Distribution of anticancer antibiotic daunomycin in the rat heart and kidney revealed by immunocytochemistry using monoclonal antibodies

Two monoclonal antibodies (ADM-1-11 and 79-31 mAbs) were raised against daunomycin (DM) conjugated to bovine serum albumin via the cross-linker N-(gamma-maleimidobutyryloxy)succinimide. The monoclonal antibodies (mAbs) specifically detected DM as well as its analogs doxorubicin and epirubicin, but did not react with other anticancer antibiotics, including pepleomycin, mitomycin C, and actinomycin D. The mAbs reacted strongly with glutaraldehyde-conjugated DM in an enzyme linked immunosorbent assay (ELISA) used as a model system for immunocytochemistry as well as in appropriately pretreated sections of tissues from animals injected with DM. No staining occurred in tissues from uninjected animals. In order to perform DM ICC a number of tissue treatment conditions critical to the detection of low molecular weight substances were employed. Uptake of DM was studied in rats after a single i.v. or i.p. administration of the drug. In the heart, accumulation of DM occurred in nuclei and in the cytoplasm. In the kidney, DM immunoreactivity accumulated in all segments of the nephron except for the proximal tubules. Since the proximal tubules are known to be where a variety of transport systems including P-glycoprotein (Pgp) and organic anion-transporting polypeptides (OATPs) in drug interactions occur, the absence of DM accumulation in these segments may reflect a transport phenomenon depending upon such transporters. The availability of methods to study sites of accumulation of DM offers possibilities for understanding toxic side effects of this drug on the heart and kidney. Moreover, the immunocytochemical methodology developed may prove useful for the localization of other low molecular weight drugs that can be fixed in situ by glutaraldehyde.     

Amine-producing endocrine-like cells in the epithelium of urethra and prostate of the guinea-pig a chemical,fluorescence histochemical,and electron microscopic study

Summary The urethra and prostate of the guinea-pig contain at least two types of endocrine-like cells in the epithelium. The predominant type is argentaffin and stores 5-hydroxytryptamine. Treatment with reserpine or a dopa decarboxylase inhibitor markedly reduces the 5-hydroxytryptamine content of this cell type. The other less numerous cell type, which is argyrophil but not argentaffin, is devoid of 5-hydroxytryptamine but can be induced to store dopamine if supplied with dopa. Both cell types occur disseminated in the urethral epithelium, whilst only the argyrophyl, non-argentaffin cell type devoid of 5-hydroxytryptamine is found in the prostate. At the ultrastructural level the argentaffin cell type contains numerous electron-dense cytoplasmic 800–1000 Å granules. These granules are argentaffin, suggesting that they are the storage site for 5-hydroxytryptamine. The cells sometimes reach the urethral lumen via a narrow neck, the apex being endowed with microvilli. This arrangement suggests that the cells are capable of responding to stimuli in the urethral lumen. Preliminary attempts to test the effect of depriving or loading guinea-pigs with water failed to induced changes in the 5-hydroxytryptamine content of the urethral endocrine-like cells.     

Purification of Functionally Sealed Cytoplasmic Side-out Plasma Membrane Vesicles from Saccharomyces cerevisiae

Highly purified plasma membrane vesicles were prepared from yeast protoplasts by a combination of osmotic lysis, differential centrifugation, and separation in an aqueous dextran/polyethylene glycol two-phase system. The vesicles were predominantly (85-90%) of cytoplasmic side-out orientation and displayed large ATP-dependent proton pumping activity which was inhibited by vanadate (100 μM) but not by bafilomycin or nitrate. The preparation presented a distinct polypeptide profile with respect to the total membrane fraction and was enriched in the 110-kDa polypeptide corresponding to the plasma membrane H⁺-ATPase. This preparation of native plasma membranes vesicles is especially suitable for functional studies in vitro.     

