Degeneration and calcification of the cervical endplate is connected with decreased expression of ANK, ENPP-1, OPN and TGF-ß1 in the intervertebral disc.

The aim of this study was to clarify the relationship between the expression of ALP, ANK, ENPP-1, OPN and TGF-ß1 in the intervertebral disc (IVD), and cervical vertebral endplate calcification and degeneration. Sixty cervical IVDs were excised from 30 human cadavers. Each cadaver was assessed macroscopically for degeneration (Thompson's classification), and then underwent histological processing, regular staining (hematoxylin and eosin, Masson-Goldner trichrome and alcian blue-PAS), immunohistochemistry (ALP, ANK, ENPP-1, OPN and TGF-ß1), microscopic degeneration grading (Boos classification), and assessment of endplate calcification. The mean age ± SD of the cadavers was 51.4 ±19.5. The percentage of endplate calcification significantly correlated with the degree of endplate and IVD degeneration graded using Boos's score (both r = 0.91; p < 0.0001). The intensity and number of stained cells per FOV markedly decreased, for ANK, ENPP-1, and TGF-ß1, with the grade of IVD degeneration, regardless of the analyzed IVD region. This was not true only for ALP, which demonstrated an increasing trend corresponding to the degree of IVD degeneration. The expression of OPN was low throughout all analyzed regions, regardless of the degree of degeneration. Modulating the expression of the abovementioned proteins, especially ANK and TGF-ß1, may be a new way to prevent degeneration and calcification of the IVD.

NITROGEN LIMITATION ADAPTATION recruits PHOSPHATE2 to target the phosphate transporter PT2 for degradation during the regulation of Arabidopsis phosphate homeostasis.

The NITROGEN LIMITATION ADAPTION (NLA) gene was initially shown to function in nitrogen limitation responses; however, recent work shows that the nla mutant hyperaccumulates Pi, phenocopying the Pi signaling mutant pho2. PHO2 encodes a putative E2 conjugase, UBC24. Here, we show that NLA is an E3 ligase that specifically requires UBC24 for polyubiquitination in Arabidopsis thaliana. Among five members of the Pht1 Pi-transporter family tested, NLA associates only with PT2 (Pht1;4). The NLA-UBC24 pair mediates polyubiquitination of PT2 but not PT1. Posttranslational decay of PT2 at high Pi is blocked in pho2 and inhibited by MG132, indicating the requirement of UBC24 and 26S proteasomes. Consistent with NLA/UBC24 function, induced NLA expression causes a UBC24-dependent decrease in PT2 levels. Confocal microscopy of fusion proteins revealed an NLA/PT2 interaction at the plasma membrane. Collectively, these results show that under Pi-replete conditions, NLA and UBC24 target the PT2 transporter for destruction. During the Pi deprivation response, NLA and PHO2 transcripts are cleaved by miR399 and miR827, respectively, and our results suggest that this downregulation relieves the posttranslational repression of PT2, allowing it to accumulate and participate in Pi uptake. Our work provides additional molecular details describing Pi signaling/homeostasis regulation by identifying NLA and UBC24 as partners and PT2 as one of their downstream targets.

The study of phosphate transporter NAPI2B expression in different histological types of epithelial ovarian cancer.

UNLABELLED: The identification of markers that are specifically expressed by different histological types of epithelial ovarian cancer (EOC) may lead to the development of novel and more specific diagnostic and therapeutic strategies. Sodium-dependent phosphate transporter NaPi2b (or MX35 ovarian cancer antigen) is a novel perspective marker of EOC. To date, the studies on NaPi2b/MX35 expression in different histological types of EOC are limited. AIM: To examine NaPi2b/MX35 expression in different histological types of epithelial ovarian tumors. METHODS: Here, we describe the analysis of NaPi2b expression in serous (n = 17), endometrioid (n = 8), and mucinous ovarian tumors (n = 3) by Western-blotting (WB), immunohistochemistry and RT-PCR. RESULTS: The results of immunohistochemical and WB analysis showed that benign and well-differentiated malignant papillary serous tumors as well as well-differentiated malignant endometriod tumors overexpress NaPi2b protein. However, no overexpression of NaPi2b was detected in benign and malignant mucinous tumors as well as in poorly differentiated endometriod tumors. Notably, the expression NaPi2b mRNA was detected in all investigated histological types of EOC. CONCLUSION: We have shown the differential expression profile of NaPi2b phosphate transporter at protein level in various histological types of epithelial ovarian cancer. This finding might facilitate the development of more effective approaches for diagnosis and treatment of this disease.

