Tissue-specific Variation Research Articles

Avian Integrin αv Subunit Associates with Multiple β Subunits and Shows Tissue-Specific Variation in Its Association

Integrins are heterodimeric cell surface receptors involved in cell adhesion to extracellular matrix proteins and in cell-cell interactions. RGD peptide-specific integrin subunit αv and its associated β subunits were isolated by GRGDSPK-Sepharose affinity chromatography. Using Western blot and immunoprecipitation techniques, the nature of this integrin complexity in the avian system was studied in comparison with that of the human system. As found in human cell systems, in chicken embryo fibroblasts the integrin αv subunit is associated with both the 90-kDa β3 and the 110-kDa β1-like subunits with high specific binding to RGD peptides. Furthermore, immunoprecipitation studies revealed the presence of an unidentified 120-kDa subunit and an 85-kDa β5 subunit associated with the αv subunit in these cells. No qualitative differences were observed in the β subunit profile associated with αv, as a function of the proliferating nonconfluent or nonproliferating confluent states of the embryonic fibroblast cultures. In adult chicken gizzard, a 110-kDa β1-like subunit and a 90-kDa subunit that does not cross-react with anti-H-β3 and anti-H-β5 are associated with the αv subunit. The structure of the novel 120-kDa subunit found in the embryonic fibroblasts and the 90-kDa subunit found in adult chicken gizzard and the ligand specificities of these novel combinations of αv are not known at present, but these integrins are RGD specific. It is hypothesized that embryonic fibroblasts, being the primordial proliferating cells, display a wider spectrum of αv-associated β subunits, whose expression may progressively be restricted or highly reduced in favor of one or more β subunits in subsequent progenies as differentiation progresses.

Genetic and dietary interactions in the regulation of HMG-CoA reductase gene expression.

Inbred strains of mice exhibit large genetic variations in hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity. A tissue-specific genetic variation between the strains BALB/c and C57BL/6, resulting in about 5-fold higher levels in hepatic reductase activity in strain C57BL/6, was examined in detail. The activity difference between these two strains could be explained entirely by differences in hepatic reductase mRNA levels. In genetic crosses, the variation segregated as a single major Mendelian element. Surprisingly, the mode of inheritance was recessive since F1 mice exhibited the BALB/c levels of enzyme activity. Despite the fact that the rates of hepatic sterol synthesis also differed between the strains by a factor of about five, the altered hepatic reductase expression did not significantly influence plasma lipoprotein levels. The response to a high cholesterol, high fat diet between the strains was remarkably different. Thus, in BALB/c mice, both hepatic reductase activity and mRNA levels were affected only slightly, if at all, by cholesterol feeding, while in strain C57BL/6 mice both were reduced more than 10-fold by cholesterol feeding. Several lines of evidence, including analysis of cis-acting regulatory elements, the nonadditive mode of inheritance, and genetic studies of the HMG-CoA reductase gene locus on mouse chromosome 13, support the possibility that the variation in reductase expression is not due to a mutation of the structural gene but, rather, is determined by a trans-acting factor controlling reductase mRNA levels. The variation provides a striking example, at the molecular level, of the importance of dietary-genetic interactions in the control of cholesterol metabolism.

Growth Phase and Tissue Specific Variations in the Multiple Murine Dihydrofolate Reductase mRNAs

Growth Phase and Tissue Specific Variations in the Multiple Murine Dihydrofolate Reductase mRNAs

Nuclear magnetic resonance relaxation studies of plant polyester dynamics. 2. Suberized potato cell wall

Measurements of the proton rotating-frame relaxation time T 1ρ (H) indicate that wound-healing involves the formation of spatially separated suberin and cell-wall domains. Suberin appears to be attached to the cell-wall domain at discrete sites, with both its methylene and phenylpropanoid groups divided into two populations having distinct chemical shifts and spin relaxation characteristics. Measurements of T 1ρ (C) and T 1 (C) in this biopolymer system sho significant site-specific and tissue-specific variations .

