Expression of non-classical islet hormone-like peptides during the embryonic development of the pancreas

The highly enriched anaerobic bacterium that couples the reductive dechlorination of tetrachloroethene to growth, previously referred to as PER-K23, was obtained in pure culture and characterized. The bacterium, which does not form spores, is a small, gram-negative rod with one lateral flagellum. It utilized only H2 as an electron donor and tetrachloroethene and trichloroethene as electron acceptors in an anaerobic respiration process; it could not grow fermentatively. Acetate served as a carbon source in a defined medium containing iron as the sole trace element, the two vitamins thiamine and cyanocobalamin, and the three amino acids arginine, histidine, and threonine. The cells contained menaquinones and b-type cytochromes. The G+C content of the DNA was 45.3 +/- 0.3 mol%. The cell wall consisted of type-A3gamma peptidoglycan with ll-diaminopimelic acid and one glycine as an interpeptide bridge. The cells are surrounded by an S-layer; an outer membrane was absent. Comparative sequence analysis of the 16S rRNA sequence showed that PER-K23 is related to gram-positive bacteria with a low G+C content of the DNA. Based on the cytological, physiological, and phylogenetic characterization, it is proposed to affiliate the isolate to a new genus, Dehalobacter, with PER-K23 as the type strain of the new species Dehalobacter restrictus.     

Expression pattern for adrenomedullin during pancreatic development in the rat reveals a common precursor with other endocrine cell types

We cloned a cDNA encoding tissue inhibitor of metalloproteinases-3 (TIMP3) from the frog Xenopus laevis. Similar to TIMP3 from other species, Xenopus TIMP3 has 188 residues including 12 conserved cysteines and Asn184, a putative site for N-linked sugars. Xenopus TIMP3 is 84% identical with human TIMP3. As shown by Northern blotting and RT-PCR, Xenopus TIMP3 mRNA is maternally inherited in eggs and midblastula (stage 8) embryos, downregulated in gastrula and then upregulated in neurula and pretailbud embryos. In select adult tissues, TIMP3 mRNA is present in heart, muscle, liver, skin, intestine and ovaries. These results suggest that TIMP3 is involved in the regulation of expression of matrix metalloproteinases in Xenopus early development and adult tissue remodeling.     

Prospective study of provocative angiograms to localize functional islet cell tumors of the pancreas

BACKGROUND: Controversy exists concerning the use of preoperative imaging studies in patients with islet cell tumors. Since 1993 we have evaluated the use of provocative angiography in patients with insulinoma or Zollinger-Ellison syndrome (ZES). METHODS: Twelve patients with a working diagnosis of insulinoma (n = 4) or ZES (n = 8) were studied. Of the eight patients with ZES, four were known to have multiple endocrine neoplasia type 1. All patients underwent conventional imaging studies followed by provocative angiography. Angiograms were graded based on the ability to detect tumor and regionalize it within the pancreas. RESULTS: Of the three patients with a working diagnosis of ZES but equivocal results of biochemical studies, none had arteriographic imaging of an islet cell tumor or a positive provocative study result (true negative result). Of the nine patients with documented islet cell tumor, seven (78%) underwent arteriographic imaging of the tumor and eight (89%) had correct regional localization by provocative angiography. Sensitivity and specificity for imaging were 78% and 100%, respectively, and for regional localization 89% and 100%, respectively. CONCLUSIONS: Provocative angiography is the localization study of choice for both gastrinoma and insulinoma. Having few false-negative results, it can be used to corroborate the diagnosis and, having few false-positive results, it detects tumor and biochemically confirms localization in nearly every patient.     

Expression of GATA-4 in migrating gonadotropin-releasing neurons of the developing mouse

Epidemiological studies indicating that nitrite inhalant abuse is a co-factor in HIV infection and in Kaposi's sarcoma are supported by recent experimental studies, described in this review. Inhalation exposure to the nitrites produce a nonspecific cytotoxicity, depleting many cells of the immune system. Apparently distinct from this cytotoxicity, inhalation of the nitrites impairs a variety of immune mechanisms, affecting both humoral and cell-mediated immunity. In addition, the inhalant-increased macrophage production of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), can directly stimulate HIV replication and can also stimulate the growth of Kaposi's sarcoma cells. Thus, nitrite inhalants may impair immune resistance to infection and actively promote viral replication and tumor growth.     

