Cytoplasmic and nuclear protein synthesis during in vitro differentiation of murine ICM and embryonal carcinoma cells

Protein syntheses during in vitro differentiation of inner cell masses (ICM) isolated from mouse blastocysts and of pluripotent embryonal carcinoma cells (ECC) were compared by two-dimensional electrophoretic analysis of [³⁵S]methionine-labeled cells. While most of the polypeptides found in ICM, ECC, and embryoid bodies (EB) derived from them were common to all four preparations, some distinct differences were noted. More polypeptides changed in intensity during the differentiation of ICM than during the differentiation of ECC. Analysis of ECC prior to differentiation revealed that only some of the polypeptides abundant in ICM were present, while at the same time, some of the polypeptides abundant in ICM-EB were being synthesized. These data indicate that ECC represent cells further advanced in development than the cells of ICM isolated from 4-day-old blastocysts. The EB derived from ECC also differ from those from ICM. Comparison of EB derived from ICM and ECC with cells of the parietal yolk sac line, PYS, indicates that all three synthesize two polypeptides abundant in EB. These two polypeptides can, therefore, be used as biochemical markers of parietal entoderm differentiation. Pluripotent ECC synthesize small amounts of characteristic EB proteins and the 10-nm filament protein (also found in PYS cells but not in EB). This indicates that small numbers of differentiated or differentiating cells are present in pluripotent ECC cultures.     

Exonuclease requirements for recombination of lambda-phage in recD mutants of Escherichia coli

Recombination of lambda red gam phage in recD mutants is unaffected by inactivation of RecJ exonuclease. Since nucleases play redundant roles in E. coli, we inactivated several exonucleases in a recD mutant and discovered that 5'-3' exonuclease activity of RecJ and exonuclease VII is essential for lambda-recombination, whereas exonucleases of 3'-5' polarity are dispensable. The implications of the presented data on current models for recombination initiation in E. coli are discussed.     

A case of canine hypodontia in an early Croatian cemetery Strance-Gorica

In the old Croatian cemetery Strance-Gorica in the Vinodol region, dating from the 9th to 11th century, osteological parts of the upper and the lower jaws with teeth were found, besides some other archeological finds. Data processing in dentistry regarding a possible presence of hypodontia was carried out on archeological finds (skeletal remains) on 27 persons available for the research. Only one case of canine hypodontia was found and described. In the remaining 26 persons no case of hypodontia was found on the relicts of the upper and lower jaws nor in other teeth groups. The frequency of hypodontia in the old Croatian cemetery Strance-Gorica was 3.7, which corresponds to the frequency of this anomaly in the 20th century population of Croatia.     

Distribution of hepatitis C virus genotypes in Croatia--a 10 year retrospective study of four geographic regions

The aim of this 10-year retrospective study was to investigate the distribution of HCV genotypes in patients with chronic hepatitis C monitored in the largest center for molecular diagnostics of HCV infection in Croatia. The study enrolled 1163 anti-HCV positive adults with detectable HCV RNA in the plasma. The patients were classified in four regions: Zagreb and surrounding continental area, Split, Slavonija and Rijeka. HCV genotyping was performed by using VERSANT HCV Genotyping Assay (LIPA) (Bayer Diagnostics, Puteaux Cedex, France). Statistical analysis was performed by using Statistica for Windows V.5.1. The majority of HCV infections in the study population were caused by genotypes 1 (58.8% of infected patients) and 3 (35.6%). Percentages of patients infected with subtypes 1b and 1a were 37.4% and 13.1%, respectively. Genotypes 2 and 4 were present in a very low percentage of patients (2.2% and 3.4%, respectively) while genotypes 5 and 6 were not detected. Analysis of regional differences in the distribution of HCV genotypes revealed similar percentages of subtype 3a and 1b infections in the Split region while the majority of infections in other regions were caused by subtype 1b. Infections with genotypes 2 and 4 were present in less than 5% of patients in all geographic regions. Analysis of an association between risk factors for infection and distribution of genotypes and subtypes in a subset of patients from the Split region confirmed the association between IVDU and subtype 3a. We conclude that the prevalence of HCV genotypes and subtypes follows the pattern of other Southern and Eastern European Countries with the predominance of subtypes 1b, 3a and 1a.     

