The Effect of Chronic Long-Term Intermittent Hypobaric Hypoxia on Bone Mineral Density in Rats: Role of Nitric Oxide

Intermittent hypoxia is the most common pattern of hypoxic exposure in humans. The effect of chronic long-term intermittent hypobaric hypoxia (CLTIHH) on bone metabolism is not investigated. We examined the effect of CLTIHH on bone metabolism and the role of nitric oxide (NO) in this process. The rats were divided into three groups in this study. The animals in groups I and II have been exposed to CLTIHH. The animals in group II were also treated with nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester. To obtain CLTIHH, rats were placed in a hypobaric chamber (430 mm Hg; 5 h/day, 5 days/week, 5 weeks). The group III (control) rats breathed room air in the same environment. At the begining of the experiments, bone mineral density (BMD) of the animals were measured, and blood samples were collected from the tail vein. After the 5-week CLTIHH period, the same measurements were repeated. Parathyroid hormone, calcium, phosphate, bone alkaline phosphatase (b-ALP), NO, interleukin 1 beta, interleukin 6, and tumor necrosis factor alpha levels were determined. The cytokines, NO levels, and BMD in CLTIHH-induced rats were higher compared with baseline and control values. The cytokines, b-ALP, and BMD increased while NO levels decreased in the group II compared with baseline values. BMD values of group II were lower than group I but higher than control group. Our results suggested that CLTIHH has positive effects on bone density. Intermittent hypoxia protocols may be developed for treatment and prevention of osteopenia and osteoporosis.     

Osteoprotective effect of soybean and sesame oils in ovariectomized rats via estrogen-like mechanism

The purpose of the present study was to investigate the osteoprotective effects of soybean oil (SbO) and sesame oil (SO) in ovarictomized (OVX) rats. The results indicated that the OVX rats exhibited a significant decrease in Ca and P level in both serum and bone, the activities of the antioxidant enzymes SOD and CAT and the antioxidant biomarker GSH accompanied with a marked increase in the oxidative stress markers MDA and PC, the inflammatory indices (TNF-α, CRP levels, WBCs counts and ACP activity) in, both, bone and serum. Supplementating the diet of the OVX rats with SbO (15 % w/w) or SO (10 % w/w) for 2 months to resulted in modulation of the alterations in all tested parameters and succeeded to restore minerals, antioxidant enzymes, antioxidant biomarkers, oxidative stress markers, inflammatory indices, and WBCs counts. It could be concluded that the consumption of diets supplemented with SbO or SO might be useful for preventing bone loss caused by estrogen deficiency in ovariectomy status.     

Ghrelin is present in teeth

Ghrelin belongs to the family of a gut-brain hormone that promotes food intake and controls energy balance. Recently, it has also been shown to regulate bone formation directly. Dental tissue shares several functional, developmental and anatomical similarities with bone, and in the present study we have investigated the presence of ghrelin in 44 human teeth using immunocytochemistry and radioimmunoassay. Both methods showed that the hormone is present in canines and molars, mainly in the odontoblasts but also in the pulp. Ghrelin could potentially play interesting physiological roles in teeth.     

Direct Determination of Phosphatase Activity from Physiological Substrates in Cells

A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP) performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min^-1 mg^-1 for PPi, to 56 ± 11 nmol min^-1 mg^-1 for AMP, to 79 ± 23 nmol min^-1 mg^-1 for beta-glycerophosphate and to 73 ± 15 nmol min^-1 mg^-1 for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole – a TNAP inhibitor- served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC₅₀ value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.     

Evidence of a Reduced and Modified Mitochondrial Protein Import Apparatus in Microsporidian Mitosomes