Functional biology of plant phosphate uptake at root and mycorrhiza interfaces.

Phosphorus (P) is an essential plant nutrient and one of the most limiting in natural habitats as well as in agricultural production world-wide. The control of P acquisition efficiency and its subsequent uptake and translocation in vascular plants is complex. The physiological role of key cellular structures in plant P uptake and underlying molecular mechanisms are discussed in this review, with emphasis on phosphate transport across the cellular membrane at the root and arbuscular-mycorrhizal (AM) interfaces. The tools of molecular genetics have facilitated novel approaches and provided one of the major driving forces in the investigation of the basic transport mechanisms underlying plant P nutrition. Genetic engineering holds the potential to modify the system in a targeted way at the root-soil or AM symbiotic interface. Such approaches should assist in the breeding of crop plants that exhibit improved P acquisition efficiency and thus require lower inputs of P fertilizer for optimal growth. Whether engineering of P transport systems can contribute to enhanced P uptake will be discussed.

Novel inter- and intrasubunit contacts between transport-relevant residues of the homodimeric mitochondrial phosphate transport protein.

Ser158 is located near the middle of the matrix loop connecting transmembrane helices C and D of the mitochondrial phosphate transport protein (PTP). The mutant Ser158Thr PTP is transport-inactive. His32 is located near the middle of transmembrane helix A and Thr79 is located 5 residues away from transmembrane helix B and its N-terminal (matrix end). Single site mutant PTPs that have either residue replaced with Ala are transport-inactive. Based on the high resolution structure of a subunit of the bovine ADP/ATP translocase, on sequence similarities between members of the mitochondrial transport protein family, and on the PTP subunit/subunit contact site between transmembrane A helices, it is now suggested that the Ser158 site is at the PTP subunit/subunit contact site. This contact site is essential for keeping the transport cycles catalyzed by the two PTP subunits 180 degrees out of phase. The data also suggest that His32 and Thr79 of the same subunit interact and couple the phosphate and the proton transport paths.

Immunohistochemical localization of candidates for vesicular glutamate transporters in the rat brain.

Vesicular glutamate transporter 1 (VGluT1) is one of the best markers for glutamatergic neurons, because it accumulates transmitter glutamate into synaptic vesicles. Differentiation-associated Na(+)-dependent inorganic phosphate cotransporter (DNPI) shows 82% amino acid identity to VGluT1, and is another candidate for vesicular glutamate transporters. Here, we report the immunocytochemical localization of DNPI and compare it with that of VGluT1 in the adult rat brain. Both DNPI and VGluT1 immunoreactivities were found mostly in neuropil, presumably in axon terminals, throughout the brain. In the telencephalic regions, intense DNPI immunoreactivity was observed in the glomeruli of the olfactory bulb, layer IV of the neocortex, granular layer of the dentate gyrus, presubiculum, and postsubiculum. In contrast, VGluT1 immunoreactivity was intense in the olfactory tubercle, layers I-III of the neocortex, piriform cortex, entorhinal cortex, hippocampus, dentate gyrus, and subiculum. In the thalamic nuclei, DNPI-immunoreactive terminal-like profiles were much larger than VGluT1-immunoreactive ones, suggesting that DNPI immunoreactivity was subcortical in origin. DNPI immunoreactivity was much more intense than VGluT1 immunoreactivity in many brainstem and spinal cord regions, except the pontine nuclei, interpeduncular nucleus, cochlear nuclei, and external cuneate nucleus. In the molecular layer of the cerebellar cortex, climbing-like fibers showed intense DNPI immunoreactivity, whereas neuropil contained dense VGluT1-immnoreactive deposits. Both DNPI and VGluT1 immunoreactivities were observed as mossy fiber terminal-like profiles in the cerebellar granular layer. DNPI and VGluT1 immunoreactivities appeared associated with synaptic vesicles in the axon terminals forming asymmetric synapses in several regions examined electron microscopically. The present results indicate that DNPI and VGluT1 are used by different neural components in most, if not all, brain regions, suggesting the complementary functions of DNPI and VGluT1.