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PubMed2006

In vitro-in vivo correlation for drugs and other compounds eliminated by glucuronidation in humans: pitfalls and promises.

Enzymes of the UDP-glucuronosyltransferase (UGT) superfamily are responsible for the metabolism of many drugs, environmental chemicals and endogenous compounds. Identification of the UGT(s) involved in the metabolism of a given compound ('reaction phenotyping') currently relies on multiple confirmatory approaches, which may be confounded by the dependence of UGT activity on enzyme source, incubation conditions, and the occurrence of atypical glucuronidation kinetics. However, the increasing availability of substrate and inhibitor 'probes' for the individual UGTs provides the prospect for reliable phenotyping of glucuronidation reactions using human liver microsomes or hepatocytes, thereby providing data directly relevant to drug metabolism in humans. While the feasibility of computational prediction of UGT substrate selectivity has been demonstrated, the development of easily interpretable and generalisable models requires further improvement in the datasets available for analysis. Quantitative prediction of the hepatic clearance of glucuronidated drugs and the magnitude of inhibitory interactions based on in vitro kinetic data is more problematic. Intrinsic clearance (CL(int)) values generated using human liver microsomes under-predict in vivo hepatic clearance, typically by an order of magnitude. In vivo clearances of glucuronidated drugs are also generally under-predicted by CL(int) values from human hepatocytes, but to a lesser extent than observed with the microsomal model. While it is anticipated that systematic analysis of the potential causes of under-prediction may provide more reliable in vitro-in vivo scaling strategies, mechanistic interpretation of in vitro-in vivo correlation more broadly awaits further advances in our understanding of the structural and cellular determinants of UGT activity.

Glucuronides↗
PubMed2006

Characterization of cycA mutants of Escherichia coli. An assay for measuring in vivo mutation rates.

Quantitative assessment of the spontaneous or induced genomic mutation rate, a fundamental evolutionary parameter, usually requires the use of well-characterized mutant selection systems. Although there is a great number of genetic selection schemes available in Escherichia coli, the selection of D-cycloserine resistant mutants is shown here to be particularly useful to yield a general view of mutation rates and spectra. The combination of a well-defined experimental protocol with the Ma-Sandri-Sarkar maximum likelihood method of fluctuation analysis results in reproducible data, adequate for statistical comparisons. The straightforward procedure is based on a simple phenotype-genotype relationship, and detects mutations in the single-copy, chromosomal cycA gene, involved in the uptake of D-cycloserine. In contrast to the widely used rifampicin resistance assay, the procedure selects mutations which are neutral in respect of cell growth. No specific genetic background is needed, and practically the entire mutation spectrum (base substitutions, frameshifts, deletions, insertions) can simultaneously be measured. A systematic analysis of cycA mutations revealed a spontaneous mutation rate of 6.54 x 10(-8) in E. coli K-12 MG1655. The mutation spectrum was dominated by point mutations (base substitutions, frameshifts), spread over the entire gene. IS insertions, caused by IS1, IS2, IS3, IS4, IS5 and IS150, represented 24% of the mutations.

Amino Acid Transport Systems↗
PubMed1989

On-line high-performance liquid chromatographic diode array spectrophotometric analysis of steroidal hormones in illegal preparations.

An on-line high-performance liquid chromatographic diode array spectroscopic analytical method for the identification of more than 60 different steroidal compounds is described. For the chromatographic separation, a gradient elution that could distinguish the esterified and non-esterified steroids in the same run on a reversed-phase C18 column, using methanol as modifier, was developed. For both types of compound an internal standard was chosen to establish reproducible relative retention times that could be used as one element of the identification; the second element is the UV spectrum, which is recorded on-line during the separation. The combination of chromatographic and UV spectroscopic recordings selects only a few probable steroids, which could be the unknown. This approach has been applied to forensic analysis of illicit preparations used in cattle-breeding, some examples of which are shown. For those steroids that are very difficult to distinguish using this procedure, because of their chromatographic and spectroscopic similarity on this system, other solvent mixtures are used in place of methanol as modifier, namely acetonitrile or tetrahydrofuran, or both, with the same solvent strength, as proposed by Snyder. In this way totally different elution patterns and separations are obtained, providing complementary information for identification, as shown by the systematic analysis on two other isoeluotropic solvent systems.

Chromatography, High Pressure Liquid↗
PubMed1997

Linkers of secondary structures in proteins.

