Characterization of a transglutaminase from scallop hemocyte and identification of its intracellular substrates

Scallop hemocytes contain a transglutaminase (TGase) that is electrophoretically different from the TGase in the adductor muscle. The optimum temperature of the hemocyte TGase was lower (about 15 degrees C), compared with the muscle TGase (35-40 degrees C). Other properties, such as the high sodium chloride (NaCl) and CaCl2 concentrations required for activation, instability in salt solutions, and the Km values against monodansylcadaverine (MDC) and succinylated casein, were similar for both enzymes. When hemocyte homogenate was incubated with MDC at 10 degrees C, MDC was incorporated into the 230 k and 100 k proteins of the hemocytes. The 100 k protein was only detected in the supernatant, the 230 k protein was insoluble, and the 210 k protein was detected in both fractions. In the absence of MDC, the 230 k, 210 k, and 100 k proteins were cross-linked by endogenous transglutaminase. The 230 k protein was most quickly cross-linked and formed huge polymers within 5 min. These results suggest that if scallop tissues are injured, hemocyte transglutaminase may be activated, initially cross-linking the insoluble hemocyte 230 k protein, followed by the 210 k and 100 k proteins, to form a cross-linked protein matrix with inter cross-linking of hemocyte sheets, to stop the bleeding.     

Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system

The renin-angiotensin system is a central component of the physiological and pathological responses of cardiovascular system. Its primary effector hormone, angiotensin II (ANG II), not only mediates immediate physiological effects of vasoconstriction and blood pressure regulation, but is also implicated in inflammation, endothelial dysfunction, atherosclerosis, hypertension, and congestive heart failure. The myriad effects of ANG II depend on time (acute vs. chronic) and on the cells/tissues upon which it acts. In addition to inducing G protein- and non-G protein-related signaling pathways, ANG II, via AT₁ receptors, carries out its functions via MAP kinases (ERK 1/2, JNK, p38MAPK), receptor tyrosine kinases [PDGF, EGFR, insulin receptor], and nonreceptor tyrosine kinases [Src, JAK/STAT, focal adhesion kinase (FAK)]. AT₁R-mediated NAD(P)H oxidase activation leads to generation of reactive oxygen species, widely implicated in vascular inflammation and fibrosis. ANG II also promotes the association of scaffolding proteins, such as paxillin, talin, and p130Cas, leading to focal adhesion and extracellular matrix formation. These signaling cascades lead to contraction, smooth muscle cell growth, hypertrophy, and cell migration, events that contribute to normal vascular function, and to disease progression. This review focuses on the structure and function of AT₁ receptors and the major signaling mechanisms by which angiotensin influences cardiovascular physiology and pathology. vascular smooth muscle; NAD(P)H oxidase; tyrosine and nontyrosine receptor kinases; endothelial dysfunction; vascular disease     

The carbonic anhydrase isoforms of Chlamydomonas reinhardtii: intracellular location, expression, and physiological roles

Aquatic photosynthetic organisms, such as the green alga Chlamydomonas reinhardtii, respond to low CO(2) conditions by inducing a CO(2) concentrating mechanism (CCM). Carbonic anhydrases (CAs) are important components of the CCM. CAs are zinc-containing metalloenzymes that catalyze the reversible interconversion of CO(2) and HCO(3)(-). In C. reinhardtii, there are at least 12 genes that encode CA isoforms, including three alpha, six beta, and three gamma or gamma-like CAs. The expression of the three alpha and six beta genes has been measured from cells grown on elevated CO(2) (having no active CCM) versus cells growing on low levels of CO(2) (with an active CCM) using northern blots, differential hybridization to DNA chips and quantitative RT-PCR. Recent RNA-seq profiles add to our knowledge of the expression of all of the CA genes. In addition, protein content for some of the CA isoforms was estimated using antibodies corresponding to the specific CA isoforms: CAH1/2, CAH3, CAH4/5, CAH6, and CAH7. The intracellular location of each of the CA isoforms was elucidated using immunolocalization and cell fractionation techniques. Combining these results with previous studies using CA mutant strains, we will discuss possible physiological roles of the CA isoforms concentrating on how these CAs might contribute to the acquisition and retention of CO(2) in C. reinhardtii.     

