Protein kinase C-dependent phosphorylation and mitochondrial translocation of aldose reductase

Although aldose reductase (AR) is a critical participant in osmoregulation, and the metabolism of glucose and aldehydes derived from lipid peroxidation, post-translational mechanisms regulating its activity have not been identified. In this paper, we report that stimulation of protein kinase C (PKC) in several cell types induces phosphorylation of AR and translocation of the phosphorylated protein to the mitochondria. In vitro, recombinant AR was directly phosphorylated by activated PKC, suggesting that AR may be an in vivo PKC substrate. Together, these observations reveal a novel link between PKC activation and the regulation of glucose and aldehyde metabolism.     

Masking of pleomorphic glycogen sites by methanolic uranyl acetate.

Rat liver tissue was fixed in 2.5% glutaraldehyde buffered with cacodylic acid (pH 7.3) for 2 hr, washed twice in buffer, and postfixed in 2% osmium tetroxide at 4 C for 1 hr. The tissue then was dehydrated, infiltrated with and embedded in Epon by routine procedures. The ultrathin sections from this tissue, when stained with spectroscopic grade methanol saturated with uranyl acetate (SMUA) for 1 min followed by aqueous lead citrate (PbCi) (Reynolds 1963) for 5 min at room temperature, showed a uniform staining of all major cellular components except glycogen. The SMUA appeared to be specific for ribonuceloprotein granules, rendering them more prominent in the cytoplasm due to the lack of glycogen staining. The question of glycogen removal from the sections due to SMUA treatment was evulated using various extractions and staining methods. It appeared that SMUA pretreatment alters the subsequent binding ability of lead salts, resulting in lack of glycogen staining, although it does not remove the glycogen from the sections.     

Synthetic peptide analogues differentially alter the binding affinities of cyclic nucleotide dependent protein kinases for nucleotide substrates

Analogues of a synthetic heptapeptide substrate corresponding to the sequence around a phosphorylation site in histone H2B [Glass, D. B. & Krebs, E. G. (1982) J. Biol. Chem. 257, 1196-1200] were used to assess interactions between the peptide substrate and the ATP binding sites of cGMP-dependent protein kinase and the catalytic subunit of cAMP-dependent protein kinase. The affinity of each protein kinase for lin-benzo-ADP was determined in the absence and presence of substrate peptide by fluorescence anisotropy titrations [Bhatnagar, D., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 6310-6317]. The Kd values of cGMP-dependent protein kinase for lin-benzo-ADP in the absence and presence of cGMP were 7.6 and 9.7 microM, respectively. Histone H2B(29-35) (Arg-Lys-Arg-Ser-Arg-Lys-Glu) had no effect on nucleotide affinity in either the absence or presence of cGMP. However, when lysine-34 located two residues after the phosphorylatable serine is replaced with an alanyl residue, the resulting [Ala34]histone H2B(29-35) and its analogue peptides interact with cGMP-dependent protein kinase and/or the nucleotide in a fashion that decreases nucleotide binding affinity approximately 3-fold. This amino acid replacement had previously been shown to cause an increase in Vmax and a decrease in the pH optimum for the phosphotransferase reaction. Replacement of positively charged residues at positions 30 and 31 of the peptide also decreased nucleotide affinity. Other analogues of histone H2B(29-35) failed to affect binding of lin-benzo-ADP to the active site of the cGMP-dependent enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence variability in homologs of the aflatoxin pathway gene aflR distinguishes species in Aspergillus section Flavi.

