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Biomedical subjects

M Graf

Publications and source records attributed to M Graf.

At least 19 recordsLinked to original sources
PubMed2003

Low L-selectin (CD62L) expression in acute myeloid leukemia correlates with a bad cytogenetic risk.

OBJECTIVES: Interactions between hemopoietic cells and the stromal microenvironment or immunoreactive cells are mediated by specific cell surface receptors. The expression of those molecules may alter the adhesive qualities (mobility and homing) as well as immune response behavior of leukemic blasts. L-Selectin (CD62L) is suggested to play a role in the redistribution and homing of hemopoietic progenitor cells to the bone marrow (BM). Down-regulation of L-selectin is responsible for mobilization of blasts from the BM into the circulation and ligation of L-selectin stimulates proliferation of progenitor cells. This could have an influence on the process of leukemia. METHOD: We have studied the expression of L-selectin on mononuclear BM cells of 36 acute myeloid leukemia (AML) patients at first diagnosis by FACS analysis using a directly fluorescein isothiocyanate conjugated antibody (clone DRE G56). RESULTS: On average the patients presented with 88% blasts in the BM. The expression tended to be higher in primary (p) AML compared with secondary (s) AML. L-Selectin was very heterogenously expressed in all FAB groups. Highest expression was found in cases with AML-M4 with four of nine cases presenting with an inv(16) karyotype. Separating our patient cohort in cytogenetic risk groups we could detect a significantly higher expression of L-selectin in cases with a 'good risk' karyotype and a very low expression in cases with a 'bad risk' karyotype (P = 0.037). Comparing patients who achieved remission after double induction therapy (responders) with patients who showed persisting disease (non-responders) we found a higher percentage of L-selectin+ cases or cells in the responder group than in the non-responder group, although the differences were not significant because of only five cases in the 'non-responder' group. Evaluating cut-off points greatest differences in relapse-free survival probabilities were found in patients who presented with > or = 30% L-selectin+ BM cells compared with cases with < 30%: 86% of cases with > or = 30% L-selectin+ cells were still in remission after a mean follow up time of only 8 months compared with only 46% in the group with < 30% L-selectin+ cells. CONCLUSIONS: We can conclude that (i) expression of L-selectin on AML blasts is variable. This reveals the great diversitiy of immunophenotypes in AML and might contribute to identify individual blast phenotypes in order to detect minimal residual disease in remission. (ii) Low L-selectin expression correlates with a bad cytogenetic risk, with a lower probability to achieve remission and with a shorter relapse-free survival time. This might reflect a decreased homing of the blasts to the BM as well as an impaired cytotoxic T-cell reaction against leukemic cells. The expression of L-selectin on leukemic blasts might be influenced by different cytokine therapies (e.g. with interferon alpha) and this might result in an altered hematologic reconstitution after cytotoxic therapies as well as in an altered immunologic recognition of blasts.

Acute Disease↗
PubMed2003

[Direct MR arthrography of the wrist- value in detecting complete and partial defects of intrinsic ligaments and the TFCC in comparison with arthroscopy].

