Generation of a scaffold free cartilage-like implant from a small amount of starting material

INTRODUCTION: An autologous cellular based treatment of a traumatic cartilage injury requires a procedure whereby a biopsy of healthy cartilage is removed from the patient and the cells isolated and expanded by monolayer passage. This increases the cell number to required levels but also leads to a de-differentiation of the cells. We aim to produce a scaffold-free, de-novo implant from a biopsy of cartilage. METHODS: Bovine chondrocytes were isolated from a small biopsy and expanded. The chondrocytic phenotype of the monolayer expanded cells was recovered during a period of culture in alginate and the effect of factors such as IGF1, TFGbeta1 and dexamethasone was investigated. RESULTS: During the alginate culture period a pre-treatment with IGF1 and dexamethasone was shown to have little effect. IGF1 however increased the glycosaminoglycan/DNA (GAG/DNA) content on day 14 to 84.95+/-5 ng/ng compared with 37.3+/-1.8 ng/ng in the controls (P<0.001). 35S labeling demonstrated an increased GAG synthesis in the presence of IGF1 (P<0.001). IGF1 also induced a increase of DNA content 1383+/-314 ng/bead compared to 512+/-19 ng/bead in the controls (P<0.001).The cells were released from the alginate and cultured in a silicon mould for a further 14 days to obtain a three dimensional implant. Releasing the cells from the alginate and casting in a mould produced an implant of defined shape which contained no foreign material. After 31 days of culture the implants contained 152.4+/-13.14 ng/ng GAG/DNA and 42.93+/-10.23 ng/ng collagen II. DISCUSSION: We believe alginate released chondrocytes provide a real alternative to artificial scaffolds.     

Mutant analyses define multiple roles for phytochrome C in Arabidopsis photomorphogenesis

The analysis of Arabidopsis mutants deficient in the A, B, D, and E phytochromes has revealed that each of these phytochrome isoforms has both distinct and overlapping roles throughout plant photomorphogenesis. Although overexpression studies of phytochrome C (phyC) have suggested photomorphogenic roles for this receptor, conclusive evidence of function has been lacking as a result of the absence of mutants in the PHYC locus. Here, we describe the isolation of a T-DNA insertion mutant of phyC (phyC-1), the subsequent creation of mutant lines deficient in multiple phytochrome combinations, and the physiological characterization of these lines. In addition to operating as a weak red light sensor, phyC may perform a significant role in the modulation of other photoreceptors. phyA and phyC appear to act redundantly to modulate the phyB-mediated inhibition of hypocotyl elongation in red light and to function together to regulate rosette leaf morphology. In addition, phyC performs a significant role in the modulation of blue light sensing. Several of these phenotypes are supported by the parallel analysis of a quadruple mutant deficient in phytochromes A, B, D, and E, which thus contains only active phyC. Together, these data suggest that phyC has multiple functions throughout plant development that may include working as a coactivator with other phytochromes and the cryptochrome blue light receptors.     

CCR5- and CXCR4-Utilizing Strains of Human Immunodeficiency Virus Type 1 Exhibit Differential Tropism and Pathogenesis In Vivo

CCR5-utilizing (R5) and CXCR4-utilizing (X4) strains of human immunodeficiency virus type 1 (HIV-1) have been studied intensively in vitro, but the pathologic correlates of such differential tropism in vivo remain incompletely defined. In this study, X4 and R5 strains of HIV-1 were compared for tropism and pathogenesis in SCID-hu Thy/Liv mice, an in vivo model of human thymopoiesis. The X4 strain NL4-3 replicates quickly and extensively in thymocytes in the cortex and medulla, causing significant depletion. In contrast, the R5 strain Ba-L initially infects stromal cells including macrophages in the thymic medulla, without any obvious pathologic consequence. After a period of 3 to 4 weeks, Ba-L infection slowly spreads through the thymocyte populations, occasionally culminating in thymocyte depletion after week 6 of infection. During the entire time of infection, Ba-L did not mutate into variants capable of utilizing CXCR4. Therefore, X4 strains are highly cytopathic after infection of the human thymus. In contrast, infection with R5 strains of HIV-1 can result in a two-phase process in vivo, involving apparently nonpathogenic replication in medullary stromal cells followed by cytopathic replication in thymocytes.     

