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.

Ovarian cancers express and process progastrin.

Gastrin synthesis in ovarian tumors has been described in a few isolated cases associated with the Zollinger-Ellison syndrome. Consequently, ovarian gastrin synthesis has been considered exceptional. In order to evaluate whether expression of gastrin in ovarian tumors indeed is rare, we examined the expression and processing of progastrin in 16 malignant and 5 benign ovarian tumors and 4 normal postmenopausal ovaria. Using a library of sequence specific radioimmunoassays, cleavage by processing-like enzymes, and gel chromatography, we found that one-half of the malignant tumors expressed significant concentrations of amidated gastrins [6.7 +/- 2.7 (SEM) pmol/g; range, 1.4-20.0 pmol/g, n = 7]. The concentrations of glycine-extended gastrins and progastrins were low (0.25 +/- 0.03 and 1.4 +/- 0.4 pmol/g, respectively) but higher than in controls and benign tumors. Chromatography showed that the majority of the bioactive gastrins was unsulfated gastrin-17. The other half of the malignant tumors expressed glycine-extended gastrins and progastrins (0.2 +/- 0.03 and 0.6 +/- 0.1 pmol/g; n = 9), but the amidation of the peptides was impaired (0.1 +/- 0.03 pmol/g). Low concentrations of glycine-extended gastrins and progastrins were detected in the normal ovarian tissues (0.2 +/- 0.05 pmol/g tissue and 0.2 +/- 0.06 pmol/g, respectively, n = 4) and in the benign tumors (0.1 +/- 0.02 pmol/g and 0.5 +/- 0.03 pmol/g; n = 5). Amidated gastrins were undetectable, except in low amounts in a single benign tumor (0.2 pmol/g tissue). The results show that postmenopausal ovaria and neoplastic ovarian tissues express the gastrin gene at peptide level. The synthesis and processing of progastrin increase considerably in malignant tumors.

Two polymorphisms in the vitamin D receptor gene--association with bone mass and 5-year change in bone mass with or without hormone-replacement therapy in postmenopausal women: the Danish Osteoporosis Prevention Study.

The significance of an interrelation between nongenetic factors and genotype effects in the regulation of bone mass is not clear. In this prospective study of 429 healthy early postmenopausal Danish women, we investigated the association between bone mineral density (BMD) and the FokI and BsmI polymorphisms in the vitamin D receptor (VDR) gene. Participants were allocated to either hormone-replacement therapy (HRT) or no treatment by randomization or personal choice. After 5 years, 332 women with unchanged treatment status were available for analyses, 98 of these women were still on HRT. No association with initial BMD or 5-year change in BMD was found for either polymorphism. In women with body mass index (BMI) < 25 (n = 282), the f allele was associated with lower BMD of the hip (p < 0.001) and forearm (p = 0.001), and the b allele was associated with lower spine BMD (p = 0.02). Comparing thin/normal weight women with overweight/ obese women of the same genotype, FF women had similar BMD at all measured sites in contrast to Ff and ff women in whom BMD, as expected, was higher in the overweight/obese women. Similar results were found for the BsmI polymorphism with no difference in BMD between BMI groups in BB women. Segregation into groups according to dietary calcium intake did not reveal any genotype association with BMD. These results provide some evidence of a modifying effect of nongenetic factors, specifically BMI, on the association between VDR genotype and BMD. High BMI may protect against lower BMD seen in association with thef or b alleles. In some genotypes (FF and BB), BMI had relatively little effect on BMD.

Pituitary adenylate cyclase activating polypeptide (PACAP) in human ovarian cancers.

