Apoptosis and expression of Bax, Bcl-x, and Bcl-2 apoptotic regulatory proteins in colorectal carcinomas, and association with p53 genotype/phenotype.

AIMS: Spontaneous apoptosis and expression of the apoptotic regulatory proteins Bax, Bcl-x, and Bcl-2 were investigated in 50 colorectal carcinomas. The p53 genotypes/phenotypes and BAX genotypes were also determined, and possible associations of these with apoptosis and/or with expression of the different apoptotic regulatory proteins were studied. METHODS: Terminal deoxynucleotidyl transferase (TdT) mediated dUTP labelling of DNA fragments was used to detect apoptotic tumour cells in sections and peroxidase immunohistochemistry was used to assess protein expression. p53 genotype/phenotype was determined using constant denaturant gel electrophoresis/immunoblotting and bax genotype was determined using polymerase chain reaction based methods. RESULTS: The distribution of tumour apoptotic indices was bimodal with a natural cut off at 1.0% (range, 0.0-5.4%); the median fraction of apoptotic tumour cells was 0.8%. Tumour apoptosis was not associated significantly with tumour DNA ploidy status. Normal mucosal tissue had less than 0.1% apoptotic cells. Staining intensities for Bax, Bcl-x, and Bcl-2 were strong; that is, equivalent to or greater than positive normal mucosal cells, in 11 of 50, 20 of 49, and 20 of 48 carcinomas. Frameshift mutations in the bax gene were detected in three of 42 tumours analysed, all of which were DNA diploid, and Bax protein expression in these tumours was absent or very low. Bax, Bcl-x, and Bcl-2 protein expression were not correlated with tumour apoptosis or tumour DNA ploidy status. p53 was expressed in 34 of 50 tumours and p53 gene mutations were detected in 22 of 29 p53 positive tumours analysed. Apoptosis was significantly lower in a greater number of p53 positive tumours than p53 negative tumours. In addition, Bcl-2 protein expression was significantly higher in a greater number of p53 positive tumours compared with p53 negative tumours. Bax and Bcl-x protein expression were not significantly associated with p53 phenotype/genotype. CONCLUSIONS: The results indicate that acquisition of a p53 phenotype is associated with lower spontaneous apoptosis and higher expression of Bcl-2. The results also suggest that p53 is not a major determinant for Bax expression in colorectal carcinomas in vivo.     

Chondroma of the bladder

 This case report describes a chondroma of the bladder in a 63-year-old woman with clinical complaints of pain in the left fossa iliaca. The lesion was a tumour with a lobulated growth pattern composed of chondrocytes embedded in a chondroid matrix. Neither mitotic figures nor increased cellularity were present. Nuclei were inconspicuous. Immunohistochemical examination showed reactivity for S100 and vimentin. Received: 22 April 1997 / Accepted: 25 August 1997     

Episodic variations of prolactin, thyroid-stimulating hormone, luteinizing hormone, melatonin and cortisol in infertile women with subclinical hypothyroidism

Preliminary data have suggested that female infertility due to corpus luteum insufficiency may be caused by subclinical hypothyroidism [exaggerated thyroid-stimulating hormone (TSH) response to thyrotrophin- releasing hormone (TRH) stimulation]. L-Thyroxine supplementation has been recommended to achieve pregnancies in subclinical hypothyroid women. This controlled study was carried out in order to investigate the biochemical diagnosis of subclinical hypothyroidism as a possible infertility factor. Five infertile patients (aged 25-36 years) with subclinical hypothyroidism (n = 4, stimulated TSH >20 microU/ml) or primary hypothyroidism (n = 1) and five healthy controls (aged 22-39 years) with normal thyroid function (stimulated TSH <15 microU/ml), regular cycles and no history of infertility were studied in the early follicular phase. In the pre-study evaluation, eight of 23 volunteers (34.8%) had to be excluded because of subclinical hypothyroidism with stimulated TSH values (TSHs) >15 microU/ml. Cycle function of patients and controls was compared by the method of LH pulse pattern analysis. Therefore blood samples were drawn every 10 min during a 24 h period. Sleep was recorded from midnight to 7 a.m. Repetition of the TRH tests at the end of the 24 h blood sampling period confirmed the difference in stimulated TSH values of the two study groups. Pulse analysis for luteinizing hormone (LH), TSH and prolactin showed no differences between patients and controls for pulse frequency, amplitude, height, length, area under curve (AUC) and the 24 h mean. Even the hypothyroid patient had a normal LH pulse pattern. Additional measurement of melatonin in pooled sera every 30 min gave the well-documented diurnal profiles during day and night for both groups. Patients had significantly higher melatonin values at seven time points during the night. Peaks for LH, TSH, prolactin and cortisol were correlated with the sleep stages wake, rapid eye movement, 1 + 2 and 3 + 4. We concluded that corpus luteum insufficiency in female infertility cannot be explained by subclinical hypothyroidism and thus should not be treated with L-thyroxine for fertility reasons.      

