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PubMed1994

Protein kinase C isoforms in human and rat colonic mucosa.

The protein kinase C (PKC) family of enzymes plays a key role in the regulation of cellular events, including cell proliferation and differentiation. Work from our laboratory has shown that the effects of dietary fat and fiber on colonic cell proliferation were positively correlated with membrane/cytosol PKC activity ratios (Chapkin et al., 1993, J. Nutr. 123, 649-655). The presence and subcellular distribution of specific PKC isoforms in rat and human colon were therefore determined in cytosolic and membrane extracts. Tissue extracts were probed with antibodies to individual PKC isoforms. PKC alpha, beta, delta, epsilon, and zeta were detected in both rat and human colonic mucosa, while PKC eta was detected in human colonic mucosa only. PKC alpha, beta, and zeta were predominantly localized in the cytosolic fraction, whereas the majority of PKC delta, epsilon, and eta were found in the membrane-associated fraction. Presence of mRNA for individual PKC isoforms was determined by reverse transcriptase PCR (RT-PCR). Using rat colonic mucosa, mRNA for PKC alpha, beta, delta, epsilon, eta, and zeta were detected by RT-PCR with identity confirmed by sequencing. The relative steady-state levels of PKC isoforms in human colon adenocarcinoma as compared with normal colonic mucosa were determined, with adenocarcinomas having higher amounts of cytosolic PKC beta, delta, epsilon, eta, and zeta. PKC isoforms were also detected in viable, exfoliated colonic cells isolated from human feces, demonstrating that this noninvasive method can be utilized to examine PKC expression in colonic cells. These results demonstrate that colonic mucosa expresses both calcium-dependent (classical) and calcium-independent (novel and atypical) PKC isoforms with distinct subcellular distributions for each. The dynamics of these PKC isoforms may have implications in the development of colon carcinogenesis.

Adenocarcinoma↗
PubMed1996

Purification and properties of four phosphoenolpyruvate carboxylase isoforms from the green alga Selenastrum minutum: evidence that association of the 102-kDa catalytic subunit with unrelated polypeptides may modify the physical and kinetic properties of the enzyme.

Four isoforms of phosphoenolpyruvate carboxylase (PEPC1, PEPC2, PEPC3, PEPC4) have been purified from the green alga Selenastrum minutum. PEPC1 is a homotetramer with a subunit M(r) of 102 kDa. PEPC2, PEPC3, and PEPC4 have respective native M(r)S of approximately 984, 1186, and 1590 kDa. SDS/PAGE analysis revealed that the latter three isoforms contain polypeptides having M(r)S of 102, 73, 70, 65, and 61 kDa. Immunoblot analyses and CNBr cleavage patterns suggest that the 102-kDa polypeptide present in all four isoforms is the same PEPC catalytic subunit. Our data suggest that the three high M(r)S PEPC isoforms are heteromeric protein complexes consisting of the 102-kDa PEPC1 catalytic subunit and immunologically unrelated polypeptides. Attempts to measure other enzyme activities associated with the protein complexes gave negative results. However, PEPC1 had immunological, physical, and kinetic properties very different from those of the larger M(r) PEPC isoforms: (i) the anti-PEPC1 immune-serum was relatively inefficient for immunoprecipitating PEPC2, PEPC3, or PEPC4; (ii) immune-serum raised against a mixture of PEPC2, PEPC3, and PEPC4 had relatively weak immunoprecipitating activity toward PEPC1; (iii) PEPC1 was more heat sensitive than the other three isoforms; (iv) PEPC1 had a pH optimum of 9 versus 8.5 for the PEPC protein complexes; (v) the high Mr PEPCs had greater apparent affinity for phosphoenolpyruvate compared to PEPC1 and (vi) PEPC1 activity was far more sensitive to metabolite activators (Gln and dihydroxyacetone phosphate) and inhibitors (Asp, Glu, 2-oxoglutarate and malate). We conclude that the interaction of the PEPC catalytic subunit with unrelated polypeptides is responsible for the observed differences between PEPC1 and the high molecular mass isoforms. We propose that this interaction possibly regulates PEPC activity in vivo.

