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Analysis of ex vivo left ventricular pressure-volume relations in the isolated murine ejecting heart.

The development of microconductance technology to study cardiac pressure-volume relations in mice in vivo has significantly advanced the haemodynamic assessment of gene-modified models of cardiovascular disease. In this study, we describe the application of microconductance analysis of cardiac function to the isolated murine ejecting heart. This ex vivo model is complementary to the previously described in vivo preparation, allows assessment without confounding effects of anaesthetic or neurohumoral influences and enables careful control of cardiac loading (particularly preload). Ex vivo pressure-volume relations in the isolated murine heart are sensitive to changes in myocardial contractility induced by beta-adrenoceptor stimulation or beta-adrenoceptor blockade, as well as the effects of chronic pressure overload induced by aortic banding. We present data for both steady-state analyses of the Frank-Starling relation and for assessment of the left ventricular pressure-volume relation over variably loaded beats, which allows investigation of the end-systolic and end-diastolic pressure-volume relations. The measurement of ventricular volume in addition to pressure under carefully controlled loading conditions in the isolated ejecting heart allows a comprehensive analysis of cardiac contractile function, and provides a useful complementary model for the assessment of cardiac performance in murine models of heart disease.

Animals↗

Long-term efficacy and safety of clarithromycin treatment in patients with diffuse panbronchiolitis.

Diffuse panbronchiolitis (DPB) can now be cured with long-term erythromycin treatment. Our group conducted a prospective open trial of long-term treatment with a macrolide antibiotic, clarithromycin. We studied ten patients who were treated for 4 years with oral clarithromycin (200 mg once a day). Pulmonary function test, blood gas analysis, comprehensive improvement score, and bacterial culture of sputum were examined at 3, 6, 12 months, and at 2, 3, 4 years after the initiation of the therapy. Pulmonary function improved in most of the patients within 6 months: the forced expiratory volume in one second showed a maximal increase from a mean (SE) value of 1.74 (0.12) l at baseline to 2.31 (0.22) l at 6 months (P < 0.01) and the volume (l) of forced vital capacity also showed a maximal increase within 6 months. The partial pressure of arterial oxygen at rest significantly increased at 3-6 months. The comprehensive improvement score also reached maximum within 6 months in nine of the patients. The majority of patients have developed sputum culture in which bacteria were negative within 6 months after the therapy. All of the patients maintained a stable condition with continued therapy, and no side effects of clarithromycin were observed during the study. This prospective study demonstrated that 6-month treatment with clarithromycin might be necessary to improve the clinical conditions of patients with DPB and the drug could be safely used for a long term.

Adult↗

Characterization of lipopolysaccharide-deficient mutants of Pseudomonas aeruginosa derived from serotypes O3, O5, and O6.

Well-characterized rough mutants are important for the understanding of structures, functions, and biosynthesis of lipopolysaccharide (LPS) in gram-negative organisms. In this study, three series of Pseudomonas aeruginosa LPS-deficient mutants, namely PAC strains derived from serotype O3, AK strains derived from strain PAO1 (serotype O5), and serotype O6-derived mutants were subjected to biochemical analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining as well as immunochemical characterization using LPS-specific monoclonal antibodies. The O-side-chain deficiency among the O6-derived mutants was also examined, and three mutants, A28, R5, and H4, were subsequently chosen for the elucidation of component sugars of the core structure of serotype O6 LPS. LPS of strain A28 has L-rhamnose and proportionally higher amounts of D-glucose, a feature shared by the O5-derived mutant, strain AK1401 (previously demonstrated as a mutant with a core-plus-one O repeat). In contrast strains R5 and H4 were shown to be devoid of L-rhamnose and have low and undetectable amounts of D-glucose, respectively, which indicated their core deficiency. The LPS-deficient or -sufficient characteristics of the P. aeruginosa strains examined correlated will with serum sensitivity data. This report represents a comprehensive analysis of rough mutants derived from O3 and O5 strains that have been used by others in many studies and a first look at the core oligosaccharide region of serotype O6 LPS obtained with the O6-derived mutants generated in this study.

Antibodies, Monoclonal↗

SEARCHPKS: A program for detection and analysis of polyketide synthase domains.

