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PubMed1998

Modulation of GABA(A) receptor function by neuroactive steroids: evidence for heterogeneity of steroid sensitivity of recombinant GABA(A) receptor isoforms.

Neuroactive steroids are potent, selective allosteric modulators of gamma-aminobutyric acid type A (GABA(A)) receptor function in the central nervous system, and may serve as endogenous anxiolytic and analgesic agents. In order to study the influence of subunit subtypes of the GABA(A) receptor on modulation of receptor function by neuroactive steroids, we expressed human recombinant GABA(A) receptors in Xenopus oocytes. GABA-activated membrane current, and the modulatory effects of the endogenous neurosteroid 5alpha-pregnan-3alpha-ol-20-one (allopregnanolone) and the synthetic steroid anesthetic 5alpha-pregnan-3alpha-ol-11,20-dione (alphaxalone) were measured using two-electrode voltage-clamp recording techniques. Allopregnanolone had similar effects to potentiate GABA-activated membrane current in the alpha1beta1gamma2L and alpha1beta2gamma2L receptor isoforms. In contrast, alphaxalone was much more effective as a positive allosteric modulator on the alpha1beta1gamma2L receptor isoform. In the absence of the gamma2L subunit subtype, allopregnanolone had much greater efficacy, but its potency was decreased. Allopregnanolone was much more effective on the alpha1beta1 receptor isoform compared with the alpha1beta2 receptor isoform. The potency for alphaxalone to potentiate the GABA response was not altered in the absence of the gamma2L subunit subtype, although its efficacy was greatly enhanced. Both allopregnanolone and alphaxalone produced nonparallel leftward shifts in the GABA concentration-response relationship in the absence of the gamma2L subunit, decreasing the EC50 concentration of GABA and increasing the maximal response. Only alphaxalone increased the maximal GABA response when the gamma2L subunit subtype was present. The 3beta-pregnane isomers epipregnanolone and isopregnanolone both inhibited the ability of allopregnanolone and alphaxalone to potentiate GABA(A) receptor function. However, the degree of block produced by the 3beta-pregnane steroid isomers was dependent on the type of receptor isoform studied and the neuroactive steroid tested. Isopregnanolone, the 3beta-isomer of allopregnanolone, was significantly more effective as a blocker of potentiation caused by allopregnanolone compared with alphaxalone in all receptor isoforms tested. Epipregnanolone had a greater efficacy as a blocker at the alpha1beta2gamma2L receptor isoform compared with the alpha1beta1gamma2L receptor isoform, and also produced a greater degree of block of potentiation caused by allopregnanolone compared with alphaxalone. Our results support the hypothesis that the heteromeric assembly of different GABA(A) receptor isoforms containing different subunit subtypes results in multiple steroid recognition sites on GABA(A) receptors, which in turn produces distinctly different modulatory interactions between neuroactive steroids acting at the GABA(A) receptor. The alpha and gamma subunit subtypes may have the greatest influence on allopregnanolone modulation of GABA(A) receptor function, whereas the beta and gamma subunit subtypes appear to be most important for the modulatory effects of alphaxalone.

Animals↗
PubMed2000

MHC isoform composition and Ca(2+)- or Sr(2+)-activation properties of rat skeletal muscle fibers.

Chemically skinned single fibers from adult rat skeletal muscles were used to test the hypothesis that, in mammalian muscle fibers, myosin heavy chain (MHC) isoform expression and Ca(2+)- or Sr(2+)-activation characteristics are only partly correlated. The fibers were first activated in Ca(2+)- or Sr(2+)-buffered solutions under near-physiological conditions, and then their MHC isoform composition was determined electrophoretically. Fibers expressing only the MHC I isoform could be appropriately identified on the basis of either the Ca(2+)- or Sr(2+)-activation characteristics or the MHC isoform composition. Fibers expressing one or a combination of fast MHC isoforms displayed no significant differences in their Ca(2+)- or Sr(2+)-activation properties; therefore, their MHC isoform composition could not be predicted from their Ca(2+)- or Sr(2+)-activation characteristics. A large proportion of fibers expressing both fast- and slow-twitch MHC isoforms displayed Ca(2+)- or Sr(2+)-activation properties that were not consistent with their MHC isoform composition; thus both fiber-typing methods were needed to fully characterize such fibers. These data show that, in rat skeletal muscles, the extent of correlation between MHC isoform expression and Ca(2+)- or Sr(2+)-activation characteristics is fiber-type dependent.

