Modulation of tumor-induced angiogenesis by proteins of extracellular matrix.

The formation of new vessels is a known event in enlarging tumors. Furthermore, the metastatic potential is abrogated or reduced markedly in the absence of neovascularization. Shedding of tumor cells into the circulation is not observed until vascularization has occurred. As a result, the interruption of neovascularization could be a good target for cancer control. This research was an attempt to see if two proteins present in extracellular matrix, collagen and fibronectin (FN), could modify the tumor-induced angiogenesis. The strong angiogenic response induced by S13 tumor cells in the skin of BALB/c mice was blocked by treatment with FN and FN-derived peptides. In contrast, collagen did not modify tumor-induced angiogenesis.     

Processing of PDGF gene products determines interactions with glycosaminoglycans

The platelet derived growth factor (PDGF), a mitogen for mesenchymal cells, may be bound to and inhibited by heparin and other glycosaminoglycans. PDGF is a homo‐ or heterodimer of A‐ and B‐chains. They occur as short (A₁₀₉ and B₁₁₀) and long (A₁₂₅ and B₁₆₀) isoforms. The latter contain basic carboxyl‐terminal extensions. Dimeric A₁₂₅ binds to heparin through its basic extension in a two‐step reaction. The mechanism involves a conformational change and is consistent with a Monod–Wyman–Changeux allosteric model. Previous indirect experiments suggested that three critical amino acids (basic R¹¹¹, K¹¹⁶ and polar T¹²⁵) might be involved. Here, direct binding experiments using dimeric full‐length mutants in surface plasmon resonanse analysis showed that all three critical amino acids in an R(X)⁴K(X)⁸T‐motif contributed in a concerted manner to the high affinity binding. Mutations of these amino acids to alanine resulted in large thermodynamic changes, loss of the allosteric mechanism and order(s) of magnitude lower binding affinity. The binding mechanism and affinity of long dimeric rB were similar to the mutants. Short dimeric rA₁₀₉ and rB₁₁₀ showed 100 times lower binding affinity than rA₁₂₅. Consequently, interactions with glycosaminoglycans in tissues varies between PDGF isoforms and may influence their local accumulation and activity. Copyright © 1999 John Wiley & Sons, Ltd.     

Expression and regulation of LRP‐2/megalin in epithelial cells lining the efferent ducts and epididymis during postnatal development

Low density lipoprotein receptor‐related protein‐2/megalin (LRP‐2) is a receptor belonging to the low density lipoprotein receptor family that mediates endocytosis and lysosomal degradation of a variety of ligands including apolipoprotein J (Apo J)/clusterin/SGP‐2. LRP‐2 has been shown to be expressed regionally in the adult rat epididymis. In this study, we describe the pattern of expression of LRP‐2 in the efferent ducts and epididymis during postnatal development of the rat and examine the role of testicular luminally derived substances on its expression. The expression of LRP‐2 was analyzed immunocytochemically in tissues of normal animals ranging in age from postnatal day 7–90 and in 15‐day‐old efferent‐duct‐ligated animals sacrificed at later ages. In the efferent ducts, LRP‐2 expression, appearing as a dense band on the apical surface of the nonciliated epithelial cells, was noted as early as day 7, well before the entry of sperm, Sertoli‐cell‐derived secretory products, and high levels of androgens. Efferent duct ligation studies further revealed that expression under this condition was comparable to controls at all later ages examined, suggesting that the factor regulating its expression was not a luminally derived testicular substance. In normal untreated animals, LRP‐2 expression was not apparent at any of the ages examined in the proximal initial segment of the epididymis. By comparison, the distal initial segment, although having no LRP‐2 expression from 7–15 days, showed expression in principal cells by day 21 which intensified at days 29 and 39. However, by day 49 and at later ages (56 and 90), LRP‐2 immunoreactivity over principal cells became spotty or with weak or moderate reactivity in some cells and none in others. LRP‐2 expression in the intermediate zone, proximal caput, corpus, and cauda regions also appeared in principal cells by day 21, intensified at days 29 and 39 and persisted as such at all later ages examined, correlating with high levels of androgens shown to occur by day 39. Although LRP‐2 expression in the distal caput region was evident in principal cells at days 21 and 29, it became spotty with weak, moderate, or absent reactivity over principal cells at all later ages. These data suggest that LRP‐2 expression is under the influence of both stimulatory and region‐specific inhibitory factors. Analysis of 15‐day‐old efferent‐duct‐ligated animals at all later ages examined revealed that there was no change in LRP‐2 expression along the entire epididymis, suggesting that both the stimulatory and inhibitory factors are not luminally derived testicular substances. The observed pattern of LRP‐2 expression in all regions of the epididymis, except the distal caput region, was similar to that described for Apo J internalization by principal cells during postnatal development, showing a correlation between LRP‐2 expression and its ligand, Apo J. In summary, LRP‐2 expression in the epididymis undergoes region‐specific changes during postnatal development and appears to be influenced by both stimulatory and inhibitory factors. Mol. Reprod. Dev. 53:282–293, 1999. © 1999 Wiley‐Liss, Inc.     

