Magnetic resonance imaging focused on the ferritin heavy chain 1 reporter gene detects neuronal differentiation in stem cells

The neuronal differentiation of mesenchymal stem cells offers a new strategy for the treatment of neurological disorders.Thus,there is a need to identify a noninvasive and sensitive in vivo imaging approach for real-time monitoring of transplanted stem cells.Our previous study confirmed that magnetic resonance imaging,with a focus on the ferritin heavy chain 1 reporter gene,could track the proliferation and differentiation of bone marrow mesenchymal stem cells that had been transduced with lentivirus carrying the ferritin heavy chain 1 reporter gene.However,we could not determine whether or when bone marrow mesenchymal stem cells had undergone neuronal differentiation based on changes in the magnetic resonance imaging signal.To solve this problem,we identified a neuron-specific enolase that can be differentially expressed before and after neuronal differentiation in stem cells.In this study,we successfully constructed a lentivirus carrying the neuron-specific enolase promoter and expressing the ferritin heavy chain 1 reporter gene;we used this lentivirus to transduce bone marrow mesenchymal stem cells.Cellular and animal studies showed that the neuron-specific enolase promoter effectively drove the expression of ferritin heavy chain 1 after neuronal differentiation of bone marrow mesenchymal stem cells;this led to intracellular accumulation of iron and corresponding changes in the magnetic resonance imaging signal.In summary,we established an innovative magnetic resonance imaging approach focused on the induction of reporter gene expression by a neuron-specific promoter.This imaging method can be used to noninvasively and sensitively detect neuronal differentiation in stem cells,which may be useful in stem cell-based therapies.

Molecular cloning and mRNA expression analysis of myosin heavy chain(MyHC)from fast skeletal muscle of grass carp,Ctenopharyngodon idella

The myosin heavy chain(MyHC)is one of the major structural and contracting proteins of muscle.We have isolated the cDNA clone encoding MyHC of the grass carp,Ctenopharyngodon idella. The sequence comprises 5 934 bp,including a 5 814 bp open reading frame encoding an amino acid sequence of 1 937 residues.The deduced amino acid sequence showed 69%homology to rabbit fast skeletal MyHC and 73%–76%homology to the MyHCs from the mandarin fish,walleye pollack,white croaker,chum salmon,and carp.The putative sequences of subfragment-1 and the light meromyosin region showed 61.4%–80%homology to the corresponding regions of other fish MyHCs.The tissue-specific and developmental stage-specific expressions of the MyHC gene were analyzed by quantitative real-time PCR.The MyHC gene showed the highest expression in the muscles compared with the kidney,spleen and intestine.Developmentally,there was a gradual increase in MyHC mRNA expression from the neural formation stage to the tail bud stage.The highest expression was detected in hatching larva.Our work on the MyHC gene from the grass carp has provided useful information for fish molecular biology and fish genomics.

Induction of Cardiomyocyte Apoptosis by Anti-Cardiac Myosin Heavy Chain Antibodies in Patients with Acute Myocardial Infarction

Autoimmune is involved in the pathogenesis of ventricular remodeling in acute myocardial infarction (AMI).In the present study, we investigated the effect of anti-cardiac myosin heavy chain antibodies (AMHCA) from patients with AMI on rat cardiomyocyte apoptosis.Cardiomyocyte apoptosis was observed and measured by DNA end labeling and Annexin-Ⅴ/PI double-staining assay.The expression of apoptosis related p53 and Bcl-2 protein and the second messenger calcium were detected respectively by Western blotting, patch clamp and confocal calcium imaging.The results showed that AMHCA was able to induce cardiomyocyte apoptosis in a dose dependent manner.Apoptosis-accelerating nucleoprotein p53 was up-regulated, while apoptosis-inhibiting cytoplasmic protein Bcl-2 was down-regulated.In parallel, cytoplasmic calcium concentration was elevated.There was no effect on L-type calcium currents.It is concluded that AMHCA in patients with AMI as a novel triggering factor can induce cardiomyocyte apoptosis, which contributes to ventricular remodeling.

