Changes In Isoforms Research Articles

Proteomic Profiling Identifies Afamin as a Potential Biomarker for Ovarian Cancer

To discover and validate serum glycoprotein biomarkers in ovarian cancer using proteomic-based approaches. Serum samples from a "discovery set" of 20 patients with ovarian cancer or benign ovarian cysts or healthy volunteers were compared by fluorescence two-dimensional differential in-gel electrophoresis and parallel lectin-based two-dimensional profiling. Validation of a candidate biomarker was carried out with Western blotting and immunoassay (n = 424). Twenty-six proteins that changed significantly were identified by mass spectrometric sequencing. One of these, confirmed by Western blotting, was afamin, a vitamin E binding protein, with two isoforms decreasing in patients with ovarian cancer. Validation using cross-sectional samples from 303 individuals (healthy controls and patients with benign, borderline, or malignant ovarian conditions and other cancers) assayed by ELISA showed significantly decreased total afamin concentrations in patients with ovarian cancer compared with healthy controls (P = 0.002) and patients with benign disease (P = 0.046). However, the receiver operating characteristic areas for total afamin for the comparison of ovarian cancer with healthy controls or benign controls were only 0.67 and 0.60, respectively, with comparable figures for CA-125 being 0.92 and 0.88 although corresponding figures for a subgroup of samples analyzed by isoelectric focusing for afamin isoform 2 were 0.85 and 0.79. Analysis of a further 121 samples collected prospectively from 9 patients pretreatment through to relapse indicated complementarity of afamin with CA-125, including two cases in whom CA-125 was noninformative. Afamin shows potential complementarity with CA-125 in longitudinal monitoring of patients with ovarian cancer, justifying prospective larger-scale investigation. Changes in specific isoforms may provide further information.

Heterogeneity of α-cardiac myosin heavy chains in a small marsupial, Antechinus flavipes, and the effect of hypothyroidism on its ventricular myosins

The effect of drug-induced hypothyroidism on ventricular myosin gene expression was explored in a small marsupial, Antechinus flavipes. Pyrophosphate gel electrophoresis, SDS-PAGE and western blotting were used to analyse changes in native myosin isoforms and myosin heavy chains (MyHCs) in response to hypothyroidism. In some animals, five instead of the normal three native myosin components were found: V(1a), V(1b),V(1c), V(2) and V(3), in order of decreasing mobility. In western blots, V(1a), V(1b), and V(1c) reacted with anti-alpha-MyHC antibody, but not with anti-beta-MyHC, whereas V(2) and V(3) reacted with anti-beta-MyHC antibody. SDS-PAGE of the unusual ventricular myosins revealed three MyHC isoforms, two of which bound anti-alpha-MyHC antibody while the third bound anti-beta-MyHC antibody. We conclude that V(1a), V(1b), V(1c) are triplets arising from the dimerization of two distinct alpha-MyHC isoforms. Hypothyroidism, verified by metabolic studies, decreased alpha-MyHC content significantly (t-test, P < 0.001) from 91.6 +/- 5.9% (SEM, n = 4) in control animals to 67.2 +/- 5.7% (SEM, n = 4) in hypothyroid animals, with a concomitant increase in beta-MyHC content. We conclude that in adult marsupials, ventricular myosins are also responsive to changes in the thyroid state as found in eutherians, and suggest that evolution of the molecular mechanisms underlying this thyroid responsiveness predate the divergence of marsupials and eutherians.

Effect of thyroid hormone on the postnatal renal expression of NHE8

We previously demonstrated that there are developmental changes in proximal tubule Na(+)/H(+) exchanger (NHE) activity. There is a maturational increase in postnatal brush-border membrane (BBM) vesicle NHE3 protein abundance and decrease in NHE8 protein abundance. The purpose of this study was to determine whether thyroid hormone plays a role in the rat renal maturational isoform switch from NHE8 to NHE3 and whether thyroid hormone regulates NHE8. Administration of thyroid hormone to neonatal rats, before the normal postnatal increase in serum thyroid hormone levels at 3 wk of age, resulted in a premature increase in NHE3/beta-actin BBM protein abundance and mRNA abundance. Thyroid hormone also caused a premature decrease in BBM NHE8/beta-actin protein abundance, whereas there was no change in mRNA expression (standardized to 28s). Rats made hypothyroid from birth were studied at 28 days, after the normal maturational increase in thyroid hormone. In these hypothyroid adult rats, the maturational increase in BBM NHE3 protein abundance and NHE3 mRNA expression was prevented. In contrast, the developmental decrease in BBM NHE8 protein abundance was prevented in hypothyroid adults, but mRNA expression was unchanged in hypothyroid rats. To determine whether the effect of thyroid hormone was due to a direct epithelial effect, we studied normal rat kidney cells in culture. We recently showed that this cell line expresses NHE8, but does not express NHE3. Thyroid hormone caused a decrease in surface expression of NHE8, determined by biotinylation, but total cellular abundance remained unchanged. NHE8 activity, measured as the sodium-dependent rate of intracellular pH recovery from an acid load, was less with thyroid treatment than control. In conclusion, thyroid hormone plays a potential role in the developmental isoform change from NHE8 to NHE3 and decreases NHE8 activity.

