The localization and the isoenzymes of α- and β-Galactosidases in root tips

The localization was studied of α- and β-galactosidases in frozen sections of Ca-formol fixed root tips using simultaneous azocoupling reaction. In all species studied (Allium cepa,Cucurbita maxima, Lupinus albus, Pisum sativum, Vicia faba, Zea mays) positive results were obtained, the localization being ubiquitous (according to localization typology given here). InVicia faba andZea mays the isoenzymes of α- and β-galactosidases were revealed by means of acrylamide gel electrophoresis, using authors’ modification of Reisfeld method, in whole root tips, particular growth zones and separately in cortex and central cylinder. No differences were observed comparing stele and cortex. Whereas characteristic isoenzyme patterns were found in individual growth zones in maize, no differences appeared in broad bean. A comparison was made of thein situ localization and of the isoenzyme patterns of α- and β-galactosidases with α- and β-glucosidases. In the case of galactosidases, positive results appear with both α- and β-galactoside. The rising of pH to neutrality leads to considerable decrease in the activity of both galactosidases.     

Cell-specific expression and immunolocalization of nitric oxide synthase isoforms and soluble guanylyl cyclase α1 and β1 subunits in the ovary of fetal, neonatal and immature pigs

The present study is designed to investigate the cellular expression and immunolocalization of three different nitric oxide synthase (NOS) isoforms and soluble guanylyl cyclase (sGC) subunits in the porcine ovary. Our results showed that in the fetal and neonatal pigs, all three isoforms of NOS were mainly localized in the oocyte and showed the expression of gradual increase in the granulosa cell and theca cell with the growing follicle. In addition, subunits of the sGC, sGC α1 and β1 were mainly expressed in the granulosa cell in precious studies. The bioactivity of total NOS, eNOS, iNOS and nNOS was detected in the ovary and were higher at prenatal stages compared to postnatal stages. However, the activities of nNOS were no different between prenatal stages and postnatal stages. Taken together, our findings suggested that the NOS/sGC pathway may be involved in the follicular formation and development in the porcine ovary.     

Structural and functional insights into the heme-binding domain of the human soluble guanylate cyclase α2 subunit and heterodimeric α2β1

Soluble guanylate cyclase (sGC) mediates NO signaling for a wide range of physiological effects in the cardiovascular system and the central nervous system. The α1β1 isoform is ubiquitously distributed in cytosolic fractions of tissues, whereas α2β1 is mainly found in the brain. The major occurrence and the unique characteristic of human sGC α2β1 indicate a special role in the mediation of neuronal communication. We have efficiently purified and characterized the recombinant heme-binding domain of the human sGC α2 subunit (hsGC α2(H)) and heterodimeric α2β1 (hsGC β1(H)-α2(H)) by UV-vis spectroscopy, circular dichrosim spectroscopy, EPR spectroscopy, and homology modeling. The heme dissociation and related NO/CO binding/dissociation of both hsGC α2(H) and hsGC β1(H)-α2(H) were investigated. The two truncated proteins interact with heme noncovalently. The CO binding affinity of hsGC α2(H) is threefold greater than that of human sGC α1(H), whereas the dissociation constant k (1) for dissociation of NO from hsGC α2(H) is sevenfold larger than that for dissociation of NO from hsGC α1(H), although k (2) is almost identical. The results indicate that in comparison with the α1β1 isoform, the brain α2β1 isoform exhibits a distinctly different CO/NO affinity and binding rate in favor of NO signaling, and this is consistent with its physiological role in the activation and desensitization. Molecular modeling and sequence alignments are consistent with the hypothesis that His105 contributes to the different CO/NO binding properties of different isoforms. This valuable information is helpful to understand the molecular mechanism by which human sGC α2β1 mediates NO/CO signaling.     

