The Macrophage Inhibitor CNI-1493 Blocks Metastasis in a Mouse Model of Ewing Sarcoma through Inhibition of Extravasation

Metastatic Ewing Sarcoma carries a poor prognosis, and novel therapeutics to prevent and treat metastatic disease are greatly needed. Recent evidence demonstrates that tumor-associated macrophages in Ewing Sarcoma are associated with more advanced disease. While some macrophage phenotypes (M1) exhibit anti-tumor activity, distinct phenotypes (M2) may contribute to malignant progression and metastasis. In this study, we show that M2 macrophages promote Ewing Sarcoma invasion and extravasation, pointing to a potential target of anti-metastatic therapy. CNI-1493 is a selective inhibitor of macrophage function and has shown to be safe in clinical trials as an anti-inflammatory agent. In a xenograft mouse model of metastatic Ewing Sarcoma, CNI-1493 treatment dramatically reduces metastatic tumor burden. Furthermore, metastases in treated animals have a less invasive morphology. We show in vitro that CNI-1493 decreases M2-stimulated Ewing Sarcoma tumor cell invasion and extravasation, offering a functional mechanism through which CNI-1493 attenuates metastasis. These data indicate that CNI-1493 may be a safe and effective adjuvant agent for the prevention and treatment of metastatic Ewing Sarcoma.     

Mlc1p Is a Light Chain for the Unconventional Myosin Myo2p in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the unconventional myosin Myo2p is of fundamental importance in polarized growth. We explore the role of the neck region and its associated light chains in regulating Myo2p function. Surprisingly, we find that precise deletion of the six IQ sites in the neck region results in a myosin, Myo2-Δ6IQp, that can support the growth of a yeast strain at 90% the rate of a wild-type isogenic strain. We exploit this mutant in a characterization of the light chains of Myo2p. First, we demonstrate that the localization of calmodulin to sites of polarized growth largely depends on the IQ sites in the neck of Myo2p. Second, we demonstrate that a previously uncharacterized protein, Mlc1p, is a myosin light chain of Myo2p. MLC1 (YGL106w) is an essential gene that exhibits haploinsufficiency. Reduced levels of MYO2 overcome the haploinsufficiency of MLC1. The mutant MYO2-Δ6IQ is able to suppress haploinsufficiency but not deletion of MLC1. We used a modified gel overlay assay to demonstrate a direct interaction between Mlc1p and the neck of Myo2p. Overexpression of MYO2 is toxic, causing a severe decrease in growth rate. When MYO2 is overexpressed, Myo2p is fourfold less stable than in a wild-type strain. High copies of MLC1 completely overcome the growth defects and increase the stability of Myo2p. Our results suggest that Mlc1p is responsible for stabilizing this myosin by binding to the neck region.     

Selective Brain Cooling Reduces Water Turnover in Dehydrated Sheep

In artiodactyls, arterial blood destined for the brain can be cooled through counter-current heat exchange within the cavernous sinus via a process called selective brain cooling. We test the hypothesis that selective brain cooling, which results in lowered hypothalamic temperature, contributes to water conservation in sheep. Nine Dorper sheep, instrumented to provide measurements of carotid blood and brain temperature, were dosed with deuterium oxide (D₂O), exposed to heat for 8 days (40◦C for 6-h per day) and deprived of water for the last five days (days 3 to 8). Plasma osmolality increased and the body water fraction decreased over the five days of water deprivation, with the sheep losing 16.7% of their body mass. Following water deprivation, both the mean 24h carotid blood temperature and the mean 24h brain temperature increased, but carotid blood temperature increased more than did brain temperature resulting in increased selective brain cooling. There was considerable inter-individual variation in the degree to which individual sheep used selective brain cooling. In general, sheep spent more time using selective brain cooling, and it was of greater magnitude, when dehydrated compared to when they were euhydrated. We found a significant positive correlation between selective brain cooling magnitude and osmolality (an index of hydration state). Both the magnitude of selective brain cooling and the proportion of time that sheep spent selective brain cooling were negatively correlated with water turnover. Sheep that used selective brain cooling more frequently, and with greater magnitude, lost less water than did conspecifics using selective brain cooling less efficiently. Our results show that a 50kg sheep can save 2.6L of water per day (~60% of daily water intake) when it employs selective brain cooling for 50% of the day during heat exposure. We conclude that selective brain cooling has a water conservation function in artiodactyls.     

