Advancing cartilage tissue engineering: the application of stem cell technology

The treatment of cartilage pathology and trauma face the challenges of poor regenerative potential and inferior repair. Nevertheless, recent advances in tissue engineering indicate that adult stem cells could provide a source of chondrocytes for tissue engineering that the isolation of mature chondrocytes has failed to achieve. Various adjuncts to their propagation and differentiation have been explored, such as biomaterials, bioreactors and growth hormones. To date, all tissue engineered cartilage has been significantly mechanically inferior to its natural counterparts and further problems in vivo relate to poor integration and deterioration of tissue quality over time. However, adult stem cells--with their high rate of proliferation and ease of isolation--are expected to greatly further the development and usefulness of tissue engineered cartilage.     

Methylotrophic extremophilic yeast Trichosporon sp.: a soil-derived isolate with potential applications in environmental biotechnology

A yeast isolate revealing unique enzymatic activities and substrate-dependent polymorphism was obtained from autochthonous microflora of soil heavily polluted with oily slurries. By means of standard yeast identification procedures the strain was identified as Trichosporon cutaneum. Further molecular PCR product analyses of ribosomal DNA confirmed the identity of the isolate with the genus Trichosporon. As it grew on methanol as a sole carbon source, the strain appeared to be methylotrophic. Furthermore, it was also able to utilize formaldehyde. A multi-substrate growth potential was shown with several other carbon sources: glucose, glycerol, ethanol as well as petroleum derivatives and phenol. Optimum growth temperature was determined at 25 degrees C, and strong inhibition of growth at 37 degrees C together with the original soil habitat indicated lack of pathogenicity in warm-blooded animals and humans. The unusually high tolerance to xenobiotics such as diesel oil (>30 g/l), methanol (50 g/l), phenol (2 g/l) and formaldehyde (7.5 g/l) proved that the isolate was an extremophilic organism. With high-density cultures, formaldehyde was totally removed at initial concentrations up to 7.5 g/l within 24 h, which is the highest biodegradation capability ever reported. Partial biodegradation of methanol (13 g/l) and diesel fuel (20 g/l) was also observed. Enzymatic studies revealed atypical methylotrophic pathway reactions, lacking alcohol oxidase, as compared with the conventional methylotroph Hansenula polymorpha. However, the activities of glutathione-dependent formaldehyde dehydrogenase, formaldehyde reductase, formate dehydrogenase and unspecific aldehyde dehydrogenase(s) were present. An additional glutathione-dependent aldehyde dehydrogenase activity was also detected. Metabolic and biochemical characteristics of the isolated yeast open up new possibilities for environmental biotechnology. Some potential applications in soil bioremediation and wastewater decontamination are discussed.     

Construction of physical map and mapping of chromosomal virulence genes of the biovar 3 Agrobacterium (Rhizobium vitis) strain K-Ag-1

Most plant pathogenic Agrobacterium strains have been classified into three biovars, "biovar 1 (A. tumefaciens; Rhizobium radiobacter), biovar 2 (A. rhizogenes; R. rhizogenes) and biovar 3 (A. vitis; R. vitis)". The bacteria possess diverse types of genomic organization depending on the biovar. Previous genomic physical maps indicated difference in location of rDNA and chromosomally-coded virulence genes between biovar 1 and 2 genomes. In order to understand biovar 3 genome and its evolution in relation to the biovar 1, 2 and 3 genomes, we constructed physical map of a pathogenic biovar 3 strain K-Ag-1 in this study. Its genome consisted of two circular chromosomes (3.6 and 1.1 Mbp in length), and three plasmids (560, 230 and 70 kbp). Gene mapping based on the physical map showed presence of two rDNA loci in the larger chromosome and at least one rDNA locus in the smaller chromosome. Six chromosomal virulence genes, namely chvA, chvD, chvE, glgP, exoC and ros were found in the larger chromosome and not in the smaller chromosome. The location of rDNA loci is similar with that of biovar 1 genome, whereas the location of chromosomal virulence genes is similar with that of biovar 2 genome despite of the closer 16S-rRNA based phylogenetic relation of biovar 3 with biovar 1 than with biovar 2. Genomic PFGE RFLP analysis revealed that the K-Ag-1 strain, which was isolated on a kiwifruit plant in Japan, has the closest intra-species relation with two strains isolated from grapevine plants in Japan among eight biovar 3 strains examined. This datum suggests that the line of the strain is a major one in biovar 3 in Japan. Evolution of the genome of the strain is discussed based on the data.     

Neuroendocrine changes in the aging reproductive axis of female rhesus macaques (Macaca mulatta)

Femalerhesus macaques show monthly menstrual cycles and eventually enter menopause at approximately 25 yr of age. To help identify early biomarkers of menopause in this nonhuman primate, we monitored reproductive hormones longitudinally from aged female macaques during the transitions from premenopause to perimenopause and postmenopause and found that, indeed, elevated plasma FSH was a better predictive factor of menopause onset than age. In a second experiment, we compared reproductive hormone profiles of young adult macaques (8-10 yr old) with those of regularly cycling old macaques (approximately 24 yr old). Indwelling vascular catheters were used for remote blood collection for at least 100 consecutive days, thereby covering three complete menstrual cycles in each macaque. Plasma levels of estradiol, progesterone, LH, FSH, follicular phase inhibin B, and anti-müllerian hormone (AMH) were determined during each menstrual cycle and were averaged for each animal; group mean differences were analyzed using one-way ANOVA. Old premenopausal macaques showed regular menstrual cycles that were qualitatively indistinguishable from those of young macaques; peak plasma levels of estradiol, progesterone, and LH were not significantly different. In marked contrast, peak plasma FSH concentrations were significantly higher, while inhibin B and AMH levels were generally lower, in the old premenopausal macaques compared with those in the young macaques. These data provide further evidence that rhesus macaques serve as an excellent model to study underlying mechanisms of human menopause. Furthermore, the data suggest that an age-related change in FSH, inhibin B, and AMH secretion may be the first endocrine manifestation of the transition into perimenopause, potentially having value in predicting the onset of the perimenopausal transition.     

