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Biomedical subjects

K Chen

Publications and source records attributed to K Chen.

At least 415 records · Page 23Linked to original sources
PubMed1994

Evolutionary relationships and functional conservation among vertebrate Max-associated proteins: the zebra fish homolog of Mxi1.

In mammals, current evidence supports the view that Myc-responsive activities are regulated in part through an intracellular balance between levels of transcriptionally-active Myc/Max heterodimers and those of transcriptionally-inert Max/Max, Mad/Max and Mxi1/Max complexes. To gain insight into the roles of Mad and Mxi1 in cellular growth and differentiation and to fortify key structure-function relationships from an evolutionary standpoint, low stringency hybridization screens were used to identify potential homologs of these Max-associated proteins in the zebra fish genome. A single class of cDNA clones that cross-hybridized both to human mad and mxi1 probes was shown to encode a putative protein with significantly greater homology to mammalian Mxi1 than to Mad, particularly in the basic and helix-loop-helix (bHLH) regions. The high degree of structural relatedness between vertebrate Mxi1 proteins apparent in molecular modelling studies was consistent with the findings that the HLH/leucine zipper (LZ) region of zMxi1 exhibited the same profile of dimerization specificities as its mammalian counterpart in the two-hybrid system and that zmxi1 could, like human mxi1 (Lahoz et al., 1994), suppress the oncogenic potential of mouse c-myc in a mammalian cell. Finally, a comparison of steady-state zc-myc and zmxi1 mRNA levels during zebra fish embryogenesis demonstrated (i) high levels of zc-myc relative to zmxi1 mRNA during initiation of organogenesis, a period characterized by intense growth and active differentiation and (ii) rising levels of zmxi1 mRNA during progression towards the terminally differentiated state. These contrasting patterns of developmental expression together with the capacity of zmxi1 to repress myc-induced transformation support a model for the regulation, by Max-associated proteins, of Myc functions in the control of normal cell development and neoplastic growth.

Amino Acid Sequence↗
PubMed1994

Long-term reconstitution of mice after ex vivo expansion of bone marrow cells: differential activity of cultured bone marrow and enriched stem cell populations.

In this report, we evaluated the short-term expansion of murine bone marrow mononuclear cells (BMMNC) and enriched stem cell populations to determine the capacity of these cells for long-term rescue and engraftment to lethally irradiated recipients. In our study, nonadherent bone marrow mononuclear cell (NBM-MNC) and Thy1+Lin- stem cells populations were cultured with interleukin-3 (IL-3) or IL-3 plus stem cell factor (SCF) for periods up to 6 days. By day 6 of culture, the mononuclear cells (MNC) decreased to 6% of input cell number, whereas Thy1+Lin- cells increased by 2310%. Doses of 95,000; 100,000; 50,000; and 250,000 NBM-MNCs at 0, 1, 2, and 6 days of culture, respectively, rescued 50% of lethally irradiated mice. When 250,000 MNCs were cultured for 0, 1, 2, and 6 days, 71, 61, 100, and 50% of the animals survived lethal irradiation for greater than 24 weeks. In contrast, doses of 8,000 and 21,000 Thy1+Lin- cells cultured 0 and 1 day, respectively, yielded 50% survival rates. These same cells cultured for 6 days failed to rescue recipients even at high doses. Twenty thousand Thy1+Lin- cells cultured for 0, 1, 2, and 6 days, even in the presence of SCF, produced decreasing survival rates of 86, 43, 26, and 0%, respectively. The proliferative responses of these different populations in combination with their long-term rescue abilities indicated that the absolute number of long-term rescue units (LD50, 24 weeks) in the cultured Thy1+Lin- population decreased faster than in similarly cultured NBM-MNCs. Studies evaluating donor cell engraftment demonstrated that animals rescued with cultured Thy1+Lin- and NBM-MNCs maintained high levels of donor reconstitution [7]. The percent donor T cell engraftment did not significantly change between 2 and 17 months post-bone marrow transplantation (post-BMT). Therefore, those animals who received sufficient cells to survive lethal irradiation generally established and maintained high levels of donor engraftment. The data suggest a role for accessory cells and/or factors in the preservation of stem cell activity.

Animals↗
PubMed1994

Quantification of the extent and severity of perfusion defects in canine myocardium by PET polar mapping.

