New techniques for membrane protein crystallization tested on photosystem II core complex of Pisum sativum

The crystallization of a given protein is a hard task being even more complicated when the protein shows a hydrophobic behavior. In the case of photosynthetic proteins, the difficulty of the experiments increased due to the high light sensitivity. Aqueous solutions of photosystem II core complex (OEC PSII) of Pisum sativum were screened for crystallization conditions using standard crystallization methods. Crystal improvement was achieved by counter-diffusion technique in single capillaries of 0.2 mm inner diameter with a three-layer configuration. The use of this advanced crystallization technique—for the first time applied to the crystallization of membrane proteins—improves the reproducibility of the experiments allowing the initial crystal characterization, and facilitates the manipulation under light protection.     

Chronic fatigue syndrome is accompanied by an IgM-related immune response directed against neopitopes formed by oxidative or nitrosative damage to lipids and proteins

There is now some evidence that chronic fatigue syndrome (CFS) is accompanied by signs of oxidative stress and by a decreased antioxidant status. The aim of the present study was to examine whether CFS is accompanied by an immune response to neoepitopes of a variety of modified lipids and proteins indicating damage caused by oxidative and nitrosative stress. Toward this end we examined serum antibodies to fatty acids (oleic, palmitic and myristic acid), by-products of lipid peroxidation, i.e. azelaic acid and malondialdehyde (MDA), acetylcholine, S-farnesyl-L-cysteine, and N-oxide modified amino-acids in 14 patients with CFS, 14 subjects with partial CFS and 11 normal controls. We found that the prevalences and mean values for the serum IgM levels directed against oleic, palmitic and myristic acid, MDA, azelaic acid, S-farnesyl-L-cysteine, and the N-oxide derivates, nitro-tyrosine, nitro-phenylalanine, nitro-arginine, nitro-tryptophan, and nitro-cysteinyl were significantly greater in CFS patients than in normal controls, whereas patients with partial CFS took up an intermediate position. There were significant and positive correlations between the serum IgM levels directed against fatty acids, MDA and azelaic acid and the above N-oxide-derivates and the severity of illness (as measured by the FibroFatigue scale) and symptoms, such as aches and pain, muscular tension and fatigue. The results show that CFS is characterized by an IgM-related immune response directed against disrupted lipid membrane components, by-products of lipid peroxidation, S-farnesyl-L-cysteine, and NO-modified amino-acids, which are normally not detected by the immune system but due to oxidative and nitrosative damage have become immunogenic.     

Haemagglutination Induced by Bordetella pertussis Filamentous Haemagglutinin Adhesin (FHA) Is Inhibited by Antibodies Produced Against FHA430–873 Fragment Expressed in Lactobacillus casei

Filamentous haemagglutinin adhesin (FHA) is an important virulence factor from Bordetella pertussis related to the adhesion and spread of the bacteria through the respiratory tract. Three distinct domains have been characterized in mature FHA, and among them, the FHA_442–863 fragment was suggested to be responsible for the heparin-binding activity. In this study, we cloned the gene encoding the HEP fragment (FHA_430–873) in a Lactobacillus casei–inducible expression vector based on the lactose operon. The recombinant bacteria, transformed with the resulting construct (L. casei-HEP), were able to express the heterologous protein depending on the sugar added to the culture. Subcutaneous inoculation of L. casei-HEP in Balb/C mice, using the cholera toxin B subunit as adjuvant, induced systemic anti-HEP antibodies that were able to inhibit in vitro erythrocyte haemagglutination induced by FHA. This is the first example of a B. pertussis antigen produced in lactic acid bacteria and opens new perspectives for alternative vaccine strategies against whooping cough.     

The immunomodulation effect of allogenic blood transfusion in colorectal cancer--a new approach

The total number of 542 patients with colorectal cancer surgery have been analyzed in order to estimate the effect of receiving transfusion local recurrences, and the disease free - survival. It should be examined whether there are changes in general immunity indicators which would be connected with perioperative transfusion. A significant connection has been found between local recurrences and blood transfusion (p<0.0001), the most noticeable being in Dukes A (p =0.045), localization on rectum (p=0.036). The receiving of blood transfusion is linked significantly with disease free - survival reduction (p =0.0068; log rank), the most significant being in Dukes A stage (p =0.0123; log rank) and with localization on rectum (p=0.0231). The analysis of general immunity indicators has shown significant immunocompromitation of patients just before the surgery and this could have effect on immunomodulation caused by transfusion and just as on the treatment prognosis of colorectal carcinoma.     

