Heavy Chain mRNA Research Articles

Transforming growth factor-β1 regulates cell growth and causes downregulation of SMemb/non-muscle myosin heavy chain B mRNA in human prostate stromal cells

SMemb/non-muscle myosin heavy chain B (SMemb/NMMHC-B) is most abundantly expressed in proliferating smooth muscle cells and correlates with phenotypic changes from a contractive to a proliferative type. The stromal cells of the prostate play a crucial role in the regulation of prostatic growth and function. The aim of this study was to investigate the effects of the multifunctional cytokine transforming growth factor-beta1 (TGF-beta1) on SMemb/NMMHC-B mRNA expression and stromal cell growth. The expression of the SM2 isoform of smooth muscle myosin heavy chain (SMMHC) mRNA was also examined. Primary cultures of prostate stromal cells were established by means of an explant method from eight normal prostates. The effects of TGF-beta1 on stromal cell growth were determined by means of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide conversion assay. SMemb/NMMHC-B and SM2 mRNA expression were analyzed quantitatively by means of real-time polymerase chain reaction. In the absence of TGF-beta1, cells expressed alpha-smooth muscle actin and vimentin. After TGF-beta1 treatment, the expression of alpha-smooth muscle actin increased and cells also expressed desmin. TGF-beta1 at concentrations of 1.0, 5.0 and 10 ng/ml suppressed cell growth by 72%, 62% and 56%, respectively, downregulated SMemb/NMMHC-B mRNA expression by 71%, 52% and 38%, respectively and upregulated SM2 mRNA expression 2.1-, 3.0- and 5.3-fold, respectively. These results demonstrate that TGF-beta1 modulates the smooth muscle cell phenotype from a proliferative to a contractile type and that the inhibitory effects of TGF-beta1 on stromal cell growth correlate with downregulation of the SMemb/NMMHC-B gene.

The effect of magnetic stimulation on unloaded soleus muscle of rat: changes in myosin heavy chain mRNA isoforms

This study assessed the potential application and the effectiveness of functional magnetic stimulation (FMS) for preventing skeletal muscle atrophy in adult rats. FMS using magnetic stimulator was performed to rat soleus muscle by placing a round magnetic coil on the back of 3rd-5th lumbar vertebral level at 20 Hz frequency for 60 min/day up to 10 days. A reverse transcriptase-polymerase chain reaction was applied to evaluate relative amounts of mRNAs specific to four myosin heavy chain (MHC) isoforms [MHCIbeta, MHCIIa, MHCIIb, and MHCIId(x)] in rat soleus muscle during contractile activity by magnetic stimulation. Ten-day unloading by hindlimb suspension induced a drastic decrease of MHCIbeta and MHCIIa mRNA expressions, while MHCIIb and MHCIId(x) mRNA was not decreased. The magnetic stimulation resuscitated the down-regulation of the mRNA levels of MHCIbeta and MHCIIa. These results suggest that magnetic stimulation on acute atrophied muscles is useful for preventing the muscle atrophy.

NADH Dehydrogenase Activity and Expression of mRNA of Complex I (ND1, 51kDa, and 75kDa) in Heart Mitochondria of klotho Mouse

Mitochondrial enzyme activities and ultrastructure of mitochondria prepared from klotho mutant mice were compared with those in wild-type mice. We also measured the levels of expression of ND1, 51kDa, and 75kDa mRNA associated with the genes encoding NADH dehydrogenase and complex I and that of alpha cardiac myosin heavy chain mRNA in both groups. Mitochondrial NADH oxidoreductase activity was higher in klotho mutant mice during aging than that in wild-type mice. The area of mitochondria per unit area (300 microm2) of cell was almost constant from 4 to 7 weeks of age in both groups. A few large mitochondria were scattered between numerous small mitochondria with compact cristae and myofibrils in klotho mice from 5 weeks of age. The levels of ND1 and 75kDa mRNA were slightly high from 7 weeks of age in klotho mutant mice, whereas they were almost constant in wild-type mice, in spite of reduced expression of alpha cardiac myosin heavy chain mRNA. Our results indicate that klotho protein indirectly plays a role in diminished functional adaptability of enzymes in aged heart muscle, and is required for hypertrophy of cardiac mitochondria.

Suppression of Erythroid-Specific 5-Aminolevulinate Synthase Using Short-Interfering RNA Alters Iron Metabolism and Inhibits Terminal Differentiation of Human Erythroleukemia Cells.

