Subpopulations of rat lung fibroblasts with different amounts of type I and type III collagen mRNAs

A fluorescence-based method using the cell sorter has been devised to separate rat lung fibroblasts into subpopulations. Type I or type III collagen antiserum was used as the primary antibody to react with parent rat lung fibroblasts. This was followed by a fluorescein-conjugated secondary antibody. Specificity of the primary collagen antibody was determined using a monoclonal beta-actin antibody and purified IgG as the primary antibodies. The fluorescent shift of parent rat lung fibroblasts was optimized for the amount of primary collagen antibody and secondary fluorescein-conjugated antibody. An increase in slot blot intensity was observed for pro-alpha 1(I), pro-alpha 2(I), and pro-alpha 1(III) mRNAs with increasing amounts of cellular RNA. When precipitating with type I collagen antibodies, the total cellular steady-state levels of type I procollagen mRNAs were increased in the high intensity cells as compared with the low intensity cells. Alternately, when the type III collagen antibodies were used to precipitate the rat lung fibroblasts, the low intensity cells had increased type I procollagen mRNAs while the high intensity cells had increased type III procollagen mRNA. The subpopulations of rat lung fibroblasts after isolation using the fluorescent cell sorter were readily propagated for at least four passages.     

The Behavior of Insertions near a Site of Mitotic Gene Conversion in Yeast

In yeast, coincident gene conversion events involving the LEU1 and TRP5 loci (16 cM apart) occur at frequencies that are far greater than is expected for two independent acts of recombination. When a large plasmid (pJM53) is placed between these genes so that a direct repeat is produced, there is frequent loss of the insert among coincident convertants. Previous results strongly suggest that this is due to a separate, intrachromosomal exchange between the direct repeats rather than to excision from an extensive region of heteroduplex DNA. In this paper, we extend our genetic and molecular analysis to a plasmid insertion (pKSH) which replaces rather than duplicates the chromosomal material. The relative stabilities of pKSH and pJM53 are compared among coincident Leu+Trp+ convertants and convertants involving only one locus (LEU1). The pKSH insertion is significantly more stable in the latter which constitute a large majority of the selectable recombinants. In the former, both insertions are lost with high frequency. These results are used to argue that, while most mitotic conversion does not result from long intermediates, coincident convertants may arise from either multiple intermediates or extensive heteroduplex regions.     

A Rapid Chromosome-Mapping Method for Cloned Fragments of Yeast DNA

A rapid and generally applicable method is described for mapping a cloned yeast DNA segment to the chromosome(s) from which it originated. The method is based upon the recent finding that the integration into a yeast chromosome of a segment of the 2 mu plasmid DNA results, in heterozygous diploids, in the specific loss of genetic information from the chromosome into which the 2 mu DNA was integrated (Falco et al. 1982). After verification of the accuracy of the method using several genes whose position was known in advance, the method was used to locate the yeast actin gene, which lies on the left arm of chromosome VI, about 50 cM distal to CDC4.     

Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments.

A system of biological containment for recombinant DNA experiments in Saccharomyces cerevisiae (Brewer's/Baker's yeast) is described. The principle of containment is sterility: the haploid host strains all contain a mating-type-non-specific sterile mutation. The hosts also contain four auxotrophic mutations suitable for selection for the various kinds of vectors used. All vectors are derivatives of pBR322 which can be selected and maintained in both yeast and Escherichia coli. The system has recently been certified at the HV2 level by the National Institutes of Health.     

