scholarly journals Real time RT-PCR with a newly designed set of primers confirmed the presence of 2b and 2x/d myosin heavy chain mRNAs in the rat slow soleus muscle

2008 ◽  
pp. 973-978
Author(s):  
J Žurmanová ◽  
F Půta ◽  
R Stopková ◽  
T Soukup
Keyword(s):  
Myosin Heavy Chain ◽  
Real Time ◽  
Soleus Muscle ◽  
Heavy Chain ◽  
Protein Isoforms ◽  
Rt Pcr ◽  
Adult Rats ◽  
Cdna Sequences ◽  
Pcr Products

In order to re-evaluate the presence and relative quantity of 2b and 2x/d myosin heavy chain (MyHC) transcripts in rat slow soleus muscle by using real time RT-PCR we have compared the available relevant cDNA sequences and designed a new set of primers having similar melting temperatures, matching separate MyHC exons in the regions of maximal differences in MyHC coding sequences, and containing G or C at the 3 -end. These also yielded PCR products of corresponding length, which is an important requirement for real time RT-PCR quantification. The experiments were performed on 8-month-old inbred female Lewis strain rats used in our current study of regenerating transplanted muscles. The real time RT-PCR measurement confirmed the expression of all four MyHC mRNAs (type 1, 2a, 2x/d and 2b) in both fast extensor digitorum longus and slow soleus muscles, although in the soleus muscle of adult rats, only type 1 and 2a protein isoforms can be usually detected.

scholarly journals Comparison of Myosin Heavy Chain mRNAs, Protein Isoforms and Fiber Type Proportions in the Rat Slow and Fast Muscles

2013 ◽  
pp. 445-453 ◽  
Author(s):  
J. ŽURMANOVÁ ◽  
T. SOUKUP
Keyword(s):  
Myosin Heavy Chain ◽  
Real Time ◽  
Heavy Chain ◽  
Fiber Type ◽  
Protein Isoforms ◽  
Fiber Types ◽  
Rt Pcr ◽  
Fiber Type Composition ◽  
Type Composition

We studied the expression of myosin heavy chain isoforms at mRNA and protein levels as well as fiber type composition in the fast extensor digitorum longus (EDL) and slow soleus (SOL) twitch muscles of adult inbred Lewis strain rats. Comparison of the results from Real Time RT-PCR, SDS-PAGE and fiber type analysis showed corresponding proportions of MyHC transcripts (MyHC-1, -2a, -2x/d, -2b), protein isoforms (MyHC-1, -2a, -2x/d, -2b) and fiber types (type 1, 2A, 2X/D, 2B) in both muscles. Furthermore, we found that slow MyHC-1 mRNA expression in the SOL was up to three orders higher than that of fast MyHC transcripts. This finding can explain the predominance of MyHC-1 isoform and fiber type 1 and the absence of pure 2X/D and 2B fibers in the SOL muscle. Based on our data presenting quantitative evidence of corresponding proportions between mRNA level, protein content and fiber type composition, we suggest that the Real Time RT-PCR technique can be used as a routine method for analysis of muscle composition changes and could be advantageous for the analysis of scant biological samples such as muscle biopsies in humans.

scholarly journals Changes in myosin heavy chain mRNA and protein isoforms in single fibers of unloaded rat soleus muscle

FEBS Letters ◽  
1999 ◽  
Vol 463 (1-2) ◽  
pp. 15-18 ◽  
Author(s):  
Laurence Stevens ◽  
Bärbel Gohlsch ◽  
Yvonne Mounier ◽  
Dirk Pette
Keyword(s):  
Myosin Heavy Chain ◽  
Soleus Muscle ◽  
Heavy Chain ◽  
Protein Isoforms ◽  
Single Fibers ◽  
Rat Soleus Muscle

Heterogeneity of smooth muscle myosin heavy chain expression at the single cell level

1996 ◽  
Vol 270 (6) ◽  
pp. C1819-C1824 ◽  
Author(s):  
D. P. Meer ◽  
T. J. Eddinger
Keyword(s):  
Smooth Muscle ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Pcr Amplification ◽  
Smooth Muscle Myosin ◽  
Adult Rabbit ◽  
Rt Pcr ◽  
Chain Reactions ◽  
Pcr Products ◽  
Muscle Myosin

