Simple and efficient method for isolation and cultivation of endoscopically obtained human colonocytes

Few comparative and validated reports exist on the isolation and growth of colonoscopically obtained colonic epithelium. The aim of this study was to develop and validate a simple method for the cultivation of colonoscopically obtained colonocytes. Forty patients, who underwent routine colonoscopy and where the diagnosis of irritable bowel syndrome was later reached, were included. Seven colon biopsies were taken and incubated at varying time periods of 10-120 min and temperatures of 4-37 degrees C in a chelating buffer. The epithelium was then harvested and cultivated under three different conditions: 1) on a collagen coating, 2) embedded in a collagen gel, or 3) embedded in a gel put on a porous well insert. The effect of conditioned medium (CM), insulin, transferrin, selenium, and the oxygen content was assessed. Viability was tested by the metabolic dimethylthiazol-diphenyl-tetrazolium bromide assay, by flowcytometry, by phase contrast microscopy, and by transmission electron microscopy. Incubation at 21 degrees C for 75 min gave an optimal yield of 3 x 10(6) (2.0-3.8 x 10(6)) viable epithelial cells in intact crypts per seven biopsies. Embedding of crypts in a collagen gel put on a porous membrane was superior to the other methods applied [P < 0.003; median viability 71% (62-100%) compared with preculture values] after 24 h, which was a 160% increase in viability compared with coat-cultivated cells. CM had similar viability supporting effects to FCS. Other supplements had no effects. A simple method is presented, which makes cultivation of colonocytes obtained at endoscopy possible for up to 72 h.     

Dysfunctional MreB inhibits chromosome segregation in Escherichia coli

The mechanism of prokaryotic chromosome segregation is not known. MreB, an actin homolog, is a shape-determining factor in rod-shaped prokaryotic cells. Using immunofluorescence microscopy we found that MreB of Escherichia coli formed helical filaments located beneath the cell surface. Flow cytometric and cytological analyses indicated that MreB-depleted cells segregated their chromosomes in pairs, consistent with chromosome cohesion. Overexpression of wild-type MreB inhibited cell division but did not perturb chromosome segregation. Overexpression of mutant forms of MreB inhibited cell division, caused abnormal MreB filament morphology and induced severe localization defects of the nucleoid and of the oriC and terC chromosomal regions. The chromosomal terminus regions appeared cohered in both MreB-depleted cells and in cells overexpressing mutant forms of MreB. Our observations indicate that MreB filaments participate in directional chromosome movement and segregation.     

Pilot study of elevated levels of insulin-like growth factor-binding protein-2 as indicators of hepatocellular carcinoma

BACKGROUND/AIMS: Insulin-like growth factor-binding protein-2 (IGFBP-2) is expressed in many malignant tissues, and elevated serum levels can be indicators of tumour activity in addition to conventional tumour markers. Our aim was to evaluate the role of IGFBP-2 levels together with insulin-like growth factor (IGF)-I, IGF-II and IGFBP-3 in the diagnostic work-up of patients with hepatocellular carcinoma (HCC). METHODS: In 50 (39 males, 11 females) histologically confirmed and TNM-graded patients with HCC who had not received adjuvant chemotherapy, the basal serum levels of IGF-I, IGF-II, IGFBP-3, IGFBP-2 and alpha-fetoprotein (AFP) were measured. The median age of the patients was 66 (37-84) years, body mass index was normal (25 (35-16) kg/m2). RESULTS: The levels of IGF-I, IGF-II and IGFBP-3 were diminished, as is the case when nutrition, hepatic function and growth hormone (GH) secretion are decreased. The levels of AFP and IGFBP-2 were markedly high. In 37 cases, IGFBP-2 levels were above the age-related norm, and in 40 cases AFP levels were also elevated. In 3 cases, both AFP and IGFBP-3 were normal, and in 4 cases AFP was high but IGFBP-2 normal, whereas in 10 cases AFP was normal but IGFBP-2 was high. CONCLUSIONS: In addition to AFP, IGFBP-2 appears to be a suitable marker for the evaluation of the serological status of HCC patients. A longitudinal study during disease management is required to assess the full potential of IGFBP-2 measurements as a marker.     

