Improved Production of Heterologous Proteins by a Glucose Repression-Defective Mutant of Kluyveromyces lactis

The secreted production of heterologous proteins in Kluyveromyces lactis was studied. A glucoamylase (GAA) from the yeast Arxula adeninivorans was used as a reporter protein for the study of the secretion efficiencies of several wild-type and mutant strains of K. lactis. The expression of the reporter protein was placed under the control of the strong promoter of the glyceraldehyde-3-phosphate dehydrogenase of Saccharomyces cerevisiae. Among the laboratory strains tested, strain JA6 was the best producer of GAA. Since this strain is known to be highly sensitive to glucose repression and since this is an undesired trait for biomass-oriented applications, we examined heterologous protein production by using glucose repression-defective mutants isolated from this strain. One of them, a mutant carrying a dgr151-1 mutation, showed a significantly improved capability of producing heterologous proteins such as GAA, human serum albumin, and human interleukin-1β compared to the parent strain. dgr151-1 is an allele of RAG5, the gene encoding the only hexokinase present in K. lactis (a homologue of S. cerevisiae HXK2). The mutation in this strain was mapped to nucleotide position +527, resulting in a change from glycine to aspartic acid within the highly conserved kinase domain. Cells carrying the dgr151-1 allele also showed a reduction in N- and O-glycosylation. Therefore, the dgr151 strain may be a promising host for the production of heterologous proteins, especially when the hyperglycosylation of recombinant proteins must be avoided.     

Impaired carotenogenesis can affect organization and functionality of etioplast membranes

The effects of impaired carotenogenesis on plastid membrane organization, functionality and stability were studied in etiolated barley plants grown at 20 and 30°C. The plants were treated with norflurazon or amitrole, two herbicides affecting phytoene desaturation and lycopene cyclization, respectively. At 20°C, the amitrole-treated etioplasts, which accumulated lycopene in their inner membranes, exhibited disorganized prolamellar bodies, containing a prevalent form of non-phototransformable protochlorophyllide (Pchlide). They also showed a certain difficulty in reducing the phototransformable pigment to chlorophyllide when exposed to light, and were unable to reform the active ternary complex [protochlorophyllide–oxidoreductase (POR)–Pchlide–NADPH] when placed back in darkness. No ultrastructural alterations were found in norflurazon-treated etioplasts, with carotenogenesis inhibited at the phytoene desaturation step. In these latter organelles, Pchlide, whose forms were comparable with those of the control etioplasts, was photoreduced quickly after illumination and the ternary complex was reformed during a subsequent dark period. Thus, the impaired carotenogenesis leading to the accumulation of lycopene showed greater interference with the etioplast membrane arrangement and functionality than did the earlier interruption of the biosynthetic pathway at the phytoene level. This might be due to the different interactions of the distinct carotenoid precursors with other membrane components. However, in etioplasts of norflurazon-treated plants, a rise in growth temperature caused a partial demolition of prolamellar bodies, showing a lowered thermostability of the carotenoid-deficient membranes. This latter effect strengthens the concept that a correct and complete carotenogenesis pathway, leading to the synthesis of polar carotenoids (i.e. xanthophylls), is required for the maintenance of stable plastid membranes.     

Vocal communication in the bicoloured white-toothed shrew<Emphasis Type="Italic">Crocidura leucodon</Emphasis>

Vocal sounds of 15 individuals ofCrocidura leucodon (Hermann, 1780) emitted during agonistic and amicable interactions in male-male, female-female and male-female dyadic encounters, and intraspecific cage groups, were studied. An analysis of spectral properties, along with an examination of context and function, were performed. The sounds registered during agonistic interactions showed an increase, then a decrease in frequency, often followed a complex undulating curve. The dominant frequency (DF) was 10.7±0.05 kHz, and duration (DU) was 104.2±4.4 ms. The calls emitted by shrews at investigation and grooming often ended with chirping notes (DF=4.8±0.2 kHz, DU=23.5±1.1 ms). While clustering and huddling up to the partner, the animals produced calls consisting of short notes with a very low intensity (DF=1.0±0.07 kHz, DU=35.2±0.8 ms). Based on these finding, it can be assumed that threatening sounds, emitted in agonistic encounters, allow shrews to avoid conflicts, while those, emitted when clustering and huddling up to the partner, contribute to maintenance of group cohesiveness. The variability of frequency ranges and intensity of sounds probably reflects the territorial and gregarious behaviour ofC. leucodon and adaptation to communication in variable acoustic environments.     

Relationships between blood pressure, polymorphism of angiotensin-converting enzyme (ACE), body composition and biochemical characteristics in elderly Slovaks