Prevalence of scoliosis in school-children from Mostar, Bosnia and Herzegovina

In the school-year 2002/2003 a prospective epidemiological study was performed with the aim of evaluating the prevalence and distribution of scoliosis in the population of schoolchildren from Mostar, Bosnia and Herzegovina. The general check-up of primary-school children covered a total of 2,517 children aged 7-14. The children in which at least one positive symptom of scoliosis was found were directed to undergo orthopedic examination and--if indicated--radiography. Incorrect posture was noted in 33.4% of children, and 11.8% of children were sent to orthopedic specialist examination. The prevalence of structural scoliosis amounted to 3.1%, with the spine curvature threshold being 10 degrees. In eight children (0.32%; 1 boy and 7 girls) a curvature of 20 degrees or more was diagnosed. The most common type of curvature was the thoracal (39%) and the thoraco-lumbar (39%) while 14 children had a double curvature (17.8%). A scoliosis was detected due to here performed check-up in 83.5% of children with scoliosis. No case of serious spine deformity (45 degree or more) was recorded, due to regular general check-ups taking place biannually in this population.     

Roles of ExoI and SbcCD Nucleases in “Reckless” DNA Degradation in recA Mutants of Escherichia coli

Exponentially growing recA mutant cells of Escherichia coli display pronounced DNA degradation that starts at the sites of DNA damage and depends on RecBCD nuclease (ExoV) activity. As a consequence of this “reckless” DNA degradation, populations of recA mutants contain a large proportion of anucleate cells. We have found that both DNA degradation and anucleate-cell production are efficiently suppressed by mutations in the xonA (sbcB) and sbcD genes. The suppressive effects of these mutations were observed in normally grown, as well as in UV-irradiated, recA cells. The products of the xonA and sbcD genes are known to code for the ExoI and SbcCD nucleases, respectively. Since both xonA and sbcD mutations are required for strong suppression of DNA degradation while individual mutations have only a weak suppressive effect, we infer that ExoI and SbcCD play partially redundant roles in regulating DNA degradation in recA cells. We suggest that their roles might be in processing (blunting) DNA ends, thereby producing suitable substrates for RecBCD binding.The RecA protein plays a central role in homologous recombination and recombinational DNA repair in Escherichia coli, as well as in other bacterial species. It catalyzes the key stages of the recombination process—homologous pairing and DNA strand exchange. Cells carrying null mutations in the recA gene are completely deficient for homologous recombination and are extremely sensitive to DNA-damaging agents (for a review, see references 21, 24, and 25). Populations of recA null mutants contain a large proportion (50 to 60%) of nonviable cells, reflecting the inability of these mutants to repair spontaneously occurring DNA damage (31). Also, exponentially growing recA cells display pronounced spontaneous DNA degradation that presumably starts at the sites of DNA damage and that depends on RecBCD nuclease (ExoV) activity (5, 48). This phenotype of recA cells is aggravated after DNA-damaging treatment, such as UV irradiation (48).According to the present data, the majority of RecA-catalyzed DNA transactions in E. coli start with binding of the RecA protein onto single-stranded DNA (ssDNA) substrates. This binding is mediated by the RecBCD and/or RecFOR protein, which helps RecA to overcome hindrance imposed by the SSB protein during competition for the DNA substrate. The RecBCD and RecFOR proteins begin RecA polymerization on ssDNA, giving rise to a nucleoprotein filament that is indispensable for further recombination reactions (3, 33; reviewed in reference 44).The RecBCD enzyme is crucial for initiation of recombinational processes at double-stranded DNA (dsDNA) ends (or breaks [DSBs]) in wild-type