The Aspergillus parasiticus aflR gene, a gene that may be involved in the regulation of aflatoxin biosynthesis, encodes a putative zinc finger DNA-binding protein. PCR and sequencing were used to examine the presence of aflR homologs in other members of Aspergillus Section Flavi. The predicted amino acid sequences indicated that the same zinc finger domain, CTSCASSKVRCTKEKPACARCIERGLAC, was present in all of the Aspergillus sojae, Aspergillus flavus, and Aspergillus parasiticus isolates examined and in some of the Aspergillus oryzae isolates examined. Unique base substitutions and a specific base deletion were found in the 5' untranslated and zinc finger region; these differences provided distinct fingerprints. A. oryzae and A. flavus had the T-G-A-A-X-C fingerprint, whereas A. parasiticus and A sojae had the C-C-C-C-C-T fingerprint at the corresponding positions. Specific nucleotides at positions -90 (C or T) and -132 (G or A) further distinguished A. flavus from A. oryzae and A. parasiticus from A. sojae, respectively. A sojae ATCC 9362, which was previously designated A. oryzae NRRL 1988, was determined to be a A. sojae strain on the basis of the presence of the characteristic fingerprint, A-C-C-C-C-C-C-T. The DNAs of other members of Aspergillus Section Flavi, such as Aspergillus nomius and Aspergillus tamarii, and some isolates of A. oryzae appeared to exhibit low levels of similarity to the A. parasiticus aflR gene since low amounts of PCR products or no PCR products were obtained when DNAs from these strains were used.     

Microbial Diversity in Soil,Sand Dune and Rock Substrates of the Thar Monsoon Desert,India

A culture-independent diversity assessment of archaea, bacteria and fungi in the Thar Desert in India was made. Six locations in Ajmer, Jaisalmer, Jaipur and Jodhupur included semi-arid soils, arid soils, arid sand dunes, plus arid cryptoendolithic substrates. A real-time quantitative PCR approach revealed that bacteria dominated soils and cryptoendoliths, whilst fungi dominated sand dunes. The archaea formed a minor component of all communities. Comparison of rRNA-defined community structure revealed that substrate and climate rather than location were the most parsimonious predictors. Sequence-based identification of 1240 phylotypes revealed that most taxa were common desert microorganisms. Semi-arid soils were dominated by actinobacteria and alpha proteobacteria, arid soils by chloroflexi and alpha proteobacteria, sand dunes by ascomycete fungi and cryptoendoliths by cyanobacteria. Climatic variables that best explained this distribution were mean annual rainfall and maximum annual temperature. Substrate variables that contributed most to observed diversity patterns were conductivity, soluble salts, Ca²⁺ and pH. This represents an important addition to the inventory of desert microbiota, novel insight into the abiotic drivers of community assembly, and the first report of biodiversity in a monsoon desert system.     

Biochemical mechanism of irreversible cell injury caused by free radical-initiated reactions

Effects of oxidative stress on isolated rat ventricular myocytes were studied. Myocyte viability was determined by the ability of these cells to retain rod-shaped morphology and to exclude trypan blue. The mean life time of myocytes was quantitated using the Weibull distribution function. Superfusion with 200 M tert-butyl hydroperoxide (t-BHP) led to a time-dependent loss of cell viability, generation of the products of lipid peroxidation, oxidation of protein and non-protein thiols, a decrease in [ATP]_i and in the cellular energy charge. Dithiothreitol (DTT, 5 mM) prolonged survival of myocytes exposed to t-BHP, attenuated oxidation of protein and non-protein thiols, and preserved the energy charge. Exposure to DTT did not affect the concentration of t-BHP-generated lipid peroxidation products. Promethazine (1 M) prevented t-BHP-induced increase in the concentration of lipid peroxidation products, but did not prevent either loss of thiols or loss of cell viability. Superfusion with N-ethylmaleimide (NEM, 5 M) also led to loss of cell viability, with accompanying decreases in protein and non-protein thiols, ATP and energy charge without the accumulation of the products of lipid peroxidation. Superfusion with FeSO₄ (400 M) and ascorbate (1 mM), (Fe-Asc) did not result in loss of cell viability or a decrease protein thiols or the energy charge. Superfusion with Fe-Asc, did, however, lead to a slight decrease in the concentration of non-protein thiols and ATP and a large increase in the concentration of lipid peroxidation products. Accumulation of lipid peroxidation products induced by Fe-Asc was prevented by promethazine. These results indicate that free radical-induced irreversible cell injury results from a loss of protein thiols. Changes in the cellular energy charge and lipid peroxidation do not bear a simple relationship to the survival of cardiac myocytes under oxidative stress.     