PURPOSE: To assess the value of two-compartment magnetic resonance (MR) wrist arthrography in comparison with diagnostic arthroscopy for the evaluation of defects of the triangular fibrocartilage complex (TFCC) and intrinsic ligaments in patients with refractory wrist pain. The direct MR arthrographies were evaluated following arthroscopic classification with consideration of complete and partial defects. The distinction between these types of lesions has clinical implications for treatment procedures such as cast immobilization, arthroscopic debridement, surgical repair or partial intercarpal arthrodesis. MATERIALS AND METHODS: Seventy-five patients (25 female, 50 males, mean age 38.3 years) who suffered from refractory wrist pain without radiography evidence of carpal instability underwent two-compartment wrist MR arthrography. Under aseptic conditions a solution of gadopentate dimeglumine and iodinated contrast agent (concentration 2.5 mmol/l) was injected into the radiocarpal and midcarpal joints under fluoroscopy guidance. Using a scanner of 1.5 T field strength and a wrist-coil following sequences were acquired: coronary and sagittal T (1)-weighted spin-echo (SE) sequences (TR 500 ms, TE 25 ms, matrix 512 x 512.3 mm) and coronary fast low angle shot (FLASH) 3D sequences (TR 24 ms, TE 11 ms, matrix 256 x 256, 1.5 mm, flip angle 50 degrees). All patients underwent subsequent arthroscopy of the wrist. The direct MR arthrographies were evaluated retrospectively by two observers experienced in the diagnosis of wrist pathology. They were not aware of the clinicial, arthrographic and arthroscopic findings. Pathology of the scapholunate ligament was classified according to the guidelines of the German Society of Hand Surgery (DGH), lesions of the lunotriquetral ligament according to Hempfling and lesions of the TFCC according to Palmer. RESULTS: Twenty-five complete and 47 partial defects were detected arthroscopically (TFCC: 21/20, scapholunate ligament: 3/18, lunotriquetral ligament: 1/9). The TFCC showed a higher prevalence for degenerative lesions (11 type 2C-lesions and 20 type 2A/B lesions) than for traumatic lesions (5 type 1A lesions, 5 type 1D lesions). For direct MR arthrography, the obtained sensitivities and specificities in assessing complete defects were 96 % and 99.6 % (T (1)-weighted SE) and 92 % and 100 % (FLASH 3D), respectively. For all partial defects, sensitivities and specificities were 68.1 % and 93.3 % (T (1)-weighted SE) and 63 % and 96.1 % (FLASH 3D), respectively. For depicting partial defects of the scapholunate ligament the T (1)-weighted SE sequence (83.3/95.5 %) was superior to the FLASH 3D sequence (64.7/96.6 %), p < 0.05. For the evaluation of the TFCC (T (1)-weighted SE: 65/94.4 %, FLASH 3D: 70/94.6 %) and the lunotriquetral ligament (T (1)-weighted SE: 44/89.4 %, FLASH 3D: 44 /96.7 %), direct MR arthrography showed an insufficient correlation with arthroscopy. CONCLUSION: Direct MR arthrography proved to be of equal value compared with diagnostic arthroscopy in detecting complete defects of the intrinsic ligaments and the TFCC. The method has the potential of replacing diagnostic arthroscopy for the evaluation of the intrinsic ligaments and the TFCC. The T (1)-weighted SE sequence appeared to be superior to the FLASH 3D sequence in evaluating partial defects of the scapholunate ligament. Direct MR arthrography did not reliably detect partial defects of the TFCC and the lunotriquetral ligament.

Adult↗
PubMed2003

Early trans-tibial oedema control using polymer gel socks.

The aim of this investigation was to determine if early trans-tibial oedema control, by polymer gel socks prior to the fitting of a shrinker, could enhance trans-tibial stump volume reduction. Sixteen (16) adults, following unilateral trans-tibial amputation, were randomly allocated into 2 groups and baseline volume measurements taken of their stumps. The control group followed the normal removable rigid dressing programme, while the experimental group wore a polymer gel sock in conjunction with the removable rigid dressing. When wound healing permitted fitting of a shrinker, use of the polymer sock was discontinued and a stump volume measurement was taken. The shrinker was worn for a minimum of 2 days before casting for a prosthesis and a final stump volume measurement taken. Volumes were measured by water filling casts taken of the stump and calculated as a percentage of the baseline volume and the rate of volume reduction calculated. Two t-tests for unequal variance (2-tail, alpha=0.05) were used to compare the rate of percentage volume reduction between the groups, for the trial period of baseline to shrinker fitting and baseline to casting. A statistical difference was observed for the period of baseline to shrinker exceeding the 95% confidence limit (p<0.05). No significant difference was detected for the overall period of baseline to cast. It was concluded that early oedema control by polymer gel sock could enhance stump volume reduction, prior to a shrinker. It was also found that no volume advantage was carried over to the time of casting for a prosthesis.

