Secretion of the Hormoconis resinae glucoamylase P enzyme from Trichoderma reesei directed by the natural and the cbh1 gene secretion signal

Abstract We have isolated two alkaline phosphatases (H-AP and L-AP, for high and low molecular mass, respectively) from Pseudomonas aeruginosa PA01. These two enzymes were found to differ in mobility on sodium dodecyl sulphate polyacrylamide gels (H-AP, M _r = 51 000 and L-AP, M _r = 39 500), amino-terminal amino acid sequence and did not cross-react. Both enzymes were active as phosphomonoesterases while only L-AP demonstrated any phosphodiesterase activity. Both enzymes were purified from P. aeruginosa grown in phosphate limiting conditions using the same protocol and were identified in both periplasmic and extracellular locations. A low level of H-AP was produced constitutively whereas L-AP was produced only after induction by reduced phosphate concentration in the growth medium. An L-AP-like enzyme has been previously described, however, this is the first report of a second P. aeruginosa alkaline phosphatase.     

Use of Very Low-Calorie Diet in Preoperative Weight Loss: Efficacy and Safety

PEKKARINEN, TUULA, PERTTI MUSTAJOKI. Use of very low-calorie diet in preoperative weight loss: Efficacy and Safety. We report the efficacy of a very low-calorie diet (VLCD)-based weight reduction program in patients with morbid obesity whose elective surgery had been postponed because of being overweight. The safety of weight loss on the immune system will also be evaluated. Thirty patients (mean age, 50 years; weight, 125 kg; BMI, 44 kg/m2) were treated. The program consisted of a 7-week to 24-week VLCD period, supported by individual sessions with a therapist, and of a refeeding period of 1 month before surgery. Two patients discontinued, and the mean weight loss of the remaining 28 patients was 19. 6 kg (15% of initial weight). In 23 patients, weight loss was 10% or more of the initial weight. After weight loss, 15 patients underwent surgery, 4 patients did not need an operation, and the remaining 9 patients were not operated on for various reasons. The numbers of circulating leukocytes, neutrophils, basophils, monocytes, CD3+, CD4+, CD8+, and natural killer cells did not change significantly by the ninth week on VLCD or by the end of the program. However, there was a significant (p<0. 05) decrease in the immunoglobulinM serum concentration during the program. In conclusion, a VLCD program is suitable for preoperative weight reduction in morbid obesity and seems not to compromise the immune system.     

Assessing product environmental performance with PEF methodology: reliability,comparability, and cost concerns

Purpose

The Product Environmental Footprint (PEF) method was launched by the European Commission to harmonize the measurement of the product’s environmental performance. The PEF aims to increase the supply of green products in EU markets by lowering the cost of measuring the product’s environmental performance, enabling reliable environmental claims, and conducting proper product comparison. This study assesses whether the methodological choices of PEF enable reliable and comparable PEF studies to be conducted in a cost-efficient way.

Methods

The PEF was compared with ISO 14044, ISO/TS 14067, and the Greenhouse Gas Protocol Product Standard to identify the differences in the methodological choices. From a practical perspective, the possible challenges for PEF that could not be identified by a method comparison were obtained via interviews. The interviewees were company representatives participating in a 3-year PEF pilot phase to create Product Environmental Footprint Category Rules (PEFCR). To discover the possible advantages and disadvantages of the PEF, the methodological choices for PEF itself were assessed from the standpoints of reliability, comparability, and cost. In conclusion, some proposals for improvements have been suggested.

Results and discussion

The impact assessment phase, i.e., some of the predefined impact assessment methods, together with a selected normalization method include disadvantages as regards reliability and comparability. They are not sufficiently mature to properly reflect both the product studied and its potential environmental impact. From a cost perspective, the inventory analysis phase increases the workload due to the cutoff ban, the numerous predefined impact assessment categories, and the data quality assessment of all data. However, the predefined issues, e.g., requirements for data quality to be determined in PEFCRs, could also increase the comparability and lower the costs of the PEF study, since these need not be determined separately for each study. Additionally, the reliability maybe increased due to the data quality assessment of all data.

Conclusions

Currently, in the PEFs 3-year pilot phase, both advantages and disadvantages exist as regards reliability, costs, and comparability. Since PEF aims are important, the Commission and PEFCR developers should devote time to finding the most appropriate methodological choices and continue developing the method further. It is important to find a balance between comparability, reliability, and costs. Additionally, the current issues and characteristics of EU green products market should be taken into account when implementing PEF.