Linkers that connect repeating secondary structures fall into conformational classes based on distance and main-chain torsion clustering. A data set of 300 unique protein chains with low pairwise sequence identity was clustered into only a few groups representing the preferred motifs. The linkers of two to eight residues for the nonredundant data set are designated H-Ln-H, H-Ln-E, E-Ln-H, E-Ln-E, where n is the length, H stands for alpha-helices, and E for beta-strands. Most of the clusters identified here corroborate earlier findings. However, 19 new clusters are identified in this paper, with many of them having seven and eight residue linkers. In our first analysis, the secondary structures flanking the linkers are both interacting and noninteracting and there is no precise angle of orientation between them. A second analysis was performed on a set of proteins with restricted orientations for the flanking elements, namely, mainly alpha class of proteins with orthogonal architecture. Two definite clusters are identified, one corresponding to linkers of orthogonal helices and the other to linkers of antiparallel helices. Loops forming binding sites or involved in catalytic activity are important determinants of the function of proteins. Although the structural conservation of the residues around the catalytic triad of serine proteases has been studied widely, there has not been a systematic analysis of the conformation of the loops that contain them. Residues of the catalytic triad reside in the linkers of beta-strands, with varying lengths of more than eight residues. Here, we analyze the structural conservation of such linkers by superposition, and observe a conserved structural feature of the linkers incorporating each of the three residues of the catalytic triad.

Amino Acid Sequence↗
PubMed1995

Intervertebral variation in trabecular microarchitecture throughout the normal spine in relation to age.

The vertebral bodies of the complete spine (C-3-L-5) were removed in 26 autopsy cases without evidence for primary or secondary bone disease (13 males aged 19-79 years and 13 females aged 17-90 years). A sagittal segment through the center of all vertebral bodies was embedded undecalcified in hydroxyethylmethacrylate and processed to so-called surface stained block grindings. Histomorphometric analysis of the complete segment was performed using a computer-assisted image analysis system (IBAS 2000). The structural parameters investigated were bone volume (BV/TV) and trabecular interconnection quantificated by trabecular bone pattern factor (TBPf). A close correlation of BV/TV and TBPf was found in all vertebral bodies irrespective of vertebral region (r = 0.8, p < 0.001). This indicates that the age-related decrease of trabecular bone mass is primarily the consequence of the transformation from plates to rods and the loss of whole trabecular structures. This basic principle is valid throughout the complete spine. However, the systematic analysis of vertebral trabecular bone from C-3 to L-5 revealed a significant intervertebral variation of trabecular microarchitecture. The density of trabecular structure of cervical vertebrae is much higher than that of thoracic and lumbar vertebrae (p < 0.001). The extent of age-related loss of trabecular bone mass and structure showed a decrease within the spine from the caudal to the cranial region (p < 0.05). The loss of bone volume in individuals between the ages of 30 and 80 years in the lumbar spine was 53%, whereas in the thoracic spine the decrease was 41%, and in the cervical spine only 24%.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorptiometry, Photon↗
PubMed2005

Phylogenetic reconstruction of ancestral character states for gene expression and mRNA splicing data.

BACKGROUND: As genomes evolve after speciation, gene content, coding sequence, gene expression, and splicing all diverge with time from ancestors with close relatives. A minimum evolution general method for continuous character analysis in a phylogenetic perspective is presented that allows for reconstruction of ancestral character states and for measuring along branch evolution. RESULTS: A software package for reconstruction of continuous character traits, like relative gene expression levels or alternative splice site usage data is presented and is available for download at http://www.rossnes.org/phyrex. This program was applied to a primate gene expression dataset to detect transcription factor binding sites that have undergone substitution, potentially having driven lineage-specific differences in gene expression. CONCLUSION: Systematic analysis of lineage-specific evolution is becoming the cornerstone of comparative genomics. New methods, like phyrex, extend the capabilities of comparative genomics by tracing the evolution of additional biomolecular processes.

Alternative Splicing↗
PubMed2001

What is happening to the health of the Croatian population?