Tissue-specific variation of pea mitochondrial polypeptides detected by computerized image analysis of two-dimensional electrophoresis gels.

A tissue-specific variation of a total of 152 mitochondria-associated polypeptides is detailed for etiolated leaves, epicotyls and roots from the pea, Pisum satisum L., using computerized image analysis. These organ systems possess, respectively, 128, 100, and 96 mitochondria-associated polypeptides with 38, 8, and 10, respectively, being unique to each. Seventy-one polypeptides were observed to be present in all gels, but some of these also underwent a variety of quantifiable changes. Two polypeptides were identified that varied similarly to a polypeptide of interest (W), known to be abundant in pea epicotyls and potato tubers. It is postulated that these latter three polypeptides are therefore probably related functionally.

A specific point mutation in the mitochondrial genome of Caucasians with MELAS

The mitochondrial DNA (mtDNA) of Japanese patients suffering from the syndrome of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) exhibits a specific heteroplasmic A----G transition in the tRNA(Leu) at position 3243. In this study, we investigated mtDNA from skeletal muscle, cardiac muscle, brain, liver, diaphragm, fibroblasts and blood cells of four Caucasians with MELAS, one younger healthy sister of two MELAS patients, and eleven controls. We found that 1) the mutation was present in all investigated tissues of Caucasians with MELAS but not in controls, 2) within a single patient, the tissue-specific variation of the copy number of mutated mtDNA covered the same range as in the skeletal muscle of different patients, 3) the mutation was also present in the blood cells of the healthy sister of two MELAS siblings.

Effect of endosulfan 35 EC on glycogen metabolism in the hemolymph and tissues of a freshwater field crab Barytelphusa guerini

The effect of endosulfan (commercial grade, 35 EC) on glycogen metabolism of a freshwater field crab, Barytelphuse guerini, was studied. In a short-term exposure of 96 hr, progressive depletion of glycogen is reflected in the activity patterns of enzymes glycogen phosphorylases a and ab. A tissue-specific and time-dependant variation in tissue glucose reserves paralleled the progressive accumulation of hemolymph sugars.

Syncytiotrophoblast brush border proteins recognized by monoclonal antibody TRA-210 and rabbit anti-TLX sera

Different subsets of placental trophoblast epithelium are directly exposed to the maternal immune system during pregnancy and consequently represent major elements in allogeneic interactions. It has been proposed that the trophoblast-lymphocyte cross-reactive (TLX) alloantigen system is involved in maternal allogeneic recognition duringpregnancy. Monoclonal antibody TRA-2-10 putatively recognizes TLX antigens, but its reactivity with trophoblast and normal tissues has not been documented in detail. In this report, immunohistological investigations revealed that TRA-2-10 recognizes all subsets of trophoblast in addition to amniotic and seminal vesicle epithelia. Immunoblotting demonstrated reactivity with glycoproteins of 55 000 and 65 000 mol. mass under non-reducing conditions on various cell types. These proteins displayed tissue-specific size variations and individuals varied in the amounts expressed of the two species. On the basis of blocking and immunoprecipitation experiments, TRA-2-10 reactive antigens are recognized by rabbit anti- TLX sera and are potential TLX antigen candidates. However, TLX antigens are found in seminal plasma whilst TRA-2-10 reactive antigens are not. Both TLX and TRA-2-10 antigens appear related if not identical to membrane cofactor protein (MCP) by virtue of shared molecular characteristics and blocking- of lymphocyte binding of monoclonals to MCP by polyclonal anti- TLX. Extra-embryonic membranes are thus richly endowed with a complement regulatory protein which could facilitate their roles in protection of the fetus by avoidance of harmful maternal immune response amplification.

