Biomedical subjects

A Bakker

Publications and source records attributed to A Bakker.

At least 73 records · Page 4Linked to original sources

PubMed1997

Inhibition of HIV-1 replication using a mutated tRNALys-3 primer.

Cellular tRNALys-3 serves as the primer for reverse transcription of human immunodeficiency virus, type 1 (HIV-1). tRNALys-3 interacts directly with HIV-1 reverse transcriptase, is packaged into viral particles and anneals to the primer-binding site (PBS) of the HIV-1 genome to initiate reverse transcription. Therefore, the priming step of reverse transcription is a potential target for antiviral strategies. We have developed a mutant tRNALys-3 derivative with mutations in the PBS-binding region such that priming specificity was re-directed to the highly conserved TAR stem-loop region. This mutant tRNA retains high-affinity binding to HIV-1 reverse transcriptase, viral encapsidation, and is able to prime at both the targeted TAR sequence and at the viral PBS. Constitutive expression of mutant tRNA in T-cells results in marked inhibition of HIV-1 replication, as determined by measurements of viral infectivity, syncytium formation, and p24 production. Inhibition of retroviral replication through interference with the normal process of priming constitutes a new anti-retroviral approach and also provides a novel tool for dissecting molecular aspects of priming.

Base Sequence↗

PubMed1997

Detection of infrequent and multiple K-ras mutations in human tumors and tumor-adjacent tissues.

A sensitive method was developed and applied to examine the distribution of K-ras gene mutations in histologically differing areas of lung tissues obtained from lung cancer patients. This method, which combines polymerase chain reaction (PCR), mutation allele enrichment (MAE), and denaturing gradient gel electrophoresis (DGGE), allows detection of one K-ras mutant allele present in 10(4) to 10(5) wild-type alleles. It was applied to analyze mutations in codon 12 of the K-ras gene in 43 tissue sites microdissected from paraffin-embedded sections obtained from 8 archival cases of lung cancer, all previously shown to have codon 12 K-ras mutations by direct sequencing. In four cases, mutations were detected only in the tumor, while in the other four cases, the same mutations were also found in tissues adjacent to tumors, using the MAE + DGGE method. No mutations were detected among normal-appearing cells in areas distant from the tumors in any of the cases studied. These findings demonstrate that K-ras mutations can be detected at low frequencies in normal-appearing cells from tissues adjacent to the tumor in some lung cancer cases. In addition, this approach also allowed detection of multiple mutations in colorectal tissues obtained from colorectal cancer patients. Thus, the MAE + DGGE method may be applicable to study of K-ras mutations in premalignant or morphologically suspicious lesions in bronchial mucosa or other types of human cancer.

Base Sequence↗

PubMed1997

K-ras and p53 mutations in aberrant crypt foci and colonic tumors from colon cancer patients.

Aberrant crypt foci (ACF) are microscopic lesions which can be detected, after methylene blue staining, in the overtly normal looking colonic mucosa of cancer patients. ACF have been postulated to be precursor lesions which develop into colorectal cancer. Mutations of K-ras and p53 are two important genetic events implicated in colon carcinogenesis. Mutations in K-ras are detectable at earlier stages, while mutations in p53 are detectable at later stages of colon carcinogenesis. Our objective was to compare the nature of genetic alterations in K-ras (codon 12 and 13) and in p53 (exon 4-9) between ACF and corresponding colonic tumors from cancer patients. ACF with > or =20 crypts/focus were harvested from overtly normal looking colonic mucosa of cancer patients at a distance of (approx.) 5 cm from the site of colonic tumors. The colonic tumors and ACF samples were compared for K-ras codon 12 and 13 base pair sequence, using DNA sequencing and for p53 (exon 5-9) allelic types, using PCR-SSCP and DNA sequencing. The results demonstrated a perfect correlation in terms of the type of K-ras allele (wild or mutated) between the ACF (> or =20 crypts/focus) and corresponding colonic tumors in 11/13 cancer patients. Analyses of p53 mutations demonstrated the presence of p53 mutations in colonic carcinomas from 10/13 patients. However, p53 mutations could be detected in an ACF from only 1/13 patient. The results provides further evidence to the role of ACF as precursor to colon cancer. The presence of an identical K-ras as well as p53 mutation in an ACF and the corresponding colonic carcinoma in a patient suggests the possibility of existence of ACF that may be at a more advanced stage in the sequence of colonic tumorigenesis than others. In conclusion, the results suggest that a subset of ACF with higher multiplicity might be considered more likely to progress to more advanced lesions and should be explored as markers of colon cancer risk.

