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PubMed1988

Isoform composition and stoichiometry of the approximately 90-kDa heat shock protein associated with glucocorticoid receptors.

We have observed that the approximately 90-kDa non-steroid-binding component of nonactivated glucocorticoid receptors purified from WEHI-7 mouse thymoma cells (which has been identified as the approximately 90-kDa heat shock protein) consistently migrates as a doublet during polyacrylamide gel electrophoresis under denaturing and reducing conditions. It has recently been reported that murine Meth A cells contain a tumor-specific transplantation antigen (TSTA) which is related or identical to the approximately 90-kDa heat shock protein (Ullrich, S.J., Robinson, E.A., Law, L.W., Willingham, M., and Appella, E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3121-3125). The observation that TSTA and the approximately 90-kDa heat shock protein isolated from these cells exists as two isoforms of similar molecular mass and charge has suggested to us that the doublet we observed is also due to the existence of two isoforms. However, unlike TSTA, which appears to contain the two isoforms in similar relative abundance, nonactivated glucocorticoid-receptor complexes seem to contain predominantly the lower molecular mass isoform. We have therefore conducted this study to determine whether TSTA and the approximately 90-kDa component of glucocorticoid receptors are indeed related, to establish whether the receptor preferentially binds one isoform of the approximately 90-kDa heat shock protein, and to investigate the stoichiometry of the nonactivated receptor complex. By comparing Meth A TSTA and the approximately 90-kDa component of the receptor in their reactions with the AC88 monoclonal antibody (specific for the approximately 90-kDa heat shock protein) and a polyclonal antibody directed against Meth A TSTA, we found that these two proteins are indistinguishable and probably identical. We then used the BuGR1 (directed against the steroid-binding subunit of glucocorticoid receptors) and AC88 monoclonal antibodies to purify, respectively, receptor-associated and free approximately 90-kDa heat shock protein from WEHI-7 cells grown for 48 h with [35S]methionine to metabolically label proteins to steady state. Following analysis of the proteins by polyacrylamide gel electrophoresis under denaturing and reducing conditions, the relative amounts of the two isoforms in each sample were determined from the 35S counts and the known methionine content of each isoform. We found that approximately three-quarters of both the receptor-associated and the free approximately 90-kDa heat shock protein is present as the lower molecular weight isoform, indicating no preferential binding of either isoform in the receptor. The long-term metabolic labeling approach has also enabled us to direc

Animals↗
PubMed1988

Rapid purification of mammalian cardiac troponin T and its isoform switching in rat hearts during development.

A rapid purification of troponin T from adult hearts of various species has been developed. The purification procedure included 60 degrees C treatment of the high salt extract, ammonium sulfate fractionation, and DEAE-cellulose column chromatography. The troponin T purified from the bovine left ventricle contained two isoforms, which differed in both apparent molecular mass and isoelectric point. Both isoforms were able to bind to F-actin filaments only in the presence of tropomyosin. Monoclonal antibody JLT12 against rabbit skeletal troponin T cross-reacted with both isoforms of bovine cardiac troponin T. There was no detectable difference in the relative amount of these two isoforms among different portions (atria, right and left ventricles) of the bovine heart. The purified protein was used as an antigen to immunize mice, and a mouse antiserum with high titer and specificity to both isoforms was subsequently obtained. This antiserum also cross-reacted with cardiac troponin T from chicken, rabbit, and rat. The antibodies were further used to probe cardiac development in rats by Western blotting and immunoprecipitation. The results clearly showed that there was a switch of troponin T isoforms between hearts from 20-day-old rat embryos and hearts from 14-day-old rats. Immunoprecipitation of the in vitro translation products of poly(A)+ RNA isolated from day 5 rat hearts revealed the presence of two isoforms of troponin T, suggesting that two mRNAs coding for these two isoforms existed in the heart cells. It is of interest to not that some profound changes in the morphology and function of cardiac muscle have also been detected at this time of development. Troponin T isoform switching thus may well represent an important marker for cardiac development and function.

Actins↗
PubMed1995

Are CD44 variant isoforms involved in human tumour progression?