Isolation and Characterization of Recombinant Variable Domain of Heavy Chain Anti-idiotypic Antibodies Specific to Aflatoxin B_1

Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain （VHH）. The recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. In particular, they might be very useful for molecular mimicry. The present study demonstrated an alpaca immunized with the F（ab＇）z fragment of anti-aflatoxin B1 mAb and developed an important anti-idiotypic （anti-ld） responses. Antigen-specific elution method was used for panning private anti-ld VHHs from the constructed alpaca VHH library. The selected VHHs were expressed, renatured, purified, and then identified by a competitive enzyme-linked immunosorbent assay （ELISA）. Our findings indicated that the VHH would be an alternative tool for haptens mimicry studies.

Effect of aerobic exercise on the contractile function of gastrocnemius myosin heavy chain

Objective To study the effect of 4-6 weeks’ treadmill training of male SD rats on the contractile function of their gastrocnemius myosin heavy chain (MHC). Methods Forty male SD rats were randomly divided into control group and training group. The treadmill training of the training group rats was incessantly performed for 4-6 weeks at an intensity of about 75% VO2max (18.5-24 m/min,gradient of 0°,each training session lasting 50 minutes,twice a day). The content of gastrocnemius MHC mRNA was tested by reverse transcription polymerase chain reaction (RT-PCR),and the changes of muscle fibre and its cross-section area (CSA) were measured using immunohistochemistry. Electric stimulation tests were used to determine the maximal tension of isometric contraction of the post-training gastrocnemius. Results ① After continuous treadmill training for 4-6 weeks,we found that the content of the total MHC,MHC Ⅰ,MHC Ⅱx,MHC Ⅱa mRNAs was 105%,105%,109% and 108% of that in the resting control group,respectively,and the MHC Ⅱb mRNA content did not change significantly. The percentage of MHC Ⅰ mRNA in the total MHC mRNA increased while that of MHC Ⅱ mRNA decreased after aerobic training. ② The slow type of fibre type Ⅰ was the main part of the MHC after training and the CSA of the muscle fibres increased simultaneously. ③ The maximal tension of isometric contraction by pulse stimulation of square wave in the training group increased significantly compared with that in the control group (P<0.01). Conclusion The findings indicate that aerobic exercise may promote an increase in the contractile function of MHC.

Cloning, sequencing and analyzing of the heavy chain V region genes of human polyreactive antibodies

The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction (PCR) technique. We found that 2 and 3 mAbs utilized genes of the VHIV and VHIII families, respectively. The former 2 VH segments were in germline configuration. A common VH segment, with the best similarity of 90.1 % to the published VHIII germline genes, was utilized by 2 different rearranged genes encoding the V regions of other 3 mAbs. This strongly suggests that the common VH segment is a unmutated copy of an unidentified germline VHIII gene. All these polyreactive mAbs displayed a large NDN region (VH-D-JH junction). The entire H chain V regions of these polyreactive mAbs are unusually basic. The analysis of the charge properties of these mAbs as well as those of other poly- and mono- reactive mAbs from literatures prompts us to propose that the charged amino acids with a particular distribution along the H chain V region,especially the binding sites (CDRs), may be an important structural feature involved in antibody polyreactivity.

Alterations in myosin heavy chain isoform gene expression during the transition from compensatory hypertrophy to congestive heart failure in rats

