Is myosin light-chain phosphorylation a regulatory signal for the osmotic activation of the Na+-K+-2Cl- cotransporter?

Myosin light-chain (MLC) kinase (MLCK)-dependent increase in MLC phosphorylation has been proposed to be a key mediator of the hyperosmotic activation of the Na⁺-K⁺-2Cl^– cotransporter (NKCC). To address this hypothesis and to assess whether MLC phosphorylation plays a signaling or permissive role in NKCC regulation, we used pharmacological and genetic means to manipulate MLCK, MLC phosphorylation, or myosin ATPase activity and followed the impact of these alterations on the hypertonic stimulation of NKCC in porcine kidney tubular LLC-PK1 epithelial cells. We found that the MLCK inhibitor ML-7 suppressed NKCC activity independently of MLC phosphorylation. Notably, ML-7 reduced both basal and hypertonically stimulated NKCC activity without influencing MLC phosphorylation under these conditions, and it inhibited NKCC activation by Cl^– depletion, a treatment that did not increase MLC phosphorylation. Furthermore, prevention of the osmotically induced increase in MLC phosphorylation by viral induction of cells with a nonphosphorylatable, dominant negative MLC mutant (AA-MLC) did not affect the hypertonic activation of NKCC. Conversely, a constitutively active MLC mutant (DD-MLC) that mimics the diphosphorylated form neither stimulated isotonic nor potentiated hypertonic NKCC activity. Furthermore, a depolarization-induced increase in endogenous MLC phosphorylation failed to activate NKCC. However, complete abolition of basal MLC phosphorylation by K252a or the inhibition of myosin ATPase by blebbistatin significantly reduced the osmotic stimulation of NKCC without suppressing its basal or Cl^– depletion-triggered activity. These results indicate that an increase in MLC phosphorylation is neither a sufficient nor a necessary signal to stimulate NKCC in tubular cells. However, basal myosin activity plays a permissive role in the optimal osmotic responsiveness of NKCC. proline-alanine-rich STE20-related kinase     

Fasting-induced changes of neuropeptide immunoreactivity in the lateral septum of male rats

The objective of the present study was to determine the rostrocaudal distribution and the effect of reduced food intake (60% of the average daily food intake for 1-4 weeks) on the amount of leucine-enkephalin (Leu-enk), neuropeptide Y (NPY) and galanin (Gal) in the lateral septum of male rat brain. Using pre-embedding immunocytochemistry combined with densitometry on 60 microm serial vibratome sections we found that in control animals Leu-enk-immunoreactive elements showed an increasing density from rostral towards the medial part of the septum, then a gradual decrease towards the caudal direction. The distribution of NPY proved to be rather even along the examined sequence of sections with two smaller peaks roughly at the 1/3 and 2/3 of the rostrocaudal axis. Gal showed similar distribution but the peaks were shifted to more caudal direction. We also found that Leu-enk forms the most dense plexus followed by a moderate amount of NPY-positive axonal meshwork. Gal was present in the lowest amount along the lateral septal nuclei. The effect of reduced food intake was marked and differential in the case of the three examined peptides. During the first 2 weeks of reduced food intake NPY-immunoreactivity was upregulated as compared to the control, then it was reduced close to the control value by the 4th week. The changes in Gal immunoreactivity showed similar pattern. The average relative density of Leu-enk-immunoreactive elements immediately decreased as a result of reduced food intake for 1 week and it gradually decreased by the end of the 4th week. These results indicate that reduced food intake affects the expression of NPY, Gal and Leu-enk not only in the relevant hypothalamic neuroendocrine centres, but also in the lateral septal area.     

Cerebellar granule cells show age-dependent migratory differences in vitro

Developmental differences between cerebellar granule cells during their migratory period were revealed using dissociated granule cell cultures isolated from 4, 7, or 10 days old (P4, P7, P10) mice. Under all culture conditions, the great majority of cultivated cell populations consisted of those granule cells that had not reach their final destination in the internal granule cell layer (IGL) by the age of isolation. In vitro morphological development and the expression of migratory markers (TAG-1, astrotactin, or EphB2) showed similar characteristics between the cultures. The migration of 1008 granule cells isolated from P4, P7, and P10 cerebella and cultivated under identical conditions were analyzed using statistical methods. In vitro time-lapse videomicroscopy revealed that P4 cells possessed the fastest migratory speed while P10 granule cells retained their migratory activity for the longest time in culture. Cultures obtained from younger postnatal ages showed more random migratory trajectories than P10 cultures. Our observations indicate that despite similar morphological and molecular properties, migratory differences exist in granule cell cultures isolated from different postnatal ages. Therefore, the age of investigation can substantially influence experimental results on the regulation of cell migration.     