The transmembrane glycoprotein CD44 exists in a variety of isoforms generated by alternative splicing of the pre-mRNA. In a rat metastasis model, certain variant isoforms (containing exon 6v) are causally involved in lung metastasis formation. We have summarized the data obtained to date on the expression of CD44 variant isoforms in human tumour progression. In non-Hodgkin lymphomas, expression of exon 6v containing isoforms is an independent prognostic factor indicating an adverse prognosis. Upregulation of exon 9v containing isoforms in gastric and renal cell carcinomas relates to a poor prognosis of patients. In colorectal carcinomas, CD44-9v isoforms are strongly expressed already in early adenomas; CD44-6v isoforms are upregulated in late adenomas along with ras and TP53 mutations. No expression of variant isoforms has been detectable in neuroblastomas, but significant downregulation of CD44s correlates inversely with tumour progression and N-myc amplification. Only in breast carcinoma has no correlation of CD44 expression with survival or any other prognostic marker been established. Evaluation of CD44 isoform expression by immunohistochemistry in cases of non-Hodgkin lymphoma, gastric, colon and renal cell carcinomas, as well as neuroblastomas, may be a useful diagnostic parameter indicating invasive processes.

Breast Neoplasms↗
PubMed1995

Anti-peptide sera against cell-CAM 105 determine high molecular-mass variants of the long isoform in rat hepatocytes.

The glycoprotein cell-CAM 105 is a member of the carcinoembryonic-antigen-(CEA)-gene family, involved in cell-cell adhesion of rat hepatocytes and expressed on the cell surface as a long (L) and a short (S) isoform with slightly differing molecular masses and isoelectric points. The cDNA of the L-isoform has been isolated and sequenced, as confirmed by the preparation of specific anti-peptide sera [Lin, S.-H., Culic, O., Flanagan, D. & Hixson, D. C. (1991) Biochem. J. 278, 155-161]. Recently, two additional cDNAs have been sequenced, which possess identical deduced primary structures, including short intracellular domains 10 amino acids in length, which differ from the cytoplasmic domain of the L-isoform specifically in the last four C-terminal amino acids. Here, we report on the production of the polyclonal antiserum [anti-(peptide 2)] by immunization with a synthetic hexapeptide (GGSGSF) corresponding to the unique intracellular C-terminal domain of these short cell-CAM 105 cDNA isoforms. This antiserum was specific in ELISA, immunoblot and immunoprecipitation assays for a protein with the same biochemical properties as the S-isoform of cell-CAM 105 expressed in rat liver. In addition, CNBr peptide maps of the S-isoform and the protein immunoprecipitated with anti-(peptide 2) serum were identical. Together, these results provide strong evidence that anti-(peptide 2) serum is specific for the S-isoform of rat liver cell-CAM 105. In immunoblot analysis on liver plasma membrane extracts prepared without collagenase perfusion, at least seven high molecular-mass proteins were observed which showed strong reactivity with mAbs against extracellular epitopes and L-isoform-specific antibodies but no reactivity with anti-(peptide 2) serum. Like the L-isoform, these proteins are expressed on the cell surface and might represent structural variants of cell-CAM 105.

Adenosine Triphosphatases↗
PubMed1995

Altered expressions of cardiac Na/K-ATPase isoforms in copper deficient rats.

OBJECTIVE: The aim was to determine if copper deficiency affects the expression of Na/K-ATPase alpha isoforms in the rat heart. METHODS: Copper deficiency was induced by placing weanling rats on a copper deficient diet for 4-5 weeks. Adult ventricular tissue, isolated ventricular myocytes, and brain stems of the control and deficient rats were compared for Cu, Zn-superoxide dismutase (CuZn-SOD) activity and for protein and mRNA contents of Na/K-ATPase alpha isoforms. RESULTS: In brain stem, where copper deficiency did not alter CuZn-SOD activity, mRNA and protein levels of alpha isoforms also remained unchanged. In ventricular tissue and ventricular myocytes, copper deficiency reduced CuZn-SOD activity, mRNAs of alpha 1 and alpha 2 isoforms, and the alpha 2 isoform protein. The alpha 1 isoform protein of ventricular tissue and its myocytes was marginally reduced by copper deficiency. CONCLUSIONS: In the rat ventricular tissue, oxidative stress resulting from copper deficiency (1) enhances the turnover of the more oxidant sensitive alpha 2 isoform to a greater extent than the turnover of the alpha 1 isoform; (2) regulates mRNA levels of alpha 1 and alpha 2 isoforms; and (3) contributes to the cardiomyopathy of copper deficiency.