A comparison of methods measuring aromatase activity in human placenta and rat ovary

The single step chromatographic product isolation method using [4-14C]-androstenedione and the tritiated water method using either [1 beta, 2 beta-3H]- or [1 beta-3H]-androstenedione have been used to determine a suitable method to measure the aromatase activity in rat ovarian 1000 g supernatant and human placental microsomes. The single step product isolation method using [4-14C]A4 reveals the presence of four distinct [4-14C]-labelled products in the rat ovary of which only the synthesis of estradiol is markedly inhibited by CGS 16949A, a well established aromatase inhibitor. In the human placenta, the formation of both [4-14C]-estrone and [4-14C]-estradiol is strongly inhibited by CGS 6949A. Therefore, in the rat ovary spurious results are obtained if accumulative radiolabelled product formation is measured without characterisation of the products. The Vmax in the rat ovary using [1 beta, 2 beta-3H]-A4 as a substrate is 13.7 pmol/h/mg compared to 2.9 pmol/h/mg when [1 beta-3H]-A4 is used. In the human placenta, the Vmax is similar using either [1 beta, 2 betat-3H]-A4 or [1 beta-3H]-A4 (1.21 and 1.27 nmol/h/mg, respectively). Consistent results are obtained for the human placenta using either the single step chromatographic product isolation method or the tritiated water method. However, in the rat ovary the more suitable method of the two used to measure the aromatase activity is the tritiated water method employing [1 beta-3H]-A4 as a substrate. Aromatase activity in the rat ovary during estrus cycle was measured using the tritiated water method employing [1 beta-3H]-A4 as a substrate. A peak of aromatase activity at proestrus was seen which returned rapidly to its basal level at estrus. Plasma estradiol concentrations were in parallel with the aromatase activity.     

Towards the mechanism of altered nutrients uptake in renal brush border membrane vesicles from pyelonephritic rats

Affinity constant (Km) of D-glucose, L-alanine, L-aspartate, L-lysine, L-proline and nutrients coupled Na+ were determined in renal brush border membrane vesicles prepared from control and pyelonephritic rats. The Km of D-glucose, amino acids and nutrients coupled Na+ was noted to be significantly increased (p less than 0.001) in experimental animals. The Vmax of D-glucose and amino acids was determined at different concentrations of nutrients keeping extravesicular Na+ constant or at different concentrations of extravesicular Na+ keeping nutrient concentration constant. In the experimental rats the Vmax decreased significantly (p less than 0.01) when compared to control. The increased Km and decreased Vmax may be one of the underlying mechanism leading to decrease in the uptake of D-glucose and amino acids.     

Ultrastructure of the pineal body of the common vampire bat, Desmodus rotundus

The type AB pineal body of the common vampire bat, Desmodus rotundus, was recessed and lobulated, was extensively vascularized and intimately related to great veins, and was unassociated with the epithalamic region. The habenular and the posterior commissures coursed anteriorly and were unassociated with the pineal. The saccular suprapineal recess of the third ventricle extended dorsally juxtaposed to the pineal body. These anatomical features are likely to make pinealectomies in the vampire more difficult to manage. The pineal parenchyma consisted of light pinealocytes surrounded by canaliculi of various sizes, often transmitting unmyelinated nerve fibers and glial processes. Desmosomes were common. The pinealocyte nuclei were large and highly infolded; characteristic cytoplasmic constituents included abundant dilated Golgi complexes associated with clear vesicles, numerous polyribosomes, few single cisternae of ribosome-studded rough endoplasmic reticulum, mitochondria, and occasional multivesicular bodies and lysosomes. Almost all pinealocytes exhibited centrioles and some, in addition, displayed basal bodies but rarely ciliary shafts. A conspicuous feature of the pinealocyte cytoplasm was the presence of branched bundles of intermediate filaments, especially in the perinuclear zone. Siderotic macrophages, lipofuscin-pigment-containing phagocytic cells, mast cells, myelin bodies, and both fenestrated and continuous capillaries were present. The perivascular compartment was densely packed with unmyelinated nerve bundles containing small to large fibers exhibiting axoaxonic densities. Other constituents of the perivascular compartment were club-shaped pinealocyte processes filled with clear vesicles, microtubules, an occasional mitochondrion, glial processes, and collagen fibers. "Synapselike" contacts were observed between the axons and pinealocyte processes. Abundant pinocytotic vesicles in the capillary endothelium indicated active pinocytosis. Myelinated nerve fibers were lacking. The pineal ultrastructure of Desmodus is in part unlike that reported for other mammals, including bats.