Cyclocreatine Suppresses Creatine Metabolism and Impairs Prostate Cancer Progression.

Prostate cancer is the second most common cause of cancer mortality in men worldwide. Applying a novel genetically engineered mouse model (GEMM) of aggressive prostate cancer driven by deficiency of the tumor suppressors PTEN and Sprouty2 (SPRY2), we identified enhanced creatine metabolism as a central component of progressive disease. Creatine treatment was associated with enhanced cellular basal respiration in vitro and increased tumor cell proliferation in vivo. Stable isotope tracing revealed that intracellular levels of creatine in prostate cancer cells are predominantly dictated by exogenous availability rather than by de novo synthesis from arginine. Genetic silencing of creatine transporter SLC6A8 depleted intracellular creatine levels and reduced the colony-forming capacity of human prostate cancer cells. Accordingly, in vitro treatment of prostate cancer cells with cyclocreatine, a creatine analog, dramatically reduced intracellular levels of creatine and its derivatives phosphocreatine and creatinine and suppressed proliferation. Supplementation with cyclocreatine impaired cancer progression in the PTEN- and SPRY2-deficient prostate cancer GEMMs and in a xenograft liver metastasis model. Collectively, these results identify a metabolic vulnerability in prostate cancer and demonstrate a rational therapeutic strategy to exploit this vulnerability to impede tumor progression. SIGNIFICANCE: Enhanced creatine uptake drives prostate cancer progression and confers a metabolic vulnerability to treatment with the creatine analog cyclocreatine.

Hemimycalins C-E; Cytotoxic and Antimicrobial Alkaloids with Hydantoin and 2-Iminoimidazolidin-4-one Backbones from the Red Sea Marine Sponge Hemimycale sp.

In the course of our continuing efforts to identify bioactive secondary metabolites from Red Sea marine sponges, we have investigated the sponge Hemimycale sp. The cytotoxic fraction of the organic extract of the sponge afforded three new compounds, hemimycalins C-E (1-3). Their structural assignments were obtained via analyses of their one- and two-dimensional NMR spectra and HRESI mass spectrometry. Hemimycalin C was found to differ from the reported hydantoin compounds in the configuration of the olefinic moiety at C-5-C-6, while hemimycalins D and E were found to contain an 2-iminoimidazolidin-4-one moiety instead of the hydantoin moiety in previously reported compounds from the sponge. Hemimycalins C-E showed significant antimicrobial activity against Escherichia coli and Candida albicans and cytotoxic effects against colorectal carcinoma (HCT 116) and the triple-negative breast cancer (MDA-MB-231) cells.

Evaluation of chronic toxicity of cyclocreatine in beagle dogs after oral gavage administration for up to 23 weeks.

Cyclocreatine (LUM-001) was evaluated for chronic toxicity (23 weeks) in beagle dogs to support clinical development in patients with creatine transporter deficiency (CTD) disorder. Deionized water (vehicle control) or cyclocreatine was administered by oral gavage twice daily (12 ± 1 h apart) at 20, 40 and 75 mg/kg/dose followed by a recovery period. Due to severe toxicity, the study was terminated earlier than the planned 39 weeks of dosing. Animals in the 20, 40 and 75 mg/kg/dose groups completed 160, 106, and 55 days of dosing, respectively, followed by 30, 55 and 106 days of a recovery period, respectively. Three (25%), 7 (58%), and 7 (58%) animals were euthanized and/or found dead in the 40, 80, and 150 mg/kg/day dose groups, respectively. Clinical signs observed were inappetence, frequent emesis, stool abnormalities, weight loss, lethargy and respiratory distress. Histopathological evaluation revealed congestion, edema, cellular infiltration, fibrin, and/or hemorrhage in the lungs of all dose groups. Additionally, animals in all cyclocreatine treatment groups had perinuclear cytoplasmic vacuoles in the heart, kidneys, skeletal and smooth muscles. After the recovery period, the vacuoles were still observed in the cardiac and renal tissues. Cyclocreatine was absorbed rapidly with mean Tmax within 1 to 2 h and half-life ranged between 2.17 and 2.79 h on Day 1, however, on the final day of dosing, it ranged between 5.80 and 8.77 h (males) and 10.3 to 13.1 h (females). To conclude, in this study the lungs, kidneys, heart, skeletal and smooth muscles were identified as the target organs of cyclocreatine toxicity in beagle dogs.

