Detecting and describing heterogeneity in meta-analysis

The investigation of heterogeneity is a crucial part of any meta-analysis. While it has been stated that the test for heterogeneity has low power, this has not been well quantified. Moreover the assumptions of normality implicit in the standard methods of meta-analysis are often not scrutinized in practice. Here we simulate how the power of the test for heterogeneity depends on the number of studies included, the total information (that is total weight or inverse variance) available and the distribution of weights among the different studies. We show that the power increases with the total information available rather than simply the number of studies, and that it is substantially lowered if, as is quite common in practice, one study comprises a large proportion of the total information. We also describe normal plots that are useful in assessing whether the data conform to a fixed effect or random effects model, together with appropriate tests, and give an application to the analysis of a multi-centre trial of blood pressure reduction. We conclude that the test of heterogeneity should not be the sole determinant of model choice in meta-analysis, and inspection of relevant normal plots, as well as clinical insight, may be more relevant to both the investigation and modelling of heterogeneity.     

Complete nucleotide sequence of the Actinomyces viscosus T14V sialidase gene: presence of a conserved repeating sequence among strains of Actinomyces spp

The nucleotide sequence of the Actinomyces viscosus T14V sialidase gene (nanH) and flanking regions was determined. An open reading frame of 2,703 nucleotides that encodes a predominately hydrophobic protein of 901 amino acids (M(r), 92,871) was identified. The amino acid sequence at the amino terminus of the predicted protein exhibited properties characteristic of a typical leader peptide. Five 12-amino-acid units that shared between 33 and 67% sequence identity were noted within the central domain of the protein. Each unit contained the sequence Ser-X-Asp-X-Gly-X-Thr-Trp, which is conserved among other bacterial and trypanosoma sp. sialidases. Thus, the A. viscosus T14V nanH gene and the other prokaryotic and eukaryotic sialidase genes evolved from a common ancestor. Southern hybridization analyses under conditions of high stringency revealed the existence of DNA sequences homologous to A. viscosus T14V nanH in the genomes of 18 strains of five Actinomyces species that expressed various levels of sialidase activity. The data demonstrate that the sialidase genes from divergent groups of Actinomyces spp. are highly conserved.     

Detecting heterogeneity of substitution along DNA and protein sequences

Regions of differing constraint, mutation rate or combination along a sequence of DNA or amino acids lead to a nonuniform distribution of polymorphism within species or fixed differences between species. The power of five tests to reject the null hypothesis of a uniform distribution is studied for four classes of alternate hypothesis. The tests explored are the variance of interval lengths; a modified variance test, which includes covariance between neighboring intervals; the length of the longest interval; the length of the shortest third-order interval; and a composite test. Although there is no uniformly most powerful test over the range of alternate hypotheses tested, the variance and modified variance tests usually have the highest power. Therefore, we recommend that one of these two tests be used to test departure from uniformity in all circumstances. Tables of critical values for the variance and modified variance tests are given. The critical values depend both on the number of events and the number of positions in the sequence. A computer program is available on request that calculates both the critical values for a specified number of events and number of positions as well as the significance level of a given data set.     

Phenotypic heterogeneity of mutational changes at a conserved nucleotide in 16 S ribosomal RNA

