Making the most of the imaging we have: using head MRI to estimate body composition.

AIM: To investigate the use of clinical head magnetic resonance imaging (MRI) in determining body composition and to evaluate how well it correlates with established measures based on abdominal computed tomography (CT). MATERIALS AND METHODS: Ninety-nine consecutive patients were identified who had undergone both brain MRI and abdominal CT within a 2-week span. Volumes of fat and muscle in the extracranial head were measured utilising several techniques by both abdominal CT and head MRI. RESULTS: MRI-based total fat volumes in the head correlated with CT-based measurements of fat in the abdomen using both single-section (r=0.64, p<0.01) and multisection (r=0.60, p<0.01) techniques. No significant correlation was found between muscle volumes in the abdomen and head. CONCLUSION: Based on the present results, head MRI-based measures may provide a useful surrogate for CT measurements of abdominal fat, particularly in patients with neurological cancers, as head MRI (and not abdominal CT) is routinely and repeatedly obtained for the purpose of clinical care for these patients.

Association of volumetric bone mineral density with abdominal aortic calcification in African ancestry men.

SUMMARY: We tested for association between cortical and trabecular volumetric bone mineral density (vBMD) with abdominal aortic calcification (AAC) prevalence in 278 Afro-Caribbean men. AAC was present in 68.3 % of the men. Greater cortical, but not trabecular, vBMD was associated with significantly decreased odds of AAC independent of traditional risk factors. INTRODUCTION: The aim of this study is to assess the prevalence and correlates of AAC in a sample of 278 Afro-Caribbean men (mean age 56) and to test for a largely unexplored association between cortical and trabecular vBMD with AAC prevalence. METHODS: Men were recruited consecutively as part of an ongoing prospective cohort study of body composition in men aged 40+. For this analysis, AAC was assessed by computed tomography of the abdomen from L3 to S1. Aortic calcium was scored using the Agatston method, and prevalence was defined as a score ≥10 to rule out false positives. Men also had BMD assessed using peripheral quantitative computed tomography at 4 % (trabecular vBMD) and 33 % (cortical vBMD) of the radius and tibia. RESULTS: Abdominal aortic calcification was present in 68.3 % of the men. Significant independent predictors of AAC prevalence were increased age, increased BMI, hypertension, and current smoking. Age was the strongest predictor, with each SD (7.8 year) increase in age conferring 2.7 times increased odds of having AAC (P < 0.0001). A one SD greater cortical, but not trabecular, vBMD was associated with a significant decreased odds of AAC prevalence independent of other traditional risk factors (OR 0.65; 95 % CI 0.45-0.92). CONCLUSIONS: Cortical vBMD is inversely associated with AAC presence. This finding suggests that there may be shared physiology between cortical bone compartment remodeling and vascular calcification.

Association of PNPLA3 SNP rs738409 with liver density in African Americans with type 2 diabetes mellitus.

AIM: Non-alcoholic fatty liver disease (NAFLD) is commonly diagnosed in patients with obesity and type 2 diabetes mellitus (T2DM), and has been associated with the single nucleotide polymorphism (SNP) rs738409 in the PNPLA3 gene. This association remains to be investigated in African Americans with T2DM, a group at lower risk for hepatic steatosis relative to European Americans with T2DM. METHODS: We examined 422 African Americans with T2DM (40.3% male; age: 56.4±9.6 years; Body Mass Index: 35.2±8.2 kg/m(2)), all with measures of liver density reflecting hepatic fat content on abdominal computed tomography, and blood glucose and lipid profiles. Associations between rs738409 and phenotypes of interest were determined using SOLAR, assuming an additive model of inheritance with covariates age, sex, BMI and use of lipid-lowering medications. RESULTS: Mean±SD liver density was 55.4±10.2 Hounsfield Units. SNP rs738409 in PNPLA3 was significantly associated with liver density (P=0.0075) and hepatic steatosis (P=0.0350), but not with blood glucose, HbA(1c), total cholesterol, triglycerides, high-density or low-density lipoprotein levels or liver function tests (P=0.15-0.96). CONCLUSION: These findings provide evidence that the PNPLA3 SNP rs738409 contributes to risk for increased liver fat content in African Americans with T2DM, an effect that appears to be independent from serum lipids. Although African Americans are less susceptible to fatty liver than European Americans, PNPLA3 appears to be a risk locus for hepatic steatosis in diabetic African Americans.