Liposomal Delivery of Cyclocreatine Impairs Cancer Cell Bioenergetics Mediating Apoptosis.

Creatine kinase (CK) enzyme overexpression has been suggested to play a role in the process of tumorigenesis and metastasis. Cyclocreatine (CCR) is a substrate analog of creatine kinase (CK), where its phosphorylated form is a poor phosphate donor in comparison with native bioenergetic molecule, creatine phosphate (Cr-P). The compound CCR has been shown to markedly inhibit the growth of a broad spectrum of cancers, both in vitro and in vivo. Intracellularly, CCR is phosphorylated by CK to yield a synthetic phosphagen [(N-phosphorylcyclocreatine (CCR ~P)], with thermodynamic and kinetic properties distinct from those of creatine phosphate (Cr-P). Distinct inhibition of tumor growth and metastasis has been attributed to CCR accumulation as CCR ~P in tumor cells, especially in those expressing a high level of CK protein, with minimal adverse effects. Unfortunately, the clinical use of CCR against malignancies is quite limited due to its amphoteric nature, which accounts for most of its extremely low membrane permeability, as well as limited oral bioavailability (BA) and poor systemic pharmacokinetics (PK).Our current work describes the encapsulation of CCR , utilizing freeze and thaw vesicles (FTV )-composed mostly of saturated PC, DOPE, and Chol-into stealth™ liposomes , postcoated with 4.5 M% PEG-PE. Following physicochemical characterization, in vitro release and cellular uptake kinetics confirmed efficient delivery of liposomal CCR (CCR-Lip), leading to intracellular accumulation of its CC-P metabolic product. Successful delivery of CCR to cancer cell effectively depleted low energetic cancer cells of ATP significantly mediating myc-induced metabolic changes. CCR-Lip showed significant antimetastatic and anticancer effectiveness against both MCF-7 and PC-3 human carcinoma models (p < 0.05-0.01), with 4- to 6-fold lower IC50 values vs. closest drug control. Such shift in bioenergetics was coupled via AMPK and phospho-p53 to the mitochondrial apoptosis effector Bak , thus inducing a cell-intrinsic mechanism to counteract uncontrolled neoplastic proliferation, in target cancer cells. Our novel liposomal delivery system of the CCR substrate analog demonstrated strong inhibition of malignant cell bioenergetics, leading to significant antineoplastic and proapoptotic actions, against different cancers.

Use of an animal model of disease for toxicology enables identification of a juvenile no observed adverse effect level for cyclocreatine in creatine transporter deficiency.

In standard general toxicology studies in two species to support clinical development, cyclocreatine, a creatine analog for the treatment of creatine transporter deficiency, caused deaths, convulsions, and/or multi-organ pathology. The potential translatability of these findings to patients was evaluated by comparing toxicity of cyclocreatine in wild-type mice to creatine transporter-deficient mice, a model of the human disease. A biodistribution study indicated greater accumulation of cyclocreatine in the brains of wild-type mice, consistent with its ability to be transported by the creatine transporter. Subsequent toxicology studies confirmed greater sensitivity of wild-type mice to cyclocreatine-induced toxicity. Exposure at the no observed adverse effect level in creatine transporter-deficient (554 µg*hr/ml) mice exceeded exposure at the maximum tolerated dose in wild-type (248 µg*hr/ml) mice. When dosed at 300 mg/kg/day for 3 months, cyclocreatine-related mortality, convulsions, and multi-organ pathology were observed in wild-type mice whereas there were no adverse findings in creatine transporter-deficient mice. Brain vacuolation was common to both strains. Although transporter-deficient mice appeared to be more sensitive, the finding had no functional correlates in this strain. The results highlight the importance of considering models of disease for toxicology in cases where they may be relevant to assessing safety in the intended patient population.

