Effect of interferon on the course of experimental E coli-induced pyelonephritis

The effect of the type I interferon on the development and process of experimental pyelonephritis caused by E. coli was studied on mice weighing 12 to 14 g. Interferon was administered intraperitoneally in a dose of 1000 units on days 3 and 7 of the disease. It was shown that the administration of the type I interferon to the mice with experimental pyelonephritis promoted rapid elimination of bacteria from the kidneys, prevented their penetration to the contralateral (intact) kidney, prevented marked macro- and microscopic damages in the kidneys, lowered the intensity of the inflammatory reaction, and increased the phagocytic activity of neutrophils and the number of the E-rosette-forming lymphocytes in the thymus. The data provided experimental grounding for clinical trials of interferon preparations in treatment of bacterial pyelonephritis.     

Big flies, small repeats: the "Thr-Gly" region of the period gene in Diptera

The region of the clock gene period (per) that encodes a repetitive tract of threonine-glycine (Thr-Gly) pairs has been compared between Dipteran species both within and outside the Drosophilidae. All the non- Drosophilidae sequences in this region are short and present a remarkably stable picture compared to the Drosophilidae, in which the region is much larger and extremely variable, both in size and composition. The accelerated evolution in the repetitive region of the Drosophilidae appears to be mainly due to an expansion of two ancestral repeats, one encoding a Thr-Gly dipeptide and the other a pentapeptide rich in serine, glycine, and asparagine or threonine. In some drosophilids the expansion involves a duplication of the pentapeptide sequence, but in Drosophila pseudoobscura both the dipeptide and the pentapeptide repeats are present in larger numbers. In the nondrosophilids, however, the pentapeptide sequence is represented by one copy and the dipeptide by two copies. These observations fulfill some of the predictions of recent theoretical models that have simulated the evolution of repetitive sequences.      

Characterization of Caenorhabditis elegans exonuclease-3 and evidence that a Mg2+-dependent variant exhibits a distinct mode of action on damaged DNA

The Caenorhabditis elegans genes, exo-3 and apn-1, encode the proteins EXO-3 and APN-1, belonging to the exo III and endo IV families of apurinic/apyrimidinic (AP) endonucleases/3'-diesterases, respectively. Homologues of EXO-3 and APN-1 in E. coli and yeast have been clearly documented to repair AP sites and DNA strand breaks with blocked 3' ends to prevent genomic instability. Herein, we purified the C. elegans EXO-3, expressed as a Gst-fusion protein in yeast, and demonstrated that it possesses strong AP endonuclease and 3'-diesterase activities. However, unlike the E. coli counterpart exonuclease III, EXO-3 shows no significant level of 3' --> 5' exonuclease activity following incision at AP sites. In addition, EXO-3 lacks the ability to directly incise DNA at the 5' side of various oxidatively damaged bases, as observed for the human counterpart Ape1, suggesting that C. elegans evolved a member with tailored functions. Importantly, a variant form of EXO-3, E68A, demonstrates altered magnesium-binding properties, and although the in vitro AP endonuclease is nearly fully recovered in the presence of MgCl2, the 3'-diesterase activity is reduced when compared to the native enzyme. We suggest that Glu68 plays a role in coordinating Mg2+ binding for the enzyme catalytic mechanism. Further analysis reveals that neither purified Gst-EXO-3 nor the E68A variant forms a readily detectable DNA-protein complex with an oligonucleotide substrate containing either an AP site or an alpha,beta-unsaturated aldehyde at its 3' end. However, if the reaction is conducted in the presence of crude extracts derived from either yeast or C. elegans embryos, only E68A forms a distinct slow migrating DNA-protein complex with each of the substrates, suggesting that Glu68 may be required to facilitate the release of EXO-3 from the incised DNA to allow entry of the remaining components of the base-excision repair pathway. Thus, the slow migrating DNA-protein complex formed by the E68A variant could be indicative of a stalled repair process with associated factor(s).     