Regulation of the density of spermatogonia in the seminiferous epithelium of the Chinese hamster: I. Undifferentiated spermatogonia

The topographical arrangement of the clones of A single, A paired, and A aligned (As, Apr, and Aal) spermatogonia on the basement membrane of seminiferous tubules of the Chinese hamster was studied. It was found that at least some of these clones are not distributed at random as clones of similar cell number were often seen in clusters. Areas were found with few or many As spermatogonia. Also, clusters of Apr spermatogonia were found, indicating that in such an area many As spermatogonia more or less synchronously formed Apr spermatogonia. Since clusters of clones of 16 Aal spermatogonia were observed, it can be concluded that these clusters of Apr spermatogonia may proliferate in at least a roughly synchronous way. It was found that over large areas the densities of undifferentiated spermatogonia may be very low or high in comparison to the mean density in the animal. Whether the ratio of self-renewal and differentiation of the stem cells changed locally in response to the high or low density of undifferentiated spermatogonia in particular areas was investigated. No indications for a regulatory mechanism to keep the density of stem cells and/or the density of undifferentiated spermatogonial clones at a certain level could be detected in the normal Chinese hamster. This lack of regulation was at least partly responsible for the widely different numbers of A1 spermatogonia that were formed in the various areas studied in stage IX.     

Molecular genetic analysis of familial early-onset Alzheimer's disease linked to chromosome 14q24.3

Genetic linkage studies have indicated that chromosome 14q24.3harbours a major locus for early-onset (onset age <65 years)Alzheimer's disease (AD3). Positional cloning efforts have identifieda novel gene S182 or presenilin 1 as the AD3 gene. We have mappedS182 in the AD3 candidate region between D14S277 and D14S284defined by genetic linkage studies in the two chromosome 14linked, early-onset AD families AD/A and AD/B. We have shownthat S182 is expressed in lymphoblasts and have determined thecomplete cDNA in both brain and lymphoblasts by RT-PCR sequencing.S182 is alternatively spliced in both brain and lymphoblastswithin a putative phosphorylation site located 5' in the codingregion. We identified two novel mutations, Ile143Thr and Gly384Alalocated in, respectively, the second transmembrane domain andin the sixth hydrophilic loop of the putative transmembranestructure of S182. As families AD/A and AD/B have a very similarAD phenotype our observation of two mutations in functionallydifferent domains suggest that onset age and severity of ADmay not be very helpful predictors of the location of putativeS182 mutations.     

Effects of a viral laryngotracheitis on the epithelial barrier of chicken airways

The effects of a virus infection on the barrier function of tracheal epithelium were compared to the effects of a chemical agent (methacholine) which selectively increases membrane permeability, and both were compared to controls. The disruption of the airway epithelium induced by the virus infection caused an increased permeation of horseradish peroxidase (HRP) through this barrier. Methacholine enhanced HRP uptake from the airway lumen to the blood as compared to controls. Visualization of HRP in the tracheal epithelium by transmission electron microscopy correlated with the radioimmunoassay measurements in the blood. Serial anti-HRP antibody titers were measured by a competitive binding technique. The antigen permeation induced by methacholine was associated with an enhanced anti-HRP antibody production. The larger increase in antigen permeation seen with the viral infection was associated with depressed anti-HRP titers. It was concluded that viral disruption of the airway epithelial barrier may contribute to an increased uptake of orally inhaled antigens. The relationship, however, between the increased antigen penetration consequent to the viral infection and the development of allergy remains unclear.     