RNA sites that contain unpaired or mismatched nucleotides can be interaction sites for other macromolecules. C1054, a virtually universally conserved nucleotide in the 16 S (small subunit) ribosomal RNA of Escherichia coli, is part of a highly conserved bulge in helix 34, which has been located at the decoding site of the ribosome. This helix has been implicated in several translational events, including peptide chain termination and decoding accuracy. Here, we observed interesting differences in phenotype associated with the three base substitutions at, and the deletion of, nucleotide C1054. The phenotypes examined include suppression of nonsense codons on different media and at different temperatures, lethality conditioned by temperature and level of expression of the mutant rRNA, ribosome profiles upon centrifugation through sucrose density gradients, association of mutant 30 S subunits with 50 S subunits, and effects on the action of tRNA suppressor mutants. Some of our findings contradict previously reported properties of individual mutants. Particularly notable is our finding that the first reported 16 S rRNA suppressor of UGA mutations was not a C1054 deletion but rather the base substitution C1054A. After constructing deltaC1054 by site-directed mutagenesis, we observed, among other differences, that it does not suppress any of the trpA mutations previously reported to be suppressed by the original UGA suppressor. In general, our results are consistent with the suggestion that the termination codon readthrough effects of mutations at nucleotide 1054 are the result of defects in peptide chain termination rather than of decreases in general translational accuracy. The phenotypic heterogeneity associated with different mutations at this one nucleotide position may be related to the mechanisms of involvement of this nucleotide, the two-nucleotide bulge, and/or helix 34 in particular translational events. In particular, previous indications from other laboratories of conformational changes associated with this region are consistent with differential effects of 1054 mutations on RNA-RNA or RNA-protein interactions. Finally, the association of a variety of phenotypes with different changes at the same nucleotide may eventually shed light on speculations about the coevolution of parts of ribosomal RNA with other translational macromolecules.     

Neuroligand receptor heterogeneity among astroglia

The clinical and histopathological findings are described in a 39-year-old female patient with two different primary ophthalmic cancers, involving the adnexa and ocular globe of the same eye. The first primary tumor was a malignant fibrous histiocytoma of the left lower eyelid aggressively invading the nasogenian region, bulbar conjunctiva, episclera, and the cornea over a 36-year follow-up period. The second primary cancer was an unsuspected choroidal malignant melanoma unexpectedly found at histology. The possible correlations between these two malignancies are discussed.     

Cloning and nucleotide sequence of a specific DNA fragment from Paracoccidioides brasiliensis

We cloned and sequenced a species-specific 110-bp DNA fragment from Paracoccidioides brasiliensis. The DNA fragment was generated by PCR with primers complementary to the rat beta-actin gene under a low annealing temperature. Comparison of the nucleotide sequence, after excluding the primers, with those in the GenBank database identified approximately 60% homology with an exon of a major surface glycoprotein gene from Pneumocystis carinii and a fragment of unknown function in Saccharomyces cerevisiae chromosome VIII. By Southern hybridization analysis, the 32P-labelled fragment detected 1.0- and 1.9-kb restriction fragments within whole-cell genomic DNA of P. brasiliensis digested with HindIII and PstI, respectively, but failed to hybridize to genomic DNAs from Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Aspergillus fumigatus, Saccharomyces cerevisiae, Pneumocystis carinii, rat tissue, or humans under low-stringency hybridization conditions. Additionally, the specific DNA fragment from three different P. brasiliensis isolates (Pb18, RP18, RP17) was amplified by PCR with primers mostly complementary to nonactin sequences of the 110-bp DNA fragment. In contrast, there were no amplified products from other fungus genomic DNAs previously tested, including Histoplasma capsulatum. To date, this is the first species-specific DNA fragment cloned from P. brasiliensis which might be useful as a diagnostic marker for the identification and classification of different P. brasiliensis isolates.     

Extreme structural heterogeneity among the members of a maize retrotransposon family

A few families of retrotransposons characterized by the presence of long terminal repeats (LTRs) have amplified relatively recently in maize and account for >50% of the genome. Surprisingly, none of these elements have been shown to cause a single mutation. In contrast, most of the retrotransposon-induced mutations isolated in maize are caused by the insertion of elements that are present in the genome at 2-50 copies. To begin to understand what limits the amplification of this mutagenic class of LTR-retrotransposons, we are focusing on five elements previously identified among 17 mutations of the maize waxy gene. One of these elements, Stonor, has sustained a deletion of the entire gag region and part of the protease domain. Missing sequences were recovered from larger members of the Stonor family and indicate that the deletion probably occurred during retrotransposition. These large elements have an exceptionally long leader of 2 kb that includes a highly variable region of approximately 1 kb that has not been seen in previously characterized retrotransposons. This region serves to distinguish each member of the Stonor family and indicates that no single element has yet evolved that can attain the very high copy numbers characteristic of other element families in maize.     