Objective To explore the molecular mechanism underlying the decreased velocity of tension rise in rat myocardium during congestive heart failure (CHF) and left ventricular hypertrophy (LVH) induced by aortic stenosis.Methods The maximum velocity of tension rise (+dT/dtmax) was measured in left ventricular papillary muscle and the mRNA level of myosin heavy chain (MHC) isoforms in the left ventricle were detected by Northern blot analysis.Results The value of +dT/dtmax in CHF and LVH group were 64.17% and 37.15% lower than sham-operated controls (Sham) (P<0.01); values in the CHF group were 42.99% lower than that of LVH (P<0.01). The level of α-MHC mRNA in LVH was not different from that of the Sham (P>0.05), but decreased significantly in CHF to 42.3% of Sham and 56.1% of LVH (P<0.01). The level of β-MHC mRNA was up-regulated by 88.3% (P<0.01) in LVH compared with Sham and the level of β-MHC in CHF was 1.5-fold and 3.7-fold higher than that in LVH and Sham respectively (P<0.01). The ratio of α-MHC/β-MHC mRNA in LVH and CHF decreased to 42.4% and 9.8% respectively of the value in Sham (P<0.01). Correlation between α-MHC/β-MHC mRNA level and +dT/dtmax was analyzed which showed that these values were positively correlated with a correlation coefficient of 0.875 (P<0.01).Conclusion The decreased ratio of α-MHC/β-MHC mRNA was the major molecular mechanism underlying the decreased +dT/dtmax in CHF and LVH myocardium. The decreased ratio of α-MHC/β-MHC mRNA in LVH was mainly due to the up regulation of β-MHC mRNA while in CHF both down regulation of α-MHC and up regulation of β-MHC were involved.

Generation of cytotoxic T lymphocytes specific for B-cell acute lymphoblastic leukemia family-shared peptides derived from immunoglobulin heavy chain framework region

Background Immunoglobulin heavy chain variable region （IgHV） is a well-characterized tumor antigen for B-cell malignancies. It can function as a target for T cell-mediated immune response. Clinical trials of IgHV protein vaccines against lymphoma have demonstrated induction of tumor-specific cytotoxic T lymphocyte （CTL） responses. However, complementary determining regions-based individual vaccines have disadvantages for wide clinical application. Although a recent study demonstrated that immunogenic peptides are derived from framework regions （FR） shared among patients with B-cell lymphoma, how to choose the appropriate peptides for each patient is still unsolved. The aim of this study was to investigate whether immunoglobulin heavy chain FR-derived peptides shared in each IgHV family are potential CTL epitopes presented by B-cell acute lymphoblastic leukemia （B-ALL）. Such CTL epitopes might be beneficial to shifting vaccination strategies against B-ALL from individual specificity to family specificity.Methods Seven IgHV gene families were amplified respectively by PCR and sequenced directly from 71 childhood B-ALL cases. Bioinformatics was applied in analyzing characteristics of sequences available and predicting HLA-A^＊0201-restricted CTL epitopes for each IgHV family. An antigen-specific T cell expansion system was used to generate peptide-specific CTLs. The cytotoxicity of CTLs against B-ALL cells was assessed in the lactate dehydrogenase release assay. Results Complete IgHV rearrangements were identified in all of the 71 B-ALL cases. All of 40 sequences available showed ≥98% homology with the nearest germline IgHV genes, indicating IgHV genes in B-ALL of germline nature. Twelve nonapeptides of high HLA-A^＊0201-binding scores were obtained from 26 productive IgHV protein sequences. Ten （83%） of the peptides were located in FR1 and FR3 shared among the corresponding IgHV family. CTLs specific for the peptide QLVQSGAEV located in FR1 （3-11） shared among the IgHV1 family could be successfully generated from peripheral blood mononuclear cells of two HLA-A^＊0201＋ healthy donors in vitro and were capable of killing HLA-matched B-ALL cell clones belonging to the IgHV1 family. Conclusion Anti-B-ALL CTLs against immunoglobulin heavy chain FR-derived peptides have family-specific cytotoxicity.

Influence of injection of Chinese botulinum toxin type A on the histomorphology and myosin heavy chain composition of rat gastrocnemius muscles