Relevance of IGF-I, IGFBP-3, and IGFBP-2 measurements during GH treatment of GH-deficient and non-GH-deficient children and adolescents

BACKGROUND: Little information is available on the relevance of parameters representing the insulin-like growth factor (IGF) system with regard to growth hormone (GH) treatment during childhood. In adults, high IGF-I levels were found to be associated with side effects and long-term risks. AIM/METHOD: Our aim was to monitor the serum levels of IGF-I, IGF-binding protein (IGFBP) 3, and IGFBP-2 during long-term GH treatment of 156 patients with GH deficiency (GHD) and of 153 non-GHD patients. We determined the extent to which the IGF parameters exceed the normal ranges and identified those parameters which are predictive of 1st-year growth. RESULTS: In prepubertal GHD children, the levels of IGF-I, IGFBP-3, and IGF-I/IGFBP-3 exceeded the 95th centile of the reference values for this age group in 2.3, 0.3, and 7.9% of the cases, respectively, whereas in prepubertal non-GHD children, the same parameters exceeded the 95th reference centile in 20.1, 3.5, and 32.2%, respectively. In pubertal GHD children IGF-I, IGFBP-3, and IGF-I/IGFBP-3 levels exceeded the 95th reference centile in 11.1, 1.5, and 15.4%, respectively. In pubertal non-GHD children, these levels also exceeded the 95th centile in 26.7, 7.0, and 41.4%, respectively. In both GHD and non-GHD groups, however, some patients had IGF parameters which were below the reference values. Our analysis showed that, in both groups, in addition to maximum GH, all IGF parameters (IGF-I, IGFBP-3, IGF-I/IGFBP-3 ratio, IGFBP-2 or derivatives) significantly extend the scope of a calculated model for predicting 1st-year height velocity. CONCLUSION: For reasons of safety and optimization of GH therapy, it is essential to follow up IGF-I, IGFBP-3, and IGFBP-2 levels regularly during childhood.     

Experience with growth hormone therapy in Turner syndrome in a single centre: low total height gain, no further gains after puberty onset and unchanged body proportions

The experience gained since 1987, through observation of 85 girls with Turner syndrome under growth hormone (GH) treatment, has enabled the analysis of one of the largest cohorts. Our results show that age, karyotype and height reflect the heterogeneity of the patients examined at our growth centre. In 47 girls, followed over 4 years on GH (median dose 0.72 IU/kg/week), the median age was 9.4 years and mean height SDS was -3.55 (Prader) and -0.14 (Turner-specific), while height and other anthropometrical parameters [weight, body mass index, sitting height (SH), leg length (LL) SH/LL, head circumference, arm span] were documented and compared to normative data as well as to Turner-specific references established on the basis of a larger (n = 165) untreated cohort from Tübingen. The latter data are also documented in this article. Although there was a trend towards normalization of these parameters during the observation period, no inherent alterations in the Turner-specific anthropometric pattern occurred. In 42 girls who started GH treatment at a median age of 11.8 years, final height (bone age >15 years) was achieved at 16.7 years. The overall gain in height SDS (Turner) from start to end of GH therapy was 0.7 (+/- 0.8) SD, but 0.9 (+/- 0.6) SD from GH start to onset of puberty (spontaneous 12.2 years, induced 13.9 years) and -0.2 (+/- 0.8) from onset of puberty to end of growth. Height gain did not occur in 12 patients (29%) and a gain of > 5 cm was only observed in 16 patients (38%). Height gain correlated positively with age at puberty onset, duration, and dose of GH, and negatively with height and bone age at the time GH treatment started. Final height correlated positively with height SDS at GH start and negatively with the ratio of SH/LL (SDS). We conclude that, in the future, GH should be given at higher doses, but oestrogen substitution should be done cautiously, owing to its potentially harmful effect on growth. LL appears to determine height variation in Turner syndrome and the potential to treat short stature successfully with GH.     

Mass spectrometry unravels disulfide bond formation as the mechanism that activates a molecular chaperone

The heat shock protein Hsp33 is a very potent molecular chaperone with a distinctive mode of functional regulation; its activity is redox-regulated. In its reduced form all six cysteinyl residues of Hsp33 are present as thiols, and Hsp33 displays no folding helper activity. Exposure of Hsp33 to oxidizing conditions like H(2)O(2), however, rapidly converts Hsp33 into an efficient molecular chaperone. Activated Hsp33 binds tightly to refolding intermediates of chemically denatured luciferase and suppresses efficiently their aggregation in vitro. Matrix-assisted laser desorption/ionization-mass spectrometry peptide mapping in combination with in vitro and on target protein chemical modification showed that this activation process of Hsp33 is accompanied by the formation of two intramolecular disulfide bonds within Hsp33: Cys(232)-S-S-Cys(234) and Cys(265)-S-S-Cys(268). Cys(141), although not involved in disulfide bond formation, was found highly reactive toward chemical modifications. In contrast, Cys(239) is readily accessible under reducing conditions but becomes poorly accessible though still reduced when Hsp33 is in its active state. This indicates a significant conformational change during the activation process of Hsp33. Mass spectrometry, thus, unraveled a novel molecular mechanism by which alteration of the disulfide bond structure, as a result of changes in the cellular redox potential, results in the activation of a molecular chaperone.