Changes In Molecular Forms Research Articles

Genomic evaluation during permeability of indomethacin and its solid dispersion

Drug resistance was first identified in cancer cells that express proteins known as multidrug resistance proteins that extrude the therapeutic agents out of the cells resulting in alteration of pharmacokinetics, tissue distribution, and pharmacodynamics of drugs. To this end studies were carried out to investigate the role of pharmacological inhibitors and pharmaceutical excipients with a primary focus on P-glycoprotein (P-gp). The aim of this study was to investigate holistic changes in transporter gene expression during permeability upon formulation of indomethacin as solid dispersion. Initial characterization studies of solid dispersion of indomethacin showed that the drug was dispersed within the carrier in amorphous form. Analysis of permeability data across Caco-2 monolayers revealed that drug absorption increased by 4-fold when reformulated as solid dispersion. The last phase of the work involved investigation of gene expression changes of transporter genes during permeability. The results showed that there were significant differences in the expression of both ATP-binding cassette (ABC) transporter genes as well as solute carrier transporter (SLC) genes suggesting that the inclusion of polyethylene glycol as well as changes in molecular form of drug from crystalline to amorphous have a significant bearing on the expression of transporter network genes resulting in differences in drug permeability.

Evidence of the Involvement of Asparagine Deamidation in the Formation of Cyclodextrin Glycosyltransferase Isoforms in Paenibacillus sp. RB01

Cyclodextrin glycosyltransferase (CGTase) from Paenibacillus sp. RB01 and its recombinant enzyme exhibit three isoforms (I, II, and III) with the same apparent size but different charge. Here, we demonstrate for the first time that the deamidation of labile Asns causes the change in molecular forms of CGTase. The faster increase in number of isoforms was observed upon incubation in deamidation buffer at the more alkaline pH. The increase in levels of isoform II and III over time correlated with the increase in isoaspartate, a unique deamidation product. The predicted labile Asns were individually mutated to Asp, then the selected mutant and wild type isoforms were tryptic digested and labile Asns were investigated by MALDI-TOF. From the results, Asn427 was the most susceptible residue for deamidation, followed by Asn336, Asn415, and Asn567. In addition, Gln389 might also share a role. The advantage of using appropriate CGTase isoform in cyclodextrin production is reported.

Natural oxidation of black carbon in soils: Changes in molecular form and surface charge along a climosequence

The aim of this work was to investigate changes in molecular form and surface charge of black carbon (BC) due to long-term natural oxidation and to examine how climatic and soil factors affect BC oxidation. Black C was collected from 11 historical charcoal blast furnace sites with a geographic distribution from Quebec, Canada, to Georgia, USA, and compared to BC that was newly produced (new BC) using rebuilt historical kilns. The results showed that the historical BC samples were substantially oxidized after 130 years in soils as compared to new BC or BC incubated for one year. The major alterations by natural oxidation of BC included: (1) changes in elemental composition with increases in oxygen (O) from 7.2% in new BC to 24.8% in historical BC and decreases in C from 90.8% to 70.5%; (2) formation of oxygen-containing functional groups, particularly carboxylic and phenolic functional groups, and (3) disappearance of surface positive charge and evolution of surface negative charge after 12 months of incubation. Although time of exposure significantly increased natural oxidation of BC, a significant positive relationship between mean annual temperature (MAT) and BC oxidation (O/C ratio with r = 0.83; P < 0.01) explained that BC oxidation was increased by 87 mmole kg C −1 per unit Celsius increase in MAT. This long-term oxidation was more pronounced on BC surfaces than for entire particles, and responded 7-fold stronger to increases in MAT. Our results also indicated that oxidation of BC was more important than adsorption of non-BC. Thus, natural oxidation of BC may play an important role in the effects of BC on soil biogeochemistry.

Altered glycosylation of acetylcholinesterase in Creutzfeldt–Jakob disease

Changes in the glycosylation pattern of brain proteins have been associated with Creutzfeldt-Jakob disease (CJD). We have investigated the glycosylation status of acetylcholinesterase (AChE) by lectin binding assay. Our data show that in lumbar CSF from definite and probable sporadic CJD cases AChE activity is lower compared with that in age-matched controls. We also show, for the first time, that AChE glycosylation is altered in CJD CSF and brain. Unlike Alzheimer's disease, in which an alteration in both the glycosylation and levels of AChE molecular forms is observed, the abnormal glycosylation of AChE in CJD appears to be unrelated to changes in molecular forms of this enzyme. These findings suggest that altered AChE glycosylation in CJD may be a consequence of the general perturbation of the glycosylation machinery that affects prion protein, as well as other proteins. The diagnostic potential of these changes remains to be explored.