Chlorophyta↗
PubMed1996

Inhibition of nitric oxide synthase isoforms by porphyrins.

Protoporphyrin IX inhibits citrulline formation by all three nitric oxide synthase isoforms in a manner reversible by dilution. Zinc protoporphyrin IX, by contrast, produces a time- and concentration-dependent inactivation of all three nitric oxide synthase isoforms, not reversible by dilution. The inhibition of citrulline formation by protoporphyrin IX occurs with IC50 values of 0.8, 4, and 5 microM for the nNOS, iNOS, and eNOS isoforms, respectively. Inhibition by N-methyl-protoporphyrin IX occurs at IC50 values of 6, 5, and 8 microM for the nNOS, iNOS, and eNOS isoforms, respectively. Inhibition of nitric oxide synthase by protoporphyrin IX is a multisite, positively cooperative inhibition that exhibits a Hill coefficient of 2.3 for the iNOS isoform. Protoporphyrin IX reduces the maximal velocity of citrulline formation for both the iNOS and nNOS isoforms without altering the K(m) for the arginine substrate or the EC50 value for the tetrahydrobiopterin cofactor. Protoporphyrin IX inhibits the arginine-independent NADPH oxidase activity of nNOS with an IC50 value of 1 microM but has no effect on cytochrome c reductase activity at concentrations as high as 30 microM. At concentrations of 10 and 20 microM, protoporphyrin IX inhibits NO formation by cytokine-induced murine RAW 264.7 cells; however, these inhibitions are accompanied by significant cellular cytotoxicity. Coproporphyrins I and III, uroporphyrins I and III, and porphobilinogen, intermediates in the biosynthesis of heme that accumulate in hepatic porphyrias, are ineffective as inhibitors of the nitric oxide synthase isoforms. Since protoporphyrin IX is the immediate biosynthetic precursor of heme that accumulates in hepatic protoporphyria, iron deficiency anemia, and lead poisoning, protoporphyrin IX inhibition of nitric oxide synthase may contribute to the pathophysiology of these conditions.

Animals↗
PubMed2000

Purification and properties of multiple isoforms of a novel thiol methyltransferase involved in the production of volatile sulfur compounds from Brassica oleracea.

Five functional isoforms of a novel plant thiol methyltransferase from the leaves of cabbage (Brassica oleracea L.) were purified to electrophoretic homogeneity. Pooled, partly purified preparations of the enzyme were previously shown to methylate thiol compounds released upon the hydrolysis of glucosinolates. The enzyme could also accept halide ions as substrates. The estimated molecular masses of the purified isoforms ranged between 26 and 31 kDa. The three most abundant isoforms of the enzyme could all catalyze the S-adenosyl-l-methionine-dependent methylation of thiocyanate, a number of organic thiols and iodide. However, the kinetic properties of these forms toward various substrates differed widely. None of the isoforms examined methylated the O- and N-equivalents of the thiol substrates. The three isoforms also had distinct pH optima, covering the range from 5 to 9. Their kinetic analysis indicated that they shared a sequential substrate binding mechanism and an Ordered Bi Bi mechanism for substrate binding and product release. Partial internal amino acid sequence from one isoform showed high similarity to an Arabidopsis EST of unknown function, and to a recently cloned methyl chloride transferase from Batis maritima. The differences in the pH optima and kinetic properties of the isoforms suggest that each may methylate a specific substrate or a narrow group of substrates under cellular conditions.

Brassica↗
PubMed1996

Alternative RNA splicing-generated cardiac troponin T isoform switching: a non-heart-restricted genetic programming synchronized in developing cardiac and skeletal muscles.