Serotonergic Innervations of the Orbitofrontal and Medial-prefrontal Cortices are Differentially Involved in Visual Discrimination and Reversal Learning in Rats.

Cross-species studies have identified an evolutionarily conserved role for serotonin in flexible behavior including reversal learning. The aim of the current study was to investigate the contribution of serotonin within the orbitofrontal cortex (OFC) and medial prefrontal cortex (mPFC) to visual discrimination and reversal learning. Male Lister Hooded rats were trained to discriminate between a rewarded (A+) and a nonrewarded (B-) visual stimulus to receive sucrose rewards in touchscreen operant chambers. Serotonin was depleted using surgical infusions of 5,7-dihydroxytryptamine (5,7-DHT), either globally by intracebroventricular (i.c.v.) infusions or locally by microinfusions into the OFC or mPFC. Rats that received i.c.v. infusions of 5,7-DHT before initial training were significantly impaired during both visual discrimination and subsequent reversal learning during which the stimulus-reward contingencies were changed (A- vs. B+). Local serotonin depletion from the OFC impaired reversal learning without affecting initial discrimination. After mPFC depletion, rats were unimpaired during reversal learning but slower to respond at the stimuli during all the stages; the mPFC group was also slower to learn during discrimination than the OFC group. These findings extend our understanding of serotonin in cognitive flexibility by revealing differential effects within two subregions of the prefrontal cortex in visual discrimination and reversal learning.

Cyclocreatine treatment ameliorates the cognitive, autistic and epileptic phenotype in a mouse model of Creatine Transporter Deficiency.

Creatine Transporter Deficiency (CTD) is an inborn error of metabolism presenting with intellectual disability, behavioral disturbances and epilepsy. There is currently no cure for this disorder. Here, we employed novel biomarkers for monitoring brain function, together with well-established behavioral readouts for CTD mice, to longitudinally study the therapeutic efficacy of cyclocreatine (cCr) at the preclinical level. Our results show that cCr treatment is able to partially correct hemodynamic responses and EEG abnormalities, improve cognitive deficits, revert autistic-like behaviors and protect against seizures. This study provides encouraging data to support the potential therapeutic benefit of cyclocreatine or other chemically modified lipophilic analogs of Cr.

Evaluation of chronic toxicity of cyclocreatine, a creatine analog, in Sprague Dawley rat after oral gavage administration for up to 26 weeks.

Cyclocreatine (LUM-001), a creatine analog, was evaluated for its nonclinical toxicity in Sprague Dawley (SD) rats. Deionized water as a vehicle control article or cyclocreatine was administered by oral gavage twice daily (approximately 12 ± 1 h apart) at 30, 100 and 300 mg/kg/dose levels in rats up to 26 weeks followed by a 28-day recovery period. Due to an increased incidence of seizures, the 600 mg/kg/day dose group males were dosed only for 16-weeks followed by a 14-week recovery period. Thirteen males and four females from 600 mg/kg/day dose group were sacrificed at interim on Day 113 to study plausible brain lesions and not due to moribundity. There was a dose dependent increase in the number of seizure incidences in ≥60 mg/kg/day males and 600 mg/kg/day females. Microscopically, higher incidences of vacuoles in the brain at 600 mg/kg/day in both sexes, thyroid follicular atrophy and follicular cell hypertrophy at ≥200 mg/kg/day in males and 600 mg/kg/day in females, and seminiferous tubular degeneration and/or interstitial edema in testes at ≥200 mg/kg/day were observed. Mean plasma half-life of cyclocreatine was between 3.5 and 6.5 h. In conclusion, chronic administration of cyclocreatine by oral gavage in Sprague Dawley rats induced the seizures and microscopic lesions in the brain, testes and thyroid. Based on the results of this study the highest tested dose of 600 mg/kg/day (mean Cmax of 151.5 µg/mL; AUC0-24 of 1970 h*µg/mL) was considered the maximum tolerated dose (MTD) in SD rats.

Cyclocreatine Transport by SLC6A8, the Creatine Transporter, in HEK293 Cells, a Human Blood-Brain Barrier Model Cell, and CCDSs Patient-Derived Fibroblasts.