Changes in expression and microheterogeneity of the genetic variants of human α1-acid glycoprotein in malignant mesothelioma

Human α₁-acid glycoprotein (AAG), an acute-phase plasma protein, is heterogeneous in the native state and polymorphic in the desialylated state. The AAG heterogeneity is mainly explained by a variable glycan chain composition in its five glycosylation sites. The AAG polymorphism is due to the presence of genetic variants. Three main variants are observed for AAG, ORM1 F1, ORM1 S and ORM2 A, which have a separate genetic origin. In this paper, we have used different isoelectric focusing (IEF) methods and chromatography on immobilized metal affinity adsorbent to study the relative occurrence of the genetic variants of AAG in relation to changes in microheterogeneity, in plasma and pleural effusions of patients with malignant mesothelioma (MM). The results were compared to those obtained with the variants in plasma of healthy individuals. Significant changes in variant distribution were observed in the MM samples, that corresponded to a rise in the proportion of the ORM1 variants and a fall in that of the ORM2 variant. However, the concentration in MM plasma increased for both variants. The AAG in MM plasma and effusion fluids was found to be more heterogeneous on IEF than AAG of healthy plasma. The evidence of stronger concentrations of both the high and low pI forms of AAG in the MM samples suggested two kinds of changes in charge heterogeneity. These two changes were shown to be attributed to different variants — i.e. the high pI forms to ORM1 F1 and S and the low pI forms to ORM2 A, after fractionation of AAG by chromatography on immobilized copper(II) ions. These results indicate specific changes in both the expression and glycosylation for each AAG variant, according to its separate genetic origin, in MM.     

G protein β1γ2 subunits purification and their interaction with adenylyl cyclase

A preliminary study on the interaction of G protein (guanine triphosphate binding pro- tein) β1γ2 subunits and their coupled components in cell signal transduction was conducted in vitro. The insect cell lines, Sf9 (Spodoptera frugiperda) and H5 (Trichoplusia ni) were used to express the recombinant protein Gβ1γ2. The cell membrane containing Gβ1γ2 was isolated through affinity chromatography column with Ni-NTA agarose by FPLC method, and the highly purified protein was obtained. The adenylyl cyclase 2 (AC2) activity assay showed that the purified Gβ1γ2 could significantly stimulate AC2 activity. The interaction of β1γ2 subunits of G protein with the cytoplasmic tail of various mammalian adenylyl cyclases was monitored by BIAcore technology using NTA sensor chip, which relies on the phenomenon of surface plasmon resonance (SPR). The experiments showed the direct binding of Gβ1γ2 to the cytoplasmic tail C2 domain of AC2. The specific binding domain of AC2 with Gβ1γ2 was the same as AC2 activity domain which was stimulated by β1γ2.     

Ultracytochemical localizations of adenylate cyclase,guanylate cyclase and cyclic 3′,5′-nucleotide phosphodiesterase activity on the trophoblast in the human placenta

Summary Ultracytochemical localizations of cyclic nucleotide-metabolizing enzymes, namely adenylate cyclase (AC), guanylate cyclase (GC) and cyclic 3,5-nucleotide phosphodiesterase (PDE), have been demonstrated in the human term placenta. AC activity was found positive on the basal plasma membrane of the syncytiotrophoblast and on the pinocytotic vesicle of the fetal capillary endothelial cell. GC activity was observed to be strong on the plasma membrane of the microvilli of the syncytiotrophoblast. The cAMP PDE activity was shown positive both on the basal plasma membrane and on the microvillous membrane, while cGMP PDE activity was exclusively confined to the microvilli of the syncytiotrophoblast. These observations suggest that the syncytiotrophoblast plays an important role in the cyclic nucleotide metabolism in the human term placenta and that there might be significant functional differences between its basal plasma membrane and its microvillous membrane.     

Cloning and analysis of the cDNA encoding the rod G-protein transducin α, β1 and γ1 subunits from the canine retina

The canine (Canis familiaris) retinal rod transducin (G_T) α, β1 and γ1 subunits were sequenced. Cloning of the cDNAs was accomplished by polymerase chain reaction (PCR) using degenerate and wild type retinal cDNA libraries as templates. The deduced amino acid sequences were highly similar to rod transducins from other species: G_Tα differed by 5 amino acids from the corresponding human sequence, whereas β1 and γ1 were identical to human sequences. The coding sequence of rod transducin was evaluated as a possible cause for the recessively inherited retinal rod-cone degeneration: there were no nucleotide differences between the wild type and retinal degenerate strains.     