Erythroid-specific 5-aminolevulinate synthase (ALAS2) is the first and the rate limiting enzyme for heme biosynthesis in erythroid cells. ALAS2 plays a critical role in hemoglobin synthesis and erythrocyte maturation, since targeting the ALAS2 gene results in embryonic death in mice because of severe anemia. In humans, heritable mutations of the ALAS2 gene are responsible for X-linked sideroblastic anemia (XLSA). However, the effect of suppressed expression of ALAS2 on erythroid cell differentiation has not been examined in human cells. We therefore addressed this question, by stably suppressing ALAS2 mRNA with short-interfering RNA (siRNA) in a human erythroleukemia cell line, YN-1. After cloning of cells expressing low ALAS2 (ALAS2low cells), cells were induced to undergo erythroid differentiation by treatment with transforming growth factor beta1 (TGF-β1). Gene expression profiles of induced and uninduced cells were examined, including genes involved in globin synthesis and iron metabolism. Hemoglobin production, as judged by o-dianisidine staining, was significantly lower in ALAS2low cells than in control cells both before and after erythroid differentiation. Both alpha and gamma globin mRNA levels were also reduced in ALAS2low cells, compared with control cells. Decreased heme synthesis as well as reduced globin production in ALAS2low erythroid cells are consistent with our previous findings in murine erythroleukemia cells studied by antisense technology (Meguro K, et al. Blood 86:940–948, 1995), and extends our previous conclusion on the critical role of ALAS2 in heme and globin formation to human erythroid cells. Transferrin receptor (TFR) mRNA level was decreased in ALAS2low cells, and remained low following TGF-β1 treatment, whereas its level was increased in control cells during erythroid differentiation, which reflects enhanced iron uptake by differentiated control cells. Decreased TFR mRNA level in ALAS2low cells may suggest iron accumulation, since TFR mRNA is known to be unstable when intracellular iron level is increased. Notably, mitochondrial ferritin (MtF) mRNA level was decreased in control cells after differentiation, reflecting utilization of mitochondrial iron for heme synthesis, but it did not change in ALAS2low cells following TGF-β1 treatment. As accumulation of MtF protein is known to occur in iron-overloaded erythroid cells of patients with XLSA, our finding also suggests that there may be intramitochondrial iron accumulation in ALAS2low cells even after differentiation. In contrast to MtF mRNA, the level of cytosolic ferritin heavy chain mRNA was similar both in ALAS2low cells and control cells. These findings suggest that MtF levels, rather than cytosolic ferritin levels, may be a sensitive and specific indicator for iron accumulation in mitochondria. This study shows the critical role of ALAS2 not only in heme synthesis and hemoglobin formation, but also in iron metabolism in erythroid cells during their cell differentiation. An ALAS2low erythroid cell line, such as ALAS2-suppressed YN-1, will provide a good model for the study of relationship between heme biosynthesis and iron metabolism during terminal differentiation of human erythroid cells.

Changes in detrusor smooth muscle myosin heavy chain mRNA expression following spinal cord injury in the mouse.

Smooth muscle myosin heavy chain (SMMHC) isoform composition has been shown to be developmentally regulated and to be associated with functional changes in smooth muscle activity. In this study, we sought to determine expression patterns of SMMHC isoforms in a murine model of spinal cord injury (SCI) and to compare these expression patterns to neurologic, cytometric, and morphometric findings. Baseline cystometry was performed on adult, female mice followed by either thoracic spinal cord transection (SCI) or sham operation (Sham). At 1, 3, or 6 weeks postoperatively neurologic evaluation and cystometry were performed, bladders were harvested, and expression patterns of SMMHC isoforms (SM1 vs. SM2 and SMA vs. SMB) were assessed by RT-PCR. Morphometrics utilizing computer-assisted color image analysis was also performed on all bladders. There was a significant increase in bladder weight and capacity 1 week following SCI which normalized over time, however, morphometric analysis did not reveal an alteration in tissue composition amongst the three groups. One week following SCI, SM1 was predominantly expressed over SM2 and began to normalize at 3 weeks. This coincided with the emergence of reflex voiding and detrusor overactivity. SMA was expressed following SCI only, and the number of bladders found to express SMA decreased with increasing duration since SCI. Smooth muscle myosin heavy chain mRNA expression patterns appear to be affected by SCI. We believe the induction of SMA may be a factor in altered bladder function following injury.