Sequence of deltaF508 CFTR allele identified at present is lacking in medieval specimens from Central Poland. Preliminary results

deltaF508 is the most common (70%) among over 1000 mutations of the gene encoding ATP-regulated chloride channel, namely CFTR--cystic fibrosis transmembrane regulator. The time which passed from the calculated mutation event was anticipated on the basis of the frequency of contemporary haplotypes, but not on its direct identification. The presence of three base pairs deletion in the ancient DNA (aDNA) isolated from skeletal remains of the Middle Ages origin was investigated. Teeth excavated in the area of three sites located in Central Poland were processed for a DNA. 6 out of 82 samples did not produce amplificable fragments of DNA. Although the number of specimens analyzed was sufficient to confirm the presence of the rare mutation, the deltaF508 CFTR sequence was not found in the remains of individuals living back 35 - 45 generations. The absence of the mutated allele in the particular geographic region cannot state for the status of mutated allele throughout the country, especially at times when migrations were limited and movements of people were more area restricted than at present days.     

Analysis of mercury levels in historical bone material from syphilitic subjects--pilot studies (short report)

The aim of the present work is to study the remains of seven individuals with typical symptoms of tertiary syphilis in terms of mercury content in bones, thereby verifying whether they were subjected to treatment and, if they were, how long their organisms were exposed to toxic mercury fumes. Mercury was used, mainly in the Middle Ages and in the early modern period, as a preventive measure in case of individuals suffering from syphilis, a venereal disease, and also leprosy. Syphilitic patients treated this way should demonstrate increased concentration of mercury in their bones. The skeletons studied in the present work originate from various archaeological sites in southern and north-central Poland. The analyses concerned individuals with diagnosed syphilis as well as healthy individuals who constituted the control group. The analyses were performed by the LA-ICP-MS technique, with the use of laser Nd: YAG, Macro, 266 nm, New Wave, USA, coupled with Spectrometer Elan DRC-e Perkin Elmer, USA. The content analysis of the studied bone material revealed with high probability that the contact method of mercurial treatment was used only in the case of two women from north-central Poland, deceased at the turn of the 15th century at the earliest.     

A developmentally regulated lipocalin-like gene is overexpressed in Tomato yellow leaf curl virus-resistant tomato plants upon virus inoculation, and its silencing abolishes resistance

To discover genes involved in tomato resistance to Tomato yellow leaf curl virus (TYLCV), we previously compared cDNA libraries from susceptible (S) and resistant (R) tomato lines. Among the genes preferentially expressed in R plants and upregulated by TYLCV infection was a gene encoding a lipocalin-like protein. This gene was termed Solanum lycopersicum virus resistant/susceptible lipocalin (SlVRSLip). The SlVRSLip structural gene sequence of R and S plants was identical. SlVRSLip was expressed in leaves during a 15-day window starting about 40?days after sowing (20?days after planting). SlVRSLip was upregulated by Bemisia tabaci (the TYLCV vector) feeding on R plant leaves, and even more strongly upregulated following whitefly-mediated TYLCV inoculation. Silencing of SlVRSLip in R plants led to the collapse of resistance upon TYLCV inoculation and to a necrotic response along the stem and petioles accompanied by ROS production. Contrary to previously identified tomato lipocalin gene DQ222981, SlVRSLip was not regulated by cold, nor was it regulated by heat or salt. The expression of SlVRSLip was inhibited in R plants in which the hexose transporter gene LeHT1 was silenced. In contrast, the expression of LeHT1 was not inhibited in SlVRSLip-silenced R plants. Hence, in the hierarchy of the gene network conferring TYLCV resistance, SlVRSLip is downstream of LeHT1. Silencing of another gene involved in resistance, a Permease-I like protein, did not affect the expression of SlVRSLip and LeHT1; expression of the Permease was not affected by silencing SlVRSLip or LeHT1, suggesting that it does not belong to the same network. The triple co-silencing of SlVRSLip, LeHT1 and Permease provoked an immediate cessation of growth of R plants upon infection and the accumulation of large amounts of virus. SlVRSLip is the first lipocalin-like gene shown to be involved in resistance to a plant virus.     

Cytopathology of Tomato torrado virus Infection in Tomato and Nicotiana benthamiana

Electron microscopy studies were carried out to investigate the cytopathological changes induced in tomato leaves by Tomato torrado virus (ToTV) that infects tomato plants worldwide causing severe necrotic symptoms. Plants infected with one of the Polish isolates of ToTV were used for cytopathological research. The results revealed severe cellular alterations, especially in Solanum lycopersicum. Moreover, it was shown that crystalline aggregates of virions occurred not only within the phloem cells as it has been previously reported.