Foreign gene expression in Hansenula polymorpha. A system for the synthesis of small functional peptides

 We describe the synthesis and purification of two functional peptides, namely human insulin-like growth factor II (IGF-II) and Xenopus laevis magainin II in Hansenula polymorpha after their synthesis as hybrid proteins fused to the C terminus of endogenous amine oxidase. The hybrid genes, placed under control of the H. polymorpha alcohol oxidase promoter (P_AOX), were integrated into the genomic alcohol oxidase locus, yielding stable production strains. High-level synthesis of the fusion proteins, exceeding 20% of total cellular protein, was obtained when the transformed strains were grown in methanol-limited chemostat cultures; when expressed by itself, i.e. in the absence of the amine oxidase gene, IGF-II could not be recovered from crude cell extracts, probably as a result of rapid proteolytic degradation. Accumulation in peroxisomes did not significantly affect the IGF-II protein stability when expressed in the absence of the carrier protein. Apparently, fusion to the large (±78 kDa) amine oxidase carrier particularly stabilizes the peptides and prevents them from proteolysis. After partial purification, the fusion partners were readily separated by factor Xa treatment. Received: 16 June 1995 / Accepted: 20 September 1995     

A scaffoldless technique for self-generation of three-dimensional keratinospheroids on liquid crystal surfaces

We describe a new scaffold-free three-dimensional (3D) cell culture model using cholesteryl ester based lyotropic liquid crystal (LC) substrates. Keratinocytes were deposited randomly on the LC surface where they self-assembled into 3D microtissues or keratinospheroids. The cell density required to form spheroids was optimized. We investigated cell viability using dead/live cell assays. The adhesion characteristics of cells within the microtissues were determined using histological sectioning and immunofluorescence staining. Fourier transform infrared spectroscopy (FTIR) was used to characterize the biochemistry of the keratinospheroids. We found that both cells and microtissues could migrate on the LC surface. The viability study indicated approximately 80% viability of cells in the microtissues up to 20 days of culture. Strong intercellular adhesion was observed in the stratification of the multi-layered microspheroids using field emission-scanning electron microscopy (FE-SEM) and histochemical staining. The cytoskeleton and vinculins of the cells in the microtissues were expressed diffusely, but the microtissues were enriched with lipids and nucleic acids, which indicates close resemblance to the conditions in vivo. The basic 3D culture model based on LC may be used for cell and microtissue migration studies in response to cytochemical treatment.     

Bioethanol production from the hydrolysate of Palmaria palmata using sulfuric acid and fermentation with brewer’s yeast

Seaweeds, particularly species of red macroalgae, are promising resources for bioethanol production because of their exceptionally high carbohydrate content. Of 20 seaweeds evaluated, Palmaria palmata (Rhodymenia palmata) contained the highest carbohydrate content (469.8 mg g^?1 seaweed) with a carrageenan content of 354 mg g^?1 seaweed. Such a high carrageenan content makes the high-volume production of bioethanol feasible. Acid hydrolysis of P. palmata in 0.4 M H₂SO₄ at 125 °C for 25 min released 27 mg of glucose, 218.4 mg of reducing sugars, and 127.6 mg of galactose per gram of seaweed. Ethanol fermentation of these hydrolysis products using an inoculum concentration of 1.5 mg mL^?1 at 30 °C and 72 h in a shaking incubator at 130 rpm yielded 17.3 mg of ethanol per gram of seaweed.     

突尼斯非洲跳鼠（Jaculus jaculus）和埃及跳鼠J.orientalis）群体的等位酶多态及遗传分化

运用16种酶蛋白编码的23个遗传座位对突尼斯非洲跳鼠(Jaculus jaculus)和埃及跳鼠(J.orientalis)自然群体的遗传变异和分化进行了电泳分析.结果表明,与其他啮齿动物等哺乳动物的相关数据比较,发现这两个种群体的遗传变异水平较低.非洲跳鼠群体的观测杂合度(Hobs)为0.08-0.19,多态座位百分比(P)为26.2%-45.2%,每个座何的平均等位基因数(A)为1.1-1.4;埃及跳鼠的Hobs为0.10-0.15,P为29.3%-44.1%,A为1.1-1.7.两个种群体各自的遗传分化程度较低(非洲跳鼠和埃及跳鼠的Fst分别为0.0017和0.0019).而两个种群体间的Fst为0.607(P＜0.05),表明两个种之间高度的遗传分化.本研究支持这两个种分类地位的合法性,并强调了地理因素(环境类犁和生物气候阶段)对两个种遗传结构的影响.     

