Vascular Channel Formation by Osteosarcoma Cells in Vitro: Vasculogenic Mimicry

Objective: To observe whether there is evidence for vascular channel formation by osteosarcoma cellsin vitro and to illustrate mechanism of vasculogenic mimicry in osteosarcoma.Methods: Osteosarcoma cell lines (U-2OS) were tested for their ability to form tubular networks in three-dimensional culture containing type I collagen. The structures of the tubular networks were observed under a phase contrast microscope and an electron microscope.Results: Observation under light microscopy and electron microscopy showed that high aggressive osteosarcoma cells line (U-2OS) formed networks containing channels when grown in three-dimensional culture containing type I collagen in the absence of endothelial cells or fibroblasts.Conclusion: These observations strongly suggest that aggressive osteosarcoma cells may generate vascular channels that facilitate tumor perfusion independent of tumor angiogenesis and have the ability of vasculogenic mimicry. Key words osteosarcoma cells line - vasculogenesis mimicry - angiogenesis - 3-dimensional cultures This study was supported in part by the National Natural Sciences Foundation of China (No. 30271314).

Growth of the Crystals of Nitrogenase MnFe Protein

Under a suitable condition of crystallization, dark brown short rhombohedron crystals could be obtained from nitrogenase MnFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmann grown in Mn-containing but Mo- and NH3-free medium. The possibility of crystallization, and number, size and quality of crystals were obviously dependent on concentrations of NaCl, MgCl2, PEG 8000,Tris and Hepes buffer and on methods for crystallization. PEG concentration affected on the shape of the crystals. The optimal, concentrations of the chemicals for crystallization of MnFe protein were slightly different from those for crystallization of Delta nifZ MoFe protein from a nifZ deleted strain of Azotobacter vinelandii. SDS-PAGE showed that the protein from the dissolved crystals was almost the same as MnFe protein before crystallization, indicating that the crystal was formed from MnFe protein.

Crystalline Growth of Nitrogenase CrFe Protein

Under a suitable condition of crystallization, dark brown rhombohedron crystals (the lengths of the longest two diagonals were 0.25 and 0.12 mm, respectively) could be obtained from nitrogenase CrFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmann grown in Cr-containing but NH3-free Medium, The possibility of crystallization, as well as the. number, size and quality of crystals obviously depended on the concentrations of PEG 8000, MgCl2, NaCl, Tris and Hepes buffer, and methods of crystallization. The optimum concentrations of the chemicals for crystallization of CrFe protein were slightly different from those for crystallization of MnFe protein from UW3 grown in Mn and DeltanifZ MoFe protein from a nifZ deleted strain of A. vinelandii. The crystal seemed to be formed from CrFe protein.

The Arabidopsis PRCl-like ring-finger proteins are necessary for repression of embryonic traits during vegetative growth

Polycomb group genes play crucial roles in the maintenance of the transcriptionally silenced state of genes for proper cell differentiation in animals and plants. While components of the polycomb repressive complex2 （PRC2） are evolutionarily conserved and their functions are extensively studied in plants, PRCI differs considerably between animals and plants, and its functions in plants are as yet not well described. Previous studies have identified the Arabidopsis AtRINGla and AtRINGlb as homologues of the animal PRC1 subunit RING1. Here, we show that the Atringla Atringlb double mutant exhibits derepression of embryonic traits during vegetative growth. Accordingly, several key regulatory genes involved in embryogenesis and stem cell activity are ectopically expressed in the mutant. Furthermore, we show that the mutant phenotypes and increased expression of regulatory genes are enhanced by the PRC2 mutant c/f. Finally, we show that three homologues of the animal PRCl-subunit ring-finger protein BMI1, AtBMIIa, AtBMIlb and AtBMIlc, can bind with AtRINGla or AtRINGIb, and in addition, AtBMIlc can bind with LHP1. The Atbmila Atbmilb double mutant shows derepression of embryonic traits similar to that of the Atringla Atringlb double mutant. Interestingly, expression levels of AtBMIla, AtBMIlb and AtBMIlc are elevated in the Atringla Atringlb mutant and those of AtBMIlc, AtRINGla and AtRINGlb are elevated in the Atbmila Atbmilb mu- tant, suggesting a self-regulatory feedback mechanism. Taken together, our results illuminate crucial functions of the PRCl-like ring-finger components in stable repression of embryonic traits and regulatory genes for proper somatic growth.