UNLABELLED: This study validates perfusion defect extent and severity as derived by PET polar maps in vivo against measurements derived from radiolabeled microspheres. METHODS: In seven open-chest dogs, either the left anterior descending (n = 11) or left circumflex coronary artery (n = 13) were ligated sequentially from distal to proximal. After each occlusion, gated PET images were acquired with 13N-ammonia (20 mCi) while radiolabeled microspheres were administered into the left atrium. The transaxial PET images were reoriented into left ventricular short-axis cuts, including the apex, and polar maps were generated from circumferential activity profiles. PET polar maps were then compared with polar maps derived from microspheres after normal databases for 13N-ammonia and for microspheres were established. Nitrogen-13 or microsphere activities of less than 1.5 s.d. below the mean were defined as hypoperfused. RESULTS: The extent (percent of left ventricular mass) and mean severity of the hypoperfused myocardium in the postmortem microsphere measurements ranged from 3% to 69% and 3% to 58%, respectively. The estimated extent by summed PET and by microspheres correlated by y = 4.95 + 0.95x (r = 0.91, s.e.e. = 0.085, p < 0.001) and mean severity by y = 5.52 + 0.87x (r = 0.85, s.e.e. = 0.101, p < 0.001). The extent and severity were similar for summed and gated PET studies. CONCLUSION: The current study validated a polar map approach that provides accurate, quantitative assessment of the extent and severity of myocardial perfusion defects in vivo. Gating did not yield an improved correlation between PET and microsphere measurements. Thus, ungated PET images can be used to assess accurately the extent and severity of perfusion defects.

Ammonia↗
PubMed1994

Bidirectional promoter of human monoamine oxidase A (MAO A) controlled by transcription factor Sp1.

The core promoter region of human monoamine oxidase (MAO) A has been identified in the two 90 bp repeat sequences, which can be further divided into four imperfect tandem repeats, each containing an Sp 1 binding site in the reversed orientation. Gel retardation and DNase 1 footprinting assays identified Sp 1 to be the major transcription factor binding to MAO A core promoter. In addition, positive association has been observed between cellular Sp1 concentration and MAO A promoter or catalytic activity, indicating that Sp1 is a controlling factor for human MAO A expression. DNA fragments from MAO A core promoter exhibit promoter activity in both orientations in a transient transfection assay, using human growth hormone as the reporter gene. A DNA probe isolated from upstream of the core promoter detected positive signals in a Northern analysis, suggesting that the reverse promoter activity may endogenously transcribe a new gene located upstream of MAO A.

Base Sequence↗
PubMed1994

Comparison of renal hemodynamic effect of ramiprilat to captopril; possible role of kinins.

The purpose of this study was to compare a lipophilic angiotensin-converting enzyme (ACE) inhibitor, ramiprilat, to the hydrophilic agent, captopril, with respect to its efficacy in decreasing blood pressure (BP) and increasing renal blood flow (RBF). Fifty-seven anesthetized rabbits were instrumented for monitoring BP and RBF. Separate groups of experiments were conducted in which the inhibitors were given i.v. and intrarenal i.a. In Group I, ramiprilat (1 mg/kg and 0.5 mg/kg/hr i.v.) and captopril (2 mg/kg and 1 mg/kg/hr i.v.) decreased BP and increased RBF to the same extent. Both ACE inhibitors abolished the pressor effect and greatly attenuated the renal vasoconstrictor response to exogenous angiotensin I. When the ACE inhibitors were administered in graded doses i.a. (Group II), they caused a similar increase in RBF and decrease in BP as when given i.v. Ramiprilat was about twice as potent as captopril by either route of administration. In the presence of an i.a. infusion of the bradykinin antagonist HOE 140 (Group IV), the effect of ramiprilat and captopril given i.a. on BP and RBF was not different than in Group II. In Group V, the angiotensin II receptor antagonist, losartan, decreased BP and increased RBF. There was little further effect on RBF seen with ramiprilat, but captopril caused some additional renal vasodilatation after losartan. A small further fall in BP was obtained with both agents after losartan. In conclusion, ramiprilat and captopril, despite marked solubility differences, produced a similar renal hemodynamic effect and decrease in BP. No significant difference was seen in the presence of bradykinin antagonism.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Connexin mutations in X-linked Charcot-Marie-Tooth disease.

