Can social media intervention improve physical activity of medical students?

OBJECTIVES: Physical activity level decreases during young adulthood. As social media are nowadays widely used and are included into many people's daily routines, the interventions on these websites have the possibilities to be integrated into those routines without becoming a burden. The aim of this study was to assess physical activity level among first- and fifth-year medical students and social media intervention with the aim to improve physical activity among them. STUDY DESIGN: Prospective longitudinal study was conducted during October of 2016 at the Faculty of Medicine, University of Belgrade, Serbia. The study included 375 first- and fifth-year students. METHODS: At the baseline, students filled in the questionnaire and were asked to join a Facebook discussion group. The intervention consisted of motivation for physical activity through motivational pictures, texts, and discussions. The second assessment was carried out after one month. Based on the reported physical activity level, students were divided into groups: sufficient (>600 metabolic equivalent [MET]-minutes/week) and insufficient physical activity (≤600 MET-minutes/week). RESULTS: Total of 85.4% of students were sufficiently active at the baseline, whereas 90.4% were sufficiently active after one month. Multivariate logistic regression analysis showed that students who were part of the Facebook group (odds ratio [OR]: 3.51, 95% confidence interval [CI]: 1.46-8.43) and students who had sufficient physical activity at the baseline (OR: 5.44, 95% CI: 2.44-12.13) had a higher likelihood to be sufficiently active after one month. CONCLUSION: Social media are shown to be valuable in health-promoting interventions and can be used for interventions targeting lifestyle change among young adults.

Time-dependent reduction of structural complexity of the buccal epithelial cell nuclei after treatment with silver nanoparticles.

Recent studies have suggested that silver nanoparticles (AgNPs) may affect cell DNA structure in in vitro conditions. In this paper, we present the results indicating that AgNPs change nuclear complexity properties in isolated human epithelial buccal cells in a time-dependent manner. Epithelial buccal cells were plated in special tissue culture chamber / slides and were kept at 37°C in an RPMI 1640 cell culture medium supplemented with L-glutamine. The cells were treated with colloidal silver nanoparticles suspended in RPMI 1640 medium at the concentration 15 mg L⁻¹. Digital micrographs of the cell nuclei in a sample of 30 cells were created at five different time steps: before the treatment (controls), immediately after the treatment, as well as 15 , 30 and 60 min after the treatment with AgNPs. For each nuclear structure, values of fractal dimension, lacunarity, circularity, as well as parameters of grey level co-occurrence matrix (GLCM) texture, were determined. The results indicate time-dependent reduction of structural complexity in the cell nuclei after the contact with AgNPs. These findings further suggest that AgNPs, at concentrations present in today's over-the-counter drug products, might have significant effects on the cell genetic material.

Comparison of cartilage histopathology assessment systems on human knee joints at all stages of osteoarthritis development.

OBJECTIVE: To compare the MANKIN and OARSI cartilage histopathology assessment systems using human articular cartilage from a large number of donors across the adult age spectrum representing all levels of cartilage degradation. DESIGN: Human knees (n=125 from 65 donors; age range 23-92) were obtained from tissue banks. All cartilage surfaces were macroscopically graded. Osteochondral slabs representing the entire central regions of both femoral condyles, tibial plateaus, and the patella were processed for histology and Safranin O - Fast Green staining. Slides representing normal, aged, and osteoarthritis (OA) tissue were scanned and electronic images were scored online by five observers. Statistical analysis was performed for inter- and intra-observer variability, reproducibility and reliability. RESULTS: The inter-observer variability among five observers for the MANKIN system showed a similar good Intra-class correlation coefficient (ICC>0.81) as for the OARSI system (ICC>0.78). Repeat scoring by three of the five readers showed very good agreement (ICC>0.94). Both systems showed a high reproducibility among four of the five readers as indicated by the Spearman's rho value. For the MANKIN system, the surface represented by lesion depth was the parameter where all readers showed an excellent agreement. Other parameters such as cellularity, Safranin O staining intensity and tidemark had greater inter-reader disagreement. CONCLUSION: Both scoring systems were reliable but appeared too complex and time consuming for assessment of lesion severity, the major parameter determined in standardized scoring systems. To rapidly and reproducibly assess severity of cartilage degradation, we propose to develop a simplified system for lesion volume.

Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro.

Cell therapies for articular cartilage defects rely on expanded chondrocytes. Mesenchymal stem cells (MSC) represent an alternative cell source should their hypertrophic differentiation pathway be prevented. Possible cellular instruction between human articular chondrocytes (HAC) and human bone marrow MSC was investigated in micromass pellets. HAC and MSC were mixed in different percentages or incubated individually in pellets for 3 or 6 weeks with and without TGF-beta1 and dexamethasone (±T±D) as chondrogenic factors. Collagen II, collagen X and S100 protein expression were assessed using immunohistochemistry. Proteoglycan synthesis was evaluated applying the Bern score and quantified using dimethylmethylene blue dye binding assay. Alkaline phosphatase activity (ALP) was detected on cryosections and soluble ALP measured in pellet supernatants. HAC alone generated hyaline-like discs, while MSC formed spheroid pellets in ±T±D. Co-cultured pellets changed from disc to spheroid shape with decreasing number of HAC, and displayed random cell distribution. In -T-D, HAC expressed S100, produced GAG and collagen II, and formed lacunae, while MSC did not produce any cartilage-specific proteins. Based on GAG, collagen type II and S100 expression chondrogenic differentiation occurred in -T-D MSC co-cultures. However, quantitative experimental GAG and DNA values did not differ from predicted values, suggesting only HAC contribution to GAG production. MSC produced cartilage-specific matrix only in +T+D but underwent hypertrophy in all pellet cultures. In summary, influence of HAC on MSC was restricted to early signs of neochondrogenesis. However, MSC did not contribute to the proteoglycan deposition, and HAC could not prevent hypertrophy of MSC induced by chondrogenic stimuli.

Centrally applied ghrelin affects feeding dynamics in male rats.

Ghrelin is 28-amino acid peptide, which is produced mainly in the stomach. Since plasma ghrelin are strictly dependent on food intake, this hormone has significant effects on appetite and energy balance. The aim of this investigation was to examine the effects of centrally applied ghrelin on feeding dynamism by measuring the approaches to food container and the amount of food and water intake within 2 hours immediately after ghrelin or PBS injections. Body weight was obtained daily, while ending retroperitoneal (RP) and epididymal (EPI) white adipose tissue (WAT) contents as well as blood levels of leptin and insulin were measured. Five injections of rat ghrelin or PBS (n = 8 per group) were administered once per day (1 microg = 0.15 nmol of ghrelin in 5 microL of PBS), into lateral cerebral ventricle (ICV) of free feeding adult male rats. Results showed that in the first and the second 30-min intervals number of approaches to food container were significantly increased already after the 2(nd) ICV ghrelin application (p < 0.05), by 50% and 67% respectively, in comparison with control rats. Centrally applied ghrelin increased body weight after the 2(nd) injection till the end of treatment (p < 0.05), which was followed by increased food and water intake (p < 0.05). At the end of treatment, RP and EPI WAT contents were increased (by 221%, p < 0.01 and 82%, p < 0.05, respectively). Serum insulin levels were elevated (by 41%, p < 0.05) while serum leptin levels were decreased (by 75%, p < 0.05). These data and the available literature strongly support the opinion that repetitive subnanomolar doses of central ghrelin administration play essential role in food initiation and feeding dynamics in freely feeding adult male rats.

Consummatory behavior and metabolic indicators after central ghrelin injections in rats.