Protective effect of prion protein via the N‐terminal region in mediating a protective effect on paraquat‐induced oxidative injury in neuronal cells

Transmissible spongiform encephalopathies are a group of neurodegenerative disorders caused by a posttranslational, conformational change in the cellular isoform of the prion protein (PrP(C)) into an infectious, disease-associated form (PrP(Sc)). Increasing evidence supports a role for PrP(C) in the cellular response to oxidative stress. We investigated the effect of oxidative stress mediated by paraquat exposure on SH-SY5Y neuroblastoma cells. A loss of mitochondrial membrane potential and subsequent reduction in ATP production were demonstrated in untransfected SH-SY5Y cells, an effect that was ameliorated by the expression of PrP(C). Cells expressing either PrP-DeltaOct, which lacks the octapeptide repeats, or PrP-DA, in which the N-terminus is tethered to the membrane, showed increased sensitivity to paraquat compared with cells expressing wild-type PrP(C) as shown by reduced viability, loss of their membrane integrity, and reduced mitochondrial bioenergetic measurements. Exposure of prion-infected mouse SMB15S cells to paraquat resulted in a reduction in viability to levels similar to those seen in the untransfected SH-SY5Y cells. However, "curing" the cells with pentosan sulfate restored the viability to the level observed in the SH-SY5Y cells expressing PrP(C). These data would indicate that the molecular mechanism promoting cellular resistance to oxidative stress had been compromised in the infected SMB15S cells, which could be reinstated upon curing. Our study supports the hypothesis that PrP(C) expression protects cells against paraquat-induced oxidative injury, demonstrates the significance of the N-terminal region of the protein in mediating this protective effect, and also shows that the biochemical consequences of prion infection may be reversed with therapeutic intervention.

Developmental changes in proximal tubule NaCl transport

The proximal tubule reabsorbs two thirds of the filtered NaCl in an isoosmotic fashion. In the adult proximal tubule, active NaCl transport is mediated by the parallel operation of Na(+)/H(+) and Cl(-)/base exchangers, and a substantive amount of chloride transport occurs passively across the paracellular pathway. Studies in the neonatal proximal tubule have resulted in unexpected results. The isoform of the Na(+)/H(+) exchanger mediating proximal tubule sodium absorption, NHE3, is virtually absent in the neonatal rat kidney. NHE8, an isoform of the Na(+)/H(+) exchange, in low abundance on the apical membrane of the adult proximal tubule, is present in high abundance in the neonatal segment. Whereas chloride permeability is high in the adult, favoring passive paracellular chloride flux, the neonatal proximal tubule is virtually impermeable to chloride ions. This is again due to a developmental change in isoforms of proteins forming the tight junction. The permeability properties of epithelia are due to a family of tight junction proteins called claudins. Claudins 6 and 9 are expressed in the neonatal proximal tubule at a time when chloride permeability is low, but these claudin isoforms are virtually absent in the adult segment. The causes for these postnatal changes in proximal tubular transport and developmental isoform changes are also discussed in this review.

