Tunicamycin reduces Na(+)-K(+)-pump expression in cultured skeletal muscle.

The purpose of this study was to examine effects of tunicamycin (TM), which inhibits core glycosylation of the beta-subunit, on functional expression of the Na(+)-K+ pump in primary cultures of embryonic chick skeletal muscle. Measurements were made of specific-[3H]-ouabain binding, ouabain-sensitive 86Rb uptake, resting membrane potential (Em), and electrogenic pump contribution to Em (Ep) of single myotubes with intracellular microelectrodes. Growth of 4-6-day-old skeletal myotubes in the presence of TM (1 microgram/ml) for 21-24 hr reduced the number of Na(+)-K+ pumps to 60-90% of control. Na(+)-K+ pump activity, the level of resting Em and Ep were also reduced significantly by TM. In addition, TM completely blocked the hyperpolarization of Em induced in single myotubes by cooling to 10 degrees C and then re-warming to 37 degrees C. Effects of tunicamycin were compared with those of tetrodotoxin (TTX; 2 x 10(-7) M for 24 hr), which blocks voltage-dependent Na+ channels. TM produced significantly greater decreases in ouabain-binding and Em than did TTX, findings that indicate that reduced Na(+)-K+ pump expression was not exclusively secondary to decreased intracellular Na+, the primary regulator of pump synthesis in cultured muscle. Similarly, effects of TM were significantly greater than those of cycloheximide, which inhibits protein synthesis by 95%. These findings demonstrate that effects were not due to inhibition of protein synthesis. We conclude that glycosylation of the Na(+)-K+ pump beta-subunit is required for full physiological expression of pump activity in skeletal muscle.     

Crystal structures of alpha-lytic protease complexes with irreversibly bound phosphonate esters

The structures of the complexes with alpha-lytic protease of both phosphorus stereoisomers of N-[(2S)-2-[[[(1R)-1-[N-[(tert-butyloxycarbonyl)-L-alanyl-L-alanyl- L-prolyl]amino]-2-methylpropyl]-phenoxyphosphinyl]oxy]propanoyl]- L-alanine methyl ester, an analogue of the peptide Boc-Ala-Ala-Pro-Val-Ala-Ala where Val is replaced with an analogous phosphonate phenyl ester and the subsequent Ala is replaced with lactate, have been determined to high resolution (1.9 A) by X-ray crystallography. Both stereoisomers inactivate the enzyme but differ by a factor of 2 in the second-order rate constant for inactivation [Sampson, N. S., & Bartlett, P. A. (1991) Biochemistry (preceding paper in this issue)]. One isomer (B) forms a tetrahedral adduct in which the phosphonate phenyl ester is displaced by the active site serine (S195) and interacts with the enzyme across seven substrate recognition sites that span both sides of the scissile bond. Seven hydrogen bonds are formed with the enzyme, and 510 A2 of hydrophobic surface area is buried when the inhibitor interacts with the enzyme. Although two hydrogen bonds are gained by incorporation of two residues on the C-terminal side of the scissile bond into the inhibitor, there is very little adjustment in the structure of the enzyme in this region. Surprisingly, the active site histidine (H57) does not interact with the phosphonate, apparently because the phosphonate lacks negative charge in or near the oxyanion hole, and instead, the side chain rotates out of the active site cleft and hydrogen bonds with solvent. The other isomer (A) forms a mixture of two different tetrahedral adducts in the active site, both covalently bonded to Ser 195. One adduct, at approximately 58% occupancy, is exactly the same in structure as the complex formed with isomer B, and the other adduct, at 42% occupancy, has lost the two residues C-terminal to the scissile bond by hydrolysis. In the lower occupancy structure, His 57 does not rotate out of the active site and forms a hydrogen bond with the phosphonate oxygen instead. The structures of both complexes were insensitive to pH. As very little change in structure accompanies the histidine rotation, the complex with isomer B provides an excellent mimic for the structure of the transition state (or high-energy reaction intermediate) that spans both sides of the scissile bond.     

Isolation and partial characterization of proteoglycans from sheep lung parenchyma.

Greater than 90% of the proteoglycans of sheep lung parenchyma, as measured by uronic acid, were solubilized employing a sequential procedure with guanidine hydrochloride, dithiothreitol and Triton X-100. The amounts solubilized were 68.7%, 16.2% and 5.9%, respectively. The guanidine hydrochloride extract was chromatographed using DEAE-cellulose in urea and eluted with increasing concentrations of NaCl. A major fraction (containing a 6.5-fold enrichment of uronic acid) was obtained with 0.5 M NaCl and further purified by Sepharose Cl-6B chromatography in guanidine hydrochloride. To demonstrate the presence of protein-linked glycosaminoglycans, the void volume peak containing protein and uronic acid was digested with papain and rechromatographed. Evidence for the presence of proteoglycans was obtained by observing an almost complete loss of uronic acid in the void volume and the appearance of a uronic acid peak in the included volume, migrating in the same area as single-chain glycosaminoglycans. Electrophoretic migration and disappearance of bands in electrophoresis after digestion with specific mucopolysaccharide lyases indicated that the small amount of uronic acid remaining in the void volume was hyaluronic acid whereas the included volume contained hyaluronic acid, heparan sulfate, chondroitin sulfates and/or dermatan sulfate.     

