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Biomedical subjects

J Rao

Publications and source records attributed to J Rao.

At least 19 recordsLinked to original sources
PubMed2001

The chaperone protein 14-3-3eta interacts with the nicotinic acetylcholine receptor alpha 4 subunit. Evidence for a dynamic role in subunit stabilization.

By using the large cytoplasmic domain of the nicotinic acetylcholine receptor (AChR) alpha4 subunit as a bait in the yeast two-hybrid system, we isolated the first cytosolic protein, 14-3-3eta, known to interact directly with neuronal AChRs. 14-3-3eta is a member of a family of proteins that function as regulatory or chaperone/ scaffolding/adaptor proteins. 14-3-3eta interacted with the recombinant alpha4 subunit alone in tsA 201 cells following activation of cAMP-dependent protein kinase by forskolin. The interaction of 14-3-3eta with recombinant alpha4 subunits was abolished when serine 441 of the alpha4 subunit was mutated to alanine (alpha4(S441A)). The surface levels of recombinant wild-type alpha4beta2 AChRs were approximately 2-fold higher than those of mutant alpha4(S441A)beta2 AChRs. The interaction significantly increased the steady state levels of the alpha4 subunit and alpha4beta2 AChRs but not that of the mutant alpha4(S441A) subunit or mutant alpha4(S441A)beta2 AChRs. The EC50 values for activation by acetylcholine were not significantly different for alpha4beta2 AChRs and alpha4(S441A)beta2 AChRs coexpressed with 14-3-3eta in oocytes following treatment with forskolin. 14-3-3 coimmunopurified with native alpha4 AChRs from brain. These results support a role for 14-3-3 in dynamically regulating the expression levels of alpha4beta2 AChRs through its interaction with the alpha4 subunit.

14-3-3 Proteins↗
PubMed2001

Injury to the ligamentum teres. Mechanism, findings, and results of treatment.

In this article we discuss the role of the ligamentum teres in relation to the hip joint. The anatomy of the ligamentum teres is used to help with an understanding of the mechanism of the ligament's actions. This action and the modes of injury to the ligament are explored. We describe a simple classification system for injuries to the ligamentum teres, such that Group I are complete tears to the ligament; Group II are partial tears; and Group III are degenerative tears. As more experience is gained with the diagnosis and treatment of injuries to this integral structure, the clearer will be our understanding of the role of the ligamentum teres in the hip joint.

Arthroscopy↗
PubMed2001

Outer dense fiber proteins are dominant postobstruction autoantigens in adult Lewis rats.

Obstruction of the male reproductive tract commonly results in generation of antisperm autoantibodies. However, only a few of the sperm autoantigens recognized by these antibodies have been characterized. To identify postobstruction rat sperm autoantigens, sperm proteins were separated by two-dimensional(2-D) gel electrophoresis. Spots corresponding to proteins that were stained by at least 50% of postvasectomy rat sera on 2-D Western blots were removed from polyacrylamide gels and microsequenced by tandem mass spectrometry. From a total of 21 spots, 12 contained peptides that matched solely to either of two outer dense fiber proteins, odf1 or odf2. Six additional spots contained peptides comprising odf1 or odf2 and were accompanied by peptides representing other proteins. Only three spots lacked outer dense fiber peptides but did contain sequences of other known proteins. The results indicate that the outer dense fiber proteins odf1 and odf2 are dominant postobstruction autoantigens because they were detected in the majority of the immunoreactive protein spots examined. Possible explanations for this observation include the abundance of outer dense fiber proteins in spermatozoa, slow solubility, which may provide a sustained supply of antigen, and testis-specific expression during spermiogenesis.

Amino Acid Sequence↗
PubMed2001

Successful use of eptifibatide as an adjunct to coronary stenting in a patient with abciximab-associated acute profound thrombocytopenia.

