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Biomedical subjects

A M Lew

Publications and source records attributed to A M Lew.

At least 73 records · Page 4Linked to original sources
PubMed1993

Cell type-specific expression of the pituitary transcription activator pit-1 in the human pituitary and pituitary adenomas.

Pit-1 is a transcription factor that has been shown to be critical for pituitary-specific activation of the GH and PRL genes. In rodents and humans, differentiation and/or maintenance of somatotroph, lactotroph, and thyrotroph phenotypes are dependent on expression of a functional pit-1 gene. In rodents, Pit-1 protein is detectable in only these three cell types; however, pit-1 mRNA transcripts appear to be present at comparable levels in all adenohypophysial cell types, suggesting that translational controls may dictate the pattern of Pit-1 expression. We examined the distribution of pit-1 transcripts in the human pituitary and pituitary adenomas. All tumors were characterized by immunocytochemistry, electron microscopy, and tissue culture for accurate classification. Northern blot analysis demonstrated abundant levels of pit-1 mRNA in somatotroph, mammosomatotroph, and lactotroph adenomas. Two clinically silent adenomas that expressed TSH as well as gonadotropins contained detectable levels of pit-1 mRNA. No pit-1 expression was otherwise detected in corticotroph, gonadotroph, null cell, or oncocytic adenomas. In situ hybridization localized pit-1 mRNA transcripts in adenomas that contained GH, PRL, or TSH, but not in adenomas composed of other cell types. Pit-1 mRNA was also localized to selected subpopulations of the human nontumorous adenohypophysis that contained immunoreactivity for GH, PRL, and/or TSH. Pit-1 protein immunoreactivity was detected in the nuclei of adenomas that expressed pit-1 mRNA, but not in those that were negative for pit-1 mRNA; it was also localized only in cells containing GH, PRL, or TSH beta in the nontumorous adenohypophysis. These data demonstrate selective expression of the human pit-1 gene in adenohypophysial cell types responsible for GH, PRL, and/or TSH synthesis and are consistent with a predominantly pretranslational regulatory mechanism for Pit-1 expression in the human.

Adenoma↗
PubMed1993

FoLT PCR: a simple PCR protocol for amplifying DNA directly from whole blood.

FoLT (formamide low temperature) PCR is a protocol for amplifying DNA directly from whole blood without any preparative steps. Up to 10% (vol/vol) whole blood can be added directly into the tube containing the PCR mixture. There is no need for transfers, centrifugations, pre-boiling or any preparative step. It involves the use of formamide (18% vol/vol) as well as reduced incubation temperatures (cycles of 85 degrees, 40 degrees, 60 degrees C). The type of anticoagulant used was critical: sodium heparin or EDTA being superior to lithium or fluoride heparin. Our studies indicate that FoLT PCR probably works by reducing the amount of protein coagulation and allowing more DNA template to be accessible for amplification. The sensitivity of FoLT PCR is such that a single copy gene from 5.5 nucleated cells in 1 microliter of whole blood can be detected.

Anticoagulants↗
PubMed1992

Chimaeric protein A/protein G and protein G/alkaline phosphatase as reporter molecules.

The IgG binding domains of staphylococcal protein A and streptococcal protein G were expressed as a chimaera using the pGEX vector which has been advocated because its fusion proteins tend to be soluble and easily isolated on immobilised glutathione. This chimaera was soluble and abundant (yield = 18 mg/l of bacterial culture) and was tested by double diffusion in agarose and by ELISA. It was found to bind IgG of all species that either parent could bind. It was superior to protein A or protein G in binding mouse Ig. A chimaera of protein G and alkaline phosphatase was also constructed and found to be soluble and abundant (yield = 20 mg/l of bacterial culture). This protein could be used as a secondary reagent in ELISA at 5 micrograms/ml for human, rabbit and mouse and at 25 micrograms/ml for sheep.

Alkaline Phosphatase↗
PubMed1992

A soluble recombinant fusion protein of the transmembrane envelope protein of equine infectious anaemia virus for ELISA.

The use of the bacterial expression vector, pGex, to produce an abundant, soluble fusion protein of gp45 from equine infectious anaemia virus is described. Purification of the recombinant protein was achieved by one step affinity chromatography on immobilized glutathione using competitive elution so no harsh conditions were required. This provides a readily available antigen that is defined, plentiful and cheap. Yields of 3.5 mg of purified soluble protein/litre of bacterial culture were obtained. This antigen was found to be suitable for ELISA. Background reactivity to either the glutathione-S-transferase (GST) fusion partner by immune sera or the EIA-GST fusion protein by normal sera were negligible.

Amino Acid Sequence↗
PubMed1991

Inhibitory control of prolactin and Pit-1 gene promoters by dopamine. Dual signaling pathways required for D2 receptor-regulated expression of the prolactin gene.

