Peroxisomes and reactive oxygen species,a lasting challenge

Oxidases generating and enzymes scavenging H₂O₂ predestine peroxisomes (PO) to a pivotal organelle in oxygen metabolism. Catalase, the classical marker enzyme of PO, exhibits both catalatic and peroxidatic activity. The latter is responsible for the staining with 3,3′-diamino-benzidine, which greatly facilitated the visualization of the organelle and promoted further studies on PO. d-Amino acid oxidase catalyzes with strict stereospecificity the oxidative deamination of d-amino acids. The oxidase is significantly more active in the kidney than in liver and more in periportal than pericentral rat hepatocytes. Peroxisomes in these tissues differ in their enzyme activity and protein concentration not only in adjacent cells but even within the same one. Moreover, the enzyme appears preferentially concentrated in the central region of the peroxisomal matrix compartment. Urate oxidase, a cuproprotein catalyzing the oxidation of urate to allantoin, is confined to the peroxisomal core, yet is lacking in human PO. Recent experiments revealed that cores in rat hepatocytes appear in close association with the peroxisomal membrane releasing H₂O₂ generated by urate oxidase to the surrounding cytoplasma. Xanthine oxidase is exclusively located to cores, oxidizes xanthine thereby generating H₂O₂ and O₂ ^– radicals. The latter are converted to O₂ and H₂O₂ by CuZn superoxide dismutase, which has been shown recently to be a bona fide peroxisomal protein. Presented at the 50th Anniversary Symposium of the Society for Histochemistry, Interlaken, Switzerland, October 1-4, 2008.     

Electron microscopic cytochemical localization ofα-hydroxyacid oxidase in rat kidney cortex

Summary The substrate specificity of-hydroxyacid oxidase in the rat kidney has been investigated cytochemically by the cerium technique and biochemically with a luminometric assay applied to isolated renal peroxisomes. Rat kidneys were fixed by perfusion via the abdominal aorta with a low concentration (0.25%) of glutaraldehyde. Vibratome sections were incubated for 60 min at 37°C in a medium containing 3 mM CeCl₃, 100 mM NaN₃ and 5 mM of an-hydroxyacid in 0.1M Pipes or 0.1M Tris-maleate buffer both adjusted to pH 7.8. Ten aliphatic -hydroxyacids with chain lengths between 2 and 8 carbon atoms and two aromatic substrates were tested. The -hydroxyacid oxidase in the kidney exhibited a markedly different substrate specificity than the corresponding enzyme in the liver. Thus glycolate gave a negative reaction while two aromatic substrates, mandelic acid and phenyllactic acid, stained prommently. With aliphatic substrates a stronger reaction was obtained in Pipes than in theTris-maleate buffered incubation media. The best reaction in the kidney was obtained with hydroxybutyric acid. These cytochemical findings were confirmed by the luminometric determination of the oxidase activity in isolated purified peroxisome fractions. By electron microscopy the electron dense reaction product of cerium perhydroxide was found in the matrix of peroxisomes in the proximal tubules. The intensity of reaction varied markedly in neighbouring epithelial cells but also in different peroxisomes within the same cell. Thus heavily stained particles were seen next to lightly reacted ones. These observations establish the substrate specificity of -hydroxyacid oxidase in the rat kidney and demonstrate the marked heterogeneity in the staining of renal peroxisomes for this enzyme.     

Are the renin-containing granules of juxtaglomerular epithelioid cells modified lysosomes?

Summary Mature secretory granules of epithelioid cells — the so-called renin granules — exhibit certain properties, which in this particular combination are expressed only by lysosomes: Renin granules have autophagic capabilities; they react to the application of lipidosis-inducing, lysosomotropic substances by the gradual accumulation of polar lipids; all secretory granules of epithelioid cells contain acid phosphatase until maturity; and exogenous tracers reach renin granules without labeling the Golgi complex. Several functional implications can therefore be considered. Hydrolytic enzymes, constitutive elements of the granule matrix, might either cleave inactive prorenin to yield active renin within the granules or, by unspecific hydrolysis of renin, participate in the regulation of the overall quantity of secretory product. Autophagic phenomena, the involvement of renin granules in the traffic of exogenous tracers, and the build-up of polar lipids following experimental interference with lipid catabolism indicate a large turnover of membrane material in renin granules. They also suggest that cytoplasmic and extracellular fluid gains access to the granule content and may thus be involved there in the regulation of biochemical reactions by changing the intragranular milieu or via signal molecules.In addition to the lysosome-like properties of epithelioid cell secretory granules, the secretory product, renin, as a carboxyl protease, is structurally related to other acidic proteases. In the case of cathepsin D, even functional similarities exist.These studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System.     

