Biomedical subjects

A H Johnsen

Publications and source records attributed to A H Johnsen.

At least 19 recordsLinked to original sources

PubMed1992

Identification of cholecystokinin/gastrin peptides in frog and turtle. Evidence that cholecystokinin is phylogenetically older than gastrin.

Peptides homologous to mammalian cholecystokinin (CCK) and gastrin in brain, antrum, and small intestine of an amphibian (the bullfrog, Rana catesbeiana) and a reptile (the turtle, Pseudomys scripta) were characterized. All tissues contained peptides reacting with antisera specific for the carboxyamidated C-terminal tetrapeptide common for CCK and gastrin. Extracts of all tissues, except the turtle antrum, also reacted with an antiserum specific for mammalian sulfated CCK, while no extract contained peptides reacting with an antiserum specific for mammalian gastrin. Both species contained predominantly small acidic forms in the brain and larger less acidic forms in the antrum and intestine. The antral peptides of both species were identified. The largest frog gastrin was a 47-residue peptide: DLLASLTHEQ KQLIMSQLLP ELLSELSNAE DHLHPMRDRD YAGWMDF.NH2. The largest turtle gastrin was a 52-residue peptide: DLLEALSQDQ KLLMAKFLPH IYAELANREG NWHEDAALRP LHDHDYPGWM DF.NH2. They display 51% similarity. Having Tyr in position 7 from the C-terminus, both resemble structurally mammalian CCK rather than gastrin, which suggests that CCK is phylogenetically older than gastrin. The turtle antral peptide contains a Tyr followed by Pro as in chicken gastrin. Thus, apparently at the stage of reptiles, a route different from the mammalian was taken in order to evolve a specific gastrin function.

Amino Acid Sequence↗

PubMed1992

Human red cell acid phosphatase (ACP1): the primary structure of the two pairs of isozymes encoded by the ACP1A and ACP1C alleles.

The Af, As, Cf and Cs isozymes encoded by the human red cell acid phosphatase ACP1*A and ACP1*C alleles, respectively, have been sequenced. All four isozymes consist of a single non-glycosylated peptide chain (157 residues), acetylated at the amino-terminal alanine residue. Each f isozyme differs from the corresponding s isozyme over the sequence segment 40-73, while the remaining four-fifth of the molecules are identical. These findings are consistent with results for the Bf and Bs isozymes encoded by the common ACP1*B allele and confirm that the presence of a specific f or s segment is a common property to ACP1 isozymes. This supports our hypothesis that f and s isozymes are generated by alternative splicing of exons in the primary RNA transcript. Cf and Cs are identical in sequence with Bf and Bs, respectively. Thus, the ACP1*B and ACP1*C alleles encode exactly the same pair of isozymes, the only difference at the protein level being the ratio of f and s isozyme. Af and As differ from the Bf and Bs isozymes by a single substitution at residue 105; Arg and Gln, respectively. These observations explain the electrophoretic identity of the B and C isozyme pairs and the higher P(i) of the A isozyme pair.

Acid Phosphatase↗

PubMed1992

Isolation, structure, and activity of -Phe-Met-Arg-Phe-NH2 neuropeptides (designated calliFMRFamides) from the blowfly Calliphora vomitoria.

Thirteen neuropeptides varying in length from 7 to 11 residues and ending C-terminally in -Phe-Met-Arg-Phe-NH2 (calliFMRFamides 1-13) and one dodecapeptide ending in -Met-Ile-Arg-Phe-NH2 (calliMIRFamide 1) have been isolated from thoracic ganglia of the blowfly Calliphora vomitoria. Different repeating patterns of amino acid sequences enable the peptides to be arranged into distinct groups. One such group of five nonapeptides has the sequence Xaa-Pro-Xaa-Gln-Asp-Phe-Met-Arg-Phe-NH2. Three peptides in this group, with the N-terminal tripeptide sequences Thr-Pro-Gln-, Thr-Pro-Ser-, and Ser-Pro-Ser-, are able to induce fluid secretion from the isolated salivary gland of Calliphora at a concentration of 0.1 to 1 nM. However, two other members of this group with the N-terminal tripeptide sequences Lys-Pro-Asn- and Ala-Pro-Gly-, the latter being the most abundant peptide isolated, were inactive in this assay, as were all the other peptides isolated. This indicates that the N terminus (in addition to the C terminus as previously found for FMRFamides of other organisms) is crucial for at least some biological activities.

Amino Acid Sequence↗

PubMed1992

Human neutrophil gelatinase: a marker for circulating blood neutrophils. Purification and quantitation by enzyme linked immunosorbent assay.

