonpreferred diets comprising leaf segments from endophyte-infected ryegrass and switchgrass contained parasitoid larvae with retarded development. Similarly, development of parasitoid larvae was retarded in hosts feeding on artificial diet containing diterpenes and alkaloids ofA. loliiorigin. Several diterpenes incorporated into the diet reduced survival of the parasitoid larvae. Attack rate of parasitoids was reduced when the quality of potential host weevils was compromised by confinement on nonpreferredA. lolii-infected ryegrass or without food for 14 days.     

Immunohistochemical localization of adenosine 3':5'-cyclic monophosphate in female ixodid tick Amblyomma americanum (L.) salivary glands

Localization of adenosine 3':5'-cyclic monophosphate (cyclic AMP) in alveoli of salivary glands of female Amblyomma americanum (L.) was accomplished with an indirect immunofluorescent technique. Little cyclic AMP fluorescence was seen in Type I alveoli in glands of unfed females but considerable fluorescence was seen in Type I alveoli of glands obtained from females that had fed. The most intense cyclic AMP fluorescence was observed in complex granular cells of Type II and III alveoli in glands of unfed females and glands from females in early stages of tick feeding. In the latter stages of tick feeding an increase in fluorescence in Type III alveoli was observed in cells near the lumen, possibly adluminal interstitial or transformed granular cells.     

Kappa-chain constant-region gene sequences in genus Rattus: coding regions are diverging more rapidly than noncoding regions

We have determined the nucleotide sequence of a 1,200-base pair (bp) genomic fragment that includes the kappa-chain constant-region gene (C kappa) from two species of native Australian rodents, Rattus leucopus cooktownensis and Rattus colletti. Comparison of these sequences with each other and with other rodent C kappa genes shows three surprising features. First, the coding regions are diverging at a rate severalfold higher than that of the nearby noncoding regions. Second, replacement changes within the coding region are accumulating at a rate at least as great as that of silent changes. Third, most of the amino acid replacements are localized in one region of the C kappa domain--namely, the carboxy-terminal "bends" in the alpha-carbon backbone. These three features have previously been described from comparisons of the two allelic forms of C kappa genes in R. norvegicus. These data imply the existence of considerable evolutionary constraints on the noncoding regions (based on as yet undetermined functions) or powerful positive selection to diversify a portion of the constant-region domain (whose physiological significance is not known). These surprising features of C kappa evolution appear to be characteristic only of closely related C kappa genes, since comparison of rodent with human sequences shows the expected greater conservation of coding regions, as well as a predominance of silent nucleotide substitutions within the coding regions.      

System for DNA sequencing with resolution of up to 600 base pairs

A system capable of resolving about 500 bases is of interest for sequencing of longer DNA molecules. Studies on further optimization of resolution on DNA sequencing gels were carried out. The effect of physico-chemical properties of gels and buffers on resolution were tested, e.g. ionic strength and pH of buffers, different buffer systems, acrylamide concentration, crosslinker concentration, type of crosslinker, temperature of polymerization, denaturing conditions, gel length and thickness. Tested were as well different running conditions like electric field, gel temperature, dimension of sample slots. Gels 0.1-0.2 mm thick and up to 1.2 m long were cast and tested routinely. Gel lengths of 60-70 cm (for sequencing up to 350-400 bases) to about 100 cm (above 400 bases) are practicable. Little is gained in resolution by increasing the gel length from 1 to 1.2 m. Resolution was improved using 0.1 mm thick gels, at a higher pH value of 8.6-8.8, and molarity increased to 0.2 M. The sequencing pattern in the region of higher bases could be better resolved on a twice-magnified picture of that region on the autoradiogram. With the long gels (70-120 cm), it is advantageous to obtain the sequence overlap by running in parallel gels of different concentrations, without re-application of samples, all loaded at the same time. Buffer chamber for running of two of three gels and thermostating plates up to 1.2 m long were designed. In this way four to six thermostated gels can be run from a power supply with two inputs. Three 1 m long gels (concentrations: 4%, 6%, 12-16%) are loaded with several samples of DNA to be sequenced and run in parallel without re-application of the samples. With good samples, the sequence overlap from the gels could be counted up to 500 base pairs, with exceptionally good samples closer to 600 bases. At present this number seems to be near the limit of the resolving power of the polyacrylamide gels.