Structure of corneal scar tissue: an X-ray diffraction study.

Full-thickness corneal wounds (2 mm diameter) were produced in rabbits at the Schepens Eye Research Institute, Boston. These wounds were allowed to heal for periods ranging from 3 weeks to 21 months. The scar tissue was examined using low- and wide-angle x-ray diffraction from which average values were calculated for 1) the center-to-center collagen fibril spacing, 2) the fibril diameter, 3) the collagen axial periodicity D, and 4) the intermolecular spacing within the collagen fibrils. Selected samples were processed for transmission electron microscopy. The results showed that the average spacing between collagen fibrils within the healing tissue remained slightly elevated after 21 months and there was a small increase in the fibril diameter. The collagen D-periodicity was unchanged. There was a significant drop in the intermolecular spacing in the scar tissues up to 6 weeks, but thereafter the spacing returned to normal. The first-order equatorial reflection in the low-angle pattern was visible after 3 weeks and became sharper and more intense with time, suggesting that, as healing progressed, the number of nearest neighbor fibrils increased and the distribution of nearest neighbor spacings reduced. This corresponded to the fibrils becoming more ordered although, even after 21 months, normal packing was not achieved. Ultrastructural changes in collagen fibril density measured from electron micrographs were consistent with the increased order of fibril packing measured by x-ray diffraction. The results suggest that collagen molecules have a normal axial and lateral arrangement within the fibrils of scar tissue. The gradual reduction in the spread of interfibrillar spacings may be related to the progressive decrease in the light scattered from the tissue as the wound heals.     

GDNF family receptor alpha1 phenotype of spermatogonial stem cells in immature mouse testes

Spermatogonial stem cells (SSCs) are essential for spermatogenesis, and these adult tissue stem cells balance self-renewal and differentiation to meet the biological demand of the testis. The developmental dynamics of SSCs are controlled, in part, by factors in the stem cell niche, which is located on the basement membrane of seminiferous tubules situated among Sertoli cells. Sertoli cells produce glial cell line-derived neurotrophic factor (GDNF), and disruption of GDNF expression results in spermatogenic defects and infertility. The GDNF signals through a receptor complex that includes GDNF family receptor alpha1 (GFRA1), which is thought to be expressed by SSCs. However, expression of GFRA1 on SSCs has not been confirmed by in vivo functional assay, which is the only method that allows definitive identification of SSCs. Therefore, we fractionated mouse pup testis cells based on GFRA1 expression using magnetic activated cell sorting. The sorted and depleted fractions of GFRA1 were characterized for germ cell markers by immunocytochemistry and for stem cell activity by germ cell transplantation. The GFRA1-positive cell fraction coeluted with other markers of SSCs, including ITGA6 and CD9, and was significantly depleted of KIT-positive cells. The transplantation results confirmed that a subpopulation of SSCs expresses GFRA1, but also that the stem cell pool is heterogeneous with respect to the level of GFRA1 expression. Interestingly, POU5F1-positive cells were enriched nearly 15-fold in the GFRA1-selected fraction, possibly suggesting heterogeneity of developmental potential within the stem cell pool.     

Antitumour and antimalarial activity of artemisinin–acridine hybrids

Artemisinin–acridine hybrids were prepared and evaluated for their in vitro activity against tumour cell lines and a chloroquine sensitive strain of Plasmodium falciparum. They showed a 2–4-fold increase in activity against HL60, MDA-MB-231 and MCF-7 cells in comparison with dihydroartemisinin (DHA) and moderate antimalarial activity. Strong evidence that the compounds induce apoptosis in HL60 cells was obtained by flow cytometry, which indicated accumulation of cells in the G1 phase of the cell cycle.     

Segmental norepinephrine and dopamine levels in cat spinal cord

Tissue levels of norepinephrine in the uninjured cat were determined by a fluorimetric or enzymatic radiometric assay technique. There were no significant differences of NE levels between the two techniques. The highest concentrations of NE were found in the cervical (.170 ug/g) and lumbar (.232 ug/g) enlargements and appear to be related to the relative amount of grey and white matter present at these levels. Dopamine levels, as determined by the enzymatic radiometric assay technique, were found to be low in those spinal segments measured (less than 0.030 ug/g). DA appears to be similarly distributed as norepinephrine in the spinal segments. Barbiturate or nitrous oxide anesthetic agents did not result in significant differences in NE determinations at cervical or thoracic segments. Variability of reported NE and DA levels from other studies appears to be related to differences in assay technique.     