Cloning and nucleotide sequence of a full-length cDNA encoding Ascaris suum malic enzyme

The nucleotide sequence of a full-length cDNA encoding NAD(+)-malic enzyme from the parasitic nematode Ascaris suum was determined. The entire sequence of 2269 bases comprises a 5'-leader, a single open reading frame of 1851 bases, and the complete 3'-noncoding region of 340 bases. The first 12 amino acids of the translated sequence are hydrophobic, typical of mitochondrial translocation signals, and do not appear in the purified mature protein. The mature protein contains 605 amino acids and has a molecular mass of 68,478 Da. The amino acid sequences of tryptic peptides from the purified protein and also the N-terminal sequence show excellent correspondence with the translated nucleotide sequence. Comparison of the amino acid sequence of the ascarid protein with the human and rat liver NAD(+)-malic enzymes reveals highly conserved regions interrupted with long stretches of lesser homologous sequences. Structural motifs such as the putative nucleotide binding domains and also the malate binding site are clearly identified by alignment of the three protein sequences.     

Molecular analysis of sequence heterogeneity among genes encoding decorin binding proteins A and B of Borrelia burgdorferi sensu lato

Immunization of mice with Borrelia burgdorferi decorin binding protein A (DbpA), one of two gene products of the dbpBA locus, has been shown recently to confer protection against challenge. Hyperimmune DbpA antiserum killed a large number of B. burgdorferi sensu lato isolates of diverse phylogeny and origin, suggesting conservation of the protective epitope(s). In order to evaluate the heterogeneity of DbpA and DbpB and to facilitate defining the conserved epitope(s) of these antigens, the sequences of the dbpA genes from 29 B. burgdorferi sensu lato isolates and of the dbpB genes from 15 B. burgdorferi sensu lato isolates were determined. The predicted DbpA sequences were fairly heterogeneous among the isolates (58.3 to 100% similarity), but DbpA sequences with the highest similarity tended to group into species previously defined by well-characterized chromosomal markers. In contrast, the predicted DbpB sequences were highly conserved (96.3 to 100% similarity). Substantial diversity in DbpA sequence was seen among isolates previously shown to be killed by antiserum against a single DbpA, suggesting that one or more conserved protective epitopes are composed of noncontiguous amino acids. The observation of individual dbpA alleles with sequence elements characteristic of more than one B. burgdorferi sensu lato species was consistent with a role for genetic recombination in the generation of dbpA diversity.     

16S-23S and 23S-5S intergenic spacer regions of lactobacilli: nucleotide sequence, secondary structure and comparative analysis

Lactobacilli have been used as industrial starters for a long time, but in several cases their identification was, and still is, neither easy nor reliable. The aim of the present work was to examine whether the intergenic spacer regions could be of value in the identification of Lactobacillus species. For that purpose, the polymerase chain reaction (PCR) was used to amplify 16S-23S and 23S-5S spacer regions of Lactobacillus (L.) acidophilus, L. delbrueckii subsp. bulgaricus, L. casei, L. helveticus and L. curvatus. The PCR products were directly sequenced, and two forms of ribosomal RNA (rrn) operons were identified in each species studied: one with tandem tRNA(Ile)/tRNA(Ala) genes and the other one without tRNA genes. Our study revealed that the rrn operons of Lactobacillus species studied comprise the genes of 16S, 23S and 5S rRNA, in that order. Only the tRNA genes and the rRNA processing stems are highly conserved in spacer regions of lactobacilli. The divergence between the lactobacilli spacer region sequences arises from insertions and deletions of short sequences. These sequences could be interesting candidates for the development of species-specific probes. Theoretical RNA/RNA secondary structure models of the interaction between the two spacer region sequences were constructed. In conclusion, the two spacer region sequences may prove to be a useful alternative to 16S and 23S rDNA sequencing for designing species-specific probes and for establishing phylogenetic relationships between closely related species such as L. curvatus and L. casei or L. acidophilus and L. helveticus.     

