Rhinanthus serotinus (Schönheit) Oborny (Scrophulariaceae): immunohistochemical and ultrastructural studies of endosperm chalazal haustorium development

Chalazal endosperm haustorium in Rhinanthus serotinus consists of a single large binucleate cell. It originates from the primary endosperm cell dividing transversely into two unequal cells: a smaller micropylar cell and a larger chalazal cell. The chalazal cell undergoes a single mitotic division, then lengthens significantly during development and functions as a chalazal endosperm haustorium. In this paper, immunofluorescent techniques, rhodamine phalloidin assay, and electron microscopy were used to examine the actin and tubulin cytoskeleton during the development of the chalazal haustorium. During the differentiation stage, numerous longitudinally oriented bundles of microfilaments ran along the axis of transvacuolar strands in haustorium. Microtubules formed intensely fluorescent areas near the nuclear envelope and also formed radial perinuclear microtubule arrays. In the fully differentiated haustorium cell, the actin cytoskeleton formed dense clusters of microfilaments on the chalazal and micropylar poles of the haustorium. Numerous microfilament bundles occurred near wall ingrowths on the chalazal wall. There were numerous clusters of microfilaments and microtubules around the huge lobed polytenic haustorial nuclei. The microfilaments were oriented longitudinally to the long axis of the haustorium cell and surrounded both nuclei. The microtubules formed radial perinuclear systems which were appeared to radiate from the surface of the nuclear envelope. The early stage of degeneration of the chalazal haustorium was accompanied by the degradation of microtubules and disruption of the parallel orientation of microtubules in the chalazal area of the cell. The degree of vacuolization increased, autophagous vacuoles appeared and the number of vesicles decreased.     

HsdR subunit of the type I restriction-modification enzyme EcoR124I: biophysical characterisation and structural modelling

Type I restriction-modification (RM) systems are large, multifunctional enzymes composed of three different subunits. HsdS and HsdM form a complex in which HsdS recognizes the target DNA sequence, and HsdM carries out methylation of adenosine residues. The HsdR subunit, when associated with the HsdS-HsdM complex, translocates DNA in an ATP-dependent process and cleaves unmethylated DNA at a distance of several thousand base-pairs from the recognition site. The molecular mechanism by which these enzymes translocate the DNA is not fully understood, in part because of the absence of crystal structures. To date, crystal structures have been determined for the individual HsdS and HsdM subunits and models have been built for the HsdM-HsdS complex with the DNA. However, no structure is available for the HsdR subunit. In this work, the gene coding for the HsdR subunit of EcoR124I was re-sequenced, which showed that there was an error in the published sequence. This changed the position of the stop codon and altered the last 17 amino acid residues of the protein sequence. An improved purification procedure was developed to enable HsdR to be purified efficiently for biophysical and structural analysis. Analytical ultracentrifugation shows that HsdR is monomeric in solution, and the frictional ratio of 1.21 indicates that the subunit is globular and fairly compact. Small angle neutron-scattering of the HsdR subunit indicates a radius of gyration of 3.4 nm and a maximum dimension of 10 nm. We constructed a model of the HsdR using protein fold-recognition and homology modelling to model individual domains, and small-angle neutron scattering data as restraints to combine them into a single molecule. The model reveals an ellipsoidal shape of the enzymatic core comprising the N-terminal and central domains, and suggests conformational heterogeneity of the C-terminal region implicated in binding of HsdR to the HsdS-HsdM complex.     

Immunoexpression of P16INK4a, Rb and TP53 proteins in bronchiolar columnar cell dysplasia (BCCD) in lungs resected due to primary non-small cell lung cancer

Lung cancer is the leading cause of death worldwide. High mortality comes out mainly of the fact that majority of the cases are diagnosed in advanced stadium. An expanded diagnostics of precancerous conditions would certainly contribute to lowering the mortality rate. Many of the molecular changes accompanying the multistep cancer development could be observed using the immunohistochemistry method. In this paper we describe the morphology and cell cycle proteins immunoexpression of the novel probable preinvasive lesion - bronchiolar columnar cell dysplasia (BCCD). Thirty cases of BCCD selected out of 193 patients population, treated for primary non-small cell lung cancer were investigated. Loss of P16INK4a protein was observed in 70% of all cases and was statistically significant in patients with adenocarcinoma. Two cases show abnormal cytoplasmic localization of this protein. TP53 protein accumulates in 26.7% of all BCCD. Rb protein was active in 48.3% of the BCCD cases. In two cases we observed differentiation of the cells composing BCCD into multilayer epithelium of the squamous type, which occurs with formation of desmosomes. We suppose that BCCD may be preneoplastic lesion leading to adenocarcinoma as well as to peripheral squamous cell lung cancer.     