Electrophoretic karyotype of Cercospora kikuchii.

Classical genetic analyses are not possible with the phytopathogenic fungus Cercospora kikuchii since no sexual stage has been identified. To facilitate gene mapping and to develop an understanding of the genome organization of C. kikuchii, an electrophoretic karyotype has been obtained using contour-clamped homogeneous electric field gel electrophoresis (CHEF). Eight chromosomes, two of which migrate as a doublet, have been separated into seven bands ranging from 2.0 to 5.5 Mb. Using this determination of chromosome number and size, the total genome size of C. kikuchii is estimated to be 28.4 Mb. In addition, genes encoding tubulin, ribosomal DNA, and four previously isolated light-enhanced cDNAs from C. kikuchii were assigned to chromosomes by Southern-hybridization analysis of CHEF blots.

Transformation of the Fungal Soybean Pathogen Cercospora kikuchii with the Selectable Marker bar.

An improved transformation protocol, utilizing selection for resistance to the herbicide bialaphos, has been developed for the plant pathogenic fungus Cercospora kikuchii. Stable, bialaphos-resistant transformants are recovered at frequencies eight times higher than those achieved with the previous system that was based on selection for benomyl resistance. In addition to C. kikuchii, this improved method can also be used to transform other species of Cercospora.

Migration properties of circular DNAs using orthogonal-field-alternation gel electrophoresis.

The migration of a series of supercoiled plasmids ranging in size from 4 to 91 kilobases (kb) has been analyzed by orthogonal-field-alternation gel electrophoresis (OFAGE). These circular DNAs enter OFAGE gels and are resolved over the same region of the gel as linear DNAs from 260 to 2200 kb. Furthermore, a distinct triphasic migration pattern was observed for the supercoiled DNAs. The migration of plasmids between 6 and 20, and 60 and 91 kb is inversely proportional to size, whereas the mobilities of plasmids between 20 and 60 kb increase with size. Unlike linear DNA molecules, the relative mobilities of these plasmids are constant over a broad range of pulse times, from 10 to 120s. Electrophoresis of supercoiled, relaxed, and nicked open circular forms as well as topoisomers of small plasmids shows that the extent of supercoiling has a dramatic effect on plasmid migration on OFAGE. Several practical applications for exploiting the different migration properties of circular and linear DNA molecules on OFAGE are presented.

Analysis of amplified DNAs from drug-resistant Leishmania by orthogonal-field-alternation gel electrophoresis. The effect of size and topology on mobility.

Amplified extrachromosomal DNAs from antifolate-resistant Leishmania are 30-75 kilobase (kb) supercoiled molecules that resolve on orthogonal-field-alternation gel electrophoresis (OFAGE) gels. These DNAs comigrate with smaller supercoiled plasmids (7-8 kb), and their mobility is not a simple function of their size. The properties of the amplified DNAs were investigated to determine if an unusual structure accounts for the observed mobility of the amplified DNAs by OFAGE; however, their topological properties were similar to those of standard Escherichia coli plasmids. The migration of a series of supercoiled plasmids ranging in size from 6 to 91 kb was analyzed by OFAGE, and a triphasic pattern was observed. The mobilities of plasmids between 20 and 60 kb increase with size, whereas the migration of plasmids between 6 and 20 and 60 and 91 kb is inversely proportional to size. Like smaller plasmids, the large supercoiled DNAs show a pulse time-independent mobility by OFAGE. The mobility of amplified DNA from Leishmania is in accord with that of the plasmid markers. Therefore, it is primarily the size of the amplified extrachromosomal DNAs from Leishmania, rather than an unusual superhelical density or topological structure, that results in the previously unexplained migration pattern.

Extrachromosomal elements in the lower eukaryote Leishmania.

Extrachromosomal DNA elements have been identified in wild-type populations of the parasitic protozoan Leishmania. Elements from L. major and L. tropica were detected using orthogonal-field-alternation-gel electrophoresis. They are nonhomologous, supercoiled circular DNA molecules derived from different chromosomes in the Leishmania genome. Electron microscopy revealed that the elements have very similar physical properties; both are 80-kilobase supercoiled DNA molecules that contain large inverted repeat structures. The extrachromosomal DNAs are amplified in the Leishmania populations and show a fluctuation in copy number, from undetectable to around 20 copies per cell. After exposure of the L. tropica population to the drug methotrexate (MTX), a second amplified DNA was observed that is homologous to the extrachromosomal DNA found in L. major. Furthermore, wild-type Leishmania populations containing extrachromosomal DNA adapt more readily to MTX selection than populations with no amplified DNA. From these observations, there appears to be a relationship between the presence of extrachromosomal elements in wild-type Leishmania and the genesis and maintenance of MTX resistance in these organisms.