Reduced activity of the red-cell sodium-potassium pump in human obesity

Looking for evidence of reduced energy use in the cells of obese persons, we measured the numbers of sodium-potassium-pump units in erythrocytes from a group of 21 obese human subjects and found them to be reduced by 22 per cent as compared with those of nonobese controls (P <0.001). The cation-transport activity of the pump, as measured by 86rubidium uptake by the cells, we also reduced in parallel with decrease in pump units. An increased concentration of sodium in the red cells of obese subjectes was also found (9.6 +/- 0.7 vs. 7.1 +/- 0.6 mmol per liter of cells; P<0.01). This finding demonstrates independently the physiologic importance of reduced numbers of sodium-pump units and reduced pump activity as measured by ouabain binding and rubidium transport, respectively. The magnitude of the reduction in the number of pump units was found to be negatively correlated with the percentage of ideal body weight (r = 0.56, P<0.001); this observation suggests a possible role of abnormalities of the sodium pump in the pathophysiology of obesity.     

Rapid detection of tuberculous and non-tuberculous mycobacteria by polymerase chain reaction amplification of a 162 bp DNA fragment from antigen 85

A polymerase chain reaction (PCR) assay was developed for detection of mycobacteria using amplification of a 162 bp region of the genes coding for the mycobacterial antigen 85 complex. Strains belonging to theMycobacterium tuberculosis complex were further differentiated from non-tuberculous mycobacteria by hybridization of the PCR derived Southern blot with an internal oligonucleotide probe and washing under stringent conditions. The method allowed rapid and sensitive detection of mycobacterial DNA in uncultured clinical samples. PCR results obtained forMycobacterium tuberculosis in 206 specimens from 180 untreated patients gave a sensitivity of 93.9% and a specificity of 94.3% compared with the culture. PCR detected DNA fromMycobacterium tuberculosis in seven samples from patients with clinically evident tuberculosis in whom culture was negative. The results suggest that this PCR assay could be used for early and specific diagnosis of tuberculosis.     

Post-tetanic potentiation increases energy cost to a higher extent than work in rat fast skeletal muscle

We studied the effects of (post-tetanic) potentiation on myosin light chain (MLC-2) phosphorylation, work and energy cost in skeletal muscle. Experiments were performed using in situ medial gastrocnemius muscles of male Wistar rats, which were electrically stimulated through the severed sciatic nerve. One group of muscles was first potentiated with an isometric tetanus before a series of 10 concentric contractions (PRC). A second group performed the same series of contractions without previous potentiation (RC). Following the last contraction the muscles were rapidly frozen and excised after which the high-energy phosphate content, lactate concentration and the level of MLC-2 phosphorylation were measured. The results indicate that PRC muscles had a higher (P < 0.05) total work output 144.5 ± 17.0 (SD) (n = 6) vs. 121.6 ± 11.4 (SD) (n = 6) mJ and level of MLC-2 phosphorylation (49.2 ± 7.3 vs. 40.8 ± 3.6%) than RC muscles. The energy cost of the series of concentric contractions in the PRC muscles (9.8 ± 1.9 μmol∼P/muscle) was significantly higher (P < 0.05) than the energy cost in the RC muscles (6.2 ± 0.97 μmol∼P/muscle). It was shown that the relative increase in energy cost of PRC muscles was higher (P < 0.05) than in total work output. It is proposed that the relative high increase in energy cost is the direct result of the increase in muscle performance rather than a property of potentiation. This revised version was published online in July 2006 with corrections to the Cover Date.