A theoretical study on effect of the initial redox state of cytochrome b559 on maximal chlorophyll fluorescence level (F(M)): implications for photoinhibition of photosystem II

In this work, we extended the reversible radical pair model which describes energy utilization and electron transfer up to the first quinone electron acceptor (Q(A)) in photosystem II (PSII), by redox reactions involving cytochrome (cyt) b559. In the model, cyt b559 accepts electrons from the reduced primary electron acceptor in PSII, pheophytin, and donates electrons to the oxidized primary electron donor in PSII (P680+). Theoretical simulations of chlorophyll fluorescence rise based on the model show that the maximal fluorescence, F(M), increases with an increasing amount of initially reduced cyt b559. In this work we applied, the first to our knowledge, metabolic control analysis (MCA) to a model of reactions in PSII. The MCA was used to determine to what extent the reactions occurring in the model control the F(M) level and how this control depends on the initial redox state of cyt b559. The simulations also revealed that increasing the amount of initially reduced cyt b559 could protect PSII against photoinhibition. Also experimental data, which might be used to validate our theory, are presented and discussed.     

Alterations in the subcellular distribution of 17β-estradiol dehydrogenase in porcine endometrial cells over the course of the estrous cycle

The uteri of German landrace gilts slaughtered at different days of the cycle were processed for immunocytochemistry and biochemical analyses. Plasma was collected for hormone assays. The monoclonal antibody F1 against the structure-bound 17-estradiol dehydrogenase of porcine endometrial epithelium was applied to rehydrated paraffin sections either as a direct, peroxidase-linked probe or in combination with a fluorescing secondary antibody. The oxidation of estradiol was measured in homogenates of tissue powdered in liquid nitrogen. Immunoreactivity was restricted to endometrial epithelium. In the glandular epithelium, faint dots of fluorescence became visible at day 4, which apparently coalesced to spherical structures of 2–4 m diameter at the cell basis between days 11 through 17 before disappearing by day 18. A similar distribution was observed for the oxidation products of diaminobenzidine beginning with a faint uniform staining and followed by the appearance of intensely stained basal bodies persisting until day 17. Essentially the same time course was seen in the luminal epithelium but with a different distribution. Immunoreactive material amassed in the apical region of the cells, but the conspicuous aggregations were absent. Time course and intensities of the immunological responses are matched by the enzymatic activity measured in parallel. Both correlate with the plasma progesterone levels, suggesting an induction of the enzyme by the hormone. An involvement of the cytoskeleton in the sequence of subcellular distribution patterns is discussed.     

Prolidase activity in fibroblasts is regulated by interaction of extracellular matrix with cell surface integrin receptors

Prolidase (EC 3.4.13.9) is a ubiquitously distributed imidodipeptidase that catalyzes the hydrolysis of C-terminal proline or hydroxyproline containing dipeptides. The enzyme plays an important role in the recycling of proline for collagen synthesis and cell growth. An increase in enzyme activity is correlated with increased rates of collagen turnover indicative of extracellular matrix (ECM) remodeling, but the mechanism linking prolidase activity and ECM is poorly understood. Thus, the effect of ECM-cell interaction on intracellular prolidase activity is of special interest. In cultured human skin fibroblasts, the interaction with ECM and, more specifically, type I collagen mediated by the β₁ integrin receptor regulates cellular prolidase activity. Supporting evidence comes from the following observations: 1) in sparse cells with a low amount of ECM collagen or in confluent cells in which ECM collagen was removed by collagenase (but not by trypsin or elastase) treatment, prolidase activity was decreased; 2) this effect was reversed by the addition of type I collagen or β₁ integrin antibody (agonist for β₁ integrin receptor); 3) sparse cells (with typically low prolidase activity) showed increased prolidase activity when grown on plates coated with type I collagen or on type IV collagen and laminin, constituents of basement membrane; 4) the relative differences in prolidase activity due to collagenase treatment and subsequent recovery of the activity by β₁ integrin antibody or type I collagen treatment were accompanied by parallel differences in the amount of the enzyme protein recovered from these cells, as shown by Western immunoblot analysis. Thus, we conclude that prolidase activity responded to ECM metabolism (tissue remodeling) through signals mediated by the integrin receptor. J. Cell. Biochem. 67:166–175, 1997. Published 1997 Wiley-Liss, Inc.     

Homeostasis of the protonmotive force in phosphorylating mitochondria

The relationship between the respiration rate and the magnitude of the electrochemical proton potential (Δμ_H⁺) in rat liver mitochondria was investigated. (1) Under the active-state conditions, the action of inhibitors of either phosphorylation (oligomycin) or respiration (rotenone, malonate) on the respiration and Δμ_H⁺ was measured. Both inhibitors diminished the respiration, whereas rotenone resulted in a decrease of Δμ_H⁺, and oligomycin produced an increase of this potential. The effect of the inhibitors was much more pronounced on the respiration rate than on Δμ_H⁺; for example, the excess of oligomycin produced a 90% inhibition of the respiration while Δμ_H⁺ was changed only by 9%. (2) Under the resting-state conditions, small concentrations of the uncoupler stimulated the respiration while changing Δμ_H⁺ to a relatively small extent. The uncoupler concentrations which doubled and tripled the respiration rate produced only 5 and 9% decrease of Δμ_H⁺, respectively. (3) The present results enabled us to propose a model describing the interrelationship between respiration and Δμ_H⁺.