Human neutrophil gelatinase was purified to apparent homogeneity. The N-terminal amino-acid sequence of the purified enzyme could be aligned to an internal part of the cDNA-derived amino-acid sequence of 92-kDa type IV collagenase from SV 40-transfected human lung fibroblasts and from a TPA differentiated monocytic cell line, U937. Total amino-acid composition of U937 and neutrophil gelatinases was identical. Gelatinase was susceptible to treatment with o- and n-glycanase, indicating that posttranslational addition of oligosaccharide side chains occurs. An enzyme-linked immunosorbent assay for gelatinase was developed using specific polyclonal rabbit antibodies. The assay was specific, sensitive, accurate, and reproducible. Ninety percent range for plasma gelatinase from normal subjects was 17.3 to 102.9 ng/ml. In patients treated with cytostatic agents for non-Hodgkin's lymphoma, there was a parallel drop in plasma gelatinase and peripheral granulocyte count. This indicates that plasma gelatinase is a marker for circulating neutrophils. Plasma gelatinase does not seem to reflect bone marrow cellularity.

Amino Acid Sequence↗

PubMed1992

Stimulus-dependent secretion of plasma proteins from human neutrophils.

In search for matrix proteins released from secretory vesicles of human neutrophils, a prominent 67-kD protein was identified in the extracellular medium of neutrophils stimulated by the chemotactic peptide, FMLP. The protein was purified to apparent homogeneity and partially sequenced. The sequence of the first 32 NH2-terminal amino acids was identical to the sequence of albumin. mRNA for human albumin could not be detected in bone marrow cells, nor could biosynthetic labeling of albumin be demonstrated in bone marrow cells during incubation with [14C]leucine. Immunofluorescence studies on single cells demonstrated the presence of intracellular albumin in fixed permeabilized neutrophils. Light microscopy of immunogold-silver-stained cryosections visualized albumin in cytoplasmic "granules." The morphology of these was determined by immunoelectron microscopy as vesicles of varying form and size. Subcellular fractionation studies on unstimulated neutrophils demonstrated the presence of albumin in the low density pre-gamma and gamma-regions that contain secretory vesicles, but are devoid of specific granules and azurophil granules. Albumin was readily released from these structures during activation of neutrophils with inflammatory mediators. Immunoblotting demonstrated the presence of immunoglobulin and transferrin along with albumin in exocytosed material from stimulated neutrophils. This indicates that secretory vesicles are unique endocytic vesicles that can be triggered to exocytose by inflammatory stimuli.

Blood Proteins↗

PubMed1991

Human red cell acid phosphatase (ACP1). The amino acid sequence of the two isozymes Bf and Bs encoded by the ACP1*B allele.

The pair of isozymes, Bf and Bs, encoded by the human red cell acid phosphatase ACP1*B allele has been sequenced. Similar but not identical primary structures were observed. Both isozymes consist of a single peptide chain of 157 amino acid residues, which is acetylated at the amino-terminal alanine residue. The Bf and Bs isozymes are not glycosylated, and the calculated molecular masses are 17,932 and 17,867 Da, respectively. They are identical except for the sequence segment 40-73, which is peculiar to the respective isozyme. This is consistent with our hypothesis that the two isozymes are generated as the result of alternative splicing of the primary RNA transcript. The finding of a signature sequence offers the basis for the characteristic differences in catalytic and molecular properties of the Bf and Bs isozymes. A high degree of homology was found between the Bs isozyme and the 18-kDa cytosolic acid phosphatase from bovine liver. No homology was observed with other sequenced proteins, and this establishes these low molecular weight acid phosphatases as products of a distinct gene family.

Acid Phosphatase↗

PubMed1991

Nondestructive amino acid analysis at the picomole level of proline-containing peptides using aminopeptidase M and prolidase: application to peptides containing tyrosine sulfate.

A micromethod for nondestructive amino acid analysis was developed. Peptides were digested with amino-peptidase M followed by prolidase. The whole procedure was performed in a microvial for the autosampler of the analyzer. The resulting method has a working range from 10 to 600 pmol, at least, of sample taken into work. The usefulness of the method was demonstrated by its ability to determine the sulfation of tyrosine preceding proline in chicken gastrin.

Amino Acid Sequence↗

PubMed1991

Human galanin: primary structure and identification of two molecular forms.

From acid/ethanol extracts of surgical specimens of human large intestine we isolated two peptides, in approximately equal amounts, that reacted with an antiserum against porcine galanin. By amino acid analysis, sequence analysis and mass spectrometry, the larger of the two peptides was found to consist of 30 amino acid residues, the sequence of which was identical to that of porcine galanin except for the following substitutions: Val16, Asn17, Asn26, Thr29 and Ser30. Unlike porcine galanin, the carboxy-terminus was not amidated. The smaller peptide corresponded to the first 19 amino acid residues counted from the N-terminus of the 30 residue peptide (again without amidation). The structural analysis was repeated on another batch of tissue with identical results. By HPLC analysis of extracts of specimens from a further 4 patients, the same peptides were identified. Thus, human galanin includes two peptides of 19 and 30 amino acids that share the sequence of the N-terminal 15 residues with other mammalian galanins, but exhibit characteristic differences in the remaining part of the molecules.