5-Methylcytosine content of DNA in blood, synovial mononuclear cells and synovial tissue from patients affected by autoimmune rheumatic diseases

The percentage of 5-methylcytosine (m5Cyt) has been determined in peripheral blood, synovial mononuclear cells and synovial tissue from patients affected by various rheumatic autoimmune diseases. The determination was performed by reversed-phase high-performance liquid chromatography. Fifteen controls were compared to twenty-one patients affected by rheumatoid arthritis and to nine patients affected by systemic lupus erythematosus. The mean percentage of m5Cyt in normal individuals was significantly higher than in the rheumatoid arthritis and systemic lupus erythematosus patients. In addition, patients with active disease showed lower values than patients in remission. This finding is in agreement with the hypothesis that DNA hypomethylation may play a role in the pathogenesis of the autoimmune diseases, resulting in altered oncogene expression. Therapy with cyclosporin A led to a decrease in the percentage of m5Cyt in three rheumatoid arthritis patients, but a rebound was observed when the cyclosporin A was suspended. The percentage of m5Cyt in the DNA of synovial tissue from four rheumatoid arthritis patients and five patients with osteoarthritis was similar; this observation confirms that, in addition to disease-specific and disease activity-specific variations, the percentage of m5Cyt may also show tissue-specific variations.

Tissue-specific variations in isoactin gene expression in the spontaneously hypertensive rat

Tissue-specific variations in isoactin gene expression in the spontaneously hypertensive rat

Chairman's Concluding Remarks

Although structurally simple, the nervous systems of parasitic helminths are biochemically complex. As well as the classical transmitters, the nervous systems of helminths contain a range of regulatory peptides and so far some 29 different peptides have been described. The only group of compounds thought to be transmitters in free-living organisms which have not yet been shown to be transmitters in helminths are the purines. There is evidence in helminths, as in vertebrates, for the co-localization, in the same cell, of classical transmitters and peptidergic molecules and for the non-neuronal occurrence of some regulatory peptides. Co-localization would effectively increase the number of transmitter types, since compounds could be released in different combinations. How such a selective release might be achieved is unknown. Different regulatory peptides are localized in different parts of the helminth nervous system, but it is quite probable that more than one peptide can occur in the same cell. Whether the relative proportions of the different peptides change under different physiological or developmental conditions is not known. The classical transmitters similarly show a differential distribution. It is presumed that the regulatory peptides in helminths, as in other organisms, are synthesized by post-translational modification of a high molecular weight precursor protein and that the peptides are not recycled but are removed from their site of action by specific peptidases. The latter are located near the receptor sites and may be membrane-bound. In vertebrates the precursor molecules usually contain several different peptides with different biological functions and processing of the precursor often shows tissue-specific variations.

Heterogeneous Expression of Poliovirus Receptor-Related Proteins in Human Cells and Tissues

Portions of the cellular receptor for poliovirus were expressed in Escherichia coli as fusion proteins with the product of the trpE gene. One of two antireceptor antisera obtained by immunizing rabbits with the fusion proteins blocked poliovirus infection. Western immunoblot analyses demonstrated that poliovirus receptor-related proteins were expressed in HeLa cells and a variety of human tissues, including those that are not sites of poliovirus replication. Tissue-specific variation in electrophoretic mobility, immunoreactivity, and subunit arrangement of poliovirus receptor-related proteins was observed. These results demonstrate that poliovirus tissue tropism cannot be explained by a limited distribution of receptor polypeptide, but may be the result of alternative splicing, posttranslational modifications, or both. In addition, the widespread but heterogeneous expression of the receptor suggests that the protein may have an important endogenous function.

Heterogeneous expression of poliovirus receptor-related proteins in human cells and tissues.