Ghrelin, an endogenous ligand for the growth-hormone-secretagogue receptor, is a 28-amino acid peptide with a post-translational acyl modification necessary for its activity. It has central nervous system actions that affect appetite, body mass and energy balance. An intracerebroventricular (ICV) injection protocol of sub-nanomolar doses of ghrelin, known to alter the morphology of ACTH and GH producing pituicytes and plasma levels of these hormones, was used to provide an overview of metabolic changes linked to energy metabolism. Variables measured were: food intake (FI), water intake (WI), fecal mass, urine volume, body weight (BW), retroperitoneal (RP) and epididymal (EPI) white adipose tissue (WAT), and changes in serum leptin, insulin, triglycerides, cholesterol, and glucose. Five injections of rat ghrelin or PBS (n=8 per group) were given ICV every 24 h (1 microg/5 muL PBS) to adult male rats. Ghrelin had a positive and cumulative effect on FI, WI and BW (p<0.05), but not feces mass or urine volume (p>0.05). Centrally applied ghrelin clearly increased RP WAT (by 235%, p<0.001), EPI WAT (by 85%, p<0.05) and serum insulin levels (by 43%, p<0.05), and decreased serum leptin levels (by 77%, p<0.05) without (p>0.05) evoking changes in blood triglyceride cholesterol, or glucose levels. These data and the available literature clearly document that exposure of the brain of normal rats, over time, to sub-nanomolar doses of ghrelin results in metabolic dysregulation culminating in increased body mass, consummatory behavior, and lipid stores as well as changes in blood leptin/insulin levels. Thus, modulation of central ghrelin receptors may represent a pharmacological approach for controlling multiple factors involved in energy balance and obesity.

Central effects of ghrelin on serum growth hormone and morphology of pituitary somatotropes in rats.

Ghrelin, an endogenous ligand for the growth hormone (GH) secretagogue receptor, was originally purified from rat stomach; subsequently, ghrelin neurons were found in the arcuate nuclei of rats. Central effects of the peptide on GH release, however, remain to be clarified. The aim of the present study was to determine the morphologic features of GH-producing pituicytes and serum GH concentration after central administration of ghrelin. Five injections of rat ghrelin or phosphate-buffered saline (PBS; n = 10 rats/group) were given every 24 hrs (1 microg of ghrelin in 5 microl of PBS) into the lateral cerebral ventricle of male rats. Significant (P < 0.05) increases in absolute and relative pituitary weights occurred in ghrelin-treated rats versus controls (58% and 41%, respectively). Morphometric parameters (i.e., the volume of GH cells, volume of their nuclei, and volume density) all significantly (P < 0.05) increased by 17%, 18%, and 19%, respectively, in the ghrelin-treated group versus controls. Terminal serum concentration of GH was significantly (P < 0.05) increased by 15% with ghrelin treatment. The results clearly document that daily nanomolar doses of ghrelin into the lateral cerebral ventricle stimulate GH cell proliferation and promote GH release. Thus, achieving pharmacologic control of central ghrelin receptors is a promising modality to modulate the actions of GH.

Structure-function analysis of the U2 snRNP-associated splicing factor SF3a.

Human splicing factor SF3a is a part of the 17 S U2 snRNP (small nuclear ribonucleoprotein), which interacts with the pre-mRNA branch site early during spliceosome formation. The SF3a subunits of 60, 66 and 120 kDa are all required for SF3a function in vitro. Depletion of individual subunits from HeLa cells by RNA interference results in a global inhibition of splicing, indicating that SF3a is a constitutive splicing factor. Structure-function analyses have defined domains necessary for interactions within the SF3a heterotrimer, association with the U2 snRNP and spliceosome assembly. Studies aimed at the identification of regions in SF3a60 and SF3a66, required for proper intracellular localization, have led to a model for the final steps in U2 snRNP biogenesis and the proposal that SF3a is incorporated into the U2 snRNP in Cajal bodies.

Domains in human splicing factors SF3a60 and SF3a66 required for binding to SF3a120, assembly of the 17S U2 snRNP, and prespliceosome formation.

