In Vivo Efficacy of Olorofim against Systemic Scedosporiosis and Lomentosporiosis.

Clinically relevant members of the Scedosporium/Pseudallescheria species complex and Lomentospora prolificans are generally resistant against currently available systemic antifungal agents in vitro, and infection due to these species is difficult to treat. We studied the in vivo efficacy of a new fungicidal agent, olorofim (formerly F901318), against scedosporiosis and lomentosporiosis in neutropenic animals. Cyclophosphamide-immunosuppressed CD-1 mice infected by Scedosporium apiospermum, Pseudallescheria boydii (Scedosporium boydii), and Lomentospora prolificans were treated by intraperitoneal administration of olorofim (15 mg/kg of body weight every 8 h for 9 days). The efficacy of olorofim treatment was assessed by the survival rate at 10 days postinfection, levels of serum (1-3)-ß-d-glucan (BG), histopathology, and fungal burdens of kidneys 3 days postinfection. Olorofim therapy significantly improved survival compared to that of the untreated controls; 80%, 100%, and 100% of treated mice survived infection by Scedosporium apiospermum, Pseudallescheria boydii, and Lomentospora prolificans, respectively, while less than 20% of the control mice (phosphate-buffered saline [PBS] treated) survived at 10 days postinfection. In the olorofim-treated neutropenic CD-1 mice infected with any of the three species, serum BG levels were significantly suppressed and fungal DNA detected in the target organs was significantly lower than in controls. Furthermore, histopathology of kidneys revealed no or only a few lesions with hyphal elements in the olorofim-treated mice, while numerous fungal hyphae were present in control mice. These results indicate olorofim to be a promising therapeutic agent for systemic scedosporiosis/lomentosporiosis, devastating emerging fungal infections that are difficult to treat with currently available antifungals.

Optimized quantification of intra-host viral diversity in SARS-CoV-2 and influenza virus sequence data.

High error rates of viral RNA-dependent RNA polymerases lead to diverse intra-host viral populations during infection. Errors made during replication that are not strongly deleterious to the virus can lead to the generation of minority variants. However, accurate detection of minority variants in viral sequence data is complicated by errors introduced during sample preparation and data analysis. We used synthetic RNA controls and simulated data to test seven variant-calling tools across a range of allele frequencies and simulated coverages. We show that choice of variant caller and use of replicate sequencing have the most significant impact on single-nucleotide variant (SNV) discovery and demonstrate how both allele frequency and coverage thresholds impact both false discovery and false-negative rates. When replicates are not available, using a combination of multiple callers with more stringent cutoffs is recommended. We use these parameters to find minority variants in sequencing data from SARS-CoV-2 clinical specimens and provide guidance for studies of intra-host viral diversity using either single replicate data or data from technical replicates. Our study provides a framework for rigorous assessment of technical factors that impact SNV identification in viral samples and establishes heuristics that will inform and improve future studies of intra-host variation, viral diversity, and viral evolution. IMPORTANCE When viruses replicate inside a host cell, the virus replication machinery makes mistakes. Over time, these mistakes create mutations that result in a diverse population of viruses inside the host. Mutations that are neither lethal to the virus nor strongly beneficial can lead to minority variants that are minor members of the virus population. However, preparing samples for sequencing can also introduce errors that resemble minority variants, resulting in the inclusion of false-positive data if not filtered correctly. In this study, we aimed to determine the best methods for identification and quantification of these minority variants by testing the performance of seven commonly used variant-calling tools. We used simulated and synthetic data to test their performance against a true set of variants and then used these studies to inform variant identification in data from SARS-CoV-2 clinical specimens. Together, analyses of our data provide extensive guidance for future studies of viral diversity and evolution.

Optimized Quantification of Intrahost Viral Diversity in SARS-CoV-2 and Influenza Virus Sequence Data.