Extreme calorie restriction and energy source starvation in Saccharomyces cerevisiae represent distinct physiological states

Cultivation methods used to investigate microbial calorie restriction often result in carbon and energy starvation. This study aims to dissect cellular responses to calorie restriction and starvation in Saccharomyces cerevisiae by using retentostat cultivation. In retentostats, cells are continuously supplied with a small, constant carbon and energy supply, sufficient for maintenance of cellular viability and integrity but insufficient for growth. When glucose-limited retentostats cultivated under extreme calorie restriction were subjected to glucose starvation, calorie-restricted and glucose-starved cells were found to share characteristics such as increased heat-shock tolerance and expression of quiescence-related genes. However, they also displayed strikingly different features. While calorie-restricted yeast cultures remained metabolically active and viable for prolonged periods of time, glucose starvation resulted in rapid consumption of reserve carbohydrates, population heterogeneity due to appearance of senescent cells and, ultimately, loss of viability. Moreover, during starvation, calculated rates of ATP synthesis from reserve carbohydrates were 2-3 orders of magnitude lower than steady-state ATP-turnover rates calculated under extreme calorie restriction in retentostats. Stringent reduction of ATP turnover during glucose starvation was accompanied by a strong down-regulation of genes involved in protein synthesis. These results demonstrate that extreme calorie restriction and carbon starvation represent different physiological states in S. cerevisiae.     

Polarized light microscopy reveals physiological and drug-induced changes in surfactant membrane assembly in alveolar type II pneumocytes

In alveolar type II (AT II) cells, pulmonary surfactant (PS) is synthetized, stored and exocytosed from lamellar bodies (LBs), specialized large secretory organelles. By applying polarization microscopy (PM), we confirm a specific optical anisotropy of LBs, which indicates a liquid-crystalline mesophase of the stored surfactant phospholipids (PL) and an unusual case of a radiation-symmetric, spherocrystalline organelle. Evidence is shown that the degree of anisotropy is dependent on the amount of lipid layers and their degree of hydration, but unaffected by acutely modulating vital cell parameters like intravesicular pH or cellular energy supply. In contrast, physiological factors that perturb this structure include osmotic cell volume changes and LB exocytosis. In addition, we found two pharmaceuticals, Amiodarone and Ambroxol, both of which severely affect the liquid-crystalline order. Our study shows that PM is an easy, very sensitive, but foremost non-invasive and label-free method able to collect important structural information of PS assembly in live AT II cells which otherwise would be accessible by destructive or labor intense techniques only. This may open new approaches to dynamically investigate LB biosynthesis - the incorporation, folding and packing of lipid membranes - or the initiation of pathological states that manifest in altered LB structures. Due to the observed drug effects, we further suggest that PM provides an appropriate way to study unspecific drug interactions with alveolar cells and even drug-membrane interactions in general.     

Microinjection of ubiquitin: intracellular distribution and metabolism in HeLa cells maintained under normal physiological conditions

Radioiodinated ubiquitin was introduced into HeLa cells by erythrocyte-mediated microinjection. Subsequent electrophoretic analyses revealed that the injected ubiquitin molecules were rapidly conjugated to HeLa proteins. At equilibrium, 10% of the injected ubiquitin was conjugated to histones and 40% was distributed among conjugates of higher molecular weight. Although the remaining ubiquitin molecules appeared to be unconjugated, the free pool of ubiquitin decreased by one-third and additional conjugates were present when electrophoresis was performed at low temperature under nonreducing conditions. Molecular weights of these labile conjugates suggest that they are ubiquitin adducts in thiolester linkage to activating enzymes. Despite the fairly rapid degradation of injected ubiquitin (t1/2 approximately 10-20 h), the size distribution of ubiquitin conjugates within interphase HeLa cells remained constant for at least 24 h after injection. The intracellular locations of ubiquitin and ubiquitin conjugates were determined by autoradiography, by differential sedimentation of subcellular fractions in sucrose, and by extraction of injected cells with buffer containing Triton X-100. Free ubiquitin was found mostly in the cytosolic or Triton X-100-soluble fractions. As expected, histone conjugates were located predominately in the nuclear fraction and exclusively in the Triton X-100-insoluble fraction. Although high molecular weight conjugates were enriched in the Triton X-100-insoluble fraction, their size distribution was similar to that of soluble conjugates. When injected HeLa cells were exposed to cycloheximide to inhibit protein synthesis, the size distribution of ubiquitin conjugates was similar to that found in untreated cells. Moreover, high molecular weight conjugates decreased less than 20% after inhibition of protein synthesis. These results indicate that most ubiquitin conjugates are not newly synthesized proteins which have been marked for destruction.     