Adult↗
PubMed2001

Dendritic hypertrophy of Stach type VI neurons within experimentally altered ileum of pigs.

Myenteric neurons were investigated morphometrically to answer the question if type-specific somal hypertrophy of type VI neurons in mechanically stressed ileum of pigs, which was known from an earlier study, is correlated with an increased dendritic arborization, that is, with dendritic hypertrophy. Muscular hypertrophy was induced in the ileum of two juvenile pigs by narrowing the gut circumference (mechanical stenosis) and by reversing a loop of ileum which results in an antiperistaltic segment (functional stenosis), respectively. After a survival time of 6 weeks, wholemounts from the pre- and poststenotic ileal regions, from the antiperistaltic segment as well as from an age matched control animal, were silver impregnated. Dendritic parameters of Stach types IV and VI neurons were recorded using a computer-aided morphometric program and analysed statistically. Type IV neurons showed no change of dendritic parameters, neither within control nor within stenosed ileal segments. In contrast, the type VI neurons displayed increased dendritic parameters within zones of muscular hypertrophy such as total dendritic length, numbers of dendrites, of dendritic branching points and of dendritic endings. We suggest that type VI neurons may participate as descending nitrergic interneurons or motorneurons in the control of muscular function, thus, undergoing plastic changes in case of experimental muscular hypertrophy. Type IV neurons which are involved in the regulation of mucosal processes were not affected by muscular hypertrophy.

Animals↗
PubMed2001

Anti-inflammatory planar chiral [2.2]paracyclophaneacetic acid enantiomers.

OBJECTIVE AND DESIGN: To elucidate if the planar chiral paracyclophane moiety conveys pharmacological activity to arylacetic acid analogs in two animal models. MATERIAL OR SUBJECTS: Female NMRI mice (6 mice/group); female Wistar rats (8 rats/group); thrombocytes from human blood. TREATMENT: The enantiomers of [2.2]paracyclophaneacetic acid were applied locally (10(-7) and 10(-6) mol/ear) and orally (10-100 mg/kg). METHODS: (a) Phorbol myristyl acetate model of acute inflammation of the inner auricle. (b) Oxazolone model of allergic contact dermatitis. (c) Carrageenan model of acute inflammation. (d) Inhibition of cyclooxygenase-1 and 12-lipoxygenase (in vitro). RESULTS: (a) PMA model: pR-(-)-[2.2]paracyclophaneacetic acid (10(-6) mmol/ear): 58% inhibition after 24 h (p < 0.05). (b) Oxazolone model: pR-(-)-[2.2]paracyclophaneacetic acid (10(-6) mmol/ear): 42% inhibition after 24 h (p < 0.05). (c) Carrageenan model: pR-(-)-[2.2]paracyclophaneacetic acid (10 mg/kg): 31.4% inhibition (paw volume 0.48 +/- 0.13 ml). (d) Cyclooxygenase-1 and 12-lipoxygenase: no inhibition at concentrations up to 10 microM. CONCLUSIONS: The easily accessible [2.2]paracyclophane moiety should find its use in medicinal chemistry as it is a pharmacophoric substituent with the interesting feature of planar chirality.

Acetates↗
PubMed2001

[Subretinal microphotodiode array as replacement for degenerated photoreceptors?].

A survey is given on the status of developments, concerning a subretinal electronic microphotodiode array that aims at replacing degenerated photoreceptors. Various prototypes have been developed, tested, and implanted in various experimental animals up to 18 months. The fact that electrical responses were recorded from the visual cortex of pigs after electrical stimulation by subretinal electrodes and the fact that responses are also recorded in-vitro in degenerated rat retinae, shows the feasibility of this approach. However, there are a number of open questions concerning the biocompatibility, the long-time stability, and the type of transmitted image to be solved before application in patients can be considered.

Animals↗
PubMed2001

Molecular cloning and expression of cERG, the ether à go-go-related gene from canine myocardium.

