Evolutionary conservation of heavy chain protein transfer between glycosaminoglycans

The bikunin proteins are composed of heavy chains (HCs) covalently linked to a chondroitin sulfate chain originating from Ser-10 of bikunin. Tumor necrosis factor stimulated gene-6 protein (TSG-6)/heavy chain 2 (HC2) cleaves this unique cross-link and transfers the HCs to hyaluronan and other glycosaminoglycans via a covalent HC•TSG-6 intermediate. In the present study, we have investigated if this reaction is evolutionary conserved based on the hypothesis that it is of fundamental importance. The results revealed that plasma/serum samples from mammal, bird, and reptile were able to form TSG-6 complexes suggesting the presence of proteins with the same function as the human bikunin proteins. To substantiate this, the complex forming protein from Gallus gallus (Gg) plasma was purified and identified as a Gg homolog of human HC2•bikunin. In addition, Gg pre-α-inhibitor and smaller amount of high molecular weight forms composed of bikunin and two HCs were purified. Like the human bikunin proteins, the purified Gg proteins were all stabilized by a protein–glycosaminoglycan–protein cross-link, i.e. the HCs were covalently attached to a chondroitin sulfate originating from bikunin. Furthermore, the complex formed between Gg HC2•bikunin and human TSG-6 appeared to be identical to that of the human proteins. Akin to human, Gg HC2 was further transferred to hyaluronan when present, and when incubated in vitro, Gg pre-α-inhibitor and TSG-6, failed to form the intermediate covalent complex, essential for HC transfer. Significantly, Gg HC2, analogous to human HC2, promoted complex formation between human HC3 and human TSG-6, substantiating the evolutionary conservation of these interactions. The present study demonstrates that the unique interactions between bikunin proteins, glycosaminoglycans, and TSG-6 are evolutionary conserved, emphasizing the physiological importance of the TSG-6/HC2-mediated HC-transfer reaction. In addition, the data show that the evolution of HC transfer is likely to predate the role of HC·HA complexes in female fertility and thus has evolved in the context of inflammation rather than fertility.     

Suppressive effects of vascular endothelial growth factor-B on tumor growth in a mouse model of pancreatic neuroendocrine tumorigenesis

Background

The family of vascular endothelial growth factors (VEGF) contains key regulators of blood and lymph vessel development, including VEGF-A, -B, -C, -D, and placental growth factor. The role of VEGF-B during physiological or pathological angiogenesis has not yet been conclusively delineated. Herein, we investigate the function of VEGF-B by the generation of mouse models of cancer with transgenic expression of VEGF-B or homozygous deletion of Vegfb.

Methodology/Principal Findings

Ectopic expression of VEGF-B in the insulin-producing β-cells of the pancreas did not alter the abundance or architecture of the islets of Langerhans. The vasculature from transgenic mice exhibited a dilated morphology, but was of similar density as that of wildtype mice. Unexpectedly, we found that transgenic expression of VEGF-B in the RIP1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis retarded tumor growth. Conversely, RIP1-Tag2 mice deficient for Vegfb presented with larger tumors. No differences in vascular density, perfusion or immune cell infiltration upon altered Vegfb gene dosage were noted. However, VEGF-B acted to increase blood vessel diameter both in normal pancreatic islets and in RIP1-Tag2 tumors.

Conclusions/Significance

Taken together, our results illustrate the differences in biological function between members of the VEGF family, and highlight the necessity of in-depth functional studies of VEGF-B to fully understand the effects of VEGFR-1 inhibitors currently used in the clinic.     

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest¹. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest^2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone^6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required¹⁰. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.Download video file.^{(56M, mov)}     

Muscle adaptations to plyometric vs. resistance training in untrained young men

The purpose of this study was to compare changes in muscle strength, power, and morphology induced by conventional strength training vs. plyometric training of equal time and effort requirements. Young, untrained men performed 12 weeks of progressive conventional resistance training (CRT, n = 8) or plyometric training (PT, n = 7). Tests before and after training included one-repetition maximum (1 RM) incline leg press, 3 RM knee extension, and 1 RM knee flexion, countermovement jumping (CMJ), and ballistic incline leg press. Also, before and after training, magnetic resonance imaging scanning was performed for the thigh, and a muscle biopsy was sampled from the vastus lateralis muscle. Muscle strength increased by approximately 20-30% (1-3 RM tests) (p < 0.001), with CRT showing 50% greater improvement in hamstring strength than PT (p < 0.01). Plyometric training increased maximum CMJ height (10%) and maximal power (Pmax; 9%) during CMJ (p < 0.01) and Pmax in ballistic leg press (17%) (p < 0.001). This was far greater than for CRT (p < 0.01), which only increased Pmax during the ballistic leg press (4%) (p < 0.05). Quadriceps, hamstring, and adductor whole-muscle cross-sectional area (CSA) increased equally (7-10%) with CRT and PT (p < 0.001). For fiber CSA analysis, some of the biopsies had to be omitted. Type I and IIa fiber CSA increased in CRT (n = 4) by 32 and 49%, respectively (p < 0.05), whereas no significant changes occurred for PT (n = 5). Myosin heavy-chain IIX content decreased from 11 to 6%, with no difference between CRT and PT. In conclusion, gross muscle size increased both by PT and CRT, whereas only CRT seemed to increase muscle fiber CSA. Gains in maximal muscle strength were essentially similar between groups, whereas muscle power increased almost exclusively with PT training.     