Synthesis of regulatory peptides has been described in a number of ovarian tumours. The recently isolated neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) is expressed transiently in steroidogenic cells of normal rat ovary. In order to evaluate if ovarian tumours also synthesize PACAP, we investigated the expression and processing of pro-PACAP in 11 serous cystadenocarcinomas, one non-differentiated carcinoma, one borderline malignant and four adenomas. Using sequence specific radioimmunoassays and gel chromatography, we found that all tumours expressed PACAP-38 immunoreactivity. Ovarian cancers contained higher (P < 0.03) concentrations (median 0.9 pmol/g, range 0.5-1.6 pmol/g, n = 13) than benign tumours (median 0.5 pmol/g, range 0.4-0.6 pmol/g, n = 4). Chromatography showed that the tumours contained PACAP-38 and PACAP-31-38 suggesting a rather complete processing. By immunohistochemistry, few scattered PACAP positive cells in small clusters or as single cells were identified in the tumours.

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.

Somatostatin in the boar reproductive system.

Somatostatin (SRIF) is a widely distributed regulatory peptide. Recently, it was shown that human seminal plasma contains high concentrations of SRIF. In order to find the cellular source of seminal SRIF we have examined the presence of SRIF in porcine urogenital tissues. The concentration of SRIF in male accessory reproductive tissues of 3-month-old pigs (N = 4) ranged from 1 to 17 nmol/kg. In the boar, only the prostate gland contained significant amounts of SRIF (median 7 nmol/kg, range 3-18 nmol/kg, N = 4). Testis and semen contained no SRIF. Gel chromatography of extracts of the male accessory sex glands and epididymis showed both SRIF-28 and SRIF-14, whereas urinary bladder contained mainly SRIF-14. In conclusion, the results show a considerable species, tissue and developmental variation in the expression of SRIF in the genitourinary tract.

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.

Vitamin D3 effects on basal and cAMP modulated expression of cholecystokinin and somatostatin genes in a rat medullary thyroid carcinoma cell line [CA-77].

The regulation of cholecystokinin and somatostatin expression by vitamin D and cyclic AMP in the rat medullary thyroid carcinoma cell line CA-77 was investigated. Treatment with 100 nmol/l vitamin D did not affect cholecystokinin mRNA and peptide concentrations significantly; somatostatin mRNA level increased 6 times and the somatostatin peptide concentration increased 2-fold after 5 days of drug treatment. Under the same experimental conditions cyclic AMP increased cholecystokinin mRNA level 4.5 times and the cellular cholecystokinin-peptide concentration 2-fold; somatostatin mRNA and peptide concentrations were not significantly changed. Cyclic AMP stimulated peptide secretion from the cells were not affected by vitamin D, but cyclic AMP mediated increase in CCK peptide concentration was significantly inhibited by vitamin D (p < 0.05).

Investigation of Helicobacter pylori ascorbic acid oxidating activity.

Helicobacter pylori sonicate was shown to oxidize ascorbic acid. Ascorbic acid oxidation was determined by chromatography combined with electrochemical detection. Water soluble ascorbic acid oxidase activity was rather independent of pH with a pH optimum around 2. By gel filtration the oxidizing activity co-eluted with an absorbency peak at 408 nm. The relative molecular mass (Mr) was approximately 14,000. It is suggested that this oxidating activity was caused by a cytochrome c-like molecule. Ascorbic acid oxidating activity could also be extracted from bacterial membranes by detergents. Gel filtration showed several forms, the major one with a Mr = 19,000. pH optimum was 6-7. Other oxidase-positive bacterial strains like Campylobacter coli, Enterobacter cloacae and Pseudomonas aeruginosa could degrade ascorbic acid. Since ascorbic acid oxidation by Helicobacter pylori whole bacterial lysates has a pH optimum in the acidic range corresponding to pH in gastric fluid, the activity of the cytochrome c-like water soluble oxidant of Helicobacter pylori seems to be primarily important for the destruction of ascorbic acid in the gastric juice of infected patients.

Human inter-alpha-trypsin inhibitor and immunologically related inhibitors investigated by quantitative immunoelectrophoresis. II. Pathological conditions.

