[Mechanism of intestinal injury induced by WNT2B high-expressed fibroblasts in Crohn's disease].

Objective: To explore the mechanism of intestinal tissue damage induced by macrophages activated by WNT2B high-expressed fibroblasts. Methods: This study involved biological information analysis, pathological tissue research and cell experimental research. The biological information of the colon tissue from the children with inflammatory bowel disease in previous study was analyzed again with single-cell sequencing. The pathological tissues were collected by colonoscopy from 10 children with Crohn's disease treated in the Department of Gastroenterology of Guangzhou Women and Children's Medical Center from July 2022 to September 2022. According to the findings of colonoscopy, tissues with obvious inflammation or ulceration were classified as the inflammatory group, while tissues with slight inflammation and no ulceration were classified as the non-inflammatory group. HE staining was performed to observe the pathological changes of the colon tissues. Macrophage infiltration and CXCL12 expression were detected by immunofluorescence. In terms of cell experiments, fibroblasts transfected with WNT2B plasmid or empty plasmid were co-cultured with salinomycin treated or non-treated macrophages, respectively; the expression of proteins through Wnt classical pathway were detected by western blotting. Macrophages treated with SKL2001 were used as the experimental group, and those with phosphate buffer as the control group. The expression and secretion of CXCL12 in macrophages were detected by quantitative Real-time PCR and enzyme-linked immunosorbent assay (ELISA). T-test or rank sum test were used for the comparison between groups. Results: Single-cell sequencing analysis suggested that macrophages were the main cells in inflammatory bowel disease colon tissue, and there was interaction between WNT2B high-expressed fibroblasts and macrophages. HE staining of the 10 patients ((9.3±3.8) years old, 7 males and 3 females) showed that the pathological score of colon tissue in the inflammatory group was higher than that in the non-inflammatory group (4 (3, 4) vs. 2 (1, 2) points, Z=3.05, P=0.002). Tissue immunofluorescence indicated that the number of infiltrating macrophages in the inflammatory group was significantly higher than that in the non-inflammatory group under high power field of view (72.8±10.4 vs.8.4±3.5, t=25.10, P<0.001), as well as the number of cells expressing CXCL12 (14.0±3.5 vs. 4.7±1.9, t=14.68, P<0.001). In cell experiments, western blotting suggested an elevated level of glycogen synthase kinase-3ß phosphorylation in macrophages co-cultured with fibroblast transfected with WNT2B plasmid, and salinmycin could reverse this change. Real-time PCR suggested that the transcription level of CXCL12 in the experimental group was higher than that in the control group (6.42±0.04 vs. 1.00±0.03, t=183.00, P<0.001), as well as the expression and secretion of CXCL12 by ELISA ((465±34) vs. (77±9) ng/L, t=13.21, P=0.006). Conclusion: WNT2B high-expressed fibroblasts can secrete WNT2B protein and activate the Wnt classical signaling pathway thus enhancing the expression and secretion of CXCL12 in macrophages, inducing the development of intestinal inflammation of Crohn's disease.

[MicroRNA-23a knockdown attenuates angiotensin â ¡ induced hypertrophy in rat H9c2 cells via activating PTEN and AMPK pathway].