Adenoma↗

PubMed1997

Molecular identification of foreign inclusions in inflammatory tissue surrounding metal implants by Fourier transform laser microprobe mass spectrometry.

Fourier transform laser microprobe mass spectrometry (FT LMMS) is a novel technique for micro-analysis of solids with a lateral resolution in the 5 microns range. One of the major advantages of the technique is the capability to perform characterisation of the molecular composition of both organic and inorganic compounds. The information is directly deduced from the signals without the aid of reference spectra. FT LMMS was applied to the characterisation of black tissue fragments in a biopsy from a patient, in which a constrained condylar nodular knee system was implanted ten years ago. The tissue contained numerous foreign giant cells with a black non-birefringent pigment in their cytoplasm. FT LMMS analysis allowed us to detect directly by means of molecular signals, that the debris consisted primarily of titanium oxide and not metallic titanium, while the implant itself only contained titanium.

Aged↗

PubMed1997

Non-puerperal lactation associated with antidepressant drug use.

AIMS: The aim of the present study was to estimate the relative risk of non-puerperal lactation in patients using antidepressants in general, and specifically for serotonergic (selective serotonin reuptake inhibitors (SSRIs) and clomipramine) and non-serotonergic antidepressants. METHODS: All suspected adverse drug reactions in women and reported from January 1986 until August 1996 to the Netherlands Pharmacovigilance Foundation, a spontaneous adverse drug reaction reporting programme, were analysed. Adverse drug reaction (ADR) reporting odds ratios, defined as the ratio of the exposure odds among reported cases of non-puerperal lactation to the exposure odds of reported other ADRs, were calculated adjusted for age and year of reporting. RESULTS: Thirty-eight cases of non-puerperal lactation were reported, of which 15 were associated with the use of antidepressant drugs. In general, antidepressants were associated with a higher risk of non-puerperal lactation in comparison with other drugs (ADR reporting odds ratio 8.3 [95%CI: 4.3-16.1]). Serotonergic antidepressants (selective serotonin reuptake inhibitors (SSRIs) and clomipramine) were associated with a higher risk (OR 12.7 [95%CI: 6.4-25.4]), whereas other antidepressants were not (OR 1.6 [95%CI: 0.2-11.6]), compared with all other drugs. CONCLUSIONS: Our results indicate that serotonergic antidepressants are associated with an approximately eight times higher risk of non-puerperal lactation compared with other antidepressants. This effect is probably mediated by an indirect inhibition effect of serotonin on the dopaminergic transmission. This finding is in line with the occurrence of other antidopaminergic effects, such as extrapyramidal symptoms, in patients using serotonergic antidepressants.

Adolescent↗

PubMed1997

The origin and molecular characterization of adenoid basal carcinoma of the uterine cervix.

Five cases of adenoid basal carcinoma (ABC) of the uterine cervix were examined for the presence of p53 tumor suppressor gene, K-ras-2 oncogene, and human papillomavirus (HPV). A topographic genotyping approach was used to search for point mutations in K-ras-2 (exon 1 and 2) and p53 (exons 5 to 8) in archival formalin-fixed tissue blocks. Minute target sites were selected from polymerase chain reaction (PCR) amplified and directly sequenced tissue sections. Tissue sections were additionally subjected to immunohistochemical staining for p53 and WAF-1 protein. Because wild type p53 induces WAF-1 gene expression, immunohistochemical staining for WAF-1 protein using monoclonal antibodies may serve as an indirect means to test for p53 mutational damage. Mutational genotype was compared to histopathologic features and immunohistochemical staining. To study the role of HPV, L1 region consensus primers were used to amplify topographic samples, followed by HPV genotyping by direct sequencing and comparison to known viral strains. ABC was found to contain HPV in all cases, proven by genotyping to be HPV type 16 in each case. The virus showed no evidence of genomic variation from prototype HPV type 16 in the L1 segment examined. No K-ras-2 point mutations were identified. p53 immunopositivity was present in all tumors, being weak and focal in 4 and strong and diffuse in 1. WAF-1 immunostaining was positive in two tumors showing weak focal p53 immunopositivity. The single strong and diffuse p53 immunopositive tumor was negative for WAF-1 and was shown to contain a missense p53 point mutation (exon 7-codon 248 tryptophan). In conclusion, ABC is characterized by the presence of HPV type 16. K-ras-2 point mutation appears to play no role in the development of this tumor. p53 gene alterations are common including wild type hyperexpression (weak focal p53 immunopositivity, WAF-1 positivity, no mutational change) and p53 point mutational damage.

