Quantifying the Assembly of Multicomponent Molecular Machines by Single-Molecule Total Internal Reflection Fluorescence Microscopy.

Large, dynamic macromolecular complexes play essential roles in many cellular processes. Knowing how the components of these complexes associate with one another and undergo structural rearrangements is critical to understanding how they function. Single-molecule total internal reflection fluorescence (TIRF) microscopy is a powerful approach for addressing these fundamental issues. In this article, we first discuss single-molecule TIRF microscopes and strategies to immobilize and fluorescently label macromolecules. We then review the use of single-molecule TIRF microscopy to study the formation of binary macromolecular complexes using one-color imaging and inhibitors. We conclude with a discussion of the use of TIRF microscopy to examine the formation of higher-order (i.e., ternary) complexes using multicolor setups. The focus throughout this article is on experimental design, controls, data acquisition, and data analysis. We hope that single-molecule TIRF microscopy, which has largely been the province of specialists, will soon become as common in the tool box of biophysicists and biochemists as structural approaches have become today.

The Many Roles of PCNA in Eukaryotic DNA Replication.

Proliferating cell nuclear antigen (PCNA) plays critical roles in many aspects of DNA replication and replication-associated processes, including translesion synthesis, error-free damage bypass, break-induced replication, mismatch repair, and chromatin assembly. Since its discovery, our view of PCNA has evolved from a replication accessory factor to the hub protein in a large protein-protein interaction network that organizes and orchestrates many of the key events at the replication fork. We begin this review article with an overview of the structure and function of PCNA. We discuss the ways its many interacting partners bind and how these interactions are regulated by posttranslational modifications such as ubiquitylation and sumoylation. We then explore the many roles of PCNA in normal DNA replication and in replication-coupled DNA damage tolerance and repair processes. We conclude by considering how PCNA can interact physically with so many binding partners to carry out its numerous roles. We propose that there is a large, dynamic network of linked PCNA molecules at and around the replication fork. This network would serve to increase the local concentration of all the proteins necessary for DNA replication and replication-associated processes and to regulate their various activities.

A molecular phylogenetic reappraisal of the Hysteriaceae, Mytilinidiaceae and Gloniaceae (Pleosporomycetidae, Dothideomycetes) with keys to world species.

A reappraisal of the phylogenetic integrity of bitunicate ascomycete fungi belonging to or previously affiliated with the Hysteriaceae, Mytilinidiaceae, Gloniaceae and Patellariaceae is presented, based on an analysis of 121 isolates and four nuclear genes, the ribosomal large and small subunits, transcription elongation factor 1 and the second largest RNA polymerase II subunit. A geographically diverse and high density taxon sampling strategy was employed, including multiple isolates/species from the following genera: Anteaglonium (6/4), Encephalographa (1/1), Farlowiella (3/1), Gloniopsis (8/4), Glonium (4/2), Hysterium (12/5), Hysterobrevium (14/3), Hysterographium (2/1), Hysteropatella (2/2), Lophium (4/2), Mytilinidion (13/10), Oedohysterium (5/3), Ostreichnion (2/2), Patellaria (1/1), Psiloglonium (11/3), Quasiconcha (1/1), Rhytidhysteron (8/3), and 24 outgroup taxa. Sequence data indicate that although the Hysteriales are closely related to the Pleosporales, sufficient branch support exists for their separation into separate orders within the Pleosporomycetidae. The Mytilinidiales are more distantly related within the subclass and show a close association with the Gloniaceae. Although there are examples of concordance between morphological and molecular data, these are few. Molecular data instead support the premise of a large number of convergent evolutionary lineages, which do not correspond to previously held assumptions of synapomorphy relating to spore morphology. Thus, within the Hysteriaceae, the genera Gloniopsis, Glonium, Hysterium and Hysterographium are highly polyphyletic. This necessitated the transfer of two species of Hysterium to Oedohysteriumgen. nov. (Od. insidenscomb. nov. and Od. sinense comb. nov.), the description of a new species, Hysterium barrianumsp. nov., and the transfer of two species of Gloniopsis to Hysterobreviumgen. nov. (Hb. smilaciscomb. nov. and Hb. constrictumcomb. nov.). While Hysterographium, with the type Hg. fraxini, is removed from the Hysteriaceae, some of its species remain within the family, transferred here to Oedohysterium (Od. pulchrumcomb. nov.), Hysterobrevium (Hb. moricomb. nov.) and Gloniopsis (Gp. subrugosacomb. nov.); the latter genus, in addition to the type, Gp. praelonga, with two new species, Gp. arciformissp. nov. and Gp. kenyensis sp. nov. The genus Glonium is now divided into Anteaglonium (Pleosporales), Glonium (Gloniaceae), and Psiloglonium (Hysteriaceae). The hysterothecium has evolved convergently no less than five times within the Pleosporomycetidae (e.g., Anteaglonium, Farlowiella, Glonium, Hysterographium and the Hysteriaceae). Similarly, thin-walled mytilinidioid (e.g., Ostreichnion) and patellarioid (e.g., Rhytidhysteron) genera, previously in the Mytilinidiaceae and Patellariaceae, respectively, transferred here to the Hysteriaceae, have also evolved at least twice within the subclass. As such, character states traditionally considered to represent synapomorphies among these fungi, whether they relate to spore septation or the ascomata, in fact, represent symplesiomorphies, and most likely have arisen multiple times through convergent evolutionary processes in response to common selective pressures.