A 72-year-old male who was given abciximab for unstable angina developed acute profound thrombocytopenia with a platelet count nadir of 6,000/mm3. He was treated with steroids and platelet transfusion. Four days later, he underwent coronary angioplasty after pretreatment with eptifibatide without development of thrombocytopenia. This suggests that the development of thrombocytopenia with abciximab is not necessarily a contraindication to subsequent use of glycoprotein (GP) IIb/IIIa receptor antagonists. Eptifibatide may be an appropriate consideration in high-risk patients who would benefit from a GP IIb/IIIa receptor antagonist, in spite of acute profound thrombocytopenia due to abciximab therapy.

Abciximab↗
PubMed2001

Effect of whole-body (18)F-FDG PET imaging on clinical staging and management of patients with malignant lymphoma.

UNLABELLED: Correct staging is important in selecting the appropriate treatment for lymphoma patients. PET imaging with (18)F-FDG is useful for staging of lymphoma as well as for monitoring of therapy. However, to our knowledge, the clinical impact of PET on staging and management of lymphoma patients has not been reported. METHODS: Standardized questionnaires were mailed to referring physicians asking them whether and how the results of PET imaging had influenced clinical staging and management of the disease in their patients. Management changes, when present, were classified as intermodality (e.g., medical to surgical, surgical to radiation, medical to no treatment) or intramodality (e.g., altered medical, surgical, or radiotherapy approach). RESULTS: The referring physicians returned 52 of 108 questionnaires (48.1%). Physicians indicated that PET led to a change in the clinical stage in 44% of patients: 21% were upstaged and 23% were downstaged. Findings of the PET examination resulted in intermodality changes in management in 42% of patients, in intramodality changes in 10%, and in a combination of the management changes in 10%. Other, not further specified, treatment changes were reported in 6% of patients. PET did not result in any management changes in only 32% of patients. CONCLUSION: This survey-based study of referring physicians indicates that FDG PET has a major impact on the management of lymphoma patients, contributing to changes in clinical stage in 44% and changes in treatment in >60% of cases.

Adolescent↗
PubMed2001

Impact of whole-body 18F-FDG PET on staging and managing patients with breast cancer: the referring physician's perspective.

UNLABELLED: FDG PET has emerged as an important clinical imaging modality for diagnosing and staging cancer. However, the impact of FDG PET on staging and managing patients with breast cancer from the referring physician's point of view is unknown. METHODS: The referring physicians of 160 breast cancer patients received standardized questionnaires inquiring if and how PET findings altered their patient's stage and their clinical management decisions. Management changes were classified as intermodality if the change was from one modality to another (e.g., medical to surgical, surgical to radiation, medical to no treatment, and vice versa) or as intramodality if the change was within the same modality (e.g., altered medical or radiotherapy approach). RESULTS: Fifty of the 160 surveys were completed (31% response rate). PET changed the clinical stage in 36% of patients (28% upstaged, 8% downstaged) and resulted in intermodality changes in 28% of patients and intramodality changes in 30% of patients. CONCLUSION: The results of this prospective survey show that FDG PET has a major impact on the management of breast cancer patients, influencing both clinical stage and management in more than 30% of patients.

Adult↗
PubMed2000

Functional interaction of human Cdc37 with the androgen receptor but not with the glucocorticoid receptor.

Cdc37 is a molecular chaperone closely associated with the folding of protein kinases. Results from studies using a yeast model system showed that it was also important for activation of the human androgen receptor (AR). Based on results from the yeast model system (Fliss, A. E., Fang, Y., Boschelli, F., and Caplan, A. J. (1997) Mol. Biol. Cell 8, 2501-2509), we initiated studies to address whether AR and Cdc37 interact with each other in animal cell systems. Our results show that Cdc37 binds to AR but not to glucocorticoid receptors (GR) synthesized in rabbit reticulocyte lysates. This binding occurs via the ligand-binding domain of the AR in a manner that is partially dependent on Hsp90 and the presence of hormone. Further studies using the yeast system showed that Cdc37 is not interchangeable with Hsp90, suggesting that it functions at a distinct step in the activation pathway. Expression of a dominant negative form of Cdc37 in animal cells down-regulates full-length AR but has very little effect on an AR truncation lacking the ligand-binding domain or full-length GR. These results reveal differences in the mechanisms by which AR and GR become active transcription factors and strengthen the notion that Cdc37 has a wider range of polypeptide clients than was realized previously.