Localization, specific activity, and molecular forms of acetylcholinesterase in developmental stages of the cestode Mesocestoides corti

The nervous system of flatworms is quite simple although there is increasing evidence indicating that it is chemically complex. Studies of the nervous system in these animals have only been performed in the larval stage or in the adult worms, which are easy to obtain in nature, while the description of the nervous system in developing stages of these organisms is missing. Mesocestoides corti is a parasitic platyhelminth whose larvae can be induced in vitro to develop to adult, sexually mature worms, opening the possibility of studying the nervous system of a flatworm in different stages of development. Here, we describe the presence, activity, location, and molecular forms of acetylcholinesterase (AChE) in different stages of development of M. corti, from the larvae to adult forms of this endoparasite, obtained in in vitro cultures after induction of the larval stage with trypsin. Our results point to AChE as a molecular marker of the nervous system in platyhelminthes. The change in molecular forms of this enzyme and the increase in its activity during development from larvae to adult worm may reflect the presence of a more complex nervous system, necessary to adjust and coordinate the movement of a much bigger structure. A relationship between the development of the reproductive apparatus in segmented and adult worms with a more complex nervous system in these stages is also apparent. Finally, our study opens the possibility of applying anti-AChE as more effective therapeutic strategies against cestode parasites.

The characterization, localization and regulation of endothelin in ovine pars intermedia

The pituitary intermediate lobe (IL) contains a single population of cells and has recently been shown to express endothelin (ET)-like peptides. The IL thus provides an excellent in vivo model to study regulation, function and processing of ET in an endocrine cell. The primary aims of the present study were to locate and characterize the precise molecular forms of ET in the ovine IL and determine if levels and/or processing of ET is under dopaminergic or other influences. We have developed a radioimmunoassay (RIA) that detects each form of ET and, when combined with reverse phase-HPLC (RP-HPLC), shows the ovine IL to contain predominantly the ET-1 isoform. In addition, using a specific anti-endothelin antiserum for immunohistochemistry (IHC), we localized ET-1 with α-melanocyte stimulating hormone (α-MSH) within the melanotroph. The effects of dopamine agonists, antagonists and hypothalamo-pituitary disconnection (HPD) on both tissue levels and processing of ET in the ovine IL were also examined. Normal sheep were treated chronically with haloperidol or bromocriptine to investigate the possibility of dopaminergic regulation of ET in the IL. In the haloperidol-treated group, plasma prolactin levels did not vary significantly from day 0 to day 8, but the bromocriptine treatment reduced prolactin levels ( t=9.4 P<0.01). Neither bromocriptine nor haloperidol, however, affected tissue ET peptide levels or forms. After HPD, the HPLC profile of pooled IL showed that ET-1 levels in the IL are slightly increased with no change in molecular forms.

Preparation of Iron Oxide Thin Films by MOCVD

Iron oxide thin films were deposited from 400 to 600°C by CVD from iron acetylacetonate (Fe(acac)3) using N2 as the transporting gas and separately introduced O2 as oxidation gas. β-Fe2O3 tended to be formed independent of O2 flow rate at 400°C. At 500 and 600°C, spinel-type Fe3O4+x was formed under low O2 flow rates. With increasing O2 flow rate, mixture of β-Fe2O3 and spinel-type Fe3O4+x was formed and the amount of β-Fe2O3 phase increased. Further increase in O2 flow rate resulted in the formation of β-Fe2O3 as a single phase. Fourier transformation infrared spectroscopy (FT-IR) measurement suggested that iron acetyl-acetonate vapor changed the molecular form by decomposition under certain condition. It was supposed that the change in molecular form influences the deposited phase of iron oxide films.

Solvent induced fluorescence of 9-chloro-10,10′-bis(dichloromethyleno)-(9′H)-10,10′-dihydro-9,9′-bianthryl (CDDB): twisted intramolecular charge transfer process

Solvent polarity induced fluorescence of 9-chloro-10,10′-bis(dichloromethyleno)-(9′H)-10,10′-dihydro-9,9′-bianthryl (CDDB) is studied in a series of homologous alcohols from methanol to pentanol and compared with the previously reported data for CDDB microcrystal and CDDB in cyclohexane. In microcrystal, the locally excited (LE) state and in non-polar solvents, intramolecular charge transfer (ICT) state are mainly responsible for fluorescence. In polar solvents, it has been proposed from the computed decomposed spectra that polarity dependent twisted intramolecular charge transfer (TICT) state also gives fluorescence along with ICT state fluorescence. The process has been discussed as per a proposed scheme. Formation of TICT state has been further confirmed from the measurement of equilibrium constants between ICT and TICT states in homologous alcohols and by analysing the kinetics of radiative and non-radiative pathways of individual ICT and TICT components. The formation mechanisms of ICT and TICT states are discussed on the basis of solvent induced change in molecular forms and charge separation due to rotation of the two moieties of CDDB along the C 9-C 9′ bond.

