Carbonic anhydrase activity in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> thylakoid membrane and fragments enriched with PSI or PSII

The procedure of isolating the thylakoids and the thylakoid membrane fragments enriched with either photosystem I or photosystem II (PSI- and PSII-membranes) from Arabidopsis thaliana leaves was developed. It differed from the one used with pea and spinach in durations of detergent treatment and centrifugation, and in concentrations of detergent and Mg²⁺ in the media. Both the thylakoid and the fragments preserved carbonic anhydrase (CA) activities. Using nondenaturing electrophoresis followed by detection of CA activity in the gel stained with bromo thymol blue, one low molecular mass carrier of CA activity was found in the PSI-membranes, and two carriers, a low molecular mass one and a high molecular mass one, were found in the PSII-membranes. The proteins in the PSII-membranes differed in their sensitivity to acetazolamide (AA), a specific CA inhibitor. AA at 5 × 10⁻⁷ M inhibited the CA activity of the high molecular mass protein but stimulated the activity of the low molecular mass carrier in the PSII-membranes. At the same concentration, AA moderately inhibited, by 30%, the CA activity of PSI-membranes. CA activity of the PSII-membranes was almost completely suppressed by the lipophilic CA inhibitor, ethoxyzolamide at 10⁻⁹ M, whereas CA activity of the PSI-membranes was inhibited by this inhibitor even at 5 × 10⁻⁷ M just the same as for AA. The observed distribution of CA activity in the thylakoid membranes from A. thaliana was close to the one found in the membranes of pea, evidencing the general pattern of CA activity in the thylakoid membranes of C3-plants.     

Species composition of Bacteroidales order bacteria in the feces of healthy people of various ages

A study of species distribution of numerically predominant Bacteroidales order isolates in feces of healthy people aged 1-33 years was accomplished using a combination of amplified ribosomal DNA restriction analysis (ARDRA) and 16S ribosomal DNA (rDNA) sequencing. It was found that the majority of isolates in all age groups belonged to species B. xylanisolvens, B. vulgatus, and B. uniformis. Members of genera Alistipes, Parabacteroides, Odoribacter, Barnesiella, and Prevotella were also detected frequently.     

Abrogated RANKL expression in properdin-deficient mice is associated with better outcome from collagen-antibody-induced arthritis

Introduction

Properdin amplifies the alternative pathway of complement activation. In the present study, we evaluated its role in the development of collagen antibody-induced arthritis (CAIA).

Methods

Arthritis was induced by intraperitoneal injection of a collagen antibody cocktail into properdin-deficient (KO) and wild-type (WT) C57BL/6 mice. Symptoms of disease were evaluated daily. The degree of joint damage was assessed histologically and with immunostaining for bone-resorption markers. Phenotypes of cell populations, their receptor expression, and intracellular cytokine production were determined with flow cytometry. Osteoclast differentiation of bone marrow (BM) precursors was evaluated by staining for tartrate-resistant acid phosphatase (TRAP).

Results

Properdin-deficient mice developed less severe CAIA than did WT mice. They showed significantly improved clinical scores and downregulated expression of bone-resorption markers in the joints at day 10 of disease. The frequencies of Ly6G⁺CD11b⁺ cells were fewer in BM, blood, and synovial fluid (SF) of KO than of WT CAIA mice. The receptor activator of nuclear factor κB ligand (RANKL) was downregulated on arthritic KO neutrophils from BM and the periphery. Decreased C5a amounts in KO SF contributed to lower frequencies of CD5aR⁺-bearing neutrophils. In blood, surface C5aR was detected on KO Ly6G⁺ cells as a result of low receptor engagement. Circulating CD4⁺ T cells had an altered ability to produce interleukin (IL)-17 and interferon (IFN)-γ and to express RANKL. In KO CAIA mice, decreased frequencies of CD4⁺ T cells in the spleen were related to low CD86 expression on Ly6G^highCD11b⁺ cells. Arthritic KO T cells spontaneously secreted IFN-γ but not IL-17 and IL-6, and responded to restimulation with less-vigorous cytokine production in comparison to WT cells. Fewer TRAP-positive mature osteoclasts were found in KO BM cell cultures.