Expression of the smooth muscle myosin heavy chain (SM-MHC) isoforms was examined in individual rabbit arterial smooth muscle cells with the use of reverse transcription-polymerase chain reactions (RT-PCR). The RT-PCR amplification protocol used oligonucleotide primers complementary to regions that flank the alternative exon that encodes the nine unique amino acids found in the carboxy terminal domain of SM2. RT-PCR products of SM1 and SM2 mRNA differ in length and electrophoretic mobility. Partial DNA sequencing of the PCR products confirmed SM1 and SM2 identity. Densitometric analyses of adjacent samples extracted from the same tissues and processed for SM2-to-SM1 protein and PCR-amplified SM2-to-SM1 RNA ratios exhibited a high correlation (R = 0.92). RT-PCR-amplified SM2-to-SM1 mRNA ratios of individual adult rabbit arterial cells ranged from 0.0 to 1.8 (n = 59), whereas multicellular vascular samples varied much less (0.4-0.6, n = 5). These results indicate that individual cells within a blood vessel differ significantly in SM-MHC expression. This difference may be important for the regulation of contraction in these vessels.

IL-7 is expressed and secreted by human skeletal muscle cells

2010 ◽  
Vol 298 (4) ◽  
pp. C807-C816 ◽  
Author(s):  
Fred Haugen ◽  
Frode Norheim ◽  
Henrik Lian ◽  
Andreas J. Wensaas ◽  
Svein Dueland ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Myosin Heavy Chain ◽  
Real Time ◽  
Satellite Cells ◽  
Heavy Chain ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Rt Pcr ◽  
Human Myotubes

In addition to generating movement, skeletal muscle may have a function as a secretory organ. The aim of the present study was to identify novel proteins with signaling capabilities secreted from skeletal muscle cells. IL-7 was detected in media conditioned by primary cultures of human myotubes differentiated from satellite cells, and concentrations increased with incubation time. By immunoblotting and real-time RT-PCR IL-7 expression was confirmed at both protein and mRNA levels. Furthermore, with immunofluorescence and specific antisera, multinucleated myotubes were found to coexpress IL-7 and myosin heavy chain. During differentiation of human myotubes from satellite cells, IL-7 expression increased at mRNA and protein levels. In contrast, mRNA expression of the IL-7 receptor was 80% lower in myotubes compared with satellite cells. Incubations with recombinant IL-7 under differentiation conditions caused ∼35% reduction in mRNA for the terminal myogenic markers myosin heavy chain 2 (MYH2) and myogenin (MYOG), suggesting that IL-7 may act on satellite cells to inhibit development of the muscle fiber phenotype. Alternative routes of cell development were investigated, and IL-7 increased migration of satellite cells by 40% after 48 h in a Transwell system, whereas cell proliferation remained unchanged. In vivo, real-time RT-PCR analysis of musculus vastus lateralis ( n = 10) and musculus trapezius ( n = 7) biopsies taken from male individuals undergoing a strength training program demonstrated that after 11 wk mean IL-7 mRNA increased by threefold ( P = 0.01) and fourfold ( P = 0.04), respectively. In conclusion, we have demonstrated that IL-7 is a novel myokine regulated both in vitro and in vivo, and it may play a role in the regulation of muscle cell development.

Time-dependent changes in myosin heavy chain mRNA and protein isoforms in unloaded soleus muscle of rat

1999 ◽  
Vol 277 (6) ◽  
pp. C1044-C1049 ◽  
Author(s):  
Laurence Stevens ◽  
Karim R. Sultan ◽  
Heidemarie Peuker ◽  
Bärbel Gohlsch ◽  
Yvonne Mounier ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
Soleus Muscle ◽  
Heavy Chain ◽  
Time Course ◽  
Mrna Level ◽  
Protein Isoforms ◽  
Time Dependent ◽  
Hindlimb Suspension ◽  
Intermediate Step ◽  
Whole Muscle

