Cytogenetics, in situ hybridization and molecular approaches in the diagnosis of cancer

BACKGROUND: Dysmotility in the gastrointestinal tract increases with age. Colonic endocrine cells play an important role in regulating intestinal secretion and motility. The objective was to study possible age-related changes in the colonic endocrine cells of an animal model. METHODS: The colonic endocrine cells in four different age groups of mice were investigated by immunocytochemistry and quantified by computerized image analysis. The ages of these groups were 1, 3, 12 and 24 months old. RESULTS: The numbers of peptide YY (PYY), enteroglucagon and serotonin immunoreactive (IR) cells in 1-month-old mice were significantly increased compared with those of 3-month-old mice. Similarly, the numbers of these cells were significantly greater in 12- and 24-month-old mice than in 3-month-old mice. The cell secretory index (CSI) of enteroglucagon and serotonin IR cells was higher in 1-, 12- and 24-month-old mice than in 3-month-old mice. There was no significant difference between the different age groups regarding the CSI of PYY IR cells, nor was there any statistical difference between females and males in all endocrine cell types regarding numbers and CSI. CONCLUSION: It is suggested that the increase in colonic endocrine cells prior to puberty might reflect the role of gut hormones in the development of the gastrointestinal tract. It is speculated further that the increase in colonic endocrine cells with ageing may compensate for increased receptor resistance and/or weakened response of effector organs. It is suggested that the increase in numbers and unchanged CSI of PYY cells with advancing age may be responsible for the slow colonic transit and constipation, both of which increase with age.     

Identification of rCop-1, a new member of the CCN protein family, as a negative regulator for cell transformation

By using a model system for cell transformation mediated by the cooperation of the activated H-ras oncogene and the inactivated p53 tumor suppressor gene, rCop-1 was identified by mRNA differential display as a gene whose expression became lost after cell transformation. Homology analysis indicates that rCop-1 belongs to an emerging cysteine-rich growth regulator family called CCN, which includes connective-tissue growth factor, CYR61, CEF10 (v-src inducible), and the product of the nov proto-oncogene. Unlike the other members of the CCN gene family, rCop-1 is not an immediate-early gene, it lacks the conserved C-terminal domain which was shown to confer both growth-stimulating and heparin-binding activities, and its expression is lost in cells transformed by a variety of mechanisms. Ectopic expression of rCop-1 by retroviral gene transfers led to cell death in a transformation-specific manner. These results suggest that rCop-1 represents a new class of CCN family proteins that have functions opposing those of the previously identified members.     

Cloning and primary structure of the chiA gene from Aeromonas caviae

The chiA gene from Aeromonas caviae encodes an extracellular chitinase, 865 amino acids long, that shows a high degree of similarity to chitinase A of Serratia marcescens. Expression in Escherichia coli yielded an enzymatically active protein from which a leader sequence was removed, presumably during transport of the enzyme across the cell membrane.     

Zinc, insulin and diabetes

Miso, a widely used Japanese fermented food was analysed for its lactic acid bacterial count on bromocresol purple agar. The binding of eight different foodborne carcinogenic heterocyclic amines to 25 bacterial isolates from miso were investigated. The heterocyclic amines used were 3-amino-1,4-dimethyl[5H]pyrido(4,3-b)indole (Trp-P-1), 3-amino-1-methyl[5H]pyrido(4,3-b)indole (Trp-P-2), 2-amino-6-methyldipyrido(1,2-a:3'2'-d)imidazole (Glu-P-1), 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), 2-amino-dimethylimidazo(4,5f)quinoline (IQ), 2-amino-3,4-dimethylimidazo(4,5-f) quinoline (MeIQ), 2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline (MeIQx), and 2-amino-3-methyl-9H-pyrido(2,3)indole (MeA alpha C). The lyophilized cells of all of the isolates exhibited high binding activity towards Trp-P-1, Trp-P-2, MeA alpha C, and PhIP, while Glu-P-1 and IQ were not effectively bound. Of the isolates tested, the strongest and weakest binders were identified as Pediococcus acidilactici 1 and 2, respectively. Lyophilized cell wall fractions, heat-treated cells, and the cytoplasmic contents of P. acidilactici 1 and 2 were analysed for their ability to bind to different mutagens. Pure cell wall and peptidoglycan showed greater binding activity than the bacterial cells. Cytoplasmic content also showed some binding, but it was much less effective. The impact of enzymes (amylase, protease, cellulase, chitinase, muraminase, and peptidase) and acetylation of Trp-P-1 and IQ on the binding action of bacteria and cell wall material were also analysed to understand the possible processes involved in the binding of lactic acid bacteria to carcinogenic heterocyclic amines.     