Binding Sites↗
PubMed2000

Neural cell response to multiple novel sites on laminin-1.

The basement membrane protein laminin-1 is a potent stimulator of neurite outgrowth for a variety of neuronal cell types. Previous studies have identified neurite outgrowth activity in several distinct regions of the laminin-1 molecule. In this study, 545 overlapping 12- to 14-mer synthetic peptides, corresponding to most of the amino acid sequence of the alpha1, beta1, and gamma1 chains of laminin-1, were screened for cell attachment and neurite outgrowth activity using primary cultures of mouse cerebellar granule neurons and two neuronal cell lines. We identified 48 peptides derived from novel regions of the laminin-1 molecule that were positive for neural cell adhesion activity. Only the cerebellar cells were found to have true neurite outgrowth activity with certain of the peptides, whereas some peptides induced short spike-like process with the cell lines. Although 23 of these peptides were active on all 3 cell types screened, 25 others showed cell-type specificity in their activity. These studies show that (1) there are multiple and distinct sites on laminin-1 for cell adhesion and neurite-like outgrowth and (2) that there are neural cell-type-specific active domains. The multiple active sites found explains, in part, the potent activity of laminin-1 on neurite outgrowth.

Amino Acid Sequence↗
PubMed2000

Inhibition of p38 MAP kinase activity up-regulates multiple MAP kinase pathways and potentiates 1,25-dihydroxyvitamin D(3)-induced differentiation of human leukemia HL60 cells.

Differentiation therapy for neoplastic diseases has potential for supplementing existing treatment modalities but its implementation has been slow. One of the reasons is the lack of full understanding of the complexities of cellular pathways through which signals for differentiation lead to cell maturation. This was addressed in this study using HL60 cells, a well-established model of differentiation of neoplastic cells. SB 203580 and SB 202190, specific inhibitors of a signaling protein p38 MAP kinase, were found to markedly accelerate monocytic differentiation of HL60 cells induced by low concentrations of 1,25-dihydroxyvitamin D(3) (1,25D(3)). Surprisingly, inhibition of p38 activity resulted in sustained enhancement of p38 phosphorylation and of its in vitro activity in the absence of the inhibitor, indicating up-regulation of the upstream components of the p38 pathway. In addition, SB 203580 or SB 202190 treatment of HL60 cells resulted in a prolonged activation of the JNK and, to a lesser extent, the ERK pathways. The data are consistent with the hypothesis that in HL60 cells an interruption of a negative feedback loop from a p38 target activates a common regulator of multiple MAPK pathways. The possibility also exists that JNK and/or ERK pathways amplify a differentiation signal provided by 1,25D(3).

Calcitriol↗
PubMed2000

Specific association of increased cyclin-dependent kinase 5 expression with monocytic lineage of differentiation of human leukemia HL60 cells.

Hematopoietic cell differentiation takes place in phenotypically recognizable stages characterized by morphology as well as by the expression of enzymes and surface markers. It is recognized that differentiation results from an interaction of environmental cues, such as cytokines and hormones, with internal cellular programs, but the precise mechanisms are not entirely clear. HL60 cells, a human acute myeloid leukemia (AML) cell line with promyelocytic features, provide a model for such studies because they behave like stem cells, which can differentiate into two different lineages, granulocytic or monocytic/macrophage, depending on the inducer. Protein levels and kinase activity of cyclin-dependent kinase 5 (Cdk5) were reported [F. Chen and G. P. Studzinski (1999) Exp. Cell Res. 249, 422, 1999] to increase in HL60 cells induced to monocytic differentiation by 1alpha,25-dihydroxyvitamin D3 (1,25D3), but the specificity of the association of Cdk5 with the monocytic phenotype has not been established. We show here that up-regulation of Cdk5 does not occur in granulocytic differentiation, whereas inhibition of Cdk5 activity by olomoucine, or its expression by a plasmid construct expressing antisense Cdk5, switches the 1,25D3-induced monocytic phenotype (a combination of positive nonspecific esterase reaction, expression of the CD14 marker, and morphology) to general myeloid phenotype (positive nitro-blue tetrazolium reaction, CD11b marker and morphology). The transcriptional up-regulation of Cdk5 by 1,25D3 was not inhibited by olomoucine. These findings show that in human myeloid cells up-regulation of Cdk5 is specifically associated with the monocytic phenotype.