Microsporidia are a group of highly adapted obligate intracellular parasites that are now recognized as close relatives of fungi. Their adaptation to parasitism has resulted in broad and severe reduction at (i) a genomic level by extensive gene loss, gene compaction, and gene shortening; (ii) a biochemical level with the loss of much basic metabolism; and (iii) a cellular level, resulting in lost or cryptic organelles. Consistent with this trend, the mitochondrion is severely reduced, lacking ATP synthesis and other typical functions and apparently containing only a fraction of the proteins of canonical mitochondria. We have investigated the mitochondrial protein import apparatus of this reduced organelle in the microsporidian Encephalitozoon cuniculi and find evidence of reduced and modified machinery. Notably, a putative outer membrane receptor, Tom70, is reduced in length but maintains a conserved structure chiefly consisting of tetratricopeptide repeats. When expressed in Saccharomyces cerevisiae, EcTom70 inserts with the correct topology into the outer membrane of mitochondria but is unable to complement the growth defects of Tom70-deficient yeast. We have scanned genomic data using hidden Markov models for other homologues of import machinery proteins and find evidence of severe reduction of this system.Microsporidia are a eukaryotic group highly adapted as obligate intracellular parasites (31, 50). They infect a diverse range of vertebrate and invertebrate animal hosts. In humans they are the cause of a number of diseases (e.g., gastroenteritis, encephalitis, and hepatitis), having their greatest impact on immunocompromised individuals, notably in children with human immunodeficiency virus (14, 31). Microsporidia are most closely related to fungi, although their high level of specialization as intracellular parasites obscured this relationship for a long time (18, 25, 30). Gene phylogenies now firmly connect these two groups, although it remains uncertain whether microsporidia are sisters to the fungi or represent a lineage derived from within fungal diversity (21, 28).A clear adaptive response to parasitism in microsporidia has been a reduction in cellular complexity. This was first recognized at an ultrastructural level with the apparent lack of peroxisomes, flagella, stacked Golgi bodies, and mitochondria (31). This reductive evolution is mirrored at a genomic level, with microsporidia containing the smallest eukaryotic genomes described to date (28, 29). The complete genomic sequence from the human microsporidian parasite Encephalitozoon cuniculi reveals a genome of only ∼2.9 Mb containing approximately 2,000 genes, in contrast to the 6,000 genes found in the genome of the model fungus Saccharomyces cerevisiae. The minimal genome of E. cuniculi has been achieved through three mechanisms in concert: (i) gene loss, resulting in broad loss of biochemical pathways and capabilities, including much basic energy metabolism and numerous anabolic pathways; (ii) gene compaction with an average intergenic space of ∼130 bp; and (iii) gene shortening, with E. cuniculi genes being on average 14% shorter than their homologues in fungi such as S. cerevisiae (28, 45). Thus, microsporidian evolution has apparently been shaped by a very strong trend to eliminate superfluous molecular and biochemical complexity.Despite earlier suppositions that microsporidia lacked mitochondria, genome and expressed sequence tag data from microsporidia suggested the presence of several proteins typically targeted to this organelle (3, 19, 20, 24, 28, 38). Immunolocalization of a mitochondrial Hsp70 to small double membrane-bound organelles in Trachipleistophora hominis provided strong evidence for the existence of a mitochondrion in microsporidia, albeit a simplified organelle that lacks cisternae (48). Annotation of genomic data from E. cuniculi provided compelling matches for only 22 proteins implicated in mitochondrial function, suggesting that the metabolism of this relict mitochondrion (or mitosome) is also significantly reduced compared to that of canonical mitochondria (28). Further, no mitochondrial genome has been retained; thus, biogenesis of this organelle is wholly dependent on nucleus-encoded proteins. Based on these 22 proteins, a major role for the mitosome is iron-sulfur cluster assembly (22, 28). No genes have been found for ATP synthesis via oxidative phosphorylation, suggesting loss of this activity in mitosomes (28, 46). While it is likely that further mitosome-targeted proteins will be identified, it is clear that compared to mitochondria from fungal relatives, which are known to import ∼1,000 proteins (40, 44), microsporidian mitosomes represent organelles with highly reduced proteomes, a feature consistent with other traits of cellular reduction.The highly reduced state of the microsporidian mitosome, requiring only a fraction of the protein diversity of other mitochondria, presents an interesting case for studying organelle biogenesis—particularly the machinery for protein import of nucleus-encoded proteins. Mitochondrial protein import has been best characterized in fungi, and in these systems most proteins are imported via four major import complexes: a TOM (translocase of the outer mitochondrial membrane), a SAM (sorting and assembly machinery), and one of two TIMs (translocase of the inner mitochondrial membrane), TIM23 or TIM22 (see Fig. ​Fig.5A)5A) (5, 36). These complexes are broadly conserved throughout fungi as well as animals (15). Mitochondrial proteins can take one of several routes to the mitochondrion via this apparatus (5, 36). Broadly, soluble matrix proteins are recognized at the TOM complex by the receptor protein Tom20 through the binding of N-terminal presequences with characteristic features (1, 5, 7, 8, 36). These proteins are passed through the pore protein Tom40 of the TOM to the TIM23 complex and then driven into the mitochondrial matrix by way of the presequence translocase-associated motor (PAM) complex, where their presequences are subsequently removed. Some membrane proteins can also be released into the inner membrane from the TIM23 complex. Mitochondrial proteins that apparently lack such an extension, notably including many of the membrane proteins, are recognized by internal sequence elements. Tom70 has a greater role in recognizing these internal signals and thus the import of hydrophobic proteins (4, 11, 32, 39, 47). Such hydrophobic proteins are often bound by cytosolic molecular chaperones (Hsp70 and/or Hsp90) en route to the mitochondrion, and Tom70 is known to independently bind to both the chaperone and the substrate protein (7, 23, 33, 52). While a measure of substrate overlap between Tom20 and Tom70 occurs, the division of responsibility between these two receptors has likely evolved in response to the wide range of substrate proteins that must be imported into mitochondria and the need to handle this complexity.Open in a separate windowFIG. 5.Schematics of the protein import machinery and pathways in yeast mitochondria (A) and E. cuniculi mitosome (B) based on identified homologues of the general fungal/animal pathways. Protein components of the yeast system were all represented by HMMs used to search the microsporidian genomic data and represent the major presequence-dependent and presequence-independent pathways. Homologues identified in E. cuniculi indicate a severely reduced import apparatus utilizing elements of the presequence-independent pathway.For microsporidia little is known of the protein import apparatus for their relict mitochondrion, the mitosome. Has the very reduced organelle proteome, in concert with a genome-wide trend of the loss of redundant or superfluous genes, resulted in a smaller and/or derived import apparatus? In this study we have investigated the microsporidian mitosome protein import apparatus from E. cuniculi in order to evaluate how this apparatus has responded to the reduction in the number of proteins required to be imported and the overall radical reduction in the number and size of proteins encoded in the nuclear genome. A putative homologue of the outer membrane receptor protein Tom70 is of particular interest as the only receptor for the TOM complex and, given the known structure of Tom70 proteins, provides a highly informative example of how proteins can be shortened in the course of genome reduction.     