Portions of the cellular receptor for poliovirus were expressed in Escherichia coli as fusion proteins with the product of the trpE gene. One of two antireceptor antisera obtained by immunizing rabbits with the fusion proteins blocked poliovirus infection. Western immunoblot analyses demonstrated that poliovirus receptor-related proteins were expressed in HeLa cells and a variety of human tissues, including those that are not sites of poliovirus replication. Tissue-specific variation in electrophoretic mobility, immunoreactivity, and subunit arrangement of poliovirus receptor-related proteins was observed. These results demonstrate that poliovirus tissue tropism cannot be explained by a limited distribution of receptor polypeptide, but may be the result of alternative splicing, posttranslational modifications, or both. In addition, the widespread but heterogeneous expression of the receptor suggests that the protein may have an important endogenous function.

Identification of mammalian and invertebrate analogues of the avian calcium-dependent cell adhesion protein N-cadherin with syntheticpeptide directed antibodies against a conserved cytoplasmic domain

N-cadherin, a 130kD transmembrane adhesive glycoprotein, is a mediator of specific cellular interactions during development. Analysis of N-cadherin at the protein level, to date, has been largely dependent upon monoclonal antibody NCD-2 which recognizes only avian N-cadherin. We produced a monospecific polyclonal antiserum, C-NCAD(838-856), to a synthetic peptide corresponding to a portion of the highly conserved c-terminal cytoplasmic domain of chick N-cadherin. Using polyacrylamide gel electrophoresis and immunoblotting to map tissue distribution we show that the antiserum detects chick N-cadherin with a similar tissue distribution as NCD-2. Unlike NCD-2, however, anti-C-NCAD(838-856) recognizes N-cadherin analogues in a wide variety of species, including mouse, human, fish and drosophila. The results of comparative immunoblot studies demonstrate similar tissue-specific patterns and apparent molecular weight variation in the chick, mouse and human. This indicates that N-cadherin structure and expression, and most likely function as well, have been highly conserved in evolution. The antiserum recognizes an epitope unique to N-cadherin which is conserved among N-cadherins from a variety of species but is absent from other members of the cadherin gene family, as no immunoreactivity was detected with tissues bearing these other cadherins. The antiserum is thus a useful tool for the phylogenetic and biochemical investigation of N-cadherin from a variety of tissue sources.

Nucleotide sequence of cDNA for nuclear encoded subunit Vb of mouse cytochrome- c oxidase

We have used a polyclonal antibody probe to isolate cDNA clones for mouse cytochrome oxidase subunit Vb from a bone marrow tumor cell mRNA library in λgt11 expression vector. The mouse cDNA contains an open reading frame of 128 amino acids, which shows 81% positional identity with the predicted amino acid sequences of the human subunit. Northern blot analysis and sequencing of cDNA from a mouse kidney library show no tissue specific variations in subunit Vb.

Cold stress during scotophase elicited differential responses in quail pineal, retinal, and serum melatonin levels

Effects of cold stress during scotophase on pineal, retinal and serum melatonin levels were examined in quails. All experimental subjects were housed under a constant room temperature of 23 +/- 2 degrees C and a daily 12 h:12 h light:dark cycle. After 1 week of adaptation, quails were exposed to 4 degrees C in darkness for 60, 120, 180 and 210 min. Immediately following their respective cold treatments, subjects were sacrificed at mid-dark and pineal, retina and serum samples were collected for melatonin radioimmunoassay. Cold stress during scotophase was found to potentiate melatonin levels in the retinas significantly. Conversely, cold exposures in dark significantly decreased melatonin levels in pineal glands and serum. Such diversified responses might be attributed to tissue specific variations in adrenergic and/or dopaminergic receptors responsible for regulating the synthesis and/or secretory mechanisms of melatonin.

