Influence of tissue preparation techniques on p53 expression in bronchial and bladder carcinomas, assessed by immunofluorescence staining and flow cytometry.

In a series of bronchial and bladder carcinomas, p53 protein expression was examined. Samples from formalin-fixed, paraffin-embedded tissue (routine-treated) were compared with parallel samples of fresh tissue and tissue fixed in paraformaldehyde and ethanol. The expression of p53 was measured by immunofluorescence staining and dual parameter flow cytometry, with simultaneous monitoring of DNA content. For each tumor, p53 fluorescence with different fixatives was expressed relative to fresh tissue. The p53 fluorescence signals were on average brighter from routine-treated tissue than from fresh tissue. The tissue fixed in paraformaldehyde showed no difference from fresh tissue. In the ethanol-fixed tissue, however, fluorescence signals were reduced by nearly 70%, and the fraction of detectable p53 positive cells in tumor tissue was reduced by more than 50%. This loss of fluorescence was probably due to a leakage of the antigen from nucleus to cytoplasm. Pepsin treatment did not influence p53 fluorescence. Within the same tumor, the S-phase fraction in p53 positive cells was significantly higher than in p53 negative cells (13.1 +/- 2.0% vs. 6.5 +/- 0.8%). This pattern was not influenced by formalin fixation or pepsin treatment. Our study demonstrates that in measuring a nuclear antigen, tissue handling may influence the results, and care should be taken to optimize the preparation procedure. Using the antibody PAb 1801, p53 expression measured in archival material is not reduced as compared to fresh tissue.     

Comparison of two cytochromes P-450 from Candida maltosa: primary structures, substrate specificities and effects of their expression in Saccharomyces cerevisiae on the proliferation of the endoplasmic reticulum

cDNAs were cloned, sequenced and expressed which encode two different cytochrome P-450 forms of the alkane-assimilating yeast Candida maltosa, designated as P-450Cm1 and P-450Cm2. The amino acid sequences deduced were about 55% identical. Expression in Saccharomyces cerevisiae resulted in the formation of intact microsomal P-450 systems catalyzing the hydroxylation of n-hexadecane and lauric acid with significantly different substrate preferences. A massive proliferation of the endoplasmic reticulum was observed in the S. cerevisiae cells which produced P-450. Depending on the P-450 form expressed, distinctly organized stacks of paired membranes appeared and occupied considerable areas of the cytoplasm. As shown by immunoelectron microscopy for P-450Cm1, the protein expressed was highly concentrated within these newly formed membrane structures.     

Human pheochromocytoma cells studied in culture contain large amounts of DSIP-like material.

Delta sleep-inducing peptide (DSIP)-like immunoreactive (LI) material has been detected in nine different human pheochromocytoma tumors by immunocytochemistry. In primary tumors subjected to indirect immunofluorescence a variable number of tumor cells (25-75%) showed positive cytoplasmic labeling after incubation with DSIP antiserum. Tumor cells grown in culture were strongly labeled by the DSIP antiserum with DSIP-LI concentrated to cell bodies. Electron microscopic immunocytochemistry (immunogold labeling) of pheochromocytoma cells demonstrated DSIP-LI over the dense core of secretory granules. The presence of DSIP-LI in several HPLC fractions from conditioned culture media indicates secretion of DSIP-LI from cultured pheochromocytoma cells. The observations suggest that DSIP-LI is synthesized and stored in secretory granules before release. The different HPLC profiles from each of the tumors may reflect differences in processing or turnover of DSIP-LI in pheochromocytoma cells.     

The stability of the alkylating derivatives of oligodeoxyribonucleotides containing a cholesterol or phenazine radical added to the 3'-termination during their interaction with Acholeplasma laidlawii PG-8]

Stability of alkylating derivatives of decathymidylates protected on the 3'-terminal by cholesterol and phenazine residues has been studied in the process of their interaction with cells of Acholeplasma laidlawii PG-8. It is shown that the studied reagents are not split by nucleases of A. laidlawii PG-8 for the time necessary for alkylation of mycoplasma biopolymers.     