Animals↗
PubMed1994

Primary structure and distribution of ryanodine-binding protein isoforms of the bullfrog skeletal muscle.

We have cloned two groups of cDNAs which encode isoforms of ryanodine-binding protein/Ca2+ release channel of the bullfrog skeletal muscle sarcoplasmic reticulum. One of the cDNA groups encodes the protein of 5,037 (or 5,031 with a deletion) amino acids with a molecular weight of 571,262 (or 570,607), which is identified as the alpha-isoform of the ryanodine-binding protein based on the amino acid sequence of three tryptic fragments of the purified protein. The other group of cDNAs encodes the protein of 4,868 amino acids with molecular weight of 553,029, which contains the sequences of three proteolytic fragments derived from the beta-isoform protein. About 70% of the amino acid sequence identity is present between alpha- and beta-isoforms of the bullfrog. The primary structure of the alpha-isoform is highly (80%) homologous to the ryanodine-binding protein cloned from rabbit skeletal muscle (type 1). The beta-isoform, on the other hand, is more than 85% identical with that from the rabbit brain (type 3), while it has only 67% overall identity with type 1. Analyses of RNA from various tissues of the bullfrog demonstrate that the beta-isoform is widely expressed, while the alpha-isoform is expressed mainly in skeletal muscle. A phylogenetic analysis of the ryanodine-binding protein/Ca2+ release channel family suggests that the various types of Ca2+ release channels have evolved from an ancestor gene. Possible differential roles of alpha- and beta-isoforms of ryanodine-binding protein in Ca2+ release mechanisms including skeletal muscle excitation-contraction coupling were discussed.

Animals↗
PubMed1994

Differential expression of synaptic vesicle protein 2 (SV2) isoforms.

The synaptic vesicle proteins SV2A and SV2B (SV2 = synaptic vesicle protein 2) are two highly related proteins belonging to a family of transporters. As a first step toward identifying the function of the SV2 proteins, we examined the expression of SV2A and SV2B in the rat brain by in situ hybridization, immunohistochemistry, and immunoprecipitation with isoform-specific antibodies. These analyses revealed that one isoform, SV2A, is expressed ubiquitously throughout the brain at varying levels. The other isoform, SV2B, has a more limited distribution with varying degrees of coexpression with SV2A. Immunoprecipitation of brain synaptic vesicles with isoform-specific antibodies followed by Western analyses suggests that both isoforms can be present on the same synaptic vesicle. The expression of the SV2 proteins did not correlate either with neurotransmitter phenotype or with the expression of other synaptic vesicle protein isoforms. SV2B expression was observed to change during development; it is more widely expressed in the immature brain and is found in cells that have yet to establish synaptic contacts. The ubiquitous and overlapping expression of the SV2s suggests that they perform a function common to all synaptic vesicles. Variable and changing coexpression of the SV2 isoforms may indicate that SV2 function is regulated by the isoform composition of synaptic vesicles. The observation that the synaptic vesicle proteins, all occurring in multiple isoforms, are differentially expressed with respect to each other indicates that up to 90 different vesicle types are possible.

Animals↗
PubMed1993

Baboon apolipoprotein A-IV. Identification of Lys76-->Glu that distinguishes two common isoforms and detection of length polymorphisms at the carboxyl terminus.