The active 17S U2 small nuclear ribonucleoprotein particle (snRNP), which binds to the intron branch site during the formation of the prespliceosome, is assembled in vitro by sequential interactions of the essential splicing factors SF3b and SF3a with the 12S U2 snRNP. We have analyzed the function of individual subunits of human SF3a (SF3a60, SF3a66, and SF3a120) by testing recombinant proteins, expressed in insect cells, in various in vitro assays. The recombinant subunits readily form the SF3a heterotrimer, where SF3a60 and SF3a66 interact with SF3a120, but not with each other. All SF3a subunits are essential for the formation of the mature 17S U2 snRNP and the prespliceosome. Single subunits engage in interactions with the 15S U2 snRNP (consisting of the 12S U2 snRNP and SF3b), and SF3a60 appears to play a major role in recruiting SF3a120 into the U2 particle. Analysis of functional domains in SF3a60 and SF3a66 identified interaction sites for SF3a120 in their N-terminal portions. C(2)H(2)-type zinc finger domains mediate the integration of SF3a60 and SF3a66 into the U2 snRNP, and we propose a model in which protein-protein interactions between the zinc finger domains and the Sm proteins, common to all spliceosomal snRNPs, contribute to the assembly of the 17S U2 snRNP. Finally, we demonstrate that all domains required for interactions within the SF3a heterotrimer and the formation of the 17S U2 snRNP are also necessary to assemble the prespliceosome.

Rel/NF-kappaB transcription factors: key mediators of B-cell activation.

The Rel/nuclear factor (NF)-kappaB family of transcription factors have been implicated in the regulation of a wide variety of genes, in particular those encoding proteins crucial to the function of the immune system. Through the use of mutant mice that lack one or more of these proteins, we have begun to examine the individual and combined roles of Rel, RelA and NF-kappaB1 in B-cell development and function. Here we outline and discuss how these transcription factors operate as differentiation stage-specific regulators of B-cell development, survival, division and immunoglobulin expression, emphasizing those Rel/NF-kappaB-regulated genes that mediate these functions.

Alpha beta TCR+ cells are a minimal fraction of peripheral CD8+ pool in MHC class I-deficient mice.

MHC class I molecules play a role in the maintenance of the naive peripheral CD8+ T cell pool. The mechanisms of the peripheral maintenance and the life span of residual CD8+ cells present in the periphery of beta 2-microglobulin-deficient (beta 2m-/-) mice are unknown. We here show that very few CD8+ cells in beta 2m-/- mice coexpress CD8 beta, a marker of the thymus-derived CD8+ T cells. Most of the CD8 alpha+ cells express CD11c and can be found in beta 2m/RAG-2 double-deficient mice, demonstrating that these cells do not require rearranged Ag receptors for differentiation and survival and may be of dendritic cell lineage. Rare CD8 alpha+CD8 beta+ cells can be detected following in vivo alloantigenic stimulation 2 wk after the adult thymectomy. Selective MHC class I expression by bone marrow-derived cells does not lead to an accumulation of CD8 beta+ cells in beta 2m-/- mice. These findings demonstrate that 1) thymic export of CD8+ T cells in beta 2m-/- mice is reduced more severely than previously thought; 2) non-T cells expressing CD8 alpha become prominent when CD8+ T cells are virtually absent; 3) at least some beta 2m-/- CD8+ T cells have a life span in the periphery comparable to wild-type CD8+ cells; and 4) similar ligands induce positive selection in the thymus and survival of CD8+ T cells in the periphery.

[Effect of various methods of treatment in chronic renal insufficiency on the quality of life in patients]. / Uticaj razlicitih nacina lecenja bolesnika s hronicnom insuficijencijom bubrega na kvalitet zivota bolesnika.

