Selective cytochemical localization of peroxidase,cytochrome oxidase and catalase in rat liver with 3,3′-diaminobenzidine

Summary In rat liver, three different enzymes with peroxidatic activity are demonstrated with modifications of the DAB-technique: peroxidase in the endoplasmic reticulum of Kupffer cells, catalase in peroxisomes and cytochrome oxidase in mitochondria. The major problem of the DAB-methods is their limited specifity so that often in tissues incubated for one enzyme the other two proteins are also stained simultaneously. We have studied the conditions for selective staining of each of these three enzymes in rat liver fixed either by perfusion with glutaraldehyde or by immersion in a modified Karnovsky's glutaraldehyde-formaldehyde fixative. The observations indicate that in perfusion fixed material selective staining can be obtained by reduction of the incubation time (5 min) and the use of optimal conditions for each enzyme. In livers fixed by immersion the distribution of the staining is patchy and irregular and usually longer incubation times (15–30 min) are required. Selective staining of peroxidase in Kupffer cells was obtained by brief incubation at room temperature in a medium containing 2.5 mM DAB in cacodylate buffer pH 6.5 and 0.02% H₂O₂. The exclusive staining for cytochrome oxidase in cristae of mitochondria was achieved after short incubation in 2.5 mM DAB in phosphate buffer pH 7.2 containing 0.05% cytochrome c. For selective demonstration of catalase in peroxisomes the tissue was incubated in 5 mM DAB in Teorell-Stenhagen (or glycine-NaOH) butffer at pH 10.5 and 0.15% H₂O₂. The prolongation of the incubation time in peroxidase medium caused marked staining of both mitochondria and peroxisomes. In the cytochrome oxidase medium longer incubations led to slight staining of peroxisomes. The catalase medium was quite selective for this enzyme so that even after incubation for 120 min only peroxisomes stained.     

Biochemical and biophysical studies on cytochrome c oxidase. XVII. An epr study of the photodissociation of cytochrome a32+ · CO

1. The photodissociation reaction of the cytochrome c oxidase-CO compound was studied by EPR at 15 °K. Illumination with white light at both room and liquid N₂ temperatures of the partially reduced cytochrome c oxidase (2 electrons per 4 metals) in the presence of CO, causes the appearance of a rhombic (g_x = 6.60, g_y = 5.37) high-spin heme signal.This signal disappears completely upon darkening of the sample and reappears upon illumination at room temperature; accordingly the photolytic process is reversible. Under these conditions, no great changes in the intensities are observed, neither of the copper signal at g = 2, nor of the low-spin heme signal at g = 3, 2.2 and 1.5.2. In the presence of ferricyanide (2 mM) and CO, both the low-spin heme signal (g = 3.0, 2.2 and 1.5) and the copper signal of the partially reduced enzyme have intensities about equal to those of the completely oxidized enzyme in the absence of CO. Upon illumination of the carboxy-cytochrome c oxidase in the presence of ferricyanide, it was found that the rhombic high-spin heme signal appears without affecting appreciably the copper of low-spin heme signals. Thus, in the presence of ferricyanide the EPR-detectable paramagnetism of the illuminated carboxy-cytochrome c oxidase is higher than in the untreated oxidized enzyme.3. The membrane-bound cytochrome c oxidase reduced with NADH in the presence of CO and subsequently oxidized with ferricyanide shows a similar rhombic high-spin heme signal (g_x = 6.62, g_y = 5.29) upon illumination at room temperature. This signal disappears completely upon darkening and reappears upon illumination at room temperature.     

In vivo radioprotection studies of 3,3′-diselenodipropionic acid,a selenocystine derivative