Clinical and pathological significance of programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) expression in high grade serous ovarian cancer

We investigated programmed cell death 1 (PD-1) / programmed cell death ligand 1 (PD-L1) expression in high grade serous ovarian cancer (HGSOC) and its relationship to tumor infiltrating lymphocytes (TIL) and prognosis. Formalin fixed paraffin embedded (FFPE) samples of 94 HGSOC cases were included in the study. Immunohistochemical analysis (CD3, CD4, CD8, PD-1 and PD-L1) was performed. Samples were analyzed for expression of immune proteins in the peritumoral stromal and intratumoral areas, scored, and expression was correlated with overall survival, stage, and age. PD-L1 staining ratio with a score greater than 0 was found to have lower survival. There were two positive staining patterns, patchy/diffuse and patchy/focal patterns, in 24 (25.5%) cases. Considering the threshold value ≥5%, we demonstrated that the PD-L1 positive cancer cell membrane immunoreactivity rate and patchy/diffuse PD-L1 expression were 9.6% (n = 9). There was statistically significant relationship between high PD-1 scores and PD-L1 cases of ≥ 5%. A statistically significant difference was found between PD-L1 staining and survival in patients with a threshold ≥ 5%. However an appropriate rate for treatment was determined in 9.6% cases. There was a statistically significant correlation between PD-1 positive TIL score and intratumoral CD3, peritumoral stromal CD3, intratumoral CD4 and intratumoral CD8 positive cells. Survival was lower in cases with higher PD-L1 positive stromal TIL score.     