Given the anatomical and physiological similarities to the human heart, canine in vivo heart models may facilitate the analysis of molecular mechanisms underlying cardiac repolarization abnormalities. The development of such models depends, however, on information about canine K+ channels responsible for the establishment of IK currents. In this context, we isolated and sequenced the reverse transcript of the canine ether à go-go-related gene (cERG). The complementary deoxyribonucleic acid (cDNA-derived cERG polypeptide consists of 1,158 amino acids, the sequence of which shows striking homology to human, rat and mouse ERG subunits (97%, 94% and 95% identity respectively). In highly conserved peptide domains like the PAS domain, the membrane-spanning segments S1, S3-S6 and the pore-forming region, there was 100% identity. Analysis of cERG transcription revealed abundant expression of cERG messenger ribonucleic acid (mRNA) in heart and brain and low expression in liver, spleen and kidney. Membrane currents recorded in Xenopus oocytes expressing cERG channels showed functional properties very similar to the human K+ channel hERG, which encodes the alpha-subunit of the cardiac rapidly activating, delayed rectifier (IKr) channel.

Animals↗
PubMed2001

Rapid desensitization of 5-HT(1A) receptors in Fawn-Hooded rats after chronic fluoxetine treatment.

Anxiety, platelet serotonin (5-HT) content and functions of the 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) were measured in Sprague--Dawley (SD) and Fawn-Hooded (FH) rats, a strain with genetically impaired 5-HT storage and reuptake system and a putative model of depression and anxiety. In addition, the effects of 7 and 16 days treatment with the selective serotonin reuptake inhibitor (SSRI) fluoxetine on 8-OH-DPAT-induced responses were studied. FH rats showed significantly higher anxiety in the social interaction test, and much lower platelet 5-HT content compared to SD rats. The efficacy of 8-OH-DPAT (15-120 microg/kg, i.v.) to induce lower lip retraction (an effect mediated by median raphe receptors) was increased in FH rats. In most FH but only a few SD rats a special neurological syndrome, clonic movement of the masseters and in-and-out movement of the eyeballs, was induced by 8-OH-DPAT, and this behaviour like other effects of 8-OH-DPAT, was completely blocked by pretreatment with the 5-HT(1A) receptor antagonist WAY-100635. In SD rats fluoxetine (10 mg/kg/day, i.p.) caused a moderate inhibition of 8-OH-DPAT-induced hypothermia, an effect mediated most likely by hypothalamic 5-HT(1A) receptors, (-19% and -40% after 7 and 16 days of fluoxetine, 24 h after the last injection, respectively). In FH rats fluoxetine caused a rapid and complete reduction in the 8-OH-DPAT-induced hypothermia (-65% and -91% after 7 and 16 days of fluoxetine, respectively). Fluoxetine caused no change in lower lip retraction but a reduction in the masseter-eyeball syndrome in both SD and FH rats. Our data provide evidence that in FH rats, median raphe 5-HT(1A) receptors are hypersensitive, and the hypothalamic 5-HT(1A) receptor desensitization, caused by SSRI antidepressants, is faster and more complete. These data support the notion that chronic treatment with SSRIs induces a desensitization of some 5-HT(1A) receptor populations, and impaired 5-HT storage and reuptake may accelerate this process.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed2001

Anxiety-like effects induced by acute fluoxetine, sertraline or m-CPP treatment are reversed by pretreatment with the 5-HT2C receptor antagonist SB-242084 but not the 5-HT1A receptor antagonist WAY-100635.

