Characterization of methanogenic and prokaryotic assemblages based on mcrA and 16S rRNA gene diversity in sediments of the Kazan mud volcano (Mediterranean Sea).

The diversity of the methyl-coenzyme reductase A (mcrA) and 16S rRNA genes was investigated in gas hydrate containing sediment from the Kazan mud volcano, eastern Mediterranean Sea. mcrA was detected only at 15 and 20 cm below seafloor (cmbsf) from a 40-cm long push core, while based on chemical profiles of methane, sulfate, and sulfide, possible anaerobic oxidation of methane (AOM) depth was inferred at 12-15 cmbsf. The phylogenetic relationships of the obtained mcrA, archaeal and bacterial 16S rRNA genes, showed that all the found sequences were found in both depths and at similar relative abundances. mcrA diversity was low. All sequences were related to the Methanosarcinales, with the most dominant (77.2%) sequences falling in group mcrA-e. The 16S rRNA-based archaeal diversity also revealed low diversity and clear dominance (72.8% of all archaeal phylotypes) of the Methanosarcinales and, in particular, ANME-2c. Bacteria showed higher diversity but 83.2% of the retrieved phylotypes from both sediment layers belonged to the delta-Proteobacteria. These phylotypes fell in the SEEP-SRB1 putative AOM group. In addition, the rest of the less abundant phylotypes were related to yet-uncultivated representatives of the Actinobacteria, Spirochaetales, and candidate divisions OP11 and WS3 from gas hydrate-bearing habitats. These phylotype patterns indicate that AOM is occurring in the 15 and 20 cmbsf sediment layers.

Mixed descending- and ascending-type thin limbs of Henle's loop in mammalian renal inner medulla.

Previous studies have generally indicated that the entire descending (DTL) and ascending thin limbs (ATL) of Henle's loops in the mammalian inner medulla exhibit structurally and functionally distinct properties. In the present study, we found that about 50% of Munich-Wistar rat inner medullary thin limbs, lying at positions distinctly above the bend, had segments exhibiting structural characteristics of DTL located immediately adjacent to segments exhibiting structural characteristics of ATL. Multiple DTL-type and ATL-type segments of variable length existed along a single straight portion of these mixed tubules. Inner medullary thin limbs with repeating, sequential expression of DTL-type and ATL-type regions were also numerous in Sprague-Dawley rats, mice, and rabbits with no evidence of sexual dimorphism. RT-PCR of microdissected segments showed that the water channel aquaporin-1 (AQP1) and the urea transporter UT-A2 were expressed in pure DTL, but not in pure ATL, and in DTL-type, but not in ATL-type, regions of mixed-type thin limbs. Immunocytochemistry revealed expression of AQP1 in cells of pure DTL, but not pure ATL, and in cells of DTL-type, but not ATL-type, regions of mixed-type thin limbs. In contrast, the chloride channel ClC-K1 was expressed in pure ATL, but not pure DTL, and in ATL-type, but not DTL-type, regions of mixed-type thin limbs. Discontinuous axial expression of AQP1, UT-A2, and ClC-K1 along the straight portion of single thin limbs indicates that these nephrons possess a more heterogeneous structure than previously recognized.

Detailed mapping of ochratoxin A reabsorption along the rat nephron in vivo: the nephrotoxin can be reabsorbed in all nephron segments by different mechanisms.