Background and objective:Botulinum toxin type A(BoNT/A)is a metalloprotease that blocks synaptic transmission via the cleavage of a synaptosomal-associated protein of 25 kDa(SNAP-25).It has gained widespread use as a treatment for cerebral palsy and skeletal muscle hypertrophy.In China,Chinese botulinum toxin type A(CBTX-A),a type of BoNT/A,is in widespread clinical use.However,the changes in the morphological and biochemical properties of treated muscles and in remote muscles from the CBTX-A injection site are relatively unknown.Therefore,we investigated the changes in histomorphology and myosin heavy chain(MyHC)isoform composition and distribution in rat gastrocnemius muscles after intramuscular injection of CBTX-A.Methods:The weakness of the injected muscles was assessed periodically to identify their functional deficiency.Muscle slices were stained with hematoxylin-eosin(HE)and adenosine triphosphatase(ATPase).MyHC isoform composition was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis(SDS-PAGE)to uncover changes in morphological and biochemical properties.Results:Our findings demonstrate that following injection of CBTX-A 5 U into rat gastrocnemius muscles,shifts in MyHC isoform composition emerged on the third day after injection and peaked in the fourth week.The composition remained distinctly different from that of the control group after the twelfth week.More specifically,there was a decrease in the proportion of the type IIb isoform and an increase in the proportions of type IIx,type IIa,and type I isoforms.Conclusions:Data revealed that CBTX-A led to a shift in MyHC composition towards slower isoforms and that the MyHC composition remained far from normal six months after a single injection.However,no noticeable remote muscle weakness was induced.

Clathrin heavy chain is essential for the development and reproduction of Locusta migratoria

Clathrin heavy chain(Chc)is a constituent of clathrin-coated vesicles and serves important functions in endocytosis and intracellular membrane trafficking but ap-pears to have physiological roles also at the organismal level.Most of what we know about Chc functions originates from studies performed in fungal or vertebrate cells.How-ever,the physiological functions of Chc in insects remain poorly understood.Here,we identified a Chc ortholog from a Locusta migratoria transcriptome database.RT-qPCR revealed that LmChc was constitutively expressed in fifth-instar nymphs.In this develop-mental stage,LmChc showed the highest expression in the ovary followed by hemolymph,testis,hindgut,midgut,and foregut.In isolated hemocytes,we detected the Chc protein in patches at the plasma membrane.To examine the role of LmChc in L.migratoria during development,RNA interference was performed by injecting dsRNA into the early fifth-instar nymphs.Silencing of LmChc caused a lethal phenotype with molting defect from nymph to adult.In addition,silencing of LmChc resulted in abnormal development of the ovaries,the size of which was significantly smaller than that in controls.Taken together,our results suggest that LmChc is a vital gene in L.migratoria that plays an important role in growth,development,and reproduction.LmChc may be used as an efficient RNAi target gene for developing dsRNA-based biological insecticides to manage insect pests.

Mutation of Arg723Gly in β-myosin heavy chain gene in five Chinese families with hypertrophic cardiomyopathy

Background Hypertrophic cardiomyopathy （HCM） is a form of cardiomyopathy with an autosomal dominant inherited disease, which is caused by mutations in at least one of the sarcomeric protein genes. Mutations in the beta-myosin heavy chain （β-MHC） are the most common cause of HCM. This study was to reveal the disease-causing gene mutations in Chinese population with HCM, and to analyze the correlation between the genotype and phenotype. Methods The exons 3 to 26 of MYH7 were amplified by PCR, and the PCR products were sequenced in five non-kin HCM patients. A 17-year-old patient was detected to be an Arg723Gly mutation carrier. Then his family was gene-screened, and the correlation between genotype and phenotype was analyzed. Results The mutation of Arg723Gly in a Chinese family with HCM was detected for the first time. With a C-G transversion in nucleotide 13 619 of the MYH7 gene, located at the essential light chain interacting region in S1, the replacement of arginine by glycine took place at amino acid residue 723. A two-dimensional echocardiogram showed moderate asymmetrical septal hypertrophy with left atria enlargement. There was no obstruction in the left ventricular outflow tract. In his family, a total of 13 individuals were diagnosed HCM and 5 of them were dead of congestive heart failure at a mean age of 66-year-old. Eight living members were all detected to carry the mutation, in which 3 developed progressive heart failure. Moreover, the heart function of the people evidently deteriorates when their age are older than 50. The mutation and the disease show co-separated. Conclusion The Arg723Gly mutation is a malignant type. In Chinese the mutation has the similar characters to the former report but has low degree malignant.