A reliable measure of similarity based on dependency for short time series: an application to gene expression networks

Background  

Microarray techniques have become an important tool to the investigation of genetic relationships and the assignment of different phenotypes. Since microarrays are still very expensive, most of the experiments are performed with small samples. This paper introduces a method to quantify dependency between data series composed of few sample points. The method is used to construct gene co-expression subnetworks of highly significant edges.     

Effects of 3-beta-diol, an androgen metabolite with intrinsic estrogen-like effects, in modulating the aquaporin-9 expression in the rat efferent ductules

Background  

Fluid homeostasis is critical for normal function of the male reproductive tract and aquaporins (AQP) play an important role in maintenance of this water and ion balance. Several AQPs have been identified in the male, but their regulation is not fully comprehended. Hormonal regulation of AQPs appears to be dependent on the steroid in the reproductive tract region. AQP9 displays unique hormonal regulation in the efferent ductules and epididymis, as it is regulated by both estrogen and dihydrotestosterone (DHT) in the efferent ductules, but only by DHT in the initial segment epididymis. Recent data have shown that a metabolite of DHT, 5-alpha-androstane-3-beta-17-beta-diol (3-beta-diol), once considered inactive, is also present in high concentrations in the male and indeed has biological activity. 3-beta-diol does not bind to the androgen receptor, but rather to estrogen receptors ER-alpha and ER-beta, with higher affinity for ER-beta. The existence of this estrogenic DHT metabolite has raised the possibility that estradiol may not be the only estrogen to play a major role in the male reproductive system. Considering that both ER-alpha and ER-beta are highly expressed in efferent ductules, we hypothesized that the DHT regulation of AQP9 could be due to the 3-beta-diol metabolite.     

Aggravation of myocardial dysfunction by injurious mechanical ventilation in LPS-induced pneumonia in rats

Background

Mechanical ventilation (MV) may cause ventilator-induced lung injury (VILI) and may thereby contribute to fatal multiple organ failure. We tested the hypothesis that injurious MV of lipopolysaccharide (LPS) pre-injured lungs induces myocardial inflammation and further dysfunction ex vivo, through calcium (Ca²⁺)-dependent mechanism.

Materials and methods

N = 35 male anesthetized and paralyzed male Wistar rats were randomized to intratracheal instillation of 2 mg/kg LPS or nothing and subsequent MV with lung-protective settings (low tidal volume (V_t) of 6 mL/kg and 5 cmH₂O positive end-expiratory pressure (PEEP)) or injurious ventilation (high V_t of 19 mL/kg and 1 cmH₂O PEEP) for 4 hours. Myocardial function ex vivo was evaluated in a Langendorff setup and Ca²⁺ exposure. Key mediators were determined in lung and heart at the mRNA level.

Results

Instillation of LPS and high V_t MV impaired gas exchange and, particularly when combined, increased pulmonary wet/dry ratio; heat shock protein (HSP)70 mRNA expression also increased by the interaction between LPS and high V_t MV. For the heart, C-X-C motif ligand (CXCL)1 and Toll-like receptor (TLR)2 mRNA expression increased, and ventricular (LV) systolic pressure, LV developed pressure, LV +dP/dt_max and contractile responses to increasing Ca²⁺ exposure ex vivo decreased by LPS. High V_t ventilation aggravated the effects of LPS on myocardial inflammation and dysfunction but not on Ca²⁺ responses.

Conclusions

Injurious MV by high V_t aggravates the effects of intratracheal instillation of LPS on myocardial dysfunction, possibly through enhancing myocardial inflammation via pulmonary release of HSP70 stimulating cardiac TLR2, not involving Ca²⁺ handling and sensitivity.