Objective: To investigate if microRNA (miR) -23a knockdown could attenuate angiotensin Ⅱ(AngⅡ) induced cardiac hypertrophy by activating phosphatase and tensin homolog deleted on chromosome ten(PTEN) and AMP-activated protein kinase(AMPK) pathway. Methods: Rat H9c2 cells were cultured in DMEM high glucose medium and put in 5% CO(2) incubator at 37 ℃(normal group). After 48 hours of culture, H9c2 cells were stimulated with 10 nmol/L AngⅡ to establish cell hypertrophy model (AngⅡgroup). The H9c2 cells were inoculated in a 6-well cell culture plate and cultured in an incubator at 37 ℃. When the confluence degree of cell growth was about 70%, the cells were transfected with different reagents, and 24 hours after transfection, 10 nmol/L AngⅡ was used to interfere with the cells. The H9c2 cells were divided into different groups according to the reagents, namely AngⅡ+anti-miR group(transfected with miR-23a inhibitor), Ang Ⅱ+NC group(transfected with miR-23a inhibitor negative control), Ang Ⅱ+anti-miR+si-PTEN group(cotransfected with miR-23a inhibitor and PTEN small interference RNA(siRNA)), and AngⅡ+anti-miR+si-NC group(cotransfected with miR-23a inhibitor and PTEN siRNA negative control). The surface area of single cell was measured by Image J software.The mRNA expression levels of α-actin 1 (ACTA1) and ß-myosin heavy chain (ß-MHC) and miR-23a were detected by quantitative real-time PCR(qRT-PCR). The expression levels of PTEN and AMPK signal pathway related proteins were detected by Western blot. In order to verify whether miR-23a targets PTEN gene, double luciferase reporter gene experiment was performed. The luciferase reporter gene vector recombinant plasmids of wild type pGL-WT-PTEN and mutant pGL-MUT-PTEN were constructed and prepared after normal sequencing. H9c2 cells was inoculated into 24-well cell culture plate and cultured overnight in 37 ℃ incubator. The cells were co-transfected with miR-23a mimic or miR-23a mimic negative control and wild type or mutant reporter gene recombinant plasmid. Forty-eight hours after transfection, firefly luciferase activity and sea kidney luciferase activity were measured, and the ratio of them was recorded as relative luciferase activity. Results: Compared with the normal group, the cell surface area, the mRNA expression levels of ACTA1, ß-MHC and miR-23a were significantly higher, while the protein expression levels of PTEN and p-AMPK were significantly lower in the Ang Ⅱ group(all P<0.05). The results of double luciferase reporter gene assay showed that the relative luciferase activity of cells co-transfected with miR-23a mimic and wild-type reporter gene recombinant plasmid was lower than that of miR-23a mimic negative control (P<0.05), and PTEN served as the target gene of miR-23a. In AngⅡ+anti-miR group the mRNA expression levels of miR-23a, ACTA1 and ß-MHC were lower, and the cell surface area was smaller, while the protein expression levels of PTEN and p-AMPK were higher than that in AngⅡ group and AngⅡ+NC group(all P<0.05). Compared with AngⅡ+anti-miR group, the cell surface area was bigger, the expression of ACTA1 and ß-MHC mRNA was up-regulated, and the protein expression levels of PTEN and p-AMPK were down-regulated in Ang Ⅱ+anti-miR+si-PTEN group(all P<0.05). Conclusion: Inhibition of miR-23a can attenuate Ang Ⅱ-induced hypertrophy in H9c2 cells through targeting PTEN and activating AMPK signaling pathway.

Knockdown of HMGB2 inhibits proliferation and invasion of renal tumor cells via the p-38MAPK pathway.

OBJECTIVE: To investigate the function of HMGB2 in renal tumor ACHN cells in vitro and in vivo and to study the underlying molecular mechanisms. PATIENTS AND METHODS: Kaplan-Meier analysis was used to study the relationship between expression of HMGB2 and prognosis of renal tumor. MTT assay was employed to examine cell proliferation and flow cytometry analysis was used to study the role of HMGB2 in cell apoptosis in ACHN cells. Transwell assays were used to explore the migration and invasion of ACHN cells. The effect of HMGB2 on tumor growth was investigated in vivo. Western blot was performed to evaluate the expression levels of p-JNK, p-ERK and p-p38MAPK. RESULTS: HMGB2 was upregulated in renal tumor and correlated with worse overall survival in renal tumor patients. Down-regulation of HMGB2 suppressed ACHN cells proliferation, invasion and migration in vitro. Moreover, down-regulation of HMGB2 inhibited tumor growth in vivo and HMGB2 exerts the oncogene function partly via the inhibition of p-p38MAPK activation. CONCLUSIONS: Our results provide novel insights into neuropathic pain and help to explore therapeutic targets in the treatment.

[Human papillomavirus infection and vaccination].

Human papillomavirus (HPV) is an infection that can be sexually transmitted and result in health consequences. Persistent high-risk HPV infection can lead to various cancers and is the essential cause of cervical cancer. HPV vaccine can prevent the HPV infection and thus the incidence of cervical cancer. In this review we introduced the prevalence of HPV infection and vaccination, and the prevention and early detection of cervical cancer. We also introduced the present knowledge and awareness of HPV infection and HPV vaccine in Chinese. Propaganda all over China should be performed on HPV vaccination to improve the vaccination rate, thus preventing the incidence of cervical cancer.

Long noncoding RNA GIHCG is a potential diagnostic and prognostic biomarker and therapeutic target for renal cell carcinoma.