Congophilic fibrils in the glomeruli with polyclonal immunoglobulin gamma staining-another cause for diagnostic overlap:A case report

BACKGROUND Glomerulopathy with fibrillary deposits is not uncommon in routine nephropathology practice,with amyloidosis and fibrillary glomerulonephritis being the two most frequently encountered entities.Renal amyloid heavy and light chain(AHL)is relatively uncommon and its biopsy diagnosis is usually limited to cases that show strong equivalent staining for a single immunoglobulin(Ig)heavy chain and a single light chain,further supported by mass spectrometry(MS)and serum studies for monoclonal protein.But polyclonal light chain staining can pose a challenge.CASE SUMMARY Herein we present a challenging case of renal AHL with polyclonal and polytypic Ig gamma(IgG)staining pattern by immunofluorescence.The patient is a 62-yearold Caucasian male who presented to an outside institution with a serum creatinine of up to 8.1 mg/dL and nephrotic range proteinuria.Despite the finding of a polyclonal and polytypic staining pattern on immunofluorescence,ultrastructural study of the renal biopsy demonstrated the presence of fibrils with a mean diameter of 10 nm.Congo red was positive while DNAJB9 was negative.MS suggested a diagnosis of amyloid AHL type with IgG and lambda,but kappa light chains were also present supporting the immunofluorescence staining results.Serum immunofixation studies demonstrated IgG lambda monoclonal spike.The patient was started on chemotherapy.The chronic renal injury however was quite advanced and he ended up needing dialysis shortly after.CONCLUSION Tissue diagnosis of AHL amyloid can be tricky.Thorough confirmation using other available diagnostic techniques is recommended in such cases.

Detection of BCL2-IGHrearrangement on paraffin-embedded tissue sections obtained from a small submucosal tumor of the rectum in a patient with recurrent follicular lymphoma

A 59-year-old woman was admitted to our hospital because of recurrent follicular lymphoma(FL).Colonoscopic examination revealed a rectal submucosal tumor(SMT)without any erosions and ulcers.In this patient,it was difficult to distinguish non-Hodgkin's lymphoma(NHL)invasion from other disorders of the colon including carcinoid tumor merely based on endoscopic findings.Histopathologic and immunohistochemical studies on biopsy specimens showed an infiltration of atypical lymphocytes that were positive for CD20 and BCL2 but negative for UCHL-1.Fluorescence in situ hybridization on paraffin-embedded tissue sections (T-FISH)identified a translocation of BCL2 with IGHgene. Based on these findings,the tumor was defined as an invasion of FL.T-FISH method is useful for the detection of a monoclonality of atypical lymphocytes in an SMT of the gastrointestinal tract,and particularly for the detection of chromosomal translocations specific to lymphoma subtypes.

Proteomic identification of tumor biomarkers associated with primary gallbladder cancer

AIM: To identify potential biomarkers of primary gallbladder cancer (PGC).

Identification of deregulated miRNAs and their targets in hepatitis B virus-associated hepatocellular carcinoma

AIM： TO identify the differentially expressed miRNAs and their targets in hepatitis B virus （HBV）-associated hepatocellular carcinoma （HCC）. METHODS： Six hundred and sixty seven human miRNAs were quantitatively analyzed by Taqman lowdensity miRNA array （TLDA） in HBV-HCC tissues. Gene ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway analyses were used to analyze the significant function and pathway of the differentially expressed miRNAs in HBV-HCC. TargetScan software was used to predict the targets of deregulated miRNAs. Western blotting and luciferase assay were performed to verify the targets of these miRNAs.RESULTS： Ten up-regulated miRNAs （miR-217, miR- 518b, miR-517c, miR-520g, miR-519a, miR-522, miR- 518e, miR-525-3p, miR-512-3p, and miR-518a-3p） and 11 down-regulated miRNAs （miR-138, miR-214, miR-214#, miR-199a-5p, miR-433, miR-511, miR-592, miR-483-3p, miR-483-5p, miR-708 and miR-1275） were identified by Taqman miRNAs array and confirmed quantitatively by reverse transcription polymerase chain reaction in HCC and adjacent non-tumor tissues. GO and KEGG pathway analysis revealed that ＂regulation of actin cytoskeleton＂ and ＂pathway in cancer＂ are most likely to play critical roles in HCC tumorigenesis. MiR- 519a and ribosomal protein S6 kinase polypeptide 3 （RPS6KA3） were predicted as the most significant can-didates by miRNA-mRNA network. In addition, cyclin D3 （CCND3） and clathrin heavy chain （CHC）, usually up-regulated in HCC tissues, were validated as the di- rect target of miR-138 and miR-199a-5p, respectively.