The selection of alkaline protease-producing yeasts from marine environments and evaluation of their bioactive peptide production

A total of 400 yeast strains from seawater, sediments, saltern mud, marine fish guts, and marine algae were obtained. The protease activity of the yeast cultures was estimated, after which four strains （HN3.11, Nllb, YF04C and HN4.9） capable of secreting extracellular alkaline protease were isolated. The isolated strains were identified as Aureobasidium pullulans, Yarrowia lipolytica, lssatchenkia orientalis and Cryptococcus cf. aureus. The optimal pH of the protease activity produced by strains HN3.11, YF04C, and HN4.9 was 9.0, while that of the protease produced by strain N1 lb was 10.0. The optimal temperature for protease activity was 45℃for strains HN3.11, N11b, and YF04C, and 50℃ for strain HN4.9. After digestion of shrimp （Penaeus vannamei） protein and spirulina （Arthospira platens＆） protein with the four crude alkaline proteases, the filtrate from spirulina （Arthrospira platensis） powder digested by the crude alkaline protease of strain HNYl 1 was found to have the highest antioxidant activity （61.4%） and the highest angiotensin I converting enzyme （ACE）-inhibitory activities （68.4%）. The other filtrates had much lower antioxidant activity and ACE-inhibitory activities.

Glutamate dehydrogenase and Na^+-K^+ ATPase expression and growth response of Litopenaeus vannamei to different salinities and dietary protein levels

Improvement in the osmoregulation capacity via nutritional supplies is vitally important in shrimp aquaculture.The effects of dietary protein levels on the osmoregulation capacity of the Pacific white shrimp(L.vannamei) were investigated.This involved an examination of growth performance,glutamate dehydrogenase(GDH) and Na+-K+ ATPase mRNA expression,,and GDH activity in muscles and gills.Three experimental diets were formulated,containing 25%,40%,and 50% dietary protein,and fed to the shrimp at a salinity of 25.After 20 days,no significant difference was observed in weight gain,though GDH and Na+-K+ ATPase gene expression and GDH activity increased with higher dietary protein levels.Subsequently,shrimp fed diets with 25% and 50% dietary protein were transferred into tanks with salinities of 38 and 5,respectively,and sampled at weeks 1 and 2.Shrimp fed with 40% protein at 25 in salinity(optimal conditions) were used as a control.Regardless of the salinities,shrimp fed with 50% dietary protein had significantly higher growth performance than other diets;no significant differences were found in comparison with the control.Shrimp fed with 25% dietary protein and maintained at salinities of 38 and 5 had significantly lower weight gain values after 2 weeks.Ambient salinity change also stimulated the hepatosomatic index,which increased in the first week and then recovered to a relatively normal level,as in the control,after 2 weeks.These findings indicate that in white shrimp,the specific protein nutrient and energy demands related to ambient salinity change are associated with protein metabolism.Increased dietary protein level could improve the osmoregulation capacity of L.vannamei with more energy resources allocated to GDH activity and expression.

TGF-β_1 inhibits connexin-43 expression in cultured smooth muscle cells of human bladder

Objective: In this research, we studied the TGF-β1 effects on connexin-43 expression in cultured human bladder smooth muscle cells. Methods: Human bladder smooth muscle cells primary cultures, with bladder tissue obtained from patients undergoing cystectomy, were intervened by recombinant human TGF-β1. Connexin-43 expression in human bladder smooth muscle cells was then examined by Western blotting and immunocytochemistry. Results: Stimulation with TGF-β1 led to significant reduction of connexin-43 immunoreactivity and coupling (P<0.0001). Connexin-43 protein expression was significantly downregulated (P<0.05). Simultaneously, low phosphorylation species of connexin-43 were particularly affected. Conclusion: Our experiments demonstrated a significant downregulation of connexin-43 by TGF-β1 in cultured human bladder smooth muscle cells. These findings support the view that TGF-β1 is involved in the pathophysiology of urinary bladder dysfunction.

Connective tissue growth factor hammerhead ribozyme attenuates human hepatic stellate cell function

AIM： To determine the effect of hammerhead ribozyme targeting connective tissue growth factor （CCN2） on human hepatic stellate cell （HSC） function.METHODS： CCN2 hammerhead ribozyme cDNA plus two self-cleaving sequences were inserted into pTriEx2 to produce pTriCCN2-Rz. Each vector was individually transfected into cultured LX-2 human HSCs, which were then stimulated by addition of transforming growth factor （TGF）-β1 to the culture medium. Semiquantitative RT-PCR was used to determine mRNA levels for CCN2 or collagen I, while protein levels of each molecule in cell /ysates and conditioned medium were measured by ELISA. Cell-cycle progression of the transfected cells was assessed by flow cytometry.RESULTS： In pTriEx2-transfected LX-2 cells, TGF-β1 treatment caused an increase in the mRNA level for CCN2 or collagen I, and an increase in produced and secreted CCN2 or extracellular collagen I protein levels, pTriCCN2-Rz-transfected LX-2 cells showed decreased basal CCN2 or collagen mRNA levels, as well as produced and secreted CCN2 or collagen I protein. Furthermore, the TGF-β1-induced increase in mRNA or protein for CCN2 or collagen I was inhibited partially in pTriCCN2-Rz-transfected LX-2 cells. Inhibition of CCN2 using hammerhead ribozyme cDNA resulted in fewer of the cells transitioning into S phase.CONCLUSION： Endogenous CCN2 is a mediator of basal or TGF-β1-induced collagen I production in human HSCs and regulates entry of the cells into S phase.