Animals↗
PubMed2002

Cardiac troponin T isoforms demonstrate similar effects on mechanical performance in a regulated contractile system.

Alteration of troponin T (TnT) isoform expression has been reported in human and animal models of myocardial failure. The two adult beef cardiac TnT isoforms (TnT(3) and TnT(4)) were isolated for comparative functional analysis. Thin filaments were reconstituted containing pure populations of the isoforms. The in vitro motility assay was used to directly compare the effect of the two TnT isoforms on force and unloaded shortening as a function of free calcium. We found no significant differences between the two isoforms in terms of calcium sensitivity, cooperativity, or maximal activation (velocity and force) as assessed in a fully calcium-regulated system. Activation by myosin strong binding was similar for thin filaments containing either of the two TnT isoforms. Whereas maximally activated velocity and cooperativity was depressed at pH 6.5, no difference between thin filaments containing the two isoforms was detected. From the small magnitude of the TnT isoform shifts detected in myocardial failure and the lack of significant mechanical effect detected in the motility assay, variable TnT isoform expression is unlikely to be any functional significance in heart failure.

Actin Cytoskeleton↗
PubMed2005

Specific measurement of o-linked core 2 sugar-containing isoforms of hyperglycosylated human chorionic gonadotropin by antibody b152.

There have been a significant number of reports on the clinical utility of measurement of 'hyperglycosylated' isoforms of the pregnancy hormone, human chorionic gonadotropin (hCG). Although there are a variety of hCG isoforms which can be termed 'hyperglycosylated', the measurements were all made using a unique antibody designated B152. This antibody was raised using a choriocarcinoma-derived form of hCG, which was hyperglycosylated with N- and O-glycans and was also 100% 'nicked' hCG. Antibody B152 was recently mapped to a linear epitope around a single O-glycan on the beta-subunit of hCG at residue number 132. Thus, the antibody can only measure isoforms of hCG that possess a core 2 type of branched O-glycan on this portion of the hCG beta-subunit. Isoforms that are hyperglycosylated in the hCG alpha-subunit or only on the N-glycans of hCGbeta will not be recognized by antibody B152. Apparently, measurements of these core 2 hCG isoforms have important clinical application in early pregnancy during which they are the predominant isoform of hCG until the 6th week of gestation. The secretory pattern of these isoforms can be used to predict the health status of the pregnancy in fertility clinics. Moreover, the measurements of these core 2 hCG isoforms are more useful than standard hCG for the prediction of Down syndrome pregnancies. The core 2 isoforms are also of important use in cancer diagnosis and monitoring since their concentration appears to correlate with malignancy.

Adult↗
PubMed2000

High levels of Lp(a) with a small apo(a) isoform are associated with coronary artery disease in African American and white men.

Elevated levels of lipoprotein(a) [Lp(a)] and the presence of small isoforms of apolipoprotein(a) [apo(a)] have been associated with coronary artery disease (CAD) in whites but not in African Americans. Because of marked race/ethnicity differences in the distribution of Lp(a) levels across apo(a) sizes, we tested the hypothesis that apo(a) isoform size determines the association between Lp(a) and CAD. We related Lp(a) levels, apo(a) isoforms, and the levels of Lp(a) associated with different apo(a) isoforms to the presence of CAD (>/=50% stenosis) in 576 white and African American men and women. Only in white men were Lp(a) levels significantly higher among patients with CAD than in those without CAD (28.4 versus 16.5 mg/dL, respectively; P:=0.004), and only in this group was the presence of small apo(a) isoforms (<22 kringle 4 repeats) associated with CAD (P:=0.043). Elevated Lp(a) levels (>/=30 mg/dL) were found in 26% of whites and 68% of African Americans, and of those, 80% of whites but only 26% of African Americans had a small apo(a) isoform. Elevated Lp(a) levels with small apo(a) isoforms were significantly associated with CAD (P:<0.01) in African American and white men but not in women. This association remained significant after adjusting for age, diabetes mellitus, smoking, hypertension, HDL cholesterol, LDL cholesterol, and triglycerides. We conclude that elevated levels of Lp(a) with small apo(a) isoforms independently predict risk for CAD in African American and white men. Our study, by determining the predictive power of Lp(a) levels combined with apo(a) isoform size, provides an explanation for the apparent lack of association of either measure alone with CAD in African Americans. Furthermore, our results suggest that small apo(a) size confers atherogenicity to Lp(a).