High error rates of viral RNA-dependent RNA polymerases lead to diverse intra-host viral populations during infection. Errors made during replication that are not strongly deleterious to the virus can lead to the generation of minority variants. However, accurate detection of minority variants in viral sequence data is complicated by errors introduced during sample preparation and data analysis. We used synthetic RNA controls and simulated data to test seven variant calling tools across a range of allele frequencies and simulated coverages. We show that choice of variant caller, and use of replicate sequencing have the most significant impact on single nucleotide variant (SNV) discovery and demonstrate how both allele frequency and coverage thresholds impact both false discovery and false negative rates. We use these parameters to find minority variants in sequencing data from SARS-CoV-2 clinical specimens and provide guidance for studies of intrahost viral diversity using either single replicate data or data from technical replicates. Our study provides a framework for rigorous assessment of technical factors that impact SNV identification in viral samples and establishes heuristics that will inform and improve future studies of intrahost variation, viral diversity, and viral evolution. IMPORTANCE: When viruses replicate inside a host, the virus replication machinery makes mistakes. Over time, these mistakes create mutations that result in a diverse population of viruses inside the host. Mutations that are neither lethal to the virus, nor strongly beneficial, can lead to minority variants that are minor members of the virus population. However, preparing samples for sequencing can also introduce errors that resemble minority variants, resulting in inclusion of false positive data if not filtered correctly. In this study, we aimed to determine the best methods for identification and quantification of these minority variants by testing the performance of seven commonly used variant calling tools. We used simulated and synthetic data to test their performance against a true set of variants, and then used these studies to inform variant identification in data from clinical SARS-CoV-2 clinical specimens. Together, analyses of our data provide extensive guidance for future studies of viral diversity and evolution.

In vivo evolution of an emerging zoonotic bacterial pathogen in an immunocompromised human host.

Zoonotic transfer of animal pathogens to human hosts can generate novel agents, but the genetic events following such host jumps are not well studied. Here we characterize the mechanisms driving adaptive evolution of the emerging zoonotic pathogen Bordetella hinzii in a patient with interleukin-12 receptor ß1 deficiency. Genomic sequencing of 24 B. hinzii isolates cultured from blood and stool over 45 months revealed a clonal lineage that had undergone extensive within-host genetic and phenotypic diversification. Twenty of 24 isolates shared an E9G substitution in the DNA polymerase III ε-subunit active site, resulting in a proofreading deficiency. Within this proofreading-deficient clade, multiple lineages with mutations in DNA repair genes and altered mutational spectra emerged and dominated clinical cultures for more than 12 months. Multiple enzymes of the tricarboxylic acid cycle and gluconeogenesis pathways were repeatedly mutated, suggesting rapid metabolic adaptation to the human environment. Furthermore, an excess of G:C > T:A transversions suggested that oxidative stress shaped genetic diversification during adaptation. We propose that inactivation of DNA proofreading activity in combination with prolonged, but sub-lethal, oxidative attack resulting from the underlying host immunodeficiency facilitated rapid genomic adaptation. These findings suggest a fundamental role for host immune phenotype in shaping pathogen evolution following zoonotic infection.

In vitro antimicrobial effect and in vivo preventive and therapeutic effects of partially purified lantibiotic lacticin NK34 against infection by Staphylococcus species isolated from bovine mastitis.

Lantibiotics are small (<5 kDa), polycyclic peptides produced by gram-positive bacteria; they are also known as gram-positive bacteriocins. The high antimicrobial activity of lacticins and the continuing appearance of antibiotic-resistant bacteria in recent years have resulted in a renewed interest in lantibiotics. A partially purified form of lacticin NK34 (a Lactococcus lactis product isolated from the Korean fermented fish jeotgal) was tested to determine its antimicrobial effects against Staphylococcus aureus (n=20) and coagulase-negative Staphylococcus (CNS, n=20) strains isolated from the raw milk of cows with subclinical bovine mastitis in the present study. The spot-on-lawn assay was used to identify the 2 strains from each group with the greatest lacticin NK34 susceptibility, and the minimal lethal dose (MLD) was measured in ICR (imprinting control region) mice. The preventive and therapeutic effects of lacticin NK34 on the mouse infection model were determined for the first time. Lacticin NK34 demonstrated antimicrobial effects in 14 of 20 (70%) S. aureus indicator strains and in 18 of 20 (90%) CNS strains. Staphylococcus aureus 69 and S. simulans 55 demonstrated the greatest susceptibility to lacticin NK34 in the spot-on-lawn assay. The S. aureus 69 MLD was measured at 1.53 x 10(9) cfu/mouse, whereas the S. simulans 55 MLD was 3.59 x 10(9) cfu/mouse. Mice infected experimentally with S. aureus 69 MLD or S. simulans 55 MLD were treated with lacticin NK34. Treated mice demonstrated an 80% survival rate (48 of 60 mice) compared with a survival rate of 7.5% (3 of 40 mice) in control mice treated with distilled water. These data suggest that lacticin NK34 might be useful in the control of bovine mastitis and systemic bacterial infection.

