Finding intron/exon splice junctions using INFO, INterruption Finder and Organizer

INFO, INterruption Finder and Organizer, has been used to find coding sequence intron-exon splice junctions in human and other DNA by comparing the six conceptual translations of the input DNA sequence with sequences in protein databanks using a similarity matrix and windowing algorithm. Similarities detected both delineate position of the gene and provide clues as to the function of the gene product. In addition to use of a standard similarity matrix and windowing algorithm, INFO uses two novel steps, the MiniLibrary and Reverse Sequence steps, to enhance identification of small exons and to improve precision of junction nucleotide delineation. Exons as small as about 30 bases can be reliably found, and > 90% of junctions are precisely identified when canonical splice junction information is used. With the MiniLibrary and Reverse Sequence steps, INFO parameters need not be optimized by the user. In comparative test runs using 19 human DNA sequences, INFO found 108 of 111 exons, with 0 reported false positives, compared with 111 exons and 51 false positives for BLASTX, 99 exons and 6 false positives for GRAIL II, 77 exons and 24 false positives for GeneMark, 61 exons and 9 false positives for GeneID, and 105 exons and 6 false positives for PROCRUSTES. The correlation coefficient for finding and positioning these 111 exons was greater than 98% for INFO. Comparable results were obtained in test runs of 13 nonhuman DNA sequences. INFO is applicable to DNA from any species, will become more robust as sequence databanks expand, and complements other heuristic approaches.     

Thrombopoietin (TPO) knockout phenotype induced by cross-reactive antibodies against TPO following injection of mice with recombinant adenovirus encoding human TPO

Adenovirus vectors have emerged as potent agents for gene transfer. Immune response against the vector and the encoded protein is one of the major factors in the transient expression following in vivo gene transfer. A single injection of an adenovirus encoding human thrombopoietin (TPO) into mice induced transient thrombocytosis, followed by a chronic immune thrombocytopenia. Thrombocytopenic mice had anti-human TPO Abs of the IgG2a and IgG1 isotypes. Thrombocytopenic mice sera neutralized more efficiently human than murine TPO, and exhibited no detectable anti-murine TPO Abs. Despite their low affinity for murine TPO, anti-TPO Abs induced a TPO knockout-like phenotype, i.e., low number of marrow megakaryocytes and of all kinds of hemopoietic progenitors. Hybridomas derived from a thrombocytopenic mouse revealed cross-reactivity of all of the secreted anti-TPO Ab isotypes. Mice subjected to myelosuppression after virus injection showed that anti-human TPO of IgG1 and IgG2a isotypes disappeared. Thus, sustained human TPO production was responsible for platelet elevation for at least 5 mo. Compelling results showed that elevated IgG2a/IgG2b ratios are always associated with thrombocytopenia, whereas low ratios are associated with tolerance or normal platelet counts. Finally, we hypothesize that in humans some chronic thrombocytopenia associated with a low TPO plasma level are due to anti-TPO Abs.     

Long-term effects of perindopril on metabolic parameters and the heart in the spontaneously hypertensive/NIH-corpulent rat with non-insulin-dependent diabetes mellitus and hypertension

The spontaneously hypertensive/NIH-corpulent (SHR/N-cp) rat is a genetic model that exhibits both non-insulin-dependent diabetes mellitus (NIDDM) and hypertension. To determine the impact of long-term treatment with the long-acting angiotensin-converting enzyme (ACE) inhibitor perindopril (PE) on the glucose metabolism, lipid levels, and heart in this model, studies were performed in three groups of SHR/N-cp rats maintained on a diet containing 54% carbohydrate with 18% sucrose and 36% starch. One group of obese rats received PE (0.5 to 1.0 mg/kg body weight/d) for 3 to 4 months, a second group of obese rats received no treatment, and a third group of lean rats were used as controls. The mean systolic blood pressure (SBP) increased gradually in both untreated obese and lean rats, with lean animals showing slightly higher levels compared with untreated obese rats. By contrast, SBP was reduced to normal levels in PE-treated obese rats throughout the treatment period. Compared with lean rats, obese rats showed significantly higher body weight and fasting serum levels of glucose, insulin, total cholesterol (TC), and triglyceride (TG). However, no significant differences were observed in these metabolic parameters between PE-treated and untreated obese rats. Plasma renin activity measured at the end of the treatment period was significantly higher in PE-treated rats compared with untreated obese and untreated lean rats. The mean heart weight and left ventricular weight, expressed in absolute terms or indexed to body weight, were significantly lower in PE-treated versus untreated obese and untreated lean rats. To further determine whether glucose metabolism is directly affected by PE treatment, in vitro glycogen synthesis was evaluated in isolated soleus muscles obtained from three additional groups of animals. The basal rate of muscle glycogen synthesis was significantly lower in obese compared with lean rats (P < .05), but did not differ between PE-treated and untreated obese rats. Maximal insulin-stimulated glycogen synthesis increased threefold in PE-treated obese rats, but this increase did not differ from the increases observed in untreated obese and lean rats. In conclusion, the present study shows that long-term PE treatment in obese SHR/N-cp rats with NIDDM and hypertension effectively controlled systemic arterial pressure and resulted in a significant reduction in left ventricular weight. However, these favorable effects of PE were not associated with significant improvement in glucose tolerance, hyperinsulinemia, and hyperlipidemia in this model. PE also had no direct stimulatory effects on either basal or insulin-mediated glycogen synthesis in the isolated soleus muscle of obese rats, perhaps because of the severe insulin-resistant state of the animals. Our results support the clinical observations that antihypertensive therapy with ACE inhibitors has neutral effects on glucose metabolism and insulin sensitivity in patients with combined hypertension and NIDDM.     