Expression patterns of bone morphogenetic proteins (Bmps) in the developing mouse tooth suggest roles in morphogenesis and cell differentiation

Bone morphogenetic proteins (BMP) are secretory signal molecules which have a variety of regulatory functions during morphogenesis and cell differentiation. Teeth are typical examples of vertebrate organs in which development is controlled by sequential and reciprocal signaling between the epithelium and mesenchyme. In addition, tooth development is characterized by formation of mineralized tissues: the bone-like dentin and cementum as well as epithelially derived enamel. We have performed a comparative in situ hybridization analysis of the expression of six different Bmps (Bmp-2 to Bmp-7) starting from initiation of tooth development to completion of crown morphogenesis when dentine and enamel matrices are being deposited. Bmps-2, -4, and -7 were frequently codistributed and showed marked associations with epithelial-mesenchymal interactions. Their expression shifted between the epithelium and mesenchyme starting from the stage of tooth initiation. They were subsequently expressed in the enamel knot, the putative signaling center regulating tooth shape. Their expression domains prior to and during the differentiation of the dentine-forming odontoblasts and enamel-forming ameloblasts was in line with functions in regulation of cell differentiation and/or secretory activities of the cells. The expression of Bmp-3 was confined to mesenchymal cells, in particular to the dental follicle cells which give rise to the cementoblasts, forming the hard tissue covering the roots of teeth. Bmp-5 was expressed only in the epithelial ameloblasts. It was upregulated as the cells started to polarize and intense expression continued in the secretory ameloblasts. Bmp-6 was expressed only weakly in the dental mesenchyme during bud and cap stages. Our results are in line with regulatory functions of Bmps at all stages of tooth morphogenesis. Bmps-2, -4, and -7 are conceivably parts of signaling networks regulating tooth initiation and shape development. They as well as Bmp-5 may be involved in the induction and formation of dentine and enamel, and Bmp-3 in the development of cementum. The remarkable overlaps in the expression domains of different Bmp genes may implicate functional redundancy and/or formation of active heterodimers between different BMPs.     

Expression and function of the type 3 complement receptor in tissues of the developing mouse

Macrophage (Mphi) expression of the leukocyte integrins has been implicated in their adhesion and migration in the adult. Little is known, however, of the expression or function of these molecules during development. This study defines the spatial and temporal sequences of expression of the type 3 complement receptor (CR3) in the developing mouse; establishes the functional efficacy of this molecule in spreading, adhesion, and phagocytosis; and investigates its role in inflammatory and constitutive migration. Expression of CR3 on monocytes occurred early compared to Mphi-restricted glycoprotein F4/80, but expression on stellate tissue Mphi appeared later than F4/80 and was transient. Expression of CR3 on resident tissue Mphi is more widespread during development, being retained on only very specific Mphi populations in the adult. Neutrophil polymorphs expressed CR3 from day 17 of gestation onward. The anti-CR3 mAb 5C6 was used to investigate the role of CR3 in adhesion, spreading, and phagocytosis by neonatal Mphi. Neonatal macrophages were found to adhere, spread, and phagocytose by CR3-dependent mechanisms, and a CR3-independent system was implicated in the spreading of neonatal Mphi. The role of CR3 in migration during development was then investigated. 5C6 had potent effects on the early stages of the migration of myelomonocytic cells to an inflammatory stimulus in vivo. Despite efficient transplacental transfer of the Ab from pregnant mother to fetus, the process by which monocytes generate populations of resident tissue Mphi was undisrupted, indicating the existence of CR3-independent mechanisms of monocyte migration during development.     

Multihormone-producing islet cell tumor of the pancreas associated with somatostatin-immunoreactive amyloid: immunohistochemical and immunoelectron microscopic studies