MMP13 is a critical target gene during the progression of osteoarthritis

Introduction

Osteoarthritis (OA) is a degenerative joint disease affecting a large population of people. The mechanism of this highly prevalent disease is not fully understood. Currently there is no effective disease-modifying treatment for OA. The purpose of this study was two-fold: 1) to investigate the role of MMP13 in the development of OA; and 2) to evaluate the efficacy of the MMP13 inhibitor CL82198 as a pharmacologic treatment for preventing OA progression.

Methods

To investigate the role of the endogenous Mmp13 gene in OA development, tamoxifen was administered to two-week-old Col2CreER;Mmp13^fx/fx (Mmp13^Col2ER) and Cre-negative control mice for five days. OA was induced by meniscal-ligamentous injury (MLI) when the mice were 10 weeks old and MLI or sham-operated joints were harvested 4, 8, 12, or 16 weeks after surgery. To evaluate the efficacy of CL82198, MLI surgery was performed on 10-week-old wild type mice. CL82198 or saline was administered to the mice daily beginning immediately after the surgery for up to 16 weeks. The joint tissues collected from both experiments were evaluated by cartilage grading, histology/histomorphometry, immunohistochemistry (IHC), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The ability of CL82198 to inhibit MMP13 activity in vitro was confirmed by ELISA.

Results

The OA progression was decelerated in Mmp13^Col2ER mice 8, 12, and 16 weeks post-surgery. Cartilage grading by blinded observers confirmed decreased articular cartilage degeneration in Mmp13^Col2ER mice at 8, 12 and 16 weeks compared to Cre-negative mice. Histomorphometric analysis demonstrated that Mmp13^Col2ER mice had a higher articular cartilage area and thickness at 12 and 16 weeks post-surgery compared to the control mice. Results of IHC revealed greater type II collagen and proteoglycan expression in Mmp13^Col2ER mice. Chondrocyte apoptosis, as determined by TUNEL staining, was higher in control mice compared to Mmp13^Col2ER mice. CL82198 inhibited MMP13 activity in conditioned media from vehicle (> 85%) or bone morphogenetic protein 2 (BMP2)-treated (> 90%) primary murine sternal chondrocytes. Intraperitoneal injection of CL82198 decelerated MLI-induced OA progression, increased type II collagen and proteoglycan levels, and inhibited chondrocyte apoptosis compared to saline treatment as determined by OA grading, histology, histomorphometry, IHC, and TUNEL staining, respectively.

Conclusions

Mmp13 is critical for OA progression and pharmacologic inhibition of MMP13 is an effective strategy to decelerate articular cartilage loss in a murine model of injury-induced knee OA.     

Neuromuscular Electrical Stimulation of the Quadriceps in Patients with Non-Small Cell Lung Cancer Receiving Palliative Chemotherapy: A Randomized Phase II Study

Background

A reduced exercise capacity is associated with increased morbidity and mortality in patients with advanced non-small cell lung cancer (NSCLC). Therapeutic exercise can be beneficial and neuromuscular electrical stimulation (NMES) of the quadriceps muscles may represent a practical approach. The primary aim of this study was to determine the acceptability of NMES of the quadriceps to patients with NSCLC used alongside palliative chemotherapy. Secondary aims explored aspects of safety and efficacy of NMES in this setting.

Methods

Patients with advanced NSCLC due to receive first-line palliative chemotherapy were randomized to usual care with or without NMES. They were asked to undertake 30 minute sessions of NMES, ideally daily, but as a minimum, three times weekly. For NMES to be considered acceptable, it was predetermined that ≥80% of patients should achieve this minimum level of adherence. Qualitative interviews were held with a subset of patients to explore factors influencing adherence. Safety was assessed according to the Common Terminology Criteria for Adverse Events. Quadriceps muscle strength, thigh lean mass, and physical activity level were assessed at baseline and after three cycles of chemotherapy.

Results

49 patients (28 male, median (IQR) age 69 (64−75) years) participated. Of 30 randomized to NMES, 18 were eligible for the primary endpoint, of whom 9 (50% [90% CI, 29 to 71]) met the minimum level of adherence. Adherence was enhanced by incorporating sessions into a daily routine and hindered by undesirable effects of chemotherapy. There were no serious adverse events related to NMES, nor significant differences in quadriceps muscle strength, thigh lean mass or physical activity level between groups.

Conclusions

NMES is not acceptable in this setting, nor was there a suggestion of benefit. The need remains to explore NMES in patients with cancer in other settings.