NAD(P)H oxidase and peroxidase activities in purified plasma membranes from cauliflower inflorescences

An NAD(P)H oxidase activity stimulated by phenolic compounds has been investigated in purified plasma membranes (pm) and in an intracellular membrane (icm) fraction depleted in plasma membranes, both obtained from a microsomal fraction from cauliflower inflorescences ( Brassica oleracea L.). The phenolic compounds salicylhydroxamic acid (SHAM), ferulic acid, coniferyl alcohol, n -propyl gallate, naringenin, kaempferol and caffeic acid all strongly stimulated the activity. Peroxidase (EC 1.11.1.7), or a peroxidase-like enzyme, was responsible for the NAD(P)H oxidase activity, which proceeded through a free-radical chain reaction and was inhibited by catalase (EC 1.11.1.6), superoxide dismutase (EC 1.15.1.1) and KCN. Most of the total activity was soluble; however, the membrane-bound activity was highly enriched in the pm compared to the icm. The catalase activity was 6 times higher in the icm-fraction than in the pm-fraction, but this was not the reason for the much lower phenol-stimulated NADH oxidase activity in the icm. Peroxidase activity measured with o -dianisidine and H₂O₂ had about the same specific activities in the pm-and icm-fractions.
Neither the phenol-stimulated NADH oxidase nor the peroxidase activity could be washed away from the pm even by 0.7 M NaCl, indicating that these activities are truly membrane-bound. SHAM as well as the other phenolic compounds capable of stimulating the NADH oxidase reaction were potent inhibitors of blue light-induced cytochrome b -reduction in the pm fraction.     

Molecular movement of the voltage sensor in a K channel

The X-ray crystallographic structure of KvAP, a voltage-gated bacterial K channel, was recently published. However, the position and the molecular movement of the voltage sensor, S4, are still controversial. For example, in the crystallographic structure, S4 is located far away (>30 A) from the pore domain, whereas electrostatic experiments have suggested that S4 is located close (<8 A) to the pore domain in open channels. To test the proposed location and motion of S4 relative to the pore domain, we induced disulphide bonds between pairs of introduced cysteines: one in S4 and one in the pore domain. Several residues in S4 formed a state-dependent disulphide bond with a residue in the pore domain. Our data suggest that S4 is located close to the pore domain in a neighboring subunit. Our data also place constraints on possible models for S4 movement and are not compatible with a recently proposed KvAP model.     

Influence of UV-B radiation and Cd2+ on chlorophyll fluorescence, growth and nutrient content in Brassica napus

The possible interaction of two stresses, UV-B radiation and cadmium, applied simultaneously, was investigated in Brassica napus L. cv. Paroll with respect of chlorophyll fluorescence, growth and uptake of selected elements. Plants were grown in nutrient solution containing CdCl2, (0, 0.5, 2 or 5 M) and irradiated with photosynthetically active radiation (PAR, 400-700 nm, 800 mol m^-2 s^-1) with or without supplemental ultraviolet-B radiation (UV-B, 280-320 nm, 15 kJ m^-2 d^-1, weighted irradiance). After 14 d of treatment, the most pronounced effects were found at 2 and 5 M CdCl2 with and without supplemental UV-B radiation. Exposure to cadmium significantly increased the amount of Cd in both roots and shoots. In addition, increases occurred in the concentrations of Fe, Zn, Cu, and P in roots, while K was reduced. In shoots the S content rose significantly both in the presence and absence of UV-B radiation, while significant increases in Mg, Ca, P, Cu, and K occurred only in plants exposed to Cd and UV-B radiation. Manganese decreased significantly under the combined exposure treatment. The rise in S content may have been due to stimulated glutathione and phytochelatin synthesis. Cadmium exposure significantly decreased root dry weight, leaf area, total chlorophyll content, carotenoid content, and the photochemical quantum yield of photosynthesis. As an estimation of energy dissipation processes in photosynthesis, non-photochemical quenching (qNPQ) was measured using a pulse amplitude modulated fluorometer. The qNPQ increased with increasing Cd, while the combination of cadmium and UV-B reduced the qNPQ compared to that in plants exposed only to cadmium or UV-B radiation. The chlorophyll a:b ratio showed a reduction with UV-B at no or low Cd concentrations (0 M, 0.5 M CdCl2), but not at the higher Cd concentrations used (2 M, 5 M CdCl2). Thus in some instances there appeared to be a UV-B and Cd interaction, while in other plants response could be attributed to either treatment alone.Keywords: Brassica napus, cadmium, ultraviolet-B radiation.      