OBJECTIVE: To assess the association between high but nondiabetic blood glucose levels and the risk of death from all causes, coronary heart disease (CHD), cardiovascular disease, and neoplasms. RESEARCH DESIGN AND METHODS: We studied the 20-year mortality of non-diabetic, working men, age 44-55 years, in three European cohorts known as the Whitehall Study (n = 10,025), the Paris Prospective Study (n = 6,629), and the Helsinki Policeman Study (n = 631). These men were identified by their 2-h glucose levels following an oral glucose tolerance test and by the absence of a prior diagnosis of diabetes. As the protocol for the oral glucose tolerance test and methods for measuring glucose differed between studies, mortality was analyzed according to the percentiles of the 2-h and fasting glucose distributions, using the Cox's proportional hazards model. RESULTS: Men in the upper 20% of the 2-h glucose distributions and those in the upper 2.5% for fasting glucose had a significantly higher risk of all-cause mortality in comparison with men in the lower 80% of these distributions, with age-adjusted hazard ratios of 1.6 (95% CI 1.4-1.9) and 2.0 (1.6-2.6) for the upper 2.5%. For death from cardiovascular and CHD, men in the upper 2.5% of the 2-h and fasting glucose distributions were at higher risk, with age-adjusted hazard ratios for CHD of 1.8 (1.4-2.4) and 2.7 (1.7-4.4), respectively. CONCLUSIONS: If early intervention aimed at lowering blood glucose concentrations can be shown to reduce mortality, it may be justified to lower the levels of both 2-h and fasting glucose, which define diabetes.     

M-cadherin distribution in the mouse adult neuromuscular system suggests a role in muscle innervation

M-cadherin belongs to the Ca(2+)-dependent cadherin family of cell adhesion molecules and was first isolated from a mouse muscle cell line cDNA library. It is specifically expressed in muscle tissue during development and is supposed to play an important role in secondary myogenesis. In the present study the expression of M-cadherin mRNA and protein and its localization were investigated in adult mouse skeletal muscle and peripheral nerve. The mRNA was abundant in embryonic legs from embryonic day (E)14 to E18. It remained expressed in new-born and adult muscles. In the adult muscle M-cadherin immunoreactivity was only detected at the neuromuscular junction, associated with perijunctional mononucleated cells and on intramuscular nerves. Peripheral nerves were also M-cadherin-positive. The molecule was found at the surface of myelinated nerve fibres where it was concentrated at the node of Ranvier. When a nerve was crushed and allowed to regenerate, M-cadherin was over-expressed at the site of nerve injury and in the distal stump. M-cadherin was also upregulated on the sarcolemma of denervated muscle fibres. Taken together, these observations point toward a much wider tissue distribution of M-cadherin than previously thought. M-cadherin might be involved not only in specific steps of myogenesis but also in some aspects of synaptogenesis, axon/Schwann cell interactions and node of Ranvier structural maintenance.     

Expression of 72-kDa gelatinase (matrix metalloproteinase-2) in the developing mouse craniofacial complex

Tissue remodelling is an important feature during embryogenesis. Although the matrix metalloproteinases are believed to participate in these processes, the relation between matrix metalloproteinases and tissue remodelling during craniofacial morphogenesis remains unclear. The purpose of the study was to look for the presence of enzymes involved in extracellular matrix degradation during craniofacial morphogenesis. Protein expression of the matrix metalloproteinase, 72-kDa gelatinase (matrix metalloproteinase-2, gelatinase A, 72-kDa type IV collagenase) was studied by gelatine zymography and by indirect immunofluorescence with conventional and confocal microscopy. In the anterior region of the developing mouse face, 72-kDa gelatinase was labelled mainly in the tips and peripheral regions of the nasal and facial prominences. Upon contact and fusion of the prominences, the staining was intensely localized to the zone of the fusion and the tips and peripheral regions of the nasal prominences and the maxilla. The labelling of 72-kDa gelatinase was also present in the peripheral regions of the mandible, second branchial arch, and the face around the developing eye. However, during lens vesicle formation, the staining of 72-kDa gelatinase was absent in the invaginated lens ectoderm. After the lens had completely detached from the surface ectoderm, the staining was resumed in the corneal epithelium and mesenchyme. Gelatine zymography was used to confirm the presence of active and latent 72-kDa gelatinase in the developing mouse craniofacial complex. Collectively, these data indicate that 72-kDa gelatinase may play a significant part in localized tissue remodelling during craniofacial morphogenesis and the aberrant expression or function of the enzyme could be involved in causing facial abnormalities.     