AIM: To describe the problems in the interpretation of Croatian mortality data and explore possible reasons for the recorded increase in mortality in the 1990-1999 period, particularly related to different methods of collection and estimation of data on deaths and population. METHODS: Numbers of recorded deaths and population estimates were first obtained from the Croatian Institute for Public Health and examined in detail. The Institute used population estimates supplied by the Croatian Statistics Bureau, which included de jure population data (including all Croatian citizens wherever they live) until 1996 and de facto population data (including only population living in Croatia at least for a year, irrespective of citizenship) since 1996. A different set of population estimates based on de facto estimates since 1992 was obtained from the Croatian Bureau of Statistics. We examined trends in age- and sex-specific death rates from major causes in 1990-1999 period, using the mortality data from the Croatian Institute for Public Health and both sets of population estimates. Lung cancer as a cause of death was examined in more detail, since it is relatively stable over short periods of time. Interviews were undertaken with key informants to identify the reasons for any discrepancies. RESULTS: In Croatia, relatively stable death rates from lung cancer in men ranged from 84/100,000 in 1990 to 79/ 100,000 in 1995. In 1996, a marked discontinuity appeared in the Croatian data, with a 14% increase compared to 1995 (from 79/100,000 to 91/100,000) and a further increase in 1999 (94/100,000), which is not credible on the basis of the natural history of lung cancer. Analysis of mortality rates with de facto population estimates showed more gradual increase from 1992-1996. Methods used to estimate population and mortality during the 1990s were inconsistent and misleading. At present, it is impossible to be certain about the true level of mortality in Croatia during 1990s, as the numerator (deaths) and denominator (population) were incompatible until 1998. CONCLUSION: Major problems in data collection would have been identified if the investigation of unexpected mortality trends in Croatia in the 1990s had been done. Systematic analysis of health patterns should be done as soon as data from the 2001 census become available. Capacities in public health should be strengthened to make this possible. This issue has received little recognition from the international donor organizations, particularly those that use health data.

Cause of Death↗
PubMed1987

Analysis of risk factors for patient and renal survival in crescentic glomerulonephritis.

A series of 57 patients with crescentic glomerulonephritis was studied retrospectively to estimate patient and renal survival and to analyze the variables that affect these end points. Renal survival was 47% at 6 months for patients with idiopathic rapidly progressive glomerulonephritis and 33% for patients with antiglomerular basement membrane disease. Most patients in these two groups presented with advanced renal insufficiency. Renal survival rates for patients with systemic illness, with or without a tissue diagnosis of vasculitis, were 93% and 100%, respectively, at 1 year. These patients had better renal function at presentation. Overall, patient survival at 5 years was 61%, and there were no significant differences among the groups. A systematic analysis of clinical, immunopathologic, and laboratory variables showed that the initial serum creatinine level and oliguria and the patient's age and oliguria were the most important indicators of renal and patient survival, respectively. Virtually all patients with a creatinine level greater than 6.0 mg/dL lost their kidneys. Five-year survival for patients greater than 60 years old was 31%, compared with 83% for those less than or equal to 60 years. Moderate-to-severe tubulointerstitial inflammation indicated worse patient and renal survival, and 24-hour urine protein excretion greater than 3 g was related to shorter renal survival. We have identified and ranked a number of variables that affect patient or renal survival in crescentic glomerulonephritis. These findings may be of help in the decision-making process of patient management and analysis of treatment modalities.

Adolescent↗
PubMed2000

Structural analysis of WW domains and design of a WW prototype.

Two new NMR structures of WW domains, the mouse formin binding protein and a putative 84.5 kDa protein from Saccharomyces cerevisiae, show that this domain, only 35 amino acids in length, defines the smallest monomeric triple-stranded antiparallel beta-sheet protein domain that is stable in the absence of disulfide bonds, tightly bound ions or ligands. The structural roles of conserved residues have been studied using site-directed mutagenesis of both wild type domains. Crucial interactions responsible for the stability of the WW structure have been identified. Based on a network of highly conserved long range interactions across the beta-sheet structure that supports the WW fold and on a systematic analysis of conserved residues in the WW family, we have designed a folded prototype WW sequence.

Amino Acid Sequence↗
PubMed2006

A combined analysis of North American case-control studies of residential radon and lung cancer.