The possible role of 5-HT1A and 5-HT2C receptors in the anxiety induced by fear, acute treatment with SSRI antidepressants or the 5-HT receptor agonist m-CPP were tested in the social interaction anxiety test in male Sprague-Dawley rats. Fluoxetine (2.5-10 mg/kg, i.p.), sertraline (15 mg/kg, i.p.) and m-CPP (0.5-2.0 mg/kg, i.p.) all had an anxiogenic-like profile (decrease in time of total social interaction and increase in self-grooming compared to vehicle) under low-light, familiar arena test conditions. All these effects were reversed by pretreatment with the highly subtype-selective 5-HT2C receptor antagonist, SB-242084 at doses of either 0.05 or 0.2 mg/kg, i.p. In contrast, the selective 5-HT1A receptor antagonist WAY-100635 (0.05 and 0.2 mg/kg, s.c.) failed to reverse SSRI-induced decrease in time of total social interaction, further, it augmented self-grooming response. SB-242084 (0.2 mg/kg) and WAY-100635 (0.05 and 0.2 mg/kg) reversed hypolocomotion caused by the SSRI antidepressants. SB-242084, tested alone against vehicle under high-light, unfamiliar arena test conditions associated with fear, caused significant anxiolysis at 0.2 mg/kg and higher doses. These results suggest that increased anxiety in rodents, and possibly, also in humans (e.g. agitation or jitteriness after SSRIs and panic after m-CPP), caused by acute administration of SSRI antidepressants or m-CPP, are mediated by activation of 5-HT2C receptors. Blockade of 5-HT1A autoreceptors may exacerbate certain acute adverse effects of SSRI antidepressants. Both 5-HT1A and 5-HT2C receptors are involved in the SSRI-induced decrease in locomotor activity. In addition, our studies confirm data that subtype-selective 5-HT2C receptor antagonists have strong anxiolytic actions.

Aminopyridines↗
PubMed2001

Restrictive dermopathy: case report, subject review with Kaplan-Meier analysis, and differential diagnosis of the lethal congenital contractural syndromes.

We report on a 34-week-old infant with restrictive dermopathy (RD), a rare lethal genodermatosis, characterized by an abnormal skin growth and differentiation with thin, tightly adherent skin that causes a dysmorphic face, generalized flexion joint contractures, and respiratory insufficiency. Kaplan-Meier analysis of 32 previously well-described infants affected with RD showed a median survival of 132 hours. Lethal congenital contractural syndromes, including Pena-Shokeir phenotype, cerebro-oculo facio-skeletal syndrome, and lethal multiple pterygium syndrome, should be considered first in the differential diagnosis. Other lethal contractural syndromes are discussed.

Contracture↗
PubMed2001

Inflammatory response during abdominal and thyroid surgery: a prospective clinical trial on mediator release.

Several studies have been demonstrated that endotoxin is a potent stimulus of the acute inflammatory response following traumatic injury. Although numerous studies have indicated that the extent of surgical intervention correlates well with the inflammatory response, the potential role of endotoxin as a trigger under those conditions still remains unknown. Therefore, the aim of this study was to elucidate whether or not the up-regulated inflammatory mediators are paralleled by increased endotoxin plasma levels during and following surgery, and whether the extent of surgical intervention represents a crucial factor under those conditions. To study this, plasma was collected at various time points during and after surgery from 52 patients subjected to abdominal surgery (i.e., major surgery) and 25 patients subjected to thyroid surgery (i.e., minor surgery). Plasma was assessed for endotoxin, endotoxin neutralizing capacity (ENC), and inflammatory mediators (leucotriene-C4 [LTC4]-, 6-keto-prostaglandin-F-1-alpha [PGF]-, thromboxane-B2 [TxB2], interleukin-6 [IL-6], and C-reactive protein [CRP]). Furthermore, splanchnic blood circulation was measured by determination of the intraluminal pH of the stomach and sigma (pHi) by intraluminal tonometry. Mesenteric lymph nodes were also collected at the time point of organ mobilization in the major surgery group and were assessed for bacterial translocation. Among all parameters investigated, endotoxin showed the most rapid changes. A significant increase in plasma levels of endotoxin and a decrease of ENC were found in the major surgery groups following induction of anesthesia and in the minor surgery groups after skin incision. Moreover, the incidence of elevated endotoxin levels was significantly higher (89% with elevated endotoxin levels) than the incidence of bacterial translocation (35% with gram-negative bacteria) in mesenterial lymph nodes of the major surgery group. pHi decreased significantly in both groups after skin incision, but no difference was observed between the major and minor surgery groups. Plasma mediators of the arachidonic acid cascade (LTC4, PGF, and TxB2) were only elevated in individual patients during and following surgery in both groups. Conversely, the post-operative increase in the acute phase mediators was significantly different in the major and minor surgery groups. IL-6 plasma levels peaked higher and earlier after major surgery than after minor surgery and the delayed increase of CRP was significantly greater in the major surgery group. In conclusion, the results indicate that plasma levels of endotoxin significantly correlate with the severity of the surgical intervention and may play an important role in inducing mediators of the acute phase reaction under such conditions.