A class-wide phylogenetic assessment of Dothideomycetes.

We present a comprehensive phylogeny derived from 5 genes, nucSSU, nucLSU rDNA, TEF1, RPB1 and RPB2, for 356 isolates and 41 families (six newly described in this volume) in Dothideomycetes. All currently accepted orders in the class are represented for the first time in addition to numerous previously unplaced lineages. Subclass Pleosporomycetidae is expanded to include the aquatic order Jahnulales. An ancestral reconstruction of basic nutritional modes supports numerous transitions from saprobic life histories to plant associated and lichenised modes and a transition from terrestrial to aquatic habitats are confirmed. Finally, a genomic comparison of 6 dothideomycete genomes with other fungi finds a high level of unique protein associated with the class, supporting its delineation as a separate taxon.

Thoracoscopic fluorescence diagnosis (TFD) of pleural malignancies: experimental studies.

BACKGROUND: Fluorescence diagnosis (FD) using the photosensitiser 5-aminolaevulinic acid (ALA) was experimentally combined with conventional video assisted thoracic surgery (VATS) to improve tumour staging in advanced lung cancer with pleural tumour spread. METHODS: A disseminated pleural carcinosis affecting the entire pleural cavity was induced by inoculation of human adenocarcinoma cells in nude rats. After 5-7 weeks of tumour growth the animals were randomised into six groups with different photosensitisation parameters. Pleural lavage was performed either with 1.5% or 3.0% ALA solution. Photosensitisation times varied were 2, 4, or 6 hours. Conventional white light VATS was first performed to evaluate tumour growth in the pleural cavity. Fluorescence illumination of the light source, the D-light, was then used to examine the site for additional tumours which were previously invisible. The tumour fluorescence intensity was measured spectrometrically and compared with normal tissue. RESULTS: Compared with conventional white light VATS alone, thoracoscopic fluorescence diagnosis (TFD) detected up to 30% additional pleural malignant lesions. The highest diagnostic sensitivity was reached 6 hours after 3.0% ALA pleural lavage. Photosensitiser accumulation in the tumour, measured indirectly by spectrometry, was up to 11 times higher than in normal tissue. CONCLUSIONS: TFD increases sensitivity of VATS for tumour staging. It may prevent unnecessary thoracotomies in cancer patients and facilitate surgical planning.

Energetic crosstalk between organelles: architectural integration of energy production and utilization.