Diverse pathways for maturation of the Na,K-ATPase β1 and β2 subunits in the endoplasmic reticulum of Madin-Darby canine kidney cells

Proper folding of the Na,K-ATPase β subunits followed by assembly with the α subunits is necessary for their export from the endoplasmic reticulum (ER). Here we examine roles of the ER lectin chaperone, calnexin, and non-lectin chaperone, BiP, in folding and quality control of the β(1) and β(2) subunits in Madin-Darby canine kidney cells. Short term prevention of glycan-calnexin interactions by castanospermine slightly increases ER retention of β(1), suggesting minor involvement of calnexin in subunit folding. However, both prolonged incubation with castanospermine and removal of N-glycosylation sites do not affect the α(1)-assembly or trafficking of β(1) but increase the amount of the β(1)-bound BiP, showing that BiP can compensate for calnexin in assisting β(1) folding. In contrast to β(1), prevention of either N-glycosylation or glycan-calnexin interactions abolishes the α(1)-assembly and export of β(2) from the ER despite increased β(2)-BiP binding. Mutations in the α(1)-interacting regions of β(1) and β(2) subunits impair α(1) assembly but do not affect folding of the β subunits tested by their sensitivity to trypsin. At the same time, these mutations increase the amount of β-bound BiP but not of β-bound calnexin and increase ER retention of both β-isoforms. BiP, therefore, prevents the ER export of folded but α(1)-unassembled β subunits. These α(1)-unassembled β subunits are degraded faster than α(1)-bound β subunits, preventing ER overload. In conclusion, folding of the β(1) and β(2) subunits is assisted predominantly by BiP and calnexin, respectively. Folded β(1) and β(2) either assemble with α(1) or bind BiP. The α(1)-bound β subunits traffic to the Golgi, whereas BiP-bound β subunits are retained and degraded in the ER.     

Ostrich α-amylase: Purification and characterization of the pancreatic isoenzymes

• 1.1. Four ostrich pancreatic α-amylase isoenzymes were isolated by isoelectric focusing, following affinity chromatography on cyclohepta-amylose-Sepharose 4B.
• 2.2. Amino acid compositions of the four isoenzymes are very similar with only one charged amino acid (Arg) being significantly different.
• 3.3. The molecular weights, as determined by SDS-PAGE and amino acid composition, are nearly identical (52–53 kDa) for all four isoenzymes.
• 4.4. The four α-amylase isoenzymes appear to be kinetically distinct enzymes with a requirement for calcium.
• 5.5. Ostrich α-amylase isoenzymes appear to be non-glycosylated and contain one free thiol group.

     

Positions of β2 and β3 subunits in the large-conductance calcium- and voltage-activated BK potassium channel

Large-conductance voltage- and Ca²⁺-gated K⁺ channels are negative-feedback regulators of excitability in many cell types. They are complexes of α subunits and of one of four types of modulatory β subunits. These have intracellular N- and C-terminal tails and two transmembrane (TM) helices, TM1 and TM2, connected by an ∼100-residue extracellular loop. Based on endogenous disulfide formation between engineered cysteines (Cys), we found that in β2 and β3, as in β1 and β4, TM1 is closest to αS1 and αS2 and TM2 is closest to αS0. Mouse β3 (mβ3) has seven Cys in its loop, one of which is free, and this Cys readily forms disulfides with Cys substituted in the extracellular flanks of each of αS0–αS6. We identified by elimination mβ3-loop Cys152 as the only free Cys. We inferred the disulfide-bonding pattern of the other six Cys. Using directed proteolysis and fragment sizing, we determined this pattern first among the four loop Cys in β1. These are conserved in β2–β4, which have four additional Cys (eight in total), except that mβ3 has one fewer. In β1, disulfides form between Cys at aligned positions 1 and 8 and between Cys at aligned positions 5 and 6. In mβ3, the free Cys is at position 7; position 2 lacks a Cys present in all other β2–β4; and the disulfide pattern is 1–8, 3–4, and 5–6. Presumably, Cys 2 cross-links to Cys 7 in all other β2–β4. Cross-linking of mβ3 Cys152 to Cys substituted in the flanks of αS0–S5 attenuated the protection against iberiotoxin (IbTX); cross-linking of Cys152 to K296C in the αS6 flank and close to the pore enhanced protection against IbTX. In no case was N-type inactivation by the N-terminal tail of mβ3 perturbed. Although the mβ3 loop can move, its position with Cys152 near αK296, in which it blocks IbTX binding, is likely favored.     