Epidemiological studies have demonstrated that several specific environmental factors and candidate genes influence the human variation in blood pressure. The aim of this study was to investigate variables associated with blood pressure; with a particular emphasis on the differences in insertion/deletion (I/D) polymorphism of the human angiotensin-converting enzyme (ACE), the body composition and the recognized risk factors for atherosclerosis among elderly males and females. A total of 374 participants (174 males and 200 females) aged from 60 to 90 years were recruited from different parts of Slovakia. The elderly were not bed-ridden, nor mentally impaired, they were able to manage their daily activities by themselves. The ACE I/D polymorphism was determined by PCR amplification of the ACE gene sequence. Body composition variables were obtained by bioelectrical impedance analysis, using the BIA 101 soft tissue-body impedance analyzer (Akern, S.r.l.). The subjects were determined to be hypertensive (blood pressure > or = 140/90 mm Hg) or normotensive (blood pressure < or = 140/90 mm Hg ). These two subgroups of males and females did not differ significantly in their mean ages. As expected, the hypertensive subjects of both sexes showed significantly higher mean values in systolic (SBP) and diastolic blood pressure (DBP), in body mass index (BMI), and in the mean values of their plasma glucose and extracellular water (ECW). The genotype distribution and allele frequencies in the whole sample (D = 0.5474, I = 0.4526) fell within the Hardy-Weinberg equilibrium. The frequency of the deleterious D allele in the normotensive (0.5532) and hypertensive (0.5516) subjects was not significantly different. The ACE I/D genotypes did not associate either with the systolic (p = 0.836) or diastolic BP (p = 0.629). From the other variables that may induce differences in blood pressure, a statistical effect was detected for glucose, Na/K, and Apo A1/ApoB ratios and physical activity on SBP, and for ApoA1, physical activity, BMI and total cholesterol on DBP.     

Web Proxy Acceleration

Numerous studies show that miss ratios at forward proxies are typically at least 40–50%. This paper proposes and evaluates a new approach for improving the throughput of Web proxy systems by reducing the overhead of handling cache misses. Namely, we propose to front-end a Web proxy with a high performance node that filters the requests, processing the misses and forwarding the hits and the new cacheable content to the proxy. Requests are filtered based on hints of the proxy cache content. This system, called Proxy Accelerator, achieves significantly better communications performance than a traditional proxy system. For instance, an accelerator can be built as an embedded system optimized for communication and HTTP processing, or as a kernel-mode HTTP server. Scalability with the Web proxy cluster size is achieved by using several accelerators. We use analytical models, trace-based simulations, and a real implementation to study the benefits and the implementation tradeoffs of this new approach. Our results show that a single proxy accelerator node in front of a 4-node Web proxy can improve the cost-performance ratio by about 40%. Hint-based request filter implementation choices that do not affect the overall hit ratio are available. An implementation of the hint management module integrated in Web proxy software is presented. Experimental evaluation of the implementation demonstrates that the associated overheads are very small.     

High-performance liquid chromatographic analysis for the determination of a novel polymer-bound camptothecin derivative (MAG-camptothecin) and free camptothecin in human plasma

A selective HPLC assay is described for the determination of free and total (free plus polymer-bound) camptothecin (CPT) in human plasma after administration of the anti-tumor drug MAG-CPT (polymer bound camptothecin). Total CPT levels were determined after hydrolysis and free CPT was extracted from acidified plasma using Oasis solid-phase extraction material. Extracts were analyzed on a Zorbax SB-C₈ analytical column, using a mixture of acetonitrile–25 mM phosphate buffer (pH 4.0) as the eluent. Detection was performed fluorimetrically. Concentrations of polymer-bound CPT were calculated by subtraction of free from total CPT. The lower limits of quantitation of the methods were 100 ng/ml for total and 1.0 ng/ml for free CPT using 50 μl and 250 μl plasma, respectively. Special attention was paid to the stability of the analytes. The presented method was successfully applied in a clinical pharmacokinetic study in our institute.     

The pro-oxidant role of protein SH groups of hemoglobin in rat erythrocytes exposed to menadione.

Menadione is selectively toxic to erythrocytes. Although GSH is considered a primary target of menadione, intraerythrocyte thiolic alterations consequent to menadione exposure are only partially known. In this study alterations of GSH and protein thiols (PSH) and their relationship with methemoglobin formation were investigated in human and rat red blood cells (RBC) exposed to menadione. In both erythrocyte types, menadione caused a marked increase in methemoglobin associated with GSH depletion and increased oxygen consumption. However, in human RBC, GSH formed a conjugate with menadione, whereas, in rat RBC it was converted to GSSG, concomitantly with a loss of protein thiols (corresponding to menadione arylation), and an increase in glutathione-protein mixed disulfides (GS-SP). Such differences were related to the presence of highly reactive cysteines, which characterize rat hemoglobin (cys beta125). In spite of the greater thiol oxidation in rat than in human RBC, methemoglobin formation and the rate of oxygen consumption elicited by menadione in both species were rather similar. Moreover, in repeated experiments under N2 or CO-blocked heme, it was found that menadione conjugation (arylation) in both species was not dependent on the presence of oxygen or the status of heme. Therefore, we assumed that GSH (human RBC) and protein (rat RBC) arylation was equally responsible for increased oxygen consumption and Hb oxidation. Moreover, thiol oxidation of rat RBC was strictly related to methemoglobin formation.     

Farmers’ genetic perceptions regarding their crop populations: An example with maize in the central valleys of Oaxaca, Mexico

Collaborative plant breeding is an approach to crop improvement that includes close attention to specific adaptation and interaction between farmers and formal plant breeders to better meet the needs of those farmers. Collegial interaction capable of making best use of the knowledge and skills of farmers and breeders will depend upon an understanding of those in terms that are relevant to each. To facilitate this interaction with the goal of making farmer selection practices more effective, the work described here sought to improve outside researchers’ understanding of farmers’ fundamental perceptions about their populations, growing environments, and expectations for response to selection. Various methods were used to accomplish this with a small sample of maize farmers in two communities in the Central Valleys of Oaxaca, Mexico. Farmers’ decisions about maize varietal type repertoires imply assessments based on genetic and environmental variation in the local context. A clear distinction was made between traits of high and low heritability and expected response to selection, however, some traits of interest to farmers such as large seed size may involve considerations other than their potential for expression in the progeny generation.