OBJECTIVE: Long noncoding RNA (lncRNA) GIHCG has been reported as an oncogene in hepatocellular carcinoma. However, the expression, roles, and clinical values of GIHCG in renal cell carcinoma (RCC) remain unclear. The aim of this study was to investigate the expression, roles, diagnostic and prognostic values of GIHCG in RCC. PATIENTS AND METHODS: The expression of GIHCG in 46 pairs of RCC tissues and adjacent normal renal tissues was measured by quantitative real-time polymerase chain reaction (qRT-PCR). GIHCG serum level in 46 RCC patients, 46 age- and sex-matched healthy controls, 20 pre- and post-surgery RCC patients was measured by qRT-PCR. The diagnostic values of serum GIHCG were evaluated by receiver operating characteristic (ROC) curves analysis. The effect of GIHCG on RCC cell proliferation was evaluated using Cell Count Kit-8 assay, and the effect of GIHCG on RCC cell migration was evaluated using transwell migration assay. RESULTS: GIHCG is upregulated in RCC tissues compared with adjacent normal renal tissues. Increased expression of GIHCG is positively correlated with advanced TNM stages, Fuhrman grades, and poor prognosis. Serum GIHCG level is also significantly upregulated in RCC patients and correlated with advanced TNM stages. Serum GIHCG could accurately discriminate RCC patients from healthy controls, and also early stage RCC patients from healthy controls. Furthermore, serum GIHCG level is positively correlated with GIHCG expression in RCC tissues. Serum GIHCG level is significantly reduced after radical resection of RCC. Functional assays showed that knockdown of GIHCG significantly represses proliferation and migration of RCC cells. CONCLUSIONS: Long noncoding RNA GIHCG would sever as a novel diagnostic and prognostic biomarker and therapeutic target for RCC.

Genetic characterization of cysteine-rich type-b avenin-like protein coding genes in common wheat.

The wheat avenin-like proteins (ALP) are considered atypical gluten constituents and have shown positive effects on dough properties revealed using a transgenic approach. However, to date the genetic architecture of ALP genes is unclear, making it impossible to be utilized in wheat breeding. In the current study, three genes of type-b ALPs were identified and mapped to chromosomes 7AS, 4AL and 7DS. The coding gene sequence of both TaALP-7A and TaALP-7D was 855 bp long, encoding two identical homologous 284 amino acid long proteins. TaALP-4A was 858 bp long, encoding a 285 amino acid protein variant. Three alleles were identified for TaALP-7A and four for TaALP-4A. TaALP-7A alleles were of two types: type-1, which includes TaALP-7A1 andTaALP-7A2, encodes mature proteins, while type-2, represented byTaALP-7A3, contains a stop codon in the coding region and thus does not encode a mature protein. Dough quality testing of 102 wheat cultivars established a highly significant association of the type-1 TaALP-7A allele with better wheat processing quality. This allelic effects were confirmed among a range of commercial wheat cultivars. Our research makes the ALP be the first of such genetic variation source that can be readily utilized in wheat breeding.

PGC-related gene variants and elite endurance athletic status in a Chinese cohort: a functional study.

This study aims to examine the association between proliferator-activated receptor γ (PGC)-gene family-related single nucleotide polymorphisms (SNPs) and elite endurance runners' status in a Chinese cohort, and to gain insights into the functionality of a subset of SNPs. Genotype distributions of 133 SNPs in PPARGC1A, PPARGC1B, PPRC1, TFAM, TFB1M, TFB2M, NRF1, GABPA, GABPB1, ERRα, and SIRT1 genes were compared between 235 elite Chinese (Han) endurance runners (127 women) and 504 healthy non-athletic controls (237 women). Luciferase gene reporter activity was determined in 20 SNPs. After adjusting for multiple comparisons (in which threshold P-value was set at 0.00041), no significant differences were found in allele/genotype frequencies between athletes and controls (when both sexes were analyzed either together or separately). The lowest P-value was found in PPARGC1A rs4697425 (P = 0.001 for the comparison of allele frequencies between elite female endurance runners and their gender-matched controls). However, no association (all P > 0.05) was observed for this SNP in a replication cohort from Poland (194 endurance athletes and 190 controls). Using functional genomics tool, the following SNPs were found to have functional significance: PPARGC1A rs6821591, rs12650562, rs12374310, rs4697425, rs13113110, and rs4452416; PPARGC1B rs251466 and rs17110586; and PPRC1 rs17114388 (all P < 0.001). This study found no significant association between PGC-related SNPs and elite endurance athlete status in the Chinese population, despite some SNPs showing potential functional significance and the strong biological rationale to hypothesize that this gene pathway is a candidate to influence endurance exercise capacity.

