接种固定化粪菌和培养基种类对体外结肠菌群发酵的影响

目的研究三种模拟结肠发酵培养基和粪菌固定化对体外结肠发酵体系菌群构成的影响,为建立高度模拟结肠体外结肠发酵提供参考。方法采集健康女性成人粪便,粪便制备悬液或用结冷胶-黄原胶固定化粪菌接种于模拟结肠发酵反应器,分别用三种培养基按0.07 m L/h稀释率进行连续发酵10 d,用末端限制性片段长度多态性(T-RFLP)与高通量测序技术分析菌群多样性,并分析发酵液短链脂肪酸含量。结果 T-RFLP分析显示,培养基类型主要影响粪菌固定体系菌群α多样性达到稳定的时间和多样性。β多样性分析显示,第10天时培养基Ⅲ悬液培养和培养基I固定化培养系统的菌群构成与粪便菌群最相近。而培养基Ⅲ在第10天达到较高而稳定的丁酸浓度。结论由于比粪菌固定培养更易操作,培养基Ⅲ悬液培养适于模拟人结肠发酵。     

The consumption of out-of-home highbrow leisure by ethnicity and national origin: attendance at museums and live theatres in Houston

Dynamics of compensation for the deprivations of segregation and discrimination, and the support of multiculturalism derived from ethnic cohesion explain the consumption of out-of-home highbrow leisure events by minority/ethnic individuals, immigrants, and their descendants as efforts toward their integration and assimilation in metropolitan areas. Using data from the Houston Area Survey, I examine whether there are any significant ethnic disparities in the attendance at museums and live theatres, which represent a relevant dimension of out-of-home highbrow leisure in Houston. I found that the odds of frequently attending museums and live theatres are lower for Anglos compared with non-Anglos, and higher for US-born individuals with at least one foreign parent compared with US-born individuals with US-born parents. These findings reveal that the audiences of museums and live theatres in Houston are already characterized by a noteworthy ethnic diversity.     

Interaction studies of novel cell selective antimicrobial peptides with model membranes and E. coli ATCC 11775

Cationic antimicrobial peptides (CAMPs) are novel candidates for drug development. Here we describe design of six short and potent CAMPs (SA-1 to SA-6) based on a minimalist template of 12 residues H+HHG+HH+HH+NH2 (where H: hydrophobic amino acid and +: charged hydrophilic amino acid). Designed peptides exhibit good antibacterial activity in micro molar concentration range (1-32 μg/ml) and rapid clearance of Gram-positive and Gram-negative bacterial strains at concentrations higher than MIC. For elucidating mode of action of designed peptides various biophysical studies including CD and Trp fluorescence were performed using model membranes. Further based on activity, selectivity and membrane bound structure; modes of action of Trp rich peptide SA-3 and template based peptide SA-4 were compared. Calcein dye leakage and transmission electron microscopic studies with model membranes exhibited selective membrane active mode of action for peptide SA-3 and SA-4. Extending our work from model membranes to intact E. coli ATCC 11775 in scanning electron micrographs we could visualize different patterns of surface perturbation caused by peptide SA-3 and SA-4. Further at low concentration rapid translocation of FITC-tagged peptide SA-3 into the cytoplasm of E. coli cells without concomitant membrane perturbation indicates involvement of intracellular targeting mechanism as an alternate mode of action as was also evidenced in DNA retardation assay. For peptide SA-4 concentration dependent translocation into the bacterial cytoplasm along with membrane perturbation was observed. Establishment of a non specific membrane lytic mode of action of these peptides makes them suitable candidates for drug development.     