Characterization of purified rat testicular transglutaminase and age-dependent changes of the enzyme activities

The Ca2+-dependent tissue transglutaminase is widely distributed in various tissues and has been reported to participate in many cellular growth and differentiation processes. In the past decade, tissue transglutaminase is also identified as a G protein, G(alphah), for intercellular signaling. To further characterize testicular transglutaminase, the rat testicular transglutaminase was purified by ammonium sulfate precipitation, DEAE ion-exchange, heparin-agarose, and GTP-agarose affinity chromatographies. This purification protocol resulted in a 8400-fold enrichment of the enzyme with a reproducible 15% yield. The purified enzyme showed as a single band of 78kDa on SDS-polyacrylamide gel. Western blot analysis using anti-liver tissue transglutaminase monoclonal antibody also recognized the enzyme, indicating it is a t-TGase in nature. The Km values of purified testicular transglutaminase for putrescine and N,N-dimethylcasein were determined to be 35 and 17 microM, respectively. Its transglutaminase cross-linking activity was strongly inhibited by EGTA, GTP, polyamines, and cystamine, as well as moderately by ATP and NaCl. The enzyme exhibited a magnesium-dependent GTP-hydrolyzing capacity, but its GTP-binding activity did not require magnesium. Furthermore, the enzyme activity was found to be closely related with the first wave of spermatogenesis. Thus, testicular transglutaminase is speculated to participate in the event of spermatogenesis. In conclusion, the purified testicular transglutaminase displays property of either the tissue-type transglutaminase, or the GTP-binding and hydrolyzing characteristics. The activity of testicular transglutaminase is age-dependent, greatly stimulated during the first wave of spermatogenesis.     

Host relations and distribution of a physiological and pathological variant of Pseudomonas morsprunorum

In a plot of sweet-cherry trees (Primus avium), leaf spot infections caused by Pseudomonas morsprunorum were more abundant on the cv. Roundel than on the normally more susceptible cv. Napoleon. Roundel also supported significantly higher leaf surface populations of the pathogen. This reversal in the relative field susceptibility of the two cultivars was associated with the presence on the trees of a variant form of P. morsprunorum that differed from typical strains in colony structure, gelatinase activity and pathological characters. The variant strains were less infective through leaf scars but more invasive in bark parenchyma than typical strains, and in branch and leaf inoculations, they showed specialization for the cv. Roundel. This specialization was reflected in the distribution of variant and typical strains on Roundel and Napoleon trees in other localities. The significance of the variant in relation to disease epidemiology and cultivar resistance is discussed. The variant is designated race 2 to distinguish it from the typical, or race 1 cherry strains of P. morsprunorum.     

Advances in analysis of glycosaminoglycans: its application for the assessment of physiological and pathological states of connective tissues

Glycosaminoglycans are a class of biological macromolecules found mainly in connective tissues as constituents of proteoglycans, covalently linked to their core protein. Hyaluronan is the only glycosaminoglycan present under its single form and possesses the ability to aggregate with the class of proteoglycans termed hyalectans. Proteoglycans are localised both at the extracellular and cellular (cell-surface and intracellular) levels and, via either their glycosaminoglycan chains or their core proteins participate in and regulate several cellular events and (patho)physiological processes. Advances in analytical separational techniques, including high-performance liquid chromatography, capillary electrophoresis and fluorophore assisted carbohydrate electrophoresis, make possible to examine alterations of glycosaminoglycans with respect to their amounts and fine structural features in various pathological conditions, thus becoming applicable for diagnosis. In this review we present the chromatographic and electromigration procedures developed to analyse and characterise glycosaminoglycans. Moreover, a critical evaluation of the biological relevance of the results obtained by the developed methodology is discussed.     

Endothelin-1: physiological and pathological roles in myometrium

Endothelin-1 (ET-1), a member of endothelin peptide family is released by many different tissues including uterine smooth muscle. ET-1 acts through ETA and ETB receptors and is implicated in a wide range of biological and pathological functions that explain the great attention of the pharmacological industry for ET-1 receptors as potential therapeutic targets in vascular pathologies and cancers. It is now well established that ET-1 is also able to regulate myometrial functions. In the present review, we focused on ET axis and related signaling pathways involved in the regulation of myometrial contraction, as well as cell proliferation and survival. Such ET-1-mediated cellular functions play a critical role in normal pregnancy, preterm birth and uterine leiomyoma.     

GTP binding and signaling by Gh/transglutaminase II involves distinct residues in a unique GTP-binding pocket

