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Biomedical subjects

L Odum

Publications and source records attributed to L Odum.

18 recordsLinked to original sources

Biosynthesis of inter-alpha-trypsin inhibitor and alpha 1-microglobulin in a human hepatoma cell line.

1. Biosynthesis of alpha 1-microglobulin and inter-alpha-trypsin inhibitor was investigated in a human hepatoma cell line HepG-2. 2. alpha 1-Microglobulin was translated as a precursor common with the light chain of inter-alpha-trypsin inhibitor. 3. alpha 1-Microglobulin was synthesized and secreted into the growth medium within 30 min. 4. Processing of inter-alpha-trypsin-inhibitor-related proteins appeared slow and incomplete. The light chain was connected via a chondroitinsulphate to a heavy chain to form a 125,000-Mr protein and secreted within 1-4 hr.

Alpha-Globulins

The expression of peptide hormones in normal cells and tumour cells.

Insight in the mechanisms of peptide hormone expression has grown explosively by elucidation of gene, mRNA and preprohormone structures for most hormone systems during the 1980s. In addition, information about the structure and substrate specificity of many prohormone processing enzymes is rapidly accumulating in these years. The preprohormones vary considerably in size and organization from poly- to monoprotein structures. According to the structural organization and sequence homology the hormones are grouped in families. The prohormones are processed to bioactive peptides by multiple enzymatic modifications during the intracellular transport from the rough endoplasmatic reticulum to the mature secretory granules. The modifications comprise different proteolytic cleavages and amino acid derivatizations. The same prohormone may be expressed in several different cell types that process the precursor in entirely different ways. Awareness of such cell-specific processing patterns is important for the understanding of ectopic synthesis in neuroendocrine tumours.

Gene Expression Regulation

Expression and processing of procholecystokinin in a rat medullary thyroid carcinoma cell line.

A rat medullary thyroid carcinoma cell line, CA-77, was shown to express the cholecystokinin (CCK) gene. Measurements using a library of sequence-specific radioimmunoassays before and after enzymic treatment of extracts and chromatographic fractions showed that the cells contained 1.0 pmol of alpha-carboxyamidated cholecystokinins/10(6) cells, 0.4 pmol of glycine-extended intermediates/10(6) cells and 1.0 pmol of further C-terminal-extended pro-CCK/10(6) cells. Gel chromatography and reverse-phase h.p.l.c. revealed both sulphated and nonsulphated CCK-8 in the cells. The growth medium contained in addition alpha-amidated CCK-33, glycine-extended CCK-8 and pro-CCK. Exposure to 0.1 microM-dexamethasone for 6 days increased the cellular content and secretion of all of the described CCK peptides by 2-3-fold. The increase was first noted after 3 days of treatment. Monensin inhibited the synthesis of alpha-carboxyamidated CCK and the secretion of all of the CCK forms measured. Colchicine at a low concentration (0.2 mumol/l) apparently increased the synthesis and secretion of alpha-carboxyamidated CCK, whereas higher concentrations inhibited CCK synthesis. Finally, chloroquine inhibited the alpha-carboxyamidation of CCK. We conclude that the CA-77 cell line is a useful tool for studies of the expression and post-translational processing of pro-CCK.

Animals

Reversible microproteinuria induced by L-aspartate infusion.

Infusion of an acidic amino acid, L-aspartate, to 10 volunteers resulted in transient, significant increases in urinary excretion of the major urinary trypsin inhibitor (p less than 0.002) and beta 2-microglobulin (p less than 0.02). Simultaneously with the proteinuria, urinary pH rose significantly (p less than 0.02). These changes appeared following the infusion and after the excretion of L-aspartate had reached the preinfusion level. Albumin excretion was unchanged indicating that the proteinuria was due to a decreased tubular reabsorption. The mechanism for the reversible tubular proteinuria is unknown. A simple pH effect due to alkalization of the urine was excluded, as NaHCO3 infusion was not followed by an increase in the excretion of the major urinary trypsin inhibitor and beta 2-microglobulin.

Adult

Expression and localization of gastrin messenger RNA and peptide in spermatogenic cells.