Protracted course of disseminated adenovirus disease with necrotizing granulomas in the liver.

A 52- year-old male with chronic lymphocytic leukemia was hospitalized with disseminated adenovirus disease. More than a month following recovery, hepatic necrotizing granulomas secondary to adenovirus were found. This case illustrates the protracted course that adenovirus disease may take and emphasizes an unusual presentation with hepatic necrotizing granulomas.

Effects of alkaline pH on the stimulation of glucose transport in rat skeletal muscle.

Alkaline pH has been reported to cause release of Ca2+ from skeletal muscle sarcoplasmic reticulum (SR). Elevation of sarcoplasmic Ca2+ concentration is thought to stimulate glucose transport in skeletal muscle. In this context, we examined the effect of alkaline pH (extracellular pH of 8.6) on 3-O-methylglucose transport in skeletal muscle. Incubation of rat epitrochlearis muscles at pH 8.6 for 45 min resulted in an approx. 3-fold increase in glucose transport activity, which was not affected by reducing Ca2+ concentration in the incubation medium and essentially completely blocked by 25 microM dantrolene, an inhibitor of SR Ca2+ release. In addition to stimulating glucose transport by itself, alkaline pH may partially inhibit the stimulation of sugar transport by insulin hypoxia and contractions, as the combined effect of alkaline pH and the maximal effect of insulin, contractions, or hypoxia on glucose transport are not different from the maximal effects of insulin, hypoxia, or contractions alone. The maximal effects of insulin and contractions, and of insulin and hypoxia, on glucose transport are normally additive in muscle. Alkaline pH completely prevented this additivity. In summary, our results show that alkaline pH stimulates glucose transport activity in skeletal muscle and provide evidence suggesting that this effect is mediated by Ca2+. They further show that alkaline pH blocks the additivity of the maximal effects of insulin and contractions or hypoxia suggesting that alkaline pH may partially inhibit the stimulation of glucose transport by insulin, contraction and hypoxia.

Conversion of oral glucose to lactate in dogs. Primary site and relative contribution to blood lactate.

We evaluated the relative contribution of oral glucose to arterial lactate and the relative role of the splanchnic bed in converting glucose to lactate in normal healthy dogs. After an oral glucose load (1.2 g/kg) spiked with [U-14C]glucose (16.9 muCi/kg; protocol 1, n = 7), arterial blood lactate increased from 0.43 +/- 0.03 mM at basal to a peak of 1.04 +/- 0.07 mM at 45 min and then slowly decreased to 0.47 +/- 0.07 mM at 240 min. Arterial blood [14C]lactate peaked at 60 min and then decreased slowly to approximately 35% of the peak at 4 h. When arterial blood lactate peaked at 45 min, the proportion of arterial lactate that was derived from oral glucose was 34 +/- 3%. The integrated area under the curve of lactate derived from exogenous glucose was 40 +/- 2% of that of total lactate. The splanchnic bed released lactate and [14C]lactate during the initial 2 h after oral [14C]glucose. Thus, the splanchnic bed apparently contributed to the conversion of exogenous glucose to lactate. In the matched experiments (protocol 2, n = 5), dogs were given the same amount of oral glucose but no [14C]glucose, and [U-14C]lactate was infused into the right atrium to match the splanchnic [14C]lactate release from the first experiment. Despite a well-matched splanchnic [14C]lactate contribution, arterial concentrations of [14C]lactate were markedly lower in protocol 2 compared with protocol 1. The integrated area under the [14C]lactate profile in protocol 2 was only 11 +/- 1% of that in protocol 1. These results indicate that the splanchnic bed is responsible for only 11% of arterial blood lactate that was derived from oral glucose. We concluded that 1) after oral glucose loading, a major portion of circulating lactate has its origin not in exogenous glucose but in endogenous sources, and 2) the splanchnic bed is not the major site of oral glucose conversion to lactate after glucose ingestion.

Fasting does not impair insulin-stimulated glucose uptake but alters intracellular glucose metabolism in conscious rats.

