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Biomedical subjects

K Lambert

Publications and source records attributed to K Lambert.

At least 19 recordsLinked to original sources
PubMed1992

Human erythrocyte protein 4.2: isoform expression, differential splicing, and chromosomal assignment.

Human protein 4.2 (P4.2) is a major membrane skeletal protein in erythrocytes. Individuals with P4.2 deficiency exhibit spherocytosis and experience various degrees of hemolytic anemia, suggesting a role for this protein in maintaining stability and integrity of the membrane. Molecular cloning of P4.2 cDNAs showed that P4.2 is a transglutaminaselike molecule in erythrocytes but lacks the essential cysteine for cross-linking activity. Two cDNA isoforms have been identified from a human reticulocyte cDNA library, with the long isoform containing a 90-base pair (bp) in-frame insertion encoding an extra 30 amino acids near the N-terminus. Characterization of the P4.2 gene suggests differential splicing as the mechanism for generating these two cDNA isoforms. The donor site for the short isoform (P4.2S) agrees better with the consensus than the donor site for the long isoform (P4.2L) does. Expression of P4.2L was detected by a long-isoform-specific antibody raised against a peptide within the 30-amino acid insert. Western blot analyses showed P4.2L to be a minor membrane skeletal protein in human erythrocytes with an apparent molecular weight (mol wt) of approximately 3 Kd larger than the major protein 4.2, P4.2S. By in situ hybridization of a full-length 2.4-kilobase (kb) cDNA to human metaphase chromosomes, the gene for P4.2 was mapped to bands q15-q21 of chromosome 15, and it is not linked to the gene for coagulation factor XIIIa (plasma transglutaminase, TGase).

Amino Acid Sequence↗
PubMed1992

Molecular cloning and characterization of human fetal liver tropomodulin. A tropomyosin-binding protein.

Human erythrocyte tropomodulin is a novel tropomyosin regulatory protein that binds to the end of erythrocyte tropomyosin and blocks heat-to-tail association of tropomyosin along actin filaments. It has been proposed to play a role in modulating the association of tropomyosin with the spectrin-actin complex in the erythrocyte membrane skeleton. Immunoscreening of a human fetal liver cDNA expression library in lambda gt11, followed by 5'-end extension by polymerase chain reaction from the same library, yielded a composite cDNA sequence of 2665 base pairs (bp). It contains a 34-bp 5'-untranslated region, a 1.6-kilobase (kb) 3'-untranslated region, and a complete open reading frame of 1077 bp that encodes a protein of 359 amino acids with a calculated molecular mass of 40.6 kDa and a pI of 4.8. Authenticity of the tropomodulin cDNA was confirmed by a complete sequence match of 49 predicted amino acids with the sequences of three tryptic peptides of the erythrocyte tropomodulin. The sequence has no internal repeats and no significant homology with any known proteins. Secondary structure predictions indicate that tropomodulin may consist of a series of seven or eight short alpha-helical segments and fold into a somewhat compact shape. The tropomyosin binding activity has been mapped to an N-terminal region containing residues 39-138. Nine independent PCR clones, five from a human reticulocyte cDNA library and four from the fetal liver cDNA library, revealed identical N-terminal 103 amino acids, suggesting that the sequence reported here may also be of erythrocyte tropomodulin. Northern analysis of human reticulocyte RNA showed two hybridizing bands of 2.7 and 1.6 kb, indicating that the 2665-bp cDNA sequence reported here was that of the longer transcript.

Amino Acid Sequence↗
PubMed1992

Sugar absorption by the biliary ductular epithelium of the rat: evidence for two transport systems.

Sugar absorption by the biliary ductular epithelium under steady-state conditions was examined using isolated perfused rat liver. The test sugar and mannitol (as a putative marker of paracellular entry) were added to the glucose-free recirculating perfusate each at a concentration of 5 mmol/L, and apparent active biliary ductular absorption equated with the change in concentration of the test sugar relative to that of mannitol. A metabolizable hexose (D-glucose), pentose (D-xylose), and three nonmetabolizable hexoses (alpha-methyl-glucoside, 3-o-methyl-glucose, and L-glucose) were used. All five monosaccharides were well absorbed at constant rates for 2 hours with apparent rates of absorption (mumol.kg body weight-1.min-1, mean +/- SE) of D-glucose, 0.24 +/- 0.01; L-glucose, 0.20 +/- 0.02; 3-o-methyl-glucose, 0.19 +/- 0.02; alpha-methyl-glucoside, 0.16 +/- 0.03; and D-xylose, 0.10 +/- 0.04. The addition of phloridzin to the perfusate inhibited D-glucose absorption in part but did not inhibit L-glucose absorption. When perfusate Na+ was replaced by N-methylglucamine, the bile-plasma ratio of mannitol remained unchanged, as did the apparent absorption rate of D-glucose and 3-o-methyl-glucose. In contrast, absorption of L-glucose and alpha-methyl-D-glucoside gradually ceased. The addition of 15 mmol/L glucose to the perfusate caused decreased bile flow and increased taurocholate concentration in bile, suggesting that glucose absorption by the biliary ductules induced water reabsorption. It is concluded that sugars are absorbed by the biliary ductular system by Na(+)-dependent and Na(+)-independent transport systems, the substrate affinities of which differ from those reported for apical membrane hexose transport systems in renal tubular and intestinal epithelia. Ductular absorption of solutes such as glucose that enter bile passively may have biological use, because ductular absorption decreases the concentration of substrates for bacterial growth in gallbladder bile. On the other hand, ductular absorption of solutes induces reabsorption of biliary water, resulting in decreased bile flow; this might contribute to cholestasis during prolonged hyperalimentation with solutions containing glucose.

