Causes of iron overload in blood donors - a clinical study.

BACKGROUND AND OBJECTIVES: Despite the obligate iron loss from blood donation, some donors present with hyperferritinaemia that can result from a wide range of acute and chronic conditions including hereditary haemochromatosis (HH). The objective of our study was to investigate the causes of hyperferritinaemia in the blood donor population and explore the value of extensive HH mutational analyses. MATERIALS AND METHODS: Forty-nine consecutive donors (f = 6, m = 43) were included prospectively from the Capital Regional Blood Center. Inclusion criteria were a single ferritin value >1000 µg/l or repeated hyperferritinaemia with at least one value >500 µg/l. All donors were questioned about their medical history and underwent a physical examination, biochemical investigations and next-generation sequencing of HH-related genes, including the HFE gene, the haemojuvelin gene (HFE2/HJV), the hepcidin gene (HAMP), the ferroportin 1 gene (SLC40A1) and the transferrin receptor 2 gene (TFR2). RESULTS: Forty of 49 donors were mutation positive with a combined 69 mutations, 54 of which were located in the HFE gene. There were 11 mutations in the TFR2 gene, two mutations in the HFE2 gene and two mutations in the HAMP gene. Only four donors had apparent alternative causes of hyperferritinaemia. CONCLUSION: HH-related mutations were the most frequent cause of hyperferritinaemia in a Danish blood donor population, and it appears that several different HH-genotypes can contribute to hyperferritinaemia. HH screening in blood donors with high ferritin levels could be warranted. HH-related iron overload should not in itself result in donor ineligibility.

The role of miRNAs in human papilloma virus (HPV)-associated cancers: bridging between HPV-related head and neck cancer and cervical cancer.

This corrects the article DOI: 10.1038/bjc.2012.109.

Increasing incidence of base of tongue cancers from 2000 to 2010 due to HPV: the largest demographic study of 210 Danish patients.

BACKGROUND: We assessed the development in the number of new base of tongue squamous-cell carcinoma (BSCC) cases per year in eastern Denmark from 2000 to 2010 and whether HPV may explain any observable increased incidence. METHODS: We performed HPV DNA PCR and p16 immunohistochemistry analysis for all (n=210) BSCCs registered in the Danish Head and Neck Cancer Group (DAHANCA) and the Danish Pathology Data Bank, and genotyped all HPV-positive specimens with amplicon-based next-generation sequencing. RESULTS: The overall crude incidence of BSCCs increased significantly (5.4% per year) during the study period. This was explained by a significant increase in the number of HPV-positive BSCCs (8.1% per year), whereas the number of HPV-negative BSCCs did not increase significantly. The overall HPV prevalence was 51%, with HPV16 as the predominant HPV type. CONCLUSIONS: The increased number of HPV-positive BSCCs may explain the increasing incidence of BSCCs in eastern Denmark, 2000-2010.

Primary hyperparathyroidism in young people. When should we perform genetic testing for multiple endocrine neoplasia 1 (MEN-1)?

CONTEXT: Multiple endocrine neoplasia (MEN-1) is a rare, autosomal dominant inherited disorder. Primary hyperparathyroidism (pHPT) is the most frequent and usually the earliest expression of MEN-1, with typical age of onset at 20-25 years. Early detection of the disease and correct treatment are therefore of great importance. CASE PRESENTATION: A 31-year-old woman with osteogenesis imperfecta was incidentally found also to have hypercalcemia and elevated PTH (pHPT). Exploratory neck surgery showed multiglandular parathyroid affection; she turned out to have MEN-1, but she was diagnosed 7 years after her debut of pHPT. OBJECTIVE AND METHODS: The aim was to search literature on indications for performing mutational analysis in young patients with pHPT and no family history of MEN-1. PubMed was searched for English language articles, and words used were: MEN1 OR MEN-1 OR MEN type 1 OR multiple endocrine neoplasia 1 OR multiple endocrine neoplasia type 1 AND Mutational analysis OR genetic testing OR testing OR Hyperparathyroidism, primary [majr]. A total of 625 abstracts were reviewed. RESULTS AND DISCUSSION: Whether to perform screening of patients with pHPT under the age of 30, 35, or 40 years is controversial. According to international guidelines from 2001, genetic testing is indicated only in patients with pHPT below the age of 30 years. However, in updated guidelines from 2012, it is suggested to perform genetic testing in patients with pHPT below the age of 30 years, but also at any age in patients presenting with multigland parathyroid disease. CONCLUSIONS: The reviewed literature and the presented case illustrate the importance of this change in international guidelines, but they also raise concern for a potential underdiagnosing of patients before year 2012.