Effects of 24-h and 48-h fasting on maximal insulin-stimulated whole-body and muscle glucose uptake, glycogen synthesis, and glycolysis were studied in conscious rats by combining the glucose clamp technique with tracer methods. Fasting decreased body weight and basal plasma glucose, plasma insulin, hepatic glucose output, and glucose clearance (P < 0.05 for all). However, maximal insulin-stimulated whole-body glucose uptake, normalized to body weight, was almost identical in fed, 24-h fasted, and 48-h fasted rats (191 +/- 8, 185 +/- 14, and 182 +/- 5 mumol.kg-1.min-1, respectively; P > 0.7). Similarly, rates of insulin-stimulated glucose uptake by four different skeletal muscles, estimated by the 2-deoxyglucose injection technique, were not different among the three groups. In contrast to glucose uptake, insulin-stimulated whole-body glycolysis was decreased significantly after fasting (36% after 48 h fasting; P < 0.05), whereas insulin-stimulated whole-body glycogen synthesis was increased (44% after 48 h fasting; P < 0.05). In fed rats, glycolysis was the major pathway for glucose metabolism during hyperinsulinemia, accounting for 60 +/- 5% of glucose uptake. This fraction was decreased significantly by fasting (P < 0.01), so that after a 48-h fast, glycolysis accounted for only 40 +/- 3% of insulin-stimulated glucose uptake and glycogen synthesis became predominant pathway, accounting for 60 +/- 3% of whole-body glucose utilization. Whole-body patterns of glucose metabolism during hyperinsulinemia were paralleled by glucose metabolism in individual muscles.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic impairment precedes insulin resistance in skeletal muscle during high-fat feeding in rats.

To examine whether impairment of intracellular glucose metabolism precedes insulin resistance, we determined the time courses of changes in insulin-stimulated glucose uptake, glycolysis, and glycogen synthesis during high-fat feeding in rats. Animals were fed with a high-fat (66.5%) diet ad libitum for 0, 2, 4, 7, or 14 days (n = 10-11 in each group) after 5 days of a low-fat (12.5%) diet. Submaximal and maximal insulin-stimulated glucose fluxes were estimated in whole body and individual skeletal muscles using the glucose clamp technique combined with D-[3-3H]glucose infusion and 2-[1-14C]deoxyglucose injection. Both submaximal and maximal insulin-stimulated glucose uptake in whole body decreased gradually with high-fat feeding. However, the decreases were minimal and not statistically significant during the initial few days (i.e., 2 and 4 days) of high-fat feeding (P > 0.05). In contrast, insulin-stimulated whole-body glycolysis (both maximal and submaximal) significantly decreased by approximately 30% with 2 days of high-fat feeding and remained suppressed thereafter (P < 0.05). Similar patterns of changes in insulin-stimulated glucose uptake and glycolysis were also observed in skeletal muscle. Insulin-stimulated glycogen synthesis and glucose-6-phosphate (G-6-P) concentrations in skeletal muscle increased significantly during the initial few days of high-fat feeding and gradually returned to control levels by day 14, suggesting that increased G-6-P concentrations were responsible for increased glycogen synthesis. Thus, suppression of insulin-stimulated glycolysis and a compensatory increase in glycogen synthesis (presumably arising from the glucose-fatty acid cycle) preceded decreases in insulin-stimulated glucose uptake in skeletal muscle during high-fat feeding. These findings suggest that the insulin resistance may develop as a secondary response to impaired intracellular glucose metabolism.

Enhanced glucose tolerance in spontaneously hypertensive rats. Pancreatic beta-cell hyperfunction with normal insulin sensitivity.

We used intravenous glucose tolerance tests in vivo and 3-O-methylglucose transport into skeletal muscle in vitro to assess glucose tolerance, pancreatic beta-cell function, and insulin action in 9- to 11-wk-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar Kyoto rats (WKY). Body weight was slightly higher in the WKY (P less than 0.001), while blood pressure was elevated in the SHR (P less than 0.001). Insulin responses to intravenous glucose after 4 or 12 h of fasting in SHR were 2-3 times the responses of WKY rats (P less than 0.001). The greater insulin responses in SHR were associated with accelerated glucose disappearance P less than 0.001 vs. WKY rats). A direct correlation (r = 0.49, P less than 0.05) between the peak plasma insulin responses to glucose and Kg values in SHR suggested that the exaggerated insulin responses contributed to the accelerated glucose disappearance in that group. 3-O-methylglucose transport rates into epitrochlearis muscles in vitro did not differ significantly between SHR and WKY groups in the absence of insulin (P less than 0.2) or in the presence of insulin at physiological (600 pM, P greater than 0.4) or pharmacological (120,000 pM, P greater than 0.9) concentrations. Thus, compared with WKY rats, SHR had exaggerated insulin responses to glucose, similar insulin-mediated glucose transport into skeletal muscle, and enhanced glucose tolerance. Our findings indicate that young, hypertensive SHR have hyperfunction of pancreatic beta-cells that is unrelated to insulin resistance. The resultant nutrient-stimulated hyperinsulinemia could play a role in the development or maintenance of elevated blood pressure in SHR.