N-Glycosyl-thiophene-2-carboxamides: synthesis, structure and effects on the growth of diverse cell types

A range of N-glycosyl-thiophene-2-carboxamides, including a 6H-thieno[2,3-c]pyridin-7-one and a bivalent compound, have been synthesised and assayed for their effects on DNA synthesis in bovine aortic endothelial cells or on the growth of synoviocytes. Per-O-acetylated analogues of the glycoconjugates were significantly more effective inhibitors when compared to their corresponding non-acetylated analogues, indicating that the lower potency observed for hydroxylated derivatives is due to less efficient transport of these compounds across the cell membrane. Thiophene-2-carboxamide was inactive as an inhibitor of bFGF induced proliferation, confirming the requirement of the carbohydrate residue for the observed biological properties. Glucose, mannose, galactose and 2-amino-2-deoxy-glucose analogues were active as were a variety of substituted thiophene derivatives; the 6H-thieno[2,3-c]pyridin-7-one conjugate was inactive. Conformational analysis of the title compounds was investigated. X-ray crystal structural analysis of four N-glucosyl-thiophene-2-carboxamides showed that the pyranose rings adopted the expected 4C1 conformations and that Z-anti structures were predominant (H1-C1-N-H anomeric torsion angle varied from -168.2 degrees to -175.0 degrees ) and that the carbonyl oxygen and sulfur of the thiophene adopted an s-cis conformation in three of the isomers. In a crystal structure of a 3-alkynyl derivative, the hydrogen atom of the NH group was directed toward the acetylene group. The distance between the hydrogen atom and acetylene carbons and angles between nitrogen, hydrogen and carbon atoms were consistent with hydrogen bonding and this was supported by IR and NMR spectroscopic studies. The geometries of thiophene-2-carboxamides were explored by density functional theory (DFT) and M?ller-Plesset (MP2) calculations and the s-cis conformer of thiophene-2-carboxamide was found to be more stable than its s-trans isomer by 0.83 kcal mol(-1). The s-cis conformer of 3-ethynyl-thiophene-2-carboxamide was 5.32 kcal mol(-1) more stable than the s-trans isomer. The larger stabilisation for the s-cis conformer in the 3-alkynyl derivatives is explained to be due to a moderate hydrogen bonding interaction between the alkyne and NH group.     

N-Glycosyl-thiophene-2-carboxamides: effects on endothelial cell growth in the presence and absence of bFGF--a significant increase in potency using per-O-acetylated sugar analogues

Inhibitors of endothelial cell proliferation are of interest in development of therapies for angiogenesis related disease. N-Glucosyl-thiophene-2-carboxamides have been synthesized and evaluated for their effects on proliferation in bovine aortic endothelial cells. Per-O-acetylated-N-glucosyl-thiophene-2-carboxamides showed improved inhibition of both serum and bFGF stimulated uptake of [(3)H]thymidine, when compared to non-acetylated analogues.     

Altering Chemosensitivity by Modulating Translation Elongation

Background

The process of translation occurs at a nexus point downstream of a number of signal pathways and developmental processes. Modeling activation of the PTEN/AKT/mTOR pathway in the Eμ-Myc mouse is a valuable tool to study tumor genotype/chemosensitivity relationships in vivo. In this model, blocking translation initiation with silvestrol, an inhibitor of the ribosome recruitment step has been showed to modulate the sensitivity of the tumors to the effect of standard chemotherapy. However, inhibitors of translation elongation have been tested as potential anti-cancer therapeutic agents in vitro, but have not been extensively tested in genetically well-defined mouse tumor models or for potential synergy with standard of care agents.

