State of art and limitations in genetic engineering to induce stable chondrogenic phenotype

Current protocols for chondrocyte expansion and chondrogenic differentiation of stem cells fail to reduce phenotypic loss and to mitigate hypertrophic tendency. To this end, cell genetic manipulation is gaining pace as a means of generating cells with stable chondrocyte phenotype. Herein, we provide an overview of candidate genes that either induce cartilage regeneration or inhibit cartilage degeneration. We further discuss in vitro, ex vivo and in vivo viral transduction and non-viral transfection strategies for targeted cells (chondrocytes, mesenchymal stem cells, induced pluripotent stem cells and synovial cells), along with the most representative results obtained in pre-clinical models and in clinical trials. We highlight current challenges and associated risks that slowdown clinical acceptance and commercialisation of gene transfer technologies.     

Protein synthesis patterns in barley embryos during germination

Summary The incorporation pattern of [¹⁴C] amino-acid into protein during the first 8 h of germination in isolated barley embryos (Hordeum vulgare) is described. Two maxima were recognised. The first, at 4 h, was entirely accounted for by scutellum activity and the second, at 8 h, coincided with active radicle elongation. An intervening minimum was situated at 5.5 h. The first peak was insensitive to actinomycin-D but the second showed a partial inhibition by this compound. Only slight changes in enzyme activity were associated with these periods of increased synthesis. Incorporation of [17-¹⁴C] kaurenoic acid into compounds co-chromatographing with gibberellins was followed over the same period in both embryos and scutella and high activity was found after only 2–4 h. It is concluded that, on the basis of protein synthetic activity, the scutellum is the most probable source of the initial gibberellin stimulus.     

REINTERPRETATION OF A CONULARIID-LIKE FOSSIL FROM THE VENDIAN OF RUSSIA

Abstract:  Vendoconularia triradiata Ivantsov and Fedonkin, recently described from Vendian (latest Proterozoic) strata of Russia, has been interpreted as a six-sided conulariid cnidarian. However, comparison of published illustrations of V .  triradiata with Palaeozoic conulariids suggests that certain key features of the anatomy of V .  triradiata should be reinterpreted. Specifically, features previously homologized with the corners of conulariid thecae may actually be homologous to the conulariid midlines. Under this new interpretation, the corners of the Vendoconularia theca were sulcate, and the midline of each face was non-sulcate and flanked by a pair of low internal carinae. This alternative set of hypotheses of homology makes the argument for a conulariid affinity for Vendoconularia stronger.     

CLADISTIC ANALYSIS OF THE SUBORDER CONULARIINA MILLER AND GURLEY, 1896 (CNIDARIA, SCYPHOZOA; VENDIAN–TRIASSIC)

Abstract: Results of a cladistic analysis of the suborder Conulariina Miller and Gurley, 1896, a major extinct (Vendian–Triassic) group of scyphozoan cnidarians, are presented. The analysis sought to test whether the three conulariid subfamilies (Conulariinae Walcott, 1886 , Paraconulariinae Sinclair, 1952 and Ctenoconulariinae Sinclair, 1952 ) recognized in the Treatise on Invertebrate Paleontology (TIP) are monophyletic. A total of 17 morphological characters were scored for 16 ingroup taxa, namely the genera Archaeoconularia, Baccaconularia, Climacoconus, Conularia, Conulariella, Conularina, Ctenoconularia, Eoconularia, Glyptoconularia, Metaconularia, Notoconularia, Paraconularia, Pseudoconularia, Reticulaconularia, Teresconularia and Vendoconularia. The extant medusozoan taxa Cubozoa, Stauromedusae, Coronatae and Semaeostomeae served as outgroups. Unweighted analysisof the data matrix yielded 1057 trees, and successive weighting analysis resulted in one of the 1057 original trees. The ingroup is monophyletic with two autapomorphies: (1) the quadrate geometry of the oral region; and (2) the presence of a mineralized (phosphatic) periderm. Within the ingroup, the clade (Vendoconularia, Teresconularia, Conularina, Eoconularia) is supported by the sinusoidal longitudinal geometry of the transverse ridges, and the much larger clade (Baccaconularia, Glyptoconularia, Metaconularia, Pseudoconularia, Conularia, Ctenoconularia, Archaeoconularia, Notoconularia, Climacoconus, Paraconularia, Reticulaconularia) is supported by the presence of external tubercles, which, however, were lost in the clade (Notoconularia, Climacoconus, Paraconularia, Reticulaconularia). As proposed by Van Iten et al. (2000) , the clade (Notoconularia, Climacoconus, Paraconularia, Reticulaconularia) is supported by the termination and alternation of the transverse ribs in the corner sulcus. The previously recognized subfamilies Conulariinae, Paraconulariinae and Ctenoconulariinae were not recovered from this analysis. The diagnostic features of Conulariinae (continuation of the transverse ornament across the corner sulcus and lack of carinae) and Ctenoconulariinae (presence of carinae) are symplesiomorphic or homoplastic, and Paraconulariinae is polyphyletic. The families Conulariellidae Kiderlen, 1937 and Conulariopsidae Sugiyama, 1942 , also recognized in the TIP, are monogeneric, and since they provide no additional phylogenetic information, should be abandoned.     