G(h) is a dual function protein. It has receptor signaling activity that requires GTP binding and Ca(2+)-activated transglutaminase (TGase) activity that is inhibited by GTP binding. G(h) shows no homology with other GTP-binding proteins, and its GTP-binding site has not been defined. Based on sequence analysis of [alpha-(32)P]GTP-photolabeled and proteolytically released internal peptide fragments, we report localization of GTP binding to a 15-residue segment ((159)YVLTQQGFIYQGSVK(173)) of the G(h) core domain. This was confirmed by site-directed mutagenesis; a G(h)/fXIIIA chimera (in which residues 162-179 of G(h) were substituted with the equivalent but nonhomologous region of the non-GTP-binding TGase factor XIIIA) and a G(h) point mutant, S171E, retained TGase activity but failed to bind and hydrolyze GTP and did not support alpha(1B)-adrenergic receptor signaling. Slight impairment of GTP binding (1.5-fold) and hydrolysis (10-fold) in the absence of altered TGase activity did not affect signaling by the mutant K173N. However, greater impairment of GTP binding (6-fold) and hydrolysis (50-fold) abolished signaling by the mutant K173L. Mutant S171C exhibited enhanced GTP binding and signaling. Thus, residues Ser(171) and Lys(173) are critical for both GTP binding and signaling but not TGase activity. Mutagenesis of residues N-terminal to Gly(170) impaired both GTP binding and TGase activity. From computer modeling of G(h), it is evident that the GTP-binding region identified here is distinct from, but interacts with, the TGase active site. Together with structural considerations of G(h) versus other GTP-binding proteins, these findings indicate that G(h) has a unique GTP-binding pocket and provide for the first time a mechanism for GTP-mediated regulation of the TGase activity of G(h).     

Role of changes in the transmembrane ion currents in pathological states of the body

Modern data of study of transmembrane ions currents (through the sodium-potassium- and calcium-channels) in the mechanism of pathological pain syndromes were reviewed. Tetrodotoxyn-resistant sodium currents, abnormal combinations of different types of sodium-channels during the inflammation, neuropathies (syndromes hyperalgesii and allodonii) and diabetic neuropathy analyzed. The role of pain sensitivity transmitters was demonstrated. Nerve Grow Factor plays the important role in the sodium-channels genes expression. Different types of transmembrane calcium currents participated in hyperalgesium and hypoalgesium analyzed. The summary mechanism of pain suggested as voltage-calcium-channels activation and transsynaptic NMDA receptors excitation that should be conductive the neurons hyperexcitation and force of synaptic transmission.     

Two-photon irradiation of an intracellular singlet oxygen photosensitizer: achieving localized sub-cellular excitation in spatially-resolved experiments

The response of a given cell to spatially-resolved sub-cellular irradiation of a singlet oxygen photosensitizer (protoporphyrin IX, PpIX) using a focused laser was assessed. In these experiments, incident light was scattered over a volume greater than that defined by the dimensions of the laser beam as a consequence of the inherent inhomogeneity of the cell. Upon irradiation at a wavelength readily absorbed by PpIX in a one-photon transition, this scattering of light eliminated any advantage accrued to the use of focused irradiation. However, upon irradiation at a longer wavelength where PpIX can only absorb light under non-linear two-photon conditions, meaningful intracellular resolution was achieved in the small spatial domain where the light intensity was high enough for absorption to occur.     

Discovery of multiple level heart-sound morphological variability resulting from changes in physiological states

Heart sounds carry information about the mechanical activity of the cardiovascular system. This information includes the specific physiological state of the subject, and short term variability related to the respiratory cycle. The interpretation of the sounds and extraction of changes in the physiological state, while monitoring short term variability is still an open problem and is the subject of this paper.We present a novel computational framework for analysis of data with multi-level variability, caused by externally induced changes. The framework presented includes an initial clustering of the first heart sound (S1) according to the morphology, and further aggregation of clusters into super-clusters. The clusters and super clusters are two methods of data segmentation, each reflecting a different level of variability in the data.The framework is applied to heart sounds recorded during laparoscopic surgeries of six patients. Procedures of this kind include anesthesia and abdominal insufflation, which together with the respiratory cycle, induce changes to the heart sound signal. We demonstrate a separation of the heart sound morphology according to different physiological states. The physiological states considered are the respiratory cycle, and the stages of the surgery. We achieve results of 90 ± 4% classification accuracy of heart beats to operation stages.The proposed framework is general and can be used to analyze data characterized by multi-level variability for various other (biomedical) applications.     

Two-photon irradiation of an intracellular singlet oxygen photosensitizer: Achieving localized sub-cellular excitation in spatially-resolved experiments

Abstract

The response of a given cell to spatially-resolved sub-cellular irradiation of a singlet oxygen photosensitizer (protoporphyrin IX, PpIX) using a focused laser was assessed. In these experiments, incident light was scattered over a volume greater than that defined by the dimensions of the laser beam as a consequence of the inherent inhomogeneity of the cell. Upon irradiation at a wavelength readily absorbed by PpIX in a one-photon transition, this scattering of light eliminated any advantage accrued to the use of focused irradiation. However, upon irradiation at a longer wavelength where PpIX can only absorb light under non-linear two-photon conditions, meaningful intracellular resolution was achieved in the small spatial domain where the light intensity was high enough for absorption to occur.