3,3′-Diselenodipropionic acid (DSePA), a diselenide and a derivative of selenocystine, was evaluated for in vivo radioprotective effects in Swiss albino mice, at an intraperitoneal dose of 2 mg/kg body wt, for 5 days before whole-body exposure to γ-radiation. The radioprotective efficacy was evaluated by assessing protection of the hepatic tissue, the spleen, and the gastrointestinal (GI) tract and survival against sub- and supralethal doses of γ-radiation. DSePA inhibited radiation-induced hepatic lipid peroxidation, protein carbonylation, loss of hepatic function, and damage to the hepatic architecture. DSePA also attenuated the depletion of endogenous antioxidants such as glutathione, glutathione peroxidase, superoxide dismutase, and catalase in the livers of irradiated mice. DSePA also restored the radiation-induced reduction in villus height, crypt cell numbers, and spleen cellularity, indicating protective effects on the GI tract and the hematopoietic system. The results from single-cell gel electrophoresis of the peripheral blood leukocytes showed that DSePA can attenuate radiation-induced DNA damage. The mRNA expression analysis of genes revealed that DSePA augmented GADD45α and inhibited p21 in both spleen and liver tissues. DSePA also inhibited radiation-induced apoptosis in the spleen and reversed radiation-induced alterations in the expression of the proapoptotic BAX and the antiapoptotic Bcl-2 genes. In line with these observations, DSePA improved the 30-day survival of irradiated mice by 35.3%. In conclusion, these findings clearly confirm that DSePA exhibits protective effects against whole-body γ-radiation and the probable mechanisms of action involve the maintenance of antioxidant enzymes, prophylactic action through the attenuation of the DNA damage, and inhibition of apoptosis.     

Genetic and biochemical studies on flavonoid 3′-hydroxylation in flowers of Petunia hybrida

Summary In flower extracts of defined genotypes of Petunia hybrida, an enzyme activity was demonstrated which catalyses the hydroxylation of naringenin and dihydrokaempferol in the 3-position. Similar to the flavonoid 3-hydroxylases of other plants, the enzyme activity was found to be localized in the microsomal fraction and the reaction required NADPH as cofactor. A strict correlation was found between 3-hydroxylase activity and the gene Ht1, which is known to be involved in the hydroxylation of flavonoids in the 3-position in Petunia. Thus, the introduction of the 3-hydroxyl group is clearly achieved by hydroxylation of C₁₅-intermediates, and the concomitant occurrence of the 3,4-hydroxylated flavonoids quercetin and cyanidin (paeonidin) in the presence of the functional allele Ht1 is due to the action of one specific hydroxylase catalysing the hydroxylation of common precursors for both flavonols and anthocyanins.     

Evidence for cytochrome oxidase subunit I and a cytochrome c--subunit II fused protein in the cytochrome 'c1aa3' of Thermus thermophilus. How old is cytochrome oxidase?

The terminal cytochrome c1aa3 of the respiratory chain of Thermus thermophilus has been isolated and purified to homogeneity by a novel procedure. The two subunit proteins (55 and 33 kDa) have been characterized chemically. Computer searches with partial amino acid sequences obtained from both subunits show that the larger subunit belongs to the cytochrome oxidase subunit I protein family while the smaller covalently heme-binding subunit is not a cytochrome c1 but appears to be a fused protein between cytochrome c and cytochrome oxidase subunit II. With respect to the 16-S rRNA-derived phylogeny of procaryotes, the results show that the genetic information for an O2-reacting cytochrome oxidase (EC 1.9.3.1) existed already in early eubacteria.     

Cytochrome oxidase activity associated with the inside of the cytoplasmic membrane in swarm cells and sheath-forming cells of Sphaerotilus natans: an ultrastructural study using 3,3′-diaminobenzidine (DAB)

Cytochrome oxidase activity has been demonstrated by electronmicroscopical cytochemical methods in swarm cells as well as in the sheathforming cells of Sphaerotilus natans. This enzyme is associated with the inside of the cytoplasmic membrane. The conditions for demonstrating this cytoplasmic membrane-associated cytochrome oxidase differ from those enabling the demonstration of the cytochrome oxidase in the polar organelle of this bacterial species. The location of this enzyme also contrasted to that generally known from other bacteria and from mitochondria.     

Solid Phase Conjugation Chemistry: Use of Alloc as a Protecting Group for 2′-Aminolinker Containing Oligonucleotides

Abstract

The drug properties of antisense and antigene oligonucleotides can he enhanced by strategic positioning of ligands capable of ameliorating these properties.^1,2 Certain ligands may improve the cellular delivery of oligomers and increase their affinity for the target gene and resistance to nucleases. The 2′-O-position is an attractive modification site.³ Oligonucleotides possessing the 2′-O-akyl modifications exhibit higher chemical stability under depurination conditions, higher stability to enzymatic cleavage by both endo- and exonucleases, and increased affinity for target mRNA. In addition, they form highly stable triple helices. Thus they promise to be versatile compounds in controlling gene expression by antisense and antigene technologies.     

