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Biomedical subjects

J L Miller

Publications and source records attributed to J L Miller.

At least 109 records · Page 6Linked to original sources
PubMed1999

Combination photoimmunotherapy and cisplatin: effects on human ovarian cancer ex vivo.

BACKGROUND: Patients with ovarian cancer that is clinically resistant to cisplatin-based chemotherapy have little hope of a cure of their disease. Photoimmunotherapy, which involves the antibody-targeted delivery of a nontoxic photosensitizer that is activated to a cytotoxic state with visible light, may offer a new treatment option. Photoimmunotherapy may be applied intraperitoneally to target disseminated tumor. We tested the hypothesis that this treatment in combination with cisplatin potentiates cytotoxicity in ovarian cancer cell lines and primary cultures of human tumors. METHODS: Five human cancer cell lines (ovarian and breast) and 19 primary cultures were studied. The primary cultures were from solid and ascites tumor samples obtained from 14 patients with ovarian cancer who were undergoing primary surgery. The photosensitizer chlorin e(6) was conjugated to the F(ab')(2) fragment of the murine monoclonal antibody OC-125, which is directed against the antigen CA 125. Cytotoxicity was measured by the microculture tetrazolium assay. Treatments consisted of cisplatin alone, photoimmunotherapy alone, and photoimmunotherapy followed by cisplatin. The fractional product method was used to assess synergy in treatment effects. Ex vivo cultured human cells exhibiting 80% or greater survival at cisplatin concentrations of 10 microM for 24 hours were defined as cisplatin resistant for this study. RESULTS: When all cell types (cisplatin sensitive and cisplatin resistant) were considered together, combination treatment yielded cytotoxicity that was, on average, 6.9 times (95% confidence interval = 1.86-11.94) greater than that of cisplatin alone (two-sided P =.023). Cisplatin-resistant cells showed a synergistic effect of the two treatments (two-sided P =.044), while cisplatin-sensitive cells showed an additive effect. CONCLUSION: These ex vivo data suggest that platinum resistance in human ovarian cancer cells may be reversible by pretreatment with OC-125-targeted photoimmunotherapy. Further studies are required to confirm the efficacy of this approach in vivo.

Antibodies, Monoclonal↗
PubMed1999

Segmental genomic replacement in embryonic stem cells by double lox targeting.

We have applied Cre-mediated double lox recombination to embryonic stem (ES) cells to facilitate repeated knock-ins at a target locus, thus helping to assure correct temporal and spatial transgene expression in mice. Using homologous recombination, we inserted a double lox cassette a few nucleo-tides before the authentic ATG start of MHL-1, the gene coding for the major subunit of the asialoglycoprotein receptor. The cassette carries a marker gene bounded by heterospecific lox sites that cannot recombine with each other, but which can undergo recombination with like sites on an incoming double lox targeting vector. Cre-mediated replacement of the lox-delimited genomic segment at MHL-1 with targeting DNA occurs at a frequency three times that of random DNA integration and is sufficiently robust that correctly targeted ES colonies can be identified by PCR screening without relying on any drug selection procedure. Moreover, double lox recombination at the MHL-1 locus is not dependent on the presence or absence of a transcriptionally active promoter at the genomic target. The strategy and vectors described here are generally applicable to designing double lox targeted knock-ins at any locus in ES cells and should prove useful in more precise molecular engineering of the mouse genome.

3T3 Cells↗
PubMed1999

Processing of the N termini of nascent polypeptide chains requires deformylation prior to methionine removal.

N-formyl-methionine termini are formed in the initiation reaction of bacterial protein synthesis and processed during elongation of the nascent polypeptide chain. We report that the formyl group must be removed before the methionine residue can be cleaved by methionine aminopeptidase. This has long been implicitly assumed, but that assumption was based on inconclusive data and was in apparent conflict with more recently published data. We demonstrate that the Salmonella typhimurium methionine aminopeptidase is totally inactive on an N-formyl-methionyl peptide in vitro, and present a detailed characterization of the substrate specificity of this key enzyme by use of a very sensitive and quantitative assay. Finally, a reporter protein expressed in a strain lacking peptide deformylase was shown to retain the formyl group confirming the physiological role of the deformylase.

Amino Acid Sequence↗
PubMed1999

Gene expression in proliferating human erythroid cells.

A complete understanding of human erythropoiesis will require a robust description of transcriptional activity in hematopoietic cells that proliferate and differentiate in response to erythropoietin (EPO). For this purpose, we cultured peripheral blood mononuclear cells in the presence or in the absence of EPO and examined the transcriptional profile of those cells arising only in response to EPO. A distinct population of CD71( +) cells that demonstrated an average of six additional doublings in suspension culture and erythroid colony formation in methylcellulose was isolated. Suppression subtractive hybridization of mRNA isolated from those cells permitted the identification of transcribed genes. A summary of 719 expressed sequence tags (ESTs) describing 505 independent transcripts is provided here with a full analysis of each EST available at http://hembase.niddk.nih.gov. Several transcripts that matched genes previously reported in the context of erythroid differentiation including 4 cell surface proteins were expressed at this developmental stage. Active chromatin remodeling was suggested by the identification of 4 histone proteins, 4 high-mobility group proteins, 13 transcription factors, and 6 genes involved in DNA recombination and repair. Numerous genes associated with leukemic translocations were also recognized including topoisomerases I and II, nucleophosmin, Translin, EGR1, dek, pim-1, TFG, and MLL. In addition to known transcripts, 44 novel EST were discovered. This transcriptional profile provides the first genomic-scale description of gene activity in erythroid progenitor cells.

Antigens, CD↗