Black or African American↗
PubMed2001

Increased expression of isoform 1 of the sarcoplasmic reticulum Ca(2+)-release channel in failing human heart.

BACKGROUND: The sarcoplasmic reticulum (SR) Ca(2+)-release channel plays a key role in the excitation-contraction coupling of cardiac myocytes. Because respective alterations have been reported in human heart failure, we investigated isoform expression of the SR Ca(2+)-release channel in human hearts from patients with terminal heart failure (dilated cardiomyopathy [DCM], n=8) and nonfailing organ donors (NF, n=8). METHODS AND RESULTS: Expression of mRNA of SR Ca(2+)-release channel isoforms in isolated human cardiomyocytes and myocardial tissue was analyzed by reverse-transcription polymerase chain reaction. Protein expression was quantified in myocardial tissue with [(3)H]-ryanodine binding and with Western blots, expressed as densitometric units per microgram of protein (DU), and cellular localization was visualized with immunohistochemistry. We found mRNA expression of isoforms 1, 2, and 3 in cardiomyocytes and myocardial tissue both in NF and DCM. Total SR Ca(2+)-release channel protein expression in NF (B(max) 2.16+/-0.43 pmol/mg protein) and in DCM (B(max) 2.33+/-0.22 pmol/mg protein) myocardium was unchanged. Expression of isoform 1 of the SR Ca(2+)-release channel was significantly (P=0.0037) increased in DCM myocardium (NF 1.97+/-0.25 versus DCM 3.37+/-0.31 DU), whereas protein expression of isoform 2 (NF 14.62+/-0.87 versus DCM 13.52+/-0.43 DU) and isoform 3 (NF 1.39+/-0.13 versus DCM 1.35+/-0.19 DU) was unchanged. All 3 isoforms of the protein could be localized in human ventricular myocytes with fluorescence immunohistochemistry. CONCLUSIONS: All 3 isoforms of the SR Ca(2+)-release channel were determined in human ventricular cardiomyocytes. Increased expression of isoform 1 of the SR Ca(2+)-release channel could contribute to impaired excitation-contraction coupling in human heart failure.

Adult↗
PubMed2001

Characterization of human FSH isoforms reveals a nonglycosylated beta-subunit in addition to the conventional glycosylated beta-subunit.

Human FSH consists of a mixture of isoforms that can be separated on the basis of differences in negative charge conferred by variations in the numbers of sialic acid residues that terminate oligosaccharide branches. Western analysis of human FSH isoforms separated by chromatofocusing revealed the presence of two human FSHbeta isoforms that differed in size. A low mol wt human FSHbeta isoform was associated with all FSH isoform fractions. A high mol wt human FSHbeta isoform was associated with the more acidic fractions and increased in relative abundance as the pI decreased. Characterization of representative human FSHbeta isoforms by mass spectrometry and automated Edman degradation revealed a low mol wt isoform that was not glycosylated. A high mol wt isoform was N-glycosylated at Asn residues 7 and 24. These results indicate that pituitary human FSH consists of two classes of molecules: those that possess a nonglycosylated beta-subunit and those that possess a glycosylated beta-subunit. Glycoprotein hormones are known to be elliptical molecules, and the beta-subunit oligosaccharides project outward from the short diameter, thereby increasing it. It is interesting to speculate that this change in shape might affect ultrafiltration rates, leading to differences in delivery rates to target tissues and elimination by filtration in the kidney.

