Expression of protein complexes and individual proteins upon transition of etioplasts to chloroplasts in pea (Pisum sativum)

The protein complexes of pea (Pisum sativum L.) etioplasts,etio-chloroplasts and chloroplasts were examined using 2D BlueNative/SDS–PAGE. The most prominent protein complexesin etioplasts were the ATPase and the Clp and FtsH proteasecomplexes which probably have a crucial role in the biogenesisof etioplasts and chloroplasts. Also the cytochrome b₆f (Cytb₆f) complex was assembled in the etioplast membrane, as wellas Rubisco, at least partially, in the stroma. These complexesare composed of proteins encoded by both the plastid and nucleargenomes, indicating that a functional cross-talk exists betweenpea etioplasts and the nucleus. In contrast, the proteins andprotein complexes that bind chlorophyll, with the PetD subunitand the entire Cyt b₆f complex as an exception, did not accumulatein etioplasts. Nevertheless, some PSII core components suchas PsbE and the luminal oxygen-evolvong complex (OEC) proteinsPsbO and PsbP accumulated efficiently in etioplasts. After 6h de-etiolation, a complete PSII core complex appeared with40% of the maximal photochemical efficiency, but a fully functionalPSII was recorded only after 24 h illumination. Similarly, thecore complex of PSI was assembled after 6 h illumination, whereasthe PSI–light-harvesting complex I was stably assembledonly in chloroplasts illuminated for 24 h. Moreover, a batteryof proteins responsible for defense against oxidative stressaccumulated particularly in etioplasts, including the stromaland thylakoidal forms of ascorbate peroxidase, glutathione reductaseand PsbS.     

Detecting small changes and additional peptides in the canine parvovirus capsid structure

Parvovirus capsids are assembled from multiple forms of a single protein and are quite stable structurally. However, in order to infect cells, conformational plasticity of the capsid is required and this likely involves the exposure of structures that are buried within the structural models. The presence of functional asymmetry in the otherwise icosahedral capsid has also been proposed. Here we examined the protein composition of canine parvovirus capsids and evaluated their structural variation and permeability by protease sensitivity, spectrofluorometry, and negative staining electron microscopy. Additional protein forms identified included an apparent smaller variant of the virus protein 1 (VP1) and a small proportion of a cleaved form of VP2. Only a small percentage of the proteins in intact capsids were cleaved by any of the proteases tested. The capsid susceptibility to proteolysis varied with temperature but new cleavages were not revealed. No global change in the capsid structure was observed by analysis of Trp fluorescence when capsids were heated between 40 degrees C and 60 degrees C. However, increased polarity of empty capsids was indicated by bis-ANS binding, something not seen for DNA-containing capsids. Removal of calcium with EGTA or exposure to pHs as low as 5.0 had little effect on the structure, but at pH 4.0 changes were revealed by proteinase K digestion. Exposure of viral DNA to the external environment started above 50 degrees C. Some negative stains showed increased permeability of empty capsids at higher temperatures, but no effects were seen after EGTA treatment.     

Leukocyte Adhesion—an Integrated Molecular Process at the Leukocyte Plasma Membrane

Leukocyte adhesion is of pivotal functional importance, because most leukocyte functions depend on cell–cell contact. It must be strictly controlled, both at the level of specificity and strength of interaction, and therefore several molecular systems are involved. The most important leukocyte adhesion molecules are the selectins, the leukocyte-specific ₂-integrins and the intercellular adhesion molecules. The selectins induce an initial weak contact between cells, whereas firm adhesion is achieved through integrin–intercellular adhesion molecular binding. Although studies during the past twenty years have revealed several important features of leukocyte adhesion much is still poorly understood, and further work dealing with several aspects of adhesion is urgently needed. In this short essay, we review some recent developments in the field.     

Molecular evolution of aromatic polyketides and comparative sequence analysis of polyketide ketosynthase and 16S ribosomal DNA genes from various streptomyces species

A 613-bp fragment of an essential ketosynthase gene from the biosynthetic pathway of aromatic polyketide antibiotics was sequenced from 99 actinomycetes isolated from soil. Phylogenetic analysis showed that the isolates clustered into clades that correspond to the various classes of aromatic polyketides. Additionally, sequencing of a 120-bp fragment from the gamma-variable region of 16S ribosomal DNA (rDNA) and subsequent comparative sequence analysis revealed incongruity between the ketosynthase and 16S rDNA phylogenetic trees, which strongly suggests that there has been horizontal transfer of aromatic polyketide biosynthesis genes. The results show that the ketosynthase tree could be used for DNA fingerprinting of secondary metabolites and for screening interesting aromatic polyketide biosynthesis genes. Furthermore, the movement of the ketosynthase genes suggests that traditional marker molecules like 16S rDNA give misleading information about the biosynthesis potential of aromatic polyketides, and thus only molecules that are directly involved in the biosynthesis of secondary metabolites can be used to gain information about the biodiversity of antibiotic production in different actinomycetes.     