Clutch size relative to tree cavity size in Northern Flickers

We analysed clutch size versus nest size in 153 broods of the Northern Flicker Colaptes auratus , a woodpecker using natural cavities in British Columbia, Canada. Larger volume cavities were less susceptible to predation and cavity size was positively associated with the age and body size of males and with the body condition of female parents. Although clutches varied between 4 and 11 eggs, and the floor area of cavities varied about 5-fold, we found no relationship between clutch size and floor area or cavity volume. To see if there were fitness consequences to clutch size relative to nest size, we examined hatching success and nestling mortality in flicker broods. Hatching success was not related to cavity size, but crowding slightly reduced nestling survival even when clutch size was controlled statistically. However, there was no effect of cavity size on the total number of nestlings fledged. Newly excavated flicker cavities were smaller than reused cavities suggesting a cost to excavation. This cost, coupled with the minimal fitness consequences of overcrowding, may explain why flickers do not adjust clutch size to cavity size.     

Reactivation of inhibited bone acid phosphatase and its significance in bone histomorphometry

Despite biochemical demonstration of acid phosphatase (AcP) activation or reactivation in bone, few attempts have been made to show similar effects histochemically. Bones from growing rats, when fixed in 4% buffered formaldehyde at room temperature and demineralized in 5% formic acid, exhibited expected inactivation of AcP. The inhibited AcP, however, was reactivated by pre-incubation of sections for 1 hr at 37 degrees C in the following buffers: 0.2 M Tris, 0.2 M glycine, 0.2 M NaHCO3, or 0.1 M borax, as well as in alkaline water, but not in 0.2 M Na2HPO4 (all at pH 9). The reactivation was (a) site-specific (e.g., osteoclasts, osteoblasts, osteocytes, and cement lines), (b) temperature- and pH-dependent, (c) unaffected by OH- or SH--binding agents or by an alkaline phosphatase inhibitor, and (d) inhibited completely by 10 mM Na2HPO4. The reactivation process, much simplified and/or more effective than with the methods previously reported, was observed in all 83 human biopsy bones embedded in methyl methacrylate and in human bones stored in cold buffered formaldehyde for 7 months. This study demonstrates a unique method for reactivating and thus localizing the inhibited AcP in bones, and suggests possible applications in bone histomorphometry.     

Complications of Gastroesophageal Reflux

An edited summary of an Interdepartmental Conference arranged by the Department of Medicine of the UCLA School of Medicine, Los Angeles. The Director of Conferences is William M. Pardridge, MD, Professor of Medicine.Several specialists have recently recognized that gastrointestinal reflux causes complications resulting in significant disease. It causes discomfort, indigestion, esophagitis, Barrett''s esophagus, and carcinoma of the esophagus. Pediatricians attribute many early pulmonary problems, and even some sudden deaths in infants, to the reflux of gastric contents. Otolaryngologists now recognize that many cases of nonbacterial, nonspecific pharyngitis and laryngitis are due to the reflux of gastrc acid secretions. Contact granuloma and cancer of the larynx may, in some instances, be secondary to nocturnal reflux. Thoracic surgeons and pulmonologists believe chronic tracheobronchitis and some cases of pulmonary disease are attributable to recurrent bathing of the respiratory epithelium by aspirated gastric contents. An awareness of the many complications of gastrointestinal reflux should lead to a multidisciplined attack on the factors responsible for these diseases.     