T cell derived HIV-1 is present in the CSF in the face of suppressive antiretroviral therapy

HIV cerebrospinal fluid (CSF) escape, where HIV is suppressed in blood but detectable in CSF, occurs when HIV persists in the CNS despite antiretroviral therapy (ART). To determine the virus producing cell type and whether lowered CSF ART levels are responsible for CSF escape, we collected blood and CSF from 156 neurosymptomatic participants from Durban, South Africa. We observed that 28% of participants with an undetectable HIV blood viral load showed CSF escape. We detected host cell surface markers on the HIV envelope to determine the cellular source of HIV in participants on the first line regimen of efavirenz, emtricitabine, and tenofovir. We confirmed CD26 as a marker which could differentiate between T cells and macrophages and microglia, and quantified CD26 levels on the virion surface, comparing the result to virus from in vitro infected T cells or macrophages. The measured CD26 level was consistent with the presence of T cell produced virus. We found no significant differences in ART concentrations between CSF escape and fully suppressed individuals in CSF or blood, and did not observe a clear association with drug resistance mutations in CSF virus which would allow HIV to replicate. Hence, CSF HIV in the face of ART may at least partly originate in CD4+ T cell populations.     

Molecular architecture of the rod domain of the Dictyostelium gelation factor (ABP120).

The Dictyostelium discoideum gelation factor is a two-chain actin-cross-linking protein that, in addition to an N-terminal actin-binding domain, has a rod domain constructed from six tandem repeats of a 100-residue motif that has an immunoglobulin fold. To define the architecture of the rod domain of gelation factor, we have expressed in E. coli a series of constructs corresponding to different numbers of gelation factor rod repeats and have characterised them by chemical crosslinking, ultracentrifugation, column chromatography, matrix-assisted laser desorption ionisation (MALDI) mass spectrometry and NMR spectroscopy. Fragments corresponding to repeats 1-6 and 5-6 dimerise, whereas repeats 1-5 and single repeats 3 and 4 are monomeric. Repeat 6 interacts weakly and was present as monomer and dimer when analysed by analytical ultracentrifugation. Proteolytic digestion of rod5-6 resulted in the generation of two polypeptides that roughly corresponded to rod5 and part of rod6. None of these polypeptides formed dimers after chemical crosslinking. Stable dimerisation therefore appears to require repeats 5 and 6. Based on these data a model of gelation factor architecture is presented. We suggest an arrangement of the chains where only the carboxy-terminal repeats interact as was observed for filamin/ABP280, the mammalian homologue of gelation factor.     

Neighbor joining algorithms for inferring phylogenies via LCA distances.

Reconstructing phylogenetic trees efficiently and accurately from distance estimates is an ongoing challenge in computational biology from both practical and theoretical considerations. We study algorithms which are based on a characterization of edge-weighted trees by distances to LCAs (Least Common Ancestors). This characterization enables a direct application of ultrametric reconstruction techniques to trees which are not necessarily ultrametric. A simple and natural neighbor joining criterion based on this observation is used to provide a family of efficient neighbor-joining algorithms. These algorithms are shown to reconstruct a refinement of the Buneman tree, which implies optimal robustness to noise under criteria defined by Atteson. In this sense, they outperform many popular algorithms such as Saitou and Nei's NJ. One member of this family is used to provide a new simple version of the 3-approximation algorithm for the closest additive metric under the iota (infinity) norm. A byproduct of our work is a novel technique which yields a time optimal O (n (2)) implementation of common clustering algorithms such as UPGMA.     

Effects of exercise training on quadriceps muscle gene expression in chronic obstructive pulmonary disease.