Molecular systematics and paleobiogeography of the South American sigmodontine rodents [published erratum appears in Mol Biol Evol 1998 Feb;15(2):224]

The murid rodent subfamily Sigmodontinae contains 79 genera which are distributed throughout the New World. The time of arrival of the first sigmodontines in South America and the estimated divergence time(s) of the different lineages of South American sigmodontines have been controversial due to the lack of a good fossil record and the immense number of extant species. The "early-arrival hypothesis" states that the sigmodontines must have arrived in South America no later than the early Miocene, at least 20 MYA, in order to account for their vast present-day diversity, whereas the "late-arrival hypothesis" includes the sigmodontines as part of the Plio-Pleistocene Great American Interchange, which occurred approximately 3.5 MYA. The phylogenetic relationships among 33 of these genera were reconstructed using mitochondrial DNA (mtDNA) sequence data from the ND3, ND4L, arginine tRNA, and ND4 genes, which we show to be evolving at the same rate. A molecular clock was calibrated for these genes using published fossil dates, and the genetic distances were estimated from the DNA sequences in this study. The molecular clock was used to estimate the dates of the South American sigmodontine origin and the main sigmodontine radiation in order to evaluate the "early-" and "late-arrival" scenarios. We estimate the time of the sigmodontine invasion of South America as between approximately 5 and 9 MYA, supporting neither of the scenarios but suggesting two possible models in which the invading lineage was either (1) ancestral to the oryzomyines, akodonts, and phyllotines or (2) ancestral to the akodonts and phyllotines and accompanied by the oryzomyines. The sigmodontine invasion of South America provides an example of the advantage afforded to a lineage by the fortuitous invasion of a previously unexploited habitat, in this case an entire continent.      

The abundance of additional N-acetyllactosamine units in N-linked tetraantennary oligosaccharides of human Tamm-Horsfall glycoprotein is a donor-specific feature

Previously, treatment of Tamm-Horsfall glycoprotein (THp) from different donors with endo-beta-galactosidase has been shown to liberate a tetra- and a Sd(a)-active pentasaccharide, concluding the presence of N-linked carbohydrate chains containing additional N - acetyllactosamine units. These type of oligosaccharides were not found in a detailed structure elucidation of the carbohydrate moiety of THp of one male donor, suggesting a donor-specific feature for these type of structures. Therefore, THp was isolated from four healthy male donors and each subjected to endo-beta-galactosidase treatment in order to release these tetra- and Sd(a)-active pentasaccharide. Differences were observed in the total amount of released tetra- and Sda-active pentasaccharide of the used donors (42, 470, 478, 718 microg/100 mg THp), indicating that the presence of repeating N-acetyllactosamine units incorporated into the N-glycan moiety of THp is donor specific. Furthermore, a higher expression of the Sd(a) determinant on antennae which display N-acetyllactosamine elongation was observed, suggesting a better accessibility for the beta-N-acetylgalactosaminyltransferase. In order to characterize the N-glycans containing repeating N- acetyllactosamine units, carbohydrate chains were enzymatically released from THp and isolated. The tetraantennary fraction, which accounts for more than 33% of the total carbohydrate moiety of THp, was used to isolate oligosaccharides containing additional N - acetyllactosamine units. Five N-linked tetraantennary oligosaccharides containing a repeating N-acetyllactosamine unit were identified, varying from structures bearing four Sd(a) determinants to structures containing no Sd(a) determinant (see below). One compound was used in order to specify the branch location of the additional N- acetyllactosamine unit, and it appeared that only the Gal-6' and Gal-8' residues were occupied by a repeating N -acetyllactosamine unit.      

Lectin-histochemical analysis of glycans in ovine and bovine near-term placental binucleate cells