Biochemical alterations in the proacrosin-acrosin system during epididymal maturation of the rat spermatozoa.

Acrosin, an acrosomal serine protease, is believed to have a role in fertilization. The enzyme is synthesized in an enzymatically inactive precursor form, proacrosin, and is processed to enzymatically active form(s). In the studies presented here, maturation-associated changes in the proacrosin-acrosin system of rat spermatozoa are reported. Acid-solubilized components of spermatozoa from caput, corpus, and cauda epididymidis were resolved on gelatin-sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and the proteolytic bands visualized by enzymography. These studies reveal the presence of one form (52 kDa), two forms (52 and 41 kDa), and four forms (52, 41, 34, and 31 kDa) in the spermatozoa from caput, corpus, and cauda, respectively. The findings suggest that the enzymatically inactive high molecular weight component (proacrosin) present in the caput spermatozoa is partially converted to the low molecular weight components (acrosin) during epididymal transit. The sensitivity of these molecular forms to an inhibitor of acrosin, p-nitrophenyl p'-guanidino benzoate (NPGB), and the fact that all four forms cross-reacted with the antibody against guinea pig testis proacrosin, suggest that these molecular forms are proacrosin-acrosin components. To understand the mechanism of the changes in molecular forms, spermatozoa from caput, corpus, and cauda regions were subjected to in vitro activation, and the acid-solubilized components resolved on gelatin-SDS-polyacrylamide gel. A smaller component of 34 kDa was generated from both the caput and corpus spermatozoa. No changes in the molecular form(s) of cauda spermatozoa were observed, even after in vitro activation for 4 hours. Inclusion of NPGB during in vitro activation blocked generation of the new molecular form from the caput spermatozoa. These studies indicate that intra-acrosomal events during epididymal transit may be important in the production of functionally mature spermatozoa.

Hyaluronates in Developing Skeletal Tissues

Hyaluronic acid (HA) is present in the extracellular matrix (ECM) as early as the time of gastrulation. At these early stages, HA is thought to organize the ECM into a hydrated, open lattice and thereby support cell movements. At later stages, when specialized tissues appear, hyaluronidase activity increases. A correlation between elevated hyaluronidase activity and deposition of cartilage-type ECM is now well established. Differentiation of cartilage may be accompanied by changes in molecular forms of HA; however, the synthesis of HA is not understood well enough to permit firm generalizations to be drawn. There is also a lack of evidence regarding specificity of the hyaluronidase that appears at the onset of chondrogenesis. Thus, while HA is a ubiquitous ECM component and its appearance has been well studied during embryogenesis, there remain large gaps in the present knowledge regarding the means by which HA interacts with embryonic cells, tissues, and other ECM components.

Molecular analysis of IgA in multiple sclerosis

IgA plays an important role in immune-mediated and anti-viral responses, and is the major antibody in external mucosal secretions. We used enzyme-linked immunosorbent assay (ELISA) and immunoblot to examine IgA in tears and parotid saliva of 21 multiple sclerosis (MS) patients and 19 normal controls. No significant difference in absolute level were noted and both groups showed a wide range in values. However, 16 of 21 MS patients had detectable changes in molecular form of thier IgA. Monomeric IgA was found in 52% of MS tears P < 0.001) and 45% of MS parotid saliva ( P < 0.05). Among normals, monomeric IgA was noted in only two saliva samples. IgA in secretions consisted of both A1 and A2 sub classes, although 14% of the monomeric MS IgA samples were restricted to a single subclass. Free secretory component was found in the secretions of all subjects with the exception of a single MS patient. However, immunoblot staining of secretory component was decreased in MS patients compared to controls. This study notes changes in IgA in two distinct external secretions in MS patients compared to controls. These changes are consistent with local mucosal inflammation. Impaired mucosal barriers have important implications in MS with regard to susceptibility to infectious agents, environmental antigen processing, and lymphocyte activation.