PURPOSE: Cyclocreatine, a creatine analog, is a candidate drug for treating patients with cerebral creatine deficiency syndromes (CCDSs) caused by creatine transporter (CRT, SLC6A8) deficiency, which reduces brain creatine level. The purpose of this study was to clarify the characteristics of cyclocreatine transport in HEK293 cells, which highly express endogenous CRT, in hCMEC/D3 cells, a human blood-brain barrier (BBB) model, and in CCDSs patient-derived fibroblasts with CRT mutations. METHODS: Cells were incubated at 37°C with [14C]cyclocreatine (9 µM) and [14C]creatine (9 µM) for specified periods of times in the presence or absence of inhibitors, while the siRNAs were transfected by lipofection. Protein expression and mRNA expression were quantified using targeted proteomics and quantitative PCR, respectively. RESULTS: [14C]Cyclocreatine was taken up by HEK293 cells in a time-dependent manner, while exhibiting saturable kinetics. The inhibition and siRNA knockdown studies demonstrated that the uptake of [14C]cyclocreatine by both HEK293 and hCMEC/D3 cells was mediated predominantly by CRT as well as [14C]creatine. In addition, uptake of [14C]cyclocreatine and [14C]creatine by the CCDSs patient-derived fibroblasts was found to be largely reduced. CONCLUSION: The present study suggests that cyclocreatine is a CRT substrate, where CRT is the predominant contributor to influx of cyclocreatine into the brain at the BBB. Our findings provide vital insights for the purposes of treating CCDSs patients using cyclocreatine.

Phosphocyclocreatine is the dominant form of cyclocreatine in control and creatine transporter deficiency patient fibroblasts.

Creatine transporter deficiency (CTD) is a metabolic disorder resulting in cognitive, motor, and behavioral deficits. Cyclocreatine (cCr), a creatine analog, has been explored as a therapeutic strategy for the treatment of CTD. We developed a rapid, selective, and accurate HILIC ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to simultaneously quantify the intracellular concentrations of cCr, creatine (Cr), creatine-d3 (Cr-d3), phosphocyclocreatine (pcCr), and phosphocreatine (pCr). Using HILIC-UPLC-MS/MS, we measured cCr and Cr-d3 uptake and their conversion to the phosphorylated forms in primary human control and CTD fibroblasts. Altogether, the data demonstrate that cCr enters cells and its dominant intracellular form is pcCr in both control and CTD patient cells. Therefore, cCr may replace creatine as a therapeutic strategy for the treatment of CTD.

Cyclocreatine protects against ischemic injury and enhances cardiac recovery during early reperfusion.

Introduction: A critical mechanism of how hypoxia/ischemia causes irreversible myocardial injury is through the exhaustion of adenosine triphosphate (ATP). Cyclocreatine (CCr) and its water-soluble salt Cyclocreatine-Phosphate (CCrP) are potent bioenergetic agents that preserve high levels of ATP during ischemia. Areas covered: CCr and CCrP treatment prior to the onset of ischemia, preserved high levels of ATP in ischemic myocardium, reduced myocardial cell injury, exerted anti-inflammatory and anti-apoptotic activities, and restored contractile function during reperfusion in animal models of acute myocardial infarction (AMI), global cardiac arrest, cardiopulmonary bypass, and heart transplantation. Medline and Embase (1970 - Feb 2019), the WIPO databank (up to Feb 2019); no language restriction. Expert opinion: This review provides the basis for a number of clinical applications of CCrP and CCr to minimize ischemic injury and necrosis. One strategy is to administer CCrP to AMI patients in the pre-hospital phase, as well as during, or after Percutaneous Coronary Intervention (PCI) procedure to potentially achieve protection of the myocardium, reduce infarcted-size, and, thus, limit the progression to heart failure. Another clinical applications are in predictable myocardial ischemia where pretreatment with CCrP would likely improve outcome and quality of life of patients who will undergo cardiopulmonary bypass for coronary revascularization and end-stage heart failure patients scheduled for heart transplantation.

Facile High-Performance Liquid Chromatography Mass Spectrometry Method for Analysis of Cyclocreatine and Phosphocyclocreatine in Complex Mixtures of Amino Acids.

