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Biomedical subjects

A Seth

Publications and source records attributed to A Seth.

At least 163 records · Page 9Linked to original sources
PubMed1991

Mutational analysis of the cytoplasmic tail of the human transferrin receptor. Identification of a sub-domain that is required for rapid endocytosis.

It has been reported that the sequence Tyr20-X-Arg-Phe23 present within the cytoplasmic tail of the transferrin receptor may represent a tyrosine internalization signal (Collawn, J.F., Stangel, M., Kuhn, L.A., Esekogwu, V., Jing, S., Trowbridge, I.S., and Tainer, J. A. (1990) Cell 63, 1061-1072). However, as Tyr20 is not conserved between species (Alvarez, E., Gironès, N., and Davis, R. J. (1990) Biochem. J. 267, 31-35), the functional role of the putative tyrosine internalization signal is not clear. To address this question, we constructed a series of 32 deletions and point mutations within the cytoplasmic tail of the human transferrin receptor. The effect of these mutations on the apparent first order rate constant for receptor endocytosis was examined. It was found that the region of the cytoplasmic tail that is proximal to the transmembrane domain (residues 28-58) is dispensable for rapid endocytosis. In contrast, the distal region of the cytoplasmic tail (residues 1-27) was found to be both necessary and sufficient for the rapid internalization of the transferrin receptor. The region identified includes Tyr20-X-Arg-Phe23, but is significantly larger than this tetrapeptide. It is therefore likely that structural information in addition to the proposed tyrosine internalization signal is required for endocytosis. To test this hypothesis, we investigated whether a heterologous tyrosine internalization signal (from the low density lipoprotein receptor) could function to cause the rapid endocytosis of the transferrin receptor. It was observed that this heterologous tyrosine internalization signal did not allow rapid endocytosis. We conclude that the putative tyrosine internalization signal (Tyr20-Thr-Arg-Phe23) is not sufficient to determine rapid endocytosis of the transferrin receptor. The data reported here indicate that the transferrin receptor internalization signal is formed by a larger cytoplasmic tail structure located at the amino terminus of the receptor.

Amino Acid Sequence↗
PubMed1991

T-cell-receptor-independent activation of cytolytic activity of cytotoxic T lymphocytes mediated through CD44 and gp90MEL-14.

CD44 is a transmembrane glycoprotein found on a variety of cells including those of myeloid and lymphoid origin. CD44 is highly conserved among various species and is involved in the homing of lymphocytes and monocytes to lymph nodes, Peyer's patches, and sites of inflammation. In the present study, we demonstrate that monoclonal antibody (mAb) 9F3, directed against murine phagocytic glycoprotein 1 (CD44) expressed on cytotoxic T lymphocytes (CTLs), can trigger the lytic activity of CTLs and redirect CTL-mediated lysis to antigen-negative Fc receptor-positive target cells. Similar redirected lysis was also inducible using mAb MEL-14, directed against the lymphocyte homing receptor for endothelium (gp90MEL-14). The redirected lysis induced by mAbs 9F3 and MEL-14 was similar to that induced by mAbs against the alpha beta T-cell receptor or CD3. In contrast, mAbs directed against CD8, CD45R, and CD11a (LFA-1, lymphocyte function-associated antigen 1) failed to evoke lytic activity. The current study demonstrates that CD44 and gp90MEL-14 molecules, in addition to participating in T-cell homing and adhesion, may play a major role in delivering the transmembrane signal to the CTL that triggers the lytic activity, even when the T-cell receptor is not occupied. Such a mechanism may account for the nonspecific tissue damage seen at sites of CTL-mediated inflammation.

Animals↗
PubMed1991

Pro-Leu-Ser/Thr-Pro is a consensus primary sequence for substrate protein phosphorylation. Characterization of the phosphorylation of c-myc and c-jun proteins by an epidermal growth factor receptor threonine 669 protein kinase.