Glutathione Transferases As Markers Of Preneoplasia And Neoplasia

Publisher Summary New markers, especially enzymes or isoenzymes, have proved useful for the detection of preneoplastic lesions in various organs of both animals and man. In addition, the description of preneoplastic marker enzymes has also played an important role in characterizing the metabolic patterns of preneoplastic cells, thus providing insights that have facilitated the analysis of carcinogenic processes. Recently, certain molecular forms of glutathione transferase (GT), one of the most important drug-metabolizing enzymes, have been reported as reliable markers for preneoplastic lesions and neoplastic tissue. This chapter reviews the known preneoplastic marker enzymes and molecular forms of GT in rat, human, and other species. Particular attention is focused on the unique properties of rat GT-P (7-7) and human GT-π, and what is known of their gene expression and possible functions. Furthermore, GT, including ligandin, have multipotential detoxifying functions and therefore, have been stimulated to study the changes in molecular forms and their roles in metabolism of carcinogens.

Evidence for a change in molecular form of DNA ligase in early development of the sea urchin Psammechinus miliaris

A change in the molecular form of DNA ligase appears when the sea urchin egg enters cleavage. Sucrose gradient analysis and DNA cellulose chromatography show that a slower migrating form (7 S) of enzyme exists in unfertilized eggs and in sperm. A faster migrating form of DNA ligase (7.8 S) is present in developing embryos as well as in artificially activated eggs. The timing of this early biochemical event has been determined, following fertilization or activation. The change in molecular form of DNA ligase has been shown to be sensitive to drugs inhibiting protein synthesis, gene transcription, or DNA replication. Consequently the appearance of the faster migrating form of enzyme is assumed to result from expression of the corresponding gene, transcription, and translation. RNA extracted from testes and from cleaving stages, assayed in vitro and in vivo, have been shown to carry the information for, respectively, 7 S and 7.8 S DNA ligase, according to the origin of the RNA.

Gamma-aminobutyric acid affects the developmental expression of neuron-associated proteins in cerebellar granule cell cultures.

The effect of gamma-aminobutyric acid (GABA) on the expression of the neuron-associated D2 and neuron-specific enolase (NSE) was studied during development in culture of cerebellar granule cells. It was found that the presence of GABA during culture development increased the overall protein content. D2 content was also increased but not above the general increase in protein whereas NSE increased above the general level of protein. The presence of GABA in the growth medium also appeared to accelerate the changes in molecular forms of D2 and NSE seen during neuronal development. This suggests that GABA promotes or accelerates the general maturation of neurons, as these two neuron-associated proteins otherwise differ from each other with respect to their subcellular localization and their physiological and biochemical properties.

Molecular forms of glutathione S-transferase and UDP-glucuronyltransferase as hepatic preneoplastic marker enzymes.

Changes in molecular forms of glutathione S-transferase (GST) and UDP-glucuronyltransferase (UDP-GT) as hepatic detoxicating enzymes were investigated during chemical hepatocarcinogenesis in the rat. The activity and the protein amount (formula; see text) itself of the GST-A form, which has fetal characteristics and is separable from other forms by CM-Sephadex column chromatography and by immunologic techniques, was much increased in gamma-glutamyl transpeptidase (gamma-GTP)-positive foci or hyperplastic nodules (HNs) induced by diethylnitrosamine and 2-fluorenylacetamide or 3'-methyl-4-dimethylaminoazobenzene. The activity of enzyme 1 (late fetal form) of UDP-GT assayed with o-aminophenol (o-GT) also increased with increased number of the foci or HNs, while the activity of enzyme 2 (neonatal form) assayed with phenolphthalein (p-GT) changed but little. The foci and HNs were stained more strongly than the nonnodular areas immunohistochemically using the antibody against purified GST-A or o-GT. The two activities were also increased in well-differentiated hepatomas, but they were decreased in moderately and poorly differentiated hepatomas, and activating enzymes such as cytochrome P-450 were markedly decreased from HN. GST-A and o-GT differ from fetal enzymes such as those of carbohydrate metabolism in that they are inducible in the short-term by drugs, including carcinogens, and they show the highest activities in HNs, and so they may be considered as hepatic preneoplastic (PN) antigens.