In previous studies we have shown that the gene encoding cholecystokinin (CCK) is expressed in spermatogenic cells of several mammalian species. In the present study we show that a gene homologous to the CCK-related hormone, gastrin, is expressed in the human testis. The mRNA hybridizing to a human gastrin cDNA probe in the human testis was of the same size (0.7 kb) as gastrin mRNA in the human antrum. By in situ hybridization the gastrinlike mRNA was localized to seminiferous tubules. Immunocytochemical staining of human testis revealed gastrinlike peptides in the seminiferous tubules primarily at a position corresponding to spermatids and spermatozoa. In ejaculated spermatozoa gastrinlike immunoreactivity was localized to the acrosome. Acrosomal localization could also be shown in spermatids with electron microscopy. Extracts of the human testis contained significant amounts of progastrin, but no bioactive amidated gastrins. In contrast, ejaculated sperm contained mature carboxyamidated gastrin 34 and gastrin 17. The concentration of gastrin in ejaculated human spermatozoa varied considerably between individuals. We suggest that amidated gastrin (in humans) and CCK (in other mammals) are released during the acrosome reaction and that they may be important for fertilization.

Animals

Non-sulphated cholecystokinin in human medullary thyroid carcinomas.

The expression of gastrin/cholecystokinin (CCK) peptides and their precursors was examined in 16 medullary carcinomas of the human thyroid. Measurements with libraries of sequence-specific radioimmunoassays before and after enzymatic cleavage of extracts and chromatographic fractions showed that the carcinomas contained 1.7 pmol carboxyamidated CCK/g tissue (median; range 0.6-21.8 pmol/g), 0.9 pmol glycine-extended precursor/g (median; range less than 0.2-2.3 pmol/g) and 2.3 pmol further COOH-terminal-extended proCCK/g (median; range 0.9-6.2 pmol/g). Neither carboxyamidated gastrins nor any progastrins could be measured. Gel and reverse-phase chromatography revealed only small molecular forms, i.e. greater than 90% of the amidated immunoreactivity eluted like non-sulphated CCK-8 or CCK-7. The results show that human medullary thyroid carcinomas synthesize CCK peptides. The predominance of non-sulphated CCK is unusual. Taken together with earlier observations from dogs and pigs, our results raise the possibility that small non-sulphated CCK peptides modulate thyroid C-cell secretion in an autocrine manner.

Amino Acid Sequence

Inter-alpha-trypsin inhibitor and pre-alpha-trypsin inhibitor in health and disease. Determination by immunoelectrophoresis and immunoblotting.

Inter-alpha-trypsin inhibitor (ITI) consists of 3 polypeptides cross-linked by chondroitin sulphate, which is o-glycosidically linked to the smallest of the polypeptides, designated bikunin. Pre-alpha-trypsin inhibitor (p alpha I) consists of bikunin and a fourth polypeptide, also associated by chondroitin sulphate. Crossed immunoelectrophoresis (CIE) of plasma, using immunoglobulins to ITI, revealed 3 precipitation-lines, two of which increased in size during disease. Molecular mass determination by polyacrylamide gel electrophoresis showed that the immunoprecipitates contained mixtures of proteins. Therefore CIE is unfit for quantitation of the individual proteins related to ITI. Immunoblotting suggested that the plasma concentrations of p alpha I and of bikunin was increased in uraemia, rheumatoid arthritis and after trauma. The plasma concentrations of ITI and of p alpha I were decreased in a patient with endocarditis.

Alpha-Globulins

Characterization of urinary proteinase inhibitors with segments of amino acids sequences identical to sequences of pancreatic secretory trypsin inhibitor.

1. Slow migrating proteinase inhibitors were isolated from pathological human urine. 2. The N-terminal amino acid sequence including 23 amino acids was identical to the one in pancreatic secretory trypsin inhibitor. 3. The slow migrating proteinase inhibitors occurred in 3 forms with different electrophoretic mobility. 4. Time of flight mass spectrometry showed that the Mw of one of the forms was 6241 while the Mw of another form was 5923. 5. The Ki of complexes with trypsin was determined to be 1 x 10(-10) M, with chymotrypsin and plasmin Ki was 1 x 10(-7) M. Elastase, kallikrein and thrombin were not inhibited.

Adult

Peptide hormone expression and precursor processing.