Critical role of TRPC6 channels in G2 phase transition and the development of human oesophageal cancer.

BACKGROUND: Oesophageal squamous cell carcinoma (OSCC) is one of the leading causes of cancer-related death worldwide. However, the mechanism by which the OSCC develops remains largely unknown. Ion channels are important for cancer development. Whether the transient receptor potential canonical (TRPC), known as the non-selective cation channels, plays a role in OSCC development is unknown. METHODS: The expression of TRPC6, a member of TRPC subfamily, was examined in samples from patients with OSCC by immunostaining and in situ hybridisation. The effects of TRPC6 channels on OSCC cell cycle progression, cell growth and in vivo tumour formation were investigated. The functional TRPC6 channels were found in OSCC cells by electrophysiology and Ca(2+) imaging analysis. RESULTS: The expression of TRPC6 at protein and mRNA levels was markedly increased in human OSCC specimens than that in normal human oesophageal tissues. Blockade of TRPC6 channels in human OSCC cells inhibited elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) and activation of Cdc2 kinase. Meanwhile, the OSCC cell cycle was arrested at G2 phase and the cell growth was suppressed. Furthermore, inhibition of TRPC6 channels suppressed in nude mice the tumour formation generated by injection of the OSCC cells. CONCLUSION: TRPC6 channels play a critical role in the development of OSCC. The [Ca(2+)](i) elevation regulated by TRPC6 channels is essential for G2 phase progression and OSCC development. These channels might be a novel target for therapeutic intervention of OSCC.

Recombinant human erythropoietin administration protects cortical neurons from traumatic brain injury in rats.

We explored the regulation of erythropoietin and erythropoietin receptor on traumatic brain injury (TBI), as well as the antiapoptotic effects of recombinant human erythropoietin (rhEPO) treatment. Female Wistar rats were randomly divided into three groups: rhEPO-treated TBI, vehicle-treated TBI, and sham-operated. TBI was induced by the Feeney free falling model. Rats were killed 5, 12, 24, 72, 120, or 168 h after TBI. Regulation of EPO, EPOR and Bcl-2 was detected by reverse transcription-polymerase chain reaction (RT-PCR), western blotting and immunofluorescence. Terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling (TUNEL) was used to assess DNA fragmentation after TBI. Induction of EPOR expression persisted for 168 h after TBI, whereas EPO was only slightly elevated for 72 h. In the rhEPO-treated TBI, Bcl-2 mRNA and protein levels were greater than in the vehicle-treated TBI. Bcl-2 mRNA peaked at 24 h and remained stable for 72-120 h. The number of TUNEL-positive cells in the rhEPO-treated TBI was far fewer than in the vehicle-treated TBI. EPOR regulation is enhanced for almost a week after TBI. Administration of rhEPO protects neurons by enhancing Bcl-2 expression, thereby inhibiting TBI-induced neuronal apoptosis.

Allelic variation of polyphenol oxidase (PPO) genes located on chromosomes 2A and 2D and development of functional markers for the PPO genes in common wheat.

Polyphenol oxidase (PPO) activity is highly related to the undesirable browning of wheat-based end products, especially Asian noodles. Characterization of PPO genes and the development of their functional markers are of great importance for marker-assisted selection in wheat breeding. In the present study, complete genomic DNA sequences of two PPO genes, one each located on chromosomes 2A and 2D and their allelic variants were characterized by means of in silico cloning and experimental validation. Sequences were aligned at both DNA and protein levels. Two haplotypes on chromosome 2D showed 95.2% sequence identity at the DNA level, indicating much more sequence diversity than those on chromosome 2A with 99.6% sequence identity. Both of the PPO genes on chromosomes 2A and 2D contain an open reading frame (ORF) of 1,731 bp, encoding a PPO precursor peptide of 577 amino acids with a predicted molecular mass of approximately 64 kD. Two complementary dominant STS markers, PPO16 and PPO29, were developed based on the PPO gene haplotypes located on chromosome 2D; they amplify a 713-bp fragment in cultivars with low PPO activity and a 490-bp fragment in those with high PPO activity, respectively. The two markers were mapped on chromosome 2DL using a doubled haploid population derived from the cross Zhongyou 9507/CA9632, and a set of nullisomic-tetrasomic lines and ditelosomic line 2DS of Chinese Spring. QTL analysis indicated that the PPO gene co-segregated with the two STS markers and was closely linked to SSR marker Xwmc41 on chromosome 2DL, explaining from 9.6 to 24.4% of the phenotypic variance for PPO activity across three environments. In order to simultaneously detect PPO loci on chromosomes 2A and 2D, a multiplexed marker combination PPO33/PPO16 was developed and yielded distinguishable DNA patterns in a number of cultivars. The STS marker PPO33 for the PPO gene on chromosome 2A was developed from the same gene sequences as PPO18 that we reported previously, and can amplify a 481-bp and a 290-bp fragment from cultivars with low and high PPO activity, respectively. A total of 217 Chinese wheat cultivars and advanced lines were used to validate the association between the polymorphic fragments and grain PPO activity. The results showed that the marker combination PPO33/PPO16 is efficient and reliable for evaluating PPO activity and can be used in wheat breeding programs aimed for noodle and other end product quality improvement.