Nerve crush induced changes in molecular forms of acetylcholinesterase in soleus and extensor digitorum muscles

The molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) extracted from the fast twitch extensor digitorum longus (EDL) and slow twitch soleus (SOL) muscles, were separated by velocity sedimentation after sciatic nerve crush. Three molecular forms were routinely separated from EDL and four from SOL muscle. In the EDL, the 4S and 10S represented the greatest amounts of AChE, and in the SOL the 12S and 16S were the major constituent forms of the enzyme. Total AChE activity in EDL and SOL muscles rapidly decreased after nerve crush. During reinnervation (2 weeks postcrush), total AChE activity in the EDL gradually recovered, whereas the SOL exhibited a 2.5-fold transient increase above control. Immediately after denervation, decreases in the three AChE forms from the EDL (4, 10, and 16S) were evident, whereas the SOL exhibited both rapid increases (4 and 10S) and decreases (12S and 16S). In both muscles the 4S form reappeared before the 16S and 10S molecular forms, suggesting that the light form, 4S, may be a precursor of the heavier molecules. Transient increases during reinnervation occurred in the 16S AChE form in both muscles; however, they were approximately five times greater in the SOL than in the EDL. In the SOL all other molecular forms showed similar increases, whereas none was seen in the EDL. Possible mechanisms are differences in synthesis and catabolism, and in release of molecular forms from these muscles.

The apparent molecular weights of human intestinal aminopeptidase, enterokinase and maltase in native duodenal fluid

The apparent molecular weights of human intestinal aminopeptidase, enterokinase and maltase in native duodenal fluid were estimated by gel chromatography on Sephadex G-200 under different conditions of operational buffer and temperature. No evidence for environmentally induced changes in molecular form was found.

Seasonal Changes in Molecular Forms of Acid Phosphatase and Ribonuclease of Potato Tubers and the Effects of Infection by Phytophthora erythroseptica

Sequential changes in total activity and molecular forms of acid phosphatase and ribonuclease from potato tubers were studied by seasonal assays and Sephadex gel filtration. Ribonuclease and p -nitrophenyl phosphatase activity fluctuated during storage of tubers, while β-glycerophosphatase declined to a low level coincident with initiation of sprout growth. In recently-lifted tubers acid phosphatase activity occurred in a single high molecular weight peak. Two new forms of lower molecular weight appeared during ageing of stored tubers. The influence of infection by a tuber-rotting fungus, Phytophthoraerythroseptica , on these seasonal changes was variable. No consistent effects on total hydrolase activities were observed, while post-infectional changes in molecular forms included a pronounced shift in the major acid phosphatase peak. The possible significance of this molecular weight change in infected samples is discussed in the light of recent evidence concerning the sub-unit structure of acid phosphatase from potato tubers.

Factors affecting hemagglutination by concanavalin A and soybean agglutinin

Abstract While investigating the effect of temperature on hemagglutination by concanavalin A, we noted three factors that seriously interfere with the usual microscopic agglutination assay and produce misleading or ambiguous results. (1) Adherence of concanavalin A-treated erythrocytes to surfaces of plastic Petri dishes, especially at (2) commonly used cell densities, effectively prevents determination of agglutination. (3) In addition, incubation times usually used may be insufficient to demonstrate agglutination. Failure to account for these factors may explain the previously reported temperature-sensitive, concanavalin A-mediated agglutination of trypsinized erythrocytes and transformed cells (Vlodavsky, I., Inbar, M. and Sachs, L., (1972) Biochim. Biophys. Acta 274, 364–369). By controlling these factors, we demonstrated that concanavalin A does agglutinate trypsinized, human erythrocytes equally well at 24 and 4 °C. Investigation of the kinetics of erythrocyte agglutination by lectins revealed that the rate of agglutination by concanavalin A is markedly slower at lower temperatures while soybean agglutinin-mediated agglutination is faster at lower temperatures. Ultracentrifugation data indicate that at low temperature concanavalin A exists partially as a dimer (mol. wt 50 000) and at warmer temperatures exists mainly as a tetramer (mol. wt 100 000). The correlation of the effect of temperature on molecular weight with the agglutinating activity of concanavalin A suggests that temperature-dependent forms of concanavalin A may determine the rate of cell agglutination by this lectin. No temperature-dependent change in molecular form was observed with soybean agglutinin.

The size and form of DNA molecules from microsporocytes of Lilium longiflorum at different stages of meiosis

DNA molecules from lysates of microsporocytes at different stages of meiosis of Lilium longiflorum were examined in the electron microscope. In all of the preparations, only linear (non-circular) molecules were found. They had a mean length of between 59 μ and 75 μ, and a range of lengths which varied from 1 μ to 115 μ, to 1 μ to 210 μ. From the data obtained, there was no indication of a change in molecular form or size of DNA molecules associated with the process of meiosis.