Electron microscopy of amplified DNA forms in antifolate-resistant Leishmania.

Three independently derived antifolate-resistant Leishmania major cell lines overproduce the bifunctional protein thymidylate synthase-dihydrofolate reductase (TS-DHFR) by amplification of a region of DNA (R-region DNA) that contains the gene for TS-DHFR. On orthogonal-field-alteration gel electrophoresis (OFAGE), the extrachromosomal R-region DNAs are circular molecules, and different forms of R-region DNA within these cell lines are resolved. The R-region DNAs migrate aberrantly on OFAGE with respect to linear DNA and supercoiled plasmid standards. We describe a method for the isolation of these R-region DNA forms from OFAGE. By electron microscopy, we show that the extrachromosomal elements are single supercoiled circular DNA molecules, and are predominantly circular monomers and dimers of the original R-region DNA amplification unit. Using OFAGE, an analysis of cloned isolates shows that individual cells may contain multiple forms of R-region DNA. Furthermore, within a given cell line, certain distinguishable forms appear to have the same size and restriction map, suggesting they may be topoisomers. The multiple forms of R-region DNA are in a dynamic state in the antifolate-resistant populations, and the relative amount of DNA in each form as well as the number of forms within each cell line change through time. As currently understood, the generation of amplified R-region DNA in L. major is summarized.

Plasmid migration using orthogonal-field-alternation gel electrophoresis.

The migration properties of a series of supercoiled plasmids ranging in size from 4 to 16 kilobases (kb) have been analyzed by orthogonal-field-alternation gel electrophoresis (OFAGE). These circular DNAs enter the gel and are well resolved. Unlike linear DNA molecules, the relative mobilities of these plasmids are constant over a wide range of pulse times, from 10 to 120 seconds, as well as over a broad range of total running times, from 6 to 24 hours. Electrophoresis of supercoiled, relaxed, and nicked open circular forms as well as topoisomers of pBR322 shows that the extent of supercoiling has a dramatic effect on plasmid migration on OFAGE. Several practical applications for exploiting the different migration properties of circular and linear DNA molecules on OFAGE are presented.

The molecular evolution of actin.

We have investigated the molecular evolution of plant and nonplant actin genes comparing nucleotide and amino acid sequences of 20 actin genes. Nucleotide changes resulting in amino acid substitutions (replacement substitutions) ranged from 3-7% for all pairwise comparisons of animal actin genes with the following exceptions. Comparisons between higher animal muscle actin gene sequences and comparisons between higher animal cytoplasmic actin gene sequences indicated less than 3% divergence. Comparisons between plant and nonplant actin genes revealed, with two exceptions, 11-15% replacement substitution. In the analysis of plant actins, replacement substitution between soybean actin genes SAc1, SAc3, SAc4 and maize actin gene MAc1 ranged from 8-10%, whereas these members within the soybean actin gene family ranged from 6-9% replacement substitution. The rate of sequence divergence of plant actin sequences appears to be similar to that observed for animal actins. Furthermore, these and other data suggest that the plant actin gene family is ancient and that the families of soybean and maize actin genes have diverged from a single common ancestral plant actin gene that originated long before the divergence of monocots and dicots. The soybean actin multigene family encodes at least three classes of actin. These classes each contain a pair of actin genes that have been designated kappa (SAc1, SAc6), lambda (SAc2, SAc4) and mu (SAc3, SAc7). The three classes of soybean actin are more divergent in nucleotide sequence from one another than higher animal cytoplasmic actin is divergent from muscle actin. The location and distribution of amino acid changes were compared between actin proteins from all sources. A comparison of the hydropathy of all actin sequences, except from Oxytricha, indicated a strong similarity in hydropathic character between all plant and nonplant actins despite the greater number of replacement substitutions in plant actins. These protein sequence comparisons are discussed with respect to the demonstrated and implicated roles of actin in plants and animals, as well as the tissue-specific expression of actin.

Divergence and differential expression of soybean actin genes.