Stem cell culture engineering - process scale up and beyond

Advances in stem cell research and recent work on clinical trials employing stem cells have heightened the prospect of stem cell applications in regenerative medicine. The eventual clinical application of stem cells will require transforming cell production from laboratory practices to robust processes. Most stem cell applications will require extensive ex vivo handling of cells, from isolation, cultivation, and directed differentiation to product cell separation, cell derivation, and final formulation. Some applications require large quantities of cells in each defined batch for clinical use in multiple patients; others may be for autologous use and require only small-scale operations. All share a common requirement: the production must be robust and generate cell products of consistent quality. Unlike the established manufacturing process of recombinant protein biologics, stem cell applications will likely see greater variability in their cell source and more fluctuations in product quality. Nevertheless, in devising stem cell-based bioprocesses, much insight could be gained from the manufacturing of biological materials, including recombinant proteins and anti-viral vaccines. The key to process robustness is thus not only the control of traditional process chemical and physical variables, but also the sustenance of cells in the desired potency or differentiation state through controlling non-traditional variables, such as signaling pathway modulators.     

Synthesis, crystal structures and, antibacterial and antiproliferative activities in vitro of palladium(II) complexes of triphenylphosphine and thioamides

Palladium(II) complexes with triphenylphosphine (PPh₃) and thioamides of the general formulae, [Pd(L)₂(PPh₃)₂]Cl₂ and [Pd(L)₂(PPh₃)₂] have been prepared and characterized by elemental analysis, IR and NMR (¹H, ¹³C and ³¹P) methods, and two of them (trans-[Pd(PPh₃)₂(Dmtu)₂]Cl₂·(H₂O)(CH₃OH)_0.5 (1) and trans-[Pd(PPh₃)₂(Mpy)₂] (2)) by X-ray crystallography; where L = thiourea (Tu), methylthiourea (Metu), N,N′-dimethylthiourea (Dmtu), tetramethylthiourea (Tmtu), 2-mercaptopyridine (Mpy), 2-mercaptopyrimidine (Mpm) and thionicotinamide (Tna). The spectral data of the complexes are consistent with the sulfur coordination of thioamides to palladium(II). The crystal structures of the complexes show that (1) has ionic character consisting of [Pd(PPh₃)₂(Dmtu)₂]⁺² cations and uncoordinated Cl⁻ ions, while (2) is a neutral complex with Mpy behaving as anionic thiolate ligand. The coordination environment around palladium in (2) is nearly regular square-planar, while in (1) the trans angles show significant distortions from 180°. The complexes were screened for antibacterial effects, brine shrimps lethality bioassay and antitumor activity. These complexes showed significant activities in most of the cases against the tested bacteria as compared to that of a standard drug. Their antitumor activity against prostate cancer cells (PC3) is comparable with doxorubicin, together with no cytotoxic effects in brine shrimps lethality bioassay study.     

Influence of culture conditions on glutathione production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A strategy of experimental design using a fractional factorial design (FFD) and a central composite rotatable design (CCRD) were carried out with the aim to obtain the best conditions of temperature (20–30°C), agitation rate (100–300 rpm), initial pH (5.0–7.0), inoculum concentration (5–15%), and glucose concentration (30–70 g/l) for glutathione (GSH) production in shake-flask culture by Saccharomyces cerevisiae ATCC 7754. By a FFD (2^5–2), the agitation rate, temperature, and pH were found to be significant factors for GSH production. In CCRD (2²) was obtained a second-order model equation, and the percent of variation explained by the model was 95%. The results showed that the optimal culture conditions were agitation rate, 300 rpm; temperature, 20°C; initial pH, 5; glucose, 54 g/l; and inoculum concentration, 5%. The highest GSH concentration (154.5 mg/l) was obtained after 72 h of fermentation.     