Cells with high and fluctuating energy demands such as cardiomyocytes need efficient systems to link energy production to energy utilization. This is achieved in part by compartmentalized energy transfer enzymes such as creatine kinase (CK). However, hearts from CK-deficient mice develop normal cardiac function under conditions of moderate workload. We have therefore investigated whether a direct functional interplay exists between mitochondria and sarcoplasmic reticulum or between mitochondria and myofilaments in cardiac cells that catalyzes direct energy and signal channeling between organelles. We used the selective permeabilization of sarcolemmal membranes with saponin to study the functional interactions between organelles within the cellular architecture. We measured contractile kinetics, oxygen consumption, and caffeine-induced tension transients. The results show that in hearts of normal mice, ATP produced by mitochondria (supplied with substrates, oxygen, and adenine nucleotides) was able to sustain calcium uptake and contractile speed. Moreover, direct mitochondrially supplied ATP was nearly as effective as CK-supplied ATP and much more effective than externally supplied ATP, suggesting that a direct ATP/ADP channeling exists between the sites of energy production (mitochondria) and energy utilization (sarcoplasmic reticulum and myofilaments). On the other hand, in cardiac cells of mice deficient in mitochondrial and cytosolic CK, marked cytoarchitectural modifications were observed, and direct adenine nucleotide channeling between mitochondria and organelles was still effective for sarcoplasmic reticulum and myofilaments. Such direct crosstalk between organelles may explain the preserved cardiac function of CK-deficient mice under moderate workloads.

Increased uncoupling proteins and decreased efficiency in palmitate-perfused hyperthyroid rat heart.

The physiological role of mitochondrial uncoupling proteins (UCPs) in heart and skeletal muscle is unknown, as is whether mitochondrial uncoupling of oxidative phosphorylation by fatty acids occurs in vivo. In this study, we found that UCP2 and UCP3 protein content, determined using Western blotting, was increased by 32 and 48%, respectively, in hyperthyroid rat heart mitochondria. Oligomycin-insensitive respiration rate, a measure of mitochondrial uncoupling, was increased in all mitochondria in the presence of palmitate: 36% in controls and 71 and 100% with 0.8 and 0.9 mM palmitate, respectively, in hyperthyroid rat heart mitochondria. In the isolated working heart, 0.4 mM palmitate significantly lowered cardiac output by 36% and cardiac efficiency by 38% in the hyperthyroid rat heart. Thus increased mitochondrial UCPs in the hyperthyroid rat heart were associated with increased uncoupling and decreased myocardial efficiency in the presence of palmitate. In conclusion, a physiological effect of UCPs on fatty acid oxidation has been found in heart at the mitochondrial and whole organ level.

Population Structure of Botryosphaeria dothidea from Pistachio and Other Hosts in California.

ABSTRACT Genetic diversity was investigated among California populations of Botryosphaeria dothidea, causal agent of Botryosphaeria panicle and shoot blight of pistachio, with random amplified polymorphic DNA (RAPD) and microsatellite-primed polymerase chain reaction (MP-PCR). We surveyed 120 isolates, 112 of which originated from the California Central Valley and included pistachio isolates (n = 52) and isolates from other plant species (n = 60). Out-group isolates (n = 8) were obtained from pistachio in Greece. There was a strong correlation (r = 0.99; P < 0.0001) between the RAPD- and MP-PCR dissimilarity data sets. Little genetic variation (haplotypic diversity [Hs] < 0.002) was detected among B. dothidea isolates collected from central and southern California pistachio plantings. We observed relatively high diversity for isolates from a northern California pistachio orchard (Hs = 0.0146), where the disease was first diagnosed, and from the Chico U.S. Department of Agriculture Germ Plasm Repository (Hs = 0.0726), where the first pistachio trees were planted in California in 1929. Isolates obtained from other hosts, especially those associated with the rare occurrence of the sexual stage of this fungus, showed the highest levels of diversity (Hs = 0.1689). Thirty-eight pistachio isolates (73.1%) had DNA fingerprints identical to 28 pycnidiospore-derived isolates (56.0%) obtained from other host species. Greenhouse inoculations demonstrated that all isolates obtained from other hosts were capable of infecting pistachio and produced characteristic disease symptomology. Thus, California populations of B. dothidea from pistachio are, for the most part, genetically uniform, with the sexual stage rare to absent. However, the rare occurrence of the sexual stage of B. dothidea on other hosts, and more importantly, the capacity of these isolates to infect pistachio, indicate that other host species may serve as sources of inoculum and genetic variation.