Barley microgreen incorporation in diet-controlled diabetes and counteracted aflatoxicosis in rats

Increased environmental pollution and unhealthy lifestyle are blamed for escalated chronic diseases. Exposure to aflatoxins was recently suggested to have a role in the increased incidence of type 2 diabetes mellitus. Diet modification and consumption of different functional food are now gaining attention, especially in diabetes management. This study investigates the effect of a diet containing barley microgreen against diabetes induced by streptozotocin with or without aflatoxin administration in rats. Barley microgreen was rich in 3′-Benzyloxy-5,6,7,4′-tetramethoxyflavone (48.8% of total) followed by 5β,7βH,10α-Eudesm-11-en-1α-ol (18.46%). Streptozotocin injection and/or aflatoxin administration significantly elevated glucose level, decreased insulin level, decreased β-cell function, deteriorated liver and kidney function parameters, and induced oxidative stress in the liver. Histopathology revealed irregular small-sized islets and decreased area % of insulin-positive beta cells in the pancreas, hepatic degeneration, nephropathy, and neuropathy in diabetic and/or aflatoxin administered rats compared to control. Barley microgreen diet fed to diabetic rats with or without aflatoxin alleviated all evaluated parameters. Barley microgreen diet also ameliorated the toxic effect of aflatoxin. In conclusion, exposure to aflatoxin aggravated diabetes and its complication. The incorporation of barley microgreen in the diet was able to control type 2 diabetes mellitus and the improved outcomes observed with barley microgreen treatments involved or occurred in conjunction with improved biomarkers of oxidative stress.     

Finding the best antibody dilution by repeated immunostaining of the same tissue section

One can find the optimal antibody dilution for immunostaining by repeated staining on the same tissue section by using a less dilute antibody for each attempt. Using secondary antibody and horseradish peroxidase conjugated to a dextran polymer, a section stained repeatedly with several dilutions of antibody appears as good as a section stained with only the last dilution.     

Repeated immunostaining of the same tissue section using alkaline phosphatase as a reporter

One can determine the best dilution of a primary antibody for immunohistochemistry that uses horseradish peroxidase conjugated to a secondary antibody by testing increasing concentrations sequentially on the same tissue section. When the same tissue section is incubated repeatedly with increasing concentrations of primary antibodies to epithelial membrane antigen, smooth muscle α-actin, or vimentin using alkaline phosphatase conjugated to a secondary antibody as the reporter, the best staining was obtained with a less concentrated primary antibody than was optimal for a single staining test. The best concentration of primary antibody for single run staining using an alkaline phosphatase reporting system is usually four times the best concentration for staining with multiple runs. The optimal concentration can be determined by denaturing the residual alkaline phosphatase and extracting residual stain by incubating the section in 4:1 diglyme:phosphate buffered saline for 20 min at 80^o C between tests of primary antibody concentrations. I tested the method for four chromogens from one supplier and one chromogen from a different supplier.     

Thermodynamic,kinetic, and structural basis for recognition and repair of 8-oxoguanine in DNA by Fpg protein from Escherichia coli

X-ray analysis does not provide quantitative estimates of the relative importance of the molecular contacts it reveals or of the relative contributions of specific and nonspecific interactions to the total affinity of specific DNA to enzymes. Stepwise increase of DNA ligand complexity has been used to estimate the relative contributions of virtually every nucleotide unit of 8-oxoguanine-containing DNA to its total affinity for Escherichia coli 8-oxoguanine DNA glycosylase (Fpg protein). Fpg protein can interact with up to 13 nucleotide units or base pairs of single- and double-stranded ribo- and deoxyribo-oligonucleotides of different lengths and sequences through weak additive contacts with their internucleotide phosphate groups. Bindings of both single-stranded and double-stranded oligonucleotides follow similar algorithms, with additive contributions to the free energy of binding of the structural components (phosphate, sugar, and base). Thermodynamic models are provided for both specific and nonspecific DNA sequences with Fpg protein. Fpg protein interacts nonspecifically with virtually all of the base-pair units within its DNA-binding cleft: this provides approximately 7 orders of magnitude of affinity (Delta G degrees approximately equal to -9.8 kcal/mol) for DNA. In contrast, the relative contribution of the 8-oxoguanine unit of the substrate (Delta G degrees approximately equal to -0.90 kcal/mol) together with other specific interactions is <2 orders of magnitude (Delta G degrees approximately equal to -2.8 kcal/mol). Michaelis complex formation of Fpg protein with DNA containing 8-oxoguanine cannot of itself provide the major part of the enzyme specificity, which lies in the k(cat) term; the rate is increased by 6-8 orders of magnitude on going from nonspecific to specific oligodeoxynucleotides.     

Excitation of System I and System II in Photosynthesis as a Possible Control Mechanism of Glycolate Metabolism in the Blue-Green Alga Anacystis nidulans

Photosynthetic enhancement studies performed at 619 nm (excitation of Systems I and II) and at 446 nm (mainly excitation of System I) revealed an 18% photosynthetic enhancement simultaneously with a 31% reduction in glycolate excretion. This observation supports the hypothesis that some glycolate may be consumed in an oxidation process associated with System I when System II is poorly excited and the supply of electrons from the water splitting process of photosynthesis is low.