Roles of syndecan-1, bcl6 and p53 in diagnosis and prognostication of immunoproliferative small intestinal disease

AIM： To evaluate roles of syndecan-1, bcl6 and p53 in diagnosis and prognostication of immunoproliferative small intestinal disease （IPSID） and to study profiles of kappa （κ） and lambda （λ） light chains and IgA heavy chain. METHODS： The study consisted of 11 cases of IPSID and similar number of controls which included 11 of normal intestinal mucosa and 11 of high grade B cell lymphoma of ileum. The parameters analyzed included clinical profiles, biochemical and other laboratory investigations, radiologic and histological findings including immunohistochemistry. RESULTS： All IPSID cases had demonstrable serum IgA heavy chain and heavy mucosal plasma cell infiltration. According to Galian＇s histological staging, there were 4 patients with stage A and 7 with stage B. κ and ;λ light chains were over-expressed in 7 patients; 1 stage A patient had H pylori-positive active gastritis and eradication of H pylori led to disease remission. Stage A biopsies had higher expression for syndecan-1, while stage B had higher expression for bcl6 and p53. Syndecan-1,κ and λ light chains and IgA heavy chain showed inverse relationship with bcl6 and p53. All patients were treated with doxycycline. CHOP regime was added in 5 patients who developed frank lymphoma. Three died of the disease due to extensive organ infiltration. CONCLUSION： Certain immunomarkers like syndecan-1,κ and λ light chains and IgA heavy chain could be of much help in identifying early stage IPSID. Stage B IPSID showed higher expression for bcl6 and p53 than stage A IPSID. bcl6 and p53 expressions correlated with a more advanced disease stage and aggressive tumour behavior.

Current application of neurofilaments in amyotrophic lateral sclerosis and future perspectives

Motor neuron disease includes a heterogeneous group of relentless progressive neurological disorders defined and characterized by the degeneration of motor neurons.Amyotrophic lateral sclerosis is the most common and aggressive form of motor neuron disease with no effective treatment so far.Unfortunately,diagnostic and prognostic biomarkers are lacking in clinical practice.Neurofilaments are fundamental structural components of the axons and neurofilament light chain and phosphorylated neurofilament heavy chain can be measured in both cerebrospinal fluid and serum.Neurofilament light chain and phosphorylated neurofilament heavy chain levels are elevated in amyotrophic lateral sclerosis,reflecting the extensive damage of motor neurons and axons.Hence,neurofilaments are now increasingly recognized as the most promising candidate biomarker in amyotrophic lateral sclerosis.The potential usefulness of neurofilaments regards various aspects,including diagnosis,prognosis,patient stratification in clinical trials and evaluation of treatment response.In this review paper,we review the body of literature about neurofilaments measurement in amyotrophic lateral sclerosis.We also discuss the open issues concerning the use of neurofilaments clinical practice,as no overall guideline exists to date;finally,we address the most recent evidence and future perspectives.

A novel triple immunoenzyme staining enables simultaneous identification of all muscle fiber types on a single skeletal muscle cryosection from normal, denervated or reinnervated rats