Maternal menopause as a predictor of anti-Mullerian hormone level and antral follicle count in daughters during reproductive age.

STUDY QUESTION: Is the ovarian reserve in a woman at a given age associated with her mother's age at menopause? SUMMARY ANSWER: We demonstrated a significant, positive association between age at maternal menopause and serum anti-Müllerian hormone (AMH) levels and antral follicle count (AFC) in daughters. The rate of decline in serum-AMH level and AFC is also associated with age at maternal menopause. WHAT IS KNOWN AND WHAT THIS PAPER ADDS: The association between menopausal age in mothers and daughters has been established through several epidemiological studies. This paper shows that early maternal menopause is related to an advanced depletion of the ovarian reserve and that late maternal menopause is related to a delayed depletion. STUDY DESIGN AND SIZE: Cross-sectional data were obtained from a prospective cohort study of 863 women. The study comprised 527 participants from this prospective cohort whose mothers' age at natural menopause was known. PARTICIPANTS, SETTING AND METHODS: Participants were recruited from female health care workers aged 20-40 years employed at Copenhagen University Hospital, Rigshospitalet, and were enrolled in the study between September 2008 and February 2010. The response rate was 52.1%. Endocrine and ovarian parameters related to reproductive ageing (AMH and AFC) were assessed by serum AMH analyses and transvaginal ovarian sonography on cycle Day 2-5. Data on reproductive history, including age at natural maternal menopause, were obtained through an internet-based questionnaire. We used an analysis of covariance model with serum-AMH and AFC as outcomes, age as the quantitative predictor and onset of maternal menopause as the categorical predictor, with further adjustments for BMI, use of oral contraceptives, participants' smoking habits and prenatal smoking exposure. MAIN FINDINGS: We found a significant effect of age at maternal menopause on both serum AMH levels (P < 0.001) and AFC (P = 0.005). Median serum-AMH concentration declined by 8.6% per year [95% confidence interval (CI): 6.4-10.8%, P < 0.001] in the group with early maternal menopausal age (≤ 45 years), by 6.8% per year (95% CI: 5.0-8.6%, P < 0.001) in the group with normal maternal menopausal age (46-54 years) and by 4.2% per year (95% CI: 2.0-6.4%, P < 0.001) in the group with late maternal menopausal age (≥ 55 years). Median AFC declined by 5.8% per year (95% CI: 4.0-7.5%, P < 0.001) in the group with early maternal menopausal age (≤ 45 years), by 4.7% per year (95% CI: 3.3-6.1%, P < 0.001) in the group with normal maternal menopausal age (46-54 years) and by 3.2% per year (95% CI: 1.4-4.9%, P < 0.001) in the group with late maternal age (≥ 55 years) at menopause. BIAS, LIMITATIONS AND GENERALIZABILITY: Information on 'age at maternal menopause' was obtained retrospectively and may be prone to recall bias and digit preference. The study population consisted of health care workers, which implies a potential selection bias. Finally, the cross-sectional nature of the data limits the generalizability. STUDY FUNDING/POTENTIAL COMPETING INTERESTS: This study was co-financed by PhD scholarships where funding was covered by the Danish Agency for Science, Technology and Innovation, Copenhagen Graduate School of Health Science (CGSHS) and the Fertility Clinic at Copenhagen University Hospital, Rigshopitalet. No competing interests are declared.