Species-Specific Detection of Monilinia fructicola from California Stone Fruits and Flowers.

ABSTRACT A set of molecular diagnostics was developed for Monilinia fructicola, causal agent of brown rot of stone fruits, capable of sensitive detection of the pathogen in planta. Species-specific repetitive sequences were identified from a partial library of 312 recombinant clones hybridized with total DNA, followed by subsequent screening for specificity. One hundred isolates, comprising 12 fungal species common to California stone fruits, were surveyed for specificity. Three clones hybridized to 60 geographically diverse M. fructicola isolates (California, Michigan, Georgia, Oregon, and Australia) to the exclusion of all other fungi surveyed, including the closely related M. laxa (n = 12). Two clones were identical and of extrachromosomal origin (pMF73 and pMF150), whereas the third (pMF210) migrated with uncut DNA. The sensitivity of all three was comparable and capable of detecting 50 pg of fungal DNA in dot blot hybridizations. Six species-specific primer pair sets were designed. They maintained the same specificity patterns observed in the initial hybridization surveys and were sensitive enough to detect 50 fg of fungal DNA template, approximately equivalent to 10 spores. The species-specific clones were capable of detecting the pathogen in planta, specifically from infected plum flowers and nectarine fruit tissue, using both hybridization- and polymerase chain reaction-based methodologies.

Magnetic resonance spectroscopy evidence of abnormal cardiac energetics in Xp21 muscular dystrophy.

OBJECTIVES: Our aim was to measure the cardiac phosphocreatine to adenosine triphosphate ratio (PCr/ATP) noninvasively in patients and carriers of Xp21 muscular dystrophy and to correlate the results with left ventricular (LV) function as measured by echocardiography. BACKGROUND: Duchenne and Becker muscular dystrophy (the Xp21 dystrophies) are associated with the absence or altered expression of dystrophin in cardiac and skeletal muscles. They are frequently complicated by cardiac hypertrophy and dilated cardiomyopathy. The main role of dystrophin is believed to be structural, but it may also be involved in signaling processes. Defects in energy metabolism have been found in skeletal muscle in patients with Xp21 muscular dystrophy. We therefore hypothesized that a defect in energy metabolism may be part of the mechanism leading to the cardiomyopathy of Xp21 muscular dystrophy. METHODS: Thirteen men with Becker muscular dystrophy, 10 female carriers and 23 control subjects were studied using phosphorus-31 magnetic resonance spectroscopy and echocardiography. RESULTS: The PCr/ATP was significantly reduced in patients (1.55+/-0.37) and carriers (1.37+/-0.25) as compared with control subjects (2.44+/-0.33; p<0.0001 for both groups). The PCr/ATP did not correlate with LV ejection fraction or mass index. CONCLUSIONS: Altered expression of dystrophin leads to a reduction in the PCr/ATP. Since this reduction did not correlate with indexes of left ventricular function, this raises the possibility of a direct link between altered dystrophin expression and the development of cardiomyopathy in such patients.

The effects of anti-hypertensive therapy on the structural, mechanical and metabolic properties of the rat aorta.

The vascular system exhibits altered growth, calcium responses and metabolism during hypertension. To relate such changes, we compared histological, tension and metabolic responses in the aorta from 32-week-old spontaneously hypertensive rats (SHRs), normotensive Wistar-Kyoto (WKY) rats, and SHRs treated with Verapamil (V) and ACE-inhibitor, Trandolapril (T) as well as a combination of the two treatments (C). Vascular hypertrophy was apparent in the SHRs. Contractile responses induced by 50 mmol/1 KCl and 2.5 mmol/1 Ca2+ were significantly lower in the SHR (64.4 mN/mm2 vs. 49.2 mN/mm2), but an associated increase in Ca2+ -sensitivity (EC50 of extracellular Ca2+ (mumol/1): SHR, 456 vs. WKY, 616) normalised tension generating ability. All treatments led to significant decreases in blood pressure, although only T and C treated animals became normotensive with concomitant normalisation of vascular hypertrophy. An increase in oxygen consumption was apparent in the SHR aorta, which was associated with significant differences in the activities of key metabolic enzymes. Anti-hypertensive treatment normalised many of the metabolic parameters, with the C therapy being the most efficacious. We conclude that the treatment of hypertension by combined therapy leads to a better normalisation of structural, contractile, and metabolic parameters in the SHR, than either treatment alone and that metabolic changes with the pathology are resolved with appropriate therapy.

