Determination of some significant batch culture conditions affecting acetyl-xylan esterase production by Penicillium notatum NRRL-1249

Background

The methylotrophic yeast, Pichia pastoris, offers the possibility to generate a high amount of recombinant proteins in a fast and easy way to use expression system. Being a single-celled microorganism, P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. A simple and direct method for the selection of high-producing clones can dramatically enhance the whole production process along with significant decrease in production costs.

Results

A visual method for rapid selection of high-producing clones based on mannanase reporter system was developed. The study explained that it was possible to use mannanase activity as a measure of the expression level of the protein of interest. High-producing target protein clones were directly selected based on the size of hydrolysis holes in the selected plate. As an example, the target gene (9elp-hal18) was expressed and purified in Pichia pastoris using this technology.

Conclusions

A novel methodology is proposed for obtaining the high-producing clones of proteins of interest, based on the mannanase reporter system. This system may be adapted to other microorganisms, such as Saccharomyces cerevisiae for the selection of clones.     

A morphometric study of the protective effect of cryotherapy on oral mucositis in cancer patients treated with 5-fluorouracil

We investigated cytological changes in oral mucosa smears from patients treated with cryotherapy to determine whether cryotherapy prevented mucositis caused by 5-fluorouracil (5-FU) therapy. Patients with gastrointestinal malignancies were divided into four groups; control patients before 5-FU therapy, patients after 5-FU therapy without cryotherapy, patients with cryotherapy before 5-FU therapy and patients with cryotherapy after 5-FU therapy. Oral mucosa samples from all patients were assessed at the beginning and on day 14 of chemotherapy. We used exfoliative cytology to evaluate cellular changes in the oral mucosa that were caused by 5-FU. Smears from each patient were stained using the Papanicolaou method and analyzed using stereology. Smears were taken from each group before and after 5-FU infusion. We found that nuclear volume was decreased significantly in cells of the 5-FU therapy after cryotherapy patients compared to the 5-FU therapy before cryotherapy patients. We also found significantly decreased cytoplasmic volumes in the 5-FU therapy after cryotherapy patients compared to the 5-FU therapy before cryotherapy patients. The results of cytomorphometric estimations revealed that cryotherapy may be used to prevent damage to oral tissue and may decrease the frequency and duration of oral mucositis caused by 5-FU.     

Poaceae pollen in the atmosphere of Zagreb (Croatia), 2002 – 2005

Pollen of grasses (Poaceae) is one of the most important airborne allergen sources worldwide. The aim of the study was to determine diurnal, day‐to‐day, weekly, monthly and annual pollen variation, and the effect of selected meteorological parameters on atmospheric pollen concentrations in Zagreb. A preliminary study was carried out during four seasons (2002 – 2005), using a 7‐day VPPS 2000 Hirst volumetric pollen trap. Total annual grass pollen concentrations in Zagreb were constant (2 673 – 3 074 p.g.m^?3), with a relative drop in 2004 (1 196 p.g.m^?3). The beginning of the grass pollen season is usually observed when the average daily temperature exceeds 13.5°C (max. daily temp. 19.5°C), without precipitation. In all four study years the absolute peak of concentrations occurs in the second half of May. The length of pollen season during the study period was 157 – 173 days. A high variability was observed in the maximal concentration and in the number of days when grass pollen concentration exceeded the threshold value of 30 p.g.m^?3 (9 – 40 days). Statistically significant correlations were found between airborne grass pollen concentrations and air temperature, level of precipitation and relative humidity.     

Mono- and binuclear non-heme iron chemistry from a theoretical perspective

In this minireview, we provide an account of the current state-of-the-art developments in the area of mono- and binuclear non-heme enzymes (NHFe and NHFe₂) and the smaller NHFe₍₂₎ synthetic models, mostly from a theoretical and computational perspective. The sheer complexity, and at the same time the beauty, of the NHFe₍₂₎ world represents a challenge for experimental as well as theoretical methods. We emphasize that the concerted progress on both theoretical and experimental side is a conditio sine qua non for future understanding, exploration and utilization of the NHFe₍₂₎ systems. After briefly discussing the current challenges and advances in the computational methodology, we review the recent spectroscopic and computational studies of NHFe₍₂₎ enzymatic and inorganic systems and highlight the correlations between various experimental data (spectroscopic, kinetic, thermodynamic, electrochemical) and computations. Throughout, we attempt to keep in mind the most fascinating and attractive phenomenon in the NHFe₍₂₎ chemistry, which is the fact that despite the strong oxidative power of many reactive intermediates, the NHFe₍₂₎ enzymes perform catalysis with high selectivity. We conclude with our personal viewpoint and hope that further developments in quantum chemistry and especially in the field of multireference wave function methods are needed to have a solid theoretical basis for the NHFe₍₂₎ studies, mostly by providing benchmarking and calibration of the computationally efficient and easy-to-use DFT methods.     