Ovarian reserve parameters: a comparison between users and non-users of hormonal contraception.

It remains controversial whether anti-Müllerian hormone (AMH) concentration is influenced by hormonal contraception. This study quantified the effect of hormonal contraception on both endocrine and sonographic ovarian reserve markers in 228 users and 504 non-users of hormonal contraception. On day 2-5 of the menstrual cycle or during withdrawal bleeding, blood sampling and transvaginal sonography was performed. After adjusting for age, ovarian reserve parameters were lower among users than among non-users of hormonal contraception: serum AMH concentration by 29.8% (95% CI 19.9 to 38.5%), antral follicle count (AFC) by 30.4% (95% CI 23.6 to 36.7%) and ovarian volume by 42.2% (95% CI 37.8 to 46.3%). AFC in all follicle size categories (small, 2-4 mm; intermediate, 5-7 mm; large, 8-10 mm) was lower in users than in non-users of hormonal contraception. A negatively linear association was observed between duration of hormonal-contraception use and ovarian reserve parameters. No dose-response relation was found between the dose of ethinyloestradiol and AMH or AFC. This study indicates that ovarian reserve markers are lower in women using sex steroids for contraception. Thus, AMH concentration and AFC may not retain their accuracy as predictors of ovarian reserve in women using hormonal contraception. Serum anti-Müllerian hormone (AMH) concentration is an indirect marker of the number of small follicles in the ovary and thereby the ovarian reserve. The AMH concentration is now widely used as one of the markers of the ovarian reserve in ovarian hormonal stimulation regimens. Hence the AMH concentration in a patient is used to decide the dose of the ovarian hormonal stimulation prior to IVF treatment. In some infertile patients, hormonal contraception is used prior to ovarian hormonal stimulation and therefore it is important to clarify whether serum AMH concentration is influenced by the use of sex steroids. The aim of this study was to quantify the potential effect of hormonal contraception on the ovarian function by hormonal analyses and ovarian ultrasound examination. Examinations were performed in the early phase of the menstrual cycle or the hormone-free interval of hormonal contraception. We compared the AMH concentration, the antral follicle count (AFC) and the ovarian volume in 228 users versus 504 non-users of hormonal contraception. Users of hormonal contraception had 29.8% lower AMH concentration, 30.4% lower AFC and 42.2% lower ovarian volume than non-users. These findings were more pronounced with increasing duration of hormonal contraception. No dose-response relation was found between the dose of ethinylestradiol and the impact on serum AMH and AFC. The study indicates that ovarian reserve markers are lower in women using sex steroids for contraception. Thus, serum AMH concentration and AFC may not retain their accuracy as predictors of the ovarian reserve in women using hormonal contraception.

Prospective study evaluating performance of first-trimester combined screening for trisomy 21 using repeat sampling of maternal serum markers PAPP-A and free ß-hCG.

OBJECTIVE: To prospectively evaluate the performance of first-trimester combined screening for trisomy 21 using the biochemical markers pregnancy-associated plasma protein-A (PAPP-A) and free beta-human chorionic gonadotropin (free ß-hCG) obtained before and at the time of the nuchal translucency (NT) scan. METHODS: Three fetal medicine departments in Denmark participated in the study. Screening for trisomy 21 was set up as a two-step approach with blood sampling performed before the NT scan (early sample) and again at the time of the NT scan (late sample). PAPP-A and free ß-hCG were measured on both the early and late samples. Age-standardized detection and false-positive rates for different screening protocols were calculated. RESULTS: We collected two blood samples in 27 pregnancies affected by trisomy 21 and in 3891 control pregnancies. The early samples were taken between gestational ages 8 + 0 and 13 + 6 weeks, and the late samples between 11 + 3 and 14 + 6 weeks. The median interval between the samples was 17 (range, 1-40) days. We found a significantly better estimated screening performance when using early sampling vs late sampling (P < 0.05). With a risk cut-off of 1 in 100, at the time of the risk assessment the estimated detection and false-positive rates when using the early sample were 91% (95% CI, 81-98%) and 1.6% (95% CI, 1.3-2.0%), respectively. For fixed false-positive rates the highest detection rates were achieved using both blood samples. When comparing early sampling vs double sampling there was no significant difference in screening performance. CONCLUSION: In combined first-trimester screening for trisomy 21, use of early sampling with measurement of PAPP-A and free ß-hCG before the time of the NT scan can optimize screening performance. Using maternal serum markers obtained both before and at the time of the NT scan has the potential to further improve performance, but larger studies are needed to confirm this potential.