Time-dependent changes in myosin heavy chain (MHC) isoform expression were investigated in rat soleus muscle unloaded by hindlimb suspension. Changes at the mRNA level were measured by RT-PCR and correlated with changes in the pattern of MHC protein isoforms. Protein analyses of whole muscle revealed that MHCI decreased after 7 days, when MHCIIa had increased, reaching a transient maximum by 15 days. Longer periods led to inductions and progressive increases of MHCIId(x) and MHCIIb. mRNA analyses of whole muscle showed that MHCIId(x) displayed the steepest increase after 4 days and continued to rise until 28 days, the longest time period investigated. MHCIIb mRNA followed a similar time course, although at lower levels. MHCIα mRNA, present at extremely low levels in control soleus, peaked after 4 days, stayed elevated until 15 days, and then decayed. Immunohistochemistry of 15-day unloaded muscles revealed that MHCIα was present in muscle spindles but at low amounts also in extrafusal fibers. The slow-to-fast transitions thus seem to proceed in the order MHCIβ → MHCIIa → MHCIId(x) → MHCIIb. Our findings indicate that MHCIα is transiently upregulated in some fibers as an intermediate step during the transition from MHCIβ to MHCIIa.

scholarly journals Myosin heavy-chain composition in striated muscle after tenotomy

1992 ◽  
Vol 282 (1) ◽  
pp. 237-242 ◽  
Author(s):  
A Jakubiec-Puka ◽  
C Catani ◽  
U Carraro
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Striated Muscle ◽  
Slow Muscle ◽  
Fast Muscle ◽  
Muscle Fibres ◽  
Adult Rats ◽  
Tendon Regeneration ◽  
Adult Muscle ◽  
Gastrocnemius Muscles

The myosin heavy-chain (MHC) isoform pattern was studied by biochemical methods in the slow-twitch (soleus) and fast-twitch (gastrocnemius) muscles of adult rats during atrophy after tenotomy and recovery after tendon regeneration. The tenotomized slow muscle atrophied more than the tenotomized fast muscle. During the 12 days after tenotomy the total MHC content decreased by about 85% in the slow muscle, and only by about 35% in the fast muscle. In the slow muscle the ratio of MHC-1 to MHC-2A(2S) remained almost unchanged, showing that similar diminution of both isoforms occurs. In the fast muscle the MHC-2A/MHC-2B ratio decreased, showing the loss of MHC-2A mainly. After tendon regeneration, the slow muscle recovered earlier than the fast muscle. Full recovery of the muscles was not observed until up to 4 months later. The embryonic MHC, which seems to be expressed in denervated adult muscle fibres, was not detected by immunoblotting in the tenotomized muscles during either atrophy or recovery after tendon regeneration. The influence of tenotomy and denervation on expression of the MHC isoforms is compared. The results show that: (a) MHC-1 and MHC-2A(2S) are very sensitive to tenotomy, whereas MHC-2B is much less sensitive; (b) expression of the embryonic MHC in adult muscle seems to be inhibited by the intact neuromuscular junction.

scholarly journals Circulation of bovine viral diarrhea virus – 1 (BVDV-1) in dairy cattle and buffalo farms in Ismailia Province, Egypt

2015 ◽  
Vol 9 (12) ◽  
pp. 1331-1337 ◽  
Author(s):  
Mohamed Ahmed Soltan ◽  
Rebecca P Wilkes ◽  
Mohamed Nagy Elsheery ◽  
Mahmoud Mohy Elhaig ◽  
Matthhew C Riley ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Dairy Cattle ◽  
Real Time ◽  
Bovine Viral Diarrhea ◽  
Rt Pcr ◽  
Cattle Farm ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Three Samples ◽  
Pcr Products

Introduction: Bovine viral diarrhea (BVD) is one of the most economically significant diseases in the bovine industry causing losses due to diarrhea, reproductive disorders, immunosuppression and mortalities. The aim of our investigation was to detect and subtype BVDV from calves on two dairy cattle and two buffalo farms in Ismailia province, Egypt as an indicator of BVDV infection status in the province. Methodology: A total of 298 blood samples were collected and tested using an optimized one-step, real-time multiplex Taqman-based RT-PCR. All the positive samples by the multiplex real-time RT-PCR were tested using conventional RT-PCR to amplify multiple areas of the genome for further phylogenetic analysis and subtyping. Results: Thirty one (10.4%) of the tested samples were positive for BVDV-1. Only three samples, all from a single dairy cattle farm, had enough viral RNA to be amplified by RT-PCR. The PCR products were sequenced and phylogenetic analysis revealed detection of BVDV-1b. The detected strain is closely related to worldwide BVDV-1b strains, making it difficult to trace its origin. Nucleotide and amino acid alignments of the E2 glycoprotein region of the detected strain with other BVDV-1b strains showed high divergence, with identity ranging from 81.3% to 93.6% and 85.3% to 93.6%, respectively. Conclusion: To our knowledge, this is the first report describing the circulation of BVDV-1b in Egyptian dairy cattle populations.