Animals↗
PubMed1999

Nonmuscle tropomyosin-4 requires coexpression with other low molecular weight isoforms for binding to thin filaments in cardiomyocytes.

Vertebrate tropomyosins (TMs) are expressed from four genes, and at least 18 distinct isoforms are generated via a complex pattern of alternative RNA splicing and alternative promoters. The functional significance of this isoform diversity is largely unknown and it remains to be determined whether specific isoforms are required for assembly and integration into distinct actin-containing structures. The ability of nonmuscle (TM-1, -2, -3, -4, -5(NM1), -5a or -5b) and striated muscle (skeletal muscle (&agr;)-TM) isoforms to incorporate into actin filaments of neonatal rat cardiomyocytes (NRCs) was studied using expression plasmids containing TM-fusions with GFP (green fluorescent protein) as well as with VSV- or HA-epitope tags. All isoforms, except of fibroblast TM-4, were able to incorporate into the I-band of NRCs. When TM-4 was co-transfected with other low molecular weight (LMW) isoforms of TM (TM-5, TM-5a and TM-5b), it was able to incorporate into sarcomeres of NRCs. This result was not obtained when TM-4 was co-transfected with high molecular weight (HMW) TMs (TM-1, TM-2 or skeletal muscle (&agr;)-TM). These data demonstrate that the ability of TM-4 to bind to actin filaments can be specifically influenced by its interaction with other LMW TM isoforms. In addition, cells that incorporated the muscle or nonmuscle GFP-TMs into their sarcomeres continued to beat and exhibited sarcomeric contraction. These studies provide the first in vivo demonstration of synergistic effects between TM isoforms for binding to actin filaments. These results have important implications in understanding actin filament dynamics in nonmuscle cell systems, especially during development and in transformed cells, where alterations in the ratio of different LMW isoforms might lead to changes in their interactions with actin filaments. Furthermore, these studies demonstrate that GFP-TM can be used to study thin-filament dynamics in muscle cells and actin filament dynamics in nonmuscle cells.

Actins↗
PubMed2002

Re-expression of fetal troponin isoforms in the postinfarction failing heart of the rat.

Molecular switches between the troponin T and I isoforms are known to occur in various conditions, but the results from studies of failing human hearts with various etiologies are contradictory and it is not certain whether troponin isoform changes occur. Therefore, the molecular switching of troponin isoforms during normal development and heart failure (HF) after myocardial infarction were investigated in Sprague-Dawley rats at the fetal, neonate, and normal adult stages, and in a postinfarction adult HF group. During normal development, switching from the fetal to the adult pattern of the troponin T and I isoforms was observed. Immunoblotting of postinfarction failing hearts revealed a marked increase in the fetal isoform of cardiac TnT (cTnT) (fetal/adult cTnT isoforms: normal adult = 0.61 +/- 0.09 vs postinfarction HF = 1.59 +/- 0.13, p < 0.001). Also, the amount of the adult troponin I (TnI) isoform decreased significantly in the postinfarction failing heart. In the semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) with glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as an internal standard, the mRNA of fetal cTnT increased in the postinfarction failing heart (fetal cTnT/GAPDH: control = 0.22 vs HF rat = 0.84, p < 0.05). Therefore, molecular switching of the troponin T and I isoforms occurred during the normal development of the rat, and there was re-expression of the fetal pattern of the isoforms in the postinfarction failing heart of the adult rat.

Animals↗
PubMed2023

A systematic CRISPR screen reveals redundant and specific roles for Dscam1 isoform diversity in neuronal wiring.