An alternative RNA splicing-generated cardiac troponin T (cTnT) isoform switching has been found during both avian and mammalian heart development. The present study demonstrates that the mouse cTnT isoform switching is approximately 10 days earlier than that in rat during perinatal heart development. The mouse cTnT isoform switching is also approximately 10 days earlier than the transcriptionally regulated troponin I (TnI) isoform switching, a transition that showed similar timing during mouse and rat heart development. In addition, synchronized cTnT and TnI switches are observed during both rat and chicken heart development. More interestingly, the expression of cTnT in both mouse and rat neonatal skeletal muscles showed an isoform transition pattern synchronized with the cTnT isoform switching in the neonatal heart of the corresponding species. The results suggest that the alternative RNA splicing-generated cTnT isoform switching during development is (a) not a direct response to the functional changes in perinatal myocardium and (b) not a restricted regulation in the heart, but is controlled by a species-specific programming that is synchronized in developing cardiac and skeletal muscles.

Alternative Splicing↗
PubMed1997

Two isoforms of prostaglandin EP3 receptor exhibiting constitutive activity and agonist-dependent activity in Rho-mediated stress fiber formation.

We have cloned two isoforms of the mouse prostaglandin E receptor EP3 subtype, EP3alpha and EP3beta, with different carboxyl-terminal tails, produced through alternative splicing. To determine the functional differences between the two isoforms, we examined the role of the isoforms in regulation of the actin cytoskeleton using Mardin-Darby canine kidney cells expressing these isoforms. The EP3alpha isoform constitutively induced stress fiber formation, independent of an agonist, while the EP3beta isoform agonist-dependently induced stress fiber formation. Pertussis toxin did not prevent stress fiber formation. This signaling pathway is mediated by Rho, because C3 transferase microinjection inhibited stress fiber formation. Therefore, the physiological significance of these isoforms of the EP3 receptor may lie in their different agonist dependency in Rho-mediated stress fiber formation via a pertussis toxin-insensitive G protein.

Actins↗
PubMed1994

Isoform transition of contractile proteins related to muscle remodeling with an axial gradient during metamorphosis in Xenopus laevis.

For clarification of the mechanism of hormonally and spatially regulated larval-to-adult conversion of skeletal muscle, changes in the expression of muscle contractile proteins were examined during metamorphosis of Xenopus laevis. Analysis by electrophoresis revealed that isoforms of myosin heavy chain (MHC) switched from larval to adult type and adult-specific beta-tropomyosin (TM) appeared during metamorphosis in addition to preexisting alpha-TM. Distinct regional differences in isoform transition were apparent. Isoform changes started at stage 54 in the hindlimb and at stage 57 in the body. However, no change in the tail occurred. Immunohistochemical examination was performed to analyze isoform transition in dorsal body muscle. Before metamorphosis (stage 53), only a small number of muscle fibers at the dorsomedial part of dorsal muscle expressed "adult-type" muscle proteins. During metamorphosis the adult-type area gradually expanded from dorsal to ventral slides with an anteroposterior gradient with increase in adult-type (adult-type MHC and beta-TM-positive) muscle fibers. Thus, there is a gradient in isoform transition. In addition, adult-type fibers showed smaller diameter than larval-type fibers and DNA synthesis occurred in dorsal muscle with an anteroposterior gradient before the isoform transition, suggesting that new myogenesis of adult-type fibers proceeds with a gradient during metamorphosis. Also, degenerating fibers were observed only in the larval-type area. These results suggest that isoform transition is achieved by new proliferation of adult-type myoblasts and death of preexisting larval-type fibers, not by change in gene expression within the same cell.

Animals↗
PubMed1996

Developmental profiles and ecdysteroid regulation of the mRNAs for two ecdysone receptor isoforms in the epidermis and wings of the tobacco hornworm, Manduca sexta.