Prevalence of tick-borne encephalitis virus antibodies in dogs from Denmark

Background  

Large regions of central and eastern Europe are recognized as areas where tick-borne encephalitis virus (TBEV) is endemic, including countries neighbouring Denmark. It is therefore timely and relevant to determine if TBEV infections occur in Denmark. This study investigates the presence of antibodies against TBEV in a cross-section of the Danish canine population to assess the level of exposure to TBEV and possibly identify TBEV microfoci in Denmark.     

The promoter of filamentation (POF1) protein from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> is an ATPase involved in the protein quality control process

Background

The gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene.

Results

Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo.

Conclusions

Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation.

     

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation.     

Plasma testosterone concentration, body weight, social dominance and scent-marking in male marsupial sugar gliders (Petaurus breviceps; Marsupialia: Petauridae)

Observations were made of a wild population of sugar gliders ( Petaurus breviceps ) by live-trapping and on four captive colonies to establish the relationship between blood concentration of testosterone, body weight, social dominance and the timing of births. In both the wild and in captivity one male exhibits a testosterone concentration far higher than his fellows, during the breeding season. This male is also the heaviest. In captivity this male is observed to be socially the most dominant and undertakes almost all of the scent-marking. At least three-quarters of each breeding season's births occur during the first month of the three- to four-month season (July-September/October), a passage of time during which the body weight of the most dominant male falls more than that of the subordinates. It is suggested that this species is highly polygynous, with the ecological and evolutionary benefits of high social status being immense.     

A comparative study of lectin binding to cultured chick sternal chondrocytes and intact chick sternum

Cultured chondrocytes derived from the caudal and cephalic ends of embryonic chick sterna have been compared with each other and with whole sternum, by using a panel of 21 lectins to probe the distribution of oligosaccharides in glycoconjugates of cells and matrix at various times of culture or development. On culture in collagen gels, the cells changed their morphology with time, degrading glycan in the surrounding culture medium and depositing new matrix, the glycan content of which reflected the site of origin of the cells, indicating that the glycan phenotype of both cells and matrix (‘glycotype’) was predetermined and persistent. Sterna of embryonic chicks showed unexpected complexity in their distribution pattern of glycan, containing at least six distinct regions. Major regional temporal differences were evident among saccharides terminating in α-N-acetyl galactosamine and β-galactose, while changes in glycans terminating in fucose, sialic acid and α-mannose were somewhat less marked. Subsets of complex N-glycans changed little. This revised version was published online in November 2006 with corrections to the Cover Date.     

South Asians with ulcerative colitis exhibit altered lectin binding compared with matched European cases

Ulcerative colitis is associated with abnormalities of mucin synthesis and secretion, features that may also be associated with malignant change. It has been shown that South Asians in Britain have a high incidence of ulcerative colitis but a low incidence of colorectal carcinoma compared with their European counterparts. Previous studies have demonstrated changes in colonic mucin sialylation and sulphation in both South Asian and European cases with ulcerative colitis. This was related to disease severity, but changes were also found in quiescent disease. The aim of the present study was to determine glycoconjugate expression in the colon from South Asian cases and to compare results with those from a group of affected Europeans. Glycans were identified in formalin-fixed, paraffin-embedded tissue from 17 South Asian patients with ulcerative colitis and from 11 European patients with a similar degree of colitis, by the application of 10 biotinylated lectins. These were directed against a range of sialyl, fucosyl and 2-deoxy, 2-acetamido-galactosyl sequences, using an avidin--peroxidase revealing system and semiquantitative assessment. The South Asian group showed a reduction in the binding of agglutinins from Sambucus nigra in the apical-membranous region of enterocytes, and a decrease in apical Maackia amurensis agglutinin binding. These results suggest that South Asians with ulcerative colitis show a different distribution of terminal N-acetyl neuraminyl residues, either in their α-2,6 or α-2,3 linkage, compared with their European counterparts. The changes in sialylation observed in European cases compared with normal disease-free control subjects were present in quiescent disease, but were also related to disease activity. Their absence in Asians with ulcerative colitis may imply an inherent, genetically determined variation in this group, which may also play a part in their reduced risk of subsequent malignancy