Conclusions

Our data show that the active involvement of properdin in arthritis is related to an increased proinflammatory cytokine production and RANKL expression on immune cells and to a stimulation of the RANKL-dependent osteoclast differentiation.     

Glandular trichomes of <Emphasis Type="Italic">Tussilago Farfara</Emphasis> (Senecioneae,Asteraceae)

Main conclusion

The glandular trichomes are developed on the aerial organs of Tussilago farfara ; they produce phenols and terpenoids. Smooth endoplasmic reticulum and leucoplasts are the main organelles of the trichome secretory cells. The aim of this study was to characterise the morphology, anatomy, histochemistry and ultrastructure of the trichomes in Tussilago farfara as well as to identify composition of the secretory products. Structure of trichomes located on the peduncles, bracts, phyllaries, and leaves were studied by light and electron microscopy. The capitate glandular trichomes consist of a multicellular head and a biseriate long stalk. Histochemical tests and fluorescence microscopy reveal phenols and terpenoids in the head cells. During secretory stage, the head cells contain smooth and rough endoplasmic reticulum, Golgi apparatus, diversiform leucoplasts with opaque contents in lamellae, chloroplasts, mitochondria, and microbodies. In the capitate glandular trichomes of T. farfara subcuticular cavity is absent, unlike glandular trichomes in other Asteraceae species. For the first time, content of metabolites in the different vegetative and reproductive organs as well as in the isolated capitate glandular trichomes was identified by GC–MS. Forty-five compounds, including organic acids, sugars, polyols, phenolics, and terpenoids were identified. It appeared that metabolite content in the methanol extracts from peduncles, bracts and phyllaries is biochemically analogous, and similar to the metabolites from leaves, in which photosynthesis happens. At the same time, the metabolites from trichome extracts essentially differ and refer to the above-mentioned secondary substances. The study has shown that the practical value of the aerial organs of coltsfoot is provided with flavonoids produced in the capitate glandular trichomes.

     

A polymer with a backbone of 3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid, a teichuronic acid, and a beta-glucosylated ribitol teichoic acid in the cell wall of plant pathogenic Streptomyces sp. VKM Ac-2124.

Structures of cell wall anionic polymers of the strain Streptomyces sp. VKM Ac-2124, a causative agent of potato scab, which is phylogenetically the closest to plant pathogenic species S. setonii and S. caviscabies, were studied. The strain contains three anionic glycopolymers, viz., a teichuronic acid with a disaccharide repeating unit -->6)-alpha-d-Glcp-(1-->4)-beta-d-ManpNAc3NAcA-(1-->, a beta-glucosylated polymer of 3-deoxy-d-glycero-d-galacto-non-2-ulopyranosonic acid (Kdn), and a beta-glucosylated 1,5-poly(ribitol phosphate). The strain studied is the second representative of plant pathogenic streptomycetes inducing potato scab disease, the cell wall anionic polymers of which were shown to contain a Kdn-polymer. Presumably, the presence of Kdn-containing structures in the surface regions of pathogens is essential for their efficient attachment to host plant cells.     

Altered profiles of serum neuroactive steroids in premenopausal women treated for alcohol addiction