Glycolysis supports calcium uptake by the sarcoplasmic reticulum in skinned ventricular fibres of mice deficient in mitochondrial and cytosolic creatine kinase.

Several works have shown the importance of the creatine kinase (CK) system for cardiac energetics and Ca2+ homeostasis. Nevertheless, CK-deficient mice have cardiac function close to normal, at least under conditions of low or moderate workload. To characterize possible adaptive changes of the sarcoplasmic reticulum (SR) and potential role of glycolytic support in cardiac contractility we used the skinned fibre technique to study properties of the SR and myofibrils, in control and muscle-type homodimer (MM-/mitochondrial-CK)-deficient mice. In control fibres, SR Ca2+ loading with ATP and phosphocreatine (solution PL) was significantly better than loading with ATP alone (solution AL), as determined by analysis of caffeine-induced tension transients. Loading in the presence of ATP and glycolytic intermediates (solution GL) was not significantly different from solution PL. These data indicate that Ca2+ uptake by the SR in situ depends on a local ATP:ADP ratio that is controlled by both CK and glycolytic enzymes. In CK-deficient mice, Ca2+ loading was impaired in solution PL due to the absence of CK. In solution GL, loading was significantly increased, such that calculated Ca2+ release parameters were normalized to those in control fibres in solution PL. In CK-deficient mice, fibre kinetic parameters of tension recovery were impaired after quick stretch in solution PL and were not improved in solution GL. These results show that in CK-deficient mice, at least under basal conditions, glycolysis can replace the CK system in fueling the SR Ca2+ ATPase, but not the myosin ATPase, and may in part explain the limited phenotypic alterations seen in the hearts of these mice.

Creatine uptake in isolated soleus muscle: kinetics and dependence on sodium, but not on insulin.

The increased use of creatine by athletes as a dietary supplement to improve their physical performance assumes that increased serum creatine levels will increase intracellular skeletal muscle creatine. Despite this common assumption, skeletal muscle creatine uptake awaits full characterization. Consequently, we have investigated 14C-labelled creatine uptake in isolated, incubated rat soleus (type I) muscle preparations at 37 degrees C. We found that the apparent Km for creatine uptake was 73 microM and the Vmax was 77 nmol h-1 gww-1. Creatine uptake was 82% inhibited by 2 mM beta-guanidinopropionic acid, the structural analogue of creatine. In addition, a decrease in buffer Na+ concentration, from 145 to 25 mM, reduced the rate of 14C-labelled creatine uptake by 77%, indicating that uptake is largely Na+-dependent in soleus muscle. Insulin had no effect on the rate of creatine uptake in vitro. The total creatine content was 34% lower, but the rate of creatine uptake in the presence of 100 microM extracellular creatine was 45% higher, in soleus than in extensor digitorum longus (type II) muscle. However, at 1 mM extracellular creatine, the maximal rate of uptake was not significantly different for the two muscle types, implying that soleus muscle has a lower Km for creatine uptake. We suggest that intracellular creatine levels may play a role in the regulation of skeletal muscle creatine uptake.