Interest in measuring the quality of life (QL) in relation to health care has increased enormously in recent years. This is also true for end-stage renal failure where it is important not only to provide a better survival but also the quality of that survival. The aim of this study was to assess the relative influence of different kinds of treatment on end-stage renal disease after the patients' evaluation of their overall QL. We studied 167 patients receiving conservative treatment (45), haemodialysis (44), haemodialysis and erythropoieth (36), and continuous ambulatory peritoneal dialysis (42). The patients completed an original questionnaire consisting of 37 questions divided in five groups and generating 15 QL variables: personal data (name, gender, age, basic kidney disease); sociodemographic data influenced by the illness (family history, working ability, employment status); general health characteristics (fatigue, appetite, wound healing, sleep, resistance to cold); aspects of private life that are mostly influenced by the disease (social interaction, traveling, mood, sports, sexual life), and patients subjective assessment of their condition (self care and happiness). Patients on haemodialysis showed lower levels of QL than that on peritoneal dialysis related to fatigue (p < 0.01), working ability (p < 0.05), wound healing (p < 0.05), and appetite (p < 0.01) compared to the conservative treatment. Peritoneal dialysis had also a statistically significant positive influence on fatigue (p < 0.05) compared to conservative treatment. However, erythropoletin treatment showed better results with regard to traveling (p < 0.05), resistance to cold (p < 0.01), self care (p < 0.05) and mood (p < 0.05) compared to peritoneal dialysis, and working ability (p < 0.05), fatigue (p < 0.05) and mood (p < 0.05) compared to conservative treatment and haemodialysis.

Prevention of antigen-induced microtubule organizing center reorientation in cytotoxic T cells by modulation of protein kinase C activity.

Lysis of target cells (TC) by cytotoxic T lymphocytes (CTL) is achieved by directional exocytosis of cytolytic molecules-perforin and granzymes. They are stored within lytic granules which can be readily released following antigenic stimulation. Secretion of lytic molecules appears to be controlled by protein kinase C (PKC) activity, since specific modulators of PKC activity abolish the lysis of TC. We have examined the effect of PKC modulation on some of the earliest events in the perforin/granzyme-mediated cytotoxicity. De novo synthesis of perforin mRNA, required for the refilling of granules and sustained cytotoxicity, seems to be unaltered in the presence of PKC modulators. Immunofluorescent studies of CTL-TC conjugates revealed that PKC modulation impairs reorientation of the microtubule organizing center toward the contact point with the TC, which accounts for the specific direction of lytic granules exocytosis. Thus, it appears that PKC regulates exocytosis of lytic granules by governing microtubule reorganization, one of the initial steps in perforin/granzyme-mediated cytotoxicity.

MHC class I is required for peripheral accumulation of CD8+ thymic emigrants.

MHC molecules influence the fate of T lymphocytes at two important stages of their differentiation. Recognition of self peptide/MHC complexes in the thymus determines whether immature T cells should live and mature into immunocompetent T cells or whether they should die. In the periphery, recognition of Ags presented by MHC molecules induces T cell activation, proliferation, and differentiation into effector/memory T cells. We describe in this work a third role that MHC molecules play in T cell physiology. CD8+ thymic emigrants require presence of MHC class I molecules in the periphery to seed the peripheral lymphoid organs. Numbers of CD8+ T cells are reduced severely in both the thymus and the periphery of beta2-microglobulin-deficient (beta2m[-/-]) mice. When grafted with wild-type (beta2m[+/+]) thymic epithelium, immature beta2m(-/-) T cells that populate the graft develop into functional mature CD8+ cells. However, significant numbers of peripheral CD8+ cells in grafted beta2m(-/-) mice can be observed only after injection of MHC class I-expressing cells in the periphery. Thus, naive T cells in the periphery do not passively await antigenic stimulation, but actively engage in interactions with self MHC molecules that may promote their survival.

The role of protein kinase C in CD8+ T lymphocyte effector responses.