Ecdysteroids acting through multiple isoforms of the ecdysone receptor (EcR) initiate molting and metamorphosis of insects. Two isoforms of EcR, A (this paper) and B1 (Fujiwara et al., Insect Biochem. Mol. Biol. 25, 845-856, 1995), were isolated from the tobacco hornworm, Manduca sexta, and shown to be similar to the corresponding Drosophila EcR isoforms. The developmental profiles of both EcR-A and EcR-B1 (determined by both analysis of isoform-specific mRNAs and use of monoclonal antibodies that detect either EcR-B1 or all forms), however, were different in Manduca epidermis, which produces sequentially the larval, the pupal, and the adult cuticles. EcR-B1 predominated through the larval, pupal, and early developing adult stages with an upregulation early in each molt. By contrast, EcR-A was present only at the onset of new cuticle synthesis during the larval molt, but in the pupal and adult molts was upregulated slightly later than EcR-B1 during the commitment period and was present during the predifferentiative phase. Both isoforms appeared in the larval wing discs after pupal commitment and persisted through pupal differentiation. The mRNAs for both isoforms were directly induced in larval epidermis in vitro by 20-hydroxyecdysone, but EcR-B1 mRNA accumulated more rapidly, peaking at 3 hr. In the presence of a protein synthesis inhibitor, the accumulation of EcR-B1 mRNA was slower and its subsequent decline was prevented, but the accumulation of EcR-A mRNA was unaffected. Thus, in this polymorphic epidermis both isoforms appear in every molt, with EcR-B1 present during the commitment and predifferentiative phases and then at the onset of cuticle synthesis EcR-A prevails. Additionally, EcR-A is apparently associated with the switching and predifferentiative events necessary for a new synthetic program.

Amino Acid Sequence↗
PubMed1997

Differential expression pattern of Rab-GDI isoforms during the parotid gland secretion cycle.

Rab GDP dissociation inhibitor (GDI) plays an important role in regulating the GDP/GTP cycle of small GTP binding proteins of the Rab family. It also regulates their association to membranes. The small family of Rab-GDI consists of several closely related isoforms, the functional differences between which are still unknown. Here we show that multiple GDI isoforms are expressed in rat parotid gland and that the individual GDI isoforms have a characteristic expression both at the RNA and at the protein level, during the parotid secretory cycle. GDIalpha, the major isoform in brain, is expressed throughout the secretory process and is equally distributed between cytoplasmic and membranous fractions. In contrast, an isoform related to, but different from GDIbeta is found predominantly in the cytoplasmic fraction and its expression is detected only after beta-adrenergic stimulation of the gland, at the end of the secretion phase, when exocytosis is already completed. The induction of such a GDI isoform at the beginning of the recovery stage correlates with the expression pattern of Rab1 and Rab5, but not Rab2 and Rab4. Our results suggest different functional roles for multiple GDI isoforms along the secretion and recovery phases in rat parotid gland.

Adrenergic beta-Agonists↗
PubMed1999

In vitro release of isoforms of prolactin from pituitary glands of turkey hens at different physiological stages.

To study the in vitro release of PRL isoforms, anterior pituitary glands from medium white turkeys at various physiological stages were stimulated by cVIP in a perifusion system. Pituitaries were cut into hemi-pituitaries after collection and placed into separate perifusion chambers. Medium (M199) was continuously perifused through the system and pituitaries were stimulated with cVIP (10(-7) M). Total PRL content was monitored by RIA and, the ratio of immunoreactive PRL isoforms in the perifusate was estimated by Western blotting. After exposure to X-ray film for autoradiography, the relative intensity of the bands was analyzed by densitometry. All the perifused pituitaries responded to cVIP stimulation by increasing the release of PRL. Two immunoreactive bands with relative molecular weights of 24 and 27 kDa were detected by Western blotting. The immunoreactive band corresponding to the glycosylated isoforms of PRL (27 kDa) was predominant in samples from egg-laying and incubating hens and the band corresponding to the nonglycosylated isoform (24 kDa) was predominant in samples from out-of-lay and molting stages. No changes in the ratio of isoform released were detected during cVIP stimulation. Our data clearly show that glycosylated and nonglycosylated PRL isoforms are released by the pituitary gland in vitro in the same relative proportion that was previously observed in pituitary extracts and thus are likely to reflect the secreted forms of PRL in the blood during various physiological stages. In addition, the PRL-releasing activity of VIP does not affect the ratio of isoforms secreted by the pituitary gland in vitro.