X-linked Charcot-Marie-Tooth disease (CMTX) is a form of hereditary neuropathy with demyelination. Recently, this disorder was mapped to chromosome Xq13.1. The gene for the gap junction protein connexin32 is located in the same chromosomal segment, which led to its consideration as a candidate gene for CMTX. With the use of Northern (RNA) blot and immunohistochemistry technique, it was found that connexin32 is normally expressed in myelinated peripheral nerve. Direct sequencing of the connexin32 gene showed seven different mutations in affected persons from eight CMTX families. These findings, a demonstration of inherited defects in a gap junction protein, suggest that connexin32 plays an important role in peripheral nerve.

Amino Acid Sequence↗
PubMed1993

Tracking of markers and onset of disease among HIV-1 seroconverters.

Repeated measurements on persons infected with HIV-1 indicate that infection has a dynamic impact on several markers of immune suppression and activation. The objectives of this report are: (a) to provide a statistical model for the correlation structure of serial measurements of immunological markers, and (b) to identify features of marker profiles associated with the timing of AIDS diagnoses. We analyse data obtained from 328 seroconverters participating in the Multicenter AIDS Cohort Study on whom the date of HIV-1 seroconversion is known within +/- 4.5 months. Immunological markers considered here are CD4 cell counts, serum beta 2-microglobulin and serum neopterin. The statistical model for HIV-related changes in markers consists of (1) a piecewise linear regression model for the trajectories of markers over time and (2) a two-parameter autocorrelation function that generalizes Markovian and simple random effects autocorrelation structures. Application of this model for marker measurements revealed a high degree of tracking, as the estimated autocorrelation function exhibited sub-exponential decay over time. Though current marker levels are most informative on future values, there is substantial information (memory) in previous measurements. A feature suggested by the analysis of groups formed according to the length of the AIDS-free period, is the sequential divergence of the CD4 trajectories where steeper declines occurred with a two-year lag prior to AIDS onset. For AIDS cases diagnosed 3-5 and 5-7 years after seroconversion, the rates of decline compared with those free of AIDS for at least 4 years were steeper by 95 and 46 per cent respectively at two years prior to AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Induction by EGF and interferon-gamma of tyrosine phosphorylated DNA binding proteins in mouse liver nuclei.

Intraperitoneal injection of epidermal growth factor (EGF) into mice resulted in the appearance in liver nuclei of three tyrosine phosphorylated proteins (84, 91, and 92 kilodaltons) within minutes after administration of EGF. Administration of interferon-gamma (IFN-gamma) resulted in the appearance in liver nuclei of two tyrosine phosphorylated proteins (84 and 91 kilodaltons). The 84- and 91-kilodalton proteins detected after either EGF or IFN-gamma administration were identified as the IFN-gamma activation factors (GAF). Furthermore, gel shift analysis revealed that these GAF proteins, detected after either EGF or IFN-gamma administration, specifically bound to the sis-inducible element of the c-fos promoter. Thus, GAF proteins participate in nuclear signaling in both IFN-gamma and EGF pathways.

Animals↗
PubMed1993

Molecular cloning and sequence analysis of the monkey and human tissue kallikrein genes.

Cynomolgus monkey renal kallikrein cDNA and genomic human tissue kallikrein gene were cloned. The monkey gene encodes a 257 amino acid (aa) preprokallikrein and exhibits 95% and 92% homology to the human at nucleotide (nt) and aa level, respectively. The monkey gene encodes a 233-aa mature kallikrein versus a 238-aa in human. The human kallikrein gene and urinary kallikrein both contain a Lys-162 instead of the reported Glu-162. Human, monkey and rat renal/pancreatic kallikrein genes evolve with a N-glycosylation containing domain (aa 81-87) which is absent in porcine and is non-glycosylable in mice. Only human kallikrein evolves with an additional Thr-108 and with a N-glycosylation site at aa-141.

Amino Acid Sequence↗
PubMed1993

Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide.

Random mutagenesis has been used to engineer the protease subtilisin E to function in a highly nonnatural environment--high concentrations of a polar organic solvent. Sequential rounds of mutagenesis and screening have yielded a variant (PC3) that hydrolyzes a peptide substrate 256 times more efficiently than wild-type subtilisin in 60% dimethylformamide. PC3 subtilisin E and other variants containing different combinations of amino acid substitutions are effective catalysts for transesterification and peptide synthesis in dimethylformamide and other organic media. Starting with a variant containing four effective amino acid substitutions (D60N, D97G, Q103R, and N218S; where, for example, D60N represents Asp-60-->Asn), six additional mutations (G131D, E156G, N181S, S182G, S188P, and T255A) were generated during three sequential rounds of mutagenesis and screening. The 10 substitutions are clustered on one face of the enzyme, near the active site and substrate binding pocket, and all are located in loops that connect core secondary structure elements and exhibit considerable sequence variability in subtilisins from different sources. These variable surface loops are effective handles for "tuning" the activity of subtilisin. Seven of the 10 amino acid substitutions in PC3 are found in other natural subtilisins. Great variability is exhibited among naturally occurring sequences that code for similar three-dimensional structures--it is possible to make use of this sequence flexibility to engineer enzymes to exhibit features not previously developed (or required) for function in vivo.