Nucleotide sequence and genetic analysis of the Rhodobacter capsulatus ORF6-nifUISVW gene region: possible role of Nif W in homocitrate processing

DNA sequence analysis of a 3494-bp HindIII-Bc1I fragment of the Rhodobacter capsulatus nif region A revealed genes that are homologous to ORF6, nifU, nifS, nifV and nifW from Azotobacter vinelandii and Klebsiella pneumoniae. R. capsulatus nifU, which is present in two copies, encodes a novel type of NifU protein. The deduced amino acid sequences of NifU_I and NifU_II share homology only with the C-terminal domain of NifU from A. vinelandii and K. pneurnoniae. In contrast to nifA andnifB which are almost perfectly duplicated, the predicted amino acid sequences of the two NifU proteins showed only 39% sequence identity. Expression of the ORF6-nifU _ISVW operon, which is preceded by a putative ⁵⁴-dependent promoter, required the function of NifA and the nif-specific rpoN gene product encoded by nifR4. Analysis of defined insertion and deletion mutants demonstrated that only nifS was absolutely essential for nitrogen fixation in R. capsulatus. Strains carrying mutations in nifV were capable of very slow diazotrophic growth, whereas ORF6, nifU _I and nifW mutants as well as a nifU _I/nifU_II, double mutant exhibited a Nif⁺ phenotype. Interestingly, R. capsulatus nifV mutants were able to reduce acetylene not only to ethylene but also to ethane under conditions preventing the expression of the alternative nitrogenase system. Homocitrate added to the growth medium repressed ethane formation and cured the NifV phenotype in R. capsulatus. Higher concentrations of homocitrate were necessary to complement the NifV phenotype of a polar nifV mutant (NifV^–NifW^–), indicating a possible role of NifW either in homocitrate transport or in the incorporation of this compound into the iron-molybdenum cofactor of nitrogenase.     

The genetic locus controlling supernodulation in soybean (Glycine max L.) co-segregates tightly with a cloned molecular marker.

Summary The genetic locus (nts) controlling nitrate-tolerant nodulation, supernodulation, and diminished autoregulation of nodulation of soybean (Glycine max (L.) Merill) was mapped tightly to the pA-132 molecular marker using a restriction fragment length polymorphism (RFLP) detected by subclone pUTG-132a. The nts (nitrate-tolerant symbiotic) locus of soybean was previously detected after its inactivation by chemical mutagenesis. Mutant plant lines were characterized by abundant nodulation (supernodulation) and tolerance to the inhibitory effects of nitrate on nodule cell proliferation and nitrogen fixation. The large number of RFLPs between G. max line nts382 (homozygous for the recessive nts allele) and the more primitive soybean G. soja (P1468.397) allowed the detection of co-segregation of several diagnostic markers with the supernodulation locus in F₂ families. We located the nts locus on the tentative RFLP linkage group E about 10 cM distal to pA-36 and directly next to marker pA-132. This very close linkage of the molecular marker and the nts locus may allow the application of this clone as a diagnostic probe in breeding programs as well as an entry point for the isolation of the nts gene.     

Immunohistochemical Analysis of 1,25-dihydroxyvitamin D3 Receptor in Cervical Carcinoma