Plasma free fatty acids decrease insulin-stimulated skeletal muscle glucose uptake by suppressing glycolysis in conscious rats.

The effects of elevated plasma free fatty acid (FFA) levels on insulin -stimulated whole-body and skeletal muscle glucose transport, glucose uptake, glycolysis, and glycogen synthesis were studied in conscious rats during hyperinsulinemic-euglycemic clamps with (n = 26) or without (n = 23) Intralipid and heparin infusion. Whole-body and skeletal muscle glucose uptake, glycolysis, and glycogen synthesis were estimated using D-[3-3H]glucose and 2-[14C]deoxyglucose (study 1), and glucose transport activity was assessed by analyzing plasma kinetics of L-[14C]glucose and 3-O-[3H]-methylglucose (study 2). Plasma FFA levels decreased during the clamps without intralipid but increased above basal during the clamps with Intralipid infusion (P < 0.01 for both). Elevated plasma FFA levels decreased insulin-stimulated whole-body glucose uptake by approximately 15% and approximately 20% during physiological and maximal insulin clamps, respectively (P < 0.01). Similarly, insulin-stimulated glucose uptake was also decreased in individual skeletal muscles with Intralipid infusion (P < 0.05). The most profound effect of elevated plasma FFA levels was a 30-50% suppression of insulin-stimulated glycolysis in whole body and individual skeletal muscles in both clamps. In contrast, physiological insulin-stimulated glycogen synthesis was increased with elevated plasma FFA levels in whole body and individual skeletal muscles (P < 0.05). Glucose-6-phosphate (G-6-P) levels were increased in soleus and extensor digitorum longus (EDL) muscles with Intralipid infusion in both clamps (P < 0.05). Intralipid infusion did not alter the time profiles of plasma L-glucose and 3-O-methylglucose after an intravenous injection during maximal insulin clamps, and compartmental analysis indicated no significant effect of elevated FFA levels on glucose transport activity in insulin-sensitive tissues (P > 0.05). Thus, elevated plasma FFA decreased insulin-stimulated glucose uptake in skeletal muscle by suppressing glycolysis and increasing G-6-P levels. These findings suggest that the classic glucose-fatty acid cycle was the predominant mechanism underlying the inhibitory effect of FFA on skeletal muscle glucose uptake.

Free fatty acids induce peripheral insulin resistance without increasing muscle hexosamine pathway product levels in rats.

To evaluate the role of the hexosamine biosynthesis pathway (HBP) in fat-induced insulin resistance, we examined whether fat-induced insulin resistance is additive to that induced by increased HBP flux via glucosamine infusion and, if so, whether such additive effects correlate with muscle HBP product levels. Prolonged hyperinsulinemic (approximately 550 pmol/l) euglycemic clamps were conducted in conscious overnight-fasted rats. After the initial 150 min to attain steady-state insulin action, rats received an additional infusion of saline, Intralipid, glucosamine, or Intralipid and glucosamine (n = 8 or 9 for each) for 330 min. At the conclusion of clamps, skeletal muscles (soleus, extensor digitorum longus, and tibialis anterior) were taken for the measurement of HBP product levels. Intralipid and glucosamine infusions decreased insulin-stimulated glucose uptake (Rd) by 38 and 28%, respectively. When the infusions were combined, insulin-stimulated Rd decreased 47%, significantly more than with Intralipid or glucosamine alone (P < 0.05). The glucosamine-induced insulin resistance was associated with four- to fivefold increases in muscle HBP product levels. In contrast, the Intralipid-induced insulin resistance was accompanied by absolutely no increase in HBP product levels in all of the muscles examined. Also, when infused with glucosamine, Intralipid decreased insulin action below that with glucosamine alone without changing HBP product levels. In a separate study, short-term (50 and 180 min) Intralipid infusion also failed to increase muscle HBP product levels. In conclusion, increased availability of plasma free fatty acids induces peripheral insulin resistance without increasing HBP product levels in skeletal muscle.