Animals↗
PubMed1998

Structure of the Drosophila projectin protein: isoforms and implication for projectin filament assembly.

The protein composition of the various muscle types in Drosophila melanogaster has been studied quite thoroughly and the analysis has revealed many differences involving the usage of muscle specific isoforms of a given protein, as well as the presence of proteins restricted to one muscle type. Drosophila projectin, the giant protein component of the third filament is quite unusual as it not only shows specific isoforms in various muscle types, but these isoforms are located at different sarcomeric locations, I band in the IFM and A band in synchronous muscles. This may suggest distinct functions for the projectin protein in various muscles, as well as a different set of protein interactions for each projectin isoform. Projectin is encoded by a single gene and the isoforms were proposed to be the result of alternative splicing of a primary transcript. Here, we report the nearly complete sequence of Drosophila projectin, as well as the possible splicing patterns used to generate different isoforms. The overall domain organization in projectin is composed of repeated motifs I and II in a few specific patterns, similar to its Caenorhabditis homolog, twitchin. Sequence similarity between twitchin and projectin further suggests how some domains may possibly be important for protein interactions and/or functions. Alternative splicing operates at the COOH terminus, leading to a shorter projectin protein lacking some of the terminal motifs II and unique sequence. These isoforms are discussed in view of projectin differential size and localization.

Alternative Splicing↗
PubMed1997

Phospholipase C isoforms delta 1 and delta 3 from human fibroblasts. High-yield expression in Escherichia coli, simple purification, and properties.

Phospholipase C isoforms delta 1 and delta 3 (PLC delta 1 and delta 3) were expressed in Escherichia coli using the cDNA sequences from human fibroblasts. The enzymes were also expressed with the sequence Met-Gly-His6-Ser-Gly-Leu-Phe-Lys-Arg, a hexahistidine sequence followed by a Kex2 protease cleavage site, denoted as "-H6K2," attached to their amino termini. PLC delta 1, PLC delta 1-H6K2, PLC delta 3, PLC delta 3-H6K2 all expressed in highly active form. The H6K2-bearing isoforms were each purified to homogeneity in a single step, with yields of 90-100%, using agarose-iminodiacetic acid-Ni columns and imidazole buffer as eluting agent. Yields in terms of activity increased as the temperature of expression was decreased. Expression at 16 degrees C for 72 h yielded 33 mg of pure PLC delta 1-H6K2 and 13 mg of pure PLC delta 3-H6K2 per liter of culture. Removal of H6K2 from both isoforms with Kex2 protease resulted in little or no loss of activity. Expression of PLC isoforms bearing -H6K2 at the amino terminus resulted in about 12 times more activity than expression of the isoforms lacking -H6K2. PLC delta 3 is much less stable than PLC delta1. Successful purification and storage of PLC delta 3 depends on a suitable stabilizing medium. Both isoforms require 0.3 microM calcium ion for half-maximum activity. The specific activities of the isoforms expressed with and without -H6K2 are the same, as are their calcium saturation curves.

Amino Acid Sequence↗
PubMed1991

Myosin heavy chain isoforms and smooth muscle function.

Using isoform specific antibodies we have verified the presence of two distinct muscle type myosin heavy chain isoforms in rat uterine muscle. We have shown that an endogenous protease can cleave a small 4 kDa region from the C-terminal of the SM1 isoform which generates a pSM1 species which comigrates with the SM2 isoform on low density SDS gels. While this cleavage can complicate isoform identification, more importantly, this cleavage was associated with a substantial increase in the actomyosin ATPase. Thus we have identified a domain at the C-terminal which may be involved in regulation of the ATPase activity. Interestingly, it is at this C-terminal, tail region of the smooth muscle myosin molecule where the only known isoform specific sequence differences are located. In skinned smooth muscle fibers of rat uterine muscle, we have also shown that differences in myosin heavy chain distribution, induced by beta-estradiol treatment of ovariectomized rats, are correlated with changes in unloaded shortening velocity. Thus our work suggests that the functional significance of myosin heavy chain isoforms in smooth muscle may be similar to that observed in striated muscle.