Mechanism of Gender-Specific Differences in Aortic Stiffness With Aging in Nonhuman Primates

Our hypothesis was that the changes in vascular properties responsible for aortic stiffness with aging would be greater in old male monkeys than old female monkeys. We analyzed the effects of gender differences in aging on in vivo measurements of aortic pressure and diameter and on extracellular matrix of the thoracic aorta in young adult (age, 6.6+/-0.5 years) versus old adult (age, 21.2+/-0.2 years) monkeys (Macaca fascicularis). Aortic stiffness, as represented by the pressure strain elastic modulus (Ep), increased more in old male monkeys (5.08+/-0.81; P<0.01) than in old females (3.06+/-0.52). In both genders, collagen density was maintained, collagen-bound glycation end products increased, and collagen type 1 decreased. However, elastin density decreased significantly (from 22+/-1.5% to 15+/-1.2%) with aging (P<0.05) only in males. Furthermore, only old males were characterized by a decrease (P<0.05) in collagen type 3 (an isoform that promotes elasticity) and an increase in collagen type 8 (an isoform that promotes the neointimal migration of vascular smooth muscle cells). In contrast to the data in monkeys, collagen types 1 and 3 both increased significantly in aging rats. There are major species differences in the effects of aging on aortic collagen types 1 and 3. Furthermore, because alterations in collagen density, collagen content, hydroxyproline, and collagen advanced glycation end products were similar in both old male and female monkeys, these factors cannot be responsible for the greater increase in stiffness in old males. However, changes in collagen isoforms and the decrease in elastin observed only in old males likely account for the greater increase in aortic stiffness.

Novel conformation-sensitive antibodies specific to three- and four-repeat tau

Two types of tau isoform, three- and four-repeat tau, are found in neurofibrillary tangles—a pathological hallmark of tauopathies. Which isoform is deposited in the affected tissues depends on the tauopathy. To study how and which tau isoforms contribute to neuronal degeneration, we have developed and characterized two novel conformation-sensitive antibodies, T3R and T4R. Two closely related synthetic peptides, PGGGKVQIVYK and PGGGSVQIVYK, respectively, were designed as antigens. The isoform-specific residues, 305K in three-repeat tau or 305S in four-repeat tau, and the PHF6 motif (VQIVYK) were identified as critical sequences. Despite the high similarity of the antigens, there was no cross-reactivity between T3R and T4R. Furthermore, T3R and T4R showed reduced binding to the thioflavin-positive β-structural form of their target. These features may enable these antibodies to act as novel indicators that allow us to observe and evaluate conformational changes in each distinct isoform of tau.

Paradoxical Effects Of Endurance Training And Chronic Hypoxia On Soleus Myofibrillar ATPase Activity

PURPOSE: The aim of this study was to determine the changes in soleus myofibrillar ATPase (m-ATPase) activity and MHC isoform expression after endurance training and/or chronic hypoxic exposure. METHODS: Dark Agouti rats were randomly divided into four groups: control, normoxic sedentary (N, n = 6); normoxic trained (NT, n = 6); hypoxic sedentary (H, n = 6) and hypoxic trained (HT, n = 6) group. Rats lived and trained in normoxia at 760 mmHg (N and NT), or hypobaric hypoxia at 550 mmHg; (≊ 2800 m) (H and HT). Training consisted of 5 sessions/week of treadmill run at 80% of maximal aerobic velocity (MAV) for five weeks. MAV was reached when the rat was not able to follow the incremen increase in speed imposed by the driven wheel and turned in the wheel more than three times. M-ATPase activity was measured by Rapid Flow Quench technique; myosin subunits were analyzed with mono- and two-dimensional gel electrophoresis. RESULTS: Endurance training significantly increased m-ATPase (P < 0.05), although no significant change in MHC isoform occurred in normoxia, there was a fast-to-slow shift in MHC isoform in hypoxia. Chronic hypoxia depressed m-ATPase (P < 0.05) in spite of a slow-to-fast transition in MHC isoform distribution. Indeed, the rate constants of m-ATPase (kF; s-1) for N and NT are respectively 0.032 ± 0.002 s-1 and 0.046 ± 0.003 s-1, while for H and HT 0.031 ±0.001 s-1 and 0.034 ± 0.004 s-1. CONCLUSION: Dissociation between variations in m-ATPase and changes in MHC isoform expression were observed. Changes in fraction of active myosin heads, in myosin light chain isoform (MLC) distribution or in MLC phosphorylation were not sufficient to explain the variations in m-ATPase. Myosin post-translational modifications or changes in other myofibrillar proteins may therefore be responsible for the observed variations in m-ATPase activity.