Drosophila melanogaster Down syndrome cell adhesion molecule 1 (Dscam1) encodes 19,008 diverse ectodomain isoforms via the alternative splicing of exon 4, 6, and 9 clusters. However, whether individual isoforms or exon clusters have specific significance is unclear. Here, using phenotype-diversity correlation analysis, we reveal the redundant and specific roles of Dscam1 diversity in neuronal wiring. A series of deletion mutations were performed from the endogenous locus harboring exon 4, 6, or 9 clusters, reducing to 396 to 18,612 potential ectodomain isoforms. Of the 3 types of neurons assessed, dendrite self/non-self discrimination required a minimum number of isoforms (approximately 2,000), independent of exon clusters or isoforms. In contrast, normal axon patterning in the mushroom body and mechanosensory neurons requires many more isoforms that tend to associate with specific exon clusters or isoforms. We conclude that the role of the Dscam1 diversity in dendrite self/non-self discrimination is nonspecifically mediated by its isoform diversity. In contrast, a separate role requires variable domain- or isoform-related functions and is essential for other neurodevelopmental contexts, such as axonal growth and branching. Our findings shed new light on a general principle for the role of Dscam1 diversity in neuronal wiring.

Animals↗
PubMed2000

Growth hormone isoforms in newborns and postpartum women.

The neonatal and postpartum periods are characterized by alterations in pituitary GH secretion. We have investigated the proportion of circulating non-22kDa GH isoforms in newborns, in women within the early postpartum phase (just after the disappearance of placental GH from the maternal circulation) and in women during late postpartum (during the somatotroph recovery phase). We studied 10 newborns (7 males; 3 females; median postnatal age, 45h), who had been admitted because of polycythaemia, 10 women in the early postpartum phase (median, 48h after delivery; range, 42-54h), 18 women in the late postpartum phase (median, 10 weeks after delivery; range, 3-25 weeks) and 9 healthy non-pregnant women. The proportion of non-22kDa GH isoforms was determined by the 22kDa GH exclusion assay, which is based on immunomagnetic extraction of 22kDa GH from serum, and quantitation of non-22kDa GH isoforms using a polyclonal GH assay. In newborns, non-22kDa GH isoforms were measured in two arterial blood samples obtained with a 5-6h interval. In the other groups, serum samples were obtained 40min after an i.v. bolus administration of the GH secretagogue, GH releasing peptide-1 (GHRP-1). In newborns, the median proportion of non-22kDa GH isoforms was 10% (range, 7. 2-19.4%) and the values were similar in samples collected at different times. In early postpartum women, total GH levels after GHRP-1 were lower and the proportion of non-22kDa GH isoforms was higher compared with the values in non-pregnant and late-postpartum women. In late postpartum, there was a partial recovery of GH response to GHRP-1, as shown by an increment in total GH levels, which was associated with a decrease in the fraction of non-22kDa GH isoforms. In conclusion, we found that (i) the proportion of non-22kDa GH isoforms in the newborn is comparable to that in the adult (non-pregnant women), (ii) in early postpartum, the non-22kDa fraction is high within the small pool of readily releasable GH, (iii) in late postpartum, recovery of pituitary GH responsiveness is associated with a relative decrease in the release of non-22kDa GH isoforms.

Adult↗
PubMed2001

Distinctive effects of rat fibroblast growth factor-2 isoforms on PC12 and Schwann cells.

Fibroblast growth factor-2 (FGF-2) is an important modulator of cell growth and differentiation and stimulates cell survival of various cells including neurons. Rat FGF-2 occurs in three isoforms, a low molecular weight 18 kD and two high molecular weight forms (21, 23 kD), representing alternative translation products from a single mRNA. The 18 kD isoform shows mainly cytoplasmatic localization, whereas the 21/23 kD FGF-2 are localized in the nucleus. In addition, the FGF-2 isoforms are differentially regulated in the sensory ganglia and peripheral nerve following nerve injury and in the adrenal medulla during post-natal development and after hormonal stimuli. The distinct intracellular distribution and differential regulation of the different FGF-2 isoforms indicate that they have unique biological roles, however, little is known about the biological effects of the high molecular weight FGF-2 isoforms. Immortalized Schwann cells and PC12 cells, which stably overexpress the different FGF-2 isoforms, showed that the different endogenous-overexpressed FGF-2 isoforms lead to dramatic modifications in cell proliferation and survival, when tested in serum-free and serum-containing medium. In contrast, application of recombinant FGF-2 isoforms on normal PC12 and immortalized Schwann cells results in similar biological effects on the proliferation and survival of the cells. Furthermore, we investigated the potential regulatory effects of endogenous-overexpressed and exogenous-applied FGF-2 isoforms on the mRNA level of the FGF-2 receptors and, additionally, on the tyrosin hydroxylase mRNA expression in PC12 cells.