A growth factor-stimulated (MAP2-related) protein kinase, ERT, that phosphorylates the epidermal growth factor receptor at Thr669 has been purified from KB human tumor cells by Northwood and co-workers (Northwood, I. C., Gonzalez, F. A., Wartmann, M., Raden, D. L., and Davis, R. J. (1991) J. Biol. Chem. 266, 15266-15276). The ERT protein kinase has a restricted substrate specificity, and the structural determinants employed for substrate recognition by this enzyme have not been defined. As an approach toward understanding the specificity of substrate phosphorylation, we have used an in vitro assay to identify additional substrates for the ERT protein kinase. In this report we describe two novel substrates: (a) the human c-myc protein at Ser62 and (b) the rat c-jun protein at Ser246. Alignment of the primary sequences surrounding the phosphorylation sites located within the epidermal growth factor receptor (Thr669), Myc (Ser62), and Jun (Ser246) demonstrated a marked similarity. The observed consensus sequence was Pro-Leu-Ser/Thr-Pro. We propose that this sequence forms part of a substrate structure that is recognized by the ERT protein kinase.

Amino Acid Sequence↗
PubMed1991

Evaluation of a theoretical Doppler index to noninvasively estimate peak dP/dt using continuous wave Doppler ultrasound of ascending aortic flow in man.

A theoretical formula for calculation of peak dP/dt was derived using parameters obtained from continuous wave Doppler echocardiography signals of aortic blood flow. The direct proportional relationship between the main variables of this formula and invasively measured peak dP/dt was validated in 20 patients undergoing routine diagnostic cardiac catheterization. Doppler signals of aortic flow were obtained simultaneously to invasive pressure recordings with a 2 MHz continuous wave transducer via the suprasternal echocardiographic window. The Doppler signals were recorded on magnetic tape and measurements were made with digital calipers by two independent, blinded observers. The following parameters were measured: peak velocity (V) and time from onset of ejection to peak velocity (T). V2/T, the variable of the derived formula, was calculated for each of the observer's measured parameters and showed a very high interobserver correlation. The two observers' measurements of each parameter were averaged for each patient and the resulting mean was used in calculating the V2/T and mean acceleration. 4. A good correlation of V2/T with invasively measured peak dP/dt was obtained. Our derived index of left ventricular function showed a higher correlation with peak dP/dt compared to other Doppler indices of ventricular function. V2/T may provide a noninvasive method for estimating peak dP/dt.

Aorta↗
PubMed1991

Changes in Doppler indices of cardiac function during and after percutaneous transluminal coronary angioplasty.

OBJECTIVE: To assess the sensitivities of Doppler indices to changes in global cardiac function during and after controlled myocardial ischaemia induced by coronary angioplasty. DESIGN: Continuous wave Doppler signals of aortic flow were recorded during coronary angioplasty. The following Doppler indices of cardiac function were measured before, during, and after balloon inflation: V (peak velocity), MA (mean acceleration), V2/T (T = time from onset to peak ejection), and MD (minute distance corrected for baseline heart rate). SETTING: A tertiary care cardiological unit in a university hospital. PATIENTS: Sixteen patients undergoing coronary angioplasty of the left anterior descending coronary artery. Eight patients had multivessel disease. MAIN OUTCOME MEASURES: The primary outcome measures were planned before data collection began. RESULTS: 12 patients showed a significant fall of three or more Doppler indices from their baseline values during balloon inflation. This occurred in all patients with multivessel disease. The Doppler indices V2 T, MD, V, and MA fell by 43.7%, 37.7%, 27.4%, and 23% respectively from their baseline values (p less than 0.0001). The relative sensitivities of the Doppler indices to ischaemia were V2/T greater than MD (p less than 0.02), MD greater than (p less than 0.001), and V greater than MA (p less than 0.01). The impairment of global left ventricular function resulting from brief balloon inflation during single vessel angioplasty was reversible in all the patients. CONCLUSIONS: The Doppler indices V2/T, MD, V, and MA are all sensitive, in order of magnitude, to falls in global cardiac function resulting from ischaemia. They may prove useful for assessing cardiac function during ischaemia in the clinical setting.

Adult↗
PubMed1990

Macrophages but not B cells from aged mice are defective in stimulating autoreactive T cells in vitro.

In the present study the effect of aging on the capacity of Ia+ cells to stimulate autoreactive T cells in the syngeneic mixed lymphocyte reaction (SMLR) was investigated. Using young CD4+ T cells as responders, it was observed that unseparated whole spleen cells from aged mice had normal stimulatory activity comparable to that of young spleen cells. Interestingly, however, when purified splenic adherent cells (SAC) enriched for macrophages or splenic B cells were used as stimulators, aged SAC but not aged B cells were found to be defective in stimulating autoreactive T cells. This defect in aged SAC was not due to decreased expression of Ia antigens since the percentage of Ia+ SAC and density of Ia antigen expression was similar in both young and old mice. Also, the B cells from aged mice expressed normal levels of Ia antigens. Aged SAC, when mixed with young SAC could also actively suppress the normal SMLR. However, this suppression was not due to increased prostaglandin production but was found to be associated with interleukin-1 (IL-1) regulation, inasmuch as addition of exogenous IL-1 could completely reconstitute the defective stimulatory activity of aged SAC and also abolished the suppressor activity of the SAC. Aged mice also demonstrated an intrinsic defect in the CD4+ T cells responding in the SMLR. Together, our studies on the SMLR demonstrate an age-related defect in responder autoreactive T cells and in stimulator splenic macrophages but not in the stimulatory activity of B cells.