The role of miRNAs in human papilloma virus (HPV)-associated cancers: bridging between HPV-related head and neck cancer and cervical cancer.

BACKGROUND: Although the role of human papilloma virus (HPV) in cervical squamous cell carcinoma (CSCC) is well established, the role in head and neck SCC (HNSCC) is less clear. MicroRNAs (miRNAs) have a role in the cancer development, and HPV status may affect the miRNA expression pattern in HNSCC. To explore the influence of HPV in HNSCC, we made a comparative miRNA profile of HPV-positive (HPV+) and HPV-negative (HPV-) HNSCC against CSCC. METHODS: Fresh frozen and laser microdissected-paraffin-embedded samples obtained from patients with HPV+/HPV- HNSCC, CSCC and controls were used for microarray analysis. Differentially expressed miRNAs in the HPV+ and HPV- HNSCC samples were compared with the differentially expressed miRNAs in the CSCC samples. RESULTS: Human papilloma virus positive (+) HNSCC had a distinct miRNA profile compared with HPV- HNSCC. Significantly more similarity was seen between HPV+ HNSCC and CSCC than HPV- and CSCC. A set of HPV core miRNAs were identified. Of these especially the miR-15a/miR-16/miR195/miR-497 family, miR-143/miR-145 and the miR-106-363 cluster appear to be important within the known HPV pathogenesis. CONCLUSION: This study adds new knowledge to the known pathogenic pathways of HPV and substantiates the oncogenic role of HPV in subsets of HNSCCs.

Different miRNA signatures of oral and pharyngeal squamous cell carcinomas: a prospective translational study.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs, which regulate mRNA translation/decay, and may serve as biomarkers. We characterised the expression of miRNAs in clinically sampled oral and pharyngeal squamous cell carcinoma (OSCC and PSCC) and described the influence of human papilloma virus (HPV). METHODS: Biopsies obtained from 51 patients with OSCC/PSCC and 40 control patients were used for microarray analysis. The results were correlated to clinical data and HPV status. Supervised learning by support vector machines was employed to generate a diagnostic miRNA signature. RESULTS: One hundred and fourteen miRNAs were differentially expressed between OSCC and normal oral epithelium, with the downregulation of miR-375 and upregulation of miR-31 as the most significant aberrations. Pharyngeal squamous cell carcinoma exhibited 38 differentially expressed miRNAs compared with normal pharyngeal epithelium. Differences in the miRNA expression pattern of both normal epithelium and SCC were observed between the oral cavity compared with the pharynx. Human papilloma virus infection revealed perturbations of 21 miRNAs, most significantly in miR-127-3p and miR363. A molecular classifier including 61 miRNAs was generated for OSCC with an accuracy of 93%. CONCLUSION: MicroRNAs may serve as useful biomarkers in OSCC and PSCC. The influence of HPV on miRNA may provide a mechanism for the distinct clinical behaviour of HPV-infected tumours.

Prospective investigation of serum anti-Müllerian hormone concentration in ovulatory intrauterine insemination patients: a preliminary study.