Adult↗

PubMed1997

Prognostic value of p53 and K-ras-2 topographic genotyping in endometrial carcinoma: a clinicopathologic and molecular comparison.

The predictive value of p53 and K-ras-2 mutational genotyping in determining tumor aggressiveness and survival in patients with endometrial carcinoma (EC) was retrospectively evaluated using a molecular genotyping approach on fixative treated tissue specimens. Two groups of patients with EC were selected based upon length of survival. Group A consisted of 14 patients that died within 3 years of initial diagnosis and treatment (mean survival of 1.1 years). Group B consisted of 18 patients that survived beyond 3 years (mean survival of 4.7 years). Clinicopathologic features including clinical stage, histologic type, and combined nuclear and architectural grade of each tumor were statistically analyzed with respect to oncogene/tumor suppressor gene alterations. The majority of carcinomas in group A were serous (57%), stage III or IV (93%), and high combined grade (93%). Group B consisted mostly of endometrioid (89%) and low-grade carcinomas (83%); 56.1% were stage III or IV. K-ras-2 point alterations were found in 2 (14%) and 4 (22%) patients from group A and B respectively; the spectrum of K-ras-2 genotypes was similar in both groups. p53 gene mutations were identified in 9 (64%) and 1 (6%) patient from group A and B respectively. p53 staining in group A tended to be of strong intensity and diffuse distribution, being associated with the presence of point mutations, mainly in exon 8. Only a single group B tumor exhibited point mutational change. The presence of p53 mutations strongly correlated with short survival (p <0.05) but the finding of K-ras-2 alterations did not. p53 genotyping has potential prognostic value in EC and can be used along with histopathologic type and histologic grade to identify subsets of more aggressive tumors and to guide the treatment.

Adenocarcinoma↗

PubMed1997

Sex differences in the pharmacological treatment of hypertension: a review of population-based studies.

OBJECTIVE: To summarize all available literature on sex differences in the pharmacological treatment of hypertension with respect to the percentage of hypertensive patients treated pharmacologically and the selection of antihypertensive drugs. The influences of the calendar period, age, definition of hypertension, prevalence of hypertension and country on these sex differences were examined. DATA IDENTIFICATION: A secondary analysis of data from 46 population-based studies in 22 countries on the prevalence of pharmacologically treated hypertension was conducted to estimate sex ratios for the prevalence of drug treatment for hypertension. RESULT: Overall, women with hypertension were 1.33-fold [95% confidence interval (CI) 1.32-1.34] more likely to be treated pharmacologically for hypertension than were hypertensive men. With increasing age, the female: male ratio for pharmacological treatment of hypertension decreased from 2.26 (95% CI 1.56-3.27) at ages 20-29 years to 1.22 (95% CI 1.11-1.34) at ages 60-69 years. In all countries more women than men were treated for hypertension, with the biggest difference observed in the USSR (1983-1986), where about twice as many women as men were treated for hypertension. Women more frequently used diuretics, whereas men more often used beta-blockers, angiotensin converting enzyme inhibitors and calcium antagonists. CONCLUSIONS: Hypertensive women are more often treated for hypertension than hypertensive men and their pattern of use of antihypertensive drugs differs from that of men. Further research is required in order to explain sex differences in the treatment of hypertension with respect to the prevalence of pharmacological treatment of hypertension and choice of antihypertensive drugs, and to investigate the consequences of this difference for long-term outcomes.