Increased cardiac workload by closure of the ductus arteriosus leads to hypertrophy and apoptosis rather than to hyperplasia in the late fetal period.

It is generally thought that adult mammalian cardiomyocytes compensate for an increased workload by hypertrophy, whereas fetal myocardium grows by cellular proliferation. We analyzed the response of late-fetal rat hearts upon an increased workload imposed by premature constriction of the ductus arteriosus with indomethacin. Initially the fetal heart responds by proliferative growth, as both wet weight and labeling index (bromodeoxyuridine incorporation) of the ventricles increased, whereas neither a change in the fibroblast fraction, ploidy and nucleation in the ventricles is observed. However, this hyperplastic growth is abrogated by a subsequent burst in apoptosis and followed by a hypertrophic response as based on a decrease in DNA and increase in both RNA and protein concentration. This hypertrophic growth was accompanied by a 1.4-fold increase in the volume of the cardiomyocytes. Changes in the molecular phenotype characteristic of pressure-overload hypertrophic growth accompany the process. Thus, the levels of expression of beta-myosin heavy chain and atrial natriuretic factor mRNA increased, of sarcoplasmic/endoplasmic reticulum ATPase (SERCA2) mRNA decreased, and of alpha-myosin heavy chain, phospholamban, and calsequestrin mRNA did not change. In situ hybridization showed that the pattern of mRNA expression changed first in the right ventricular wall and subsequently in the left ventricular free wall as well. It is concluded that pressure-overload imposed on the late-fetal heart induces limited proliferative growth complemented by extensive hypertrophic growth.

Protein kinase Cepsilon overexpression alters myofilament properties and composition during the progression of heart failure.

We report characterization of a transgenic mouse that overexpresses constitutively active protein kinase Cepsilon in the heart and slowly develops a dilated cardiomyopathy with failure. The hemodynamic, mechanical, and biochemical properties of these hearts demonstrate a series of temporal events that mark the progression of the disease. In the 3-month transgenic (TG) animals, contractile properties and gene expression measurements are normal, but an increase in myofibrillar Ca2+ sensitivity and thin filament protein phosphorylation is noted. At 6 months, there is a decrease in the myofibrillar Ca2+ sensitivity, a significant increase in beta-myosin heavy chain mRNA and protein, normal cardiac function, but a blunted response to an inotropic challenge. The transition at 9 months is especially interesting because age-related changes appear to contribute to the decline in function seen in the TG heart. At this point, there is a decline in baseline function and maximum tension produced by the myofibrils, which is coincident with the onset of atrial myosin light chain isoform re-expression in the ventricles. In the 12-month TG mice, there is clear hemodynamic and geometric evidence of failure. Alterations in the composition of the myofibrils persist but the phosphorylation of myosin light chain 2v is dramatically different at this age compared with all others. We interpret these data to implicate the disruption of the myofibrillar proteins and their interactions in the propagation of dilated cardiac disease.

Myosin heavy chain composition of rat middle ear muscles

ObjectiveTo quantitatively analyze myosin heavy chain (MHC) mRNA composition in two rat middle ear muscles (the tensor tympani and stapedius) using competitive polymerase chain reaction (PCR).Material and MethodsAn exogenous template, including oligonucleotide sequences specific for the seven rat MHCs (2A, 2B, 2X, 2L/EOM, embryonic, neonatal and β-cardiac) as well as β-actin, was constructed and used as the competitor.ResultsThe tensor tympani and stapedius contained all MHC isoforms except 2L. The tensor tympani contained approximately equal proportions of 2X (40.4%±6.5%) and 2A (34.0%±1.3%) MHCs, with a smaller percentage of 2B (16.6%±1.5%) and neonatal (7.5%±0.6%) MHCs, while β-cardiac and embryonic MHCs were minimally expressed. The stapedius contained predominantly 2X (58.0%±4.2%) and 2A (32.3%±6.7%) MHCs, with a smaller percentage of 2B (7.4%±0.2%) and β-cardiac (1.9%±0.1%) MHCs. Neonatal and embryonic MHCs were detected at very low levels.ConclusionThese results suggest that two middle ear muscles, which are mainly composed of two fast-twitching myosins (2X and 2A MHCs), contract fast and are fatigue-resistant.