This preliminary prospective study investigated serum anti-Müllerian hormone (AMH) through correlations to other basal parameters (123 patients) and according to ovarian response to 75 IU recombinant follicle-stimulating hormone (rFSH)/day (62 patients) in ovulatory patients' first rFSH treatment cycle before intrauterine insemination. Mean age of the patients was 33 years. Serum AMH significantly correlated to age (r=-0.38), antral follicle count (AFC) (r=0.68), ovarian volume (r=0.40), FSH (r=-0.31), (P<0.001) and cycle length (r=0.26, P=0.004). Serum AMH median (interquartile range; IQR) was 8.5 pmol/l (1.9-15.1) in hyporesponders (one mature follicle) versus 10.7 (7.3-17.3) in normal responders (2-3 follicles, with a maximum of two follicles 18 mm and no need for dose reduction) and 13.4 (4.4-24.2) in hyperresponders (>2-3 mature follicles or dose reduction). There was a significant trend over response groups for body weight (P=0.005), body mass index (P=0.035), AFC (P=0.031) and FSH (P=0.001). Serum AMH median (IQR) was 10.6 pmol/l (6.9-18.2) in the 23 patients who achieved an ongoing pregnancy versus 10.5 (5.9-17.2) in the 100 non-pregnant women. Serum AMH may not be the best marker of the ovarian response in these patients.

Lipoprotein lipase and endothelial lipase in human testis and in germ cell neoplasms.

The aim of this study was to investigate endothelial lipase (EL, LIPG) and lipoprotein lipase (LPL) mRNA and protein expression in normal human testis and testicular germ cell tumours (GCT). Both EL and LPL were expressed in normal seminiferous tubules and in the interstitial compartment. EL mRNA and protein were found in all germ cells as well as in Sertoli and Leydig cells. EL mRNA was abundant in pre-invasive carcinoma in situ (CIS) cells and GCTs, and EL protein was present in the cytoplasm of these cells. LPL mRNA was also relatively abundant in germ cells, Sertoli cells, CIS cells and GCTs. The LPL protein, however, was restricted to the cell membranes of pachytene spermatocytes and spermatids in normal tubules, absent from CIS cells and scarcely represented in tumours. The distribution of LPL protein in non-seminomas resembled the distribution of OCT3/4, a marker of embryonal carcinoma. The results suggest that both EL and LPL participate in the supply of nutrients and steroidogenesis in the testes, and that especially EL may be important for the supply of cholesterol for testosterone production in the Leydig cells. The partial cellular separation of the expression of the two lipases in normal testis suggests the existence of distinct biological roles, perhaps developmentally regulated, as indicated by the LPL expression in GCTs with embryonic features. A high expression of EL and abundance of lipid in tubules with CIS may have a diagnostic value.

Achlorhydria is associated with gastric microbial overgrowth and development of cancer: lessons learned from the gastrin knockout mouse.

Gastrin and gastrin receptor-deficient mice have been used for genetic dissection of the role of gastrins in maintaining gastric homeostasis and control of acid secretion. The gastrin knockout mice are achlorhydric due to inactivation of the ECL and parietal cells. Moreover, this achlorhydria is associated with intestinal metaplasia and bacterial overgrowth, which ultimately leads to the development of gastric tumours. The association between progastrin, progastrin-derived processing intermediates and colorectal carcinogenesis has also been examined through genetic or chemical cancer induction in several mouse models, although the clinical relevance of these studies remains unproven. While others have focused on models of increased gastrin production, the present review describes the lessons learned from gastrin-deficient mice. Study of these mice helps our understanding of how dysregulation of gastrin secretion may be implicated in human disease.

On the tissue-specific processing of procholecystokinin in the brain and gut--a short review.