Genome-wide exploration of silicon (Si) transporter genes, <Emphasis Type="Italic">Lsi1</Emphasis> and <Emphasis Type="Italic">Lsi2</Emphasis> in plants; insights into Si-accumulation status/capacity of plants

Silicon (Si) is a nonessential, beneficial micronutrient for plants. It increases the plant stress tolerance in relation to its accumulation capacity. In this work, root Si transporter genes were characterized in 17 different plants and inferred for their Si-accumulation status. A total of 62 Si transporter genes (31 Lsi1 and 31 Lsi2) were identified in studied plants. Lsi1s were 261–324 residues protein with a MIP family domain whereas Lsi2s were 472–547 residues with a citrate transporter family domain. Lsi1s possessed characteristic sequence features that can be employed as benchmark in prediction of Si-accumulation status/capacity of the plants. Silicic acid selectivity in Lsi1s was associated with two highly conserved NPA (Asn-Pro-Ala) motifs and a Gly-Ser-Gly-Arg (GSGR) ar/R filter. Two NPA regions were present in all Lsi1 members but some Ala substituted with Ser or Val. GSGR filter was only available in the proposed high and moderate Si accumulators. In phylogeny, Lsi1s formed three clusters as low, moderate and high Si accumulators based on tree topology and availability of GSGR filter. Low-accumulators contained filters WIGR, AIGR, FAAR, WVAR and AVAR, high-accumulators only with GSGR filter, and moderate-accumulators mostly with GSGR but some with A/CSGR filters. A positive correlation was also available between sequence homology and Si-accumulation status of the tested plants. Thus, availability of GSGR selectivity filter and sequence homology degree could be used as signatures in prediction of Si-accumulation status in experimentally uncharacterized plants. Moreover, interaction partner and expression profile analyses implicated the involvement of Si transporters in plant stress tolerance.     

Effects of brassinosteroids on barley root growth,antioxidant system and cell division

Homobrassinolide (HBR), which is one of the most biologically active forms of Brassinosteroids (BRs), was used to examine the potential effects of hormone on root germination, antioxidant system enzymes and cell division of barley (Hordeum vulgare L.). Seeds were germinated between filter papers in 0.1, 0.5 and 1.0 μM HBR-supplemented distilled water for 48 h at dark with their controls. HBR application increased especially the primary root growth significantly with increasing concentrations when compared with the control materials and reached two fold increase in 1.0 μM HBR treated material. Treated and untreated control group roots were fixed in 1:3 aceto-alcohol and aceto-orcein preparations were made. Roots treated with HBR showed more mitotic activity, mitotic abnormalities and significant enlargements at the root tips when compared with control material. HBR application decreased total soluble protein content, superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6) and peroxidase (EC 1.11.1.11) activities significantly at 1.0 μM HBR concentration. Data presented here is one of the first detailed analyses of HBR effect on barley root development.     

Decreased peroxidase activity in transgenic tobacco and its effect on lignification

An antisense gene construct of a peroxidase gene (Shpx6a) from a tropical pasture legume Stylosanthes humilis was transferred into tobacco cells via Agrobacterium tumefaciens to test whether peroxidase activity could be decreased and what effect this would have on lignification. A large number of tobacco cell lines were regenerated on selective media and stable integration of the transgene was confirmed in randomly selected putative transformants. Analyses of the primary transgenic plants and their progeny (T ₁) demonstrated that the total peroxidase activity was significantly decreased (up to 36%) as compared to that measured in untransformed control plants. Importantly, reduction in peroxidase activity is accompanied by decreases (up to 23%) in lignin content in several transgenic lines.     

Agronomical and chemical characterization of spearmint (Mentha spicata L.) originating in Turkey

The essential oil properties of spearmint (Mentha spicataL.), one of the most important spice plants, were studied and the essential oil components determined using gas chromatography. The essential oil content of wild-grown spearmint in the region was found to range from 1.00% to 2.00%, and two chemotypes were identified, one high in carvone (49.53-80.65%) and the other in pulegone (44.9-49.23%). Agronomic and essential oil properties of cultivated landraces ofM. spicata were also investigated under field conditions during the 1999 vegetation period. The examined spearmint landraces showed a great variability for each character studied, including yield and essential oil components. The crop was harvested twice during the vegetation period, and the essential oil content of the landraces varied from 0.90 to 2.70% in the first harvest and from 1.00 to 3.00% in the second one. Carvone was constantly present as the predominant essential oil in landraces, except for one sample, which was high in linalool (82.80%). Superior landraces with carvone contents were discovered; their maximum content reached 79.70% in the first cutting and 82.97% at the second cutting. The superior landraces were assayed for future improvement studies.