Real and Legal Nutritional Alternative （e.g. Application of Free Amino Acids） to Replace Forbidden Doping Substances to Produce Excellent Sport Performance

Peformance enhancing drugs are widely used today in different sport fields, and therefore the successful anti-doping activity should be based not only on regular doping controls, but also it should show the real alternative： how to replace the forbidden doping substances with legal and effective supplements. The paper deals with application proposition of protein concentrates, free AA （amino acids）, HMB （hydroxy-methyle-butyrate）, creatine and camitine. If the athlete is involved in strength and power sport （e.g. throwing events in track and field, olympic weightlifting etc.）, the explosive and maximum strength is of primary importance. For strength athletes, these mentioned substances are those legal preparates, supplements, which can help effectively in performance improvement of sport results. The main reason is that using protein concentrates, AAs, HMB, creatine and camitine, the anabolic （and anticatabolic） effect will enhance the protein biosynthesis of the organism, improve the aerob and anaerob capacity of the athletes, activate and stimulate the own hormonal system. And the final result is creating higher loadability because of the faster recovery, and higher performance level on the competition. Of course, the athletes--as a minimum requirement--need an adequate nutrition （good balanced nourishment）, as well, with appropriate application and supplementation of all vitamins and essential minerals.

Grasshoppers Sphenarium Purpurascens Ch Source of Proteins and Essential Amino Acids

Protein malnutrition is quite common worldwide; its deficiency has adverse effects in all organs. Edible insect＇s traditional cultural food, commonly consumed in rural communities, now intake is also being incorporated in some countries. Grasshoppers comprise proteins, the building blocks of AA （amino acids）, that are one of the five classes of complex biomolecules found in cells and tissues that play an important role in human metabolism and nutrition. This research was conducted to determine proximal composition of Sphenarium purpurascens Ch, grasshoppers, focused on proteins and AA. Sampling was performed during autumn, 2013, in Oaxaca State. Dry raw adult insects were ground and proceeded to macro molecules proximal analysis, protein by Kjeldahl AOAC （Association of Official Analytical Chemists） method and essential AA in a Beckman 6300 equipment. Obtained data were： total proteins 71.56%; lipids 8.14%; minerals 3.52%; fiber 7.48%; soluble carbohydrates 9.30%. Essential AA （mg/16 g-N）： isoleucine 4.5; leucine 8.1; lysine 5.4; methionine ＋ cysteine 5.1; phenylalanine ＋ tyrosine 8.5; threonine 3.5; tryptophan 0.6; valine 5.5. Sphenarium purpurascens Ch, Grasshoppers represent a good source of proteins and essential AA that can grow almost everywhere, available mostly all year round and affordable to all social groups, thus being a good option to improve human health.

Effects of acupuncture on nutritional status in patients in a persistent vegetative state:a prospective randomized controlled study

Objective To explore the effects of acupuncture on nutritional status in patients in a persistent vegetative state.Methods A prospective randomized controlled trial was designed.A total of 66 patients in a persistent vegetative state were randomized into a control group and an observation group,with 33 cases in each group.The control group was given conventional treatment plus enteral nutrition support.The observation group was treated with additional Tiao Shen Jian Pi acupuncture therapy(acupuncture for spirit-regulating and spleen-invigorating)based on the same interventions in the control group.Both groups were treated for 8 weeks.The levels of total protein(TP),prealbumin(PA),albumin(Alb),and hemoglobin(Hb)were measured before and after treatment.The upper arm circumference and skinfold thickness of triceps brachii were measured.And the intestinal flora and fecal short-chain fatty acids contents were determined.Results After treatment,the levels of TP,PA,Alb,and Hb in the control group were decreased(P<0.05),while in the observation group,compared with those before treatment,the levels of TP,PA,Alb,and Hb had no statistical differences(P>0.05),and the levels were all higher than those in the control group(P<0.05).The upper arm circumference and skinfold thickness of triceps brachii in both groups decreased(P<0.05),and the values of these two items in the observation group were higher than those in the control group(P<0.05).In the control group,the contents of Bifidobacterium and Lactobacillus in feces decreased(P<0.05),and the content of Enterococcus increased(P<0.05).In the observation group,the contents of Bifidobacterium and Lactobacillus in feces increased(P<0.05),and the content of Enterococcus decreased(P<0.05).The differences between the two groups were statistically significant(P<0.05).In the control group,the total content of fecal short-chain fatty acids and the contents of acetic acid and butyric acid in feces decreased(P<0.05).In the observation group,the total content of fecal short-chain fatty acids and the contents of acetic acid and butyric acid in feces increased(P<0.05)and were all higher than those in the control group(P<0.05).Conclusion Acupuncture can improve nutrition-related blood indicators in patients in a persistent vegetative state and delay the decrease of upper arm circumference and skinfold thickness of triceps brachii,which may be related to the regulation of intestinal flora and fecal short-chain fatty acids contents.