The rice Rim2 transcript accumulates in response to Magnaporthe grisea and its predicted protein product shares similarity with TNP2-like proteins encoded by CACTA transposons.

A rice transcript, Rim2, was identified that accumulated in both incompatible and compatible interactions between rice and Magnaporthe grisea. The Rim2 transcript also accumulated in response to treatment with a cell wall elicitor derived from M. grisea. A 3.3-kb RIM2 cDNA clone was isolated and is predicted to encode a protein of 653 amino acids, which shares 32 55% identity with TNP2-like proteins encoded by CACTA transposons of other plants. A 1.05-kb segment of the Rim2 sequence shows 82% nucleotide sequence identity with sequences flanking the A1 and C members of the rice Xa21 disease resistance gene family. The 5'-upstream region of Rim2 was cloned and the transcriptional start sites were identified. The 5' and 3' noncoding termini of Rim2 are AT-rich. A cis-element showing similarity to a sequence that mediates defense-associated transcriptional activation of the tobacco retrotransposon Tnt1, and four motifs that fit the consensus sequence of the elicitor-responsive elements in the promoters of the parsley PR-1 genes were found in the 5'-upstream region. Four imperfect tandem repeats were identified in the 3' noncoding terminus. Southern analysis with genomic DNA from different rice species indicated that Rim2 is present in 3-4 copies per genome. These results suggest that Rim2 may be one component of a large CACTA-like element, whose transcript accumulates in response to attack by pathogens.

ATP consumption and efficiency of human single muscle fibers with different myosin isoform composition.

Chemomechanical transduction was studied in single fibers isolated from human skeletal muscle containing different myosin isoforms. Permeabilized fibers were activated by laser-pulse photolytic release of 1.5 mM ATP from p(3)-1-(2-nitrophenyl)ethylester of ATP. The ATP hydrolysis rate in the muscle fibers was determined with a fluorescently labeled phosphate-binding protein. The effects of varying load and shortening velocity during contraction were investigated. The myosin isoform composition was determined in each fiber by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. At 12 degrees C large variations (three- to fourfold) were found between slow and fast (2A and 2A-2B) fibers in their maximum shortening velocity, peak power output, velocity at which peak power is produced, isometric ATPase activity, and tension cost. Isometric tension was similar in all fiber groups. The ATP consumption rate increased during shortening in proportion to shortening velocity. At 12 degrees C the maximum efficiency was similar (0.21-0.27) for all fiber types and was reached at a higher speed of shortening for the faster fibers. In all fibers, peak efficiency increased to approximately 0.4 when the temperature was raised from 12 degrees C to 20 degrees C. The results were simulated with a kinetic scheme describing the ATPase cycle, in which the rate constant controlling ADP release is sensitive to the load on the muscle. The main difference between slow and fast fibers was reproduced by increasing the rate constant for the hydrolysis step, which was rate limiting at low loads. Simulation of the effect of increasing temperature required an increase in the force per cross-bridge and an acceleration of the rate constants in the reaction pathway.

Structural responses to the photolytic release of ATP in frog muscle fibres, observed by time-resolved X-ray diffraction.