Structure–activity studies on the upstream splice junction of a semisynthetic intein

Protein trans-splicing by split inteins holds great potential for the chemical modification and semisynthesis of proteins. However, the structural requirements of the extein sequences immediately flanking the intein are only poorly understood. This knowledge is of particular importance for protein labeling, when synthetic moieties are to be attached to the protein of interest as seamlessly as possible. Using the semisynthetic Ssp DnaB intein both in form of its wild-type sequence and its evolved M86 mutant, we systematically varied the sequence upstream of the short synthetic Int^N fragment using both proteinogenic amino acids and unnatural building blocks. We could show for the wild-type variant that the native N-extein sequence could be reduced to the glycine residue at the (?1) position directly flanking the intein without significant loss of activity. The glycine at this position is strongly preferred over building blocks containing a phenyl group or extended alkyl chain adjacent to the scissile amide bond of the N-terminal splice junction. Despite their negative effects on the splicing yields, these unnatural substrates were well processed in the N–S acyl shift to form the respective thioesters and did not result in an increased decoupling of the asparagine cyclization step at the C-terminal splicing junction. Therefore, the transesterification step appeared to be the bottleneck of the protein splicing pathway. The fluorophore 7-hydroxycoumarinyl-4-acetic acid as a minimal N-extein was efficiently ligated to the model protein, in particular with the M86 mutant, probably because of its higher resemblance to glycine with an aliphatic c-α carbon atom at the (?1) position. This finding indicates a way for the virtually traceless labeling of proteins without inserting extra flanking residues. Due to its overall higher activity, the M86 mutant appears most promising for many protein labeling and chemical modification schemes using the split intein approach.     

Routes of electron transfer in beef heart cytochrome c oxidase: is there a unique pathway used by all reductants?

Cytochrome c oxidase oxidizes several hydrogen donors, including TMPD (N,N,N',N'-tetramethyl-p-phenyl-enediamine) and DMPT (2-amino-6,7-dimethyl-5,6,7,8-tetrahydropterine), in the absence of the physiological substrate cytochrome c. Maximal enzyme turnovers with TMPD and DMPT alone are rather less than with cytochrome c, but much greater than previously reported if extrapolated to high reductant levels and (or) to 100% reduction of cytochrome a in the steady state. The presence of cytochrome c is, therefore, not necessary for substantial intramolecular electron transfer to occur in the oxidase. A direct bimolecular reduction of cytochrome a by TMPD is sufficient to account for the turnover of the enzyme. CuA may not be an essential component of the TMPD oxidase pathway. DMPT oxidation seems to occur more rapidly than the DMPT--cytochrome a reduction rate and may therefore imply mediation of CuA. Both "resting" and "pulsed" oxidases contain rapid-turnover and slow-turnover species, as determined by aerobic steady-state reduction of cytochrome a by TMPD. Only the "rapid" fraction (approximately 70% of the total with resting and approximately 85% of the total with pulsed) is involved in turnover. We conclude that electron transfer to the a3CuB binuclear centre can occur either from cytochrome a or CuA, depending upon the redox state of the binuclear centre. Under steady-state conditions, cytochrome a and CuA may not always be in rapid equilibrium. Rapid enzyme turnover by either natural or artificial substrates may require reduction of both and two pathways of electron transfer to the a3CuB centre.     

Mathematical Model for Optimal Use of Sulfadoxine-Pyrimethamine as a Temporary Malaria Vaccine

In this paper, we introduce a deterministic malaria model for determining the drug administration protocol that leads to the smallest first malaria episodes during the wet season. To explore the effects of administering the malaria drug on different days during the wet season while minimizing the potential harmful effects of drug overdose, we define 40 drug administration protocols. Our results fit well with the clinical studies of Coulibaly et al. at a site in Mali. In addition, we provide protocols that lead to smaller number of first malaria episodes during the wet season than the protocol of Coulibaly et al.     