Cholecystokinin (CCK) is a major peptide hormone in the gut and a major peptide transmitter in the brain. Its synthesis requires endoproteolytic cleavage of proCCK at several mono- and dibasic sites by prohormone convertases (PCs). Of these, PC1 and PC2 are expressed in cerebral neurons and intestinal endocrine cells. Characteristically, however, the processing of proCCK varies markedly between the brain and the gut. In neurons, CCK-8 is always the predominating form, whereas the endocrine gut cells (I-cells) contain a mixture of small and larger CCK-peptides of which CCK-33 or CCK-22 often predominate. The role of PC1 and PC2 in the processing of proCCK have now been examined by measuring the concentrations of prohormone, processing intermediates and amidated end-products in jejunal and cerebral extracts of PC1 and PC2 deficient mice and corresponding wild type controls. The PC1 null mice revealed a pattern opposite to that of the PC2 null mice, in whom only the cerebral processing of proCCK was affected. Thus PC1 knockouts reveal a severe block in the processing of intestinal proCCK. Accordingly, the intestinal concentration of proCCK was many fold increased, and also the concentrations of different processing intermediates were raised; but the concentration of bioactive, alpha-amidated and O-sulfated CCK was reduced to a few percent of normal. The only bioactive CCK peptide in the gut of PC1 deficient mice was CCK-22, and it was present only in trace amounts. The cerebral processing of proCCK was, however, not at all affected by the lack of PC1 - in sharp contrast to the effect of PC2. The results show that the tissue-specific processing of proCCK to a large extent can be explained by prohormone convertases, as PC1 plays a decisive role in the maturation of hormonal CCK in the gut, whereas PC2 governs the processing of brain proCCK.

Effect of interleukin 1beta on the HPA axis in H1-receptor knockout mice.

OBJECTIVES AND METHODS: Circulating cytokines such as interleukin-1 (IL-1), and tumor necrosis factor-alpha as well as lipopolysaccharide (LPS) are potent ACTH secretagogues, acting via stimulation of corticotropin-releasing hormone (CRH) and vasopressinergic neurons in the paraventricular nucleus of the hypothalamus (PVN). Histamine (HA) has been shown to stimulate ACTH secretion in rats, an effect in part mediated by CRH and arginine vasopressin (AVP). We have previously shown that inhibition of neuronal HA synthesis or central blockade of H(1) receptors (H(1)R) decreased the ACTH response to LPS in male rats. To further elucidate the role of neuronal HA in cytokine-induced activation of the HPA axis, we compared the effect of H(1)R knockout on IL-1beta-induced ACTH secretion in adult male mice. RESULTS: In H(1)R knockout mice, ACTH secretion increased from basal levels of 261 to 492 pmol/l in response to IL-1beta whereas the cytokine-induced ACTH secretion increased from 140 to 406 pmol/l in wild-type mice. Plasma corticosterone (CORT) rose from basal levels of 99 to 831 nmol/l in knockout mice upon IL-1beta stimulation, whereas in wild-type mice CORT levels rose from 112 to 841 nmol/l. There was no significant difference in IL-1beta-stimulated plasma ACTH or CORT levels between wild-type and knockout mice. Furthermore, there was no significant difference in basal or IL-1beta-stimulated hypothalamic levels of histamine and tele-methyl-histamine between wild-type and knockout mice. HDC gene expression was significantly lower in knockout mice than in wild-type mice both under basal and IL-1beta-stimulated conditions, while there were no significant differences in CRH gene expression in the PVN in knockout mice under basal and IL-1beta-stimulated conditions. Increased basal expression of AVP in the PVN of knockout mice was observed in this study compared to wild-type mice. CONCLUSION: We conclude that the lack of the gene for histamine H(1)R does not seem to be crucial for the ACTH and CORT response to IL-1beta, either due to possible functional compensation in the H(1)R knockout mouse or due to activation of pathways other than the neuronal histaminergic system.

Lessons from the gastrin and gastrin receptor knockout mice.