DNA sequence analysis as well as genomic blotting experiments using cloned soybean actin DNA sequences as probes show that large sequence heterogeneity exists among members of the soybean actin multigene family. This heterogeneity suggested that the members of this family might be diverged in function and/or regulation. Five of the six soybean actin gene family members examined are shown to be significantly more diverged from one another than members of other known actin gene families. This high level of divergence was utilized in the preparation of actin gene-specific probes in the analysis of the complexity and expression of these members of the soybean actin gene family. Hybridization studies indicate that the six soybean actin genes fall into three classes with a pair of genes in each class. These six genes account for all but two actin gene fragments detected in the soybean genome. We have compared the relative steady state mRNA levels of these classes of soybean actin genes in three organs of soybean. We find that actin genes SAc6 and SAc7 are most highly expressed accounting for 80% of all actin mRNA with respect to the six soybean actin genes examined. Actin genes SAc3 and SAc1 are expressed at intermediate and low levels respectively; and SAc2 and SAc4 are expressed at barely detectable levels. Four of the six soybean actin genes appear to be expressed at the same level in root, shoot and hypocotyl. SAc3 and SAc7 genes appear to be more highly expressed in shoot and 2,4-dichlorophenoxyacetic acid-induced hypocotyl than in root and hypocotyl.(ABSTRACT TRUNCATED AT 250 WORDS)

Genes encoding actin in higher plants: intron positions are highly conserved but the coding sequences are not.

We have isolated actin genes from genomic libraries of two highly diverged plants, maize and soybean. The complete nucleotide sequences of a maize actin gene, MAc1, and a soybean actin gene, SAc1, were determined. The nucleotide sequences of these two actin genes and of a previously sequenced soybean actin gene were compared with the actin gene sequences from a wide spectrum of evolutionarily diverged eukaryotes. Some striking features pertinent to the evolution and function of the plant actin gene families have emerged. The deduced amino acid sequence of the plant actins resembles both cytoplasmic- and muscle-specific actins. DNA sequence analysis as well as genomic blotting experiments using cloned actin sequences as probes show that large sequence heterogeneity exists among members of the plant actin multigene families and between genes from two highly diverged plant species. The sequences of the first nine amino acids at the amino terminal end of the plant actins are far more conserved between distant plant actins than the corresponding sequences in distantly related animal actin genes, suggesting a unique and conserved function for the NH2 terminal sequence in higher plants. The soybean and maize actin genes examined each contain three introns in precisely the same positions, quite contrary to the divergent placement of introns observed in animal, protozoan, and fungal actins. The position of the first intron in soybean and maize actin genes corresponds precisely to the position of an intron found in a nematode actin gene. The position of the second intron coincides with one found in rat and chicken skeletal actin genes. These data suggest that the numerous introns found in all actins are of ancient origin. The degree of silent substitution and replacement substitution was compared among plant actin genes and to those of animal, protozoan, and yeast actin genes. It is clear that the silent substitution sites are saturated among all the genes compared, whereas the replacement sites have diverged in only 5-17% of their possible positions. By these criteria the most distant animal actins are only 6% diverged. The three plant actin genes examined are 8-10% diverged in replacement sites from each other and approximately 14% diverged in replacement sites from any of the animal actins examined. The data in this manuscript suggest that the families of soybean and maize actin genes may have diverged from a single common ancestral actin gene long before the divergence of monocots and dicots.

Complete nucleotide sequence of a soybean actin gene.

Soybean contains a small multigene family of actin-related sequences. We have determined the complete nucleotide sequence of a soybean actin gene carried on the recombinant plasmid pSAc3. As deduced from the nucleotide sequence, this soybean actin is composed of 376 amino acids. Compared to other eukaryotic actins, pSAc3 actin has a deletion of one amino acid between residues 118 and 122. The initiator methionine is followed by alanine, which is not found at this position in other eukaryotic actins. pSAc3 actin differs, in primary sequence, more from fungal and animal actins than any of the known nonplant actins differ from each other. pSAc3 actin appears to be related to both cytoplasmic and muscle specific actins in the location of specific NH(2)-terminal amino acids. The coding sequence is interrupted by three small introns, each less than 90 base pairs long. The splice junctions are similar to those found in other eukaryotic genes, suggesting the presence of a similar splicing apparatus in higher plants. Introns 1 and 3 interrupt the reading frame after codons 20 and 355, respectively. Intron 2 splits a glycine codon at position 151. None of these intron positions is conserved relative to the positions of introns in other actin genes examined.