Structural changes in the sarcoplasmic reticulum membrane of skeletal muscles at the early stage of x-ray irradiation]

A single X-ray irradiation of the rabbit hindlimbs in a dose of 0.24 C/kg evokes a decrease in fluorescence of the ANS probe bound with membranes of the sarcoplasmatic reticulum as a result of the decrease of binding sites, binding constant as well as the quantum output of the probe. A decrease in fluorescence of tryptophan residues of Ca-ATPase localized in membranes and attenuation of interaction of its SH-group with dithionitrobenzoic acid has been also observed at early postradiation terms (1 and 24 h). The obtained results evidence for structural rearrangements occurring in membranes of the sarcoplasmic reticulum under the effect of ionizing radiation. Changes in conformation of CA-ATPase molecules contribute much to this process.     

Cloning and inactivation of a chromosomal copy of the imidazole glycerophosphate dehydratase (HIS) gene from Hansenula polymorpha]

A gene for imidazole glycerophosphate dehydratase (HIS) has been selected from the library of Hansenula polymorpha genes by complementation of Escherichia coli hisB mutations or his3 mutation of Saccharomyces cerevisiae. Inactivation of the gene in Hansenula polymorpha strain 48V, as well as Leu(-)-Leu+ conversion of phenotype and arising of antibiotic G418 resistance resulted from transformation of the strain by the linear DNA molecules of the cloned HIS-gene with the in vitro inserted LEU2 gene from Saccharomices cerevisiae and KmR gene. The Southern hybridization analysis of the Leuf+His- G418R clones representing 1-2% of transformants population together with the DNA analysis of the monospore clones from the hybrid Leu+His-G418R transformant tetrad and tester strain have revealed the locus specific nature of the clones.     

The use of thin slices of myocardium for recording the currents across single ion channels]

Thin cardiac slices (100-200 microns) from newborn (1-14 days old) rat heart ventricles were used for patch clamp recordings. High resistance seals (10-50 GOhms) between patch-clamp pipettes and the membrane of cardiac cells as well as classical patch-clamp configurations can be achieved on this preparation without any enzymatic treatment of tissue. Resisting potential for cardiac cells measured in whole-cell configuration ranged between -30 and -65 mV. Averaged sodium currents and single inward rectifying potassium elementary currents recorded in cell-attached mode displayed basic features similar to those previously reported for isolated rat ventricular cells. Application of the method described here in cardiac electrophysiology will allow patch-clamp studies on heart cells without the complicated procedures of cell isolation. In addition, the uncertainty associated with enzyme treatment can be avoided. In future, this technique could be a new tool for studying electrophysiological properties of heart cells in situ.     

Decalcification by perfusion. A new method for rapid softening of temporal bones.

We describe a new technique, decalcification by perfusion, for the softening of bony tissue. The blood circulatory system was perfused in 16 rats via a cannula through the left heart ventricle with a fixative followed by New Decalc (an acidic demineralizer) for 30-240 minutes. Perfusion decalcification for 120 minutes softened all heads and middle ear specimens could be easily sampled and prepared for studies by both light and electron microscope. For comparison, a conventional immersion technique required 72 hours of decalcification to accomplish softening. The perfusion technique considerably reduced the time needed to decalcify the tissue and preserved the morphology better than did the immersion procedure.     

Albofungin formation by a strain isolated as a result of fusion of protoplasts of organisms producing aminoglycoside antibiotics

Strain 344 synthesizing an antibiotic complex was isolated after fusion of the protoplasts of Streptomyces monomycini producing monomycin and Streptomyces kanamyceticus producing kanamycin. The major component of the complex was identified with albofungin and the minor one was suggested to be chloralbofungin. In the cultures of strain 344 variants forming monomycin were detected. After regeneration of the protoplasts of the parent strains there were isolated no stable clones synthesizing antibiotics differing from monomycin and kanamycin.     

Microbial colonization and succession in the large intestine of newborn infants during their stay with mothers at the maternity hospital]

Formation of microflora in the large intestine of 5-day old infants was studied in one of the Moscow maternity homes. The up-to-date procedures for isolation and identification of aerobic and anaerobic organisms were used in the study and the findings were processed on a computer. In the newborns of the maternity home of the "mother-infant" type there was observed colonization of the large intestine with aerobic and anaerobic organisms. A wave-like dynamics in the formation of the symbiotic microflora was revealed. It reflected the phenomenon of the microbial succession in the infants. The attempts to detect microbial interference between the species colonizing the large intestine showed that it was extremely rare in the 5-day old infants. This was likely the reason of the low intestine resistance to the colonization in the newborns which in its turn defined the frequent colonization of the intestine mucosa with S. aureus and the organisms of the Klebsiella, Enterobacter and Citrobacter group.