Effects of Pronounced Weight Loss on Adiponectin Oligomer Composition and Metabolic Parameters

Adiponectin is an adipocytokine secreted into circulation in three isoforms. The aim of the study was to investigate changes of adiponectin isoforms during profound weight loss and its relation to anthropomorphometric and metabolic parameters. Thirteen severely obese female subjects were examined before and 1 year after surgical treatment. Total adiponectin was determined by radioimmunosorbent assay, and oligomer composition was detected by nondenaturing Western blot. BMI decreased substantially (p < 0.001), which was associated with an increase of total adiponectin from 12.9 +/- 5.9 to 14.3 +/- 6.1 microg/mL (p = 0.055). Medium molecular weight (MMW) adiponectin increased from 7.5 +/- 3.6 to 9.1 +/- 4.1 microg/mL (p = 0.009), whereas high (HMW) and low molecular weight adiponectin remained unchanged. Delta values of total adiponectin correlated significantly with Delta values of anthropometric parameters. Similar correlations were found for Delta values of MMW (Delta weight: r(2) = 0.4132, p = 0.0178; Delta BMI: r(2) = 0.3319, p = 0.0393; Delta fat mass: r(2) = 0.5202, p = 0.0054). Thus, profound weight loss was associated with an increase in total adiponectin, which was mainly and consistently caused by increases in MMW adiponectin (p = 0.009). These changes result in a shift from low molecular weight to MMW and HMW adiponectin isoforms, which may be related to improvements in both anthropometric and metabolic parameters.

Dynamic changes in conduction velocity and gap junction properties during development of pacing-induced heart failure

End-stage heart failure (HF) is characterized by changes in conduction velocity (CV) that predispose to arrhythmias. Here, we investigate the time course of conduction changes with respect to alterations in connexin 43 (Cx43) properties and mechanical function during the development of HF. We perform high-resolution optical mapping in arterially perfused myocardial preparations from dogs subjected to 0, 3, 7, 14, and 21 days of rapid pacing to produce variable degrees of remodeling. CV is compared with an index of mechanical function [left ventricular end-diastolic pressure (LVEDP)] and with dynamic changes in the expression, distribution, and phosphorylation of Cx43. In contrast to repolarization, CV was preserved during early stages of remodeling (3 and 7 days) and significantly reduced at later stages, which were associated with marked increases in LVEDP. Measurements of differentially phosphorylated Cx43 isoforms revealed early, sustained downregulation of pan-Cx43 that preceded changes in CV and LVEDP, a gradual rise in a dephosphorylated Cx43 isoform to over twofold baseline levels in end-stage HF, and a late abrupt increase in pan-Cx43, but not dephosphorylated Cx43, lateralization. These data demonstrate that 1) CV slowing occurs only at advanced stages of remodeling, 2) total reduction of pan-Cx43 is an early event that precedes mechanical dysfunction and CV slowing, 3) changes in Cx43 phosphorylation are more closely associated with the onset of HF, and 4) Cx43 lateralization is a late event that coincides with marked CV reduction. These data reveal a novel paradigm of remodeling based on the timing of conduction abnormalities relative to changes in Cx43 isoforms and mechanical dysfunction.

Stem/progenitor cells in amphibian limb and spinal cord regeneration

Amphibian regeneration may replace tissue via homotypic cells, dedifferentiation, transdifferentiation or stem cells. The mechanism may vary with life cycle stage. Expression of Notch signaling components suggests a role for stem cells. We examine the role of stem cells in regeneration-competent and incompetent stages of Xenopus laevis (clawed frog) tadpoles, juvenile and adult axolotls (the Mexican salamander Ambystoma mexicanum) and the regeneration-deficient axolotl mutant short-toes. Notch1 signaling is a key pathway in maintaining stem cell populations. The stem cell marker Musashi (Msi) is an RNA-binding protein inhibiting translation of Numb to maintain Notch1 signaling. Isoforms of Msi1 and Msi2 are expressed in temporal and spatial patterns that correlate with regeneration behavior. Msi-1 is expressed in specific tissue compartments in regeneration-competent Xenopus limb and spinal cord and decreases in incompetent tadpole and adult Xenopus tissues. Msi2 isoforms change between competent and incompetent Xenopus stages. Rapidly growing juvenile axolotls show high levels of Msi1 in both cord and limb, while intact adult axolotl tissues shows low levels of Msi1 that are upregulated following injury. Msi expression is also lost in limbs of the axolotl limb regeneration mutant short-toes, but maintained in the regeneration competent mutant spinal cord. Support: Indiana 21st Century Research and Technology Fund ; W.M. Keck Foundation.