Aging↗
PubMed1990

An objective computer system for the quantification of artery stenoses.

We have developed a low cost, clinically usable system for the objective assessment of the severity of coronary artery stenoses from single view angiograms. The system is based on a desktop computer with incorporated frame grabber. Images are captured by means of a video camera. The user selects a region of interest which encompasses the stenosis. Facilities are provided for automatic or manual definition of the artery centre line and edges. The computer then calculates the artery diameter and cross-sectional area by videodensitometry along profile lines which are orthogonal to the long axis of the artery. These results can be expressed numerically as a percentage stenosis when compared to a normal region of the artery. The image is corrected for geometric distortion using a grid test object. The image grey scale is corrected by means of a ramp test object such that a pixel value is proportional to the attenuator thickness. The ramp is placed on the patient during the X-ray examination and an iterative technique has been developed for subtracting the underlying structures from the superimposed ramp image. The system has been assessed using test objects constructed in Perspex which simulate arteries of known cross-sectional area and stenoses of known severity.

Algorithms↗
PubMed1990

Autoreactive T cell clones isolated from normal and autoimmune-susceptible mice exhibit lymphokine secretory and functional properties of both Th1 and Th2 cells.

Recent studies have suggested the existence of two mutually exclusive subpopulations of T helper (Th) cells in the murine immune system, called Th1 which produces interleukin (IL)-2 and interferon (IFN)-gamma but not IL-4 and Th2 which secretes IL-4 and IL-5 but not IL-2. Also, functionally, Th1 cells generally activate the macrophages and mediate delayed-type hypersensitivity whereas Th2 cells provide help efficiently to B cells. In the present study, we investigated the lymphokine secretory properties of two well-characterized autoreactive (self-Ia reactive) T cell clones isolated from normal DBA/2 mice and autoimmune-susceptible MRL-lpr/lpr mice. It was observed that both the autoreactive T cell clones, following activation, produced IL-2, IL-4, and IFN-gamma. They induced hyper-Ia expression and cell proliferation in syngeneic B cells as well as activated the macrophages to exhibit tumoristatic properties. Both clones could also induce T-T network interaction in which syngeneic naive CD4+ T cells responded directly to stimulation with autoreactive T cell clones. The T-T interaction was demonstrable in 1-month-old MRL-lpr/lpr mice prior to the onset of the autoimmune disease but not in 6-month-old mice having lymphadenopathy and autoimmune disease. Unlike Th1 and Th2 cells which upon antigenic stimulation respond to exogenous IL-2 and IL-4, the autoreactive T cell clones responded only to IL-2 but not to IL-4. Our data suggest the existence of a unique subset of immunoregulatory CD4+ Th cells having the lymphokine secretory and functional properties of both the murine Th1 and Th2 subsets.

Animals↗
PubMed1990

Sustained improvement in left ventricular function after successful coronary angioplasty.

The short and long term effects of successful percutaneous transluminal coronary angioplasty on left ventricular function, at rest and on exercise were investigated in 49 patients. Thirty-four had had no previous infarction (group 1) and 15 had (group 2). Technetium-99m gated blood pool images were obtained at rest and during exercise before, six weeks after, and a mean of fifteen months after successful angioplasty. Before angioplasty the mean (SD) ejection fraction fell significantly on exercise in both groups from 58 (10)% to 53 (13)% in group 1 and from 48 (10)% to 40 (16)% in group 2. This change was paralleled by a worsening wall motion score (from 0.6 (0.4) to 1.6 (1.2) in group 1 and from 2.3 (1.9) to 3.3 (2.4) in group 2). Six weeks after the procedure there was little change in resting ejection fraction but it increased significantly on exercise (to 62 (11)% in group 1 and to 53 (13)% in group 2). There was a concomitant significant improvement in the exercise wall motion score (to 0.4 (0.6) in group 1 and to 1.8 (1.1) in group 2). This improvement in exercise ejection fraction and wall motion was maintained at later follow up with no significant deterioration in either variable and a clearly sustained improvement in ejection fraction (60 (10)% in group 1 and 51 (10)% in group 2) and wall motion score (0.2 (0.2) in group 1 and 1.3 (0.8) in group 2) compared with values before angioplasty. The initial improvement in left ventricular function on exercise after successful angioplasty was maintained for at least 9-24 months both in patients with previous myocardial infarction and in those without.