Cohort studies have consistently shown underground miners exposed to high levels of radon to be at excess risk of lung cancer, and extrapolations based on those results indicate that residential radon may be responsible for nearly 10-15% of all lung cancer deaths per year in the United States. However, case-control studies of residential radon and lung cancer have provided ambiguous evidence of radon lung cancer risks. Regardless, alpha-particle emissions from the short-lived radioactive radon decay products can damage cellular DNA. The possibility that a demonstrated lung carcinogen may be present in large numbers of homes raises a serious public health concern. Thus, a systematic analysis of pooled data from all North American residential radon studies was undertaken to provide a more direct characterization of the public health risk posed by prolonged radon exposure. To evaluate the risk associated with prolonged residential radon exposure, a combined analysis of the primary data from seven large scale case-control studies of residential radon and lung cancer risk was conducted. The combined data set included a total of 4081 cases and 5281 controls, representing the largest aggregation of data on residential radon and lung cancer conducted to date. Residential radon concentrations were determined primarily by a-track detectors placed in the living areas of homes of the study subjects in order to obtain an integrated 1-yr average radon concentration in indoor air. Conditional likelihood regression was used to estimate the excess risk of lung cancer due to residential radon exposure, with adjustment for attained age, sex, study, smoking factors, residential mobility, and completeness of radon measurements. Although the main analyses were based on the combined data set as a whole, we also considered subsets of the data considered to have more accurate radon dosimetry. This included a subset of the data involving 3662 cases and 4966 controls with a-track radon measurements within the exposure time window (ETW) 5-30 yr prior to the index date considered previously by Krewski et al. (2005). Additional restrictions focused on subjects for which a greater proportion of the ETW was covered by measured rather than imputed radon concentrations, and on subjects who occupied at most two residences. The estimated odds ratio (OR) of lung cancer generally increased with radon concentration. The OR trend was consistent with linearity (p = .10), and the excess OR (EOR) was 0.10 per Bq/m3 with 95% confidence limits (-0.01, 0.26). For the subset of the data considered previously by Krewski et al. (2005), the EOR was 0.11 (0.00, 0.28). Further limiting subjects based on our criteria (residential stability and completeness of radon monitoring) expected to improve radon dosimetry led to increased estimates of the EOR. For example, for subjects who had resided in only one or two houses in the 5-30 ETW and who had a-track radon measurements for at least 20 yr of this 25-yr period, the EOR was 0.18 (0.02, 0.43) per 100 Bq/m3. Both estimates are compatible with the EOR of 0.12 (0.02, 0.25) per 100 Bq/m3 predicted by downward extrapolation of the miner data. Collectively, these results provide direct evidence of an association between residential radon and lung cancer risk, a finding predicted by extrapolation of results from occupational studies of radon-exposed underground miners.

Adult↗
PubMed2004

Prospective documentation and analysis of the pre- and early clinical management in severe head injury in southern Bavaria at a population based level.

Treatment of patients suffering from severe head injury is so far restricted to general procedures, whereas specific pharmacological agents of neuroprotection including hypothermia have not been found to improve the outcome in clinical trials. Albeit effective, symptomatic measures of the preclinical rescue of patients (i.e. stabilization or reestablishment of the circulatory and respiratory system) or of the early clinical care (e.g. prompt diagnosis and treatment of an intracranial space occupying mass, maintenance of a competent circulatory and respiratory system, and others) by and large constitute the current treatment based on considerable organizational and logistical efforts. These and other components of the head injury treatment are certainly worthwhile of a systematic analysis as to their efficacy or remaining deficiencies, respectively. Deficits could be associated with delays of providing preclinical rescue procedures (e.g. until intubation of the patient or administration of fluid). Delays could also be associated in the hospital with the diagnostic establishment of intracranial lesions requiring prompt neurosurgical intervention. By support of the Federal Ministry of Education and Research and under the auspices of the Forschungsverbund Neurotraumatology, University of Munich, a prospective system analysis was carried out on major aspects of the pre- and early clinical management at a population based level in patients with traumatic brain injury. Documentation of pertinent data was made from August 1998 to July 1999 covering a catchment area of Southern Bavaria (5.6 mio inhabitants). Altogether 528 cases identified to suffer from severe head injury (GCS < or = 8 or deteriorating to that level within 48 hrs) were enrolled following admission to the hospital and establishment of the diagnosis. Further, patients dying on the scene or during transport to the hospital were also documented, particularly as to the frequency of severe head injury as underlying cause of mortality. The analysis included also cases with additional peripheral trauma (polytrauma). The efficacy of the logistics and organization of the management was studied by documentation of prognosis-relevant time intervals, as for example until arrival of the rescue squad at the scene of an accident, until intubation and administration of fluid, or upon hospital admission until establishment of the CT-diagnosis and commencement of surgery or transfer to the intensive care unit, respectively. The severity of cases studied in the present analysis is evident from a mortality of far above 40% of cases admitted to the hospital, which was increased by about 20% when including prehospital mortality. The outcome data notwithstanding, the emerging results demonstrate a high efficacy of the pre- and early clinical management, as indicated by a prompt arrival of the rescue squad at the scene, a competent prehospital and early clinical management and care, indicative of a low rate of avoidable complications. It is tentatively concluded on the basis of these findings that the patient prognosis is increasingly determined by the manifestations of primary brain damage vs. the development of secondary complications.