Animals↗
PubMed2003

Two novel fully functional isoforms of CX3CR1 are potent HIV coreceptors.

We identified two novel isoforms of the human chemokine receptor CX3CR1, produced by alternative splicing and with N-terminal regions extended by 7 and 32 aa. Expression of the messengers coding these isoforms, compared with that of previously described V28 messengers, is lower in monocytes and NK cells, but higher in CD4(+) T lymphocytes. CX3CR1 and its extended isoforms were expressed in HEK-293 cells and compared for expression, ligand binding, and cellular responses. In steady state experiments, all three CX3CR1 isoforms bound CX3CL1 with similar affinity. In kinetic binding studies, however, k(on) and k(off) were significantly greater for the extended CX3CR1 isoforms, thereby suggesting that the N-terminal extensions may alter the functions induced by CX3CL1. In signaling studies, all three CX3CR1 isoforms mediated agonist-dependent calcium mobilization, but the EC(50) was lower for the extended than for the standard isoforms. In addition, chemotactic responses for these extended isoforms shifted left, also indicating a more sensitive response. Finally, the longer variants appeared to be more potent HIV coreceptors when tested in fusion and infection assays. In conclusion, we identified and characterized functionally two novel isoforms of CX3CR1 that respond more sensitively to CX3CL1 and HIV viral envelopes. These data reveal new complexity in CX3CR1 cell activation and confirm the critical role of the N-terminal domain of the chemokine receptors in ligand recognition and cellular response.

Alternative Splicing↗
PubMed1999

Truncated isoforms inhibit [3H]prazosin binding and cellular trafficking of native human alpha1A-adrenoceptors.

We have identified from human liver eight alpha(1A)-adrenoceptor (alpha(1A)-AR) splice variants that were also expressed in human heart, prostate and hippocampus. Three of these alpha(1A)-AR isoforms (alpha(1A-1)-AR, alpha(1A-2a)-AR and alpha(1A-3a)-AR) gave rise to receptors with seven transmembrane domains (7TMalpha(1A)-AR). The other five (alpha(1A-2b)-AR, alpha(1A-2c)-AR, alpha(1A-3c)-AR, alpha(1A-5)-AR and alpha(1A-6)-AR) led to truncated receptors lacking transmembrane domain VII (6TMalpha(1A)-AR). The 7TMalpha(1A)-AR isoforms transiently expressed in COS-7 cells bound [(3)H]prazosin with high affinity (K(d) 0.2 nM) and mediated a noradrenaline (norepinephrine)-induced increase in cytoplasmic free Ca(2+) concentration, whereas the 6TMalpha(1A)-AR isoforms were incapable of ligand binding and signal transduction. Immunocytochemical studies with N-terminal epitope-tagged alpha(1A)-AR isoforms showed that the 7TMalpha(1A)-AR isoforms were present both at the cell surface and in intracellular compartments, whereas the 6TMalpha(1A)-AR isoforms were exclusively localized within the cell. Interestingly, in co-transfected cells, each truncated alpha(1A)-AR isoform inhibited [(3)H]prazosin binding and cell-surface trafficking of the co-expressed 'original' 7TMalpha(1A-1)-AR. However, there was no modification of either the [(3)H]prazosin-binding affinity or the pharmacological properties of alpha(1A-1)-AR. Immunoblotting experiments revealed that co-expression of the alpha(1A-1)-AR with 6TMalpha(1A)-AR isoforms did not impair alpha(1A-1)-AR expression. Therefore the expression in human tissues of many truncated isoforms constitutes a new regulation pathway of biological properties of alpha(1A)-AR.