E. coli (a set of reactions known as the RecBCD pathway) (9, 43, 44). Upon recognizing a blunt or nearly blunt dsDNA end and binding to it, RecBCD acts as a combination of powerful helicase and nuclease, thus unwinding and simultaneously degrading both strands of the DNA duplex. After encountering a specific octanucleotide sequence designated Chi, the strong 3′-5′ nuclease activity of the enzyme is attenuated and a weaker 5′-3′ nuclease activity is upregulated (1). This Chi-dependent modification allows RecBCD to create a long 3′ ssDNA tail and to direct the loading of RecA protein onto it (2, 3). In vivo data suggest that this transition of RecBCD from a nuclease to a recombinase mode of action requires the presence of the RecA protein, suggesting that the two proteins might interact (27).In wild-type E. coli cells, the RecFOR protein complex works predominantly on DNA gaps, which may arise in chromosomes due to replication forks passing over the noncoding lesions (e.g., UV-induced pyrimidine dimers) or may be present in replication forks stalled at different obstacles in DNA (44). On the other hand, the RecFOR complex has an important role in recBC sbcBC(D) mutant cells, replacing the RecA-loading activity of RecBCD during recombination reactions starting from dsDNA ends. Recombination reactions mediated by RecFOR proteins are termed the RecF (or RecFOR) pathway (44).Cells mutated in the recB and/or recC gene exhibit strong deficiency in conjugational and transductional recombination, as well as in the repair of DSBs (8, 21). These defects can be rectified by extragenic sbcB and sbcC(D) suppressor mutations that inactivate two nucleases, thus enabling full efficiency of the RecF pathway on dsDNA ends (21, 44). The sbcB gene (also designated xonA) encodes exonuclease I (ExoI), the enzyme that digests ssDNA in the 3′-5′ direction (23). The sbcC and sbcD genes encode subunits of the SbcCD nuclease, which acts both as an endonuclease that cleaves hairpin structures and as an exonuclease that degrades linear dsDNA molecules (10, 11). Inactivation of either of the two subunits leads to the loss of SbcCD enzyme activity (18).The exact mechanism of activation of the RecF pathway by sbc mutations is not completely understood. A plausible explanation is that inactivation of ExoI and SbcCD nucleases is necessary to prevent the degradation of recombinogenic 3′ DNA ends created in a RecBCD-independent manner (8, 23, 38, 45, 46). It was recently shown that the sbcB15 mutant allele (encoding a protein without nucleolytic activity) (37) is a better suppressor of the RecBCD⁻ phenotype than an sbcB deletion (50), suggesting that some nonnucleolytic activity of ExoI may also contribute to the efficiency of the RecF pathway (46, 50).ExoI and SbcCD are usually viewed as enzymes with inhibitory roles in recombination due to their deleterious actions on the RecF pathway. However, some results suggest that these enzymes could also have stimulatory roles in recombination reactions proceeding on the RecBCD pathway. Genetic experiments with UV-irradiated E. coli cells indicated that ExoI and SbcCD might be involved in blunting radiation-induced DNA ends prior to RecBC(D) action (38, 45, 46). Such a role of ExoI and SbcCD seems to be particularly critical in recD recF mutants, in which the majority of DSB repair depends on the RecBC enzyme (38). It was also suggested that the blunting roles of the two nucleases may be required during conjugational recombination (16, 46).In this work, we studied the effects of sbcB (xonA) and sbcD mutations on DNA degradation occurring spontaneously in exponentially growing recA mutant cells, as well as on DNA degradation induced in recA mutants by UV irradiation. We have demonstrated that in both cases DNA degradation is strongly reduced in recA mutants that carry in addition a combination of xonA and sbcD null mutations. The results described in this paper suggest that ExoI and SbcCD play partially redundant roles in regulating DNA degradation in recA cells.     