SEARCHPKS is a software for detection and analysis of polyketide synthase (PKS) domains in a polypeptide sequence. Modular polyketide synthases are unusually large multi-enzymatic multi-domain megasynthases, which are involved in the biosynthesis of pharmaceutically important natural products using an assembly-line mechanism. This program facilitates easy identification of various PKS domains and modules from a given polypeptide sequence. In addition, it also predicts the specificity of the potential acyltransferase domains for various starter and extender precursor units. SEARCHPKS is a user-friendly tool for correlating polyketide chemical structures with the organization of domains and modules in the corresponding modular polyketide synthases. This program also allows the user to extensively analyze and assess the sequence homology of various polyketide synthase domains, thus providing guidelines for carrying out domain and module swapping experiments. SEARCHPKS can also aid in identification of polyketide products made by PKS clusters found in newly sequenced genomes. The computational approach used in SEARCHPKS is based on a comprehensive analysis of various characterized clusters of modular polyketide synthases compiled in PKSDB, a database of modular polyketide synthases. SEARCHPKS can be accessed at http://www.nii.res.in/searchpks.html.

Computer Graphics↗

Risk/risk trade-offs in pesticide regulation: an exploratory analysis of the public health effects of a ban on organophosphate and carbamate pesticides.

Efforts to reduce pesticide-related risks to consumers and farmworkers often neglect the possibility that measures to reduce the target risk may introduce or enhance countervailing risks. These may arise from substitute pesticides or pest-control practices, from increased levels of pests or pest-related hazards, from increased levels of toxic natural pesticides in plants, from increased costs and decreased consumption of health-enhancing fruits and vegetables, or from direct income effects on consumers and farmers. The effect of the countervailing risks may partially or completely offset the reduction in the target risk. A risk-trade-off analysis was conducted of a potential ban on the use of organophosphate and carbamate (OP/Carbamate) insecticides in U.S. agriculture. Although this scenario is extreme, it has the analytic virtue of dispensing with the infinite number of "next-best" OP/Carbamates that might be substituted for specific combinations of crops and pests should only selected uses be banned. The analysis relies on detailed descriptions of the alternative pesticides and pest-control measures that would be used for each of 14 major crops. The effects of pest-control cost changes on prices and consumption and effects on consumer and producer incomes are projected using a general-equilibrium economic model. Several countervailing risks that may be significant were found, including acute toxicity to farmworkers from substitute pesticides, cancer and noncancer risks from substitute pesticides, and mortality induced by changes in disposable income. Other countervailing risks are more difficult to estimate or weigh. Potential increases in natural plant pesticides following an OP/Carbamate ban are discussed but data are lacking to quantify the effects. Changes in diet following the ban have both positive and negative effects, and the ultimate change is difficult to estimate. Although a net risk cannot be estimated, several approaches were illustrated that would be useful in risk-trade-off analyses. Key factors complicating comprehensive analysis of risk/risk trade-offs for pesticides were also identified, including data gaps and shortcomings of current risk assessment methods.

Agriculture↗

Comparative salivary gland transcriptomics of sandfly vectors of visceral leishmaniasis.

BACKGROUND: Immune responses to sandfly saliva have been shown to protect animals against Leishmania infection. Yet very little is known about the molecular characteristics of salivary proteins from different sandflies, particularly from vectors transmitting visceral leishmaniasis, the fatal form of the disease. Further knowledge of the repertoire of these salivary proteins will give us insights into the molecular evolution of these proteins and will help us select relevant antigens for the development of a vector based anti-Leishmania vaccine. RESULTS: Two salivary gland cDNA libraries from female sandflies Phlebotomus argentipes and P. perniciosus were constructed, sequenced and proteomic analysis of the salivary proteins was performed. The majority of the sequenced transcripts from the two cDNA libraries coded for secreted proteins. In this analysis we identified transcripts coding for protein families not previously described in sandflies. A comparative sandfly salivary transcriptome analysis was performed by using these two cDNA libraries and two other sandfly salivary gland cDNA libraries from P. ariasi and Lutzomyia longipalpis, also vectors of visceral leishmaniasis. Full-length secreted proteins from each sandfly library were compared using a stand-alone version of BLAST, creating formatted protein databases of each sandfly library. Related groups of proteins from each sandfly species were combined into defined families of proteins. With this comparison, we identified families of salivary proteins common among all of the sandflies studied, proteins to be genus specific and proteins that appear to be species specific. The common proteins included apyrase, yellow-related protein, antigen-5, PpSP15 and PpSP32-related protein, a 33-kDa protein, D7-related protein, a 39- and a 16.1- kDa protein and an endonuclease-like protein. Some of these families contained multiple members, including PPSP15-like, yellow proteins and D7-related proteins suggesting gene expansion in these proteins. CONCLUSION: This comprehensive analysis allows us the identification of genus- specific proteins, species-specific proteins and, more importantly, proteins common among these different sandflies. These results give us insights into the repertoire of salivary proteins that are potential candidates for a vector-based vaccine.