Reconstitution of ATP-dependent calcium transport from streptococci

Membrane vesicles of three streptococcal strains (Streptococcus faecalis, Streptococcus lactis, and Streptococcus sanguis) were extracted with octyl-beta-D-glucoside in the presence of Escherichia coli lipid and glycerol. For reconstitution, the detergent extract was mixed with bath-sonicated E. coli lipid, in the presence of octyl-beta-D-glucoside, and proteoliposomes were formed by a 25-fold dilution. ATP-dependent calcium accumulation by proteoliposomes was comparable to that found in parent vesicles. Recovery of this calcium transport activity was dependent on the inclusion of an osmolyte protein stabilant (glycerol, etc.) during solubilization. The properties of ATP-driven calcium transport were studied in the reconstituted system. In proteoliposomes, ATP-linked calcium accumulation was not affected by the protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, or by the ionophores, valinomycin and nigericin, in the presence of potassium, or by N,N'-dicyclohexylcarbodiimide, an inhibitor of the F0F1-ATPase. On the other hand, calcium transport was completely blocked by micromolar levels of orthovanadate; half-maximal inhibitions were observed at 0.4, 4, and 4 microM vanadate, for S. faecalis, S. lactis, and S. sanguis, respectively. This marked sensitivity to orthovanadate suggests operation of an E1E2-type ion-motive pump. These data demonstrate that, in a reconstituted system, calcium transport is not linked to an ATP-dependent proton circulation via the F0F1-ATPase, but rather is driven by a calcium-translocating ATPase. Thus, calcium extrusion from the cytosol of enteric, lactic acid, or oral streptococci is mediated by an ATP-linked process analogous to the ion-motive ATPases of eukaryotic membranes.     

Bacterial anion exchange. Use of osmolytes during solubilization and reconstitution of phosphate-linked antiport from Streptococcus lactis

Membranes of Streptococcus lactis were solubilized with 1.1% octyl-beta-D-glucopyranoside in the presence of 0.37% acetone/ether-washed phospholipid from several sources. After adding excess Escherichia coli phospholipid as bath-sonicated liposomes, phosphate:sugar phosphate antiport was reconstituted in proteoliposomes by a 25-fold dilution in 0.1 M KPi (pH 7). Assays of 32Pi:Pi exchange showed that antiport was subject to an inactivation which varied in severity according to the lipid present at solubilization. Recovery of Pi-linked exchange was improved by the presence of 10-20% glycerol or other osmolyte during extraction. The osmolytes tested in this regard have included polyols (glycerol, erythritol, xylitol, sorbitol), sugars (glucose, trehalose), and two amino acids (glycine, proline). Each gave 10--20-fold increased recoveries of 32Pi:Pi antiport compared to controls using only detergent and lipid; these precautions were not required for the efficient reconstitution of F0F1-ATPase. Antiport in the artificial system was studied most carefully when glycerol was the stabilizing additive. For that case, the Kt values for Pi or 2-deoxyglucose 6-phosphate transport (275 and 25 microM, respectively) were the same as in native membranes. Maximal rates of Pi and 2-deoxyglucose 6-phosphate transport (200 and 42 nmol/min/mg of protein, respectively) and the turnover number for Pi exchange (25--50/s) suggested that antiporters were recovered without loss of activity. We conclude that the quantitative aspects of bacterial anion exchange are amenable to study in an artificial system, and that the use of osmolytes as general stabilants can be a valuable adjunct to current techniques for reconstitution of integral membrane transport proteins.     

Dilute acid hydrolysis of paper birch: kinetics studies of xylan and acetyl-group hydrolysis

Batch hydrolysis kinetics of paper birch (Betula papyrifera) xylan and its associated acetyl groups in dilute sulfuric acid have been measured for acid concentrations of between 0.04 and 0.18M and temperatures of between 100 and 170 degrees C. Only 5% of the cellulose was hydrolyzed for up to 85% xylan removal. Rate data were correlated well by a parallel reaction model based on the existence of reactive and resistant xylan portions. The resulting rate equation predicts the experimental xylan concentrations in the residue to within 10%. Hydrolysis of xylan-associated acetyl groups was found to occur at the same rate as that of xylan, except at 100 degrees C, where acetyl is released preferentially. No effect of acid concentration on the rate of acetyl removal relative to that of xylan was evident.