Angioplasty, Balloon, Coronary↗
PubMed1990

Isoforms of the human ets-1 protein: generation by alternative splicing and differential phosphorylation.

The ets-1 gene belongs to the ets gene family (ets-1, ets-2, erg, and elk) and is homologous to the v-ets oncogene found in the avian leukemia virus E26. The ets-1 gene products were characterized using a specific monoclonal antibody developed against a bacterially expressed v-ets protein. The ets-1 gene product in the human T-cell line CEM was found to consist of at least six species: four major species with apparent molecular weights of 51 kDa (p51), 48 kDa (p48), 42 kDa (p42), and 39 kDa (p39); and two minor species of 52 kDa (pp52) and 49 kDa (pp49), which are demonstrated to be the phosphorylated forms of p51 and p48, respectively. All of the ets-1 proteins are related to each other and are considered products of the ets-1 gene. Subcellular localization showed that the pp52 and p51 are found mainly in the cytoplasm, while p48 and p39 are found mainly in the nucleus. Specific antibodies against various exons of ets-1 showed that both p42 and p39 lack a region corresponding to exon VII. Polymerase chain reaction analyses revealed the presence of an additional RNA product that corresponds to mRNA lacking exon VII. These results suggest that the human ets-1 gene encodes multiple proteins that are generated by at least two distinct mechanisms: alternative splicing of mRNA and protein phosphorylation.

Amino Acid Sequence↗
PubMed1990

The c-ets-1 proto-oncogene has oncogenic activity and is positively autoregulated.

The proto-oncogene ets-1 is a member of the ets family of genes that share homology with the viral oncogene, v-ets, of the avian leukemia virus E26. By using expression vectors, we demonstrate that the ets-1 gene transforms NIH3T3 cells and the ets-1 transfected cells form colonies in soft agar and induce tumors in nude mice. We have also determined that the ets-1 protein contains homology with the helix-loop-helix motif of the HLH family proteins, but lacks the basic domain upstream of helix I. Transfection of the NIH3T3 cells with ets-1 vectors results in the activation of the endogenous ets-1 gene. Using hybridization probes that can distinguish between transcripts from endogenous and exogenous templates, we show that the endogenous ets-1 gene is activated by the expression of the transfected exogenous ets-1. In contrast, the expression of transfected ets-2 has no effect on the endogenous ets-1 gene expression. The results indicate that the ets-1 proto-oncogene is positively autoregulated by its product.

Amino Acid Sequence↗
PubMed1989

A specific defect in the proliferative capacity of B cells from old mice stimulated with autoreactive T cells.

B lymphocytes from aged mice were found to be defective in their ability to proliferate in response to stimulation with an autoreactive T cell clone D1.4. The differentiative response leading to antibody secretion was also impaired in the auto D1.4 T cell-stimulated B cells from old mice in comparison to similarly stimulated B cells from young mice. The B cells from old mice were competent in activating the autoreactive T cells such that the T cells were induced to proliferate. The B cell defect appears to be restricted to a certain phase of B cell activation, since old mouse B cells responded to the auto D1.4 T cells by increasing cell surface Ia as well as size, but failed to incorporate tritiated thymidine. The responsiveness to interleukin-4 was found to be similar between B cells from young and old mice. It appeared that the B cells from old mice are specifically defective in progressing from the G0 phase of cell cycle into the S phase when stimulated with the auto D1.4 T cells.

Aging↗
PubMed1989

Total coronary arterial occlusion: real or apparent?

We describe a case in which the angiographic appearance of total occlusion of the left anterior descending coronary artery was not due to a complete anatomical obstruction, but due to competitive and retrograde flow in the distal segment of the artery from collaterals via the contralateral vessel. This case has implications on our current practice and results of coronary angioplasty in total coronary arterial occlusion.

