Protein kinase A type II-α regulatory subunit regulates the response of prostate cancer cells to taxane treatment

In the last decade taxane-based therapy has emerged as a standard of care for hormone-refractory prostate cancer. Nevertheless, a significant fraction of tumors show no appreciable response to the treatment, while the others develop resistance and recur. Despite years of intense research, the mechanisms of taxane resistance in prostate cancer and other malignancies are poorly understood and remain a topic of intense investigation. We have used improved mutagenesis via random insertion of a strong promoter to search for events, which enable survival of prostate cancer cells after Taxol exposure. High-throughput mapping of the integration sites pointed to the PRKAR2A gene, which codes for a type II-α regulatory subunit of protein kinase A, as a candidate modulator of drug response. Both full-length and N-terminally truncated forms of the PRKAR2A gene product markedly increased survival of prostate cancer cells lines treated with Taxol and Taxotere. Suppression of protein kinase A enzymatic activity is the likely mechanism of action of the overexpressed proteins. Accordingly, protein kinase A inhibitor PKI (6–22) amide reduced toxicity of Taxol to prostate cancer cells. Our findings support the role of protein kinase A and its constituent proteins in cell response to chemotherapy.     

Evidence for independent recruitment of zeta-crystallin/quinone reductase (CRYZ) as a crystallin in camelids and hystricomorph rodents

Zeta-crystallin/quinone reductase (CRYZ) is an NADPH oxidoreductase expressed at very high levels in the lenses of two groups of mammals: camelids and some hystricomorph rodents. It is also expressed at very low levels in all other species tested. Comparative analysis of the mechanisms mediating the high expression of this enzyme/crystallin in the lens of the Ilama (Lama guanacoe) and the guinea pig (Cavia porcellus) provided evidence for independent recruitment of this enzyme as a lens crystallin in both species and allowed us to elucidate for the first time the mechanism of lens recruitment of an enzyme- crystallin. The data presented here show that in both species such recruitment most likely occurred through the generation of new lens promoters from nonfunctional intron sequences by the accumulation of point mutations and/or small deletions and insertions. These results further support the idea that recruitment of CRYZ resulted from an adaptive process in which the high expression of CRYZ in the lens provides some selective advantage rather than from a purely neutral evolutionary process.      

Evidence of independent gene duplications during the evolution of archaeal and eukaryotic family B DNA polymerases

Eukaryotes and archaea both possess multiple genes coding for family B DNA polymerases. In animals and fungi, three family B DNA polymerases, alpha, delta, and epsilon, are responsible for replication of nuclear DNA. We used a PCR-based approach to amplify and sequence phylogenetically conserved regions of these three DNA polymerases from Giardia intestinalis and Trichomonas vaginalis, representatives of early-diverging eukaryotic lineages. Phylogenetic analysis of eukaryotic and archaeal paralogs suggests that the gene duplications that gave rise to the three replicative paralogs occurred before the divergence of the earliest eukaryotic lineages, and that all eukaryotes are likely to possess these paralogs. One eukaryotic paralog, epsilon, consistently branches within archaeal sequences to the exclusion of other eukaryotic paralogs, suggesting that an epsilon-like family B DNA polymerase was ancestral to both archaea and eukaryotes. Because crenarchaeote and euryarchaeote paralogs do not form monophyletic groups in phylogenetic analysis, it is possible that archaeal family B paralogs themselves evolved by a series of gene duplications independent of the gene duplications that gave rise to eukaryotic paralogs.      

Changes in the epithelial permeability of the large intestine of mice in the process of carcinogenesis

1,2-dimethylhydrazine (DMH), a colon carcinogen, being injected weekly to BALB/c mice, inhibits an active sodium transport, increases the transepithelial passive ion permeation and decreases ion selectivity in the descending colon. A single DMH injection leads to the same alterations, manifested for a month, followed by normalization of all the parameters to the control value. Distinctive, wavy changes in electrophysiological parameters were noted after a single injection of "non-colon" carcinogen 7,12-dimethyl-benz(alpha)antracen. It is supposed that the prolonged drop in active sodium transport, transepithelial resistance and ion selectivity are specific reactions of the colonic epithelium to carcinogenic treatment with DMH.     