Manipulation of β-glucuronidase for use as a reporter in vacuolar targeting studies

It has been documented that when furnished with an endomembrane signal sequence for the endoplasmic reticulum, -glucuronidase (GUS) is N-glycosylated, resulting in the nearly complete loss of enzymatic activity. To enable use of -glucuronidase as a reporter protein in secretory and vacuolar targeting studies, one of the two putative N-linked glycosylation sites within the GUS gene was altered by site-directed mutagenesis. The second N-linked glycosylation site was not altered because sequence analysis of nucleotide sequences around the second putative glycosylation site revealed that the published sequence was incorrect, and that no such site existed.     

Design,synthesis, and biological activity of 5′-phenyl-1,2,5,6-tetrahydro-3,3′-bipyridine analogues as potential antagonists of nicotinic acetylcholine receptors

Starting from a known non-specific agonist (1) of nicotinic acetylcholine receptors (nAChRs), rationally guided structural-based design resulted in the discovery of a small series of 5′-phenyl-1,2,5,6-tetrahydro-3,3′-bipyridines (3a–3e) incorporating a phenyl ring off the pyridine core of 1. The compounds were synthesized via successive Suzuki couplings on a suitably functionalized pyridine starting monomer 4 to append phenyl and pyridyl substituents off the 3- and 5-positions, respectively, and then subsequent modifications were made on the flanking pyridyl ring to provide target compounds. Compound 3a is a novel antagonist, which is highly selective for α3β4 nAChR (K_i = 123 nM) over the α4β2 and α7 receptors.     

Use of a feedback-insensitive α subunit of anthranilate synthase as a selectable marker for transformation of rice and potato

A selection system based on a mutant rice gene for a feedback-insensitive subunit of anthranilate synthase (OASA1D) was developed for the transformation of rice and potato. Expression of OASA1D conferred resistance to the tryptophan analog 5-methyltryptophan (5MT) in transformed cells of rice and potato. The selection system based on OASA1D and 5MT was associated with a high transformation efficiency, a short time frame for the generation of transgenic plants, simple culture procedures, and it was as effective as hygromycin B selection in rice (monocotyledon) and kanamycin selection in potato (dicotyledon). Transgenic rice and potato plants established by 5MT selection had normal morphology and accumulated tryptophan when OASA1D was expressed under the control of a constitutive promoter. These results demonstrate the efficacy of OASA1D as a selectable marker and they suggest that the 5MT selection system based on this gene will prove applicable to a wide range of plant species and culture procedures.     

Partial purification of the superoxide-generating system of macrophages. Possible association of the NADPH oxidase activity with a low-potential (−247 mV) cytochrome b

NADPH oxidase activity was solubilized by detergent treatment of subcellular particles obtained from guinea-pig peritoneal macrophages stimulated with phorbol myristate acetate. Gel filtration of the material containing the NADPH oxidase activity gave two peaks of proteins, one of which eluted with the void and the other with the included volume of an AcA 22 column. The material eluted in the void volume contained more than 50% of the NADPH oxidase activity and less than 10% of the NAD(P)H cytochrome c reductase activity. A b-type cytochrome with peaks of absorption at 558, 528 and 426 nm was also enriched in the fraction which contained the NADPH oxidase activity. The distribution of flavoproteins as revealed by the measurement of FAD was different from that of NADPH oxidase and cytochrome b, and followed the elution profile of NADH cytochrome c reductase. Studies in subcellular particles showed that the b cytochromes of mitochondria and endoplasmic reticulum reduced by selective biochemical means accounted for only a minor part of the total b-type cytochromes and that the new cytochrome b previously described in neutrophils is the major chromophore also in macrophages. Oxidation-reduction midpoint potential of the partially purified cytochrome b was shown to be −247 mV. Association of cytochrome b with the NADPH oxidase activity and its very low E_m7.0 makes it a suitable candidate to be part of the superoxide-generating system also in macrophages.     