Ochratoxin A (OTA) is a widespread nephrotoxin excreted to a substantial degree via the kidney. Previously we showed that [3H]OTA can be reabsorbed along the rat nephron in vivo (Zingerle et al., 1997). In this study we investigated in detail the contribution of different nephron segments to [3H]OTA reabsorption and determined the possible mechanisms involved by microinfusion and microperfusion experiments. At pH 6 (approximately 94% of OTA neutral), OTA is reabsorbed in all nephron segments investigated. The estimated fractional reabsorptions (FR) at a tubular load of 20 fmol/min are: proximal convoluted tubule (PCT), 14.8%; proximal straight tubule (PST), 27.4%; ascending limb of Henle's loop (ALH), 13.6%; distal tubule (DT), 11.6%; collecting duct (CD), 24.6%; terminal CD, 22.0%. At pH 8 (approximately 10% of OTA neutral) FR are as follows: PCT, 0%; PST, 25.9%; ALH, 14.0%; DT, 3.2%; CD, 8.2%. Thus, OTA reabsorption in PST and ALH in pH-independent. Reabsorption in PST but not in DT or CD was inhibited by sulfobromophthalein, a substrate of the apical organic anion carrier. L-Phenylalanine did not reduce OTA reabsorption. After intravenous injection of unlabeled OTA, resulting in a plasma concentration of approximately 10(-5) mol/l, the FR of [3H]OTA during early proximal microinfusion was reduced slightly. From our results we conclude: 1) OTA can be reabsorbed in all nephron segments investigated. 2) Under physiological conditions the predominant sites of reabsorption are PST, ALH and terminal CD. 3) Reabsorption in PST and ALH is not pH-dependent. 4) pH-independent reabsorption in PST is mediated by the apical organic anion transporter (OAT-K1), whereas pH-dependent reabsorption in PCT is mediated by H(+)-dipeptide cotransporter(s). 5) Reabsorption also takes place during natural exposure, i.e., when OTA is present in plasma and renal tissue. 6) The high FR in ALH and CD explains, at least in part, the preferential impairment of postproximal functions and the accumulation in renal inner medulla and papila.

The endolymphatic duct and sac of the rat: a histological, ultrastructural, and immunocytochemical investigation.

A study of the ultrastructure, vascularization, and innervation of the endolymphatic duct and sac of the rat has been performed by means of light- and electron-microscopic and immunocytochemical methods. Two different types of epithelial cells have been identified: the ribosome-rich cell and the mitochondria-rich cell. These two cell types make up the epithelium of the complete endolymphatic duct and sac, although differences in their quantitative distribution exist. The morphology of the ribosome-rich cells varies between the different parts of the endolymphatic duct and sac; the morphology of the mitochondria-rich cells remains constant. According to the epithelial composition, vascularization, and structural organization of the lamina propria, both duct and sac are subdivided into three different parts. A graphic reconstruction of the vascular network supplying the endolymphatic duct and sac shows that the vascular pattern varies among the different parts. In addition, the capillaries of the duct are of the continuous types, whereas those supplying the sac are of the fenestrated type. Nerve fibers do not occur within the epithelium of the endolymphatic duct and sac. A few nerve fibers regularly occur in the subepithelial compartment close to the blood vessels; these fibers have been demonstrated in whole-mount preparations by the application of the neuronal marker protein gene product 9.5. Single beaded fibers immunoreactive to substance P and calcitonin-gene related peptide are observed within the same compartment. Dopamine-beta-hydroxylase-immunoreactive axons are restricted to the walls of arterioles. Morphological differences between the different portions of the endolymphatic duct and sac are discussed with regard to possible roles in fluid absorption and immunocompetence.

Topography and distribution of nerve fibers in the posterior longitudinal ligament of the rat: an immunocytochemical and electron-microscopical study.

The distribution and immunocytochemical characterization of nerve fibers and their terminals in the posterior longitudinal ligament of the rat lumbar vertebral column was studied in whole-mount preparations and serial semithin and ultrathin sections. Differences in the localization, distribution pattern and density of peptidergic and catecholaminergic nerve fibers were found in the vertebral and intervertebral regions of the posterior longitudinal ligament. For immunocytochemistry, free floating specimens were incubated with primary antibodies against protein gene product 9.5, substance P, calcitonin gene-related peptide, dopamine-beta-hydroxylase, vasoactive intestinal polypeptide and neuropeptide Y together with the avidin-biotin-peroxidase method. In whole-mount preparations, the neural marker protein gene product 9.5 is immunostained in all unmyelinated nerve fibers in the posterior longitudinal ligament, thus giving a panoramic view of the nerve fiber plexus. The most striking nerve fiber plexus is localized in the intervertebral region. In this region, the posterior longitudinal ligament is rich in capillaries that form a dense plexus within its ventral part and extend to the outer layer of the annulus fibrosus. The peptidergic and catecholaminergic innervation of the posterior longitudinal ligament is discussed in the context of pain syndromes related to the vertebral column and degenerative lumbar spine diseases.