Adventures with ABC-proteins: highly conserved ATP-dependent transporters

The general properties of ABC transporters, from bacteria to humans, including a brief history of their initial discovery, are considered. ABC transporters, one of the largest protein super families and vital for human health, are in toto responsible for the transport of an enormous range of molecules from ions (CFTR) or anti-tumour drugs (Pgp/MDR) to large polypeptides. Nevertheless, all ABC transporters are powered by a conserved ATPase the ABC or NBD domain, using in all probability the same basic mechanism of action for the hydrolysis of ATP and its coupling to the transport process. Based on recent high resolution structures of several NBDs and an intact transporter, a model of how dimers of these important proteins function will be discussed, with particular attention to HlyB, the ABC transporter from E. coli.     

Enhanced biodegradation of transformer oil in soils with cyclodextrin – from the laboratory to the field

The use cyclodextrins for the intensification of bioremediation by improving the mobility and bioavailability of contaminants has recently been studied. In this work, the role of randomly methylated -cyclodextrin in the bioremediation of soils contaminated with transformer oil was studied both in bench scale bioreactors and through field experiments. The aims of this research were to (a) establish the scientific background of a cyclodextrin-based soil bioremediation technology, (b) demonstrate its feasibility and effectiveness in the field, and (c) develop an integrated methodology, consisting of a combination of physical, chemical, biological and ecotoxicological analytical methods, for efficiently monitoring the technology performances. The stepwise increasing scale of the experiments and the application of the integrated analytical methodology supported the development of a scientifically established new technology and the identification of the advantages and the limitations of its application in the field. At each phase of the study, randomly methylated -cyclodextrin was found to significantly enhance the bioremediation and detoxification of the transformer oil-contaminated soils employed by increasing the bioavailability of the pollutants and the activity of indigenous microorganisms.     

Mitochondrial DNA of ancient Cumanians: culturally Asian steppe nomadic immigrants with substantially more western Eurasian mitochondrial DNA lineages

The Cumanians were originally Asian pastoral nomads who in the 13th century migrated to Hungary. We have examined mitochondrial DNA from members of the earliest Cumanian population in Hungary from two archeologically well-documented excavations and from 74 modern Hungarians from different rural locations in Hungary. Haplogroups were defined based on HVS I sequences and examinations of haplogroup-associated polymorphic sites of the protein coding region and of HVS II. To exclude contamination, some ancient DNA samples were cloned. A database was created from previously published mtDNA HVS I sequences (representing 2,615 individuals from different Asian and European populations) and 74 modem Hungarian sequences from the present study. This database was used to determine the relationships between the ancient Cumanians, modern Hungarians, and Eurasian populations and to estimate the genetic distances between these populations. We attempted to deduce the genetic trace of the migration of Cumanians. This study is the first ancient DNA characterization of an eastern pastoral nomad population that migrated into Europe. The results indicate that, while still possessing a Central Asian steppe culture, the Cumanians received a large admixture of maternal genes from more westerly populations before arriving in Hungary. A similar dilution of genetic, but not cultural, factors may have accompanied the settlement of other Asian nomads in Europe.     

Gene expression profiling in murine autoimmune arthritis during the initiation and progression of joint inflammation

We present here an extensive study of differential gene expression in the initiation, acute and chronic phases of murine autoimmune arthritis with the use of high-density oligonucleotide arrays interrogating the entire mouse genome. Arthritis was induced in severe combined immunodeficient mice by using adoptive transfer of lymphocytes from proteoglycan-immunized arthritic BALB/c mice. In this unique system only proteoglycan-specific lymphocytes are transferred from arthritic mice into syngeneic immunodeficient recipients that lack adaptive immunity but have intact innate immunity on an identical (BALB/c) genetic background.     

Multiple metabolic hits converge on CD36 as novel mediator of tubular epithelial apoptosis in diabetic nephropathy

Background

Diabetic nephropathy (DNP) is a common complication of type 1 and type 2 diabetes mellitus and the most common cause of kidney failure. While DNP manifests with albuminuria and diabetic glomerulopathy, its progression correlates best with tubular epithelial degeneration (TED) and interstitial fibrosis. However, mechanisms leading to TED in DNP remain poorly understood.

Methods and Findings

We found that expression of scavenger receptor CD36 coincided with proximal tubular epithelial cell (PTEC) apoptosis and TED specifically in human DNP. High glucose stimulated cell surface expression of CD36 in PTECs. CD36 expression was necessary and sufficient to mediate PTEC apoptosis induced by glycated albumins (AGE-BSA and CML-BSA) and free fatty acid palmitate through sequential activation of src kinase, and proapoptotic p38 MAPK and caspase 3. In contrast, paucity of expression of CD36 in PTECs in diabetic mice with diabetic glomerulopathy was associated with normal tubular epithelium and the absence of tubular apoptosis. Mouse PTECs lacked CD36 and were resistant to AGE-BSA-induced apoptosis. Recombinant expression of CD36 in mouse PTECs conferred susceptibility to AGE-BSA-induced apoptosis.

Conclusion

Our findings suggest a novel role for CD36 as an essential mediator of proximal tubular apoptosis in human DNP. Because CD36 expression was induced by glucose in PTECs, and because increased CD36 mediated AGE-BSA-, CML-BSA-, and palmitate-induced PTEC apoptosis, we propose a two-step metabolic hit model for TED, a hallmark of progression in DNP.