Differential gene expression in the jellyfish Aurelia aurita

The body of Aurelia aurita, as well as other diploblasts, consists of two epithelial layers: ectodermal and gastral epithelium. These two tissues are separated by mesoglea, or extracellular matrix. In most coelenterates mesoglea is acellular. In A. aurita mesogleal cells are scattered in mesoglea. Differential display PCR was used to compare mRNA pools from ectodermal epithelium, gastral epithelium and mesoglea. 4 novel gene fragments were cloned and sequenced. According to RTPCR results, one of these fragments is differentially expressed in the ectodermal epithelium.     

Molecular genetic typing of methicillin-resistant Staphylococcus aureus strains, isolated in hospitals of different regions of the Russia and Belarus

The study of the length of the amplification products of the coagulase gene with the subsequent restriction analysis (the method of PCR--restrictive fragment length polymorphism, or RFLP) was used for typing 90 S. aureus strains. Among the strains under study, 78 were methicillin-resistant S. aureus (MRSA) strains, including 74 obtained in 1986 - 2002 in hospitals of different cities of the Russia and Belarus, as well as epidemic strains EMRSA-1, -2, -3, -12, obtained from the National Laboratory of Health, London (UK). The use of this method made it possible to type all the strains under study, which were differentiated into 9 groups by means of endonuclease Sfo1 and 7 groups by means of Alu1. Majority of clinical MRSA strains, belonged, according to the type of restriction, to groups 4 and 5. The study of the coagulase gene by the method of PCR - RFLP made it possible: to analyze the epidemic situation in hospitals for a period of several years; to compare the properties of strains isolated in different hospitals; to establish the genetic relationship of strains, isolated in 1998 - 2002, with strains, isolated in 1986 - 1990. The results of the study suggest that at least two epidemic MRSA strains, genetically similar to international strains, circulate in hospitals of Russia.     

Expression Profiles of Genes—Potential Therapy Targets—and Their Relationship to Survival in Renal Cell Carcinoma

The main mechanisms of pathogenesis of clear cell renal cell carcinoma (CCRCC) are realized through the PI3K–AKT–mTOR and Ras–RAF–ERK signaling pathways. Targeted therapy is directed primarily at the genes and their encoded products that are components of these pathways. The levels of expression and coexpression of target genes were determined, and the difference in the functioning of the genes of one of the two major signaling pathways in tumors of CCRCC patients with different life duration (more and less than 3.5 years) and the relationship of the VEGFA gene expression level with the life duration was revealed.     

Effect of competition on the demography of planktonic cladocerans — Daphnia and Diaphanosoma

Summary Isolated and mixed continuous cultures of Daphnia hyalina and Diaphanosoma brachyurum in lake water were maintained under laboratory conditions to elucidate demographic effects of competition. Population dynamics curves were obtained. Interspecific competition was revealed by the decrease in the average density of animals in the mixed versus isolated cultures and by the extinction of one species in the presence of the other. Within the first 50 days either Diaphanosoma (4 cases) or Daphnia (1 case) was the superior competitor, depending on the initial conditions. Further cultivation resulted in the extinction of Diaphanosoma in the mixed cultures. There were no statistically significant differences between the maximum rates of population increase (r _m ) in Daphnia and Diaphanosoma at the concentration of edible algae about 2·10⁵ m³ml^-1(0.293 and 0.286 days^-1, respectively). Time lags for density-dependent parameters of the populations were evaluated by means of rank cross-correlations. Regardless of the species identity the time lags of fecundity, birth rate, and the rate of population growth were significantly higher in the superior competitor. The initial conditions of culturing affected the time lags which in their turn influenced the outcome of the interaction. Enhanced competitive ability due to the maximized time lags in Daphnia was not associated with the loss of population stability. Conversely, it brought about destabilization of Diaphanosoma populations which seemed to be the ultimate cause of its extinction observed in the end of the experiment. Time lag of the population growth rate was well predicted based on the half-sum of time lags in birth and death rates (r²=0.80, P<0.001). Daphnia responded to competition with a sharp shortening of the time lags of fecundity, birth rate, and the population growth rate. It increased clutch size and showed inverse relationship between the fecundity time lag and average fecundity even though it was strongly suppressed by Diaphanosoma. The competitive ability was not related to the percentage of adults in the populations. In contrast to the current belief the major result of interspecific competition in the experiment was not a decrease in the rate of population growth but was a reduction in population time lags.     