Inter-alpha-trypsin inhibitor (I alpha I) and the immunologically related prealbumin-like-migrating proteinase inhibitor (pA-PI) were investigated by crossed immunoelectrophoresis in sera from 68 persons with myocardial infarction, neoplastic diseases, inflammatory diseases, collagenosis, cirrhosis of the liver or uremia. The concentration of pA-PI in serum increased during each of these diseases (p less than 0.01). The concentration of I alpha I was significantly decreased in patients with cirrhosis (p less than 0.01). In day to day studies of a patient with myocardial infarction, a patient with erysipelas and a postoperative patient the concentration of I alpha I was low normal to decreased in the first days of the conditions and increased thereafter to high normal values. A comparison of the concentration of pA-PI with the excretion of the immunologically identical urinary proteinase inhibitor (UPI) showed that the excretion could not be caused by simple overflow of pA-PI in the kidney. The excretion of UPI followed closely the acute-phase-response, as measured by serum C-reactive protein.

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.

Age-related hearing impairment and B vitamin status.

The aim of this study was to examine, in elderly subjects, a possible association between age-related hearing impairment and vitamin B12 or folic acid status. Ninety-one consecutive subjects with pure age-related hearing impairment, 35 males and 56 females, with a median age of 78 years, range 67-88 years, were included in the investigation. All subjects underwent a thorough evaluation, including pure-tone, speech and impedance audiometry. Blood samples were drawn for determination of B12, folic acid and homocysteine and analysed by routine laboratory measurements. No significant differences in the blood parameters as a function of gender could be demonstrated and no correlations were found between B12 or folic acid and hearing levels averaged across the range 0.5-4 kHz. A weak correlation between hearing levels and homocysteine (r = 0.03; correlation coefficient 0.004) was found; however, a comparison of the hearing levels between those with increased and normal homocysteine failed to show any significant differences. In addition, no association between B12 and the speech recognition score could be found. This investigation therefore fails to demonstrate any association between hearing level and vitamin B12 or folic acid in elderly subjects.

[Measurement of glucose and diagnosis of diabetes mellitus]. / Glukosemåling og diagnostik af diabetes mellitus.

In order to reduce the number of complications associated with type 2 diabetes mellitus, clinicians have begun to screen for asymptomatic diabetes more intensely. The American Diabetes Association has proposed new guidelines for the diagnosis of diabetes mellitus to obtain a more simple diagnostic procedure. This is now mainly based on measurements of at least two fasting venous plasma glucose concentrations. The risk of wrong conclusions are larger using the glucose tolerance test due to the large intra-individual biological variation. It would be desirable if a national consensus of using either whole blood glucose values or plasma values could be reached, in order to obtain a more simple system. As the influence of haematocrit are omitted by using plasma, it is suggested that diagnostic glucose concentrations are measured in venous plasma/serum. Capillary blood values measured in connection with treatment could be converted to plasma values.

pH optimum of the reduction of dehydroascorbic acid by dithioerytritol.

Ascorbic acid is a water soluble antioxidant, that is itself oxidized to dehydroascorbic acid. In order to detect dehydroascorbic acid by amperometri it has to be reduced to ascorbic acid beforehand. This reduction was performed with dithioerytritol at pH = 6.0 for 10 min, which is a choice between optimal reaction rate and stability of ascorbic acid. The limit of detection of ascorbic acid is 1.1 pmol making the assay useful for determination of ascorbic and dehydroascorbic acid in small tissue biopsies. Results of this method and a colorimetric method were comparable.

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.

Trypsin-inhibitory activity of ovalbumin preparations is due to ovomucoid.

Based on the amino-acid sequence it has been proposed that ovalbumin is a proteinase inhibitor. The inhibitory activity against trypsin, chymotrypsin, pancreatic elastase, plasmin, thrombin and pancreatic kallikrein was tested, but none of these proteinases were inhibited by ovalbumin. A commercial preparation of ovalbumin contained an impurity with trypsin inhibitory activity. This impurity was probably ovomucoid.