beta-Amyloid precursor protein isoforms show correlations with neurones but not with glia of demented subjects

Post-mortem cerebral cortex from 15 demented patients was specially collected to minimise autolysis and two membrane fractions and one soluble fraction were quantitatively examined for the major species of beta-amyloid precursor protein (APP) of high apparent molecular mass (> or = 80 kDa) together with the major mRNA species encoding APP isoforms. The number of pyramidal neurones and astrocytes, putative biochemical indices of interneurones and pyramidal neurones, and choline acetyl transferase activity were also determined. Multiple regression analysis has been used to investigate intercorrelations of APP species with biochemical and morphometric measures, free of any effects of confounding demographic variables. Subjects with Alzheimer's disease showed a loss of cholinergic activity and D-aspartate uptake compared with patients with other causes of dementia. The major finding of the study is that measures of neurones rather than astrocytes most closely correlate with the concentration of APP. Pyramidal cell numbers were positively correlated with mRNA for APP695. APP in the soluble fraction showed a negative correlation with pyramidal cell numbers and cholinergic activity. These results indicate that neurones within the cerebral cortex are the major source of APP, and that secretion of APP is dependent upon cortical pyramidal neuronal activity and cholinergic activity.     

Sternal traction after open heart operation: an effective alternative to delayed sternal closure

BACKGROUND: Routine closure of the sternum after cardiovascular surgical procedures sometimes causes severe cardiac depression because of a tamponade-like reduction in ventricular filling, leading to cardiogenic shock. Leaving the sternal halves apart, sealing the mediastinum by simply approximating the skin or using a prosthetic patch, and then performing delayed sternal closure in several days is a widely practiced life-saving maneuver. METHODS: Described herein is an experience with 5 patients with severe cardiac output depression of the type usually treated by delayed sternal closure. Instead, upward (outward) traction was applied to the anterior chest while the sternum was primarily closed. Traction was maintained with full-thickness chest wall sutures. RESULTS: The traction sutures were removed successfully in the intensive care unit between 1 and 4 days postoperatively, after appropriate vigorous treatment of postbypass myocardial enlargement and pulmonary distention and edema. CONCLUSIONS: This method of sternal traction allows physiologic improvement equivalent to delayed sternal closure in some patients and obviates the need for returning to the operating room to close the sternum in the early postoperative period.     

Lineage- and stage-specific expression of runt box polypeptides in primitive and definitive hematopoiesis

Translocations involving the human CBFA2 locus have been associated with leukemia. This gene, originally named AML1, is a human homologue of the Drosophila gene runt that controls early events in fly embryogenesis. To clarify the role of mammalian runt products in normal and leukemic hematopoiesis, we have studied their pattern of expression in mouse hematopoietic tissues in the adult and during ontogeny using an anti-runt box antiserum. In the adult bone marrow, we found expression of runt polypeptides in differentiating myeloid cells and in B lymphocytes. Within the erythroid lineage, runt expression is biphasic, clearly present in the erythroblasts of early blood islands and of the fetal liver, but absent in the adult. Biochemical analysis by Western blotting of fetal and adult hematopoietic populations shows several runt isoforms. At least one of them appears to be myeloid specific.     

Contrasting patterns of nucleotide sequence variation at the glucose dehydrogenase (Gld) locus in different populations of Drosophila melanogaster

We have analyzed nucleotide sequence variation at the Glucose dehydrogenase (Gld) locus from four populations of Drosophila melanogaster from four continents. All four population samples show a significant reduction in silent variation compared to the neutral expectation. The levels of silent variation across all four populations are consistent with the predictions of the background selection model; however, Zimbabwe has a remarkably low level of variation. In the face of dramatically reduced silent polymorphism, an amino acid variant, leading to the common allozyme polymorphism at Gld, remains in low to intermediate frequency in all non-African samples. In the Chinese population sample, the ratio of replacement to silent variation is significantly elevated compared to the neutral expectation. The difference in patterns of variation across these population samples suggests that selection on Gld (or the Gld region) has been different in the Chinese population than in the other three.     

A mouse molecular mimic of human vascular adhesion protein-1

Human vascular adhesion protein-1 (VAP-1) is an endothelial sialoglycoprotein which exists in forms of Mr 90000 and 170000 and mediates lymphocyte binding to vessels under shear. VAP-1 is functionally defined by an inhibitory mouse mAb 1B2. A large-scale immunoaffinity purification of VAP-1 from human tonsil lysates was performed to determine the protein sequence for VAP-1 cDNA cloning. A dominant protein of molecular weight 90000 was obtained which yielded an N-terminal sequence of 20 amino acids which bore no significant identity to any protein sequence in the data banks. A mouse mAb (5B11) against a synthetic peptide from this sequence was raised and found to stain tissues in an identical manner to mAb 1B2, to inhibit lymphocyte adhesion to endothelial cells and to recognize VAP-1. Later, the N-terminal sequence obtained from the 1B2 immunoprecipitations was found to be identical to a mouse cyclophilin C associated protein (mCyCAP) subsequently published by others. We show here by several criteria at the protein and DNA level that VAP-1 is distinct from mCyCAP. Moreover, we elucidate the mechanism which results in binding of mCyCAP to mAb 1B2 during antibody synthesis in hybridoma cells and the sequelae of co-precipitation of mCyCAP during the immunoaffinity chromatography. Binding of mCyCAP to a mouse mAb has not been described before and suggests a new function for this molecule in immunoglobulin synthesis and/or secretion. Moreover, these data indicate that the N-terminal peptide of mCyCAP is a molecular mimic of a functionally important epitope of VAP-1.