Lipid accumulation in the ovaries of a marine shrimp penaeus semisulcatus (de haan)

By the end of oocyte development, the ovaries of Penaeus semisulcatus have accumulated almost equal amounts (approximately 16 mg lipid g-1 protein) of phospholipids and triacylglycerols. The phospholipids consist mainly of phosphatidylcholine (75-80 %) and phosphatidylethanolamine (20-25 %). Approximately 30 % of the total fatty acid content of both phospholipids and triacylglycerols is made up of polyunsaturated fatty acids. In fractions obtained by centrifugation of ovarian homogenates, most of the increase in levels of ovarian lipids during ovarian maturation was associated with an increase in triacylglycerol levels in the floating fat fraction and of phospholipids in the infranatant fraction. The presence of polyunsaturated fatty acids in the ovaries indicates the occurrence of lipid transport to the ovary during oocyte maturation. The gradual decrease in the relative abundance of polyunsaturated fatty acids as the ovaries matured supports previously published results suggesting intra-ovarian synthesis of saturated and mono-unsaturated fatty acids. Most of the lipids found in the female haemolymph (64.8 %) were recovered in the high-density lipoprotein fraction after density ultracentrifugation. The haemocyanin fraction recovered from this stage of fractionation contained substantial amounts of lipid (16.8 %) that could be removed by further sequential centrifugation at a higher NaBr density, leaving less than 0.9 % of the total haemolymph lipids associated with this fraction. While 16.2 % of the lipids were recovered from the very high-density lipoprotein fractions, these lipoproteins carried only 64-89 microg lipid mg-1 protein compared with 538.9 microg lipid mg-1 protein in the high-density lipoprotein fraction, indicating that the high-density lipoproteins are more likely to be the main transporters of lipids to the ovary. However, the contribution of very high-density lipoproteins to lipid transport cannot be ruled out at this stage. In this study, we present two models for lipid transport to the ovary based on the abundance of phospholipids and triacylglycerols in the haemolymph and on the amounts of polyunsaturated fatty acids accumulated within the ovary during vitellogenesis.

Serum bone sialoprotein in patients with primary breast cancer is a prognostic marker for subsequent bone metastasis.

Bone sialoprotein (BSP) is a noncoflagenous bone matrix protein that is important for both mineralization and cell-cell interactions. Tissue studies in primary breast cancers have shown that immunohistochemical expression of BSP is associated with a high incidence of bone metastases in the course of the disease. We used a RIA to investigate the importance of serum BSP as a marker for subsequent bone metastases. Between 1994 and 1996, preoperative blood samples were collected from 388 consecutive patients with nonmetastatic breast cancer and from 30 control patients with benign breast disease. Serum BSP concentrations were measured in a blinded fashion by RIA. The cutoff for elevated serum BSP values was 24 ng/ml, ie., two SDs above the normal mean value. Serum BSP was correlated with the risk of metastasis and analyzed with regard to its prognostic value. After a median follow-up period of only 20 months, 28 patients had developed metastases. Fourteen patients had bone metastases only, 9 visceral metastases only, and 5 a combination of osseous and visceral metastases. Of the 19 women with skeletal metastases, 17 had preoperative serum BSP values in excess of 24 ng/ml (median BSP values: 48.3 ng/ml for isolated metastatic bone disease, 30.6 ng/ml for combined metastases), whereas none of the women with visceral metastases only had elevated serum BSP concentrations (median BSP value: 12.3 ng/ml). The median serum BSP value in the control group (benign breast disease) was 8.8 ng/ml serum BSP; levels correlated with the size of the primary tumor, but not with any other prognostic factors. Using a multivariate regression analysis, serum BSP was found to be the most important independent prognostic factor for the development of skeletal metastasis (P < 0.001; relative risk, 94); its specificity was 96.7%, and its sensitivity was 89.5%. Our study shows that patients with preoperatively elevated serum BSP levels are at high risk of subsequent bone metastases in the first years after primary surgery. The mechanism of BSP in the pathogenesis of skeletal metastases is unclear. Because BSP contains an integrin recognition sequence, its expression in tumor cells may facilitate their adhesion to the bone surface. However, it is possible that a proportion of circulation BSP is derived from normal or tumor-induced bone turnover. Breast cancer patients with elevated serum BSP levels may benefit from osteoprotective adjuvant therapy with bisphosphonates.

Functional coupling of creatine kinases in muscles: species and tissue specificity.