Amino Acid Sequence↗

Expression dynamics of the tomato rbcS gene family during development.

The tomato rbcS gene family is composed of five genes (rbcS1, 2, 3A, 3B, and 3C) that are differentially expressed during tomato development. Nuclear run-on transcription assays and RNA analysis were used to determine the contribution of transcriptional and post-transcriptional regulation to the accumulation of mRNA from the five rbcS genes in tomato seedlings, leaves, and fruit. We found that the qualitative pattern of mRNA accumulation is regulated at the transcriptional level and that, in general, there is a correlation of rates of rbcS transcription with overall rbcS mRNA abundance in fruit and leaves. Although transcriptional control is a primary determinant for rbcS gene expression in tomato, examination of relative transcription rates and mRNA accumulation of each rbcS gene demonstrated that there is also significant post-transcriptional control of rbcS gene expression during organ development. Individual rbcS mRNAs, which have highly conserved coding sequences and differ only in their 5' and 3' untranslated sequences, have different stabilities. We showed that both transcription and stability of individual rbcS mRNAs are altered in different organs and by the developmental program within these organs as well as by exposure to light. Together, the results provide a comprehensive analysis of the extent of transcriptional and post-transcriptional control that operates within the rbcS gene family during plant development.

Base Sequence↗

Analysis of high-frequency signal-averaged ECG measurements.

Analysis of high frequency (150-250 Hz) in the signal-averaged electrocardiogram (SAECG) is one of the emerging methods for detecting vessel patency in acute myocardial infarction following thrombolytic therapy and angioplasty. Root-mean-square voltage (RMSV) of the filtered QRS has been used in earlier studies to detect reperfusion; however, previous analysis indicated that RMSV is sensitive to residual noise in the SAECG and errors in QRS delineation (onset/offset). A new measurement is proposed, high-frequency energy (HFQE), and the robustness of the RMSV and HFQE was evaluated for simulated errors in QRS delineation. In this study, two measures (RMSV and HFQE) were tested on 24 control subjects and 21 patients undergoing thrombolytic therapy. Results indicate that unfiltered QRS duration is more stable than filtered QRS duration for the control subjects and patients and that HFQE had less fluctuation than RMSV in thrombolytic therapy patients. In the control group, HFQE was sensitive to the amplitude variation of the filtered SAECG. Therefore, another new measurement is proposed high-frequency integral of absolute value (HFAV), for reducing the sensitivity to amplitude changes in the filtered SAECG. This new feature was tested on control subjects and was found to be more stable than HFQE. In the thrombolitic therapy group, HFAV provided similar information as HFQE. These three measurements-RMSV, HFQE, and HFAV-provide a comprehensive analysis of the high-frequency SAECG for detecting vessel patency and reocclusion. Relative merits of these measures need to be evaluated on a larger database of patients undergoing thrombolysis and angioplasty for acute myocardial infarction.

Angioplasty, Balloon, Coronary↗

No association between 12 dopaminergic genes and schizophrenia in a large Dutch sample.

It has been suggested that genes involved in dopamine neurotransmission contribute to the pathogenesis of schizophrenia. However, reported associations of the disorder with genetic markers in dopaminergic genes have yielded inconsistent results. Possible explanations are differences in phenotyping, genetic heterogeneity, low marker informativity, and the use of small sample sizes. Here, we present a two-stage analysis of 12 dopaminergic genes in a large sample of Dutch schizophrenic patients. To reduce genetic heterogeneity, only patients with at least three Caucasian grandparents of Dutch ancestry were ascertained. An efficient genotyping strategy was used, in which polymorphic microsatellite markers were first screened for association in DNA pools. Promising results were followed up by individual genotyping in an extended sample. The pooled samples consisted of 208 schizophrenic patients and 288 unmatched control individuals. For each of the genes, more than one microsatellite marker was selected where possible, either intragenic or close to the gene. After correcting for multiple testing, significantly different allele frequencies were detected for DRD5 marker D4S615. Subsequently, we individually genotyped this particular marker and another DRD5 marker, as well as a DRD3 marker that could not be analyzed using the pooling strategy. This was done in an extended sample of 282 schizophrenic patients and a control sample of 585 individuals. In this second stage of the study, we found no association between these three markers and schizophrenia. The results of our comprehensive analysis provide no evidence for association between schizophrenia and 12 dopaminergic genes in a large Dutch sample.