Amino Acid Sequence↗

PubMed1991

Oxidation/reduction explains heterogeneity of pancreatic somatostatin.

Somatostatin 14 (SS 14) has been isolated from pancreatic extracts, but open gel filtration immunoreactive SS often elutes in two peaks. We isolated both peaks, but upon sequence analysis only authentic SS 14 could be identified. By further gel filtration experiments it turned out that both synthetic and extractable SS appeared homogeneous at neutral pH 7.5, but showed an additional, earlier peak in acetic acid. After addition of mercaptoethanol, all of the SS eluted at this earlier position regardless of the pH. We conclude that partial reduction/oxidation of SS explains the heterogeneity.

Amino Acid Sequence↗

PubMed1991

Cionin, a protochordean hybrid of cholecystokinin and gastrin: biological activity in mammalian systems.

The protochordean octapeptide cionin is structurally a hybrid of mammalian cholecystokinin (CCK) and gastrin, and thus their possible common ancestor. To determine whether cionin behaves like CCK or gastrin, we examined its effect on canine fundic somatostatin cells and on porcine and bovine gallbladder muscles. Cionin released somatostatin with a potency (ED50 0.15 nM) and efficacy (14.8% of cell content) similar to that of CCK-8 (ED50 0.12 nM, efficacy 16.7%). The efficacies but not the potencies of CCK-8 and cionin differed from those of sulfated gastrin (0.12 nM, 9.7%), nonsulfated gastrin (0.20 nM, 9.4%), and nonsulfated CCK-8 (0.30 nM, 10.4%). CCK and gastrin stimulated contractions of porcine gallbladder muscle strips in a concentration-dependent manner with no differences in efficacy but with characteristic differences in potency. CCK-8 and cionin displayed similar potencies of ED50 2.0 and 2.6 nM; both were significantly different from the ED50 of 0.4 microM for sulfated gastrin and 2.3 microM for nonsulfated gastrin. CCK radioligand binding to membrane-enriched preparations of porcine and bovine gallbladder muscularis was specific and of high affinity. The equilibrium data revealed that binding of CCK and gastrin peptides best fit a single site. CCK-8 and cionin displayed similar affinities [Kd 0.5 nM (porcine), 0.5 nM (bovine, CCK) vs. Kd 0.8 and 0.9 nM (cionin), respectively]. These differed again significantly from Kd 0.6 and 1.5 microM (sulfated gastrin) and 0.7 and 0.2 microM (nonsulfated gastrin). The results show that cionin behaves like CCK rather than gastrin in mammals.

Amino Acid Sequence↗

PubMed1991

Doxorubicin in combination with verapamil in advanced colorectal cancer. A phase II trial.

The possibility that verapamil might increase the sensibility of colorectal carcinomas to doxorubicin was studied in 24 patients without previous cytotoxic chemotherapy. The treatment started with oral verapamil, which was escalated up to the individual maximum tolerable dose (defined by prolongation of P-Q in ECG, fall in blood pressure, or dizziness). The median maximum tolerable dose was 600 mg (range 320-1,440 mg). During continued verapamil administration the patients then got weekly infusions of doxorubicin, 25 mg/m2. The median number of doxorubicin courses was 8 (range 2-22). Among 21 patients evaluable for response and toxicity two partial remissions occurred but no complete remission. The study did not indicate enhanced cardiac, hematological or non-hematological toxicity from the combined treatment.

Adult↗

PubMed1991

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↗

PubMed1990

Galanin in the porcine pancreas.

Galanin, a 29 amino acid neuropeptide, was recently isolated from pig intestine. We studied the localization, nature and effect of galanin in pig pancreas. Galanin immunoreactive nerve fibers were regularly found in the pancreas. A peptide chromatographically similar to synthetic galanin was identified in pancreas extracts. The effect of galanin on the endocrine and exocrine secretion was studied in isolated pancreases, perfused with a synthetic medium containing 3.5, 5 or 8 mmol/l glucose and synthetic galanin (10(-10)-10(-8) mol/l). There was no effect on the basal exocrine secretion. The output of insulin, glucagon, somatostatin and pancreatic polypeptide (PP) was measured in the effluent. There was no effect on PP secretion. At a perfusate glucose concentration of 5 mmol/l, galanin at 10(-9) mol/l increased insulin secretion by 55 +/- 14% (mean +/- S.E.M., n = 5) of basal secretion, and at 10(-8) mol/l by 58 +/- 27% (n = 6). At 8 mmol/l glucose, insulin secretion increased by 25 +/- 10% (n = 6) and 62 +/- 17% (n = 8). At 5 mmol/l glucose glucagon secretion was increased by 15 +/- 3% (n = 5) by galanin at 10(-9) mol/l and by 29 +/- 11% (n = 5) by galanin at 10(-8) mol/l, and at 8 mmol/l glucose by 66 +/- 27% and 41 +/- 25%. Somatostatin secretion was inhibited to 72 +/- 2% (n = 5) of basal secretion by galanin at 10(-9) mol/l and to 65 +/- 7% (n = 7) at galanin at 10(-8) mol/l, both at 5 mmol/l glucose. At 8 mmol/l the figures were 83 +/- 6% and 70 +/- 10%. Insulin secretion in response to square wave increases in glucose concentration from 3.5 to 11 mmol/l (n = 5) increased 2-fold during simultaneous perfusion with galanin (10(-8) mol/l).