Amino Acid Sequence↗
PubMed1993

Epidermal growth factor stimulates the tyrosine phosphorylation of SHC in the mouse.

Previous studies have demonstrated that the administration of epidermal growth factor (EGF) to neonatal mice results in the tyrosine phosphorylation of multiple substrates in all organs examined (Donaldson, R. W., and Cohen, S. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 8477-8481). One of these substrates, a 55-kDa protein, was shown to associate with the receptor for EGF (EGFR). We now report the identification of this receptor-associated protein as SHC. Immunoprecipitation and Western blotting analyses have revealed that SHC associates only with the activated EGFR. In the absence of EGF stimulation, SHC exists in the liver as a cytoplasmic monomer. Intraperitoneal injection of EGF results, within minutes, in the translocation of 50-80% of SHC to the liver plasma membrane. The membrane-associated SHC was found to be tyrosine-phosphorylated; the subsequent release of SHC from the membrane correlated with a tyrosine dephosphorylation. We conclude that SHC is a physiological substrate that appears to participate in the in vivo signaling response to EGF.

Animals↗
PubMed1993

Moderately severe osteogenesis imperfecta associated with substitutions of serine for glycine in the alpha 1(I) chain of type I collagen.

We have examined the type I collagen protein, RNA, and cDNA of 2 children with moderately severe (type IV) osteogenesis imperfecta (OI). They have in common a non-lethal form of OI with ambulatory potential, overmodification of type I collagen protein, and a substitution of serine for glycine in the collagen chain produced by one alpha 1(I) allele. The first child (Marini et al.: J Biol Chem 264:11893-11900, 1989) is now 7 years old, with the height of a 3-year-old. Her course includes significant remodeling of lower long bones and 4 femur fractures. She walks independently. A mishmatch was detected in her alpha 1(I) mRNA using RNA/RNA hybrids; it was demonstrated to be due to a G-->A point mutation in one allele of alpha 1(I), resulting in the substitution of serine for glycine 832. The second child is now 6 1/2 years old, with the height of 1 1/2-year-old. Her history includes significant bowing of femurs and tibias, 6 femur fractures, S-curve scoliosis, compression of all lumbar vertebrae, and limited short-distance walking with braces. Her alpha 1(I) mRNA has also been studied by RNA hybrid analysis; there is a single G-->A change in one alpha 1(I) allele causing the substitution of serine for gly 352. Both children have moderately severe OI. However, the serine substitution at gly 352 is associated with a more severe phenotype then is the serine substitution at gly 832. Compared to substitutions described in other cases of OI, the serine 352 is located in the middle of a cluster of cysteine substitutions associated with non-lethal OI.(ABSTRACT TRUNCATED AT 250 WORDS)

Child↗
PubMed1993

Inhibition of human platelet glycoprotein IIB/IIIA binding to fibrinogen by tumor cell membrane proteins.

Immunochemical and functional characteristics of tumor cell membrane proteins and human platelet glycoproteins were studied. Immunoblotting revealed that membrane proteins of a cultured breast tumor cell line (BT-20) had three protein bands, which were each recognized by monoclonal antibodies to human platelet glycoprotein Ib, IIb, and IIIa, suggesting some immunochemical similarities between the tumor cell membrane proteins and platelet glycoproteins. The monoclonal antibodies failed to bind to an extract of a lung tumor cell line (A549). Neither tumor extract induced platelet aggregation. However, tumor-associated antigens isolated from the breast tumor cells markedly inhibited platelet glycoprotein IIb/IIIa binding to fibrinogen. In contrast, tumor-associated antigens from the lung tumor cells had no effect. These results suggest that tumor cells which have immunological and/or structural similarities to platelets may affect hemostasis and coagulation in vivo.

Binding, Competitive↗