Time courses of changes in hepatic and skeletal muscle insulin action and GLUT4 protein in skeletal muscle after STZ injection.

To determine the relative time courses of changes in peripheral and hepatic insulin action and skeletal muscle GLUT4 protein levels after a streptozotocin (STZ) injection in rats, we performed hyperinsulinemic (14-18 nM), euglycemic (7.5 mM) clamps in control (n = 8) and diabetic rats at 1 (n = 7), 3 (n = 8), 7 (n = 8), and 14 (n = 6) days after intraperitoneal STZ (65 mg/kg). Basal plasma glucose concentrations increased from 8.1 +/- 0.2 mM in control rats to 23.5 +/- 1.2 mM 1 day after STZ (P < 0.01) and remained constant thereafter. Basal plasma insulin levels were approximately 35% of control levels in all STZ groups (P < 0.01). Insulin-stimulated whole-body glucose uptake decreased significantly as early as one day after STZ injection (P < 0.01), resulting predominantly from a decrease in whole-body glycolysis. Insulin action to suppress hepatic glucose output was normal on day 1 after STZ but impaired markedly on day 3 and thereafter (P < 0.01). Insulin-stimulated glucose uptake in individual skeletal muscles was not altered until day 7 after STZ, and the magnitudes of decreases in skeletal muscle insulin action on days 7 and 14 were not fully accounted for by the decreases in GLUT4 protein level measured from the same muscles. Our data indicate that there is a temporal hierarchy in the development of insulin resistance in STZ-induced diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular screening for Rickettsia, Anaplasmataceae and Coxiella burnetii in Rhipicephalus sanguineus ticks from Malaysia.

A total of 44 Rhipicephalus sanguineus ticks collected from 23 dogs from Malaysia were screened for Rickettsia, Anaplasmataceae and Coxiella burnetii. Coxiella burnetii was detected in 59% (26/44) of ticks however Rickettsia and Anaplasmataceae were not detected in any of the ticks. In order to genotype the strains of C. burnetii, multispacer sequence typing (MST) was carried out using three different spacers. One of the spacers; Cox2 successfully amplified a fragment for which the full length sequence of 397 bp was obtained. The sequenced product revealed only a single nucleotide difference with the Cox2.3 type sequence.

Electrophilic cyclizations of vinylcyclopropanols to tethered aldehydes.

[reaction: see text]. The intramolecular, stereoselective addition of 1-vinylcyclopropanols to tethered aldehydes has been achieved under mild conditions. Thus, sequential application of the titanium-mediated cyclopropanation of alpha,beta-unsaturated esters and the electrophilic cyclization of the aldehyde-tethered cyclopropanol products provides the facile formation of carbocyclic rings.

Glucose transporters and maximal transport are increased in endurance-trained rat soleus.

Voluntary wheel running induces an increase in the concentration of the regulatable glucose transporter (GLUT4) in rat plantaris muscle but not in soleus muscle (K. J. Rodnick, J. O. Holloszy, C. E. Mondon, and D. E. James. Diabetes 39: 1425-1429, 1990). Wheel running also causes hypertrophy of the soleus in rats. This study was undertaken to ascertain whether endurance training that induces enzymatic adaptations but no hypertrophy results in an increase in the concentration of GLUT4 protein in rat soleus (slow-twitch red) muscle and, if it does, to determine whether there is a concomitant increase in maximal glucose transport activity. Female rats were trained by treadmill running at 25 m/min up a 15% grade, 90 min/day, 6 days/wk for 3 wk. This training program induced increases of 52% in citrate synthase activity, 66% in hexokinase activity, and 47% in immunoreactive GLUT4 protein concentration in soleus muscles without causing hypertrophy. Glucose transport activity stimulated maximally with insulin plus contractile activity was increased to roughly the same extent (44%) as GLUT4 protein content in soleus muscle by the treadmill exercise training. In a second set of experiments, we examined whether a swim-training program increases glucose transport activity in the soleus in the presence of a maximally effective concentration of insulin. The swimming program induced a 44% increase in immunoreactive GLUT4 protein concentration. Glucose transport activity maximally stimulated with insulin was 62% greater in soleus muscle of the swimmers than in untrained controls. Training did not alter the basal rate of 2-deoxyglucose uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

CD24+ cells from hierarchically organized ovarian cancer are enriched in cancer stem cells.