Exercise capacity and training response are limited in chronic obstructive pulmonary disease (COPD), but the extent to which this is related to altered skeletal muscle function is not fully understood. To test the hypothesis that muscle gene expression is altered in COPD, we performed needle biopsies from the vastus lateralis of six COPD patients and five sedentary age-matched healthy men, before and after 3 mo of exercise training. RNA was hybridized to Affymetrix U133A Genechip arrays. In addition, peak O(2) uptake and other functional parameters (e.g., 6-min walk) were measured before and after training. The 6-min walk test increased significantly following training in both groups (53.6 +/- 18.6 m in controls, P = 0.045; 37.1 +/- 6.7 m in COPD, P = 0.002), but peak O(2) uptake increased only in controls (19.4 +/- 4.5%, P = 0.011). Training significantly altered muscle gene expression in both groups, but the number of affected genes was lower in the COPD patients (231) compared with controls (573). Genes related to energy pathways had higher expression in trained controls. In contrast, oxidative stress, ubiquitin proteasome, and COX gene pathways had higher expression in trained COPD patients, and some genes (e.g., COX11, COX15, and MAPK-9) were upregulated by training only in COPD patients. We conclude that both COPD and control subjects demonstrated functional responses to training but with somewhat different patterns in muscle gene expression. The pathways that are uniquely induced by exercise in COPD (e.g., ubiquitin proteasome and COX) might indicate a greater degree of tissue stress (perhaps by altered O(2) and CO(2) dynamics) than in controls.     

The yeast RNA gene products are essential for mRNA splicing in vitro

The yeast rna mutations (rna2-rna11) are a set of temperature-sensitive mutations that result in the accumulation of intron-containing mRNA precursors at the restrictive temperature. We have used the yeast in vitro splicing system to investigate the role of products of the RNA genes in mRNA splicing. We have tested the heat lability of the in vitro mRNA splicing reaction in extracts isolated from mutant and wild-type cells. Extracts isolated from seven of the nine rna mutants demonstrated heat lability in this assay, while most wild-type extracts were stable under the conditions utilized. We have also demonstrated that heat inactivation usually results in the specific loss of an exchangeable component by showing that most combinations of heat-inactivated extracts from different mutants complement one another. In three cases (rna2, rna5, and rna11), the linkage of the in vitro defect to the rna mutations was ascertained by a combination of reversion, tetrad, and in vitro complementation analyses. Furthermore, each heat-inactivated extract was capable of complementation by at least one fraction of the wild-type splicing system. Thus many of the RNA genes are likely to code for products directly involved in and essential for mRNA splicing.     

Plasminogen activator activity in differentiating rat myoblasts

Summary Primary cultures of skeletal muscle cells secrete plasminogen activator (PA) activity to the conditioning medium and display membrane-bound PA. Growth of these cells in culture in presence of 10^-7 M dexamethasone resulted in a marked reduction of the membranal and secreted PA activity. The hormone also reduced cytosolic creative phosphokinase (CPK) activity and cytosolic protein content. However, cell viability and their ability to undergo fusion were uneffected. The extent of hormone-induced reduction in PA activity depended on the time and extend of exposure. Maximal suppression was obtained by exposing the cells to dexamethasone during the first 4 days of culture. The medium conditioned with dexamethasone-treated cells did not inhibit plasmin, endogenous PA or exogenous PA. Exposure of the conditioned medium from hormone-treated cells to sodium dodecyl sulphate (SDS) or trypsin restored the activity to values observed in media from cells not exposed to the hormone. Acidification of the medium failed to reactivate the enzyme. The myogenic cell line L-8 also displayed membrane-bound PA activity, which was of a comparable magnitude in both fusing and non-fusing L-8 cells. However, in contrast to the primary cultures, exposure of L-8 cells to dexamethasone had no effect on their PA activity whether studied under conditions which allowed or prohibited fusion. The present findings imply that PA has no conducive role in the process of fusion associated with maturation of skeletal muscle cells.Abbreviations CPK Creative phosphokinase - PA Plasminogen activator - PBS Phosphate buffered saline - SDS Sodium dodecyl sulphate - TCA Trichloroacetic acid     

Repeated sequences on mitochondrial DNA ofSpirodela oligorhiza

Summary Mitochondrial DNA ofSpirodela oligorhiza (duck weed) was analyzed with restriction enzymes. The genome size appears to be at least 250 kbp. Four different PstI fragments were cloned. These four clones contain a sequence which is reiterated about 100-fold on theSpirodela mitochondrial DNA. Hybridization analysis showed that a similar sequence is present onZea mays mitochondrial DNA, although much less reiterated here. The presence of these reiterated sequences might contribute to the physical heterogeneity of plant mitochondrial DNA.