Adult↗

PubMed1997

A meta-analysis of the treatment of panic disorder with or without agoraphobia: a comparison of psychopharmacological, cognitive-behavioral, and combination treatments.

To compare short-term efficacy of benzodiazepines, antidepressants, psychological panic management, exposure in vivo, and combination treatments in panic disorder with or without agoraphobia (PA), a meta-analysis was conducted. Included were 106 studies, pertaining to 222 treatment conditions, 5,011 patients at pretest and 4,016 at posttest. Pre/post effect sizes Cohen's d were calculated within the treatment conditions for four clinical variables: panic, agoraphobia, depression, and general anxiety. Seven large treatment conditions were used in the main analyses: high-potency benzodiazepines, antidepressants, psychological panic management, exposure in vivo, pill-placebo combined with exposure, antidepressants combined with exposure, and psychological panic management combined with exposure in vivo. First, these treatments were compared with a control condition, consisting of pill-placebo, attention placebo, and waiting list. Next, a comparison was made between the treatments. Antidepressants, psychological panic management, high-potency benzodiazepines, and antidepressants combined with exposure in vivo were superior to the control condition for panic attacks. Exposure in vivo alone was not effective for panic attacks. With regard to agoraphobic avoidance, all seven treatments were superior to the control condition. When comparing the various treatment conditions, no differences concerning panic attacks were found. For agoraphobic avoidance, the combination of antidepressants with exposure in vivo was superior to the other conditions. The combination of antidepressants with exposure in vivo is the most potent short-term treatment of PA.

Adult↗

PubMed1997

A comparison of paroxetine, clomipramine and placebo in the treatment of panic disorder. Collaborative Paroxetine Panic Study Investigators.

The aim of this 12-week, double-blind, parallel group, placebo-controlled study was to compare paroxetine with clomipramine in 367 patients with DSM-III-R defined panic disorder. Efficacy assessments included the daily panic attack diary, the Clinical Global Impression Scale, the Hamilton Anxiety Rating Scale, the Marks Sheehan Phobia Scale and the Sheehan Disability Scale. Paroxetine produced significant improvements compared with placebo in various measurements of panic attack frequency, and was as effective as clomipramine. However, paroxetine appeared to have a more rapid onset of action than clomipramine in reducing the number of panic attacks to zero. There was an equivalent improvement with both paroxetine and clomipramine in the supportive efficacy variables which assessed associated aspects of therapeutic improvement. Significantly more adverse effects were reported in the clomipramine group compared with the paroxetine group, while there was no difference between the paroxetine and placebo groups.

Adolescent↗

PubMed1997

Impact of dosage frequency on patient compliance.

OBJECTIVE: To evaluate the impact of dosage frequency on the compliance of patients who receive their medicines from community pharmacies. RESEARCH DESIGN AND METHODS: Each month, patients received a supply of their medication in a Medication Event Monitoring Systems container, which registered each opening of the package. At the end of the study, the patients received a short questionnaire. The subjects were 91 diabetic patients using oral antidiabetic agents. Patients taking insulin and those who were unable to collect their medicines from the pharmacy were excluded from the study. Compliance was defined as the percentage of doses taken during the observation period. Another parameter used was compliance with the prescribed regimen, defined as the percentage of days in which the number of tablets were taken as prescribed. As a last parameter, compliance with the prescribed dose intervals was used. RESULTS: Compliance is influenced by the frequency of doses. The compliance for this group of patients is 74.8%, with an average of 79% in the case of a dose once daily and 38% in the case of a dose three times daily. The predominant type of noncompliance in all groups was dose omissions. However, more than one-third of the patients used more doses than prescribed. Overconsumption is a frequently made mistake by patients on a one-dose daily schedule. CONCLUSIONS: The reduction of dose frequency may decrease total noncompliance, but at the same time, it increases the risk of overconsumption. Reducing the frequency does not automatically result in a better therapeutic schedule. The choice of once or twice daily should depend on the therapeutic range of the drug.