Various protein isoforms have been identified for human apolipoprotein A-IV (apoA-IV). However, investigations of their physiological effects have been limited because of low frequencies for many of the apoA-IV variants. Recent discovery of extensive variation in baboon apoA-IV using isoelectric focusing (IEF) makes this primate species an excellent model for genetic studies of apoA-IV. In this study, the molecular basis for net charge differences between two common apoA-IV isoforms (I and E) was determined by cloning and sequencing of intestinal cDNAs from homozygous baboons. An A-->G substitution was found in the third amphipathic repeat of the E isoform. This substitution causes a Lys-->Glu substitution at amino acid position 76 (Lys76-->Glu), adding two negative charges to the E isoform compared to the I isoform, consistent with their relative mobilities on IEF gels. Restriction isotyping was used to identify the substitution in leukocyte DNA from 15 baboons that had been typed by IEF, thus verifying Lys76-->Glu as the basis for the charge differences between the I and E isoforms. Physiological effects of the Lys76-->Glu substitution on high density lipoprotein-C levels were investigated in 431 baboons carrying the E and I isoforms. These studies revealed that the I isoform was associated with higher levels of high density lipoprotein-C on a high cholesterol, saturated fat diet (p = 0.04). The cDNA sequences showed that the carboxyl terminus of baboon apoA-IV contains a region of hydrophilic repeats (Glu-Gln-X-Gln) that is the largest yet found in any species (nine repeats compared to three to five repeats in human, mouse, and rat). A common length polymorphism was identified that inserts a single amino acid to form a five amino acid repeat. This is the first report of this type of length variation (insertion of a single amino acid rather than insertion of an entire repeat) in this region. In addition, a rare variant was found that inserts an entire four-amino-acid repeat, similar to the human apoA-IV-0 isoform.

Amino Acid Sequence↗
PubMed1993

Similar alternative splicing events generate two sarcoplasmic or endoplasmic reticulum Ca-ATPase isoforms in the crustacean Artemia franciscana and in vertebrates.

A second isoform of the Artemia franciscana sarcoplasmic or endoplasmic reticulum Ca-ATPase has been identified through the isolation of cDNA clones. This isoform differs from the previously identified one only at the C-terminal end of the protein. The last 6 amino acids of the former isoform change to 30 hydrophobic amino acids in the newly identified isoform that have the potentiality of being an additional transmembrane domain. The two A. franciscana isoforms are highly homologous to the two isoforms coded by the mammalian and bird SERCA2 gene, except that the C-terminal extensions share their hydrophobic character but have no significant amino acid homology. The isolation of genomic clones coding for this region of the gene shows that both isoforms arise from the same gene by alternative splicing. The donor splicing site of the penultimate exon can either be recognized and fused to the last exon, giving rise to the mRNA coding for the shortest protein, or remain unrecognized, in which case a polyadenylation site is recognized before the last exon of the gene and the mRNA coding for the largest protein is originated. The alternative splicing used to originate the two isoforms is similar in Artemia and vertebrates. The conservation of the alternative splicing between species so distant in evolution suggests an important physiological role for the existence of the two isoforms of the protein.

Alternative Splicing↗
PubMed1993

Sequence, expression pattern, intracellular localization, and targeted disruption of the Dictyostelium myosin ID heavy chain isoform.

The complete sequence of the Dictyostelium myosin ID (DMID) heavy chain isoform has been determined from cDNA and genomic clones. Like the DMIB isoform characterized previously, the DMID isoform is up-regulated during starvation-induced chemotactic aggregation, and its 124-kDa heavy chain contains the tail domain sequences that correspond to both the membrane and second actin-binding sites. An antibody that is specific for the DMID isoform was found to stain the actin-rich pseudopods at the leading edge of migrating cells. Protein microsequencing data reveals that the myosin I isoform localized to leading edge pseudopods in a previous study (Fukui, Y., Lynch, T. J., Brzeska, H., and Korn, E. D. (1989) Nature 341, 328-331) was DMIB, indicating that DMID and DMIB also colocalize and that both should influence the dynamics of actin-rich cortical structures. This and other data indicate that the DMID and DMIB isoforms are closely related and are distinct from the DMIA and DMIE isoforms, which possess truncated tail domains and are not up-regulated during chemotactic aggregation. Cells in which the DMID gene was rendered nonfunctional by targeted gene disruption do not show obvious behavioral defects, suggesting that another myosin I isoform(s) (possibly DMIB) might compensate for DMID. Finally, Southern blot data indicate that Dictyostelium may contain as many as nine myosin I isoforms.