Starvation Response of Saccharomyces cerevisiae Grown in Anaerobic Nitrogen- or Carbon-Limited Chemostat Cultures

Anaerobic starvation conditions are frequent in industrial fermentation and can affect the performance of the cells. In this study, the anaerobic carbon or nitrogen starvation response of Saccharomyces cerevisiae was investigated for cells grown in anaerobic carbon or nitrogen-limited chemostat cultures at a dilution rate of 0.1 h⁻¹ at pH 3.25 or 5. Lactic or benzoic acid was present in the growth medium at different concentrations, resulting in 16 different growth conditions. At steady state, cells were harvested and then starved for either carbon or nitrogen for 24 h under anaerobic conditions. We measured fermentative capacity, glucose uptake capacity, intracellular ATP content, and reserve carbohydrates and found that the carbon, but not the nitrogen, starvation response was dependent upon the previous growth conditions. All cells subjected to nitrogen starvation retained a large portion of their initial fermentative capacity, independently of previous growth conditions. However, nitrogen-limited cells that were starved for carbon lost almost all their fermentative capacity, while carbon-limited cells managed to preserve a larger portion of their fermentative capacity during carbon starvation. There was a positive correlation between the amount of glycogen before carbon starvation and the fermentative capacity and ATP content of the cells after carbon starvation. Fermentative capacity and glucose uptake capacity were not correlated under any of the conditions tested. Thus, the successful adaptation to sudden carbon starvation requires energy and, under anaerobic conditions, fermentable endogenous resources. In an industrial setting, carbon starvation in anaerobic fermentations should be avoided to maintain a productive yeast population.     

A unique tetrameric structure of deer plasma haptoglobin--an evolutionary advantage in the Hp 2-2 phenotype with homogeneous structure

Similar to blood types, human plasma haptoglobin (Hp) is classified into three phenotypes: Hp 1-1, 2-1 and 2-2. They are genetically inherited from two alleles Hp 1 and Hp 2 (represented in bold), but only the Hp 1-1 phenotype is found in almost all animal species. The Hp 2-2 protein consists of complicated large polymers cross-linked by alpha2-beta subunits or (alpha2-beta)n (where n>or=3, up to 12 or more), and is associated with the risk of the development of diabetic, cardiovascular and inflammatory diseases. In the present study, we found that deer plasma Hp mimics human Hp 2, containing a tandem repeat over the alpha-chain based on our cloned cDNA sequence. Interestingly, the isolated deer Hp is homogeneous and tetrameric, i.e. (alpha-beta)4, although the locations of -SH groups (responsible for the formation of polymers) are exactly identical to that of human. Denaturation of deer Hp using 6 m urea under reducing conditions (143 mmbeta-mercaptoethanol), followed by renaturation, sustained the formation of (alpha-beta)4, suggesting that the Hp tetramers are not randomly assembled. Interestingly, an alpha-chain monoclonal antibody (W1), known to recognize both human and deer alpha-chains, only binds to intact human Hp polymers, but not to deer Hp tetramers. This implies that the epitope of the deer alpha-chain is no longer exposed on the surface when Hp tetramers are formed. We propose that steric hindrance plays a major role in determining the polymeric formation in human and deer polymers. Phylogenetic and immunochemical analyses revealed that the Hp 2 allele of deer might have arisen at least 25 million years ago. A mechanism involved in forming Hp tetramers is proposed and discussed, and the possibility is raised that the evolved tetrameric structure of deer Hp might confer a physiological advantage.