6-Ketoprostaglandin F1 alpha↗
PubMed2001

Multiple effects of codon usage optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type 1 Gag protein.

We have analyzed the influence of codon usage modifications on the expression levels and immunogenicity of DNA vaccines, encoding the human immunodeficiency virus type 1 (HIV-1) group-specific antigen (Gag). In the presence of Rev, an expression vector containing the wild-type (wt) gag gene flanked by essential cis-acting sites such as the 5'-untranslated region and 3'-Rev response element supported substantial Gag protein expression and secretion in human H1299 and monkey COS-7 cells. However, only weak Gag production was observed from the murine muscle cell line C2C12. In contrast, optimization of the Gag coding sequence to that of highly expressed mammalian genes (syngag) resulted in an obvious increase in the G+C content and a Rev-independent expression and secretion of Gag in all tested mammalian cell lines, including murine C2C12 muscle cells. Mice immunized intramuscularly with the syngag plasmid showed Th1-driven humoral and cellular responses that were substantially higher than those obtained after injection of the Rev-dependent wild-type (wt) gag vector system. In contrast, intradermal immunization of both wt gag and syngag vector systems with the particle gun induced a Th2-biased antibody response and no cytotoxic T lymphocytes. Deletion analysis demonstrated that the CpG motifs generated within syngag by codon optimization do not contribute significantly to the high immunogenicity of the syngag plasmid. Moreover, low doses of coadministered stimulatory phosphorothioate oligodeoxynucleotides (ODNs) had only a weak effect on antibody production, whereas at higher doses immunostimulatory and nonstimulatory ODNs showed a dose-dependent suppression of humoral responses. These results suggest that increased Gag expression, rather than modulation of CpG-driven vector immunity, is responsible for the enhanced immunogenicity of the syngag DNA vaccine.

AIDS Vaccines↗
PubMed2000

Rev-independent expression of synthetic gag-pol genes of human immunodeficiency virus type 1 and simian immunodeficiency virus: implications for the safety of lentiviral vectors.

The safety of lentiviral vectors for clinical applications is still a major concern. The gag-pol expression plasmids and the lentiviral vectors used in previous studies contain homologous regions, which constitute a risk for recombination events. Synthetic gag-pol genes of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) were therefore constructed, in which the codon usage was optimized for expression in human cells without altering the amino acid sequences. The synthetic gag-pol genes allowed efficient expression of these genes in the absence of Rev and the 5' untranslated leader region. Both the HIV-1 and the SIV synthetic gag-pol expression plasmids could mediate transduction of an SIV vector into nondividing human cells with titers of about 10(6) transducing units/ml. Similar titers were obtained with a four-plasmid vector-packaging system based on HIV-1. Using a biological assay, homologous recombination events between the synthetic gag-pol expression plasmids and an SIV vector were undetectable and in comparison with a previously used gag-pol expression plasmid at least approximately 100-fold less frequent. By eliminating regions of homology and sequences involved in packaging, synthetic gag-pol genes should improve the safety profile of lentiviral vectors.

5' Untranslated Regions↗
PubMed2000

Experimental hypertrophy of myenteric neurones in the pig: a morphometric study.