Diabetes Mellitus, Type 2↗

PubMed1997

Sporadic and Thorotrast-induced angiosarcomas of the liver manifest frequent and multiple point mutations in K-ras-2.

Hepatic angiosarcoma (HA) is an uncommon neoplasm associated with known etiologic factors in 25% to 42% of cases. It is, however, one of the most common sarcomas found in the liver. The aim of this study was to find was to find mutations in the K-ras-2 oncogene in sporadic and Thorotrast (TT)-induced HA. Point mutations in K-ras-2 were sought in archival, formalin-fixed tissue blocks from 24 patients with angiosarcoma. Of these, 19 cases were sporadic and 5 were TT-induced. Mutational analysis was performed by topographic microdissection with PCR amplification followed by genotyping. Specific mutations were determined by two independent methods: (a) direct sequencing of the PCR product confirmed by rePCR and by using a different sequencing primer, and (b) PCR-based selective enrichment of mutant DNA by endonuclease digestion followed by heteroduplex DNA analysis using denaturing gradient gel electrophoresis. Eleven K-ras-2 point mutations were detected in 7 of 24 (29%) tumors, including 5 of 19 (26%) sporadic HA and 2 of 5 (40%) TT-induced HA. There were seven G:C > A:T and four G:C > T:A mutations. All seven mutated tumors contained a codon 12-aspartate amino acid substitution. In addition, a second codon 12-cysteine mutant cell population was present in one of two codon 12-aspartate mutated TT-induced HA and in three of five codon 12-aspartate sporadic tumors. Of these four tumors, three contained both aspartate and cysteine mutations and were composed of multiple nodules; the fourth was a single mass. Seventeen tumors had multiple nodules; whereas 5 had a K-ras-2 mutation, 12 were wild-type. The molecular pathology of both sporadic and TT-induced HA is characterized by a high rate of K-ras-2 mutations characteristic of oxidative damage (ie, G:C > A:T and G:C > T:A mutations) resulting in two mutated population sets: codon 12 GGT > GAT and GGT > TGT (glycine to aspartic acid and cysteine). This is, to date, the first study to characterize the K-ras-2 gene mutations within human sporadic and TT-induced HA by direct sequence analysis and denaturing gradient gel electrophoresis. These data further support the hypothesis linking adduct-forming vinyl chloride exposure to HA containing a much higher frequency of K-ras-2 mutations and a mutational spectrum characteristic of chloroethylene oxide, a carcinogenic metabolite of vinyl chloride.

Adult↗

PubMed1997

Rab3 dissociation and clathrin-mediated endocytosis, two key steps in the exo-endocytotic pathway of large dense-cored vesicles in primary cultures of superior cervical ganglia.

In this study we have used primary cultures of porcine superior cervical ganglia as a model system to study exo-endocytosis in sympathetic neurons. Pure neuronal cultures with a defined noradrenergic phenotype can be obtained when antimitotics are included in the culture medium, and the high yield from prenatal piglets allows a biochemical approach in addition to morphological studies. Release of large dense-cored vesicles (LDCVs) was visualized by exposing stimulated neurons to anti-dopamine-beta-hydroxylase (D beta H) prior to fixation. Double immunofluorescent staining revealed that synaptotagmin was colocalized with anti-D beta H labeled exocytotic spots, but not the small GTP-binding protein rab3. Density gradient centrifugation of a postmitochondrial supernatant of cultured cells confirmed the dissociation of rab3 from a population of mature LDCVs. As expected, rab3 did not reassociate with the lighter D beta H-positive membrane fraction in the gradient representing internalized LDCV membranes. The fluid phase marker horseradish peroxidase colocalized with retrieved LDCV membranes. Indirect immunofluorescence demonstrated the colocalization of clathrin with D beta H-positive exocytotic spots on the plasma membrane. At the ultrastructural level, depolarization of neurons resulted in the abrupt loss of LDCVs and concomitant appearance of clathrin-coated vesicles and coated omega profiles. The size distribution of coated structures overlapped strongly with that of LDCVs. Taken together, these data clearly suggest that two key regulatory events in the process of exo-endocytosis, i.e. rab3 dissociation and clathrin-mediated internalization, are not only reminiscent of small synaptic vesicles, but also are a feature of the LDCV pathway at the presynaptic plasma membrane of sympathetic neurons.

