Myosin heavy chain mRNA transform to faster isoforms in immobilized skeletal muscle: a quantitative PCR study

Jänkälä, Heidi, Veli-Pekka Harjola, NielsErik Petersen, and Matti Härkönen. Myosin heavy chainmRNA transform to faster isoforms in immobilized skeletal muscle: aquantitative PCR study. J. Appl.Physiol. 82(3): 977-982, 1997.A quantitative polymerase chain reaction (PCR) method was used to measure the quantities of type I, IIa, IIx, and IIb myosin heavy chain (MHC) mRNAin total RNA preparations of the soleus, gastrocnemius, and plantarismuscles of normal and hindlimb-immobilized rats. Type IIx and even typeIIb MHC mRNA were demonstrated at extremely low levels in normalsoleus, 2.1 ± 0.4 × 10⁵and 5.0 ± 0.2 × 10⁵molecules of mRNA per microgram total RNA, respectively. Immobilization for 1 wk significantly altered the gene expression of MHC isoforms. Insoleus, both type IIx and IIb MHC genes became significantly upregulated, 24-fold (P < 0.005) and 2.6-fold (P < 0.05),respectively. In gastrocnemius, the level of type IIa MHC mRNAdecreased by 51% (P < 0.01) and thelevel of type IIx MHC mRNA increased by 140%(P < 0.05). In plantaris, the levelof type IIa MHC mRNA decreased by 58%(P < 0.005). In conclusion,immobilization changed the MHC mRNA profile in three different types ofskeletal muscle toward faster isoforms. The quantitative results permitreliable evaluation of changes in mRNA levels.

     

High serum fasting peptide YY (3-36) is associated with obesity-associated insulin resistance and type 2 diabetes

We studied whether serum fasting levels of active form of peptide YY (PYY), PYY(3-36), are associated with obesity and related phenotypes. The study population consisted of 428 patients with coronary artery disease and diagnosed type 2 diabetes and 440 patients with coronary artery disease but without evidence of diabetes from the ARTEMIS study. The patients were recruited from the consecutive series of patients undergoing coronary angiography in the Oulu University Hospital. The patients without diabetes underwent a 2-hour oral glucose tolerance test. PYY(3-36) levels were analyzed by human PYY(3-36) specific radioimmunoassay. Result suggested that when PYY(3-36) tertiles were considered, high serum fasting PYY(3-36) concentration was associated with high body mass index, waist circumference, hemoglobin A1c, fasting blood glucose, leptin, triglyceride (p for all p ≤ 0.001), serum insulin (p=0.013) and with a low high-density lipoprotein cholesterol (p=0.004) concentrations in the analyses adjusted for age, sex and study group. The link high PYY(3-36)-high insulin level was evident in subjects with normal glucose tolerance (p<0.05). The prevalence of diabetes was 72%, 46% and 30% in the highest, medium and lowest PYY(3-36) tertile (p<0.001). The PYY(3-36) concentrations (after adjustment for age, sex and body mass index) were higher in type 2 diabetics compared to subjects with impaired fasting glucose, impaired glucose tolerance and normal glucose tolerance (p<0.001 for trend). In conclusion, fasting PYY(3-36) concentrations in type 2 diabetic subjects are high. Although high PYY(3-36) is strongly linked to obesity and associated insulin resistance, the relation between PYY(3-36) and type 2 diabetes is independent of body fatness.     

Laminin α1-Chain Shows a Restricted Distribution in Epithelial Basement Membranes of Fetal and Adult Human Tissues

Two novel monoclonal antibodies were raised and used to study the expression of laminin (Ln) α1-chain in developing and adult human tissues. In both fetal and adult kidney, a distinct immunoreactivity was seen in basement membranes (BM) of most proximal tubules but not in the distal tubular or glomerular BM or in the basal laminae of blood vessels. Immunoprecipitation of metabolically labeled cultured human renal proximal tubular cells showed an abundant production and deposition of Ln α1-chain to the extracellular matrix, suggestive of an epithelial origin of kidney Ln-1. Quantitative cell adhesion experiments with JAR choriocarcinoma cells showed that purified human Ln-1 is a good substrate for cell adhesion that it is differently recognized by integrin receptors when compared to mouse Ln-1. In fetal and adult testes immunoreactivity was solely confined to BM of the seminiferous epithelium. In the airways BM-confined reaction was only seen in fetal budding bronchial tubules (16–19 weeks) at the pseudoglandular stage of development. In the skin a distinct immunoreactivity was confined to BM of developing hair buds but not in epithelial BMs of adult epidermis or of epidermal appendages. In other adult tissues, immunoreactivity was found in BMs of thyroid, salivary, and mammary glands as well as in BMs of endometrium and endocervix, but not of ectocervix or vagina. No immunoreactivity was found in BMs of most of the digestive tract, including the liver and pancreas, except for BMs of esophageal submucosal glands and duodenal Brunner's glands. In fetal specimens, BMs of the bottoms of the intestinal and gastric glands were positive. Basal laminae of blood vessels were generally negative for Ln α1 chain with the exception of specimens of both fetal and adult central nervous system in which immunoreactivity for Ln α1 chain was prominently confined to capillary walls. The results suggest that outside the central nervous system, Ln α1 chain shows a restricted and developmentally regulated expression in BMs of distinct epithelial tissues.     