The effect of mechanical load on integrin subunits α5 and β1 in chondrocytes from mature and immature cartilage explants

Articular cartilage is subjected to cyclic compressive stresses during joint loading. There is increasing experimental evidence that this loading is essential for the chondrocytes to maintain the functionality of the cartilage extracellular matrix (ECM) and that members of the integrin family of transmembrane receptors may play an important role in signal mechanotransduction between the ECM and chondrocytes. Of particular interest are the integrin subunits 5 and 1, which are known to form the receptor for fibronectin, an important ECM protein, and to be involved in mechanotransduction as well as in the regulation of cytokine production. In this study, we measured the amounts of the integrin subunits 5 and 1 in chondrocytes from young (immature) and adult (mature) bovine articular cartilage explants which were subjected to a continuously applied cyclic compressive stress of 1 MPa for 6 and 24 h. The integrin content per chondrocyte was measured immediately after load cessation by flow cytometry following matrix digestion to release the cells. We found that a mechanical stress induced an increase in the number of integrin subunit 5 in immature and mature cartilage but not in the integrin subunit 1 content. The integrin contents were greatest after 6 h of loading and returned to control levels after 24 h of unloading. The results of this study supply further experimental evidence that chondrocytes respond to changes in their mechanical environment and that the integrin 51 may act as a mechanical signal transducer between the chondrocyte and the ECM for the modulation of cellular physiology.This work was supported by NIH grant AR45748 to PAT and the HSS MacArthur Cartilage Fund.     

MUC1 in human and murine mammary carcinoma cells decreases the expression of core 2 β1,6-N-acetylglucosaminyltransferase and β-galactoside α2,3-sialyltransferase

A good correlation between the expression of mucin1 (MUC1) and T antigen was found in breast cancer tumors and breast cancer cell lines, especially after treatment with neuraminidase. The association between the appearance of T antigen and the overexpression of MUC1 was further confirmed by transfecting MDA-MB-231 cells and murine 4T1 mammary carcinoma cells with cDNA for MUC1 and using an RNAi approach to inhibit the expression of MUC1 gene in T47D cells. Furthermore, we discovered that in 4T1 cells which express the sialyl Le(X) antigen, overexpression of MUC1 caused not only appearance of T antigen, but also loss of the sialyl Le(X) structure. As the observed changes in O-glycan synthesis can be associated with changes in the expression of specific glycosyltransferases, core 1 β1,3-galactosyltransferase, core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT1) and β-galactoside α2,3-sialyltransferase (ST3Gal I), we studied their expression in parental, vector-transfected and MUC1-transfected MDA-MB-231 and 4T1 cells as well as T47D cells transduced with small hairpin RNA targeted MUC1 mRNA. It was found that the expression of C2GnT1 and ST3Gal I is highly decreased in MUC1-expressing MDA-MB-231 and 4T1 cells and increased in T47D cells with suppressed expression of MUC1. Therefore, we found that changes in the structure of O-linked oligosaccharides, resulting in the occurrence of T antigen, are at least partially associated with MUC1 overexpression which down-regulates the expression of C2GnT1 and ST3Gal I. We showed also that the overexpression of MUC1 in 4T1 cells changes their adhesive properties, as MUC1-expressing cells do not adhere to E-selectin, but bind galectin-3.     