Chorionic binucleate cells (BNC) occur in several ruminants including cow, deer, goat and sheep. They migrate through the chorionic tight junction to fuse with uterine epithelial cells and discharge their granules into maternal connective tissue. We have compared the BNC of near-term, resin-embedded, ovine and bovine placentae using 15 biotinylated lectins and an avidinperoxidase revealing system. There was pronounced conservation of saccharides between the two species. Several sub-types of N-glycan were present, with highly branched structures being abundant, as shown by Galanthus nivalis, Pisum sativum and Phaseolus vulgaris (leuko) agglutinins. Among the non-reducing terminal saccharides conserved were GalNAc1,3(Fuc1,2)-Gal1,4GlcNAc1-, GalNAc1,6Gal1-, Gal1,4GlcNAc-and Gal1,3GalNAc1- shown by Dolichos biflorus, Wisteria floribunda, Erythrina cristagalli, and Maclura pomifera agglutinins, respectively. Arachis hypogaea and Glycine max agglutinins tended to bind to bovine BNC at different stages of maturity, while fucosyl residues detectable by Tetragonolobus purpureus and Ulex europaeus-1 agglutinins were not observed in either species. The only major difference related to sialyl residues, with 2,3-linked sialic acid being present in bovine (Maackia amurensis, Limax flavus) and 2,6 sialic acid being present in ovine (Sambucus nigra agglutinin) cells. This conservation of glycan may be related to glycosylation of peptide hormones in the granules, and may thus be important in the targeting of these hormones to their receptors.     

Ribosomal RNA sequence phylogeny is not congruent with ascospore morphology among species in Ceratocystis sensu stricto

The genus Ceratocystis sensu stricto includes important fungal pathogens of woody and herbaceous plants. This genus is distinguished from species in Ceratocystis sensu lato by the presence of Chalara anamorphs. Ascospore shape has been used extensively in delineating Ceratocystis taxa, which show a large variety of ascospore shapes. Sequence analysis of one region of he 18S ribosomal RNA subunit and two regions of the 28S ribosomal RNA subunit showed that there was a majority of multiple substitutions at nucleotide sites and that there was a low transition/transversion ratio, T = 0.72. Both of these results suggest that these are well established, old species. Ascospore morphology, for the most part, was not congruent with the molecular phylogeny, and the use of morphological characters may be misleading in the taxonomy of these species.      

Membrane compartmentalized ATP and its preferential use by the Na, K-ATPase of human red cell ghosts

This paper describes work which begins to define the molecular organization in the region of the membrane that comprises the functional domain of the Na:K pump. The membrane-bound phosphoglycerate kinase (PGK) and Na, K-ATPase appear to be directly linked via a compartmentalized form of ATP. Evidence for the membrane pool of ATP is based on the labeling characteristics of the phosphoproteins by [γ-(32)P]ATP of ghosts incubated under various conditions. Preincubation of ghosts in the presence of ATP at 37 degrees C, but not at 0 degrees C, completely obscures the formation of the Na-phosphoprotein in ghosts washed and subsequently incubated in the presence of [gamma-(32)P]ATP. In contrast to the Na component, the Mg component of phosphorylation is only slightly altered by preincubation with ATP. ATPase activity measured as (32)P(i) liberated during the subsequent incubation at 0 degrees C, reflects completely the differential effects of preincubation with ATP on (32)P incorporation into phosphoprotein. ATP placed within the pool by preincubation can be removed by operating the Na, K-ATPase or the PGK reaction in the reverse direction by use of exogenous substrates. Alternatively, the membrane pool of ATP can be formed also from exogenous substrates by running the PGK reaction in the forward direction. These results, while providing direct support for a membrane compartment of ATP, also indicate the location of this compartment in relation to the PGK and the Na, K-ATPase. In addition, these results also imply that the Mg and Na components are different enzymatic entities since substrate ATP can be derived from separate sources.     

Plasma fenfluramine levels,weight loss,and side effects.

Fifty women with refractory obesity received fenfluramine for 20 weeks. Every two weeks details of weight change, drug dose, degree of anorexia, and any side effects were recorded and plasma was obtained for fenfluramine and norfenfluramine measurements. Of the 41 patients available for final analysis 26 achieved a maximum plateau dose of 160 mg/day. Plasma fenfluramine concentrations did not correlate with the degree of anorexia or with the incidence of side effects other than the severity of dream disturbance. There was a highly significant relation between weight loss and plasma fenfluramine and norfenfluramine concentrations and also between weight loss and the presence of sustained anorexia. Women who achieved mean plateau concentrations over 200 ng/ml lost a mean 8.8 kg while those with concentrations less than 100 ng/ml lost a mean of only 2.1 kg. When fenfluramine is prescribed in refractory obesity the dose should be increased stepwise until either satisfactory weight loss is achieved or troublesome side effects appear.