The immunohistochemical localization and expression of 1,25-dihydroxyvitamin D₃ receptors (VDR) has been investigated in normal human cervical tissue (n = 15) and in cervical carcinomas (n = 23). VDR immunoreactivity (monoclonal antibody 9A7r354;) was compared with the staining patterns of transglutaminase K, cytokeratin 10 and Ki-67 in these tumours. Moderate to strong nuclear immunoreactivity for VDR was detected in almost all cervical carcinomas analysed. VDR staining was homogeneous, with no visual differences between individual tumour cells. Some 60% of normal cervical tissues revealed weak immunoreactivity for VDR. In normal cervical tissue, nuclear VDR staining was confined to the lower cervical layers, predominantly to the basal cell layer. Both the intensity of VDR immunostaining and the number of VDR-positive cells were up-regulated in cervical carcinomas compared with normal cervical tissue. No visual correlation wa s found for the coexpression of VDR with markers of proliferation and differentiation. Our findings indicate that: (1) cervical tissue may be a new target organ for therapeutically applied vitamin D analogues; (2) VDR is up-regulated at the protein level in cervical carcinomas compared with normal cervical tissue; (3) up-regulation of VDR in cervical carcinoma is induced not exclusively by alterations in epithelial differentiation or proliferation, but by different, unknown mechanisms; and (4) calcitriol and new vitamin D analogues exerting fewer calcaemic side-effects may be promising new drugs for the treatment or chemoprevention of metastasizing cervical carcinomas as well as of cervical precancerous lesions.     

Morphology and anatomy ofCaloplaca coralligera (Teloschistaceae) as adaptation to extreme environmental conditions in the maritime Antarctic

The anatomical and morphological structure of the lichenCaloplaca coralligera (Hue.)Zahlbr. was investigated in connection with mechanisms of colonization and adaptation to the special conditions of maritime Antarctic.Caloplaca coralligera seems to be endemic to the antarctic region. The lichen is unique in its morphology, growing like a crustose lichen with a prothallus, but the very thallus being composed of many vertical, frutescent branches not higher than large isidia. Due to fusions of the distal parts of the branches the morphology is characterized by air-filled cavities between the erect thallus parts where the phycobionts are located only close to the outermost surface. But also anatomically a system of air-filled cavities is developed by a net-like structure of hyphae. This may result in an insulation from temperature exchanges with the rocky substrate. It is speculated that this peculiar thallus structure might be advantageous to a lichen growing on compact substrates by buffering the diurnal temperature extremes which are characteristic for rocks under a strong radiation exchange with the atmospheric surrounding.Dedicated to Prof. DrO. L. Lange on the occasion of his retirement and of his 65th birthday.     

Specific Transport of Inorganic Phosphate and C3- and C6-Sugar-Phosphates across the Envelope Membranes of Tomato (Lycopersicon esculentum) Leaf-Chloroplasts,Tomato Fruit-Chloroplasts and Fruit-Chromoplasts

Plastidial envelope membranes were isolated from tomato (Lycopersicon esculentum) leaves and green and red tomato fruits by isopycnic discontinuous sucrose density gradient centrifugation. Solubilized envelope membrane proteins were reconstituted into liposomes. Transport measurements revealed that the phosphate translocator from tomato leaves transports inorganic phosphate, 3-phosphoglycerate and triosephosphates. The phosphate translocators of green and red fruit plastids catalyze, in addition to the transport of these substrates, also the transport of glucose-6-phosphate, glucose-1-phosphate and phosphoenolpyruvate.     

Long-term variability of winter nitrate concentrations in the Northern Wadden Sea driven by freshwater discharge,decreasing riverine loads and denitrification

The hypothesis of a recent reversal in the eutrophication of the Wadden Sea and the potential of inshore waters in denitrification is explored. Salinity, temperature and nitrate concentrations in the List Tidal Basin (Northern Wadden Sea) have been measured about twice weekly since 1984. Salinity has a clear seasonal cycle with lowest salinities of about 27 in late winter and highest salinities of about 31 in summer. Mean annual deviations from the long-term mean salinity correlate significantly with riverine freshwater discharge. Winter nitrate concentrations are generally high (about 50 μM on average). The major part of the variability is related to salinity (∼35%). Temperature had a minor impact (∼1%). Superimposed on this, a long-term decrease of about 1 μM per year was found. Together, these processes account for about 45% of the nitrate variability. The long-term decrease of about 2% per year is similar to continental riverine trend in total nitrogen loads. In contrast to the List Tidal Basin, salinity explained more than 90% of nitrate variability in the off-shore German Bight. Salinity (30) normalised winter nitrate data of the German Bight also show a long-term decreasing trend. Most of the List Tidal Basin data are either on or below the nitrate–salinity relation found in the German Bight. This observation suggests that denitrification has a major impact on the winter nitrate concentrations in the Northern Wadden Sea compared to the German Bight. It is hypothesised that a large part of the unexplained variability is related to weather-dependent changes in residence time of tidal water masses in the Wadden Sea and circulation patterns within the German Bight.