Acetylcholine↗

PubMed1996

Morphologic, histologic, and molecular similarities between adenocarcinomas arising in the gastric stump and the intact stomach.

BACKGROUND: Patients with gastric remnants resulting from partial resections have an increased risk for carcinoma. It is unclear whether adenocarcinoma arising in the gastric stump (GSca) differs from intact stomach carcinoma (Gca). The goal of this study was to examine the pathologic and molecular features of GSca and compare them with Gca. METHODS: Adjacent nonmalignant areas and tumors from 14 patients who were 19-55 years postgastrectomy (mean, 32.1 years) were compared with 14 Gca by pathologic and molecular analysis. Formalin fixed, paraffin embedded specimens were immunohistochemically stained for p53 followed by topographic genotyping. Exons 5-8 were amplified by the polymerase chain reaction and directly sequenced. RESULTS: No differences were noted between the two groups regarding gender, types of metaplasia, dysplasia, morphology, or histologic tumor type. However, a higher incidence of cystic dilatation and foveolar hyperplasia were present in GSca. p53 gene point mutations occurred in 5 of 14 (35.7%) GSca patients. GSca p53 mutations included missense point mutations (G:A transitions in four patients and G:C transversion in one patient) with allelic loss. In four of the five patients with p53 mutations, the same mutation was also observed in the adjacent area. p53 point mutations were present in 4 of 14 Gca (28.6%), in exons 5, 6, and 8. In one case, the same mutation was also detected in the adjacent nonmalignant mucosa. CONCLUSIONS: Similarities in clinical, pathologic, and molecular features between GSca and Gca suggest the possibility that they share similar mechanisms of carcinogenesis. p53 gene alterations in premalignant areas may denote a possible early role of this gene in gastric carcinoma.

Adenocarcinoma↗

PubMed1996

Luminal foam cell accumulation is associated with smooth muscle cell death in the intimal thickening of human saphenous vein grafts.

BACKGROUND: Occlusion of saphenous vein grafts is a major problem after coronary artery bypass graft surgery. Diffuse intimal thickening develops in all implanted aortocoronary saphenous vein grafts within 6 months to 1 year. In some regions of the thickened intima, foam cells accumulate along the luminal margin. This particular morphology resembles the morphology of unstable atherosclerotic plaques as they occur in coronary arteries. In the present study, we focused on the possible topographic relation between luminal foam cell accumulation and cell death of smooth muscle cells (SMCs) within the adjacent thickened intima. METHODS AND RESULTS: Segments of occluded and suboccluded implanted human aortocoronary saphenous vein grafts were obtained during reintervention coronary artery bypass graft surgery in 30 patients. In the regions of the vein grafts with luminal foam cell accumulation, the percentage of SMC alpha-actin immunoreactive area of the superficial intimal thickening was 6 +/- 1.4%, which was different from the 17.6 +/- 2.3% of the deep intimal thickening. A strong negative correlation between the number of foam cell nuclei and the percentage of SMC alpha-actin immunoreactive area in the adjacent superficial intimal thickening was present (r = -.77, P < .001). Within the superficial intimal thickening, cytoplasmic and DNA fragmentation could be detected, which points to apoptotic cell death. A fraction of the cytoplasmic fragments fitted the ultrastructural characteristics of matrix vesicles and showed pronounced calcium and phosphorus accumulation as demonstrated with the use of x-ray microanalysis. CONCLUSIONS: The close spatial relation among foam cell accumulation, pronounced intimal SMC loss, and cell death suggests the presence of a foam cell-derived factor that can induce cell death in the SMC population of the intimal thickening. The depletion of the intimal SMC population could promote plaque rupture and thrombotic complications in the grafts.

Cell Death↗

PubMed1996

Negative regulation of gene expression from the HTLV type II long terminal repeat by Rex: functional and structural dissociation from positive posttranscriptional regulation.