The gastric hormone gastrin was first recognized for its ability to induce acid secretion. Following the purification and subsequent development of specific radioimmunoassays for gastrin, it was also shown to be a regulator of oxyntic mucosal growth. To examine the importance of gastrin or its receptors during development in general and for gastric physiology specifically both have been knocked out. Gastrin and gastrin receptor knockout mice are viable, develop without any gross abnormalities, and are fertile. Even though gastrin acts as a growth factor during hypergastrinemia there was no general atrophy of the gastric mucosa in the knockout mice. However, the maturation of both parietal and ECL cells was disturbed and the number of parietal cells was reduced. Basal acid secretion was impaired and rendered the parietal cells unresponsive to secretagogues. Outside the stomach the mice had no apparent phenotype. However, studies have suggested that progastrin and glycine-extended proforms of gastrin may have biological importance, but these results are still circumstantial and identification of the implicated receptors will be crucial for further studies.

E2F repression by C/EBPalpha is required for adipogenesis and granulopoiesis in vivo.

The C/EBPalpha transcription factor is required for differentiation of adipocytes and neutrophil granulocytes, and controls cellular proliferation in vivo. To address the molecular mechanisms of C/EBPalpha action, we have identified C/EBPalpha mutants defective in repression of E2F-dependent transcription and found them to be impaired in their ability to suppress cellular proliferation, and to induce adipocyte differentiation in vitro. Using targeted mutagenesis of the mouse germline, we show that E2F repression-deficient C/EBPalpha alleles failed to support adipocyte and granulocyte differentiation in vivo. These results indicate that E2F repression by C/EBPalpha is critical for its ability to induce terminal differentiation, and thus provide genetic evidence that direct cell cycle control by a mammalian lineage-instructive transcription factor couples cellular growth arrest and differentiation.

Attenuated processing of proglucagon and glucagon-like peptide-1 in carboxypeptidase E-deficient mice.

The maturation of many peptide hormones is attenuated in carboxypeptidase E (CPE)-deficient fat/fat mice, leading to a slowly developing, adult-onset obesity with mild diabetes. To determine the contribution of the hormones generated from the proglucagon precursor to this phenotype, we studied the tissue-specific processing of glucagon and glucagon-like peptide-1 (GLP-1) in these mice. In all tissues examined there was a great reduction in mature amidated GLP-1. Furthermore, a lack of CPE attenuates prohormone convertase processing of proglucagon in both the pancreas and the intestine. These findings suggest that defects in proglucagon processing together with other endocrine malfunctions could contribute to the diabetic and obesity phenotype in fat/fat mice.

Structure and developmental expression of the mouse CCK-B receptor gene.

Cholecystokinin (CCK) and gastrin exert their effects through two receptors, the CCK-A and CCK-B receptors. We have cloned the mouse CCK-B receptor gene (Cckbr) and determined its complete genomic structure, nucleotide sequence, and tissue-specific expression pattern. Cckbr is divided into five exons spanning 11 kb. A primer extension assay was used to map the transcription initiation site to 199 bp upstream of the translational start site. Rapid amplification of cDNA ends was used to localize the 3' end downstream of an atypical polyadenylation site (GATAAA). Mouse Cckbr transcripts were most abundant in brain and stomach, but were also detected in colon, kidney, ovary, and pancreas. Prenatal expression of both CCK-A and CCK-B receptors in various tissues was analyzed by RT-PCR. The expression pattern was similar to the adult pattern, suggesting that receptor transcription is an early event in gastrointestinal development.

Impaired feedback of gastric functions in carboxypeptidase E-deficient mice.

Carboxypeptidase E deficiency as seen in the fat/fat mice is associated with reduced antral somatostatin content but tripling of the progastrin product. Thus, fat/fat mice are able to maintain normal tissue concentrations of bioactive alpha-amidated gastrin in spite of grossly attenuated progastrin processing. After induction of achlorhydria, however, neither the amount of alpha-amidated gastrin nor the total progastrin product increased in the fat/fat mice. This is contrary to what is seen in wild-type mice. Furthermore, the synthesis of antral somatostatin and fundic chromogranin A is also abnormal. Hence the results suggest a breakdown in the feedback loop that regulates gastric acid secretion.