Creatine kinase (CK) isoenzymes are present in all vertebrates. An important property of the creatine kinase system is that its total activity, its isoform distribution, and the concentration of guanidino substrates are highly variable among species and tissues. In the highly organized structure of adult muscles, it has been shown that specific CK isoenzymes are bound to intracellular compartments, and are functionally coupled to enzymes and transport systems involved in energy production and utilization. It is however, not established whether functional coupling and intracellular compartmentation are present in all vertebrates. Furthermore, these characteristics seem to be different among different muscle types within a given species. This study will review some of these aspects. It has been observed that: (1) In heart ventricle, CK compartmentation and coupling characterize adult mammalian cells. It is almost absent in frogs, and is weakly present in birds. (2) Efficient coupling of MM-CK to myosin ATPase is seen in adult mammalian striated muscles but not in frog and bird heart where B-CK is expressed instead of M-CK. Thus, the functional efficacy of bound MM-CK to regulate adenine nucleotide turnover within the myofibrillar compartment seems to be specific for muscles expressing M-CK as an integral part of the sarcomere. (3) Mi-CK expression and/or functional coupling are highly tissue and species specific; moreover, they are subject to short term and long term adaptations, and are present late in development. The mitochondrial form of CK (mi-CK) can function in two modes depending on the tissue: (i) in an <> and (ii) in an <>. The mode of action of mi-CK seems to be related to its precise localization within the mitochondrial intermembrane space, whereas its amount might control the quantitative aspects of the coupling. Mi-CK is highly plastic, making it a strong candidate for fine regulation of excitation-contraction coupling in muscles and for energy transfer in cells with large and fluctuating energy demands in general. (4) Although CK isoforms show a binding specificity, the presence of a given isoform within a tissue or a species only, does not predict its functional role. For example, M-CK is expressed before it is functionally compartmentalized within myofibrils during development. Similarly, the presence of ubiquitous or sarcomeric mi-CK isoforms, is not an index of functional coupling of mi-CK to oxidative phosphorylation. (5) Amongst species or muscles, it appears that a large buffering action of the CK system is associated with rapid contraction and high glycolytic activity. On the other hand, an oxidative metabolism is associated with isoform diversity, increased compartmentation, a subsequent low buffering action and efficient phosphotransfer between mitochondria and energy utilization sites. It can be concluded that, in addition to a high variation of total activity and isoform expression, the role of the CK system also critically depends on its intracellular organization and interaction with energy producing and utilizing pathways. This compartmentation will determine the high cellular efficiency and fine specialization of highly organized and differentiated muscle cells.

Maintained coupling of oxidative phosphorylation to creatine kinase activity in sarcomeric mitochondrial creatine kinase-deficient mice.

The importance of mitochondrial creatine kinase (mi-CK) in oxidative muscle was tested by studying the functional properties of in situ mitochondria in saponin-skinned muscle fibres from sarcomeric mi-CK-deficient (mutant) mice. Biochemical analyses showed that the lack of mi-CK in mutant muscle was associated with a decrease in specific activity of MM-CK in mutant ventricle, and increase in mutant soleus (oxidative) muscle. Lactate dehydrogenase activity and isoenzyme analysis showed an increased glycolytic metabolism in mutant soleus. No change was observed in ventricular muscle. In control animals, the apparent K(m) of mitochondrial respiration for ADP in ventricle and soleus (232 +/- 36 and 381 +/- 63 microM, respectively) was significantly reduced in the presence of creatine (52 +/- 8 and 45 +/- 12 microM, respectively). There was no change in the K(m) in oxidative fibres from mutant mice (258 +/- 27 and 399 +/- 66 microM, respectively) compared with control, though surprisingly, it was also significantly decreased in the presence of creatine (144 +/- 8 and 150 +/- 27 microM, respectively) despite the absence of mi-CK. It is proposed that in mutant (and perhaps normal) oxidative tissue, cytosolic MM-CK can relocate to the outer mitochondrial membrane, where it is coupled to oxidative phosphorylation by close proximity to porin, and the adenine nucleotide translocase. Such an effect can preserve the functioning of the CK shuttle and the energetic properties of mi-CK deficient tissue.