Long-term alcohol consumption results in menstrual irregularities due to the inhibition of progesterone secretion. Some progesterone metabolites, including three pregnanolone isomers (PI), abate, while pregnenolone sulfate (PregS) and dehydroepiandrosterone sulfate (DHEAS) increase, alcohol tolerance. The rationale of this study was to evaluate how the neuroactive steroids reflect the impaired progesterone formation in premenopausal women treated for alcohol addiction, and whether detoxification therapy could restore female reproductive functions and psychosomatic stability by reinstatement of the steroid biosynthesis. Accordingly, serum allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one (P3alpha5alpha)), pregnanolone (P3alpha5beta), isopregnanolone (P3beta5alpha) and epipregnanolone (P3beta5beta), progesterone, PregS, pregnenolone, 17alpha-hydroxy-pregnenolone (Preg17), 17alpha-hydroxy-progesterone (Prog17), DHEA, DHEAS, cortisol and estradiol were measured in 20 women during the therapy (start, 3 days, 14 days, 1 month, 4 months), and in 17 controls, using GC-MS or RIA and evaluated by age-adjusted ANCOVA with status and phase of the menstrual cycle (PMC) as factors, and status-PMC interaction. The patients exhibited depressed progesterone, Prog17, PI, and estradiol, a decreased progesterone/pregnenolone ratio, a decreased ratio of neuroinhibiting P3alpha5alpha to neuroactivating PregS, and an elevated PregS and PregS/pregnenolone ratio. The treatment mostly restored the indices. The reduction of neuroinhibiting pregnanolone isomers in the patients is primarily associated with the impairment in ovarian steroid biosynthesis. Nevertheless, changes in enzyme activities connected with the formation of PI and the influence of altered physiological requirements on the balance between endogenous neuroinhibiting and neuroactivating steroids are also likely. The reinstatement of serum estradiol, progesterone, and PI during the therapy demonstrates its favorable effect on both reproductive functions and the psychosomatic stability of the patients.     

Association of Polymorphic Markers of the Antioxidant Enzyme Genes with Diabetic Polyneuropathy in Type 1 Diabetes Mellitus

The allele and genotype frequency distributions of polymorphic markers of genes coding for antioxidant enzymes were compared for type 1 diabetes mellitus patients with or without diabetic polyneuropathy (DPN). The groups (total 180 patients) had nonoverlapping (polar) phenotypes. Group DPN+ included 86 patients with DPN and diabetic record no more than 5 years. Control group DPN– included patients without DPN and diabetic record of at least 10 years. Comparative analysis with Fisher's exact test revealed a significant difference in allele and genotype frequency distributions of the (–262) polymorphic marker of the CAT gene. Polymorphic markers C1167T of the CAT gene, Pro/Leu of the GPX1 gene, 0/+ of the GSTT1 gene, and 0/+ of the GSTM1 gene showed no significant difference in allele or genotype frequency distribution. On this evidence, these markers were not associated with DPN in the sample examined.     

HSP70-inducible hNIS-IRES-eGFP reporter imaging: response to heat shock

A retroviral vector pQHSP70/hNIS-IRES-eGFP (pQHNIG70) was constructed containing the hNIS-IRES-eGFP dual-reporter genes under the control of an inducible human heat shock protein (HSP)70 promoter and RG2-pQHSP70/hNIS-IRES-eGFP (RG2-pQHNIG70) transduced cells were generated. Heat-induced expression of both reporter genes in RG2-pQHNIG70 cells was validated by enhanced green fluorescent protein (eGFP) fluorescence-activated cell sorter, in vitro radiotracer assays, and immunoblot and immunocytochemistry. A 2.2- to 6.1-fold ((131)I(-)), a 6.1- to 14.4-fold ((99m)TcO(4)(-)), and a 5.1- to 39-fold (fluorescence) increase above baseline was observed in response to graded hyperthermia (39-43 degrees C). Increases in eGFP fluorescence and radiotracer uptake were first noted at 6 hours, reached a maximum at 24 hours, and fell toward baseline at 72 hours. A stable ratio of radiotracer uptake to eGFP fluorescence and to heat shock protein (HSP)70 protein was demonstrated over a wide range of expression levels, induced by different levels of heating. We also demonstrate that the local application of heat on RG2-pQHNIG70 xenografts can effectively induce hNIS and eGFP gene expression in vivo and that this expression can be efficiently visualized by fluorescence, scintigraphic, and micro-positron emission tomography imaging. Endogenous HSP70 protein and reporter expression was confirmed by postmortem tissue evaluations (immunoblot and immunohistochemistry). The pQHNIG70 reporter system can be used to study stress and drug responses in transduced cells and tissues.