Targeted disruption of spermidine/spermine N1-acetyltransferase gene in mouse embryonic stem cells. Effects on polyamine homeostasis and sensitivity to polyamine analogues

We have generated mouse embryonic stem cells with targeted disruption of spermidine/spermine N(1)-acetyltransferase (SSAT) gene. The targeted cells did not contain any inducible SSAT activity, and the SSAT protein was not present. The SSAT-deficient cells proliferated normally and appeared to maintain otherwise similar polyamine pools as did the wild-type cells, with the possible exception of constantly elevated (about 30%) cellular spermidine. As expected, the mutated cells were significantly more resistant toward the growth-inhibitory action of polyamine analogues, such as N(1),N(11)-diethylnorspermine. However, this resistance was not directly attributable to cellular depletion of the higher polyamines spermidine and spermine, as the analogue depleted the polyamine pools almost equally effectively in both wild-type and SSAT-deficient cells. Tracer experiments with [C(14)]-labeled spermidine revealed that SSAT activity is essential for the back-conversion of spermidine to putrescine as radioactive N(1)-acetylspermidine and putrescine were readily detectable in N(1),N(11)-diethylnorspermine-exposed wild-type cells but not in SSAT-deficient cells. Similar experiments with [C(14)]spermine indicated that the latter polyamine was converted to spermidine in both cell lines and, unexpectedly, more effectively in the targeted cells than in the parental cells. This back-conversion was only partly inhibited by MDL72527, an inhibitor of polyamine oxidase. These results indicated that SSAT does not play a major role in the maintenance of polyamine homeostasis, and the toxicity exerted by polyamine analogues is largely not based on SSAT-induced depletion of the natural polyamines. Moreover, embryonic stem cells appear to operate an SSAT-independent system for the back-conversion of spermine to spermidine.     

Prevalence of cold-related complaints,symptoms and injuries in the general population: the FINRISK 2002 cold substudy

The prevalence of cold-related complaints and symptoms in the general population has remained unknown. As part of the nationwide FINRISK 2002 health survey performed in Finland, 8,723 people aged 25–64 years filled in a questionnaire asking about the number of hours spent weekly in cold air, their sensations during cold exposure, cold-related complaints, symptoms of diseases, and degradation of performance. Cold thermal sensations at +5°C to −5°C were reported by 35% of men and 46% of women. Almost all subjects reported at least some cold-related complaints, most commonly musculoskeletal pain (men 30%, women 27%), followed by respiratory (25% / 29%), white finger (15% / 18%) and episodic peripheral circulation symptoms (12% / 15%). Decreased mental or physical performance in cold was reported by 75% of men and 70% of women, most commonly impairing manual dexterity and tactile sense. With declining temperature, the first symptom to emerge was pain in the elbow or the forearm (at −3°C), followed by increased excretion of mucus from the lungs (−5°C), while most other symptoms appeared only at lower temperatures of −15°C to −20°C. Most symptoms showed little or no association with the weekly duration of exposure, with the exception of cold-induced pain at most sites. Although, in general, Finns are well adapted to the cold climate, the high prevalence of cold-related complaints poses a challenge to the health care system in terms of decreased performance and the possibility that such symptoms predict more serious health effects, such as increased mortality. This work was carried out in the framework of the FINADAPT project (Assessing the adaptive capacity of the Finnish environment and society under a changing climate).     

G-protein-coupled receptor domain overexpression in Halobacterium salinarum: long-range transmembrane interactions in heptahelical membrane proteins

The aminergic alpha(2b)-adrenergic receptor (alpha(2b)-AR) third intracellular loop (alpha(2b)-AR 3i) mediates receptor subcellular compartmentalization and signal transduction processes via ligand-dependent interaction with G(i)- and G(o)- proteins. To understand the structural origins of these processes we engineered several lengths of alpha(2b)-AR 3i into the third intracellular loop of the proton pump bacteriorhodopsin (bR) and produced the fusion proteins in quantities suitable for physical studies. The fusion proteins were expressed in the Archaeon Halobacterium salinarum and purified. A highly expressed fusion protein was crystallized from bicelles and diffracted to low resolution on an in-house diffractometer. The bR-alpha(2b)-AR 3i(203-292) protein possessed a photocycle slightly perturbed from that of the wild-type bR. The first half of the fusion protein photocycle, correlated with proton release, is accelerated by a factor of 3, whereas the second half, correlated with proton uptake, is slightly slower than wild-type bR. In addition, there is a large decrease in the pK(a), (from 9.6 to 8.3) of the terminal proton release group in the unphotolyzed state of bR-alpha(2b)-AR 3i as deduced from the pH-dependence of the M-formation. Perturbation of a cytoplasmic loop has thus resulted in the perturbation of proton release at the extracellular surface. The current work indicates that long-range and highly coupled intramolecular interactions exist that are capable of "transducing" structural perturbations (e.g., signals) across the cellular membrane. This gene fusion approach may have general applicability for physical studies of G-protein-coupled receptor domains in the context of the bR structural scaffold.