Muscle unloading induces slow to fast transitions in myofibrillar but not mitochondrial properties. Relevance to skeletal muscle abnormalities in heart failure.

Muscle deconditioning is a common observation in patients with congestive heart failure (CHF), chronic obstructive pulmonary disease, neuromuscular diseases or prolonged bed rest. To gain further insight into metabolic and mechanical properties of deconditioned slow-twitch (soleus) or fast-twitch (EDL) skeletal muscles, we induced experimental muscle deconditioning by hindlimb suspension (HS) in rats for 3 weeks. Cardiac muscle was also studied. Besides profound muscle atrophy, increased proportion of fast type II fibers as well as fast myosin isoenzymes, we found decreased calcium sensitivity of Triton X-100 skinned fiber bundles of soleus muscle directed towards the fast muscle phenotype. Glycolytic enzymes such as hexokinase and pyruvate kinase were increased, and the LDH isoenzyme pattern was clearly shifted from an oxidative to an anaerobic profile. Creatine kinase (CK) and myokinase activities were increased in HS soleus towards EDL values. Moreover, the M-CK mRNA level was greatly increased in soleus, with no change in EDL. However, oxygen consumption rate assessed in situ in saponin skinned fibers (12.5 +/- 0.8 in C and 15.1 +/- 0.9 micromol O2/min/g dw in HS soleus compared to 7.3 +/- 1.3 micromol O2/min/g dw in control EDL), as well as mitochondrial CK (mi-CK) and citrate synthase activities, were preserved in HS soleus. Following deconditioning no change in Km for ADP of mitochondrial respiration, either in the absence (511 +/- 92 in C and 511 +/- 111 microM in HS soleus compared to 9 +/- 4 microM in control EDL) or presence of creatine (88 +/- 10 in C and 95 +/- 16 microM in HS soleus compared to 32 +/- 9 microM in control EDL), was found. The results show that muscle deconditioning induces a biochemical and functional slow to fast phenotype transition in myofibrillar and cytosolic compartments of postural muscle, but not in the mitochondrial compartment, suggesting that these compartments are differently regulated under conditions of decreased activity.

The utilisation of creatine and its analogues by cytosolic and mitochondrial creatine kinase.

We have investigated the utilisation of four analogues of creatine by cytosolic Creatine Kinase (CK), using 31P-NMR in the porcine carotid artery, and by mitochondrial CK (Mt-CK), using oxygen consumption studies in isolated heart mitochondria and skinned fibers. Porcine carotid arteries were superfused for 12 h with Krebs-Henseleit buffer at 22 degrees C, containing 11 mM glucose as substrate, and supplemented with either 20 mM beta-guanidinopropionic acid (beta-GPA), methyl-guanidinopropionic acid (m-GPA), guanidinoacetic acid (GA) or cyclocreatine (cCr). All four analogues entered the tissue and became phosphorylated by CK as seen by 31 P-NMR, Inhibition of oxidative metabolism by 1 mM cyanide after accumulation of the phosphorylated analogue resulted in the utilisation of PCr, beta-GPA-P, GA-P and GA-P over a similar time course (approximately 2 h), despite very different kinetic properties of these analogues in vitro. cCr-P was utilised at a significantly slower rate, but was rapidly dephosphorylated in the presence of both 1 mM iodoacetate and cyanide (to inhibit both glycolysis and oxidative metabolism respectively). The technique of creatine stimulated respiration was used to investigate the phosphorylation of the analogues by Mt-CK, Isolated mitochondria were subjected to increasing [ATP], whereas skinned fibres received a similar protocol with increasing [ADP]. There was a significant stimulation of respiration by creatine and cCr in isolated mitochondria (decreased K(m) and increased Vmax vs control), but none by GA, mGPA or beta-GPA (also in skinned fibres), indicating that these latter analogues were not utilised by Mt-CK. These results demonstrate differences in the phosphorylation and dephosphorylation of creatine and its analogues by cytosolic CK and Mt-CK in vivo and in vitro.