Animals↗
PubMed1996

Cloning and characterization of an IGF-1 isoform expressed in skeletal muscle subjected to stretch.

To ascertain if IGF-1 is a regulator of local muscle growth, total RNA was extracted from rabbit muscle induced to undergo rapid hypertrophy using active stretch and from control muscles. This was analysed by Northern hybridization with a 280 base pair probe containing sequences derived from exons 3 and 4 of the insulin-like growth factor 1 gene. Two types of insulin-like growth factor 1 mRNA were shown to be strong expressed in the stretched muscles. In situ hybridization using the same probe (280 base pair) showed that IGF-1 is strongly expressed in muscle that is induced to grow rapidly and is expressed in the muscle fibres themselves. Using RT-PCR a single insulin-like growth factor 1 isoform cDNA (IGF-1Ea) could be cloned from the normal resting muscles. However, an additional isoform of insulin-like growth factor 1 (insulin-like growth factor 1Eb) was found to be expressed in stretched muscle undergoing hypertrophy. The E domain sequence of the additional isoform differs from the liver insulin-like growth factor 1Ea by the presence a 52 base pair insert. This changes the reading frame of the derived carboxyl-terminal resulting in a different precursor insulin-like growth factor 1 isoform. This insulin-like growth factor 1 mRNA probably encodes the precursor insulin-like growth factor 1 isoform that is responsible for local muscle growth regulation in response to mechanical stimulation. To confirm that alternative splicing of the insulin-like growth factor 1 gene occurs in muscle in response to physical activity, oligonucleotide primers were made which specifically amplify the cDNAs of two isoforms (insulin-like growth factors 1Ea and Eb) in the human as well as the rabbit. Following altered physical activity for 2 h to 6 days, appreciable levels of insulin-like growth factor 1Eb (in human the Ec) isoform were detected in skeletal muscle by using RT-PCR. In contrast very little if any of this splice variant could be detected in control muscle not subjected to stretch or extra physical activity.

Animals↗
PubMed1987

Differential effects of carbohydrate intake on cardiac myosin isoform expression in normal weanling and adult rats.

Dietary manipulations involving high carbohydrate feeding increase V1 cardiac myosin isoform expression in hormonally deficient rats. The purpose of this study was to determine if extremes in dietary carbohydrate availability could alter cardiac myosin isoform patterns in normal weanling and adult rats. Three and six weeks of dietary manipulations (either high or low carbohydrate diets) failed to change calcium-activated myofibril ATPase activity, calcium regulated myofibril ATPase activity, or the myosin isoform distribution in the adult. In contrast, a four week, high carbohydrate diet reduced calcium activated myosin ATPase activity by 33%, calcium regulated myofibril ATPase activity by 10%, and V1 isoform expression by 66% in weanling rats. Although the low carbohydrate diet caused no change in the myosin ATPase properties, it decreased V1 isoform expression by 17%. These results show that carbohydrate availability can alter cardiac myosin isoform expression in normal rats, but only at weanling age. The reason for this age-related contrast in response to dietary manipulations is unknown at this stage. The dietary manipulations may have acted directly on the heart by creating a state of malnutrition, or indirectly, by altering some developmental process which links maturation of the sympathetic nervous system with myosin isoform expression.

Adenosine Triphosphatases↗
PubMed1995

Immunohistochemical analysis of C-protein isoforms in cardiac and skeletal muscle of the axolotl, Ambystoma mexicanum.