Muscular hypertrophy in the ileum of two pigs aged 6 weeks was induced using two different surgical techniques, narrowing of the gut circumference (mechanical stenosis) and segmental reversal of an ileal loop which results in a persistent antiperistalsis of that segment (functional stenosis). These pigs were sacrificed 5-6 weeks postoperatively. Cross sections through the gut wall at various distances from the operation sites revealed marked muscular hypertrophy in the pre-stenotic regions and in the reversed segment. Whole mounts from pre- and post-stenotic, as well as reversed ileal regions, were silver- impregnated. The corresponding ileal region of a third, nonoperated pig served as control. Using a computer-aided morphometric device, somal areas of five morphological neurone types were measured at various distances orally and anally from the operation sites and along the control ileum. Values between hypertrophic and nonhypertrophic zones as well as between two corresponding zones of nonoperated ileum were compared statistically. Along the control ileum, values revealed no differences in soma sizes. Within the experimentally altered material, somal areas of type VI neurones showed marked hypertrophy related to the sites of muscular hypertrophy whereas the other types remained constant throughout (II, IV in segmental reversal) or showed slightly larger somal areas within the post-stenotic, nonhypertrophied zones (I, V, IV in stenosis). Additionally, within the reversed segment, neuronal perikarya of type I, II, IV and V neurones were larger as compared to the neighbouring regions. However, this enlargement of perikarya within the reversed segment may not be correlated with muscular hypertrophy but rather with the transections of intramural axons before reversing this segment. The results suggest that morphologically distinct neurone types may play different roles within the mechanically stressed small intestine and possibly also in the coordination of normal muscular function.

Animals↗
PubMed2000

Concerted action of multiple cis-acting sequences is required for Rev dependence of late human immunodeficiency virus type 1 gene expression.

Based on the human immunodeficiency virus type 1 (HIV-1) gag gene, subgenomic reporter constructs have been established allowing the contributions of different cis-acting elements to the Rev dependency of late HIV-1 gene products to be determined. Modification of intragenic regulatory elements achieved by adapting the codon usage of the complete gene to highly expressed mammalian genes resulted in constitutive nuclear export allowing high levels of Gag expression independent from the Rev/Rev-responsive element system and irrespective of the absence or presence of the isolated major splice donor. Leptomycin B inhibitor studies revealed that the RNAs derived from the codon-optimized gag gene lacking AU-rich inhibitory elements are directed to a distinct, CRM1-independent, nuclear export pathway.

Base Sequence↗
PubMed2000

Characterization of a virtually full-length human immunodeficiency virus type 1 genome of a prevalent intersubtype (C/B') recombinant strain in China.

A molecular epidemiology study was conducted among more than 100 human immunodeficiency virus type 1 (HIV-1) subtype C seropositive intravenous drug users (IDUs) from China. Genotyping based on the envelope C2V3 coding region revealed the highest homology of the most prevalent virus strains circulating throughout China to subtype C sequences of Indian origin. Based on these results, a virtually full-length genome representing the most prevalent class of clade C strains circulating throughout China was directly amplified from peripheral blood mononuclear cells of a selected HIV-infected IDU and subcloned. Sequence analysis identified a mosaic structure, suggesting extensive intersubtype recombination events between genomes of the prevalent clade C and (B')-subtype Thai virus strains of that geographic region. Recombinant Identification Program analysis and phylogenetic bootstrapping suggested that there were 10 breakpoints (i) in the gag-pol coding region, (ii) in vpr and at the 3' end of the vpu gene, and (iii) in the nef open reading frame. (B')-sequences therefore include (i) several insertions in the gag-pol coding region; (ii) 3'-vpr, the complete vpu gene, and the first exons of tat and rev; and (iii) the 5' half of the nef gene. Breakpoints located in the vpr/vpu coding region as well as in the nef gene of 97cn54 were found at almost identical positions of all subtype C strains isolated from IDUs living in different areas of China, suggesting a common ancestor for the C/B' recombinant strains. More than 50% of well-defined subtype B-derived cytotoxic T-lymphocyte epitopes within Gag and Pol and 10% of the known epitopes in Env were found to exactly match sequences within in this clade C/B' chimeric reference strain. These results may substantially facilitate a biological comparison of clade C-derived reference strains as well as the generation of useful reagents supporting vaccine-related efforts in China.

Amino Acid Sequence↗