Single short-term signal that enhances cooperative activity of old rat hepatocytes acts for several days

Primary cultures of rat hepatocytes were studied in serum-free medium. Ultradian protein synthesis rhythm was used as a marker of overall cell synchronization and cooperation amongst the population. The level of synchronization was determined by amplitudes of the rhythm. Low synchronization of old rat hepatocytes can be enhanced by addition of either gangliosides or phenylephrine to the medium. Incubation of cultures with gangliosides lasted for 2.5 h, while action of phenylephrine was only for 2 min. The amplitude of protein synthesis rhythm was increased 1.5-2 times. In cultures transferred to a fresh normal medium, this increased amplitude was observed for at least 2-3 days. Thus, both gangliosides and phenyleprine are triggers, which, as shown earlier, initiated calcium-dependent processes in the cytoplasm. The results are discussed in the light of concept of the cell self-organization by a direct cell-cell communication.     

Physiological and morphological characteristics of the rhythmic contractions of the amnion in veiled chameleon (Chamaeleo calyptratus) embryogenesis

A morpho-functional study of the veiled chameleon (Chamaeleo calyptratus) amnion was performed as part of a general comparative study of amnion rhythmic contractions (ARC) in reptile and bird embryogenesis. Eggs incubated at 27.5 degrees C were used at different developmental stages from day 80 (D80) to D184 for the recording of ARC using a force transducer. Slow ARC, about 1 min in duration, were revealed from D88 (stage 31) to the near-hatching stages (incubation time was 183-198 days). The frequency and amplitude of slow ARC increased significantly towards the end of incubation. This ARC enhancement correlated with the differentiation of smooth muscle elements in the amniotic membrane from single spindle-shaped cells to complex "star-like" structures and with the development of the actin fibers, revealed by phalloidin, in the amniotic muscle layer. Short-term changes in temperature influenced ARC and heart rate (HR). Cooling to 25 degrees C from the control (27.5 degrees C) resulted in a significant decrease in both ARC frequency and HR. Heating to 30 degrees C significantly increased the embryonic HR, but not ARC frequency. Within the temperature range from 25 to 30 degrees C the temperature effect on ARC frequency and HR was reversible.     

Effect of catabolic plasmids on physiological parameters and efficiency of oil destruction by Pseudomonas bacteria

The ability of microbial degraders of polycyclic aromatic hydrocarbons to grow at 24°C in liquid mineral medium supplemented with oil as the sole source of carbon and energy was studied. Growth characteristics (CFU) and the level of oil destruction by plasmid-bearing and plasmid-free strains were determined after seven days of cultivation. The presence of catabolic plasmids in the degrader strains, including rhizosphere pseudomonads, was shown to increase cell growth and enhance the level of oil degradation. Strain Pseudomonas chlororaphis BS1391 bearing plasmid pBS216 was found to be the most effective oil degrader.     

Differences in certain immunological indices among persons who have had cholera and among Vibrio carriers]

Blood sera of persons, who sustained cholera of different degree of severity, and of vibrio-carriers, and also chromatographic fractions obtained in separation of the mentioned sera on DEAE-cellulose were investigated in the reaction of agglutination and the indirect hemagglutination test. Immunological response in the vibrio carriers was realized by the microglobulin, and in the convalescents--by the macroglobulin type. There was also revealed an association between the severity of the course of cholera and the prevalence in the blood serum of one or another type of specific antibodies: in severe forms of the disease the antibodies of the IgM-class were detected with the greatest frequency, and microglobulin antibodies--in vibrio carriers. The detected differences in the immunological status of those who sustained cholera and of vibrio-carriers could be used as an additional differential sign in the diagnosis of the mentioned conditions.     

Effect of a pH change in the medium and cytoplasm of cultured cells on the rhythm of protein synthesis

A circahoral rhythm of protein syntheses similar to that in monolayer hepatocytes was discovered in cell culture of Chinese hamster fibroblasts. Studies on the effects of pH changes in the culture medium and cultured cells on different parameters of protein synthesis showed some pH-dependent changes of predecessor pool and of its incorporation intensity into proteins. At the same time changes in a relative incorporation of the predecessor into proteins (with a correction for the pool) were insignificant. This value characterizing the productivity of protein synthesis does not seem to be directly associated with pH changes in the cells. The mean period of the rhythm of protein synthesis and intracellular pH was not changed with medium pH alterations.     

Optimization of melting analysis with TaqMan probes for detection of KRAS,NRAS, and BRAF mutations

The TaqMan probes that have been long and effectively used in real-time polymerase chain reaction (PCR) may also be used in DNA melting analysis. We studied some factors affecting efficiency of the approach such as (i) number of asymmetric PCR cycles preceding DNA melting analysis, (ii) choice of fluorophores for the multiplex DNA melting analysis, and (iii) choice of sense or antisense TaqMan probes for optimal resolution of wild-type and mutant alleles. We also determined ΔT_m (i.e., the temperature shift of a heteroduplex relative to the corresponding homoduplex) as a means of preliminary identification of mutation type. In experiments with serial dilution of mutant KRAS DNA with wild-type DNA, the limit of detection of mutant alleles was 1.5–3.0%. Using DNA from both tumor and formalin-fixed paraffin-embedded tissues, we demonstrated a high efficiency of TaqMan probes in mono- and multiplex mutation scanning of KRAS, NRAS (codons 12, 13, and 61), and BRAF (codon 600) genes. This cost-effective method, which can be applied to practically any mutation hot spot in the human genome, combines simplicity, ease of execution, and high sensitivity—all of the qualities required for clinical genotyping.