Rare CD8+ T cells present in beta2microglobulin-deficient (beta2m-/-) mice reject allogeneic tumors but not syngeneic wild-type tumors. The lack of syngeneic tumor rejection in vivo is correlated with a partial response of beta2m-/- CD8+ cell lines to syngeneic tumor cells in vitro. This partial response is characterized by perforin/granzyme-mediated cytolytic activity in the absence of cytokine secretion or proliferation. Allogeneic tumors induce cytolysis, cytokine secretion, and proliferation. Cytokine secretion may therefore be an important effector mechanism for tumor rejection by CD8+ T cells. To determine the missing signaling events needed for cytokine secretion as well as the events inducing the isolated cytotoxic response, we attempted to restore cytokine secretion of beta2m-/- CD8+ cells to syngeneic MHC class I. Phorbol ester and syngeneic tumor cells acted synergistically to induce full responsiveness of beta2m-/- CD8+ cells. However, this synergistic induction of cytokine secretion used a different pathway than that induced by alloantigen. Protein kinase C (PKC) inhibitor prevented the syngeneic class I plus PMA-induced cytokine secretion, but not allo-class I-induced cytokine secretion. In contrast to the PKC independent alloantigen-induced cytokine secretion, cytolysis of both allogeneic and syngeneic targets was PKC dependent. The differential dependence of effector functions on PKC activation was also found in beta2m+/+ CD8+ T cells. Thus, two distinct signaling pathways (PKC dependent and PKC independent) may ultimately converge to induce cytokine secretion in CD8+ cells. The TCR engagement-initiated pathway is PKC independent, whereas the phorbol ester-activated pathway is PKC dependent.

Lack of an effect of the efficiency of RNA 3'-end formation on the efficiency of removal of either the final or the penultimate intron in intact cells.

Evidence exists from studies using intact cells that intron removal can be influenced by the reactivity of upstream and downstream splice sites and that cleavage and polyadenylation can be influenced by the reactivity of upstream splice sites. These results indicate that sequences within 3'-terminal introns can function in the removal of upstream introns as well as the formation of RNA 3' ends. Evidence from studies using intact cells for an influence of RNA 3'-end formation on intron removal is lacking. We report here that mutations within polyadenylation sequences that either decrease or increase the efficiency of RNA 3'-end formation have no effect on the efficiencies with which either the 3'-terminal or the penultimate intron is removed by splicing. Northern (RNA) blot hybridization, RNase mapping, and an assay that couples reverse transcription and PCR were used to analyze the effects of deletions and a substitution of the polyadenylation sequences within the human gene for triosephosphate isomerase (TPI). TPI pre-mRNA harbors six introns that are constitutively removed by splicing. Relative to normal levels, each of the deletions was found to reduce the nuclear and cytoplasmic levels of TPI mRNA, increase the nuclear level of unprocessed RNA 3' ends, and decrease the nuclear level of processed RNA 3' ends. The simplest interpretation of these data indicates that (i) the rate of 3'-end formation normally limits the amount of mRNA produced and (ii) the deletions decrease and the substitution increases the efficiency of RNA 3'-end formation. While each of the deletions and the substitution altered the absolute levels of intron 6-containing, intron 5-containing, intron 6-free, and intron 5-free RNAs, in no case was there an abnormal ratio of intron-containing to intron-free RNA for either intron. Therefore, at least for TPI RNA, while the efficiency of removal of the 3'-terminal intron influences the efficiency of removal of either the 3'-end formation, the efficiency of RNA 3'-end formation does not influence the efficiency of removal of either the 3'-terminal or penultimate intron. The dependence of TPI RNA 3'-end formation on splicing may reflect the suboptimal strengths of the corresponding regulatory sequences and may function to ensure that TPI pre-mRNA is not released from the chromatin template until it has formed a complex with spliceosomes. If so, then the independence of TPI RNA splicing on 3'-end formation may be rationalized by the lack of a comparable function.

Upstream introns influence the efficiency of final intron removal and RNA 3'-end formation.