Creatine transporter deficiency (CTD) is caused by a defect in the X-linked creatine transporter SLC6A8 gene leading to severe neurologic and physiologic conditions. Cyclocreatine and phosphocyclocreatine supplementation is seen as a potential treatment, but the presence of these compounds within commercially available dietary supplements presents the risk of self-medication. High-performance liquid chromatography-mass spectrometry (HPLC-MS) is an excellent technique to assess composition of complex amino acid mixtures. Herein, we have developed a facile HPLC-MS method using a cyano column in hydrophilic interaction liquid chromatography (HILIC) mode with isocratic elution over 4 min to identify the main components of two commercially available dietary supplements. The relative standard deviation (RSD) for retention time and extracted ion integrated area are <0.3% and 4%, respectively, showing excellent reproducibility. Cyclocreatine and phosphocyclocreatine were not detectable within the dietary supplements, even at ppm levels, demonstrating the power and importance of the developed HPLC-MS method in analyzing complex mixtures.

Mitochondrial and calcium perturbations in rat CNS neurons induce calpain-cleavage of Parkin: Phosphatase inhibition stabilizes pSer65Parkin reducing its calpain-cleavage.

Mitochondrial impairment and calcium (Ca++) dyshomeostasis are associated with Parkinson's disease (PD). When intracellular ATP levels are lowered, Ca++-ATPase pumps are impaired causing cytoplasmic Ca++ to be elevated and calpain activation. Little is known about the effect of calpain activation on Parkin integrity. To address this gap, we examined the effects of mitochondrial inhibitors [oligomycin (Oligo), antimycin and rotenone] on endogenous Parkin integrity in rat midbrain and cerebral cortical cultures. All drugs induced calpain-cleavage of Parkin to ~36.9/43.6 kDa fragments. In contrast, treatment with the proinflammatory prostaglandin J2 (PGJ2) and the proteasome inhibitor epoxomicin induced caspase-cleavage of Parkin to fragments of a different size, previously shown by others to be triggered by apoptosis. Calpain-cleaved Parkin was enriched in neuronal mitochondrial fractions. Pre-treatment with the phosphatase inhibitor okadaic acid prior to Oligo-treatment, stabilized full-length Parkin phosphorylated at Ser65, and reduced calpain-cleavage of Parkin. Treatment with the Ca++ ionophore A23187, which facilitates Ca++ transport across the plasma membrane, mimicked the effect of Oligo by inducing calpain-cleavage of Parkin. Removing extracellular Ca++ from the media prevented oligomycin- and ionophore-induced calpain-cleavage of Parkin. Computational analysis predicted that calpain-cleavage of Parkin liberates its UbL domain. The phosphagen cyclocreatine moderately mitigated Parkin cleavage by calpain. Moreover, the pituitary adenylate cyclase activating peptide (PACAP27), which stimulates cAMP production, prevented caspase but not calpain-cleavage of Parkin. Overall, our data support a link between Parkin phosphorylation and its cleavage by calpain. This mechanism reflects the impact of mitochondrial impairment and Ca++-dyshomeostasis on Parkin integrity and could influence PD pathogenesis.

Tyrosine Phosphorylation of Mitochondrial Creatine Kinase 1 Enhances a Druggable Tumor Energy Shuttle Pathway.

How mitochondrial metabolism is altered by oncogenic tyrosine kinases to promote tumor growth is incompletely understood. Here, we show that oncogenic HER2 tyrosine kinase signaling induces phosphorylation of mitochondrial creatine kinase 1 (MtCK1) on tyrosine 153 (Y153) in an ABL-dependent manner in breast cancer cells. Y153 phosphorylation, which is commonly upregulated in HER2+ breast cancers, stabilizes MtCK1 to increase the phosphocreatine energy shuttle and promote proliferation. Inhibition of the phosphocreatine energy shuttle by MtCK1 knockdown or with the creatine analog cyclocreatine decreases proliferation of trastuzumab-sensitive and -resistant HER2+ cell lines in culture and in xenografts. Finally, we show that cyclocreatine in combination with the HER2 kinase inhibitor lapatinib reduces the growth of a trastuzumab-resistant HER2+ patient-derived xenograft. These findings suggest that activation of the phosphocreatine energy shuttle by MtCK1 Y153 phosphorylation creates a druggable metabolic vulnerability in cancer.