Potential involvement of rainbow trout thrombocytes in immune functions: a study using a panel of monoclonal antibodies and RT-PCR

The functional relationship between fish and mammalian thrombocytes is relatively unknown. In this study, a panel of monoclonal antibodies (mAbs) was used to investigate the functional properties of rainbow trout thrombocytes. The mAbs recognize cell-surface molecules on thrombocytes with molecular weights ranging from 17 to 160 kDa. Flow cytometric and immuno-electron microscopic analyses demonstrate that these molecules are expressed at different levels and that surface expression increased upon activation with bovine collagen. Two of these cell-surface molecules (17 and 21 kDa) were directly involved in collagen-induced aggregation of thrombocytes since aggregation was blocked upon pre-treatment with mAbs that recognize the two surface markers. Interestingly, the percentage of thrombocytes in blood increased after stimulation using different antigens. The transcriptional profile of trout thrombocytes was then examined after immuno-magnetic enrichment using the described mAbs to assess potential roles of trout thrombocytes in immune functions. Trout thrombocytes express components of the MHC class Ia pathway, IL1β, TNFα, TGFβ, the interleukin receptor common γ chain as well as CXC and CC chemokines. MHC class IIB and TNFα were expressed at low levels in resting thrombocytes. No evidence was found for the expression of TCRαβ, Ig heavy chain, CD8α or CK1 mRNA. Taken together, these results suggest that rainbow trout thrombocytes express molecules involved in activation, aggregation and genes encoding proteins, that are involved in antigen presentation and immune regulation.

Cardiac Enlargement in a Mouse Model of Intestinal Cancer

1233 Cancer-induced cachexia is a life-threatening wasting syndrome characterized by reduced body mass and decreased antioxidant defense systems. This condition is often associated with the activation of the immune system and also heart failure. PURPOSE: To characterize cardiac muscle remodeling in a cachexic mouse model induced by intestinal tumors. The APC+/− mouse used in this study has a high incidence of intestinal tumor formation. We hypothesized that these cachexic mice would have altered heart remodeling. METHODS: Female C57BL6 (CON, n = 7) and APCmin+/− (MIN, n = 7) mice, 6–8 months of age, were monitored for daily food intake. Animals were sacrificed and liver, heart, and tibias were excised. Liver catalase (CAT) activity was measured spectrophotometrically. Cardiac α-myosin heavy chain and 18S mRNA abundance were analyzed by RT-PCR. RESULTS: CON and MIN ate similarly (p>0.05), but MIN BW was decreased by 20.5% (p<0.05). MIN CAT was decreased 54% compared to CON. The heart weight:tibia length ratio was significantly increased 27% in MIN compared to CON (p<0.05). Cardiac α-myosin heavy chain, corrected with 18S and normalized to CON was decreased 40% in MIN (p = 0.045). CONCLUSIONS: Cachexic female APCmin+/− mice, as described by decreased body weight with normal eating behavior as well as decreased catalase activity, suffer from cardiac hypertrophy with altered remodeling. While the stimulus of cardiac enlargement in this cachexic model is not certain, blood volume expansion and atrial natriuretic peptide signaling may be important mechanisms for the induction of this cardiac enlargement. This study was funded by the Colorectal Cancer COBRE program from the National Institute of Health/USC.

Cardiac Enlargement in a Mouse Model of Intestinal Cancer

1233 Cancer-induced cachexia is a life-threatening wasting syndrome characterized by reduced body mass and decreased antioxidant defense systems. This condition is often associated with the activation of the immune system and also heart failure. PURPOSE: To characterize cardiac muscle remodeling in a cachexic mouse model induced by intestinal tumors. The APC+/− mouse used in this study has a high incidence of intestinal tumor formation. We hypothesized that these cachexic mice would have altered heart remodeling. METHODS: Female C57BL6 (CON, n = 7) and APCmin+/− (MIN, n = 7) mice, 6–8 months of age, were monitored for daily food intake. Animals were sacrificed and liver, heart, and tibias were excised. Liver catalase (CAT) activity was measured spectrophotometrically. Cardiac α-myosin heavy chain and 18S mRNA abundance were analyzed by RT-PCR. RESULTS: CON and MIN ate similarly (p>0.05), but MIN BW was decreased by 20.5% (p<0.05). MIN CAT was decreased 54% compared to CON. The heart weight:tibia length ratio was significantly increased 27% in MIN compared to CON (p<0.05). Cardiac α-myosin heavy chain, corrected with 18S and normalized to CON was decreased 40% in MIN (p = 0.045). CONCLUSIONS: Cachexic female APCmin+/− mice, as described by decreased body weight with normal eating behavior as well as decreased catalase activity, suffer from cardiac hypertrophy with altered remodeling. While the stimulus of cardiac enlargement in this cachexic model is not certain, blood volume expansion and atrial natriuretic peptide signaling may be important mechanisms for the induction of this cardiac enlargement. This study was funded by the Colorectal Cancer COBRE program from the National Institute of Health/USC.