Cancer stem cells (CSCs) have been identified in solid tumors and cancer cell lines. In this study, we isolated a series of cancer cell clones, which were heterogeneous in growth rate, cell cycle distribution and expression profile of genes and proteins, from ovarian tumor specimens of a patient and identified a sub-population enriched for ovarian CSCs defined by CD24 phenotype. Experiments in vitro demonstrated CD24(+) sub-population possessed stem cell-like characteristics of remaining quiescence and more chemoresistant compared with CD24(-) fraction, as well as a specific capacity for self-renewal and differentiation. In addition, injection of 5 x 10(3) CD24(+) cells was able to form tumor xenografts in nude mice, whereas equal number of CD24(-) cells remained nontumorigenic. We also found that CD24(+) cells expressed higher mRNA levels of some 'stemness' genes, including Nestin, beta-catenin, Bmi-1, Oct4, Oct3/4, Notch1 and Notch4 which were involved in modulating many functions of stem cells, and lower E-cadherin mRNA level than CD24(-) cells. Altogether, these observations suggest human ovarian tumor cells are organized as a hierarchy and CD24 demarcates an ovarian cancer-initiating cell population. These findings will have important clinical applications for developing effective therapeutic strategies to treat ovarian cancer.

Patterns of glycogen turnover in liver characterized by computer modeling.

We used a computer model of liver glycogen turnover to reexamine the data of Devos and Hers, who reported the time course of accumulation in and loss from glycogen of label originating in [1-14C]galactose injected at different times after the start of refeeding of 40-h fasted mice or rats. In the present study computer representation of individual glycogen molecules was utilized to account for growth and degradation of glycogen according to specific hypothetical patterns. Using this model we could predict the accumulation and localization within glycogen of labeled glucose residues and compare the predictions with the previously published data. We considered three specific hypotheses of glycogen accumulation during refeeding: 1) simultaneous, 2) sequential, and 3) accelerating growth. Hypothetical patterns of glycogen degradation were 1) ordered and 2) random degradation. The pattern of glycogen synthesis consistent with experimental data was a steadily increasing number of growing glycogen molecules, whereas during degradation glycogen molecules are exposed to degrading enzymes randomly, rather than in a specific reverse order of synthesis. These patterns predict the existence of a specific mechanism for the steadily increasing "seeding" of new glycogen molecules during synthesis.

Prolonged suppression of glucose metabolism causes insulin resistance in rat skeletal muscle.

To determine whether an impairment of intracellular glucose metabolism causes insulin resistance, we examined the effects of suppression of glycolysis or glycogen synthesis on whole body and skeletal muscle insulin-stimulated glucose uptake during 450-min hyperinsulinemic euglycemic clamps in conscious rats. After the initial 150 min to attain steady-state insulin action, animals received an additional infusion of saline, Intralipid and heparin (to suppress glycolysis), or amylin (to suppress glycogen synthesis) for up to 300 min. Insulin-stimulated whole body glucose fluxes were constant with saline infusion (n = 7). In contrast, Intralipid infusion (n = 7) suppressed glycolysis by approximately 32%, and amylin infusion (n = 7) suppressed glycogen synthesis by approximately 45% within 30 min after the start of the infusions (P < 0.05). The suppression of metabolic fluxes increased muscle glucose 6-phosphate levels (P < 0.05), but this did not immediately affect insulin-stimulated glucose uptake due to compensatory increases in other metabolic fluxes. Insulin-stimulated whole body glucose uptake started to decrease at approximately 60 min and was significantly decreased by approximately 30% at the end of clamps (P < 0.05). Similar patterns of changes in insulin-stimulated glucose fluxes were observed in individual skeletal muscles. Thus the suppression of intracellular glucose metabolism caused decreases in insulin-stimulated glucose uptake through a cellular adaptive mechanism in response to a prolonged elevation of glucose 6-phosphate rather than the classic mechanism involving glucose 6-phosphate inhibition of hexokinase.