Dopamine↗

The human mu opioid receptor gene: 5' regulatory and intronic sequences.

The human mu opioid receptor (hMOR) interacts with endogenous and exogenous ligands to mediate its characteristic effects, reward, dependence, and analgesia. Specifically binding morphine, it represents the target of the most valuable pain killer in contemporary medicine. Analysis of its structure, regulation, and expression will elucidate molecular processes involved in opioid/morphine-induced actions. Thus we have contributed significant information on the genomic organization of hMOR, extending the previously known cDNA sequence information (2162 bp) up to a total of 6968 bp: we have determined 2412 bp of 5' regulatory region, identified one major and three minor transcription initiation sites 216, 285, 358, and 373 bp upstream from the translation start codon, as well as potential binding sites for transcriptional regulatory factors, including putative cis-acting enhancer motifs for a glucocorticoid response element, cAMP response elements, activator proteins 1, and Yin Yang-1 boxes. Moreover, we have analyzed the 5' and 3' nucleotide sequences of introns 1 and 3 and the complete sequence of intron 2. In addition to the classical consensus sequences involved in RNA splicing, we have identified intronic repeats (A/T GGG) found to regulate alternative splicing, mutations of which cause human disease. A similar genetic variant is observed in the hMOR gene. Taken together, the sequence information presented will allow comprehensive analysis of this gene for allelic variations associated with vulnerability to drug abuse or individual differences in opiate mediated analgesia.

Base Sequence↗

Smokers have less reductions in probing depth than non-smokers following nonsurgical periodontal therapy.

DATA SOURCES: Studies were located using Medline, Embase and the Cochrane Central Register of Controlled Clinical Trials, as well as searching bibliographies of identified references, review articles and consensus statements by hand. STUDY SELECTION: As the habit of smoking cannot be randomised, studies included were both controlled clinical trials and arms of randomised controlled trials investigating the effects of nonsurgical periodontal treatment, which reported results separately for smokers and nonsmokers. Other inclusion criteria were that studies should assess otherwise healthy people who had been diagnosed with chronic or adult periodontitis and that the patient was the unit of analysis (rather than a tooth- or site-based analysis). Studies were not excluded on the basis of quality, only on whether they fulfilled the inclusion criteria for entry. DATA EXTRACTION AND SYNTHESIS: General information about the paper, study characteristics, outcome measures, treatment characteristics and quality assessment was extracted independently, in duplicate. Where disagreement arose, this was resolved by discussion. Meta-analysis of data was performed and heterogeneity between studies was investigated using meta-analysis regression. RESULTS: Out of the 330 studies initially identified, 13 were considered eligible for inclusion. No studies reported data on tooth loss. For all sites, the reduction in probing depth (PD) was 0.13 mm greater in nonsmokers than in smokers (six studies) and there was no evidence to suggest that the studies were dissimilar in their estimates of this result (no evidence of heterogeneity; P>0.05). For sites with an initial PD of 5.00 mm (eight studies), a random-effects meta-analysis indicated a weighted mean difference in PD reduction of 0.43 mm favouring nonsmokers (95% confidence interval (CI), 0.24-0.63; P<0.001). Because of significant heterogeneity between studies, only a cautious observation can be made but, with one exception, all studies produced a summary estimate favouring nonsmokers. Meta-analysis of the two studies that compared the change in clinical attachment level between nonsmokers and ex-smokers, who had given up their habit, in sites with an initial PD of +/-5.00 mm, showed a difference in clinical attachment level gain of 1.34 mm favouring the nonsmokers (95% CI, 0.65-2.03; P<0.001; chi(2) test for heterogeneity, 7.47 with 1 degree of freedom; P=0.006). In both of these analyses, the degree of heterogeneity is a cause for concern. Bleeding was assessed after therapy in seven studies but meta-analysis was not performed because of great heterogeneity in the methods used to assess bleeding. No statistically significant differences in bleeding were found between smokers and nonsmokers either at baseline or after therapy in most of the studies. One study found significantly less bleeding in smokers than in nonsmokers at baseline and another found a reduced response in terms of bleeding in smokers than in nonsmokers. Two studies evaluating the change in bleeding in ex-smokers found no statistically significant difference after treatment. No data were reported for any of the included studies on patient-centred outcomes such as quality of life, ease of maintenance, changes in aesthetic appearance, or patient experience. CONCLUSIONS: Following nonsurgical periodontal therapy, people who smoke will experience less reduction in PD than nonsmokers. There is no evidence of a difference in gain in clinical attachment between smokers and nonsmokers or a reduction of bleeding on probing between smokers and nonsmokers. Differences in study design and lack of data precluded an adequate and complete pooling of data for a more comprehensive analysis. In short-term studies, it is unclear whether people who stop smoking will respond as favourably to nonsurgical therapy as those who have never been smokers. Progress in understanding the effects of smoking on periodontal therapy will require the evaluation of objective measures of smoking such as nicotine exposure and exhaled carbon monoxide in place of sole reliance on patient-reported information.