A standardized laboratory and surgical method for in vitro culture isolation and expansion of primary human Tenon’s fibroblasts

Good manufacturing practices guidelines require safer and standardized cell substrates especially for those cell therapy products to treat ocular diseases where fibroblasts are used as feeder layers. However, if these are unavailable for stem cells culturing, murine fibroblasts are regularly used, raising critical issues as accidentally transplanting xenogenous graft and adversely affecting stem cell clinical trials. Moreover, human fibroblasts play a significant role in testing novel ophthalmologic drugs. Accordingly, we developed a standardized laboratory and surgical approach to isolate normal and undamaged Tenon’s fibroblasts to implement the setting up of banks for both stem cells-based ocular cell therapy and in vitro drug testing. A 2–3 cm² undamaged Tenon’s biopsy was surgically obtained from 28 patients without mutually correlated ocular diseases. Nineteen dermal biopsies were used as control. Fibroblasts were isolated with or without collagenase, cultured in autologous, fetal bovine or AB serum, tested for viability by trypan blue, vimentin expression and standardized until passage 6. Successful Tenon’s fibroblasts isolation was age dependent (P = 0.001) but not sex, pathology or surgery related. A significant rate of successful cultures were obtained when biopsies were not digested by collagenase (P = 0.013). Moreover, cultures in autologous or fetal bovine serum had comparable proliferative properties (P = 0.77; P = 0.82). Through our surgical and laboratory standardized procedure, we elucidated for the first time key points of this human primary culture system, the role of the autologous serum, comparing Tenon’s and dermal fibroblasts. Our protocol may be clinically useful to reduce the risk above mentioned and may be potentially more effective for ophthalmological clinical purposes.     

MECHANICAL ANALYSIS OF THE ROUNDHOUSE KICK ACCORDING TO HEIGHT AND DISTANCE IN TAEKWONDO

Competition regulation in taekwondo has experienced several changes during the last few years, for example, kicks to the head score more points than kicks to the chest. In addition, some external factors such as the height of target and execution distance seem to affect the kick performance. The aim of this study was to analyse selected biomechanical parameters (impact force, reaction time, and execution time) according to the height and execution distance in two different male groups (experts (n = 12) and novices (n = 21)). Athletes kicked twice from every execution distance (short, normal and long) and towards two different heights of target (chest and head) in a random order. Novices kicked to the head with a longer reaction time than to the chest (p < 0.05) but experts were able to kick with similar performance for both heights. From short and normal distances experts kicked with similar performance; whereas from the normal distance novices had longer reaction and execution time than from the short distance (p < 0.05). In conclusion, in counterattacking situations, experts should perform the roundhouse kick to the head instead of to the chest, because it produces better scores with similar performance; whereas novice athletes should avoid kicking to the head because they are not able to kick with similar performance. Moreover, it is recommended that during counterattacks higher-level taekwondo athletes should intend to kick from normal distances.     

Retinoic Acid Specifically Enhances Embryonic Stem Cell Metastate Marked by Zscan4

Pluripotency confers Embryonic Stem Cells (ESCs) the ability to differentiate in ectoderm, endoderm, and mesoderm derivatives, producing the majority of cell types. Although the majority of ESCs divide without losing pluripotency, it has become evident that ESCs culture consists of multiple cell populations with different degrees of potency that are spontaneously induced in regular ESC culture conditions. Zscan4, a key pluripotency factor, marks ESC subpopulation that is referred to as high-level of pluripotency metastate. Here, we report that in ESC cultures treated with retinoic acid (RA), Zscan4 ESCs metastate is strongly enhanced. In particular, we found that induction of Zscan4 metastate is mediated via RA receptors (RAR-alpha, RAR-beta, and RAR-gamma), and it is dependent on phosphoinositide-3-kinase (PI3K) signaling. Remarkably, Zscan4 metastate induced by RA lacks canonical pluripotency genes Oct3/4 and Nanog but retained both self-renewal and pluripotency capabilities. Finally we demonstrated that the conditional ablation of Zscan4 subpopulation is dispensable for both endoderm and mesoderm but is required for ectoderm lineage. In conclusion, our research provides new insights about the role of RA signaling during ESCs high pluripotency metastate fluctuation.     

Common dandelion: a review of its botanical,phytochemical and pharmacological profiles

Phytochemistry Reviews - Phytoalimurgy is a term that derives from Greek and Latin by combination of the words φυτόν, which meaning plant, and alimenta urgentia, indicating...