Transfer RNA genes from the hyperthermophilic Archaeon, Methanopyrus kandleri.

Genes encoding the Leu (GAG), Ser (UGA), Gln (UUG) and Lys (UUU) tRNAs have been cloned and sequenced from the deep sea hyperthermophilic Archaeon, Methanopyrus kandleri. Sequences conforming to the TATA box element established for methanogen promoters are located upstream of the tRNA(Gln) and tRNA(Lys) genes. All four of the tRNA genes appear to encode the 3' terminal CCA residues of the mature tRNA. These methanogen tRNAs are predicted to contain most, but not all, invariant residues and are characterized by a high level of G + C base pairing, consistent with the 98 degrees C optimum growth temperature of M. kandleri.     

Polyunsaturated fatty acid incorporation into plasmalogens in plasma membrane of glioma cells is preceded temporally by acylation in microsomes.

Plasmalogens (1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) are major phospholipids in many tissues and cells, particularly of neural origin. Using cultured C6 glioma cells and subcellular fractions isolated on Percoll gradients we investigated selectivity for esterification of several polyunsaturated fatty acids (PUFA) in the sn-2 position of plasmalogens compared to [1-14C]hexadecanol, representative of de novo synthesis of the ether-linked sn-1 position. In whole cells at a final concentration of 105 microM PUFA, 2-4 nmol plasmalogen/mg protein was labeled in 4 h and 10-14 nmol in 24 h, representing 8-15% and 35-50%, respectively, of initial plasmalogen mass. Incorporation of label from hexadecanol was lower than PUFA incorporation (20:5(n-3) greater than 20:4(n-6) greater than 18:3(n-3) much greater than 18:2(n-6)) suggesting deacylation-reacylation at the sn-2 position. Plasmalogens accounted for 50% of total cell ethanolamine phospholipids and 75% in plasma membrane. Using a novel, improved method for extraction of subcellular fractions containing Percoll, plasma membrane also was enriched in plasmalogen relative to microsomes (107.4 +/- 5.2 vs. 40.0 +/- 2.9 nmol/mg protein). Selectivity for esterification at the sn-2 position of plasmalogens with respect to chain length and unsaturation of the fatty acyl chain was similar in both subcellular fractions and reflected that of whole cells. Labeling of plasma membrane with PUFA and fatty alcohol lagged behind that of microsomes. Chase experiments in cells prelabeled with [1-14C]18:3(n-3) for 2 h showed no significant reduction of label in plasmalogen of any subcellular fraction although accumulation of label in the microsomal fraction was slowed initially. Reduction of plasmalogen label (40-50%) did occur in microsomes and plasma membrane when cells prelabeled for 24 h were switched to chase medium with or without chase fatty acid. Our data suggest that esterification of PUFA to plasmalogen may occur at the endoplasmic reticulum with subsequent translocation to plasma membrane resulting in accumulation of relatively stable pools of plasmalogen that are not readily accessible for deacylation-reacylation exchange with newly appearing PUFA. Alternatively, deacylation-reacylation may occur in a more stable phospholipid pool within the plasma membrane but would involve a slower process than at the endoplasmic reticulum.     

The sequence of the major protein stored in ovine ceroid lipofuscinosis is identical with that of the dicyclohexylcarbodiimide-reactive proteolipid of mitochondrial ATP synthase.

The ceroid lipofuscinoses are a group of neurodegenerative lysosomal storage diseases of children and animals that are recessively inherited. In diseased individuals fluorescent storage bodies accumulate in a wide variety of cells, including neurons. Previous studies of these bodies isolated from tissues of affected sheep confirmed that the storage occurs in lysosomes, and showed that the storage body is mostly made of a single protein with an apparent molecular mass of 3500 Da with an N-terminal amino acid sequence that is the same as residues 1-40 of the c-subunit (or dicyclohexylcarbodi-imide-reactive proteolipid) of mitochondrial ATP synthase. In the present work we have shown by direct analysis that the stored protein is identical in sequence with the entire c-subunit of mitochondrial ATP synthase, a very hydrophobic protein of 75 amino acid residues. As far as can be detected by the Edman degradation, the stored protein appears not to have been subject to any post-translational modification other than the correct removal of the mitochondrial import sequences that have been shown in other experiments to be present at the N-terminal of its two different precursors. No other protein accumulates in the storage bodies to any significant extent. Taken with studies of the cDNAs for the c-subunit in normal and diseased sheep, these results indicate that the material that is stored in lysosomes of diseased animals has probably entered mitochondria and has been subjected to the proteolytic processing that is associated with mitochondrial import. This implies that the defect that leads to the lysosomal accumulation concerns the degradative pathway of the c-subunit of ATP synthase. An alternative, but less likely, hypothesis is that for some unknown reason the precursors of subunit c are being directly mis-targeted to lysosomes, where they become processed to yield a protein identical with the protein that is normally found in the mitochondrial ATP synthase assembly, and which then accumulates.     