Transcription of the prolactin gene is suppressed by dopaminergic activation of D2 receptors in pituitary lactotrophs. The mechanism of signal transduction at the nuclear level and the cell surface was examined in the dopamine-responsive GH4ZR7 cell line. Dopamine treatment caused a 40-50% decrease in endogenous prolactin mRNA that was specifically blocked by addition of (-)-sulpiride. To define dopamine-responsive elements, plasmids containing 5'-regulatory regions of the prolactin gene fused to the coding sequences for luciferase were transiently or stably transfected into GH4ZR7 cells. Chimeric transcripts initiated at the authentic transcription start site were regulated in a promoter-selective manner; dopamine or the agonist bromocryptine inhibited prolactin promoter (position -422) activity by 70%, but had no regulatory effects on other cellular or viral promoters. A shorter prolactin promoter (position -78) or a prolactin TATAA box linked to heterologous binding sites for transcription factor Pit-1 was sufficient to confer dopamine inhibition (40%). In addition to the prolactin promoter, we found that dopamine inhibited transcriptional activity of the Pit-1 promoter (positions -258 to +8) by 60%. Surprisingly, deletion of two cAMP response elements in the Pit-1 promoter only partially eliminated dopamine responsiveness. These data suggest that sequences in the Pit-1 promoter between positions -92 and +8, which include an autoregulatory Pit-1-binding site and the TATAA box, are sufficient for negative regulation. In this study, we also examined the signal transduction pathways that link D2 receptor activation and the inhibition of prolactin gene transcription. We found, as suggested in earlier studies, that a dopamine-dependent decrease in cAMP may be important for mediating negative regulation of transcription. However, high extracellular K+ concentrations that prevent dopamine effects on membrane potential and [Ca2+]i, but not cAMP levels, completely blocked dopamine regulation of the prolactin promoter. This suggests that two distinct signaling pathways initiated at D2 receptors may be required for transcriptional regulation of the prolactin gene.

Bromocriptine↗
PubMed1991

Recombinant fusion proteins of protein A and protein G with glutathione S-transferase as reporter molecules.

The regions encoding the IgG-binding domains of protein A (PA) and protein G (PG) were cloned into the bacterial expression vector pGEX. Both proteins were expressed in Escherichia coli as fusion proteins with glutathione S-transferase (PA-GST and PG-GST) and were found to be soluble, abundant and easily purified in one step from the bacterial lysate by affinity chromatography on immobilized glutathione. Yields of 50 mg/litre of cultures were obtained. Both purified fusion proteins were shown to be functional in a variety of immunochemical procedures. In radial diffusion tests, PA-GST precipitated IgG from human, squirrel monkey, rabbit, dog, cat and pig but not mouse, sheep, goat, cow, horse or chicken. PG-GST formed precipitin bands with IgG from human, rabbit, mouse, pig, sheep, goat, cow and horse but not squirrel monkey, dog, cat and chicken IgG. The fusion proteins were shown to function as effective detection reagents in ELISA and Western blotting. Glutathione agarose beads with bound fusion protein were shown to be useful for immunoprecipitation.

Base Sequence↗
PubMed1991

The polymerase chain reaction and related techniques.

The polymerase chain reaction, and other methods for rapidly amplifying DNA or RNA are versatile tools that can be used in diverse applications that include HLA typing, analyzing the T-cell repertoire, constructing chimaeric antibodies and quantifying cytokine expression. The sensitivity of the polymerase chain reaction has allowed many cellular events to be investigated in vitro and in vivo. Many currently emerging adaptions of the basic technique are of interest to both the research and the clinical immunologist.

Animals↗
PubMed1990

Chromosomal rearrangements and point mutations in the DHFR-TS gene of Plasmodium chabaudi under antifolate selection.

Selection of the rodent malaria Plasmodium chabaudi with low levels of the antifolate drug pyrimethamine has previously been shown by us to result in duplication of the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene by a duplication of chromosome 7 and subsequent rearrangements. We have selected this resultant parasite line with large doses of pyrimethamine and analysed the DHFR-TS gene and chromosomes for any changes. Increased drug pressure has resulted in reappearance of a chromosome with the same structure as chromosome 7 from DS the parent line. Sequencing of the DHFR gene from each of the chromosomes has identified a single point mutation that results in a serine to asparagine change at position 106. This is the equivalent mutation that has been identified as the key residue in the mechanism of resistance to pyrimethamine in Plasmodium falciparum. There is no apparent increase in transcription of the DHFR-TS gene and the large increase in resistance is most likely a result of the mutation in the DHFR gene.

Animals↗
PubMed1989

Class II restriction in mice to the malaria candidate vaccine ring infected erythrocyte surface antigen (RESA) as synthetic peptides or as expressed in recombinant vaccinia.

The immune response to three peptides corresponding to the repeat regions of the malaria candidate vaccine ring infected E surface Ag (RESA) were studied. Both antibody responses and lymphocyte stimulation in mice injected with these peptides without carrier were found to be restricted to certain MHC class II haplotypes. Mice bearing IAk were strong responders to all three peptides. Mice bearing IAd were strong responders only to the 3' repeat peptides, the octamer and tetramer. Mice bearing Is or Iq did not respond to any repeat peptides. Remarkably, the pattern of genetic restriction of the antibody response to the entire RESA as expressed in vaccinia indicated that there were no other epitopes besides the three repeats. Because only one class II haplotype (i.e., k) out of five tested responded strongly to this peptide and only two out of five (i.e., k and d) responded to the octamer or tetramer, it may be difficult to achieve a good immune response against RESA in most or all humans.

Animals↗
PubMed1989

Molecular karyotyping of the rodent malarias Plasmodium chabaudi, Plasmodium berghei and Plasmodium vinckei.

The molecular karyotypes of four isolates of Plasmodium chabaudi, three of the subspecies P. chabaudi adami and one P. chabaudi chabaudi, as well as P. berghei and P. vinckei were studied by means of pulsed field gradient (PFG) gel electrophoresis. Each species appears to have 14 chromosomes, ranging in size from approximately 730 kb to greater than 2000 kb. The three P. chabaudi adami isolates did not appear any more similar to each other than to the P. c. chabaudi isolate. The chromosome locations of genes for a heat shock protein (hsp) 70, ribosomal RNA (rRNA), the precursor to the major merozoite surface proteins, dihydrofolate reductase and P. falciparum antigen 352 as well as four cloned DNA markers and a telomere probe were determined. However, a number of probes representing cloned P. falciparum antigens failed to hybridize to P. chabaudi DNA. Hence genes for malaria antigens appear to be much more divergent than genes for housekeeping functions.

Animals↗