Effect of long-term leisure time physical activity on lean mass and fat mass in girls during adolescence

The purpose of this 7-yr prospective longitudinal study was to examine if the level and consistency of leisure-time physical activity (LTPA) during adolescence affected the quantity and distribution of lean mass (LM) and fat mass (FM) at early adulthood. The study subjects were 202 Finnish girls who were 10-13 yr old at baseline. LM and FM of the total body (TB), arms, legs, and trunk were assessed by dual-energy X-ray absorptiometry. Muscle cross-sectional area (mCSA) of the left leg was assessed by peripheral quantitative computed tomography. Scores of LTPA were obtained by questionnaire. Girls were divided into four groups comprising those with consistently low (G(LL)) or consistently high (G(HH)) physical activity, or those whose physical activity changed from low to high (G(LH)), or from high to low (G(HL)), over the 7 yr of follow-up. At baseline, no differences were found in LM, FM, and FM% among the groups in any of the body segments. By the end of the study G(HH) and G(LH) had higher values of LM of the TB, arms, legs, and trunk than that of the G(HL) and G(LL) groups (P < 0.05, respectively). High FM% of the TB was associated with low level of LTPA, but no significant differences were found in the absolute amount of FM and mCSA among the LTPA groups. Our results suggest that a consistently high level of LTPA during the transition from prepuberty to early adulthood has a positive effect on lean mass gain in girls. Participating in 5 h of LTPA per week had a significant effect on FM% but not on the absolute amount of fat mass.     

Additive effects of enhanced ambient ultraviolet B radiation and increased temperature on immune function, growth and physiological condition of juvenile (parr) Atlantic Salmon, Salmo salar

Climate change models predict increased ultraviolet B (UVB) radiation levels due to stratospheric ozone depletion and global warming. In order to study the impact of these two environmental stressors acting simultaneously on the physiology of fish, Atlantic salmon parr were exposed, for 8 weeks in outdoor tanks, to different combinations of UVB radiation (depleted and enhanced) and temperature (standard rearing temperature of 14 °C or 19 °C). The immune function (plasma IgM, lysozyme activity and complement bacteriolytic activity), growth (body weight) and physiological condition (haematocrit and plasma protein concentration) of the fish were determined. Increased UVB level, regardless of water temperature, had a negative effect on immune function parameters, growth and physiological condition. Higher temperature increased plasma IgM concentration but had a negative effect on complement bacteriolytic activity under both spectral treatments. Increased temperature, irrespective of UVB level, increased fish growth but negatively affected haematocrit and plasma protein. Exposing the fish to enhanced UVB at elevated temperature increased plasma IgM concentration and slightly improved growth. However, complement activity and physiological condition parameters decreased more than when the fish were exposed to each stressor separately. The changes were mainly additive; no interactive or synergistic effects were observed. The negative impact of multiple stressors on immune function, together with predicted increases in pathogen load in warmer waters resulting from global climate change, suggest an increased risk to diseases in fishes.     

Pre-expression of a sulfhydryl oxidase significantly increases the yields of eukaryotic disulfide bond containing proteins expressed in the cytoplasm of E.coli

Background  

Disulfide bonds are one of the most common post-translational modifications found in proteins. The production of proteins that contain native disulfide bonds is challenging, especially on a large scale. Either the protein needs to be targeted to the endoplasmic reticulum in eukaryotes or to the prokaryotic periplasm. These compartments that are specialised for disulfide bond formation have an active catalyst for their formation, along with catalysts for isomerization to the native state. We have recently shown that it is possible to produce large amounts of prokaryotic disulfide bond containing proteins in the cytoplasm of wild-type bacteria such as E. coli by the introduction of catalysts for both of these processes.     

Establishment of Lipofection for Studying miRNA Function in Human Adipocytes

miRNA dysregulation has recently been linked to human obesity and its related complications such as type 2 diabetes. In order to study miRNA function in human adipocytes, we aimed for the modulation of mature miRNA concentration in these cells. Adipocytes, however, tend to be resistant to transfection and there is often a need to resort to viral transduction or electroporation. Our objective therefore was to identify an efficient, non-viral transfection reagent capable of delivering small RNAs into these cells. To achieve this, we compared the efficiencies of three transfection agents, Lipofectamine 2000, ScreenFect A and BPEI 1.2 k in delivering fluorescent-labelled siRNA into human Simpson-Golabi-Behmel syndrome (SGBS) preadipocytes and adipocytes. Downregulation of a specific target gene in response to miRNA mimic overexpression was assayed in SGBS cells and also in ex vivo differentiated primary human adipocytes. Our results demonstrated that while all three transfection agents were able to internalize the oligos, only lipofection resulted in the efficient downregulation of a specific target gene both in SGBS cells and in primary human adipocytes. Lipofectamine 2000 outperformed ScreenFect A in preadipocytes, but in adipocytes the two reagents gave comparable results making ScreenFect A a notable new alternative for the gold standard Lipofectamine 2000.