Small-scale environmental heterogeneity and the analysis of species distributions along gradients

Abstract. The observed distribution of a species along an environmental gradient is strongly affected by environmental variability within a quadrat. Because a quadrat does not represent a point along an environmental gradient, but rather a range of conditions, it is likely to contain species not typically associated with the mean conditions in the quadrat. Systematic relationships exist between a species' true distribution, the observed distribution as a function of mean quadrat environment, and the frequency distribution of the environment within that quadrat. The observed species habitat breadth increases and the observed maximum abundance decreases as within-quadrat environmental heterogeneity increases. If species distributions or beta diversities are to be compared among species or coenoclines, they should be correctedforintra-quadratheterogeneity.Wederive simple corrections for environmental heterogeneity. The distributions of hardwood forest understory species along a soil acidity gradient in the North Carolina piedmont are presented as an example.     

Lipase regulation of muscle triglyceride hydrolysis

The cellular control of intramuscular triglyceride (TG) metabolism involves two major identified lipases: hormone-sensitive lipase (HSL) and lipoprotein lipase (LPL). Recently, the presence of HSL in muscle has been unequivocally demonstrated. However, although it is thought that HSL is responsible for intramuscular TG lipolysis, direct evidence for this is lacking. There is evidence to suggest that HSL and LPL are simultaneously activated under a variety of conditions. The two muscle lipases appear to be turned on by the same signal and function as a coordinated unit in meeting the energy demands of muscle. At a time when HSL is presumably hydrolyzing endogenous TG, LPL is sent to the capillary beds in search of substrate. TG uptake from circulation is highly related to muscle LPL activity. Exercise training increases LPL activity in plasma and in parenchymal cells in muscle. These results suggest that training may increase the capacity to clear TG from circulation and that LPL might have a role in replenishing muscle TG stores that have been decreased with exercise.     

Triacylglycerol metabolism in rat skeletal muscle after exercise

The temporal relationships between triacylglycerol (TG) content and TG lipase activity in slow-twitch (STR) and fast-twitch red (FTR) muscles were determined in rats during recovery from a 2-h swim. Immediately after the exercise, plasma free fatty acid (FFA) was elevated and glycogen concentrations were decreased. TG content was decreased 40% in STR muscle and reduced 45% in FTR muscle. The TG concentration of STR muscle increased in a linear fashion throughout recovery so that control levels were reached within the first 24 h after exercise. TG lipase activity of STR muscle was elevated 36% above control immediately after the swim and continued to increase to 84% above control 24 h after the work. In STR muscle there was a net synthesis of TG, while lipase activity was elevated above that measured in muscle of control rats. TG content of FTR muscle remained 45% below control throughout the first 24 h of recovery, and TG lipase activity increased from 26% (P greater than 0.05) greater than control immediately after exercise to threefold above control 24 h after work. All parameters returned to control levels by 48 h of recovery. These data indicated that a net TG synthesis occurs in STR muscle when lipolytic activity is elevated. In FTR muscle, however, a gradual increase in TG lipase activity that occurs during the first 24 h of recovery accompanies a TG concentration well below the control level throughout this same time frame.(ABSTRACT TRUNCATED AT 250 WORDS)     

A testcross procedure for selecting exotic strains to improve pure-line cultivars in predominantly self-fertilizing species