Association of ecto-5'-nucleotidase with specific cell types in the adult and developing rat olfactory organ

A unique feature of the olfactory epithelium is its ability to give rise to new sensory neurons throughout life and also following injury. Cells at the basal side of the epithelium serve as neurogenic progenitor cells. The enzyme ecto-5'-nucleotidase is expressed at the surface of developing nerve cells and is regarded as a marker of neural development. To study the expression pattern of the enzyme, we analyzed its distribution in the adult and developing rat olfactory organ. Labeling is restricted to specific cell types and varies between the epithelia investigated. At the basal side of the olfactory epithelium, activity of 5'-nucleotidase is associated specifically with the dark/horizontal basal cells. Neither the light/globose basal cells, which are the immediate precursors of the sensory receptor cells, nor subsets of potentially immature olfactory receptor cells are labeled. On the other hand, microvillar cells dispersed at the lumenal side of the epithelium contain 5'-nucleotidase activity. The enzyme is also present at the inner lining of the ducts of Bowman's glands as they traverse the epithelium. Within the respiratory epithelium, activity of 5'-nucleotidase is associated with basal cells as well as with the epithelial surface. During development, 5'-nucleotidase is initially limited to the respiratory epithelium, including its basal cells. Dark/horizontal basal cells of the olfactory epithelium, which are positive for 5'-nucleotidase, first appear at the border of the respiratory epithelium, suggesting that they might originate from immigrating basal cells of the respiratory epithelium. Within the vomeronasal organ, labeling is largely restricted to the receptor-free epithelium. Although the functional role of 5'-nucleotidase in the olfactory system needs to be further defined, the distribution of the enzyme can be used successfully as a marker for defined cell types.     

Expression of functional MHC class II molecules by a mouse pro-B cell clone

OBJECTIVE: Pooled bronchoalveolar lavage fluid (BALF), the return of lavage, contains both bronchial and alveolar material which differ from each other. Artifacts may be created by filtering, centrifuging and washing cells before cytopreparation. This study presents reference values of healthy volunteers for the alveolar sample, ALF, cytopreparation being performed without filtration or centrifugation. METHODS: Eighteen healthy, non-smoking volunteers underwent a standard bronchoalveolar lavage using 10 aliquots of 20 ml of saline. Excluding the return of the first and second aliquots, the rest were pooled and examined cytologically, immunocytochemically and biochemically. The mean, standard deviation, and 95% confidence limits were calculated for the following variables: amount of return, estimated content of epithelial lining fluid (ELF), total and differential cell counts on filter and cytocentrifuge (CCF) preparations, computed cell counts per unit volume of ALF, distribution of lymphocyte subgroups CD3+CD2, CD4, CD8, CD19, CD25 and CD57, and the ratio of CD4 to CD8, the amounts of lymphocytes in the same subgroups per volume of ALF, and the concentrations of total protein, albumin, immunoglobulins A, G and M, hyaluronic acid, eosinophilic cationic protein (ECP), procollagen III aminoterminal propeptide (PCP) and beta 2-microglobulin in ALF and in ELF, as well as the ratios of the concentrations of the solutes in ALF to the same in serum. RESULTS: The 95% confidence limits of means for the most important variables were as follows: estimated ELF content 0.42-0.74%; total cells in ALF 76.6-143.0 x 10(6) l-1; distribution of inflammatory cells on filter and CCF slides: macrophages 74.9-83.6 and 81.4-90.1%, lymphocytes 13.1-22.5 and 8.1-16.4%, and neutrophils 1.0-4.1 and 0.7-2.7%, respectively; distribution of lymphocyte subsets: CD3+CD2 85.6-90.6%, CD4 44.3-53.1%, CD8 26.9-35.8%; concentration of solutes in ALF: total protein 44.8-61.3 mg l-1, albumin 15.4-22.2 mg l-1, IgA 1.8-3.4 mg l-1, IgG 3.1-6.1 mg l-1, IgM 0.05-0.26 mg l-1, hyaluronic acid 8.8-11.1 micrograms l-1, ECP 0.19-0.77 micrograms l-1, PCP 0.005-0.58 micrograms l-1, beta 2-microglobulin 62.2-81.5 micrograms l-1. CONCLUSIONS: Our results show that excluding the bronchial sample from ALF of volunteer subjects and omitting filtering and washing before cytopreparation produces cytologic, immunocytochemical and biochemical reference values with reasonable 95% confidence limits to be used in clinical settings.     