Amino Acid Sequence↗
PubMed1993

Two isoforms of prostaglandin E receptor EP3 subtype. Different COOH-terminal domains determine sensitivity to agonist-induced desensitization.

We recently identified two isoforms of mouse prostaglandin (PG) E receptor EP3 subtype, EP3 alpha and EP 3 beta, which are produced by alternative splicing and different only in the carboxyl-terminal domain (Sugimoto, Y., Negishi, M., Hayashi, Y., Namba, Y., T., Honda, A., Watabe, A., Hirata, M., Narumiya, S., and Ichikawa, A. (1993) J. Biol. Chem. 268, 2712-2718). We examined here agonist-induced desensitization of the two isoforms using Chinese hamster ovary cells stably expressing these isoforms. Exposure of the EP3 alpha isoform to PGE2 for 30 min did not change maximal response but increased PGE2 concentration needed to inhibit forskolin-induced cAMP accumulation in the cells. Further exposure of this isoform to PGE2 suppressed the maximal response as well as sensitivity to PGE2 in a time-dependent manner; after 24-h exposure, it elicited only 50% of the maximal response of the control cells. Consistent with these results, short term exposure sequestered the EP3 alpha isoform away from the cell surface and long term incubation decreased the total receptor number in the cells. In contrast, exposure of the EP3 beta isoform to PGE2 did not affect its dose-response curve for PGE2, and no sequestration or decrease in the receptor number was observed in this isoform. Thus, alternative splicing produced the two isoforms with different carboxyl-terminal domains, which are different in sensitivity to agonist-induced desensitization.

Alternative Splicing↗
PubMed1996

Expression and activation of protein kinase C isoforms in a human megakaryocytic cell line.

Megakaryocytes undergo a unique differentiation program, becoming polyploid through repeated cycles of DNA synthesis without concomitant cell division. We have shown previously that phorbol 12-myristate 13-acetate (PMA) induces the Dami human megakaryocytic cell line to become polyploid and to express platelet-specific proteins, including von Willebrand factor (vWF) and glycoprotein Ib (GpIb). Phorbol esters are thought to regulate gene expression principally through the activation of protein kinase C (PKC), a family of structurally related kinases with potentially unique activation requirements and substrate specificities. A survey of PKC isoforms in Dami cells revealed that, by both Western and Northern analyses, PKC isoforms alpha, beta, delta, epsilon, eta, theta, and zeta were reproducibly detected. PKC-gamma was not detected. In order to define the role of individual PKC isoforms in megakaryocytic maturation, PMA and 2-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), a putative selective activator of the PKC-beta 1 isotype, were compared for their effects on Dami cell maturation. Treatment with either dPPA or PMA caused Dami cells to cease proliferating, to become polyploid, and to express vWF. We also examined dPPA and PMA for their ability to activate and to downregulate expression of different PKC isoforms. Fifteen-minute treatment with PMA resulted in the translocation of PKC isoforms alpha, epsilon, and theta from the cytosolic to the membrane fraction; twenty-four hour treatment resulted in the downregulation of these isoforms. In contrast, dPPA was found to be a potent activator of PKC-epsilon alone and exhibited weaker effects on alpha and theta. These data suggest that PKC isoforms beta, delta, eta, and zeta, which appear not to be activated by either phorbol ester, are unlikely to be primarily involved in megakaryocytic maturation in response to these agents. The isoforms that are translocated by both phorbol esters-PKC isoforms alpha and theta, and particularly epsilon-are more likely to transduce the signals that stimulate Dami cell differentiation.

Cell Differentiation↗
PubMed1997

Protein kinase C isoforms undergo quantitative variations during rat spermatogenesis and are selectively retained at specific spermatozoon sites.