DETECTION AND DIFFERENTIATION OF THE VACCINE STRAIN AND FIELD ISOLATES OF DUCK HEPATITIS A VIRUS TYPE 1 USING REAL-TIME RT-PCR AND HIGH RESOLUTION MELTING ASSAYS

2015 ◽  
Vol 41 (04) ◽  
pp. 229-235
Author(s):  
Kuang-Po Li ◽  
Shan-Chia Ou ◽  
Jui-Hung Shien ◽  
Poa-Chun Chang
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Virus Type ◽  
Vaccine Strain ◽  
Hepatitis A ◽  
High Resolution Melting ◽  
Hepatitis A Virus ◽  
Rt Pcr ◽  
Duck Hepatitis

Duck hepatitis A virus type 1 (DHAV-1) infection is a highly contagious and fatal disease of young ducklings. A live attenuated vaccine strain designated as 5886 has been used in Taiwan for the control of DHAV-1. Although several molecular biological methods are reported for diagnosis of DHAV-1 infection, none of them is able to discriminate between the vaccine strain and field viruses of DHAV-1. In the present study, a real-time reverse transcriptase polymerase chain reaction (RT-PCR) and high resolution melting (HRM) assay was developed for rapid detection and differentiation between the vaccine strain and field viruses of DHAV-1. This assay is highly specific for DHAV-1 and the detection limit is about 100 copies of the viral RNA. Experiments using fecal samples collected from ducklings experimentally infected with DHAV-1 showed that DHAV-1 could be detected in fecal samples as early as 6 h post-infection. In summary, a real-time RT-PCR and HRM assay is developed in this study and this assay could be valuable for diagnosis and surveillance of DHAV-1 infection in the field.

Distribution of myosin heavy chain isoforms in non-weight-bearing rat soleus muscle fibers

1996 ◽  
Vol 81 (6) ◽  
pp. 2540-2546 ◽  
Author(s):  
Robert J. Talmadge ◽  
Roland R. Roy ◽  
V. Reggie Edgerton
Keyword(s):  
Myosin Heavy Chain ◽  
Soleus Muscle ◽  
Heavy Chain ◽  
De Novo ◽  
Weight Bearing ◽  
Muscle Fibers ◽  
Myosin Heavy Chain Isoforms ◽  
Hindlimb Suspension ◽  
Type I ◽  
Rat Soleus Muscle

Talmadge, Robert J., Roland R. Roy, and V. Reggie Edgerton.Distribution of myosin heavy chain isoforms in non-weight-bearing rat soleus muscle fibers. J. Appl. Physiol. 81(6): 2540–2546, 1996.—The effects of 14 days of spaceflight (SF) or hindlimb suspension (HS) (Cosmos 2044) on myosin heavy chain (MHC) isoform content of the rat soleus muscle and single muscle fibers were determined. On the basis of electrophoretic analyses, there was a de novo synthesis of type IIx MHC but no change in either type I or IIa MHC isoform proportions after either SF or HS compared with controls. The percentage of fibers containing only type I MHC decreased by 26 and 23%, and the percentage of fibers with multiple MHCs increased from 6% in controls to 32% in HS and 34% in SF rats. Type IIx MHC was always found in combination with another MHC or combination of MHCs; i.e., no fibers contained type IIx MHC exclusively. These data suggest that the expression of the normal complement of MHC isoforms in the adult rat soleus muscle is dependent, in part, on normal weight bearing and that the absence of weight bearing induces a shift toward type IIx MHC protein expression in the preexisting type I and IIa fibers of the soleus.

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