     

Morphological Differentiation Towards Neuronal Phenotype of SH-SY5Y Neuroblastoma Cells by Estradiol,Retinoic Acid and Cholesterol

Human SH-SY5Y neuroblastoma cells maintain their potential for differentiation and regression in culture conditions. The induction of differentiation could serve as a strategy to inhibit cell proliferation and tumor growth. Previous studies have shown that differentiation of SH-SY5Y cells can be induced by all-trans-retinoic-acid (RA) and cholesterol (CHOL). However, signaling pathways that lead to terminal differentiation of SH-SY5Y cells are still largely unknown. The goal of this study was to examine in the RA and CHOL treated SH-SY5Y cells the additive impacts of estradiol (E₂) and brain-derived neurotrophic factor (BDNF) on cell morphology, cell population growth, synaptic vesicle recycling and presence of neurofilaments. The above features indicate a higher level of neuronal differentiation. Our data show that treatment for 10 days in vitro (DIV) with RA alone or when combined with E₂ (RE) or CHOL (RC), but not when combined with BDNF (RB), significantly (p < 0.01) inhibited the cell population growth. Synaptic vesicle recycling, induced by high-K⁺ depolarization, was significantly increased in all treatments where RA was included (RE, RC, RB, RCB), and when all agents were added together (RCBE). Specifically, our results show for the first time that E₂ treatment can alone increase synaptic vesicle recycling in SH-SY5Y cells. This work contributes to the understanding of the ways to improve suppression of neuroblastoma cells’ population growth by inducing maturation and differentiation.     

Associative Nitrogen Fixation by Klebsiella spp.: Adhesion Sites and Inoculation Effects on Grass Roots

Adhesion sites on grass roots for Klebsiella strains carrying type 3 or type 1 fimbriae or both were determined. Adhesion of the strains to the roots of Poa pratensis and Festuca rubra was highly localized; the bacteria adhered strongly to root hairs and with a markedly lower efficiency to the surface of the zone of elongation and to the root cap mucilage. No adhesion to the epidermal cells between root hairs was observed. The adhesion sites were identical for the type 3- and 1-fimbriated bacteria and for P. pratensis, F. rubra, and Trifolium pratense. Inoculation of P. pratensis seedlings with Klebsiella pneumoniae strain As resulted in morphological changes in plant roots. The roots of infected plants were heavily covered with root hairs, which often were deformed and branched.     

Root-Associated N2 Fixation (Acetylene Reduction) by Enterobacteriaceae and Azospirillum Strains in Cold-Climate Spodosols

N₂ fixation by bacteria in associative symbiosis with washed roots of 13 Poaceae and 8 other noncultivated plant species in Finland was demonstrated by the acetylene reduction method. The roots most active in C₂H₂ reduction were those of Agrostis stolonifera, Calamagrostis lanceolata, Elytrigia repens, and Phalaris arundinacea, which produced 538 to 1,510 nmol of C₂H₄·g⁻¹ (dry weight)· h⁻¹ when incubated at pO₂ 0.04 with sucrose (pH 6.5), and 70 to 269 nmol of C₂H₄· g⁻¹ (dry weight)·h⁻¹ without an added energy source and unbuffered. Azospirillum lipferum, Enterobacter agglomerans, Klebsiella pneumoniae, and a Pseudomonas sp. were the acetylene-reducing organisms isolated. The results demonstrate the presence of N₂-fixing organisms in associative symbiosis with plant roots found in a northern climatic region in acidic soils ranging down to pH 4.0.     

Human Saliva-Derived Exosomes: Comparing Methods of Isolation

ExoQuick-TC^TM (EQ), a chemical-based agent designed to precipitate exosomes, was calibrated for use on saliva collected from healthy individuals. The morphological and molecular features of the precipitations were compared with those obtained using the classical, physical-based method of ultracentrifugation (UC). Electron microscopy and immunoelectron microscopy with anti-CD63 showed vesicular nanoparticles surrounded by bi-layered membrane, compatible with exosomes in EQ, similar to that observed with UC. Atomic force microscopy highlighted larger, irregularly shaped/aggregated EQ nanoparticles that contrasted with the single, round-shaped UC nanoparticles. ELISA (performed on 0.5 ml of saliva) revealed a tendency for a higher expression of the specific exosomal markers (CD63, CD9, CD81) in EQ than in UC (p>0.05). ELISA for epithelial growth factor receptor, a non-exosomal-related marker, showed a significantly higher concentration in EQ than in UC (p=0.04). Western blotting of equal total-protein concentrations revealed bands of CD63, CD9 and CD81 in both types of preparations, although they were less pronounced in EQ compared with UC. This may be related to a higher fraction of non-exosomal proteins in EQ. In conclusion, EQ is suitable and efficient for precipitation of salivary exosomes from small volumes of saliva; however, EQ tends to be associated with considerably more biological impurities (non-exosomal-related proteins/microvesicles) as compared with UC.     

Glucoamylase P gene of Hormoconis resinae: Molecular cloning, sequencing and introduction into Trichoderma reesei

The glucoamylase P gene of the fungus Hormoconis resinae has been cloned and sequenced from a genomic library. The gene consists of a 2153-bp protein coding region including three introns. The usual number of introns in cloned fungal glucoamylase genes has been four and in some cases five. Two of the glucoamylase P gene introns contain a sequence resembling the consensus sequence found near the 3' splice site in the introns of the fungus Trichoderma reesei cellobiohydrolase 1 (cbh1) gene. The H. resinae glucoamylase P gene, under its own promoter, was introduced into T. reesei, but no expression could be detected.