Atmospheric conditions during high ragweed pollen concentrations in Zagreb,Croatia

We examined the atmospheric conditions favourable to the occurrence of maximum concentrations of ragweed pollen with an extremely high risk of producing allergy. Over the 2002–2009 period, daily pollen data collected in Zagreb were used to identify two periods of high pollen concentration (> 600 grains/m³) for our analysis: period A (3–4 September 2002) and period B (6–7 September 2003). Synoptic conditions in both periods were very similar: Croatia was under the influence of a lower sector high pressure system moving slowly eastward over Eastern Europe. During the 2002–2009 period, this type of weather pattern (on ~ 70% of days), in conjunction with almost non-gradient surface pressure conditions in the area (on ~ 30% of days) characterised days when the daily pollen concentrations were higher than 400 grains/m³. Numerical experiments using a mesoscale model at fine resolution showed successful multi-day simulations reproducing the local topographic influence on wind flow and in reasonable agreement with available observations. According to the model, the relatively weak synoptic flow (predominantly from the eastern direction) allowed local thermal circulations to develop over Zagreb during both high pollen episodes. Two-hour pollen concentrations and 48-h back-trajectories indicated that regional-range transport of pollen grains from the central Pannonian Plain was the cause of the high pollen concentrations during period A. During period B, the north-westward regional-range transport in Zagreb was supplemented significantly by pronounced horizontal recirculation of pollen grains. This recirculation happened within the diurnal local circulation over the city, causing a late-evening increase in pollen concentration.     

人HCN4通道的滞后现象:影响窦房结起搏的潜在决定因素(英文)

超极化活化环核苷酸门控(hyperpolarization-activated cyclic-nucleotide-gated,HCN)通道参与调制心脏跳动的节律和速率。与HCN1和HCN2有所不同,慢通道HCN4可能不存在电压依赖的滞后现象。本研究采用单细胞膜片钳方法,在稳定转染hHCN4的HEK293细胞上进行电生理记录,观察hHCN4通道是否存在滞后现象,以及cAMP对其的调制作用;同时采用实时定量RT-PCR方法检测窦房结和心房组织中HCNs的表达。电压钳实验结果显示hHCN4电流(Ih)激活随着保持电位超极化的变化而向去极化方向移动。三角电位变化钳(triangular ramp)和动作电位钳的结果也显示了hHCN4的滞后现象。cAMP增加Ih电流幅度,且使电流激活向去极化方向移动,从而改变内源性hHCN4滞后行为。RT-PCR结果显示,人窦房结组织主要表达HCN4,占75%,HCN1占21%,HCN2占3%,HCN3占0.7%。以上结果提示,人窦房结组织主要表达HCN4亚型,hHCN4的Ih存在电压依赖性的滞后现象,且受cAMP调制。由此推断,hHCN4通道的滞后现象可能在窦房结起搏活动中起到了关键作用。     

Structural basis of TRAPPIII‐mediated Rab1 activation

The GTPase Rab1 is a master regulator of the early secretory pathway and is critical for autophagy. Rab1 activation is controlled by its guanine nucleotide exchange factor, the multisubunit TRAPPIII complex. Here, we report the 3.7 Å cryo‐EM structure of the Saccharomyces cerevisiae TRAPPIII complex bound to its substrate Rab1/Ypt1. The structure reveals the binding site for the Rab1/Ypt1 hypervariable domain, leading to a model for how the complex interacts with membranes during the activation reaction. We determined that stable membrane binding by the TRAPPIII complex is required for robust activation of Rab1/Ypt1 in vitro and in vivo, and is mediated by a conserved amphipathic α‐helix within the regulatory Trs85 subunit. Our results show that the Trs85 subunit serves as a membrane anchor, via its amphipathic helix, for the entire TRAPPIII complex. These findings provide a structural understanding of Rab activation on organelle and vesicle membranes.     

Spawning seasons of Rasbora tawarensis (Pisces: Cyprinidae) in Lake Laut Tawar,Aceh Province,Indonesia

Background  

Rasbora tawarensis is an endemic freshwater fish in Lake Laut Tawar, Aceh Province, Indonesia. Unfortunately, its status is regarded as critical endangered with populations decreasing in recent years. To date no information on the spawning activities of the fish are available. Therefore, this study provides a contribution to the knowledge on reproductive biology of R. tawarensis especially on spawning seasons as well as basic information for conservation of the species.