Update on Pneumocystis carinii f. sp. hominis typing based on nucleotide sequence variations in internal transcribed spacer regions of rRNA genes

Few data exist concerning expiratory muscle function in amyotrophic lateral sclerosis (ALS). We studied 26 patients with ALS (16 with respiratory symptoms and 10 without) and measured the maximal static expiratory mouth pressure (MEP), the gastric pressure during a maximal cough (Cough Pga), and the gastric pressure after magnetic stimulation of the lower thoracic nerve roots (Tw Pga). These measurements were related to the ability to generate transient supramaximal flow during a cough (cough spikes), to arterialized capillary blood gases, and to inspiratory muscle strength. Vocal cord motion was examined endoscopically in 11 of the 16 symptomatic patients. Expiratory muscle weakness was related to inability to generate cough spikes with a threshold effect such that spikes were absent for Cough Pga < 50 cm H2O (p = 0.009) or Tw Pga < 7 cm H2O (p = 0.006) and was usually associated with inspiratory muscle weakness. However, in multivariate analysis, PaCO2 was only significantly associated with the maximal sniff esophageal pressure (p = 0.02). Symptomatic patients had significantly lower inspiratory muscle strength, whereas, of the expiratory muscle tests, only Tw Pga was significantly lower (p = 0.0009) in symptomatic patients. Abnormal vocal cord motion was observed in two of the 11 patients examined. We conclude that abdominal muscle weakness in ALS, when substantial, results in an inability to generate transient supramaximal flow during a cough. However, the primary determinant of both ventilatory failure and respiratory symptoms seems to be inspiratory muscle weakness.     

Antigenic and differentiative heterogeneity among human glioblastomas

Increasing evidence suggests that in mammals, astrocytes are a heterogenous family of cells all of which share certain properties, but differ in lineage, biochemical and functional aspects. It seems likely that glioblastomas, arising from glial precursors, may also represent a family of related but distinct cell types. We have examined the antigenic characteristics and differentiative potential of 7 different human glioblastoma cell lines in vitro. All the cell lines were labeled with a monoclonal antibody 7B11 which labels all classes of astrocytes and their precursors in the rat CNS. U138MG and Tm3 cells expressed antigens on their surfaces recognized by the monoclonal antibodies A2B5 and HNK-1. When grown in serum-free medium in the presence of cAMP and theophylline, U138MG cells assumed a process-bearing morphology and some cells expressed the Gal-C antigen specific for oligodendrocytes. Under identical conditions, Tm3 cells converted to process-bearing cells, some of which expressed glial fibrillary acidic protein (GFAP) specific for astrocytes. Other cell lines with similar antigenic characteristics did not respond similarly to cAMP and theophylline. Finally, A2781 cells were GFAP immunoreactive and unlabeled by either A2B5 or HNK-1 antibodies. These observations suggest that individual glioblastoma cell lines may be derived from distinct glial precursor cells in the vertebrate CNS.     

Complete nucleotide sequence of the Plasmodium vivax 6 kb element

A small survey confirms that epidural infusions are often prepared by clinicians and solutions may be changed each day because of fears of microbiological contamination. We have assessed the microbiological safety of six mixtures of diamorphine (0.01-1.0%) and bupivacaine (0.1-0.5%) representing the spectrum of clinically useful concentrations for use as extradural infusions. The solutions were studied for antibacterial activity against common contaminants of fluids: Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis and a coagulase negative Staphylococcus sp. A saline control was included. Challenge experiments used an inoculum of approximately 5.0 x 10(6) cfu/ml. Mixtures were incubated at 30 degrees C for up to 7 days. Viable counts of all organisms decreased with time for all the formulations tested. Formulations containing 0.5% bupivacaine were rapidly bactericidal, and increasing diamorphine concentrations increased this effect. Formulations with 0.5% bupivacaine had more activity than those with 0.1% bupivacaine. Mixtures of diamorphine and bupivacaine in concentrations used clinically have bactericidal activity against commonly encountered skin organisms. The common practice of changing every day epidural infusions containing these drugs is unnecessary.     