Ultrasound evaluation of extracranial carotid artery lesions in Parkinsonian patients

The purpose of this investigation was to determine the atherosclerotic changes in patients with vascular parkinsonism and in patients with idiopathic Parkinson's disease, in order to evaluate the possible influence of the extracranial pathology of carotid arteries in developing lacunar cerebral infarcts. Degree of stenosis and plaque morphology of the extracranial part of carotids in both group of patients were evaluated by color Doppler flow imaging ultrasound investigation and the results were compared. We selected two matched groups of patients with parkinsonism: 22 patients with vascular parkinsonism, and 28 with idiopathic Parkinson's disease.The atherosclerotic changes found in patients with Parkinson's disease showed mild carotid lesions with mostly stable calcified plaques and lesser risk for embolic cerebral intravascular events contrary to the higher degree of carotid stenosis found in patients with vascular parkinsonism with mostly mixed plaques prone to embolization. Therefore, we suggest performing ultrasonographic examination of the extracranial part of carotid arteries in all patients with parkinsonism to assess risk of vascular accidents originating from carotid lesions. That would enable adequate treatment of parkinsonism and prevent further occurrence of intracranial vascular changes.     

Chromotherapy of macular degeneration with transitions lenses and green-yellow medical filters and special programme for psychoorganic disturbances

Optical spectrum of the sunlight consists of visible or chromatic spectrum, with the range of wavelengths of electromagnetic vibrations from 7700 to 3900 AU, and the invisible spectrum: infrared and ultraviolet. Chromatic spectrum gives rise to the sensation of colour, capable for simulating specialized retinal photoreceptors and is perceptible as light. This rule of perception of the particular range of the optical spectrum goes mainly for man, while particular deviations, more or less, are applicable to the rest of animal and plant life. The optical part of the spectrum belongs to nonionizing radiation. It created the life on the Earth, maintaining it nowadays and even threatening the human organ of vision, because the retina had not been yet adequately accommodated through evolution with its photoreactive metabolism. Human retina is very sensitive about possible harmful influence of ultraviolet and blue light even today in evolution, but also phototoxic on complete strong visible light. In their clinical and experimental work on animals, the authors prove with their own patent (P 20020077A)-Vojnikovi? B&D, and in collaboration with Essilor Optic Austria GmbH, that particular medical filters in the range of green-yellow colour especially (565 to 570 nm), and in combination with "Transitions" successfully threat macular degeneration-AMD, slowing down its progression and having positive psychoorganic effect on the depressive mood of such patients with threatened sight. Full attention has been paid to the design of medical filter, so the periphery of the lens plays a positive role in blood concentration of melatonin, while the central part stimulates the sight and the concentration of serotonine. Thus the physiological balance of melatonin and serotonin and the stability of psychophysical disturbances have been achieved.     

From teratocarcinomas to embryonic stem cells and beyond: a history of embryonic stem cell research

We are currently facing an unprecedented level of public interest in research on embryonic stem cells, an area of biomedical research that until recently was small, highly specialized and of limited interest to anyone but experts in the field. Real and imagined possibilities for the treatment of degenerative and other diseases are of special interest to our rapidly ageing population; real and imagined associations of stem cells to cloning, embryos and reproduction stir deeply held beliefs and prejudices. The conjunction of these factors could explain the recent sudden interest in embryonic stem cells but we ought to remember that this research has a long and convoluted history, and that the findings described today in the scientific and popular press are firmly grounded in research that has been going on for several decades. Here I briefly recapitulate this fascinating history.     

Intravenous immunoglobulin ameliorates ITP via activating Fc gamma receptors on dendritic cells

Despite a more than 20-year experience of therapeutic benefit, the relevant molecular and cellular targets of intravenous immunoglobulin (IVIg) in autoimmune disease remain unclear. Contrary to the prevailing theories of IVIg action in autoimmunity, we show that IVIg drives signaling through activating Fc gamma receptors (Fc gammaR) in the amelioration of mouse immune thrombocytopenic purpura (ITP). The actual administration of IVIg was unnecessary because as few as 10(5) IVIg-treated cells could, upon adoptive transfer, ameliorate ITP. IVIg did not interact with the inhibitory Fc gammaRIIB on the initiator cell, although Fc gammaRIIB does have a role in the late phase of IVIg action. Notably, only IVIg-treated CD11c+ dendritic cells could mediate these effects. We hypothesize that IVIg forms soluble immune complexes in vivo that prime dendritic-cell regulatory activity. In conclusion, the clinical effects of IVIg in ameliorating ITP seem to involve the acute interaction of IVIg with activating Fc gammaR on dendritic cells.