Amino Acid Sequence↗
PubMed2000

Characterization of AMP-activated protein kinase gamma-subunit isoforms and their role in AMP binding.

The AMP-activated protein kinase (AMPK) cascade plays an important role in the regulation of energy homeostasis within the cell. AMPK is a heterotrimer composed of a catalytic subunit (alpha) and two regulatory subunits (beta and gamma). We have isolated and characterized two isoforms of the gamma subunit, termed gamma2 and gamma3. Both gamma2 (569 amino acids) and gamma3 (492 amino acids) have a long N-terminal domain which is not present in the previously characterized isoform, gamma1. As with gamma1, mRNA encoding gamma2 is widely expressed in human tissues, whereas significant expression of gamma3 mRNA was only detected in skeletal muscle. Using isoform-specific antibodies, we determined the AMPK activity associated with the different gamma isoforms in a number of rat tissues. In most tissues examined more than 80% of total AMPK activity was associated with the gamma1 isoform, with the remaining activity being accounted for mainly by the gamma2 isoform. Exceptions to this were testis and, more notably, brain where all three isoforms contributed approximately equally to activity. There was no evidence for any selective association between the alpha1 and alpha2isoforms and the various gamma isoforms. However, the AMP-dependence of the kinase complex is markedly affected by the identity of the gamma isoform present, with gamma2-containing complexes having the greatest AMP-dependence, gamma3 the lowest, and gamma1 having an intermediate effect. Labelling studies, using the reactive AMP analogue 8-azido-[(32)P]AMP, indicate that the gamma subunit may participate directly in the binding of AMP within the complex.

AMP-Activated Protein Kinases↗
PubMed2001

Identification of rat H3 receptor isoforms with different brain expression and signaling properties.

We identified the cDNAs of three functional rat H3 receptor isoforms (H3A, H3B, and H3C) and one nonfunctional truncated H3 receptor (H3T). The H3A, H3B, and H3C receptor isoforms vary in the length of their third intracellular loop; the H3B and H3C receptor lack 32 and 48 amino acids, respectively. Transient expression of the H3A, H3B, and H3C receptors in COS-7 cells results in high affinity binding for the H3 antagonist [125I]iodophenpropit, which is displaced by selective H3 agonists and antagonists. The three isoforms differentially couple to the Gi protein-dependent inhibition of adenylate cyclase or stimulation of p44/p42 mitogen activated protein kinase (MAPK), a new signaling pathway for the H3 receptor. Whereas the H3A receptor was less effective in inhibiting forskolin-induced cAMP production compared with the H3B or H3C receptor, this isoform was more effective in the stimulation of p44/p42 MAPK. The H3 receptor isoforms also displayed differential CNS expression in key areas involved in regulation of sensory, endocrine, and cognitive functions. A differential H3 receptor isoform expression was seen in, for example, hippocampus, where a characteristic dorsoventral distribution was revealed. Differential H3 receptor expression was also characteristic for the cerebellum, indicating possible histaminergic regulation of motor functions. The identification of these new H3 receptor isoforms and their specific signaling properties adds a new level of complexity to our understanding of the role of histamine, and the H3 receptor in brain function. The heterogeneous distribution of the isoforms suggests that H3 receptor isoform-specific regulation is important in several brain functions.

Adenylyl Cyclases↗
PubMed2003

The human T-cell factor-4 gene splicing isoforms, Wnt signal pathway, and apoptosis in renal cell carcinoma.