Triple immunofluorescence staining has recently been developed to simultaneously identify all muscle fibers on a single cryosection which is helpful for clinical and basic research, but it has disadvantages such as fast photobleaching and unclear outlines of muscle fibers. Triple immunoenzyme staining（TIE） is likely to avoid these disadvantages. In this study, we aimed to establish a sensitive and specific TIE technique to identify fiber types in normal, denervated, and reinnervated rat muscles, and to develop a systematic sampling method for muscle fiber quantification. Tibialis anterior and soleus from normal, denervated, and reinnervated Lewis rat hind limbs were used. Five consecutive cryosections were cut from each muscle, including one for TIE and four for single immunoenzyme staining（SIE）. The TIE was performed using the polymerized reporter enzyme staining system for the first two antigens（A4.74 for My HC-IIA, BA-F8 for My HC-I） and alkaline phosphatase staining system for the third antigen（BF-F3 for My HC-IIB）, followed by corresponding detective systems and respective chromogens. The type of muscle fibers was quantified by systematic sampling at 12.5%, 25%, 33% and 50% of all muscle fibers, and was compared with that acquired from counting all the fibers（100%）. All muscle fiber phenotypes, including pure and hybrid, could be simultaneously identified on a single TIE cryosection with clear outlines. The fiber types on TIE slides matched well with their respective counterpart on the consecutive SIE slides with a 95% match rate. Systematic sampling of 12.5% fibers could represent the true fiber type distribution of the entire muscle section. Our results suggest that novel TIE can effectively visualize fiber types in normal, denervated or reinnervated rat muscles.

Restricted nutrient intake does not alter serum-mediated measures of implant response in cell culture

Background： During nutritional stress, reduced intake may reduce the efficacy of anabolic implants. This study was conducted to evaluate basic cellular responses to a growth promotant implant at two intake levels. Methods： Sixteen crossbred steers （293 ± 19.3 kg） were used to evaluate the impact of anabolic implants in either an adequate or a restricted nutritional state. Steers were trained to individual Calan gates, and then randomly assigned to 1 of 4 treatments in a 2 × 2 factorial arrangement. Treatments consisted off presence or absence of an anabolic growth implant （Revalor-XS, 200 mg TBA and 40 mg estradiol; IMPLANT or CONTROL） and a moderate energy, pelleted, starting cattle diet fed at either 2.0 × or 1.0 × maintenance energy （NEM） requirements （HIGH or LOW）. Serum （d O, 14, and 28） was used for application to bovine muscle satellite cells. After treatment with the serum （20% of total media） from the trial cattle, the satellite cells were incubated for 72 h. Protein abundance of myosin heavy chain （MHC）, phosphorylated extracellular signal-related kinase （phospho-ERK）, and phosphorylated mammalian target of rapamycin （phospho-mTOR） were analyzed to determine the effects of implant, intake, and their interaction （applied via the serum）. Results： Intake had no effect on MHC （P = 0.85） but IMPLANT increased （P 〈 0.01） MHC abundance vs. CONTROL. Implant status, intake status, and the interaction had no effect on the abundance of phospho-ERK （P〉0.23）. Implanting increased phospho-mTOR （P 〈 0.01） but there was no effect （P 〉 0.51） of intake or intake x implant. Conclusions： The nearly complete lack of interaction between implant and nutritional status indicates that the signaling molecules measured herein respond to implants and nutritional status independently. Furthermore, results suggest that the muscle hypertrophic effects of anabolic implants may not be mediated by circulating IGF-1.

Studies on the role of microtubules in myofibrillogenesis

Co-localization of mierotubule (MT) and muscle myosin (MHO) myofibril immunofluoresoence in developing myotubes of chicken' skeletal muscle cultures was observed by using double staining of tubulin and MHO indirect immunofluorescence. 12-o-tetradeeanoyl-phorbol-13-acetate (TPA) selectively and reversibly blocks mycfibrillogenesis and alters the morphology of myotubes into myosacs where MTs are present in radiating pattern,when the arrested myogenic cells recover and start myofibrillogenesis after released from TPA, prior to the emergence of myofibrils, the pre-existing MTs become bipolarly aligned ooincidently with the tubular restoration of cell shape. Single nascent myofibrils overlapping with MTs extend into the base of growth tips where MTs go farther to the end of the tips. That MT might act as scaffold in guiding the bipolar elongation of the growing myofibrils was suggested. Taxol and ooloemid disturbed MT polymerization and disposition, and interfered with the normal spatial assembly of myofibrils in developing myotubes.