Changes in PDE4D Isoforms in the Hippocampus of a Patient With Advanced Alzheimer Disease

Changes in PDE4D Isoforms in the Hippocampus of a Patient With Advanced Alzheimer Disease

Plasma membrane Ca2+-ATPase expression during colon cancer cell line differentiation

The differentiation of colon cancer cell lines is associated with changes in calcium homeostasis. Concomitantly there are changes in the expression of some calcium transporters and G-protein-coupled receptors, which are capable of altering cytosolic-free calcium levels. Recent studies associate alterations in calcium transporter expression with tumourigenesis, such as changes in specific isoforms of the plasma membrane calcium ATPase (PMCA) in breast cancer cell lines. In this study, we examined the expression of PMCA isoforms in the HT-29 colon cancer cell line using two methods of differentiation (sodium butyrate-mediated and spontaneous post-confluency induced differentiation). Our studies show that differentiation of HT-29 colon cancer cells is associated with the up-regulation of the PMCA isoform PMCA4 but no significant alteration in PMCA1. These results suggest that PMCA4 may be important and have a specific role in colon cells as well as being significant in colon cancer tumourigenesis.

Effect of herbicides on glutathione S-transferases in the earthworm, Eisenia fetida

Earthworms have been studied as a readily available, easily maintainable and cheap test species for assessing chemical pollution, and may be an alternative to in vivo rodent bioassays. The current investigation aims to characterize detoxification enzymes in Eisenia fetida and stress response against two herbicides with different modes of action, namely, fenoxaprop and metolachlor. Herbicides were applied to soil containing earthworms. Animals were then collected, sacrificed and shock-frozen. Extracted protein was analyzed for glutathione S-transferase (GST) activity using CDNB (1-chloro-2,4-dinitrobenzene), DCNB (1,2-dichloro-4-nitrobenzene), pNBC (p-nitrobenzylchloride), PNOBC (p-nitrobenz-o-ylchloride) and selected herbicides. GST isoenzymes were partially purified by affinity chromatography and molecular weights were estimated by SDS-PAGE. In E. fetida protein extracts, GST activity towards model compounds ranked as CDNB>DCNB>PNBOC>PNBC. Fluorodifen was not conjugated at all, but fenoxaprop and metolachlor were conjugated at low rates. Furthermore, the GST isoenzyme pattern changed during the incubation with herbicides, either due to stress or as a defense reaction. After incubation with monochlorobimane, a strong fluorescence of the intestinal tract and the intersegments was observed, indicating organ-specific GST induction. According to the author's knowledge, here, for the first time, evidence is presented that E. fetida GST are also capable of conjugating a wider range of xenobiotic substrates. Different forms of GST were observed and changes in GST isoforms due to the herbicide treatment were also noticed. GST conjugation rates varied between different herbicides used in this experiment. It might be assumed that herbicides may well be detoxified by earthworms, to a certain extent, but that they are also potent stress factors influencing the detoxification system of the animal. High doses or long exposure might lead to deleterious effects on earthworms and limit their survival rate. The use of the animals as bioindicators for herbicides and herbicide residues seems very promising, but is surely influenced by the lack of detoxification for some compounds. Conjugation of several xenobiotics with model substances and herbicides is proven in the earthworm E. fetida. However, E. fetida has only limited capabilities of detoxifying herbicidal compounds. Different isoforms of GST were involved and altered in their activity after treatment. The accumulation of GS-conjugates and their determination via fluorescence microscopy is a quick and secure, additional marker for exposure that should be further developed to complement existing biotests. The described methods and endpoints might help to understand the complex reaction of earthworms towards herbicides and lead to an adapted test methodology.

Dual role of thyroid hormones in rat soleus muscle MyHC isoform expression

We have analyzed the influence of altered thyroid hormone levels on changes of MyHC protein isoforms and their mRNA transcripts in the soleus muscle of 2-, 4- and 7-month-old euthyroid (EU), hypothyroid (HY) and hyperthyroid (TH) female inbred Lewis strain rats (methimazole and T3 treatment started 3 to 4 weeks after birth). We have found that the content of the dominant MyHC 1 isoform gradually increased in the EU rats and that this increase was more progressive in the HY rats at all three stages. On the other hand, in the TH rats the content of MyHC 1 isoform was the highest in the 2-month-old rats and it decreased with an increasing length of T3 treatment. The content of the minor 2a MyHC isoform followed the opposite pattern. In contrast to the protein isoforms, the MyHC mRNA transcripts remained at similar levels. Nevertheless, in general, the MyHC 1 mRNA level was decreased and MyHC 2a transcript increased in the TH rats, while the opposite changes occurred in the HY rats. Our results thus suggest that in the rat soleus muscle, both increased and decreased levels of thyroid hormones speed up the formation of an adult slow phenotype which is demonstrated by the precocious appearance of the slow MyHC 1 isoform, but opposite to the hypothyroid status, a longer T3 application promotes the expression of the faster MyHC 2a isoform.