Variations in lymphokine generation by individual lymph nodes draining human malignant tumors

Summary Individual lymph nodes draining tumors vary in their degree of immunological activity. Cell suspensions from tumor-free nodes located relatively near to tumors are spontaneously less reactive and respond poorly to exogenous stimulation by mitogens and lymphokines. Diminished spontaneous uptake of tritiated thymidine by lymph node cells not exposed to exogenous stimulation suggests that tumor-proximate immune suppression exists in vivo and is not purely a laboratory artefact. The present study was undertaken to explore that possibility further. Fluid in which cell suspensions from tumor-free nodes were prepared, and supernatants from short-term cultures of nodes located at different distances from tumors were compared for their capacity to inhibit the in vitro migration of the human lymphoblastoid cell line QIMR-WIL. Inhibitory activity of fluids from individual nodes was related to their position relative to the tumor and their immune competence, assessed by the responses to mitogens of cell suspensions prepared from them. Cell suspension fluids from 92/111 nodes (83%) significantly inhibited the migration of QIMR-WIL, at a level similar (44±14%) to that induced by the supernatants of mixed lymphocyte cultures (43±17%). Fluids from the nodes of melanoma patients were more inhibitory than those from breast cancer patients (49±12% and 37±13%, respectively,P = 0.003). The inhibitory activity of the different nodes of individual node groups varied significantly in 25 of 33 patients (76%), the node nearest the tumor generating least inhibitory activity (indexing the greatest immune suppression) in 20 of these 25 patients (80%). The strength of migration-inhibitory activity was concordant with the responsiveness to mitogen stimulation in up to 14 of 18 patients (78%). Studies of molecular size and heat stability indicated that the inhibitory factors had characteristics consistent with common migration-inhibitory lymphokines such as leukocyte-migration-inhibitory factor, macrophage-inhibitory factor and interleukin-2. Our findings further support the hypothesis that lymph nodes nearest to tumors are relatively immune-suppressed in vivo.Supported by grants CA 29938 and CA 43658, awarded by the National Cancer Institute, DHHS and a grant from the Candle Foundation, Los Angeles     

Changes in the heart of neonatal rats after cryptosporidial gastroenteritis of different degrees of severity

Disturbances at the childhood age increase risk of appearance of cardiovascular disease decades later. The nature of this interconnection called ontogenetic programming is not completely understood. Valuable source of knowledge about mechanisms of ontogenetic programming are data of interspecies study of biology of the body life cycles understanding on the triggers and mechanisms are the cross-species comparative data on life-cycle and heart aerobic capacity. Taken into account the interspecies differences, these data allow finding the correct direction of experimental investigations. Results of studies of almost 100 homoiothermal species have shown the slow growth and a high loading on the heart at postnatal development to decrease its aerobic capacity in adults. Basing on these data, we suggested that the neonatal infectious gastroenteritis causing tachyarrhythmia, malabsorption, and the growth deceleration might lead to pathologic changes in the heart. Our task was to evaluate the effect of cryptosporidial gastroenteritis of different degrees of severity on heart of neonatal rats. By using methods of Real-Time PCR, immunocytochemistry, image analysis, and study of atrial septum (ostuim primum), we have established that a gradual increase of intensity of infestation with Cryptosoridium parvum oocysts causes sharp changes corresponding to “all-or-nothing” response. At a weak infestation the atrial septum was close (like in control), while significant changes in expression of isoforms of heavy chains of α- and β- myosin were absent. At the intermediate and severe infestation, in the atrial septum the foramen ovale was visualized and there were observed the cardiac atrophy and a strong shift of ration of expression of myosin heavy chains toward the low-velocity of β-chain. Thus, by disturbing the frequency-strength ratios and causing the outflow of resources from the formed heart, the neonatal cryptosporidiosis produces pathological changes of the organ molecular and anatomical structure. Our results can be interest to evolutionary biologists and physicians, as they show the importance of knowledge of evolutionary-comparative investigations for search for novel risk factors of heart diseases and demonstrate interconnection between gastroenteritis, pathology of atrial septum, and a change of composition of the main contractile proteins in cardiomyocytes.