Effects of diet consistency on the expression of insulin-like growth factors (IGFs), IGF receptors and IGF binding proteins during the development of rat masseter muscle soon after weaning

A soft diet facilitates the development of faster-type fibres in rat masseter muscle in the 9 days after weaning compared with a hard diet. To determine whether insulin-like growth factors (IGFs), IGF receptors (IGFRs) and IGF binding proteins (IGFBPs) are involved in this fibre-type alteration, the expression of myosin heavy chain (MHC), IGF, IGFR and IGFBP mRNAs in the masseter muscle of rats fed a hard or soft diet for 9 days after weaning was analysed using competitive, reverse transcriptase–polymerase chain reaction. A soft diet decreased the expression of MHC IIa (slower type) by 70%, but increased the expression of MHC IIx (intermediate type) and IIb (faster type) by 80 and 582%, respectively, compared with a hard diet. These findings verified that a soft diet facilitates the development of faster-type fibres in rat masseter muscle compared with a hard diet. A soft diet induced reductions of 25–76% ( P<0.05–0.01) in the expression of IGF-I, IGF-II, IGFR2, IGFBP4 and IGFBP6 compared with a hard diet, but induced a 25% ( P<0.05) increase only in expression of IGFBP3. These findings suggest that the changes in expression of IGF-I, IGF-II, IGFR2, IGFBP3, IGFBP4 and IGFBP6 are associated with the fibre-type alteration of rat masseter muscle in response to diet consistency soon after weaning.

Endothelin-1-induced cardiac hypertrophy is inhibited by activation of peroxisome proliferator-activated receptor-alpha partly via blockade of c-Jun NH2-terminal kinase pathway.

Peroxisome proliferator-activated receptor-alpha (PPAR-alpha) is a lipid-activated nuclear receptor that negatively regulates the vascular inflammatory gene response by interacting with transcription factors, nuclear factor-kappaB, and AP-1. However, the roles of PPAR-alpha activators in endothelin (ET)-1-induced cardiac hypertrophy are not yet known. First, in cultured neonatal rat cardiomyocytes, a PPAR-alpha activator, fenofibrate (10 micromol/L), and PPAR-alpha overexpression markedly inhibited the ET-1-induced increase in protein synthesis. Second, fenofibrate markedly inhibited ET-1-induced increase in c-Jun gene expression and phosphorylation of c-Jun and JNK. These results suggest that this PPAR-alpha activator interferes with the formation and activation of AP-1 protein induced by ET-1 in cardiomyocytes. Third, fenofibrate significantly inhibited the increase of ET-1 mRNA level by ET-1, which was also confirmed by luciferase assay. Electrophoretic mobility shift assay revealed that fenofibrate significantly decreased the ET-1-stimulated or phorbol 12-myristate 13-acetate-stimulated AP-1 DNA binding activity, and the nuclear extract probe complex was supershifted by anti-c-Jun antibody. Fourth, 24 hours after aortic banding (AB) operation, fenofibrate treatment significantly inhibited left ventricular hypertrophy and hypertrophy-related gene expression pattern (ET-1, brain natriuretic peptide, and beta-myosin heavy chain mRNA) in AB rats. These results suggest that PPAR-alpha activation interferes with the signaling pathway of ET-1-induced cardiac hypertrophy through negative regulation of AP-1 binding activity, partly via inhibition of the JNK pathway in cultured cardiomyocytes. We also revealed that fenofibrate treatment inhibited left ventricle hypertrophy and phenotypic changes in cardiac gene expression in AB rats in vivo.