Trial Registration

Current Controlled Trials ISRCTN 42944026 www.controlled-trials.com/ISRCTN42944026     

A Long-Term Assessment of the Variability in Winter Use of Dense Conifer Cover by Female White-Tailed Deer

Backgound

Long-term studies allow capture of a wide breadth of environmental variability and a broader context within which to maximize our understanding of relationships to specific aspects of wildlife behavior. The goal of our study was to improve our understanding of the biological value of dense conifer cover to deer on winter range relative to snow depth and ambient temperature.

Methodology/Principal Findings

We examined variation among deer in their use of dense conifer cover during a 12-year study period as potentially influenced by winter severity and cover availability. Female deer were fitted with a mixture of very high frequency (VHF, n = 267) and Global Positioning System (GPS, n = 24) collars for monitoring use of specific cover types at the population and individual levels, respectively. We developed habitat composites for four study sites. We fit multinomial response models to VHF (daytime) data to describe population-level use patterns as a function of snow depth, ambient temperature, and cover availability. To develop alternative hypotheses regarding expected spatio-temporal patterns in the use of dense conifer cover, we considered two sets of competing sub-hypotheses. The first set addressed whether or not dense conifer cover was limiting on the four study sites. The second set considered four alternative sub-hypotheses regarding the potential influence of snow depth and ambient temperature on space use patterns. Deer use of dense conifer cover increased the most with increasing snow depth and most abruptly on the two sites where it was most available, suggestive of an energy conservation strategy. Deer use of dense cover decreased the most with decreasing temperatures on the sites where it was most available. At all four sites deer made greater daytime use (55 to >80% probability of use) of open vegetation types at the lowest daily minimum temperatures indicating the importance of thermal benefits afforded from increased exposure to solar radiation. Date-time plots of GPS data (24 hr) allowed us to explore individual diurnal and seasonal patterns of habitat use relative to changes in snow depth. There was significant among-animal variability in their propensity to be found in three density classes of conifer cover and other open types, but little difference between diurnal and nocturnal patterns of habitat use.

Conclusions/Significance

Consistent with our findings reported elsewhere that snow depth has a greater impact on deer survival than ambient temperature, herein our population-level results highlight the importance of dense conifer cover as snow shelter rather than thermal cover. Collectively, our findings suggest that maximizing availability of dense conifer cover in an energetically beneficial arrangement with quality feeding sites should be a prominent component of habitat management for deer.     

A New Pollen Type in the Winteraceae

Pollen of Zygogynum balansae and Z. pomiferum is described with the aid of the scanning electron microscope. Other members of the primitive ranalean family Winteraceae hitherto investigated have pollen in permanent tetrads, but these two species have solitary grains. Sculpturing is similar on the distal and proximal faces of each grain, excluding the distal apertural zone. The exine pattern resembles that on distal faces of tetrads of other Zygogynum species investigated, including Z. bailloni, the pollen of which is illustrated. Although Zygogynum and Exospermum have rather similar pollen, with sculpturing differing from other Winteraceae in consisting of a less coarse reticulum, pollen of Exospermum stipitatum is clearly distinct from the Zygogynum species investigated, as seen in the scanning electron microscope. It is not clear whether or not the solitary pollen type has evolved from the tetrad form.     

Pollen characteristics of New Zealand Gnaphalieae (Compositae) and their taxonomic significance II. Tem

Pollen ultrastructure has been investigated in 35 taxa of Gnaphalieae (Compositae), predominantly from New Zealand. Pollen grains of all taxa examined are tectate‐columellate and caveate. The characters regarded as particularly distinctive include variability of columellar form and internal organisation of the columellae. The importance of pollen characters in the classification of the New Zealand Gnaphalieae is discussed. The species of Haastia, characterised by senecioid pollen and internal tecta, do not belong to the Gnaphalieae. In the species with helianthoid pollen and sub‐columellae five different columellae types can be distinguished.     

A remarkable short-snouted horned dinosaur from the Late Cretaceous (late Campanian) of southern Laramidia

The fossil record of centrosaurine ceratopsids is largely restricted to the northern region of western North America (Alberta, Montana and Alaska). Exceptions consist of single taxa from Utah (Diabloceratops) and China (Sinoceratops), plus otherwise fragmentary remains from the southern Western Interior of North America. Here, we describe a remarkable new taxon, Nasutoceratops titusi n. gen. et sp., from the late Campanian Kaiparowits Formation of Utah, represented by multiple specimens, including a nearly complete skull and partial postcranial skeleton. Autapomorphies include an enlarged narial region, pneumatic nasal ornamentation, abbreviated snout and elongate, rostrolaterally directed supraorbital horncores. The subrectangular parietosquamosal frill is relatively unadorned and broadest in the mid-region. A phylogenetic analysis indicates that Nasutoceratops is the sister taxon to Avaceratops, and that a previously unknown subclade of centrosaurines branched off early in the group''s history and persisted for several million years during the late Campanian. As the first well-represented southern centrosaurine comparable in age to the bulk of northern forms, Nasutoceratops provides strong support for the provincialism hypothesis, which posits that Laramidia—the western landmass formed by inundation of the central region of North America by the Western Interior Seaway—hosted at least two coeval dinosaur communities for over a million years of late Campanian time.