Dual effect of heparin on Fe2+-induced cardiolipin peroxidation: implications for peroxidation of cytochrome c oxidase bound cardiolipin

The effect of heparin on peroxidation of cardiolipin (CL) initiated by ferrous iron was studied in vitro using detergent-solubilized CL, liposomal CL, or CL bound to isolated cytochrome c oxidase (CcO). Heparin increased both the rate and the extent of CL peroxidation for detergent-solubilized CL and for CcO-bound CL. The effect of heparin was time- and concentration-dependent as monitored by the formation of conjugated dienes or thiobarbituric acid reactive substances. The results showed great similarity between the effect of heparin and the effect of certain iron chelators, such as ADP, on phospholipid peroxidation. Heparin increased the peroxidation of CcO-bound CL only when tertiary butyl hydroperoxide was also present. The enzyme activity of the resulting CcO complex decreased 25 %, in part due to peroxidation of functionally important CL. In contrast to peroxidation of detergent-solubilized CL, peroxidation of liposomal CL was inhibited by heparin, suggesting that the effect of heparin and ferrous iron depends on their proximity to the acyl chains of CL.     

Immobilization of lysine oxidase on a gold–platinum nanoparticles modified Au electrode for detection of lysine

A commercial lysine oxidase (LyOx) from Trichoderma viride was immobilized covalently onto gold nanoparticles (AuNPs) and platinum nanoparticles (PtNPs) electrodeposited onto Au electrode using 3-aminopropyltriethoxy silane (3-APTES) and glutaraldehyde cross linking chemistry. A lysine biosensor was fabricated using LyOx/3-APTES/AuNPs-PtNPs/Au electrode as a working electrode, Ag/AgCl (3 M KCl) as standard electrode and Pt wire as auxiliary electrode connected through a potentiostat. The enzyme electrode was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The cumulative effect of AuNPs and PtNPs showed excellent electrocatalytic activity at low applied potential for detection of H₂O₂, a product of LyOx reaction. The sensor showed its optimum response within 4 s, when polarized at 0.2 V vs. Ag/AgCl in 0.1 M phosphate buffer, pH 7.5 at 30 °C. The linear range and detection limit of the sensor were 1.0–600 μM and 1.0 μM (S/N = 3), respectively. Biosensor measured lysine level in sera, milk and amino acid tablet, which correlated well with those by standard HPLC method. The enzyme electrode lost 50% of its initial activity after 200 uses over a period of 4 months.     

Structure–activity studies on the side chain of a simplified analog of aplysiatoxin (aplog-1) with anti-proliferative activity

We have recently developed a simplified analog of aplysiatoxin (aplog-1) as an activator of protein kinase C (PKC) with anti-proliferative activity like bryostain 1. To identify sites in aplog-1 that could be readily modified to optimize therapeutic performance and to develop a molecular probe for examining the analog’s mode of action, substituent effects on the phenol ring were systematically examined. Whereas hydrophilic acetamido derivatives were less active than aplog-1 in inhibiting cancer cell growth and binding to PKCδ, introduction of hydrophobic bromine and iodine atoms enhanced both biological activities. The anti-proliferative activity was found to correlate closely with molecular hydrophobicity, and maximal activity was observed at a log P value of 4.0–4.5. On the other hand, an induction test with Epstein–Barr virus early antigen demonstrated that these derivatives have less tumor-promoting activity in vitro than aplog-1 regardless of the hydrophobicity of their substituents. These results would facilitate rapid preparation of molecular probes to examine the mechanism of the unique biological activities of aplog-1.     

Birds girdling activity on exotic tree species as a form of adaptive behavior?

Four tree species in the Kostelec n. ?. l. arboretum (Czech Republic) have been repeatedly damaged by Dendrocopos medius. The unique aspect of this otherwise common behavior called girdling consists in regularly visiting the same trees every spring, although there are more than 1.200 tree species within the arboretum. We monitored transpiration, leaf phenology and the chemical composition of the xylem sap of girdled and nongirdled trees. Spectral analysis revealed slightly higher amounts of sugars, especially saccharose, in Cladrastis Raf. as the most regularly girdled tree among other conditions, comparing girdled to non-girdled trees. Higher transpiration rates were not confirmed in connection with girdling—quite the opposite—Cladrastis Raf. as the most highly favored tree for girdling showed the lowest transpiration rates (in average 6 kg water per day within spring months) compared to other non-girdled trees. We presume that the birds do not choose a particular tree on the basis of any visible or chemical traits but they examine many trees within their territory. Afterwards they probably remember the position of trees whose xylem sap starts to flow early in the spring compared to other trees, as their transpiration stream is enriched with sweet organic substances that represent an advantage for the forthcoming nesting period.