Comment↗

Structure-function analysis of the active sites of complement receptor type 1.

Two functionally distinct but homologous sites in complement receptor type 1 (CR1) (CD35) were further characterized by homologous substitution mutagenesis of two CR1 derivatives, each containing one site. In both sites, reducing negative and/or increasing positive charge augmented interaction with iC3/C3b and C4b, supporting a role of ionic forces in the binding reaction. In one case, substitution of Asp at the end of complement control protein repeat (CCP) 2 with an Asn transformed the protein, with negligible cofactor activity and iC3 binding, into a mutant with activities similar to native CR1. Consequently, this protein, one-fourth the size of CR1, is a therapeutic candidate for a complement inhibitor. Another important observation is that the residues between two CCPs contribute to activity, probably because they influence positioning of one CCP relative to the next. The initial characterization of the third CCP of an active site led to identification of three peptides necessary for binding. In line with earlier findings for the first two CCPs, interactions with iC3/C3b are similar but not identical to those with C4b, implying overlapping but distinct binding domains. Moreover, changes in cofactor activity usually, but not always, parallel alterations in binding, indicating that these two activities are separable. We also mapped epitopes for a blocking and a function enhancing monoclonal antibody. Their effects can be explained by epitope location. The first antibody binds near functionally important residues. The second may shield inhibitory (negatively charged) residues. These results represent a comprehensive analysis of the active sites of CR1, which is built of modules found in more than 50 mammalian proteins.

Amino Acid Sequence↗

A century of research on the Planctomycetota bacterial phylum, previously known as Planctomycetes.

One hundred years after planctomycetes were discovered and 50 years since the first isolate was successfully cultured, this bacterial phylum remains enigmatic in many ways. In the last few decades, a significant effort to characterize new isolates has resulted in >150 described species, allowing a more comprehensive analysis of their features. However, metagenomic studies reveal that a diverse group of planctomycetes has yet to be cultured and characterized, and that many biological surprises are yet to be revealed. This is the case for the recently discovered phagotrophic Candidatus Uabimicrobium, which challenges our understanding of the distinction between prokaryotes and eukaryotes. The unique biology of planctomycete cells, such as their ability to divide without the FtsZ protein, their complex structure and characteristic morphology, their relatively large genomes containing many genes with unknown function, and their variable metabolic capabilities, imposes significant barriers for researchers. Although ubiquitous, the precise ecological roles of planctomycetes in various environments are still not fully understood. However, their distinctive metabolism opens the door to a large number of potential biotechnological applications, which are beginning to be unveiled. In this article, we first review the historical milestones in planctomycetes research and describe the pioneers of the field. We then describe the controversies and their resolutions, we highlight the past discoveries and current interrogations related to planctomycetes, and discuss the ongoing challenges that hinder a comprehensive understanding of their biology. We end up with directions for exploring the biology and ecological roles of these fascinating organisms.

Bacteria↗

HIV-1 Vpr: genetic diversity and functional features from the perspective of structure.