Increased expression of SERCA2 mRNA in hearts of genetically obese Zucker rats after a moderate treadmill running

Aim. – Analyze the effects of exercise training on cardiac sarco endoplasmic reticulum calcium ATPase (SERCA) 2 gene expression of genetically obese Zucker rats, an animal model of non-insulin dependent diabetes mellitus (NIDDM). Methods. – Twenty male and 20 female Zucker fatty rats ran 30–50 min/d at 35–40% VO 2max, 6 d/week for 13 weeks. Semi-quantitative RT-PCR associated with specific restriction fragment analysis was used to detect and quantify the relative expression of SERCA mRNA isoforms. Results. – Fatty rats had an enhanced expression of SERCA2 and phospholamban (PLB) mRNA levels in ventricles. Moreover, fatty rats exposed to a moderate treadmill running exercise for 13 weeks exhibited a significant increase in ventricles SERCA2 mRNA levels, whereas, no change occurred in control lean rats. Exercise training produced a significant induction of α-myosin heavy chain mRNA levels only in fatty rats. Conclusion. – We hypothesize that exercise training may improve the cardiac function in diabetic rats by increasing the mRNA expression of SERCA2 since it is known that NIDDM rats have a reduced left ventricular diastolic compliance, and that exercise training improves the ventricular function in diabetic rats.

Swimming exercise in infancy has beneficial effect on the hearts in cardiomyopathic Syrian hamsters

The phenotypic expression of cardiomyopathy is greatly influenced by extrinsic factors other than intrinsic genetic defects, such as environmental stress. Exercise is assumed to be an important extrinsic factor, since sudden death is sometimes seen during exercise in young patients with hypertrophic cardiomyopathy (HCM). However, the long-term effects of mild exercise on phenotypic expression in cardiomyopathy remain unclear. To evaluate the effects of exercise performed during infancy or adolescence in cardiomyopathic patients, cardiomyopathic Syrian hamsters (BIO14.6) were subjected to swimming. BIO14.6 and age-matched congenic normal hamsters (CN) as controls were divided into three groups: sedentary (Sed), and trained during infancy (Inf) and during adolescence (Ado). Histological and biochemical analysis of 41-week-old hamsters revealed that (1) the relative level of beta-myosin heavy chain mRNA was significantly lower in the Inf group than in the Sed and Ado groups of BIO14.6. The level in the Inf group of BIO14.6 was compatible with that in the age-matched Sed group of the CN strain; (2) in BIO14.6, degenerative mitochondrial change in the cardiomyocytes was not seen in the Inf group while it was common in the Sed and Ado groups; (3) calcineurin phosphatase activity in the swimming group in 10-week-old CN was significantly higher than that of the age-matched sedentary group, and was as much as that of the swimming and sedentary groups in 10- and 41-week-old BIO14.6.

Maternal methamphetamine administration during pregnancy influences on fetal rat heart development

Methamphetamine (MAP) is one of the most abused drugs in Japan. The rate of MAP abuse by young women has recently reached more than 50 percent in adolescents. A major health concern is that these women will continue to use MAP during pregnancy. The purpose of this study was to investigate whether MAP administered to the mother during pregnancy would change the expression of α- and β- myosin heavy chain (MHC) mRNA in rat neonatal hearts, as detected by quantitative RT-PCR. In addition, morphological changes in the rat neonatal ventricles were examined. Pregnant rats were injected intraperitoneally with MAP (1 mg/kg/day) starting at day 0 of gestation and ending at day 21. There was a significant increase in α-MHC mRNA expression in the neonatal ventricular muscle in the experimental group compared with the control at postnatal day (P) 0 and 5. α-MHC mRNA expression in both groups was similar after P9. β-MHC mRNA expression was similar in both groups at P0. Postnatal β-MHC mRNA expression decreased rapidly, but significant alteration was not detected. Neonatal rats at P0 exhibited some cardiac changes, including hypertrophy, degeneration, and disarrangement of myofibers, but these lesions disappeared by P14. We conclude that chronic maternal administration of MAP changes the α- and β-MHC mRNA expression pattern in fetal and neonatal hearts, correlating with abnormal development, plasma level of hormones, and myocardial damage. At the same time, it is indicated that neonatal cardiomyocytes have reversibility.

Semiquantitative evaluation of mRNAs for the membranous form of immunoglobulin heavy chain is useful for investigating the etiology in CVID.

