Donald B. Thomason

Impact of genetic polymorphisms of the β2‐adrenergic receptor on albuterol bronchodilator pharmacodynamics

To determine whether genetic polymorphisms of the β2‐adrenergic receptor gene affect the relationship between albuterol (INN, salbutamol) plasma concentrations and the forced expiratory volume in 1 second (FEV1) in subjects with moderate asthma.

Parvalbumins and Muscle-relaxation - a Computer-simulation Study

SummaryThe distribution of Ca2+ and Mg2+ among the ‘regulatory’ cation binding sites of troponin (T-sites) and the strong, Ca2+-Mg2+ binding sites of troponin and parvalbumins (P-sites) in the sarcoplasm of a muscle was calculated. At rest, 60% of the T-sites were metal free, while 92% of the P-sites were loaded with Mg2+.In response to a Ca2+ pulse, troponin-calcium (T-Ca) complexes were rapidly formed, while the binding of Ca2+ to P-sites was limited by the slow rate of dissociation of the parvalbumin-magnesium (P-Mg) complexes. Muscle activation was not prevented by a high content of parvalbumins.Parvalbumin and the sarcoplasmic reticulum (SR) pump were complementary relaxing factors that removed Ca2+ from the cytosol and from the T-sites. Parvalbumins dominated the first part of relaxation, while the action of the SR was essential to ensure the return to a very low level of free Ca2+ ion and of T-Ca. After relaxation, a large fraction of the Ca2+ pulse was still bound to parvalbumins and returned slowly to the SR during the recovery.When the SR activity was reduced, the presence of parvalbumins preserved a fast rate of relaxation, at least for a few contractions. This may have a high adaptive value in cold-blooded animals.

Mechanical, morphological and biochemical adaptations of bone and muscle to hindlimb suspension and exercise

The influences of weightbearing forces on the structural remodeling, matrix biochemistry, and mechanical characteristics of the rat tibia and femur and surrounding musculature were examined by means of a hindlimb suspension protocol and highly intensive treadmill running. Female, young adult, Sprague-Dawley rats were designated as either normal control, sedentary suspended, or exercise suspended rats. For 4 weeks, sedentary suspended rats were deprived of hindlimb-to-ground contact forces, while the exercise suspended rats experienced hindlimb ground reaction forces only during daily intensive treadmill training sessions. The suspension produced generalized atrophy of hindlimb skeletal muscles, with greater atrophy occurring in predominantly slow-twitch extensors and adductors, as compared with the mixed fiber-type extensors and flexors. Region-specific cortical thinning and endosteal resorption in tibial and femoral diaphyses occurred in conjunction with decrements in bone mechanical properties. Tibial and femoral regional remodeling was related to both the absence of cyclic bending strains due to normal weightbearing forces and the decrease in forces applied to bone by antigravity muscles. To a moderate extent, the superimposed strenuous running counteracted muscular atrophy during the suspension, particularly in the predominantly slow-twitch extensor and adductor muscles. The exercise did not, however, mitigate changes in bone mechanical properties and cross-sectional morphologies, and in some cases exacerbated the changes. Suspension with or without exercise did not alter the normal concentrations of collagen, phosphorus, and calcium in either tibia or femur.

Molecular and functional evidence for Na+-K+-2Cl− cotransporter expression in rat skeletal muscle

Doubt has been raised about the expression of a functional Na+-K+-2Cl-cotransporter in rat skeletal muscle. In this study we present molecular and functional evidence for expression of a protein having the characteristics of a cotransporter. RT-PCR of RNA isolated from rat soleus muscle with primers to a conserved putative membrane-spanning domain resulted in a single product of predicted size. Sequencing of the product showed that it bears >90% homology with known rodent NKCC1 (BSC2) cotransporters. RNase protection assay of RNA isolated from the rat soleus muscle also identified this sequence. Immunologic detection of the cotransporter with two different antibodies indicated the presence of cotransporter protein, perhaps more than one, in blots of total muscle protein. Immunohistochemical detection by confocal microscopy localized the majority of expression of the protein to the muscle fibers. Functional studies of cotransport activity also indicate the appropriate sensitivity to inhibitors and ion dependence. Taken together, these data support the presence and function of Na+-K+-2Cl-cotransporter activity in the soleus muscle of the rat.

High-throughput sequencing of the DBA/2J mouse genome

BackgroundThe DBA/2J mouse is not only the oldest inbred strain,but also one of the most widely used strains. DBA/2Jexhibits many unique anatomical, physiological, andbehavior traits. In addition, DBA/2J is one parent of thelarge BXD family of recombinant inbred strains [1]. Thegenome of the other parent of this BXD family—C57BL/6J—has been sequenced and serves as themouse reference genome [2]. We sequenced the gen-ome of DBA/2J using SOLiD and Illumina highthroughput short read protocols to generate a compre-hensive set of ~5 million sequence variants segregatingin the BXD family that ultimately cause developmental,anatomical, functional and behavioral differences amongthese 80+ strains.ResultsWe generated approximately 13.2 and 38.9× whole-gen-ome short reads of DBA/2J females using Illumina GA2and ABI SOLiD massively parallel DNA sequencingplatforms. Comparing to the C57BL/6J reference gen-ome sequence, we identified over 4.5 million singlenucleotide polymorphisms (SNPs), including 84 non-sense and ~11,000 missense mutations, 78% of whichare novel. We also detected ~568,000 insertions anddeletions (indels) within single short reads and ~9,400between mate-paired reads. Approximately 300 inver-sions were detected by SOLiD mate-pair reads, 46 ofwhich span at least one exon. In addition, we identified~22,000 copy number variants (CNVs) in the range of1 Kb to 100 Kb (Figure 1).ConclusionOur study generates the first consensus sequence for theDBA/2J and creates a compendium of sequence andstructural variations that will be used by the communityof researchers who study complex traits in mouse mod-els.Thesequencedataprovideanovelresourcewithwhich to initiate reverse genetic analysis of complex