Of the several proteins located within sarcomeric A-bands, C-protein, a myosin binding protein (MyBP) is thought to regulate and stabilize thick filaments during assembly. This paper reports the characterization of C-protein isoforms in juvenile and adult axolotls, Ambystoma mexicanum, by means of immunofluorescent microscopy and Western blot analyses. C-protein and myosin are found specifically within the A-bands, whereas tropomyosin and alpha-actin are detected in the I-bands of axolotl myofibrils. The MF1 antibody prepared against the fast skeletal muscle isoform of chicken C-protein specifically recognizes a cardiac isoform (Axcard1) in juvenile and adult axolotls but does not label axolotl skeletal muscle. The ALD66 antibody, which reacts with the C-protein slow isoform in chicken, local- izes only in skeletal muscle of the axolotl. This slow axolotl isoform (Axslow) displays a heterogeneous distribution in fibers of dorsalis trunci skeletal muscle. The C315 antibody against the chicken C-protein cardiac isoform identifies a second axolotl cardiac isoform (Axcard2), which is present also in axolotl skeletal muscle. No C-protein was detected in smooth muscle of the juvenile and adult axolotl with these antibodies.

Actins↗
PubMed1990

Further data on the selective expression of Ly-5 isoforms.

Ly-5 (CD45) glycoproteins of the mouse, expressed by all or most hematopoietic cell lineages and specified by a single Ly-5 gene, range in size from isoform T200 of T cells (the smallest), in which exons 4, 5, and 6 are not represented, to isoform B220 of B cells (the largest), in which all three of these optional exons are represented. The main purpose of the present study, utilizing the polymerase chain reaction (PCR), was to ascertain whether known isoforms of intermediate size are generated by single or dual usage of optional exons 4, 5, and 6. Transcripts representing all eight isoforms predictable from varied use of three exons were observed among a diverse panel of nine B-cell tumors in culture, but there was no evident concordance with known contrasting differential features that distinguish members of the B-cell tumor panel. No two B tumors exhibited the same variety of transcripts and the relative quantities of transcripts expressed varied greatly from tumor to tumor. Cloning of B-cell tumors did not alter their distinctive transcript patterns. Separation methods (sodium dodecyl sulfate polyacrylamide gel electrophoresis; SDS-PAGE) did not suffice to segregate all corresponding expressed isoforms but did establish that transcripts representing usage of a single optional exon and of two optional exons were actually translated, which supports a provisional inference that all eight isoforms exist. The considerable diversity of B-cell transcript phenotypes was not seen among seven T-cell leukemias, two cytolytic T-cell lines, and three Th 1 helper T-cell lines, all of which displayed a uniform phenotype comprising major expression of the T200 transcript (no optional exon) and minor expression of a transcript employing exon 5. However, a panel of five cloned Th2 T-cell lines, which represent a second and functionally different branch of the helper/inducer T-cell category, exhibited a characteristic transcript pattern which distinguished them from a panel of three Th1 T-cell lines. The major transcript in the Th2 lines was also T200, but the Th2 lines showed higher representation of transcripts containing optional exons. A single Th2 clone expressed an unusual transcript suggesting a potential isoform not compounded simply by varied inclusion of the three identified optional exons. After activation of the helper T-cell lines with concanavalin A (Con A), expression of transcripts containing optional exons appeared to decrease.

Animals↗
PubMed1996

Cytokine-induced selective increase of high-molecular-weight bFGF isoforms and their subcellular kinetics in cultured rat hippocampal astrocytes.

Cytokines such as interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha) and epidermal growth factor (EGF) are probable factors responsible for up-regulation of basic fibroblast growth factor (bFGF) expression in reactive astrocytes following brain damage, however the effect of these cytokines on the expression of each bFGF-isoform has not been elucidated. Western blot analysis revealed the expression of 18, 22 and 24-kD bFGF isoforms in cultured rat hippocampal astrocytes, and the expression of high molecular weight (HMW)-isoforms (22 and 24-kD isoforms) but not of 18-kD isoform was selectively increased by cytokines. Immunofluorescent analysis demonstrated that bFGF content in the cytoplasm of astrocytes is initially increased by cytokines followed by nuclear targeting and localization in agreement with the previous evidence that HMW-isoforms possess a nuclear targeting signal. The present results suggest the important role of HMW-bFGF isoforms in the response of nervous tissue to injury.

Animals↗