Craniocerebral Trauma↗
PubMed2000

Viscoelastic finite-element analysis of a lumbar motion segment in combined compression and sagittal flexion. Effect of loading rate.

STUDY DESIGN: A study using a validated viscoelastic finite-element model of a L2-L3 motion segment to identify the load sharing among the passive elements at different loading rates. OBJECTIVE: To enhance understanding concerning the role of the loading rate (i.e., speed of lifting and lowering during manual material handling tasks) on the load sharing and safety margin of spinal structures. SUMMARY OF BACKGROUND DATA: Industrial epidemiologic studies have shown that jobs requiring a higher speed of trunk motion contribute to a higher risk of industrial low back disorders. Consideration of the dynamic loading characteristics, such as lifting at different speeds, requires modeling of the viscoelastic behavior of passive tissues. Detailed systematic analysis of loading rate effects has been lacking in the literature. METHODS: Complex flexion movement was simulated by applying compression and shear loads at the top of the upper vertebra while its sagittal flexion angle was prescribed without constraining any translations. The lower vertebra was fixed at the bottom. The load reached its maximum values of 2000 N compression and 200 N anterior shear while L2 was flexed to 10 degrees of flexion in the three different durations of 0.3, 1, and 3 seconds to represent fast, medium, and slow movements, respectively. The resisted bending moment, gross load-displacement response of the motion segment, forces in facet joints and ligaments, stresses and strains in anulus fibrosus, and intradiscal pressure were compared across different rates. RESULTS: The distribution of stress and strain was markedly affected by the loading rate. The higher loading rate increased the peak intradiscal pressure (12.4%), bending moment (20.7%), total ligament forces (11.4%), posterior longitudinal ligament stress (15.7%), and anulus fiber stress at the posterolateral innermost region (17.9%), despite the 15.4% reduction in their strain. CONCLUSIONS: Consideration of the time-dependent material properties of passive elements is essential to improving understanding of motion segment responses to dynamic loading conditions. Higher loading rate markedly reduces the safety margin of passive spinal elements. When the dynamic tolerance limits of tissues are available, the results provide bases for the guidelines of safe dynamic activities in clinics or industry.

Elasticity↗
PubMed1999

Disentangling ethical and psychological issues--a guide for oncologists.

The rapid growth of bioethics has injected a new style of analysis into medicine. It requires philosophical rigor, yet is deeply embedded in human situations that frustrate abstract thinking and are laced with subjective factors. These interlaced ethical and psychological components can lead to conflicts and dilemmas. Doctors, as experts and decision-makers, play a key role, but will benefit from additional skills to disentangle these situations. This paper notes ways in which patients, families and caregivers are newly vulnerable and delineates how ethical dilemmas and psychological issues mold or frustrate decision-making. To help physicians manage such cases, a method of systematic analysis, the 'situational diagnosis', and a related hierarchy of interventions, is described and illustrated with case examples.

Adaptation, Psychological↗
PubMed1993

Electrophoretic analysis of protein-induced DNA bending and twist changes.

DNA fragments with a varied phasing of two intrinsic bends at their ends and a tet operator in-between were constructed to determine Tet repressor-induced twist changes in the operator DNA. These distance variants show a sinusoidal dependence of their electrophoretic mobilities on the phasing of their bends in polyacrylamide gels. Complex formation with Tet repressor leads to a displacement of the first minimum, indicating an unwinding of the tet operator DNA. Model calculations were performed to assess the contribution of Tet repressor-induced DNA bending to this result. They revealed that the amplitude of the electrophoretic mobilities of the distance variants may be used as a parameter to separate the effects of Tet repressor-induced twist change and bending. The systematic analysis presented here may help to improve the quantitative interpretation of gel shifts and promote the use of this highly sensitive method to gain structural information about protein-DNA complexes.

Antiporters↗
PubMed1984

Flow-cytometric analysis of DNA content in postmortem tissue.