Both replication bypass fidelity and repair efficiency influence the yield of mutations per target dose in intact mammalian cells induced by benzo[a]pyrene-diol-epoxide and dibenzo[a,l]pyrene-diol-epoxide

Mutations induced by polycyclic aromatic hydrocarbons (PAH) are expected to be produced when error-prone DNA replication occurs across unrepaired DNA lesions formed by reactive PAH metabolites such as diol epoxides. The mutagenicity of the two PAH-diol epoxides (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and (+/-)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DBPDE) was compared in nucleotide excision repair (NER) proficient and deficient hamster cell lines. We applied the (32)P-postlabelling assay to analyze adduct levels and the hprt gene mutation assay for monitoring mutations. It was found that the mutagenicity per target dose was 4 times higher for DBPDE compared to BPDE in NER proficient cells while in NER deficient cells, the mutagenicity per target dose was 1.4 times higher for BPDE. In order to investigate to what extent the mutagenicity of the different adducts in NER proficient cells was influenced by repair or replication bypass, we measured the overall NER incision rate, the rate of adduct removal, the rate of replication bypass and the frequency of induced recombination in the hprt gene. The results suggest that NER of BPDE lesions are 5 times more efficient than for DBPDE lesions, in NER proficient cells. However, DBPDE adducts block replication more efficiently and also induce 6 times more recombination events in the hprt gene than adducts of BPDE, suggesting that DBPDE adducts are, to a larger extent, bypassed by homologous recombination. The results obtained here indicate that the mutagenicity of PAH is influenced not only by NER, but also by replication bypass fidelity. This has been postulated earlier based on results using in vitro enzyme assays, but is now also being recognized in terms of forward mutations in intact mammalian cells.     

Barley alpha-amylase Met53 situated at the high-affinity subsite -2 belongs to a substrate binding motif in the beta-->alpha loop 2 of the catalytic (beta/alpha)8-barrel and is critical for activity and substrate specificity.

Met53 in barley alpha-amylase 1 (AMY1) is situated at the high-affinity subsite -2. While Met53 is unique to plant alpha-amylases, the adjacent Tyr52 stacks onto substrate at subsite -1 and is essentially invariant in glycoside hydrolase family 13. These residues belong to a short sequence motif in beta-->alpha loop 2 of the catalytic (beta/alpha)8-barrel and site-directed mutagenesis was used to introduce a representative variety of structural changes, Met53Glu/Ala/Ser/Gly/Asp/Tyr/Trp, to investigate the role of Met53. Compared to wild-type, Met53Glu/Asp AMY1 displayed 117/90% activity towards insoluble Blue Starch, and Met53Ala/Ser/Gly 76/58/38%, but Met53Tyr/Trp only 0.9/0.1%, even though both Asp and Trp occur frequently at this position in family 13. Towards amylose DP17 (degree of polymerization = 17) and 2-chloro-4-nitrophenyl beta-d-maltoheptaoside the activity (kcat/Km) of all mutants was reduced to 5.5-0.01 and 1.7-0.02% of wild-type, respectively. Km increased up to 20-fold for these soluble substrates and the attack on glucosidic linkages in 4-nitrophenyl alpha-d-maltohexaoside (PNPG6) and PNPG5 was determined by action pattern analysis to shift to be closer to the nonreducing end. This indicated that side chain replacement at subsite -2 weakened substrate glycon moiety contacts. Thus whereas all mutants produced mainly PNPG2 from PNPG6 and similar amounts of PNPG2 and PNPG3 accounting for 85% of the products from PNPG5, wild-type released 4-nitrophenol from PNPG6 and PNPG and PNPG2 in equal amounts from PNPG5. Met53Trp affected the action pattern on PNPG7, which was highly unusual for AMY1 subsite mutants. It was also the sole mutant to catalyze substantial transglycosylation - promoted probably by slow substrate hydrolysis - to produce up to maltoundecaose from PNPG6.     

Cell specific effects of polyunsaturated fatty acids on 5-aminolevulinic acid based photosensitization.

The purpose of this study was to examine whether the dietary components n-6 and n-3 polyunsaturated fatty acids (PUFAs) may potentiate the effect of photodynamic therapy (PDT) in human cancer cell lines by enhancing the lipid peroxidation. The effects of the porphyrin precursor 5-aminolevulinic acid (5-ALA) and light (320 < lambda < 440 nm, 33 W m(-2)), with or without docosahexaenoic acid (DHA) or arachidonic acid (AA), were tested in the colon carcinoma cell lines SW480 and WiDr, the glioblastoma cell line A-172 and the lung adenocarcinoma cell line A-427. The production of endogenous protoporphyrin IX (PpIX) varied substantially between the cell lines and was approximately 4-fold higher in WiDr as compared with SW480. Cell killing by 5-ALA-PDT also varied between the cell lines, but without clear correlation with PpIX levels. Treatment with DHA or AA (10 or 70 microM, 48 or 72 h) in combination with 5-ALA-PDT (1 or 2 mM) enhanced the cytotoxic effect in A-172 and A-427 cells, but not in SW480 and WiDr cells. While 5-ALA-PDT alone increased the lipid peroxidation in A-172 and WiDr cells only, 5-ALA-PDT plus PUFAs increased the lipid peroxidation substantially in all four cell lines. Interestingly, alpha-tocopherol (50 microM, 48 h) strongly reduced lipid peroxidation after all treatments in all cell lines, while cytotoxicity was only reduced substantially in A-427 cells. This demonstrates that induction of lipid peroxidation is not a general mechanism responsible for the cytotoxicity of 5-ALA-PDT, although it may be important in cell lines with an inherent sensitivity to lipid peroxidation products. Thus, the mechanisms of cell growth inhibition/cell killing by PDT are complex and cell specific.