HTLV-2 Tax Immortalizes Human CD4+ Memory T Lymphocytes by Oncogenic Activation and Dysregulation of Autophagy

Human T cell leukemia virus type 1 and type 2 (HTLV-1 and -2) are two closely related retroviruses with the former causing adult T cell leukemia. HTLV-2 infection is prevalent among intravenous drug users, and the viral genome encodes the viral transactivator Tax, which is highly homologous to the transforming protein Tax from HTLV-1. However, the link between HTLV-2 infection and leukemia has not been established. In the present study, we evaluated the activity of HTLV-2 Tax in promoting aberrant proliferation of human CD4 T lymphocytes. Tax2 efficiently immortalized CD4⁺ memory T lymphocytes with a CD3/TCRαβ/CD4/CD25/CD45RO/CD69 immunophenotype, promoted constitutive activation of PI3K/Akt, IκB kinase/NF-κB, mitogen-activated protein kinase, and STAT3, and it also increased the level of Mcl-1. Disruption of these oncogenic pathways led to growth retardation and apoptotic cell death of the Tax2-established T cell lines. We further found that Tax2 induced autophagy by interacting with the autophagy molecule complex containing Beclin1 and PI3K class III to form the LC3⁺ autophagosome. Tax2-mediated autophagy promoted survival and proliferation of the immortalized T cells. The present study demonstrated the oncogenic properties of Tax2 in human T cells and also implicated Tax2 in serving as a molecular tool to generate distinct T cell subtype lines.     

Molecular and cellular effects of Tamm-Horsfall protein mutations and their rescue by chemical chaperones

Correct folding of a nascent polypeptide in the lumen of the endoplasmic reticulum (ER) into a three-dimensional conformation is a crucial step in the stability, intracellular trafficking, and targeting to the final destination of a protein. By transiently and stably expressing human-relevant mutants of Tamm-Horsfall protein in polarized Madin-Darby canine kidney cells, we show here that a cysteine-altering mutation in the evolutionally conserved cysteine-rich domain had more severe defects in ER exit and surface translocation and triggered more apoptosis than a cysteine-altering mutation outside the domain. Both mutants were able to specifically bind and trap the wild-type Tamm-Horsfall protein (THP) and prevent it from exiting the ER and translocating to the cell surface. This explains at least partly why in patients with THP-associated diseases there is a marked urinary reduction of both the mutant and the wild-type THP. Exposure of mutant-expressing cells to low temperature (30 °C), osmolytes (glycerol, trimethylamine N-oxide, and dimethyl sulfoxide), and the Ca(2+)-ATP inhibitor thapsigargin only slightly relieved ER retention and increased surface targeting of the mutants. In contrast, sodium 4-phenylbutyrate and probenecid, the latter a uricosuric drug used clinically to treat gout, markedly reduced ER retention of the mutants and increased their surface translocation and secretion into the culture media. The rescue of the THP mutants was associated with the restoration of the level and subcellular localization of cytosolic chaperone HSP70. Our results reveal intricate mechanistic details that may underlie THP-associated diseases and suggest that novel therapeutics enhancing the refolding of THP mutants may be of important value in therapy.     

Cdc6 protein activates p27KIP1-bound Cdk2 protein only after the bound p27 protein undergoes C-terminal phosphorylation

In mammalian cells Cdk2 activity during the G(1)-S transition is mainly controlled by p27(KIP1). Although the amount and subcellular localization of p27 influence Cdk2 activity, how Cdk2 activity is regulated during this phase transition still remains virtually unknown. Here we report an entirely new mechanism for this regulation. Cdc6 the AAA+ ATPase, known to assemble prereplicative complexes on chromosomal replication origins and activate p21(CIP1)-bound Cdk2, also activated p27-bound Cdk2 in its ATPase and cyclin binding motif-dependent manner but only after the p27 bound to the Cdk2 was phosphorylated at the C terminus. ROCK, which mediates a signal for cell anchorage to the extracellular matrix and activates the mTORC1 cascade as well as controls cytoskeleton assembly, was partly responsible for C-terminal phosphorylation of the p27. In vitro reconstitution demonstrated ROCK (Rho-associated kinase)-mediated phosphorylation of Cdk2-bound p27 at the C terminus and subsequent activation of the Cdk2 by Cdc6.