Biomarkers↗
PubMed2000

Dendritic cells armed with anti-CD3 mAbs reduce pulmonary metastases, prolong survival, and engender antitumor effector cells demonstrable by adoptive transfer.

BACKGROUND: Dendritic cells (DCs) pulsed with tumor cells or peptides are effective antitumor agents in a number of tumor models. In light of our earlier demonstration that T-cell signaling via the CD3 proteins induces cytolytic activity and constrains tumor progression, we equipped DCs pulsed with tumor cells with anti-CD3 mAbs and tested their antitumor efficacy in a murine renal cell cancer pulmonary metastasis model. METHODS: We investigated the antitumor efficacy of DCs pulsed with whole irradiated tumor cells (DC/R) or DCs pulsed with irradiated tumor cells and armed with anti-CD3 mAbs (DC/R/anti-CD3 mAbs). Experimental end points included the number of pulmonary metastases and survival of tumor-inoculated mice. RESULTS: Our studies demonstrate that arming tumor-pulsed DCs with anti-CD3 mAbs results in a superior outcome compared to that from tumor-pulsed DCs alone in terms of reduction in the number of pulmonary metastases and survival times. Furthermore, adoptive transfer experiments revealed that the splenocytes from DC/R/anti-CD3 mAbs-treated mice are superior to splenocytes from DC/R-treated mice in reducing renal cancer pulmonary metastases in severe combined immunodeficient (SCID) beige mice. CONCLUSION: Our data suggest that the therapeutic efficacy of DCs pulsed with tumor cells can be augmented by arming them with anti-CD3 mAbs. DC-based treatment regimens that currently are being pursued in clinical trials might be improved by equipping such cells with anti-CD3 mAbs.

Adoptive Transfer↗
PubMed2000

Control of estrogen receptor ligand binding by Hsp90.

The molecular chaperone Hsp90 interacts with unliganded steroid hormone receptors and regulates their activity. We have analyzed the function of yeast and mammalian Hsp90 in regulating the ability of the human estrogen receptor (ER) to bind ligands in vivo and in vitro. Using the yeast system, we show that the ER expressed in several different hsp82 mutant strains binds reduced amounts of the synthetic estrogen diethylstilbestrol compared to the wild type. This defect in hormone binding occurs without any significant change in the steady state levels of ER protein. To analyze the role of mammalian Hsp90, we synthesized the human ER in rabbit reticulocyte lysates containing geldanamycin, an Hsp90 inhibitor. At low concentrations of geldanamycin we observed reduced levels of hormone binding by the ER. At higher concentrations, we found reduced synthesis of the receptor. These data indicate that Hsp90 functions to maintain the ER in a high affinity hormone-binding conformation.

Animals↗
PubMed2000

Content analysis of critical incidents to redesign a university walk-in clinic.

The UCLA walk-in clinic was scheduled to move into a new building in late 1997. This prompted the reevaluation of its existing service delivery system. A study was conducted among patients to understand their expectations and perceptions of the clinic's performance. The study was successful in identifying trends and problems and it was instrumental in developing proposals for a new patient flow system to be implemented in the new facility.

Ambulatory Care↗
PubMed2000

Strain-dependent variability in immune response to chicken riboflavin carrier protein in mice with different haplotypes.