Regulation of human T cell leukemia virus type II (HTLV-II) gene expression by Rex is mediated by cis-acting elements in the 5' viral long terminal repeat (LTR). Rex acts posttranscriptionally to enhance cytoplasmic accumulation of incompletely spliced viral mRNAs encoding structural proteins. We report a distinct negative regulatory function mediated by Rex affecting expression from the viral 5' LTR. Using both LTR-driven CAT reporters and a full-length HTLV-II proviral construct, we demonstrate that Rex decreases total cellular levels of LTR-containing mRNA in a dose-dependent manner. Negative regulation is an independent function as demonstrated by structural and functional dissociation from Rex positive posttranscriptional regulation. This negative regulatory action was dependent on nuclear localization sequences, but did not require the previously defined Rex-responsive element (RxRE). Negative regulation was observed in T cell lines but not in B cell lines, suggesting the involvement of cell type-specific factors distinct from those involved in posttranscriptional regulation. An internal deletion mutant of Rex removing aa 38-80 retained the ability to repress, but did not posttranscriptionally increase expression, while negative regulation requires a previously uncharacterized carboxy-terminal region (aa 154-170). These findings suggest that Rex may serve two simultaneous functions: to decrease overall levels of transcribed viral mRNA, and to facilitate nuclear to cytoplasmic export of mRNAs encoding structural proteins. The negative regulatory function of Rex may play a role in viral latency.

Base Sequence↗

PubMed1996

Nuclear appearance of a factor that binds the CD28 response element within the interleukin-2 enhancer correlates with interleukin-2 production.

Activation of T lymphocytes requires the combined signaling of the T cell receptor and costimulatory molecules such as CD28. The ability of T cells to produce interleukin-2 (IL-2) is a critical control point in T lymphocyte activation. The IL-2 enhancer contains a functional motif named CD28 response element (CD28RE) that serves a role as a target for mitogenic T cell activation signals. The CD28RE sequence reveals similarity to the consensus kappaB binding motif. Here we demonstrate that CD28RE binds an inducible protein with a molecular mass of approximately 35 kDa called nuclear factor of mitogenic-activated T cells (NF-MATp35) that is clearly different from the known NF- kappaB/Rel family members. Induction of NF-MATp35 was shown to depend on de novo protein synthesis and was restricted to T cells that received a mitogenic combination of T cell stimuli, not necessarily including CD28 signaling. Nonmitogenic T cell stimulation did not result in appearance of NF-MATp35. These results indicate that mitogenic combinations of T cell activation signals are integrated at the level of NF-MATp35 induction. Similar to its effect on IL-2 production, cyclosporin A inhibited the induction of NF-MATp35. Taken together, these data demonstrate that the nuclear appearance of NF-MATp35 shows excellent correlation with IL-2 production, which is a unique characteristic among nuclear factors implicated in the control of IL-2 gene expression.

Base Sequence↗

PubMed1996

Specific binding of polypyrimidine tract binding protein and hnRNP A1 to HIV-1 CRS elements.

The human immunodeficiency virus (HIV) Rev and human T-cell leukemia virus (HTLV) Rex proteins regulate viral RNA processing. Both proteins act to overcome the block to viral structural gene expression, at least in part, by reversing the inhibitory effect of intronic RNA sequences, termed cis-acting repressive (CRS) sequences. Using HTLV type II (HTLV-II) as a model, we recently showed that the function of a 5' long terminal repeat (LTR) CRS correlates with in vitro binding by both polypyrimidine tract binding (PTB) protein (also known as hnRNP I) and hnRNP A1 to CRS RNA (1,2). Using radioimmunoprecipitation of proteins ultraviolet (UV) crosslinked to each HIV CRS RNA with monoclonal anti-hnRNP antibodies, we now demonstrate that hnRNP I and hnRNP A1 bind to two different HIV-1 CRS RNAs. In addition, we show that hnRNP I and hnRNP A1 binding to HIV-1 CRS RNAs can be specifically competed by HTLV-II CRS RNAs using electrophoretic mobility shift assay (EMSA)/UV crosslinking assays. Binding by both hnRNP I and hnRNP A1 to HIV-1 and HTLV-II CRS RNAs suggests a role for these proteins in CRS function that may be influenced by the Rev and Rex proteins, respectively.

Electrophoresis, Polyacrylamide Gel↗