Signal transduction elements, including protein kinase C, have been identified in mammalian spermatozoa. In order to evaluate the pattern of expression and the subcellular localization of nine different protein kinase C isoforms in the course of spermatogenesis, we utilized quantitative electron microscopy immunocytochemistry on thin sections of rat seminiferous tubules. The results indicate a progressive reduction of the protein kinase C isoforms present in the early stages of spermatogenesis, so that in late spermatids none of them is present in the nucleus, while the isoforms alpha, gamma and beta II are specifically retained in the acrosome, the isoforms beta I and zeta in the neck, and the isoform epsilon in the tail. These isoforms, except for beta II, are maintained at the same sites in spermatozoa. Western blotting analysis indicates the presence of alpha and gamma isoforms in the head subfraction, and of beta I, zeta and epsilon isoforms in the tail subfraction of spermatozoa. These findings suggest that specific protein kinase C isoforms may be functionally involved in some events of spermatozoa differentiation and, eventually, in the fertilization process.

Animals↗
PubMed1998

[Relationship between lipoprotein A, apolipoprotein A and their isoforms in parents and children].

OBJECTIVE: It has been observed that the apo(a) isoforms of low molecular weight predict the family history of premature ECV. Our objective was to study the relationship between the levels of Lp(a) and the isoforms of apo(a) in parents and children. PATIENTS AND METHODS: Twenty-four families, where at least one of the children had been diagnosed with familial or polygenic hypercholesterolemia and at least one of these children and one parent had Lp(a) > or = 30 mg/dl, were studied. They were classified according to whether Lp(a) was > or = < 30 mg/dl, with 44 and 38 subjects in each group, respectively. The total cholesterol, triglycerides, HDL and LDL, the Lp(a) and apo(a) isoforms were determined, as well as the relative concentration of the latter. RESULTS: The molecular weight average of the major isoform was lower in the group with high Lp(a) levels (592 +/- 38 vs 656 +/- 65 kD, p < 0.001). An inverse correlation between Lp(a) levels and the major isoform size was found (r = -0.522, p < 0.001). The correlation of the Lp(a) levels in the child with that of his or her "best fit" (the best fit parent was that whose level was closer to that of the child) was very significant (r = 0.61, p < 0.01), but lower than the corresponding paternal apo(a) isoform levels (r = 0.801, p < 0.01). The study of the apo(a) isoforms in parents and their children suggests not only that the isoform is inherited, but also its concentration. CONCLUSIONS: Subjects with small size apo(a) isoforms have higher Lp(a) levels. There is a better correlation between parents and children in the phenotypes of the apo(a) than in Lp(a) levels. The determination of these parameters allows the identification of a risk population.

Adolescent↗
PubMed1998

Cardiac troponin T isoforms expressed in renal diseased skeletal muscle will not cause false-positive results by the second generation cardiac troponin T assay by Boehringer Mannheim.

The purpose of this study was to determine whether the two monoclonal anti-cardiac troponin T (cTnT) antibodies (MAbs) used in the second generation cTnT assay by Boehringer Mannheim (BM, capture Ab, M11.7; detection Ab, M7) would detect cTnT isoforms expressed in human skeletal muscle in response to chronic renal disease (CRD). cTnT expression was examined in skeletal muscle biopsies obtained from 45 CRD patients, as well as nondiseased human heart (n = 3) and skeletal muscle (n = 3). cTnT proteins were resolved by modified 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with the following anti-cTnT MAbs: M11.7; M7; JS-2, Lakeland Biomedical; and 13-11, Duke University. All four antibodies detected the cTnT isoforms (Ta, Te) expressed in human myocardium. In 20 of 45 skeletal muscle biopsies, MAb M11.7 recognized its epitope in one to three proteins, molecular mass 34-36 kDa, designated Te, Td, and Tc; the strongest signal was that of Te. The same proteins were recognized by MAbs JS-2 and 13-11. The BM M7 antibody did not detect the cTnT isoforms in the molecular mass range of 34-36 kDa. However, MAb M7 did detect a cTnT isoform, molecular mass 39 kDa, in 2 of 45 biopsies. This isoform had an electrophoretic mobility similar to the predominant heart cTnT isoform, Ta. We conclude that cTnT isoforms are expressed in the skeletal muscle of CRD patients. However, given the epitopes recognized by the BM MAbs M7 and M11.7 and the variable presence of these cTnT isoforms in skeletal muscle, the second generation BM cTnT assay will not detect these isoforms if they are released from skeletal muscle into the circulation.