Active immunization of fertile female mice, rats and sub-human primates with linearized chicken riboflavin carrier protein (RCP) curtailed pregnancy suggesting that sequence-specific RCP antibodies interfere with fertilization/early embryo development. To investigate the genetic basis of variations in immunogenecity, antibody response to reduced and carboxymethylated RCP (RCM-RCP) was studied in different strains of mice of independent H-2 haplotypes. Among these, AKR (H-2k) were low or non-responders. Measurement of antibody titers in hyperimmune sera showed that among responder strains of mice, C57BL/6 (H-2b) and BALB/c (H-2d) generated higher levels of antibody compared to mice of SJLJ (H-2S). The relative affinities of these antibodies also varied depending upon the strain, with BALB/c mice showing highest affinity. Epitope mapping by pepscan ELISA revealed significant variability in determinant-specific antibody populations, with SJLJ strain lacking antibodies to N-terminal half of RCP sequence. However, four immunodominant sequential epitopes (residues 100-107, 134-141, 174-181 and 200-207) common to all the three strains of mice have been identified. Binding to these regions was not haplotype restricted although there were qualitative differences in recognition patterns. Present investigations have shown that site-specific antibodies directed towards any one of the four epitopic regions comprising of residues 3-24, 64-83, 130-147 and 200-219 in chicken RCP sequence effectively interfered with pregnancy establishment in female BALB/c mice. This implies the propensity of RCP antibodies to curtail pregnancy in the other two responder mouse strains also.

Amino Acid Sequence↗
PubMed1999

Domain requirements of DnaJ-like (Hsp40) molecular chaperones in the activation of a steroid hormone receptor.

DnaJ-like proteins function in association with Hsp70 molecular chaperones to facilitate protein folding. We previously demonstrated that a yeast DnaJ-like protein, Ydj1p, was important for activation of heterologously expressed steroid hormone receptors (Caplan, A. J., Langley, E., Wilson, E. M., and Vidal, J. (1995) J. Biol. Chem. 270, 5251-5257). In the present study, we analyzed Ydj1p function by assaying hormone binding to the human androgen receptor (AR) heterologously expressed in yeast. We analyzed hormone binding in strains that were wild type or deleted for the YDJ1 gene. In the deletion mutant, the AR did not bind hormone to the same extent as the wild type. Introduction of mutant forms of Ydj1p to the deletion strain revealed that the J-domain is necessary but not sufficient for Ydj1p action, and that other domains of the protein are also functionally important. Of three human DnaJ-like proteins introduced into the deletion mutant, only Hdj2, which displays full domain conservation with Ydj1p, suppressed the hormone binding defect of the deletion mutant. By comparison of the domains shared by these three human proteins, and with mutants of Ydj1p that were functional, it was deduced that the cysteine-rich zinc binding domain is important for Hdj2/Ydj1p action in hormone receptor function. A model for the mechanism of DnaJ-like protein action is discussed.

Binding Sites↗
PubMed1999

Binding of a dimeric derivative of vancomycin to L-Lys-D-Ala-D-lactate in solution and at a surface.

BACKGROUND: The emergence of bacteria that are resistant to vancomycin (V), a glycopeptide antibiotic, results from the replacement of the carboxy-terminal D-Ala-D-Ala of bacterial cell wall precursors by D-Ala-D-lactate. Recently, it has been demonstrated that covalent dimeric variants of V are active against vancomycin-resistant enterococci (VRE). To study the contribution of divalency to the activities of these variants, we modeled the interactions of V and a dimeric V with L-Lys-D-Ala-D-lactate, an analog of the cell-wall precursors of the vancomycin-resistant bacteria. RESULTS: A dimeric derivative of V (V-Rd-V) was found to be much more effective than V in inhibiting the growth of VRE. The interactions of V and V-Rd-V with a monomeric lactate ligand - diacetyl-L-Lys-D-Ala-D-lactate (Ac2KDADLac) - and a dimeric derivative of L-Lys-D-Ala-D-lactate (Lac-R'd-Lac) in solution have been examined using isothermal titration calorimetry and UV spectroscopy titrations; the results reveal that V-Rd-V binds Lac-R'd-Lac approximately 40 times more tightly than V binds Ac2KDADLac. Binding of V and of V-Rd-V to Nalpha-Ac-L-Lys-D-Ala-D-lactate presented on the surface of mixed self-assembled monolayers (SAMs) of alkanethiolates on gold indicates that the apparent off-rate for dissociation of V-Rd-V from the surface is much slower than that of V from the same surface. CONCLUSIONS: The results are compatible with the hypothesis that divalency is responsible for tight binding, which correlates with small values of minimum inhibitory concentrations of V and V-Rd-V.

Anti-Bacterial Agents↗