Complete nucleotide sequence of wheat yellow mosaic bymovirus genomic RNAs

The complete sequences of wheat yellow mosaic bymovirus (WYMV) RNA1 and RNA2 were determined. RNA1 is 7636 nucleotides long [excluding the 3'-poly(A)], and codes for a 269 kDa polyprotein of 2,404 amino acids which contains the capsid protein (CP) at the C terminus and seven putative nonstructural proteins. RNA2 is 3,659 nucleotides long and codes for a polyprotein of 904 amino acids which contains a 28 kDa putative proteinase and a 73 kDa polypeptide. These functional proteins are arranged as in RNA1 and RNA2 of barley yellow mosaic bymovirus (BaYMV). Comparisons with the sequence reported for the 3' half of RNA1 of wheat spindle streak mosaic bymovirus (WSSMV) from Southern France show that WYMV and WSSMV have a similar genetic organization. However, WYMV and WSSMV share only 77% amino acid sequence identity in their deduced CPs in spite of their close serological relationship, and 74% nucleotide sequence identity in their 3' non-coding regions. Thus, the sequence data indicate that WYMV and WSSMV are not strains of the same virus, which has long been suggested, but are distinct virus species within the genus Bymovirus of the family Potyviridae.     

Cloning and nucleotide sequence of amidase gene from Pseudomonas putida

The peptidergic signal substance thyrotropin-releasing hormone (TRH) is inactivated by the TRH-degrading ectoenzyme (TRH-DE), a peptidase that exhibits an extraordinary high degree of substrate specificity and other unusual characteristics. There is no other ectopeptidase known capable of degrading this tripeptideamide, and vice versa, TRH is the only known substrate of this unique enzyme. Thus, studies on this enzyme may reveal new aspects on the function of the TRH signaling system. After succeeding in purifying this enzyme to homogeneity and cloning the cDNA encoding rat TRH-DE, molecular tools became available to study the expression of this enzyme by Northern blot analysis and in situ hybridization histochemistry. The stringent and tissue-specific regulation of the adenohypophyseal TRH-DE by estradiol and thyroid hormones strongly suggests that this enzyme may act as a regulatory element modulating pituitary hormone secretion. In brain, the expression of TRH-DE is not influenced by peripheral hormones but the distinct distribution pattern, and the high activities support the concept that in this tissue TRH-DE may act as a terminator of TRH signals.     

Cloning and nucleotide sequence of mouse immunoglobulin epsilon chain cDNA

cDNA corresponding to mouse IgE heavy (epsilon) chain mRNA was cloned from mouse IgE-secreting hybridoma cells. A clone containing the epsilon cDNA insert was identified by hybridization to epsilon mRNA and subsequent translation in vitro to unprocessed epsilon chain reactive with anti-mouse IgE antibodies. This clone was used to select 20 other epsilon cDNA clones by colony hybridization. The clone containing the longest insert was selected and the epsilon cDNA insert was subjected to sequence analysis. The determined sequence is 1,279 nucleotides long and contains the coding regions for part of the constant region (C epsilon) I and all of the C epsilon 2, C epsilon 3, and C epsilon 4 domains and also the entire 3' untranslated region of epsilon mRNA. When the amino acid sequence determined from the nucleotide sequence is compared to that of human epsilon chain, significant homologies between corresponding domains of the two epsilon chains are found, including conservations in cysteine and tryptophan residues and carbohydrate attachment sites.