beta-Catenin and transcriptional factor TCF-4 (human T-cell factor-4) genes comprise the Wnt signal. The Wnt signal pathway plays an important role in malignant transformation. We hypothesize that the beta-catenin and TCF-4 gene and Wnt signal are important in the progression of renal cell carcinoma (RCC). To test this hypothesis, we investigated TCF-4 splicing isoforms, beta-catenin, and Wnt signal pathway (cyclin D1, c-myc, c-jun, and MMP7) in three RCC cell lines (A498, Caki-1, and Caki-2), 38 primary RCCs, and 29 normal kidney samples. We also analyzed the relationship between TCF-4 gene splicing isoforms, proliferation (proliferating cell nuclear antigen labeling index), and apoptosis [antiapoptotic factors (Bcl-2 and Bcl-x(L)), proapoptotic factors (Bak and Bax), and caspase-3] in RCC samples. In 38 RCC samples, four splicing isoforms of the TCF-4 gene were present in the region between exon 12 and exon 17. Thirty (79%) of 38 RCCs and all (100%) of the normal kidney samples showed mixed isoforms with both long and short reading frames in the COOH-terminal region, whereas the remaining 8 RCC samples showed only the long-form reading frame. Two COOH-terminal-binding protein sites were present only in the long-form reading frame. The eight RCCs that demonstrated only the long reading frame isoform showed early disease progression and poor prognosis. In these 8 RCC samples, down-regulation of cyclin D1, c-myc, c-jun, and MMP7 expression was observed at the mRNA level. In addition, a marked reduction of caspase-3 expression was also found at both the mRNA and the protein level. However, the beta-catenin gene was not overexpressed at the mRNA level and protein level, and mutation and deletion were not observed in exon 3. In these three renal cell lines, there was no significant difference in TCF-4 mRNA expression before and after 5-Aza-2'-deoxycytidine treatment, and there appeared to be no splicing isoforms in the region between exon 1 and exon 11. These findings suggest that alteration in beta-catenin is an infrequent event in RCC. In samples in which beta-catenin was not overexpressed, the target genes of Wnt signal were regulated through TCF-4 splicing isoforms. The imbalance between TCF-4 gene splicing isoforms with long and short reading frames is associated with RCC progression through the inhibition of the apoptotic pathway. We demonstrate for the first time that TCF-4 gene splicing isoforms and the Wnt signal pathway can induce progression of RCC by the inhibition of apoptosis and not by the induction of cell proliferation.

Adult↗
PubMed2003

Frequency distribution of apoprotein(a) isoforms in patients with diabetes mellitus and healthy subjects.

AIM: To determine the frequency distribution of apoprotein(a) isoforms in patients with insulin-dependent (IDDM) and non-insulin-dependent (NIDDM) diabetes mellitus and healthy subjects. METHOD: We separated and visualized 5 apo(a) isoforms in 40 patients with IDDM (12 men aged 48.00-/+4.59 and 28 women aged 52.37-/+8.21), 65 patients with NIDDM (26 men aged 61.88-/+9.25 and 39 women aged 60.15-/+7.98), and 182 healthy subjects, using 3-15% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by immunoblotting. RESULTS: The frequency distribution of apo(a) isoforms was very similar in patients with diabetes mellitus and the control group. Atherogenic low molecular weight (LMW) S1 apo(a) isoform was more frequent in patients with IDDM (7.5%) and NIDDM (6.15%) than in the control group (0.78%). LMW S1 apo(a) isoform in patients with IDDM (relative risk [RR], 6.86; 95% confidence interval [CI], 1.19-25.21; p<0.001) and patients with NIDDM (RR, 7.04; 95% CI, 1.40-35.40; p=0.0057) as well as high molecular weight >S4 apo(a) isoform in patients with NIDDM (RR, 2.39; 95% CI, 1.28-5.21; p=0.0067) significantly increased the risk for the development of atherosclerosis. Mean molecular weight of S3, S1, and B apo(a) isoforms was higher in patients with IDDM and NIDDM than in the healthy subjects carriers of the same isoforms, but this difference was not statistically significant. We estimated high inverse statistical correlation between apo(a) size (kDa) and plasma lipoprotein(a) concentration in all study groups, patients with IDDM (p<0.001), patients with NIDDM (p<0.001), and healthy subjects (p<0.01). CONCLUSION: Not only the increased plasma Lp(a) levels, but also apoprotein(a) isoforms may play an important role as a risk factor for the development of atherosclerosis in patients with diabetes mellitus.

Adult↗