Methods for identifying germplasm carrying alleles with the potential to improve a particular single-cross hybrid have been proposed and discussed in recent years. There is a need for similar methods to be used in breeding crops for which pure-line cultivars, rather than hybrids, are the goal. The objective of this research was to develop a method to identify germplasm lines with the potential to contribute favorable alleles not present in a specified pure line or set of pure lines. Given a set of adapted pure lines (A ₁, A ₂ ..., A _m) to be improved and a set of germplasm lines (P ₁ P ₂ ..., P _f), the procedure consists of producing all f x m possible hybrids and evaluating them along with the parents. The testcross statistic T _ij is defined by T _ij=(F _ij–A _j)+(1–) (F _ij–P _i), where A _j, P _i, and F _ij represent the performance of thej ^th adapted line, the i ^th germplasm line, and their hybrid, respectively. The statistic is the mean value of T _ij over all adapted parents A _j. If =(1/2)(1+d), where d = the mean degree of dominance, then T _ij measures the potential for alleles from P _i to improve A _j and measures the potential for alleles from P _i to improve the set A ₁, A ₂ ..., A _m. Use of data on soybean and peanut hybrids published by other researchers suggests that the value assumed for d has little effect on the P _i chosen. The ability of the T _ij and statistics to identify germplasm strains carrying rare favorable alleles should be assessed in empirical studies.Joint contribution: OARDC (Journal Articale No. 161-94), USDAARS, Iowa Agriculture and Home Economics Expriment Station (Journal Paper No. J-16109; Project 2985), and Agreculture and Agri-Food Canada. Salaries and research support for S. K. St. Martin Provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, Ohio State University     

Blurry artifacts: a retraction

Abstract. This note is to apologize for an error in the computer program used to evaluate the random data used in Fuzzy Set Ordination according to Zhang & Oxley. After correction of this error no artifacts could be detected any longer. However, the basic conclusion of the earlier critical note still stands: if one selects environmental variables after analyzing the results of a multivariate gradient analysis, and then uses these variables as input into a multiple univariate gradient analysis, the results are expected to be comparable.     

Polymorphic Admixture Typing in Human Ethnic Populations

A panel of 257 RFLP loci was selected on the basis of high heterozygosity in Caucasian DNA surveys and equivalent spacing throughout the human genome. Probes from each locus were used in a Southern blot survey of allele frequency distribution for four human ethnic groups: Caucasian, African American, Asian (Chinese), and American Indian (Cheyenne). Nearly all RFLP loci were polymorphic in each group, albeit with a broad range of differing allele frequencies (δ). The distribution of frequency differences (δ values) was used for three purposes: (1) to provide estimates for genetic distance (differentiation) among these ethnic groups, (2) to revisit with a large data set the proportion of human genetic variation attributable to differentiation within ethnic groups, and (3) to identify loci with high δ values between recently admixed populations of use in mapping by admixture linkage disequilibrium (MALD). Although most markers display significant allele frequency differences between ethnic groups, the overall genetic distances between ethnic groups were small (.066–.098), and <10% of the measured overall molecular genetic diversity in these human samples can be attributed to “racial” differentiation. The median δ values for pairwise comparisons between groups fell between .15 and .20, permitting identification of highly informative RFLP loci for MALD disease association studies.     

Multiplex PCR for detection of the heat-labile toxin gene and shiga-like toxin I and II genes in Escherichia coli isolated from natural waters.

A triplex PCR method was developed to simultaneously amplify a heat-labile toxin sequence (LT) of 258 bp, a shiga-like toxin I sequence (SLT I) of 130 bp, and a shiga-like toxin II sequence (SLT II) of 346 bp from toxigenic strains of Escherichia coli. This method was used to screen 377 environmental E. coli isolates from marine waters or estuaries located in Southern California and North Carolina for enterotoxigenic or enterohemorrhagic E. coli strains. Of the 377 E. coli screened, one isolate was found to belong to the enterotoxigenic group, since it contained a LT homologous sequence, and one isolate was found to belong to the enterohemorrhagic group, since it contained a SLT I homologous sequence. None was found to contain SLT II homologous sequences. The pathogenicity of the positive environmental E. coli isolates was confirmed by standard bioassays with Y-1 adrenal cells and Vero cells to confirm toxin production. Our results suggest that toxigenic E. coli occurs infrequently in environmental waters and that there is a low public health risk from toxigenic E. coli in coastal waters.