Angina Pectoris↗
PubMed1989

c-ets-2 protooncogene has mitogenic and oncogenic activity.

An expression vector containing the murine c-ets-2 protooncogene cDNA was introduced into NIH 3T3 cells by DNA transfection. The cells transfected with this construct showed foci of densely growing, morphologically altered cells, when grown either in low-serum (0.05%) or in serum-free medium. The c-ets-2-derived foci contained additional copies of the c-ets-2 gene, Northern blot analysis demonstrated overexpression of a c-ets-2-specific 2.5-kilobase RNA, and ets-specific antiserum recognized a 56-kDa protein. Overexpression of the c-ets-2-encoded protein stimulated cell proliferation and abolished their serum requirement. The c-ets-2 transfected cells formed colonies in semisolid medium and induced tumors in nude mice, indicating that c-ets-2 can be a transforming gene when overexpressed in these cells. This work demonstrates that a member of the c-ets gene family has transforming and mitogenic activity. In addition, the role of c-ets-2 in cell proliferation and its location in the minimal Down syndrome region on chromosome 21 implicates its involvement in the phenotypic changes associated with Down syndrome.

Animals↗
PubMed1989

Balloon occlusion during coronary angioplasty as a model of myocardial ischaemia: reproducibility of sequential inflations.

In order to evaluate the potential of balloon occlusion during coronary angioplasty as a model of myocardial ischaemia in man we have measured coronary sinus blood flow (CSBF), myocardial oxygen consumption (MVO2), lactate extraction (LER) and electrocardiographic changes in 11 patients undergoing left anterior descending artery (LAD) angioplasty. Baseline measurements were made before balloon crossing and between inflations. Four consecutive inflations each of 60 s duration were made; 5 min return to baseline was allowed between inflations. There was a significant reduction in CSBF and MVO2 (ml min-1) during inflations 2, 3 and 4 (CSBF: 121 +/- 66----94 +/- 53, 113 +/- 49----99 +/- 42, 124 +/- 66----102 +/- 41, P less than 0.02; MVO2: 11.3 +/- 6.6-9.1 +/- 3.9, 10.4 +/- 3.7-8.7 +/- 2.4, 12.2 +/- 4.4----9.4 +/- 2.8, P less than 0.05). However during the first period of balloon occlusion there were inconsistent changes in coronary flow with an overall rise in mean flow (97 +/- 35----128 +/- 80 ml min-1, P = NS) and an overall rise in mean myocardial oxygen consumption (9.6 +/- 3.8----12.5 +/- 7.5 ml min-1, P = NS). There was lactate production during all four inflations but the changes during the first one did not achieve statistical significance. These inconsistent changes during the first inflation were thought to be due to partial obstruction of the stenosis by the deflated balloon before primary dilatation. The changes due to crossing and during the first two inflations were further investigated in another group of 12 patients undergoing LAD angioplasty. Great cardiac vein flow (GCVF), CSBF, MVO2 and LER were recorded at baseline, during crossing and during the first two inflations. With the deflated balloon across the stenosis there were no changes in CSBF or MVO2 but there was a fall in GCVF (103 +/- 28----77 +/- 50, P = NS) and a significant fall in LER (77 +/- 57----16 +/- 37, P less than 0.01). Although there was a fall during the first inflation in CSBF, GCVF, MVO2 and lactate extraction none of these changes were significant. During the second inflation these changes were of greater magnitude and achieved statistical significance. While balloon occlusion during coronary angioplasty has the potential of providing a model of ischaemia in man we have found the first inflation period unreliable, due to the variable degree of occlusion by the deflated balloon. We suggest that only subsequent inflations after the primary dilatation are used for observations.(ABSTRACT TRUNCATED AT 400 WORDS)

Angioplasty, Balloon, Coronary↗
PubMed1989

Expression of human and viral ets genes in E. coli production of human ets-2-specific monoclonal antibodies.

Full-length and deletion variants of the ets proto-oncogene and v-ets oncogene of E26 virus were expressed in E. coli using the previously described expression vectors pANH1, pJL6 and pJLA16. The recombinant proteins were expressed at greater than 5% of the total cellular proteins and were characterized by Western blot analysis using ets-specific antipeptide antibodies. A number of monoclonal antibodies were raised against p35, the expressed product of the partial human ets-2 proto-oncogene construct. These monoclonal antibodies are highly specific and recognize the p56 ets-2 product from several human cell lines.

Antibodies, Monoclonal↗