Insight in the mechanisms of peptide hormone expression has grown explosively by elucidation of gene, mRNA and preprohormone structures for most hormone systems during the 1980s. The preprohormones vary considerably in size and organization from poly- to mono-protein structures. According to the structural organization and sequence homology the hormones are grouped in families. The prohormones are processed to bioactive peptides by multiple enzymatic modifications during the intracellular transport from the rough endoplasmatic reticulum to the mature secretory granules. The modifications comprise different proteolytic cleavages and amino acid derivatizations. The same prohormone may be expressed in several different cell-types that process the precursor in entirely different ways. Awareness of such cell-specific processing patterns is important for the understanding of ectopic synthesis in neuroendocrine tumours.

Animals

Immunohistochemical investigation of inter-alpha-trypsin inhibitor in the urinary tract.

The presence of inter-alpha-trypsin inhibitor in the urinary tract was investigated by immunohistochemistry. A positive reaction for inter-alpha-trypsin inhibitor was found in the proximal convoluted tubules of human kidney, probably representing the reabsorbed protein. In addition, inter-alpha-trypsin-inhibitor immunoreactivity was also demonstrated in human and rat mast cells.

Alpha-Globulins

Protein I serotype of serum-resistant versus serum-sensitive Neisseria gonorrhoeae strains.

In order to characterize serum-resistant and serum-sensitive strains of N. gonorrhoeae, the protein I serotype, auxotype, and penicillin susceptibility of 128 strains were tested. Sensitivity to the complement-dependent bactericidal activity of normal human serum was highly associated with protein I serotype (p less than 0.001). Thus 85% of serotype 1-3 strains were serum-resistant, whereas 86% of serotype 8 strains and all strains with serotypes 8 + 9 or 9 were serum-sensitive. Serum-resistance or sensitivity for a given serotype was independent of auxotype. The susceptibility to penicillin within the serotypes 1-3 was significantly associated with auxotype (p = 0.0016); all AHU- (requirement for arginine, hypoxanthine and uracil) strains had MICs of penicillin of 0.04 microgram/ml or less and were serotypes 1-3. Among the non-AHU-strains, serotype 9 was significantly more penicillin susceptible than the other serotypes (p less than 0.003).

Blood Bactericidal Activity

Indirect haemagglutination test using gonococcal pilus antigen: how useful to diagnose gonorrhoea?

In 1979 an indirect haemagglutination test (gonococcal antibody test) using gonococcal pilus antigen replaced the gonococcal complement fixation test as our routine procedure to show gonococcal antibodies. In the diagnosis of current gonorrhoea the sensitivity of the gonococcal antibody test was far superior to that of the gonococcal complement fixation test (about 55% versus 9% for first episode gonorrhoea). To evaluate the usefulness of the test result the following population groups were studied: 1376 patients undergoing medical examination for gonorrhoea (386 had gonorrhoea), 1384 healthy people aged 15-65, 54 patients with meningococcal disease, 30 children with respiratory tract infection, and 254 patients with evidence of various diseases other than neisserial infections that might be associated with symptoms of arthritis. These investigations showed that (1) non-specific positive gonococcal antibody test results occur rarely, (2) at least half the people who have had gonorrhoea remain seropositive (with titres of 1/40 to 1/160), and (3) a positive test result is more significant the younger the patient and the higher the titre. For younger people a positive test result should always be followed up by bacteriological examination; in all age groups titres of 1/320 or more should indicate medical examination for current gonorrhoea.

Adolescent

Human inter-alpha trypsin inhibitor and immunologically related inhibitors investigated by quantitative immunoelectrophoresis. I. Method and reference material.

The concentration in serum of inter-alpha trypsin inhibitor (I alpha I) and its immunologically related inhibitor, the prealbumin-like proteinase inhibitor (pA-PI), was investigated by quantitative crossed immunoelectrophoresis (CIE) in 33 healthy persons. The corresponding urinary excretion over a 4 h period of the immunologically related urinary proteinase inhibitor (UPI) was measured by rocket immunoelectrophoresis. The concentration of pA-PI increased significantly (p less than 0.001) with age whereas that of I alpha I and the urinary excretion of UPI were independent of age. There was no significantly difference in the concentration of the inhibitors in serum between the sexes, but females were found to have a lower urinary excretion of UPI than males (p less than 0.05). Furthermore we found no significant variation in the concentration of the inhibitors during the course of the day or from day to day over a 5 day period. Results obtained by rocket immunoelectrophoresis correlated well with enzymatically measured trypsin inhibitory activity.

Adult