RNA viruses are well known for the enormous genetic variation. Retroviruses share this feature with other RNA viruses, and human immunodeficiency virus type 1 (HIV-1) has been extensively investigated in this regard. Based on the DNA sequence analysis, HIV-1 has been classified into three groups; M, N, and O, with viral subtypes in each group. While the genetic variation between viral isolates has been documented throughout the genome, specifically, the env gene exhibits high variation. Analysis of the env gene from the sequential samples from HIV-1-infected patients reveals variation in the range of 1% per year. The variation observed in individual HIV-1 genes in the form of changes at the nucleotide level, as expected, should result in one of the possible scenarios: (1) no change in the amino acid, (2) conservative change in the amino acid, (3) nonconservative change in the amino acid, and (4) premature stop codon resulting in a truncated protein. Hence, it is likely that the variation may impact on the function of the protein, depending on the nature of the mutation. The goal of this review is to summarize the polymorphisms in Vpr using the available sequence information and discuss their effects on the functions of Vpr from the point of view of its structure. The data generated by several groups provide a base for understanding the consequences of natural polymorphisms in specific regions of the Vpr molecule. However, it is also clear that secondary changes (second site or compensatory mutations) may modify the effect of a specific mutation and a comprehensive analysis is needed to delineate the role of specific residues in Vpr molecule. This is an area which, we hope, will attract investigators for further studies, and may provide information for understanding the molecular basis of Vpr functions.

Acquired Immunodeficiency Syndrome↗

High-resolution fragmentary decomposition--a model-based method of non-stationary electrophysiological signal analysis.

Fragmentary decomposition (FD) is a recently developed method of non-stationary electrophysiological signal analysis addressed to mass potentials, such as electromyogram (EMG), event-related potential (ERP), evoked potential, electroencephalogram (EEG), electroretinogram, etc. Being supported by the generally accepted physiological notion that a peak is a functionally meaningful component of a mass potential, FD provides a way to avoid averaging and, instead, quantifies the component composition of complex electrophysiological signals directly from single-trials. The major computational procedures of FD include adaptive segmentation, the frequency domain component identification, and creation of the signal model as a linear aggregation of multiple components, with the generic mass potential (GMP) being the universal component template. This paper presents an improved, high-resolution FD technique which allows the resolution of overlapping sub-components and supports each identified component by an individual model. On the basis of this methodological innovation, we define two fundamental categories of multi-peak component waveforms: complex components (CC), comprised of multiple sub-components (GMPs), versus monolithic components (MC), involving a single GMP. We show that quantification of MCs and CCs from single-trial eyeblink EMG and single-trial ERP provides a more comprehensive analysis of these signals. Given single-trial eyeblink EMG, we find that the stimulus elicits strong though short-term (phasic) effects on MCs and moderate but long-lasting (tonic) effects on CCs. A new realm of single-trial ERP quantification is possible in that the MC appears as a marker of a single cognitive variable whereas the CC appears as a marker of a series of functionally related cognitive variables. The engagement of the brain in a specific cognitive task is accompanied by an increase of CCs in single-trial ERPs, which is especially informative with respect to the P3 cognitive potential. New methodology provides evidence for the three basic types of single-trial P3 sub-components: monolithic P3a, monolithic P3b, and a complex component, P3ab, which includes both P3a and P3b as sub-components.

Algorithms↗

Mitochondrial respiration and ATP production are significantly impaired in striatal cells expressing mutant huntingtin.

There is significant evidence that energy production impairment and mitochondrial dysfunction play a role in the pathogenesis of Huntington disease. Nonetheless, the specific mitochondrial defects due to the presence of mutant huntingtin have not been fully elucidated. To determine the effects of mutant huntingtin on mitochondrial energy production, a thorough analysis of respiration, ATP production, and functioning of the respiratory complexes was carried out in clonal striatal cells established from Hdh(Q7) (wild-type) and Hdh(Q111) (mutant huntingtin knock-in) mouse embryos. Mitochondrial respiration and ATP production were significantly reduced in the mutant striatal cells compared with the wild-type cells when either glutamate/malate or succinate was used as the substrate. However, mitochondrial respiration was similar in the two cell lines when the artificial electron donor TMPD/ascorbate, which feeds into complex IV, was used as the substrate. The attenuation of mitochondrial respiration and ATP production when either glutamate/malate or succinate was used as the substrate was not due to impairment of the respiratory complexes, because their activities were equivalent in both cell lines. Intriguingly, in the striatum of presymptomatic and pathological grade 1 Huntington disease cases there is also no impairment of mitochondrial complexes I-IV (Guidetti, P., Charles, V., Chen, E. Y., Reddy, P. H., Kordower, J. H., Whetsell, W. O., Jr., Schwarcz, R., and Tagle, D. A. (2001) Exp. Neurol. 169, 340-350). To our knowledge, this is the first comprehensive analysis of the effects of physiological levels of mutant huntingtin on mitochondrial respiratory function within an appropriate cellular context. These findings demonstrate that the presence of mutant huntingtin impairs mitochondrial ATP production through one or more mechanisms that do not directly affect the function of the respiration complexes.