Methodology/Principal Findings

Here, we chose four structurally different chemical inhibitors of translation elongation: homoharringtonine, bruceantin, didemnin B and cycloheximide, and tested their ability to alter the chemoresistance of Eμ-myc lymphomas harbouring lesions in Pten, Tsc2, Bcl-2, or eIF4E. We show that in some genetic settings, translation elongation inhibitors are able to synergize with doxorubicin by reinstating an apoptotic program in tumor cells. We attribute this effect to a reduction in levels of pro-oncogenic or pro-survival proteins having short half-lives, like Mcl-1, cyclin D1 or c-Myc. Using lymphomas cells grown ex vivo we reproduced the synergy observed in mice between chemotherapy and elongation inhibition and show that this is reversed by blocking protein degradation with a proteasome inhibitor.

Conclusion/Significance

Our results indicate that depleting short-lived pro-survival factors by inhibiting their synthesis could achieve a therapeutic response in tumors harboring PTEN/AKT/mTOR pathway mutations.     

Spermatocyte/spermatid-specific thioredoxin-3, a novel Golgi apparatus-associated thioredoxin, is a specific marker of aberrant spermatogenesis

Mammalian germ cells are endowed with a complete set of thioredoxins (Trx), a class of redox proteins located in specific structures of the spermatid and sperm tail. We report here the characterization, under normal and pathological conditions, of a novel thioredoxin with a germ line-restricted expression pattern, named spermatocyte/spermatid-specific thioredoxin-3 (SPTRX-3). The human SPTRX-3 gene maps at 9q32, only 50 kb downstream from the TRX-1 gene from which it probably originated as genomic duplication. Therefore, human SPTRX-3 protein comprises a unique thioredoxin domain displaying high homology with the ubiquitously expressed TRX-1. Among the tissues investigated, Sptrx-3 mRNA is found exclusively in the male germ cells at pachytene spermatocyte and round spermatid stages. Light and electron microscopy show SPTRX-3 protein to be predominately located in the Golgi apparatus of pachytene spermatocytes and round and elongated spermatids, with a transient localization in the developing acrosome of round spermatids. In addition, increased levels of SPTRX-3, possibly caused by overexpression, are observed in morphologically abnormal human spermatozoa from infertile men. In addition, SPTRX-3 is identified as a novel postobstruction autoantigen. In this report, we propose that SPTRX-3 can be used as a specific marker for diverse sperm and testis pathologies. SPTRX-3 is the first thioredoxin specific to the Golgi apparatus, and its function within this organelle might be related to the post-translational modification of proteins required for germ cell-specific functions, such as acrosomal biogenesis.     

The making of abnormal spermatozoa: cellular and molecular mechanisms underlying pathological spermiogenesis

Fertilization in mammals occurs via a series of well-defined events in the secluded environment of the female reproductive tract. The mode of selection of the fertilizing spermatozoon nevertheless remains unknown. As has become evident during in vitro fertilization by sperm microinjection into the oocyte, abnormal spermatozoa can successfully fertilize oocytes. Under these extreme conditions, post-fertilization events, early embryonic development and implantation are significantly compromised indicating that the contribution of spermatozoa extends beyond sperm penetration. Microscopic identification of normal spermatozoa is a well-standardized procedure but insights into the mechanisms that lead to aberrant sperm differentiation and into the subcellular nature of sperm abnormalities have only recently begun to be obtained. The spermatozoon is the result of a complex development in which spermatid organelles give rise to various structural components with characteristic functions. Similar to other differentiated cells, the spermatozoon has a specific pathology that is most clearly identified by ultrastructural evaluation coupled with immunocytochemistry and molecular techniques. This multidisciplinary approach allows the precise characterization of sperm abnormalities, including structural, molecular and functional aspects. We summarize here studies of the physiopathology of spermiogenesis in two abnormal sperm phenotypes of infertile men: dysplasia of the fibrous sheath and acephalic spermatozoa/abnormal head-tail attachment. The characterization of the abnormalities of the tail cytoskeleton and centrioles has uncovered aspects of the subcellular basis of pathological spermiogenesis, has suggested experimental approaches to explore the nature of these anomalies and has opened the way for genetic studies that will ultimately lead to the design of the therapeutic tools of the future.