Primary sequence and solution conformation of ferrocytochrome c-552 from Nitrosomonas europaea

Cytochrome c-552 from Nitrosomonas europaea is a 9.1-kDa monoheme protein that is a member of the bacterial cytochrome c-551 family. The gene encoding for c-552 has been cloned and sequenced and the primary sequence of the product deduced. Proton resonance assignments were made for all main-chain and most side-chain protons in the diamagnetic, reduced form by two-dimensional NMR techniques. Distance constraints (1056) were determined from nuclear Overhauser enhancements, and torsion angle constraints (88) were determined from scalar coupling estimates. Solution conformations for the protein were computed by the hybrid distance geometry-simulated annealing approach. For 20 computed structures, the root mean squared deviation from the average position of equivalent atoms was 0.84 A (sigma = 0.12) for backbone atoms over all residues. Analysis by residue revealed there were three regions clearly less well defined than the rest of the protein: the first two residues at the N-terminus, the last two at the C-terminus, and a loop region from residues 34 to 40. Omitting these regions from the comparison, the root mean squared deviation was 0.61 A (sigma = 0.13) for backbone atoms, 0.86 A (sigma = 0.12) for all associated heavy atoms, and 0. 43 A (sigma = 0.17) for the heme group. The global folding of the protein is consistent with others in the c-551 family. A deletion at the N-terminus relative to other family members had no impact on the global folding, whereas an insertion at residue 65 did affect the way the polypeptide packs against the methionine-ligated side of the heme. The effects of specific substitutions will be discussed. The structure of c-552 serves to delineate essential features of the c-551 family.     

Clinicopathological analysis and therapy in hepatitis C virus-associated nephropathy

The authors evaluated, using computer image analysis system, 15 renal biopsy specimens from patients with MCD and 15 from patients with FSGS. As a control group 5 biopsy and 10 autopsy renal specimens were used. These studies revealed, that in MCD glomeruli showed proliferative changes unrecognizable by light microscopy. In FSGS glomeruli without sclerotic changes were larger than in controls and showed glomerular cells proliferation (per unit of glomerular area) similar to MCD. FSGS in comparison with MCD showed larger glomeruli and prominent interstitial fibrosis. These observations appeared to be helpful in distinguishing MCD and recent forms of FSGS. There were no correlations between relative interstitial volume and glomerular changes in MCD as well as in FSGS. The authors concluded, that histomorphometry can't be used instead of morphological examinations of renal biopsy specimens, but it gives objective informations about progression of the illness and may be useful in differential diagnosis of selected cases of MCD and FSGS.     

Responses of human birch pollen allergen-reactive T cells to chemically modified allergens (allergoids)

BACKGROUND: Allergoids are widely used in specific immunotherapy for the treatment of IgE-mediated allergic diseases. OBJECTIVE: The aim of this study was to analyse whether a modification of birch pollen allergens with formaldehyde affects the availability of T-cell epitopes. METHODS: Efficient modification of the allergens was verified by determining IgE and IgG binding activity using ELISA inhibition tests. T-cell responses to birch pollen allergoids were analysed in polyclonal systems, using peripheral blood mononuclear cells (PBMC) of five birch pollen-allergic individuals, as well as birch pollen extract-reactive T-cell lines (TCL), established from the peripheral blood of 14 birch pollen-allergic donors. To determine whether the modification of natural (n)Bet v 1 with formaldehyde or maleic anhydride results in epitope-specific changes in T-cell reactivities, 22 Bet v 1-specific T-cell clones (TCC), established from nine additional birch pollen-allergic individuals, were tested for their reactivity with these products. RESULTS: The majority of PBMC and TCL showed a reduced response to the birch pollen extract allergoid. Bet v 1-specific TCC could be divided into allergoid-reactive and -non-reactive TCC. No simple correlation between possible modification sites of formaldehyde in the respective T-cell epitopes and the stimulatory potential of the allergoid was observed. Mechanisms of suppression or of anergy induction were excluded as an explanation for the non-reactivity of representative TCC. All TCC could be stimulated by maleylated and unmodified nBet v 1 to a similar extent. CONCLUSION: These results demonstrate differences in the availability of T-cell epitopes between allergoids and unmodified allergens, which are most likely due to structural changes within the allergen molecule.     

Comparative pharmacodynamics of CYP2B induction by DDT, DDE, and DDD in male rat liver and cultured rat hepatocytes

In this study the pharmacodynamics were characterized of rat hepatic cytochrome P-450 2B (CYP2B) induction by the pesticide DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane] and its metabolites DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene], which is bioretained, and DDD [1,1-dichloro-2,2-bis(p-chlorophenyl)ethane], which is metabolized further and therefore less prone to bioaccumulate. DDT, DDE, and DDD were each found to be pure phenobarbital-type cytochrome P-450 inducers in the male F344/NCr rat, causing induction of hepatic CYP2B and CYP3A, but not CYP1A. The ED50 values for CYP2B induction (benzyloxyresorufin O-dealkylation) by DDT, DDE, and DDD were, respectively, 103, 88, and > or = 620 ppm in diet (14 d of exposure). The efficacies (Emax values) for induction of benzyloxyresorufin O-dealkylation by DDT, DDE, and DDD were 24-, 22-, and > or = 1-fold, respectively, compared to control values. The potencies of the three congeners for CYP2B induction appeared also to be similar, with EC50 values (based on total serum DDT equivalents) of 1.5, 1.8, and > or = 0.51 microM, respectively. The EC50 values based on DDT equivalents in hepatic tissue were 15, 16, and > or = 5.9 micromol/kg liver tissue, respectively. In primary cultures of adult rat hepatocytes, DDT, DDE, and DDD each displayed ability to induce total cellular RNA coding for CYP2B (ED50 values of 0.98, 0.83, and > or = 2.7 microM, respectively). These results suggest that DDT, DDE, and DDD each possess a high degree of intrinsic CYP2B-inducing ability for rat liver, despite marked differences in bioretention among the congeners.