Myosin heavy-chain isoform distribution, fibre-type composition and fibre size in skeletal muscle of patients on haemodialysis

Chronic uraemia is associated with abnormalities in skeletal muscles, which can affect their working capacity. It is also well known that the fibre-type composition of skeletal muscles influences endurance, muscle strength and power. In this study we therefore determined the size and distribution of muscle fibres and the myosin heavy-chain (MHC) isoform composition in patients on haemodialysis (HD) in order to establish any differences with values for untrained control subjects. Muscle biopsies were obtained from the vastus lateralis muscle of 14 non-diabetic patients on HD. The size and distribution of muscle fibres were evaluated using adenosine triphosphate synthase (ATPase) histochemistry, whilst MHC isoform composition was determined in muscle homogenates using sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Values were compared to those for a group of age-, gender- and BMI-matched untrained control subjects. The aerobic work capacity of the patients was also determined. The MHC composition for I, IIA and IIX isoforms was found to be 35.3% +/- 18.2%, 35.9% +/- 7.1% and 28.9% +/- 15.6%, respectively, findings supported by the ATPase histochemically determined fibre-type composition of the vastus lateralis muscle. The mean fibre area of type 1 and 2 fibres was 3283 +/- 873 and 3594 +/- 1483 MICROm2, respectively. The MHC composition and the size of the type 1 fibres of the patients on HD were significantly different from those of the control subjects. The data demonstrate relatively fewer type 1 and consequently more type 2x fibres, with a corresponding change in MHC isoforms (MHC I and MHC IIX) in the skeletal muscle of patients on HD. Several patients on HD were found to have <15% type 1 (or relative percentage of MHC I) fibres. Such a low percentage of type 1 fibres is very rarely observed in normal untrained subjects. Chronic uraemia more severely affects the composition than the size of fibres.

Changes in the Expression of Smooth Muscle Myosin Heavy Chain mRNA following Partial Bladder Obstruction or Spinal Cord Injury in Rat: A Preliminary Study

Purpose: The smooth muscle myosin heavy chain (SMMHC) isoform composition has been actively researched in a partial bladder obstruction (PBO) or spinal cord injury (SCI) model. Even though rat is an ideal animal for studying bladder physiology, there were very few reports about the changes of the SMMHC isoforms in the PBO or SCI injured bladder of rat. We developed two polymerrase chain reaction (PCR) primer sets to amplify the isoforms and we applied the primers to the PBO and SCI rat models. Materials and Methods: Female rats had their bladder necks surgically obstructed or they were subjected to spinal cord injury. Six weeks after the event, the bladders were excised. The expression of the C-terminal (SM1 and SM2) and N-terminal (SM-A and SM-B) isoforms of SMMHC was analyzed by performing reverse transcriptase-PCR (RT-PCR). Results: The control bladder showed only the SM-B isoform in the C-terminal. However, the bladder after SCI showed an increased SM-A to SM-B ratio. In case of PBO, the ratios were variable. A decreased SM1 expression was noted in the PBO and SCI groups when compared to the control group (p<0.05). Conclusions: Our female rat models for PBO or SCI demonstrates changes in the expression of smooth muscle myosin heavy chain isoforms. We will apply this primer set for studying of rat muscular physiology in PBO or SCI model. (Korean J Urol 2007;48:522-526)

Tamoxifen effect on L-DOPA induced response complications in parkinsonian rats and primates