1. Structural changes following the photolytic release of ATP were observed in single, permeabilised fibres of frog skeletal muscle at 5-6 C, using time-resolved, low-angle X-ray diffraction. The structural order in the fibres and their isometric function were preserved by cross-linking 10-20 % of the myosin cross-bridges to the thin filaments. 2. The time courses of the change in force, stiffness and in intensity of the main equatorial reflections (1,0) and (1,1), of the third myosin layer line (M3) at a reciprocal spacing of (14.5 nm)-1 on the meridian and of the first myosin-actin layer line (LL1) were measured with 1 ms time resolution. 3. In the absence of Ca2+, photolytic release of ATP in muscle fibres initially in the rigor state caused the force and stiffness to decrease monotonically towards their values in relaxed muscle fibres. 4. In the presence of Ca2+, photolytic release of ATP resulted in an initial rapid decrease in force, followed by a slower increase to the isometric plateau. Muscle fibre stiffness decreased rapidly to approximately 65 % of its value in rigor. 5. In the absence of Ca2+, changes on the equator, in LL1 and in M3 occurred with a time scale comparable to that of the changes in tension and stiffness. 6. In the presence of Ca2+, the changes on the equator and LL1 occurred simultaneously with the early phase of tension decrease. The changes in the intensity of M3 (IM3) occurred on the time scale of the subsequent increase in force. The time courses of the changes in tension and IM3 were similar following the photolytic release of 0. 33 or 1.1 mM ATP. However the gradual return towards the rigor state began earlier when only 0.33 mM ATP was released. 7. In the presence of Ca2+, the time course of changes in IM3 closely mimicked that of force development following photolytic release of ATP. This is consistent with models that propose that force development results from a change in the average orientation of cross-bridges, although other factors, such as their redistribution, may also be involved.

Biological activities and signaling pathways of the granulin/epithelin precursor.

Growth-regulated cells, such as 3T3 mouse embryo fibroblasts (MEFs), require more than one growth factor for growth, usually the insulin-like growth factor I (IGF-I) in combination with either platelet-derived growth factor or epidermal growth factor. Singly, these growth factors cannot sustain the growth of 3T3 cells. However, if the IGF-I receptor (IGF-IR) is even modestly overexpressed, then IGF-I, by itself, stimulates the growth of MEFs in monolayer and makes them capable of forming colonies in soft agar. The granulin/epithelin precursor (GEP) has been identified as the only growth factor, thus far, that can stimulate by itself the growth of R- cells, a 3T3-like cell line in which the genes for the IGF-IR have been deleted. We have expressed GEP in R- cells and show that these cells can now grow in serum-free medium. GEP, however, cannot replace other functions of the IGF-IR, such as protection from apoptosis (anoikis) or transforming activity (colony formation in soft agar). GEP activates, in R- cells, the two signaling pathways that are known to be sufficient for IGF-I-mediated mitogenesis in cells overexpressing the IGF-IR, the mitogen-activated protein kinase and the phosphatidylinositol 3-kinase pathways. This may explain why GEP, by itself, can replace the IGF-IR for growth in monolayer cultures. It also confirms that, for transformation, other pathways must be activated besides the two pathways that are sufficient for mitogenesis.

The efficiency of contraction in rabbit skeletal muscle fibres, determined from the rate of release of inorganic phosphate.

1. The relationship between mechanical power output and the rate of ATP hydrolysis was investigated in segments of permeabilized fibres isolated from rabbit psoas muscle. 2. Contractions were elicited at 12 degrees C by photolytic release of ATP from the P3 -1-(2-nitrophenyl) ester of ATP (NPE-caged ATP). Inorganic phosphate (Pi) release was measured by a fluorescence method using a coumarin-labelled phosphate binding protein. Force and sarcomere length were also monitored. 3. ATPase activity was determined from the rate of appearance of Pi during each phase of contraction. The ATPase rate was 10.3 s-1 immediately following release of ATP and 5. 1 s-1 during the isometric phase prior to the applied shortening. It rose hyperbolically with shortening velocity, reaching 18.5 s-1 at a maximal shortening velocity > 1 ML s-1 (muscle lengths s-1). 4. Sarcomeres shortened at 0.09 ML s-1 immediately following the photolytic release of ATP and at 0.04 ML s-1 prior to the period of applied shortening. The high initial ATPase rate may be largely attributed to initial sarcomere shortening. 5. During shortening, maximal power output was 28 W l-1. Assuming the free energy of hydrolysis is 50 kJ mol-1, the efficiency of contraction was calculated from the power output at each shortening velocity. The maximum efficiency was 0.36 at a shortening velocity of 0.27 ML s-1, corresponding to a force level 51 % of that in the isometric state. 6. At the maximal shortening velocity, only 10 % of the myosin heads are attached to the thin filaments at any one time.