Quantification of cyclocreatine in mouse and rat plasma using hydrophilic-interaction ultra-performance liquid chromatography-tandem mass spectrometry.

An accurate, rapid and selective method was developed to quantify cyclocreatine in mouse and rat plasma using hydrophilic interaction (HILIC) ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The plasma samples were prepared by protein precipitation with acetonitrile:methanol (70:30). Chromatographic separation was performed on a HILIC BEH amide column (2.1mm×50mm, 1.7µm) with a 3min gradient elution at a flow rate of 0.5mL/min. For mass spectrometric detection, selected reaction monitoring (SRM) was used; the SRM transitions were m/z 144→98 and m/z 144→56 for cyclocreatine and m/z 148→102 for the internal standard (D4-cyclocreatine) in the positive ionization mode. No endogenous components interfered with the analysis of cyclocreatine and the internal standard in mouse and rat plasma. Plasma calibration curves were constructed in the range of 0.01-25µM. The correlation coefficient of the calibration curves was greater than 0.99. The mean intraday assay accuracy for all quality control (QC) replicates was between 93 and 105%. The mean intraday assay precision (CV%) was 1.9-11% for all QC levels. The HILIC-UPLC-MS/MS method was successfully applied in pharmacokinetic (PK) studies of cyclocreatine in mice and rats for the first time. After a single 30mg/kg oral administration in mice and rats, the AUC0-∞ (area under the curve) was 84.1µgh/mL and 91.7±18.0µgh/mL, respectively.

TREM2 Maintains Microglial Metabolic Fitness in Alzheimer's Disease.

Elevated risk of developing Alzheimer's disease (AD) is associated with hypomorphic variants of TREM2, a surface receptor required for microglial responses to neurodegeneration, including proliferation, survival, clustering, and phagocytosis. How TREM2 promotes such diverse responses is unknown. Here, we find that microglia in AD patients carrying TREM2 risk variants and TREM2-deficient mice with AD-like pathology have abundant autophagic vesicles, as do TREM2-deficient macrophages under growth-factor limitation or endoplasmic reticulum (ER) stress. Combined metabolomics and RNA sequencing (RNA-seq) linked this anomalous autophagy to defective mammalian target of rapamycin (mTOR) signaling, which affects ATP levels and biosynthetic pathways. Metabolic derailment and autophagy were offset in vitro through Dectin-1, a receptor that elicits TREM2-like intracellular signals, and cyclocreatine, a creatine analog that can supply ATP. Dietary cyclocreatine tempered autophagy, restored microglial clustering around plaques, and decreased plaque-adjacent neuronal dystrophy in TREM2-deficient mice with amyloid-ß pathology. Thus, TREM2 enables microglial responses during AD by sustaining cellular energetic and biosynthetic metabolism.

Spectroscopic and structural study of the newly synthesized heteroligand complex of copper with creatinine and urea.

Study of copper complex of creatinine and urea is very important in life science and medicine. In this paper, spectroscopic and structural study of a newly synthesized heteroligand complex of copper with creatinine and urea has been discussed. Structural studies have been carried out using DFT calculations and spectroscopic analyses were carried out by FT-IR, Raman, UV-vis absorption and fluorescence techniques. The copper complex of creatinine and the heteroligand complex were found to have much increased water solubility as compared to pure creatinine. The analysis of FT-IR and Raman spectra helps to understand the coordination properties of the two ligands and to determine the probable structure of the heteroligand complex. The LIBS spectra of the heteroligand complex reveal that the complex is free from other metal impurities. UV-visible absorption spectra and the fluorescence emission spectra of the aqueous solution of Cu-Crn-urea heteroligand complex at different solute concentrations have been analyzed and the complex is found to be rigid and stable in its monomeric form at very low concentrations.

In vivo (1)H MRS and (31)P MRSI of the response to cyclocreatine in transgenic mouse liver expressing creatine kinase.