Absorption↗
PubMed1990

Molecular cloning and expression of a hexameric Drosophila heat shock factor subject to negative regulation.

We report the cloning of the transcriptional activator of heat shock genes, HSF, from Drosophila. The predicted sequence of Drosophila HSF protein is surprisingly divergent from that of yeast HSF, except in regions important for DNA binding and oligomerization. A segment of the DNA binding domain of HSF bears an intriguing similarity to the putative DNA recognition helix of bacterial sigma factors, while the oligomerization domain contains an unusual arrangement of conserved hydrophobic heptad repeats. Drosophila HSF produced in E. coli under nonshock conditions forms a hexamer that binds specifically to DNA with high affinity and activates transcription from a heat shock promoter in vitro. In contrast, when HSF is expressed in Xenopus oocytes, maximal DNA binding affinity is observed only after heat shock induction. These results suggest that Drosophila HSF has an intrinsic affinity for DNA, which is repressed under nonshock conditions in vivo.

Amino Acid Sequence↗
PubMed1990

Molecular cloning of human protein 4.2: a major component of the erythrocyte membrane.

Protein 4.2 (P4.2) comprises approximately 5% of the protein mass of human erythrocyte (RBC) membranes. Anemia occurs in patients with RBCs deficient in P4.2, suggesting a role for this protein in maintaining RBC stability and integrity. We now report the molecular cloning and characterization of human RBC P4.2 cDNAs. By immunoscreening a human reticulocyte cDNA library and by using the polymerase chain reaction, two cDNA sequences of 2.4 and 2.5 kilobases (kb) were obtained. These cDNAs differ only by a 90-base-pair insert in the longer isoform located three codons downstream from the putative initiation site. The 2.4- and 2.5-kb cDNAs predict proteins of approximately 77 and approximately 80 kDa, respectively, and the authenticity was confirmed by sequence identity with 46 amino acids of three cyanogen bromide-cleaved peptides of P4.2. Northern blot analysis detected a major 2.4-kb RNA species in reticulocytes. Isolation of two P4.2 cDNAs implies existence of specific regulation of P4.2 expression in human RBCs. Human RBC P4.2 has significant homology with human factor XIII subunit a and guinea pig liver transglutaminase. Sequence alignment of P4.2 with these two transglutaminases, however, revealed that P4.2 lacks the critical cysteine residue required for the enzymatic crosslinking of substrates.

Amino Acid Sequence↗
PubMed1982

Incorporation of donor muscle precursor cells into an area of muscle regeneration in the host mouse.

Normal muscle precursor cells, prepared by the enzymatic disaggregation of neonatal mouse muscle, were implanted into an area of regenerating muscle in a genetically different inbred strain. This was done in an attempt to determine, first, whether donor muscle precursor cells prepared in this way would fuse with the developing muscle fibres of the host; and second, whether in the "mosaic" muscle fibres thus formed donor as well as host genes were expressed. As markers of the host and donor genes we used the allelic isoenzyme variants of glucose-6-phosphate isomerase (GPI). In 43 out of 60 grafts we detected the presence of a "hybrid" isoenzyme intermediate between host and donor types. This "hybrid" indicated that donor muscle precursor cells had fused with regenerating host muscle cells, and had expressed their GPI genes within the resulting mosaic muscle fibres. We have developed this technique with a view to inserting normal genes into genetically abnormal myopathic muscle.

Animals↗
PubMed1979

Growth of human diploid cells (strain MRC-5) in defined medium; replacement of serum by a fraction of serum ultrafiltrate.

A calf serum ultrafiltrate fraction permitted growth for at least 3.5 generations, including one subculture, of MRC-5 cells in defined medium in the absence of whole serum. The active material has a molecular weight of 10 000 Daltons or less. This suggests that there may be no requirement for a large macromolecular component of serum. The ultrafiltrate was assayed by maximum cell yield from a serum-limited inoculum in a defined medium containing non-limiting amounts of vitamins, amino acids, glucose, a 68-component supplement, iron and methylcellulose. The levels of vitamins, amino acids and glucose were based on quantitative measurements of uptake and the levels of the other components by minimum amount required for maximum yield in defined medium without ultrafiltrate or serum. With excess ultrafiltrate maximum cell yield was limited by the defined part of the medium, probably the supplement. The cell doubling time in defined medium with ultrafiltrate fractions was 70 h compared with 27 h in the medium with serum. Excess ultrafiltrate did not inhibit growth. The lowered growth rate is attributed to a nutritional deficiency in the supplement.

Blood↗
PubMed1975

The quantitative requirements of human diploid cells (strain MRC-5) for amino acids, vitamins and serum.

The uptakes of all essential amino acids, vitamins (except riboflavin), glucose and serum during growth of human diploid cells (MRC-5) were determined. The amino acid uptakes varied considerably with the conditions of culture. The glucose requirement is several times greater than that for mouse LS or human HeLa cells. These analytical results were used to modify the medium so as to ensure that an excess of all defined medium constituents was present and pH was not limiting during study of the serum requirements. It was then found that maximum cell populations were directly proportional to the serum concentration. Hence the growth was limited by the supply of an unknown growth factor in serum. The serum growth factor was not replaced by a mixture of over 60 vitamins, co-enzymes, hormones and other organic and inorganic compounds considered to be possible growth factors, although this mixture did not lower the growth rate and somewhat (22%) increased the yield from the serum growth factor. The unit of serum growth factor is precisely defined in terms of the amount in a standard batch of calf serum. This standard contains 10 units/ml whereas the other batch of serum used contained only 5 units/ml.

Amino Acids↗