Colonic mucin discharge by a cholinergic agonist, prostaglandins, and peptide YY in the isolated vascularly perfused rat colon

Efforts to understand the function of dystrophin, the protein product for the Duchenne muscular dystrophy gene, resulted in the purification of the dystrophin-glycoprotein complex. Over the past year several novel components of this complex have been identified. Recent studies have extended the number of muscular dystrophies associated with the oligomeric complex to six genetically distinct diseases, including three new forms of limb-girdle muscular dystrophy and one form of congenital muscular dystrophy.     

Expression of cell adhesion molecules and common acute lymphoblastic leukaemia antigen in hepatoblastoma

Hepatoblastoma is an embryonal tumour of the liver, which often contains tissue components with multidirectional differentiation. The occurrence of cell surface antigens in this tumour has not been studied systematically, and we therefore investigated 20 hepatoblastomas for the expression of common acute lymphoblastic leukaemia antigen (CALLA) and cell adhesion molecules (CAMs) in their different tissue components. Epithelial tumour cells of fetal differentiation contained E-cadherin. This protein did not occur in tumour areas with embryonal or mesenchymal differentiation. In contrast, immature embryonal and anaplastic cells expressed CALLA and the hyaluronate receptor (HCAM, CD44). Both fetal and embryonal areas stained irregularly positive for ICAM-1, which, in contrast, was not present on anaplastic cells. Immature fibrous tissue did not contain any of these molecules except for ICAM-1. However, some cells adjacent to, or enclosed in, osteoid were positive for HCAM and NCAM. Like small undifferentiated hepatoblastoma cells, primitive mesenchymal spindle-shaped cells also expressed CALLA, HCAM, and the polysialylated embryonic form of NCAM strongly. This last is not present on other epithelial or mesenchymal tumour cells. Hepatoblastoma cells of varying differentiation express distinct patterns of CAMs and CALLA. Our results give further insight into their histogenesis and cellular interactions and may explain their variable ability for invasive growth and formation of metastases.     

Different members of the fibroblast growth factor receptor family are specific to distinct cell types in the developing chicken embryo

Single-stranded RNA probes for the three chicken fibroblast growth factor (FGF) receptors, cek-1, cek-2, and cek-3, in conjunction with in situ hybridization were used to characterize the distribution of the corresponding mRNAs in the developing chicken embryo. Cek-1 was expressed diffusely in most tissues examined, whereas the expression of cek-2 and cek-3 was more restricted. The highest levels of FGF receptor expression were seen in the developing bones; in skeletal, cardiac, and smooth muscle; and in some areas of the brain. Although all three receptors were expressed in a number of the same tissues, the expression of each receptor within a given tissue was generally specific for different cell types. In addition, the distribution of each of these receptors did not correlate with the previously characterized distributions of individual FGFs. These results suggest that the members of the FGF receptor family may represent cell-type-specific receptors rather than ligand-specific receptors. Thus, the interaction between a growth factor of the FGF family and a given FGF receptor is likely to be controlled to a large extent by spatial constraints, rather than exclusively by high binding affinities.     

Widespread occurrence of three sequence motifs in diverse S-adenosylmethionine-dependent methyltransferases suggests a common structure for these enzymes

Three regions of sequence similarity have been reported in several protein and small-molecule S-adenosylmethionine-dependent methyltransferases. Using multiple alignments, we have now identified these three regions in a much broader group of methyltransferases and have used these data to define a consensus for each region. Of the 84 non-DNA methyltransferase sequences in the GenBank, NBRF PIR, and Swissprot databases comprising 37 distinct enzymes, we have found 69 sequences possessing motif I. This motif is similar to a conserved region previously described in DNA adenine and cytosine methyltransferases. Motif II is found in 46 sequences, while motif III is found in 61 sequences. All three regions are found in 45 of these enzymes, and an additional 15 have motifs I and III. The motifs are always found in the same order on the polypeptide chain and are separated by comparable intervals. We suggest that these conserved regions contribute to the binding of the substrate S-adenosylmethionine and/or the product S-adenosylhomocysteine. These motifs can also be identified in certain nonmethyltransferases that utilize either S-adenosylmethionine or S-adenosylhomocysteine, including S-adenosylmethionine decarboxylase, S-adenosylmethionine synthetase, and S-adenosylhomocysteine hydrolase. In the latter two types of enzymes, motif I is similar to the conserved nucleotide binding motif of protein kinases and other nucleotide binding proteins. These motifs may be of use in predicting methyltransferases and related enzymes from the open reading frames generated by genomic sequencing projects.