In order to determine the heterogeneity of ploidy in disseminated cancer, we assessed the suitability of postmortem tissue for flow-cytometric analysis of cellular DNA content. Forty tissue samples from various tumor-bearing sites in 12 patients with widely metastatic cancer were analyzed. In eight cases studied there was a single unimodal ploidy level in all disease sites studied. The remaining four cases revealed bimodal tumor populations present in different proportions in the various sites studied. No difference in ploidy could be detected between irradiated and nonirradiated sites in patients who had received radiation therapy. Samples taken from normal tissues all revealed diploid DNA content, suggesting that DNA fluorescence is not altered in the immediate postmortem period. Systematic analysis of cellular DNA content in autopsy material is feasible and may contribute to our understanding of patterns of metastatic spread in patients with cancer.

Autopsy↗
PubMed2006

Proteomic analysis of seed filling in Brassica napus. Developmental characterization of metabolic isozymes using high-resolution two-dimensional gel electrophoresis.

Brassica napus (cultivar Reston) seed proteins were analyzed at 2, 3, 4, 5, and 6 weeks after flowering in biological quadruplicate using two-dimensional gel electrophoresis. Developmental expression profiles for 794 protein spot groups were established and hierarchical cluster analysis revealed 12 different expression trends. Tryptic peptides from each spot group were analyzed in duplicate using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and liquid chromatography-tandem mass spectrometry. The identity of 517 spot groups was determined, representing 289 nonredundant proteins. These proteins were classified into 14 functional categories based upon the Arabidopsis (Arabidopsis thaliana) genome classification scheme. Energy and metabolism related proteins were highly represented in developing seed, accounting for 24.3% and 16.8% of the total proteins, respectively. Analysis of subclasses within the metabolism group revealed coordinated expression during seed filling. The influence of prominently expressed seed storage proteins on relative quantification data is discussed and an in silico subtraction method is presented. The preponderance of energy and metabolic proteins detected in this study provides an in-depth proteomic view on carbon assimilation in B. napus seed. These data suggest that sugar mobilization from glucose to acetyl-coenzyme A [corrected] is a collaboration between the cytosol and plastids and that temporal control of enzymes and pathways extends beyond transcription. This study provides a systematic analysis of metabolic processes operating in developing B. napus seed from the perspective of protein expression. Data generated from this study have been deposited into a web database (http://oilseedproteomics.missouri.edu) that is accessible to the public domain.

Arabidopsis↗
PubMed2006

Specific host genes required for the killing of Klebsiella bacteria by phagocytes.

The amoeba Dictyostelium discoideum shares many traits with mammalian macrophages, in particular the ability to phagocytose and kill bacteria. In response, pathogenic bacteria use conserved mechanisms to fight amoebae and mammalian phagocytes. Here we developed an assay using Dictyostelium to monitor phagocyte-bacteria interactions. Genetic analysis revealed that the virulence of Klebsiella pneumoniae measured by this test is very similar to that observed in a mouse pneumonia model. Using this assay, two new host resistance genes (PHG1 and KIL1) were identified and shown to be involved in intracellular killing of K. pneumoniae by phagocytes. Phg1 is a member of the 9TM family of proteins, and Kil1 is a sulphotransferase. The loss of PHG1 resulted in Dictyostelium susceptibility to a small subset of bacterial species including K. pneumoniae. Remarkably, Drosophila mutants deficient for PHG1 also exhibited a specific susceptibility to K. pneumoniae infections. Systematic analysis of several additional Dictyostelium mutants created a two-dimensional virulence array, where the complex interactions between host and bacteria are visualized.

Animals↗
PubMed1998

Functional analysis of the human immunodeficiency virus type 1 Rev protein oligomerization interface.

The expression of human immunodeficiency virus type 1 (HIV-1) structural proteins requires the action of the viral trans-regulatory protein Rev. Rev is a nuclear shuttle protein that directly binds to its cis-acting Rev response element (RRE) RNA target sequence. Subsequent oligomerization of Rev monomers on the RRE and interaction of Rev with a cellular cofactor(s) result in the cytoplasmic accumulation of RRE-containing viral mRNAs. Moreover, Rev by itself is exported from the nucleus to the cytoplasm. Although it has been demonstrated that Rev multimerization is critically required for Rev activity and hence for HIV-1 replication, the number of Rev monomers required to form a trans-activation-competent complex on the RRE is unknown. Here we report a systematic analysis of the putative multimerization domains within the Rev trans-activator protein. We identify the amino acid residues which are part of the proposed single hydrophobic surface patch in the Rev amino terminus that mediates intermolecular interactions. Furthermore, we show that the expression of a multimerization-deficient Rev mutant blocks HIV-1 replication in a trans-dominant (dominant-negative) fashion.

Amino Acid Sequence↗