Animals↗

PubMed1990

Cionin: a disulfotyrosyl hybrid of cholecystokinin and gastrin from the neural ganglion of the protochordate Ciona intestinalis.

We have purified an acidic octapeptide from the neural ganglion of the protochordate Ciona intestinalis by a three-step procedure including C18 Sep-Pak fractionation, MonoQ ion-exchange chromatography, and C4 reversed-phase high-performance liquid chromatography. The purification was monitored by an immunoassay specific for the alpha-carboxyamidated COOH terminus common to the mammalian brain-gut hormones, cholecystokinin and gastrin. Automated Edman degradation revealed the sequence Asn-Tyr-Tyr-Gly-Trp-Met-Asp-Phe. In accordance with the high acidity of the peptide, amino acid analysis after cleavage with aminopeptidase M showed that both tyrosyl residues are sulfated. Hence, the structure is Asn-Tyr(SO3)-Tyr(SO3)-Gly-Trp-Met-Asp-Phe-NH2, as also confirmed by identity with the synthetic disulfated peptide in different chromatographic systems. The occurrence of two consecutively sulfated tyrosyl residues after a neutral residue challenges present concepts of consensus sites for tyrosyl sulfation. We conclude that the structure of the peptide, named cionin, suits that of a common ancestor for cholecystokinin and gastrin.

Amino Acid Sequence↗

PubMed1990

Gamma-aminobutyric acid concentration in lumbar cerebrospinal fluid from patients with febrile convulsions and controls.

The cerebrospinal fluid (CSF) concentration of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) was analysed in 41 children with febrile convulsions (FC), 41 febrile controls of similar age (control group 1), and 59 controls, who had no fever and/or were outside the age range for FC (control group 2). A significant correlation between CSF-GABA and age was demonstrated for controls (1 + 2) (r = 0.63, p less than 0.00001), as well as for patients with FC (r = 0.42, p = 0.003). Patients with FC did not differ significantly from control group 1 in respect to CSF-GABA. Duration of FC was related to both CSF-GABA and age (GABA: r = -0.29, p less than 0.05; age: r = -0.32, p less than 0.05). For 56 controls (1 + 2) greater than 1 year of age, a significant negative correlation between CFC-GABA and body temperature was found (r = -0.34, p = 0.01). The low CSF-GABA in the FC-labile age group, the negative correlation of CSF-GABA to body temperature, and the negative correlation of the duration of FC to both CSF-GABA and age, all indicate that GABA could be of importance in the pathophysiology of FC.

Adolescent↗

PubMed1990

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↗

PubMed1989

Complete sequences of glucagon-like peptide-1 from human and pig small intestine.

In the small intestine, proglucagon is processed into the previously characterized peptide "glicentin" (proglucagon (PG) 1-69) and two smaller peptides showing about 50% homology with glucagon: glucagon-like peptide-1 and -2. It was assumed that the sites of post-translational cleavage in the small intestine of the proglucagon precursor were determined by pairs of basic amino acid residues flanking the two peptides. Earlier studies have shown that synthetic glucagon-like peptide-1 (GLP-1) synthesized according to the proposed structure (proglucagon 71-108 or because residue 108 is Gly, 72-107 amide) had no physiological effects, whereas a truncated from of GLP-1, corresponding to proglucagon 78-107 amide, strongly stimulated insulin secretion and depressed glucagon secretion. To determine the amino acid sequence of the naturally occurring peptide we isolated GLP-1 from human small intestine by hydrophobic, gel permeation, and reverse-phase high performance liquid chromatography. By analysis of composition and sequence it was determined that the peptide corresponded to PG 78-107. By mass spectrometry the molecular mass was determined to be 3295, corresponding to PG 78-107 amide. Furthermore, mass spectrometry of the methyl-esterified peptide showed an increase in mass compatible with the presence of alpha-carboxyl amidation. Thus, the gut-derived insulinotrophic hormone GLP-1 is shown to be PG 78-107 amide.

Amino Acid Sequence↗

PubMed1989

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↗