Adenosine Triphosphate↗

Insights into gene expression changes impacting B-cell transformation: cross-species microarray analysis of bovine leukemia virus tax-responsive genes in ovine B cells.

Large-animal models for leukemia have the potential to aid in the understanding of networks that contribute to oncogenesis. Infection of cattle and sheep with bovine leukemia virus (BLV), a complex retrovirus related to human T-cell leukemia virus type 1 (HTLV-1), is associated with the development of B-cell leukemia. Whereas the natural disease in cattle is characterized by a low tumor incidence, experimental infection of sheep leads to overt leukemia in the majority of infected animals, providing a model for studying the pathogenesis associated with BLV and HTLV-1. Tax(BLV), the major oncoprotein, initiates a cascade of events leading toward malignancy, although the basis of transformation is not fully understood. We have taken a cross-species ovine-to-human microarray approach to identify Tax(BLV)-responsive transcriptional changes in two sets of cultured ovine B cells following retroviral vector-mediated delivery of Tax(BLV). Using cDNA-spotted microarrays comprising 10,336 human genes/expressed sequence tags, we identified a cohort of differentially expressed genes, including genes related to apoptosis, DNA transcription, and repair; proto-oncogenes; cell cycle regulators; transcription factors; small Rho GTPases/GTPase-binding proteins; and previously reported Tax(HTLV-1)-responsive genes. Interestingly, genes known to be associated with human neoplasia, especially B-cell malignancies, were extensively represented. Others were novel or unexpected. The results suggest that Tax(BLV) deregulates a broad network of interrelated pathways rather than a single B-lineage-specific regulatory process. Although cross-species approaches do not permit a comprehensive analysis of gene expression patterns, they can provide initial clues for the functional roles of genes that participate in B-cell transformation and pinpoint molecular targets not identified using other methods in animal models.

Animals↗

TEL deletion analysis supports a novel view of relapse in childhood acute lymphoblastic leukemia.

PURPOSE: TEL (ETV6)-AML1 (RUNX1) chimeric gene fusions are frequent genetic abnormalities in childhood acute lymphoblastic leukemia (ALL). They often arise prenatally as early events or initiating events and are complemented by secondary postnatal genetic events of which deletion of the non-rearranged, second TEL allele is the most common. This consistent sequence of molecular pathogenesis facilitates an analysis of the clonal origins of relapse in this leukemia, which has some unusual clinical features. EXPERIMENTAL DESIGN: We compared the boundaries, by microsatellite mapping, of TEL deletions at relapse versus diagnosis in 15 informative patients. Moreover, we compared the relatedness of diagnostic and relapse clones using immunoglobulin and T-cell receptor genes rearrangements and clonotypic TEL-AML1 genomic fusion. RESULTS: Five patients retained the apparent same size TEL deletion, seven had larger deletions, and three had smaller deletions at relapse. In all of the cases evaluated, the clonal relatedness of diagnostic and relapse cells was confirmed by the retention of clonotypic TEL-AML1 genomic sequence and/or at least one identical immunoreceptor gene rearrangement. CONCLUSIONS: These data provide further evidence that TEL deletions are secondary to TEL-AML1 fusions in ALL. They are compatible with the novel idea that in at least some cases of childhood ALL, remission occurs with persistence of a preleukemic "fetal" clone, and subsequent relapse reflects the emergence of a new subclone from this reservoir after an independent "second hit," i.e., independent TEL deletion. To our knowledge, the study is the most extensive and comprehensive analysis of the relationship between diagnostic and relapse clones in childhood ALL presented thus far.

Artificial Gene Fusion↗