Hepatocyte transplantation has been explored as a therapeutic alternative to liver transplantation, but a means to monitor the success of the procedure is lacking. Published findings support the use of in vivo (31)P MRSI of creatine kinase (CK)-expressing hepatocytes to monitor proliferation of implanted hepatocytes. Phosphocreatine tissue level depends upon creatine (Cr) input to the CK enzyme reaction, but Cr measurement by (1)H MRS suffers from low signal-to-noise ratio (SNR). We examine the possibility of using the Cr analog cyclocreatine (CCr, a substrate for CK), which is quickly phosphorylated to phosphocyclocreatine (PCCr), as a higher SNR alternative to Cr. (1)H MRS and (31)P MRSI were employed to measure the effect of incremental supplementation of CCr upon PCCr, γ-ATP, pH and Pi /ATP in the liver of transgenic mice expressing the BB isoform of CK (CKBB) in hepatocytes. Water supplementation with 0.1% CCr led to a peak total PCCr level of 17.15 ± 1.07 mmol/kg wet weight by 6 weeks, while adding 1.0% CCr led to a stable PCCr liver level of 18.12 ± 3.91 mmol/kg by the fourth day of feeding. PCCr was positively correlated with CCr, and ATP concentration and pH declined with increasing PCCr. Feeding with 1% CCr in water induced an apparent saturated level of PCCr, suggesting that CCr quantization may not be necessary for quantifying expression of CK in mice. These findings support the possibility of using (31)P MRS to noninvasively monitor hepatocyte transplant success with CK-expressing hepatocytes.

Submembranous recruitment of creatine kinase B supports formation of dynamic actin-based protrusions of macrophages and relies on its C-terminal flexible loop.

Subcellular partitioning of creatine kinase contributes to the formation of patterns in intracellular ATP distribution and the fuelling of cellular processes with a high and sudden energy demand. We have previously shown that brain-type creatine kinase (CK-B) accumulates at the phagocytic cup in macrophages where it is involved in the compartmentalized generation of ATP for actin remodeling. Here, we report that CK-B catalytic activity also helps in the formation of protrusive ruffle structures which are actin-dependent and abundant on the surface of both unstimulated and LPS-activated macrophages. Recruitment of CK-B to these structures occurred transiently and inhibition of the enzyme's catalytic activity with cyclocreatine led to a general smoothening of surface morphology as visualized by scanning electron microscopy. Comparison of the dynamics of distribution of YFP-tagged CK-mutants and isoforms by live imaging revealed that amino acid residues in the C-terminal segment (aa positions 323-330) that forms one of the protein's two mobile loops are involved in partitioning over inner regions of the cytosol and nearby sites where membrane protrusions occur during induction of phagocytic cup formation. Although wt CK-B, muscle-type CK (CK-M), and a catalytically dead CK-B-E232Q mutant with intact loop region were normally recruited from the cytosolic pool, no dynamic transition to the phagocytic cup area was seen for the CK-homologue arginine kinase and a CK-B-D326A mutant protein. Bioinformatics analysis helped us to predict that conformational flexibility of the C-terminal loop, independent of conformational changes induced by substrate binding or catalytic activity, is likely involved in exposing the enzyme for binding at or near the sites of membrane protrusion formation.

A novel, broadly applicable approach to isolation of fungi in diverse growth media.

Creatinine (CRN) is a vertebrate metabolic waste product normally found in blood and urine. Previous work demonstrated that the hydrochloride salt of creatinine (CRN-HCl) acted as a potent inhibitor of bacterial replication. Creatinine hydrochloride does not inhibit the growth of yeasts or molds (i.e. fungi), making it a potentially useful addition to growth media to facilitate isolation of environmental or clinically relevant fungal species. Sabouraud dextrose agar is the current medium of choice for detection and isolation of fungi although it does not offer optimal nutritional requirements for some fungi and can permit growth of bacteria which may subsequently inhibit fungal growth and/or obscure fungal isolation. We show that CRN-HCl effectively suppresses bacterial growth in either liquid or solid agar media while allowing outgrowth of slower growing fungi using either experimentally prepared samples or environmental samples.