The expression of α-internexin and peripherin in the developing mouse pineal gland

Summary The mammalian pineal gland contains pinealocytes, interstitial glial cells, perivascular macrophages, neurons and neuron-like cells. The neuronal identity of neurons and neuron-like cells was an enigma. α-Internexin and peripherin are specific neuronal intermediate filament proteins and are expressed differentially in the CNS and PNS. We investigated the development of immunoreactivity and expression patterns of mRNAs for α-internexin and peripherin in the mouse pineal gland to determine the neuronal identity of these cells. Both α-internexin- and peripherin-immunoreactive cells were readily visualized only after birth. Both proteins were at the highest level on the postnatal day 7 (P7), rapidly declined at P14, and obtained their adult level at P21. Both protein and mRNA of α-internexin are expressed in some cells and nerve processes, but not all, of adult mouse pineal gland. Less number of peripherin immunoreactive or RNA-expressing cells and nerve processes were identified. Accumulations of α-internexin and peripherin proteins were also found in the cells from the aged pineal gland (P360). We concluded that some cells in the developing mouse pineal gland may differentiated into neurons and neuron-like cells expressing both α-internexin and/or peripherin only postnatally, and these cells possess dual properties of CNS and PNS neurons in nature. We suggested that they may act as interneurons between the pinealocyte and the distal neurons innervating the pinealocytes, or form a local circuitry with pinealocytes to play a role of paracrine regulatory function on the pinealocytes.     

Expression of integrin subunits αv and β3 in acute lung inflammation

Integrin subunits v and 3 form a dimer, v3, which is expressed on normal neutrophils and endothelium. We investigated the expression of integrin subunits v and 3 in acute lung inflammation in Sprague-Dawley rats (n=5 each) following intratracheal challenge with Escherichia coli or Streptococcus pneumoniae, which induce neutrophil recruitment through different mechanisms. Control rats (n=5) were given endotoxin-free saline. Both bacterial challenges induced similar levels of recruitment of neutrophils in lungs. Western blots showed lower expression of integrin subunits v and 3 in lungs challenged with E. coli compared to those given S. pneumoniae. Immunohistochemistry and immunogold electron microscopy localized both integrin subunits in neutrophils and endothelium in the control and treated rat lungs. Quantitative immunohistochemistry showed that E. coli-challenged rat lungs contained a lower percentage of neutrophils expressing integrin subunits v and 3 compared to those challenged with S. pneumoniae (P<0.05). We conclude that E. coli infection decreased the percentage of neutrophils expressing integrin subunits v and 3 compared to S. pneumoniae infection. These data lay the foundation for further characterization of these integrin subunits in neutrophil migration specifically in S. pneumoniae infection that utilizes molecules other than 2 integrins for neutrophil recruitment.     

Study of the residues involved in the binding of β1 to β3 subunits in the sodium channel

The voltage-gated sodium channel (VGSC) is a complex, which is composed of one pore-forming α subunit and at least one β subunit. Up to now, five β subunits are known: β1/β1A, β1B, β2, β3, and β4, encoded by four genes (SCN1B∼SCN4B). It is critical to have a deep understanding of the interaction between β1 and β3 subunits, two subunits which frequently appear in many diseases concurrently. In this study, we had screened out the new template of β1 subunit for homology modelling, which shares higher similarity to β3. Docking studies of the β1 and β3 homology model were conducted, and likely β1 and β3 binding loci were investigated. The results revealed that β1–β3 is more likely to form a di-polymer than β1–β1 based on molecular interaction analysis, including potential energy analysis, Van der Waals (VDW) energy analysis and electrostatic energy analysis, and in addition, consideration of the hydrogen bonds and hydrophobic contacts that are involved. Based on these analyses, the residues His122 and Lys140 of β1 and Glu 66, Asn 131, Asp 118, Glu 120, Glu133, Asn135, Ser 137 of β3 were predicted to play a functional role.     

Synthesis and in vitro evaluation of thiododecaborated α, α- cycloalkylamino acids for the treatment of malignant brain tumors by boron neutron capture therapy

Boron-neutron capture therapy (BNCT) is an attractive technique for cancer treatment. As such, α, α-cycloalkyl amino acids containing thiododecaborate ([B₁₂H₁₁]^2?-S-) units were designed and synthesized as novel boron delivery agents for BNCT. In the present study, new thiododecaborate α, α-cycloalkyl amino acids were synthesized, and biological evaluation of the boron compounds as boron carrier for BNCT was carried out.