For all intron-containing pre-mRNAs of higher eukaryotes that have been examined using either living cells or cell-free extracts, a functional 3' splice site within the 3'-terminal intron is required for efficient RNA 3'-end formation. The mechanism by which intron sequences facilitate RNA 3'-end formation, which is achieved by endonucleolytic cleavage and polyadenylation, is not understood. We report here that in intact cells the efficiency of RNA 3'-end formation correlates with the efficiency of final intron removal, even when the intron is normally a 5'-terminal or internal intron. Therefore, the influence of the 3'-terminal intron on 3'-end formation is likely to be attributable to the determinants of splicing efficiency, which include but are not limited to the 3' splice site. Quantitative RNase mapping and methods that couple reverse transcription and the polymerase chain reaction were used to assess the consequence to RNA 3'-end formation of intron deletions within the human gene for triosephosphate isomerase (TPI). Results indicate that the formation of TPI RNA 3' ends requires TPI gene introns in addition to the last intron, intron 6, to proceed efficiently. These additional TPI gene introns are also required for the efficient removal of intron 6. When introns 1 and 5 were engineered to be the final intron, they were found, as was intron 6, to function in RNA 3'-end formation with an efficiency that correlated with their efficiency of removal. The simultaneous deletion of the 5' and 3' splice sites of intron 6 reduced the efficiencies of both RNA 3'-end formation and the removal of intron 5, which constituted the 3'-most functional intron. Deletion of only the 3' splice site of intron 6 precluded RNA 3'-end formation but had no effect on the efficiency of intron 5 removal. Deletion of only the 5' splice site of intron 6, which resulted in exon 6 skipping (i.e., the removal of intron 5, exon 6, and intron 6 as a single unit), had no effect on the efficiencies of either RNA 3'-end formation or the removal of intron 5-exon 6-intron 6. These results indicate that sequences within the 3'-terminal intron are functionally coupled to both RNA 3'-end formation and removal of the penultimate intron via a network of interactions that form across the last two exons and, most likely, between RNA processing factors.

Distinct spectrum of N-ras mutations in aml and mds patients in yugoslavia.

Mutations that activate ras genes were demonstrated to be associated with certain types of malignancies. Multiple point mutations were predominantly found in the N-ras and occasionally in the K-ras genes. The analysis of 4 MDS, 23 AML and 11 CML patients from Yugoslavia revealed the prevalence of the N-ras mutation (83%) over K-ras mutations (17%). Although the frequencies of the N- and K-ras mutations in these patients were similar to the ones reported for patients from USA and Japan, the N-ras mutational spectra considerably differed. The prevailing type of mutation in patients from Yugoslavia was G-to-T transversion at the first position in the codon 12 of the N-ras gene. This study supports a hypothesis that different geographical and environmental factors may cause the accumulation of different type of point mutations in the same target gene.

Sequences within the last intron function in RNA 3'-end formation in cultured cells.

In cultured cells, little if any mRNA accumulates from an intronless version of the human gene for triosephosphate isomerase (TPI), a gene that normally contains six introns. By deleting introns either individually or in combinations, it was demonstrated by Northern (RNA) blot hybridization that while the deletion of a greater number of introns generally results in a lower level of product mRNA, not all introns contribute equally to mRNA formation. For example, intron 1 appeared to be dispensable, at least when the remaining introns are present, but deletion of the last intron, intron 6, reduced the level of product mRNA to 51% of normal. To determine how intron 6 contributes to mRNA formation, partial deletions of intron 6 were constructed and analyzed. Deletion of the lariat and acceptor splice sites or the donor, lariat, and acceptor splice sites, each of which precluded removal of the intron 6 sequences that remained, reduced the level of product mRNA to < 1 or 27% of normal, respectively. As measured by RNase mapping and cDNA sequencing, the decrease in mRNA abundance that was attributable to the complete and partial intron 6 deletions was accompanied by an increase in the abundance of pre-mRNA that lacked a mature 3' end, i.e., that was neither cleaved nor polyadenylated. We infer from these and other data that sequences within the final intron facilitate proper 3'-end formation, possibly through an association with the components of a productive spliceosome.

Prediction of 'hot spots' in SV40 enhancer and relation with experimental data.

A theoretical method for prediction of the points in the sequence which are relevant for its biological function, the so-called 'hot spots', is presented. This method is based on significant correlation between the spectrum of numerical presentation of any genetic sequence and its biological function. One number corresponds to the particular nucleotide, thus forming a numerical sequence. The 'hot spot' prediction has been tested on the SV40 enhancer as a model system. The SV40 enhancer was chosen because of existing detailed data about activities of systematically obtained mutants. These results have been compared with results obtained theoretically.