The notochordal sheath in amphioxus--an ultrastructural and histochemical study

The notochord and notochordal sheath of 10 adult amphioxus were investigated ultrastructurally and histochemically. The notochord in amphioxus consists of parallel notochordal cells (plates) and each plate consists of parallel thicker and thinner fibrils and numerous profiles of smooth endoplasmic reticulum situated just beneath the cell membrane. Histochemical staining shows that the notochordal plates resemble neither the connective tissue notochordal sheath nor the typical muscular structure myotomes. The notochordal sheath has a complex three-layered organization with the outer, middle and inner layer The outer and middle layer are composed of collagen fibers of different thickness and course, that correspond to collagen type I and collagen type III in vertebrates, respectively, and the inner layer is amorphous, resembles basal lamina, and is closely attached to the notochord by hemidesmosome junctions. These results confirm the presence of collagen fibers and absence of elastic fibers in amphioxus.     

Variations in mugwort (Artemisia spp.) airborne pollen concentrations at three sites in central Croatia, in period from 2002 to 2003

In spite of the low atmospheric pollen levels, Artemisia sensitisation and allergy has been reported widely. The aim of the study was to determine the length of pollen season, intradiurnal, daily and monthly pollen variation, and the effect of some meteorological parameters on atmospheric pollen concentrations in Central Croatia. Seven-day Hirst volumetric pollen and spore traps were used for pollen sampling. The Artemisia pollen season lasted from the end of July until the end of September with the highest concentrations in August. The percentage of the total pollen count ranged from 0.52% to 0.92%. The intradiurnal peak occurred between 10 a.m. and 12 a.m. Statistical analysis showed a significant correlations between higher air temperature and high pollen concentration as well as high precipitation and low pollen concentration. Results of this study are expected to help in preventing the symptoms of allergic reaction in individuals with Artemisia pollen hypersensitivity.     

Measurement of tooth extraction forces in upper incisors

A new method, applied for the first time in this research, was used for measurement of tooth extraction forces. The research has been done in a group of 50 examinees to whom the tooth extraction has been done with lower premolar forceps - forceps "13" and in the control group of 54 examinees in whom the tooth extraction has been done with upper incisive forceps - forceps "1". The measurement instrument registered the extraction forces values in both types of forceps. There was no difference in any parameters in these two groups except in used pressure. While using the forceps "13", a significantly lower tooth extraction force was measured than the force measured while using the forceps "1" (p < 0.001). This means that in clinical work we can already apply noticeably less force using the lower premolar forceps for the extraction of the upper incisors (in the moments of rotation up to 70%). These results are meaningful, because they lead to better and improved instrument solutions and working techniques.     

Influence of the temperature regime on the composition of the macrozoobenthos community in a thermal brook in Serbia

In contrast to cold and eurythermal waters, benthic communities of warm brooks in temperate regions have been inadequately studied. In order to investigate the effects of water thermal regime on the benthic communities of warm waters and their relationships with those of cold and eurythermic ones, the macrozoobenthos was studied at eight sites in the Toplica River, and at four sites in its tributary, the Termalni brook. Investigations were carried out seasonally from April 2000 to January 2001. Warm waters of the Termalni brook were characterized by specific macrozoobenthos assemblages that exhibited significant differences to the populations of eurythermal and cold waters of the Toplica River. The dominant taxa in the macrozoobenthos community of warm waters were mainly Gastropoda species. Moreover, benthic communities of warm waters were characterized by lower diversity and greater biomass in comparison with those of cold and eurythermal waters. The gradient of average annual temperatures represented the main ecological factor influencing changes of diversity and biomass along the course of the investigated Termalni brook. Inflow of warm waters at site T₆ lead to a decrease in macrozoobenthos abundance and changes in qualitative and quantitative composition of the benthocoenosis of a highland stream, but did not significantly alter diversity.     

Immunohistochemical study of cytoskeletal and extracellular matrix components in the notochord and notochordal sheath of amphioxus

A major cytoskeletal and extracellular matrix proteins of the amphioxus notochordal cells and sheath were detected by immunohistochemical techniques. The three-layered amphioxus notochordal sheath strongly expressed fish collagen type I in its outer and middle layers, while in the innermost layer expression did not occur. The amphioxus notochordal sheath was reactive to applied anti-human antibodies for intermediate filament proteins such as cytokeratins, desmin and vimentin, as well as to microtubule components (beta-tubulin), particularly in the area close to the epipharyngeal groove. Alpha-smooth muscle actin was expressed in some notochordal cells and in the area of the notochordal attachment to the sheath. Thus muscular nature of notochordal cells was shown by immunohistochemistry in tissue section. Our results confirm that genes encoding intermediate filament proteins, microtubules and microfilaments are highly conserved during evolution. Collagen type I was proven to be the key extracellular matrix protein that forms the amphioxus notochordal sheath.