Ion Channels and Insulin Secretion

We review the role of ion channels in regulating insulin secretion from pancreatic beta-cells. By controlling ion permeability, ion channels at the membrane play a major role in regulating both electrical activity and signal transduction in the beta-cell. A proximal step in the cascade of events required for stimulus-secretion coupling is the closure of ATP-sensitive K+ channels, resulting in cell depolarization. Of particular relevance is the finding that this channel is directly regulated by a metabolite of glucose, which is the primary insulin secretagogue. In addition, this channel, or a closely associated protein, contains the sulfonylurea-binding site. Another K+ channel, the Ca2(+)-activated K+ channel, may be involved in cell repolarization to create homeostasis. Voltage-dependent Ca2+ channels are activated by cell depolarization and regulate Ca2+ influx into the cell. By controlling cytosolic free-Ca2+ levels ([Ca2+]i), these channels play an important role in transducing the initial stimulus to the effector systems that modulate insulin secretion. The link between a rise in [Ca2+]i and the terminal event of exocytosis is the least-understood aspect of stimulus-secretion coupling. However, phosphorylation studies have identified substrate proteins that may correspond to those involved in smooth muscle contraction, suggesting an analogy in the processes of stimulus secretion and excitation contraction. The advent of new methodology, particularly the patch-clamp technique, has fostered a more detailed characterization of the beta-cell ion channels. Furthermore, biochemical and molecular approaches developed for the structural analysis of ion channels in other tissues can now be applied to the isolation and characterization of the beta-cell ion channels. This is of particular significance because there appear to be tissue-specific variations in the different types of ion channels. Given the importance of ion channels in cell physiology, a knowledge of the structure and properties of these channels in the beta-cell is required for understanding the abnormalities of insulin secretion that occur in non-insulin-dependent diabetes mellitus. Ultimately, these studies should also provide new therapeutic approaches to the treatment of this disease.

Tissue and strain-specific patterns of endogenous proviral hypomethylation analyzed by two-dimensional gel electrophoresis.

Proviral sequences related to the intracisternal A particle (IAP) are amplified and dispersed in the mouse genome. Their expression is associated with hypomethylation at CpG sites in the 5' long terminal repeat. We have used two-dimensional agarose gel electrophoresis to examine patterns of IAP hypomethylation in mouse DNA. The method is sensitive to both the methylation status of a conserved Hae II site in the 5' long terminal repeat and the location of the closest BamHI site in the flanking DNA upstream of each hypomethylated long terminal repeat. The method also defects restriction fragments derived from IAP elements that are themselves methylated but have an unmethylated Hae II site in their 5' adjacent DNA. DNAs from each of four inbred mouse strains (BALB/c, C3H/He, C57BL/6, and DBA/2) gave distinctive two-dimensional patterns of BamHI/Hae II restriction fragments detected by hybridization with an IAP probe. This constitutive pattern was largely conserved among several tissues of each strain, but some tissue-specific variations were observed. The site-specific hypomethylations reflected in the two-dimensional patterns were heritable properties, since DNA from progeny of an interstrain cross contained both parental sets of fragments. IAP elements may be useful endogenous reporters of genomic methylation patterns.

Tissue-specific and age-related variations in repetitive sequences of mouse extrachromosomal circular DNAs

Extrachromosomal circular (ecc) DNA was isolated from mouse brain, liver, and heart tissues at different ages. To determine the abundance of repetitive sequences in eccDNAs, preparations were probed for short-interspersed (B1 and B2), long-interspersed (L1), endogenous retroviral-like (IAP), and tandemly repeated satellite sequences (SAT) of the mouse genome. Together these sequence families comprise approximately 15% of the mouse genome. The hybridization results showed that each tissue had a characteristic pattern of repetitive sequence elements in eccDNAs, and the abundance of repetitive sequences changed as a function of age. Repetitive sequences decreased in liver and brain eccDNAs from 1 month to 8 months of age but appeared to remain stable thereafter. In contrast, repetitive sequence families in heart eccDNAs were constant from 1 month to 16 months of age but declined in 24-month-old mice. The present studies indicate that extrachromosomal sequences exhibit greater flexibility than chromosomal sequences.

Genetic and tissue-specific variation in the expression of a closely linked murine multigene family on chromosome 15 that encodes salivary and lacrimal proteins

Genetic and tissue-specific variation in the expression of a closely linked murine multigene family on chromosome 15 that encodes salivary and lacrimal proteins