Common variable immunodeficiency (CVID) is a primary antibody deficiency syndrome characterized by defective B-cell maturation and antibody formation resulting in low serum antibody levels of all immunoglobulin (Ig) isotypes. To investigate the pathogenesis of CVID, we developed a set of competitive polymerase chain reaction for membrane-bound Ig heavy chain (mHC) mRNAs for IgM, IgG and IgA. Data on three children with CVID in group A of Bryant's classification were analysed. All the three mHC mRNA levels in Patient 1 were almost same as those in healthy controls. In Patient 2, mHC mRNA for IgM was detected at a level similar to that in controls, but mHC mRNAs for IgG and IgA heavy chains were not detected. In Patient 3, all the three mHC mRNAs were undetectable. Our data suggest that a different molecular basis exists in these patients with CVID even though all belong to group A of Bryant's classification. Use of our method facilitates a better understanding of molecular events in CVID patients and may be useful for precise classifications of CVID.

Evidence for a Functional Role of Angiotensin II Type 2 Receptor in the Cardiac Hypertrophic Process In Vivo in the Rat Heart

The precise function of angiotensin II type 2 receptor (AT2-R) in the mammalian heart in vivo is unknown. Here, we investigated the role of AT2-R in cardiac pressure overload. Rats were infused with vehicle, angiotensin II (Ang II), PD123319 (an AT2-R antagonist), or the combination of Ang II and PD123319 via subcutaneously implanted osmotic minipumps for 12 or 72 hours. Ang II-induced increases in mean arterial pressure, left ventricular weight/body weight ratio, and elevation of skeletal alpha-actin and beta-myosin heavy chain mRNA levels were not altered by PD123319. In contrast, AT2-R blockade resulted in a marked increase in the gene expression of c-fos, endothelin-1, and insulin-like growth factor-1 in Ang II-induced hypertension. In parallel, Ang II-stimulated mRNA and protein expression of atrial natriuretic peptide were significantly augmented by AT2-R blockade. Moreover, PD123319 markedly increased the synthesis of B-type natriuretic peptide. Furthermore, the expression of vascular endothelial growth factor and fibroblast growth factor-1 was downregulated by Ang II only in the presence of AT2-R blockade. Our results provide evidence that AT2-R plays a functional role in the cardiac hypertrophic process in vivo by selectively regulating the expression of growth-promoting and growth-inhibiting factors.

Mouse myeloma cell line Sp2/0 multidrug-resistant variant as parental cell line for hybridoma construction.

The use of multidrug-resistant variant of Sp2/0 mouse myeloma cell line SpEBr-5 as a partner for making mouse hybridoma producing monoclonal antibodies is described here. The resulting hybridoma cell line 1F7 was characterized with a high level of monoclonal antibody production and karyotype containing all normal mouse chromosomes. 1F7 cells were separately selected for resistance to ethidium bromide (EBr) and adriamycin (ADR) and different mechanisms of drug resistance were found in these cell variants. The resistance in ADR-selected 1F7 cells was due to amplification and overexpression of mdr genes. In EBr-resistant 1F7 cells, mdr genes were overexpressed without amplification. Substantially decreased level of Topo II activity in both cell lines also suggests the existence of additional mechanisms for MDR phenotype of hybridoma cells. Finally, adriamycin-resistant 1F7 hybridoma cell variant was found to produce higher level of specific immunoglobulins due to the increased level of Iggamma(2b) heavy chain mRNA.

Differential expression of equine myosin heavy-chain mRNA and protein isoforms in a limb muscle.

The horse is one of the few animals kept and bred for its athletic performance and is therefore an interesting model for human sports performance. The regulation of the development of equine locomotion in the first year of life, and the influence of early training on later performance, are largely unknown. The major structural protein in skeletal muscle, myosin heavy-chain (MyHC), is believed to be primarily transcriptionally controlled. To investigate the expression of the MyHC genes at the transcriptional level, we isolated cDNAs encoding the equine MyHC isoforms type 1 (slow), type 2a (fast oxidative), and type 2d/x (fast glycolytic). cDNAs encoding the 2b gene were not identified. The mRNA expression was compared to the protein expression on a fiber-to-fiber basis using in situ hybridization (non-radioactive) and immunohistochemistry. Marked differences were detected between the expression of MyHC transcripts and MyHC protein isoforms in adult equine gluteus medius muscle. Mismatches were primarily due to the presence of hybrid fibers expressing two fast (2ad) MyHC protein isoforms, but only one fast (mainly 2a) MyHC RNA isoform. This discrepancy was most likely not due to differential mRNA expression of myonuclei.