Ketosis-Prone Type 2 Diabetes: Effect of Hyperglycemia on β-Cell Function and Skeletal Muscle Insulin Signaling

OBJECTIVE To determine the underlying mechanism for the severe and transient beta-cell dysfunction and impaired insulin action in obese African American patients with ketosis-prone diabetes. METHODS The effect of sustained hyperglycemia (glucotoxicity) and increased free fatty acids (lipotoxicity) on beta-cell function was assessed by changes in insulin secretion during a 20-hour glucose (200 mg/m2 per minute) and a 48-hour Intralipid (40 mL/h) infusion, respectively. Insulin-activated signaling pathways and pattern of Akt-1 and Akt-2 expression and insulin-stimulated phosphorylation were analyzed in skeletal muscle biopsy specimens. Studies were performed in an obese African American woman within 48 hours after resolution of diabetic ketoacidosis and 1 week after discontinuation of insulin treatment. RESULTS Dextrose infusion rapidly increased C-peptide levels from a baseline of 3.2 ng/mL to a mean of 7.1 +/- 0.5 ng/mL during the first 8 hours of infusion; thereafter, C-peptide levels progressively declined. Lipid infusion was not associated with any deleterious effect on insulin and C-peptide secretion. Initial in vitro stimulation of muscle tissue with insulin resulted in a substantial and selectively decreased Akt-2 expression and insulin-stimulated phosphorylation on the serine residue. Improved metabolic control resulted in 70% greater Akt expression at near-normoglycemic remission in comparison with the period of hyperglycemia. CONCLUSION Hyperglycemia, but not increased free fatty acid levels, led to progressive beta-cell dysfunction and impaired insulin secretion. Hyperglycemia was also associated with diminished skeletal muscle Akt expression and phosphorylation in an African American woman with ketosis-prone diabetes, and this defect improved notably with aggressive insulin therapy. These results indicate the importance of glucose toxicity in the pathogenesis of ketosis-prone diabetes in obese African American patients.

Pulse wave velocity and age- and gender-dependent aortic wall hardening in fowl

Before sexual maturation, chickens (Gallus gallus) show high blood pressure (BP) and neointimal plaques in the lower abdominal aortae (AbA). We investigated age/sex-related changes in pulse wave velocity (PWV), elastin, collagen, and protein levels in AbA, and cardiac morphology to determine whether PWV increases during incremental increases in BP of maturing fowl, while arterial stiffness becomes dominant with aging. PWV (m/s) was significantly greater in male chicks (6-7 weeks, 9.3+/-0.8; females, 6.1+/-0.5) and remained high in cockerels (13 weeks), young (27-28 weeks), and adults (44-66 weeks). PWV increased in prepubertal pullets (10.0+/-0.9), dropped significantly in young hens, and remained low in adults. In contrast, medial thickness, protein levels, and collagen levels increased, while elastin/collagen ratios decreased, with maturation/aging. Males had heavier ventricular mass and thicker ventricular walls than females at all ages; left ventricular thickness decreased with maturation/aging. Thus, sustained high BP may have caused progressive medial hypertrophy, increased aortic rigidity, and enlarged hearts with left ventricular dilation. PWV of AbA was already greater in male chicks at an age when both sexes have similar collagen levels and low protein levels, suggesting that a factor other than structural stiffness may be an important determinant of PWV.

Resting extracellular signal-regulated protein kinase 1/2 expression following a continuum of chronic resistance exercise training paradigms

ABSTRACT Extracellular signal-regulated protein kinase 1/2 (ERK1/2) moderates skeletal muscle growth; however, chronic responses of this protein to unique resistance exercise (RE) paradigms are yet to be explored. The purpose of this investigation was to describe the long-term response of ERK1/2 following circuit weight training (CWT), recreationally weight training (WT), powerlifting (PL) and weightlifting (WL). Independent t-tests were used to determine differences in trained groups compared to sedentary controls. Total ERK1/2 content was lower in PL and WL compared to their controls (p ≤ 0.05). Specific trained groups displayed large (WL: pERK/total-ERK; d = 1.25) and moderate (CWT: total ERK1/2; d = 0.54) effect sizes for altered kinase expression compared to controls. The results indicate ERK1/2 expression is down-regulated after chronic RE in well-trained weightlifters and powerlifters. Lower expression of this protein may be a method in which anabolism is tightly regulated after many years of high-intensity RE.

β2‐adrenergic receptor (β2AR) polymorphism affects albuterol concentration (Cp)‐response relationship in asthmatics

Clinical Pharmacology & Therapeutics (1999) 65, 144–144; doi:

High-accuracy x-ray imaging: screen, lens, and CCD

A liquid nitrogen cooled CCD TV camera from Astromed, Ltd. has been used for quantitative X-ray medical imaging. The CCD camera is coupled to a Kodak Lanex Regular X-ray intensifying screen with a 5:1 macro lens for bone mineral densitometry of the femur of a rat for a study of the development of osteoporosis. As a feasibility study of the use of the CCD for mammography, a 2:1 macro lens has been used to couple the CCD to a clear CsI(Tl) crystal, 100 mm in diameter and 3 mm thick. The spatial resolution and quantum efficiency of the system is significantly improved by replacing the Lanex Regular screen with the CsI(Tl) crystal.