The contribution of striatal protein kinase C (PKC) isoform changes in levodopa (L-DOPA) induced motor response complications in parkinsonian rats was investigated and the ability of tamoxifen, an antiestrogen with a partial PKC antagonist property, to prevent these response alterations in 6-hydroxydopamine (6-OHDA) lesioned rats as well as in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treated cynomologous monkeys was studied. Following treatment of adult male rats with L-DOPA twice daily for 3 weeks, protein levels of left (lesioned) and right (intact) striatal PKC isoforms were measured. Western blot analysis showed increased protein expression of both the novel PKC epsilon isoform and the atypical PKC lambda isoform ipsilateral to the lesion (174 ± 17% for epsilon, 140 ± 9% for lambda, of intact striatum in 6-OHDA lesioned plus chronic L-DOPA treated animals) in acute L-DOPA treated rats. No enhancement was observed in PKC immunoreactivity for other isoforms. Tamoxifen (5.0 mg/kg p.o.) significantly attenuated the L-DOPA induced augmentation of protein expression of PKC epsilon and PKC lambda, but had no effect on immunoreactivity for other PKC isoforms. In chronic L-DOPA treated parkinsonian rats, tamoxifen prevented (5.0 mg/kg p.o.) as well as ameliorated (5.0 mg/kg p.o.) the characteristic shortening in duration of motor response to L-DOPA challenge. In MPTP lesioned primates, similar to the ameliorative effect seen in rats, tamoxifen (1 and 3 mg/kg p.o) reduced the appearance of L-DOPA induced dyskinesia by 61% and 55% respectively ( p < 0.05). These results suggest that changes in specific striatal PKC isoforms contribute to the pathogenesis of L-DOPA induced motor complications and further that drugs able to selectively inhibit these signaling kinases might provide adjunctive benefit in the treatment of Parkinson's disease.

Differential regulation of myofilament protein isoforms underlying the contractility changes in skeletal muscle unloading

Weight-bearing skeletal muscles change phenotype in response to unloading. Using the hindlimb suspension rat model, we investigated the regulation of myofilament protein isoforms in correlation to contractility. Four weeks of continuous hindlimb unloading produced progressive atrophy and contractility changes in soleus but not extensor digitorum longus muscle. The unloaded soleus muscle also had decreased fatigue resistance. Along with the decrease of myosin heavy chain isoform I and IIa and increase of IIb and IIx, coordinated regulation of thin filament regulatory protein isoforms were observed: gamma- and beta-tropomyosin decreased and alpha-tropomyosin increased, resulting in an alpha/beta ratio similar to that in normal fast twitch skeletal muscle; troponin I and troponin T (TnT) both showed decrease in the slow isoform and increases in the fast isoform. The TnT isoform switching began after 7 days of unloading and TnI isoform showed detectable changes at 14 days while other protein isoform changes were not significant until 28 days of treatment. Correlating to the early changes in contractility, especially the resistance to fatigue, the early response of TnT isoform regulation may play a unique role in the adaptation of skeletal muscle to unloading. When the fast TnT gene expression was upregulated in the unloaded soleus muscle, alternative RNA splicing switched to produce more high molecular weight acidic isoforms, reflecting a potential compensation for the decrease of slow TnT that is critical to skeletal muscle function. The results demonstrate that differential regulation of TnT isoforms is a sensitive mechanism in muscle adaptation to functional demands.

PAX8 expression in human bladder cancer

PAX8 is a transcription factor with a role in ontogenesis of the urinary tract. The aim of the present investigation was to investigate PAX8 expression in normal bladder and in non-invasive urothelial tumours. Nine cases of normal urothelial mucosa, 2 cases of papillary urothelial neoplasia of low malignant potential, 12 cases of low grade non-invasive papillary urothelial carcinoma and 16 cases of high grade non-invasive papillary urothelial carcinoma were investigated by immunohistochemistry. PAX8 mRNA expression was evaluated by RT-PCR in a different set of normal bladder mucosas and tumours. In addition, PAX8 expression was evaluated by RT-PCR in bladder from 2 human embryos and in several continuous cell lines derived from bladder tumours (5637, RT-112, TCC-SUP, HT 1376). In immunohistochemical studies, PAX8 was expressed in 28 out of 30 non-invasive urothelial tumours, but not in the normal adult bladders. In RT-PCR studies, PAX8 was expressed in 13 out of 13 bladder tumours but not in the 6 normal bladder mucosa. Contrary to that in adults, PAX8 was expressed in 2 cases of bladder mucosa from 16-week-old embryos. PAX8 was expressed in all the cell lines from bladder tumours. Both in the bladder tumours and cell lines PAX8 expression was highly heterogeneous in terms of the splicing isoforms. Treatment of cell lines with sodium butyrate (NaB) induced several changes of the splicing isoforms. Therefore, only subsets of molecular events that determine the PAX8 mRNA splicing heterogeneity in bladder tumours are sensitive to NaB treatment.