Adult↗
PubMed2000

Smooth muscle myosin heavy chain isoforms and their role in muscle physiology.

Unlike vertebrate skeletal muscle, smooth muscle myosin heavy chain isoforms are encoded by a single gene. Alternative splicing of the primary transcript from a single gene generates four smooth muscle myosin heavy chain isoforms. These isoforms differ both at the carboxyl terminus (SM1 and SM2 isoforms) and at the amino terminus (SM-A and SM-B isoforms). The smooth muscle myosin heavy chain isoforms are differentially expressed during smooth muscle development and in different smooth muscle cell types. The mechanical properties of smooth muscle may be correlated with the myosin heavy chain content/isoform expression. However, the precise function of each smooth muscle myosin heavy chain isoform to muscle contraction remains to be determined. This review mainly focuses on the molecular basis of smooth muscle myosin heavy chain isoform diversity, its expression during development and disease, and its role in muscle physiology.

Amino Acid Sequence↗
PubMed2005

Rapid separation of protein isoforms by capillary zone electrophoresis with new dynamic coatings.

Many cellular functions are regulated through protein isoforms. Changes in the expression level or regulatory dysfunctions of isoforms often lead to developmental or pathological disorders. Isoforms are traditionally analyzed using techniques such as gel- or capillary-based isoelectric focusing. However, with proper electro-osmotic flow (EOF) control, isoforms with small pI differences can also be analyzed using capillary zone electrophoresis (CZE). Here we demonstrate the ability to quickly resolve isoforms of three model proteins (bovine serum albumin, transferrin, alpha1-antitrypsin) in capillaries coated with novel dynamic coatings. The coatings allow reproducible EOF modulation in the cathodal direction to a level of 10(-9) m2V(-1)s(-1). They also appear to inhibit protein adsorption to the capillary wall, making the isoform separations highly reproducible both in peak areas and apparent mobility. Isoforms of transferrin and alpha1-antitrypsin have been implicated in several human diseases. By coupling the CZE isoform separation with standard affinity capture assays, it may be possible to develop a cost-effective analytical platform for clinical diagnostics.

Animals↗
PubMed2002

Regulation and differential expression of tau mRNA isoforms as oligodendrocytes mature in vivo: implications for myelination.

Oligodendrocytes and neurons derive from the same cell type but develop distinct morphologic and functional properties as they mature in vivo. Both cells express tau protein, a developmentally regulated protein in the central nervous system. The regulation of tau has been investigated extensively in neurons but not in oligodendrocytes, so we studied regulation of tau in oligodendrocytes in vivo. The amino-derived tau isoforms consist of isoforms with zero (A0), one (A1), or two (A2) inserts. We examined the developmental regulation of tau mRNA isoforms at the amino domain by comparing tau expression in oligodendrocytes (OLGs) isolated from 1- and 20-day-old rat brain and in age-matched cortex, which abounds in neurons. In the rat brain, myelination peaks at 20 days. By using semiquantitative RT-PCR, we found that OLGs and cortex from 1-day-old rat brain largely had amino-derived tau isoforms with no insert, whereas OLGs from 20-day-old rat brain had similar levels of amino-derived tau isoforms with no insert or with one insert. We also found that 20-day-old OLGs had twofold more tau mRNA levels than younger OLGs. In contrast to OLGs from 20-day-old rat brain, age-matched cortex had comparable levels of A0, A1, and A2 tau amino-derived isoforms. Further, younger and older OLGs had a reciprocal pattern of expression of both carboxy-derived tau mRNA isoforms with either three (3R) or four (4R) repeats. In contrast, younger and older cortex expressed either 3R or 4R tau. This study showed an upregulation of tau mRNA and cell-specific tau mRNA isoform expression in OLGs forming myelin.

Aging↗