Molecular and Cellular Biochemistry

The possibility that the glucose transporter may serve as water channel is explored with the help of theoretical and experimental arguments. A model for a pore is drawn based on a hypothetical water channel structure, subject to the constraints that: molecules will bind to the channel wall in successive rings, forming a hollow sleeve; an integer number of molecules will exist in each ring; the pore radius will not be large enough to allow water molecules along its center, but will be large enough to allow glucose molecules across. The only configurations that meet these conditions exhibit either 5 or 6 water molecules abreast in each ring, with pore radii of 4.1 and 4.5 Å, respectively. The kinetic characteristics of such pores are estimated and found to conform to available evidence.

Gen myosin nhẹ kiềm cơ vân nhanh (MYL1) là một trong ba gen myosin nhẹ kiềm ở động vật có vú và mã hóa hai isoform, 1f và 3f, có vai trò quan trọng trong sự phát triển cơ vân ở phôi, bào thai và người lớn. Chúng tôi đã tách chiết gen MYL1 từ thư viện BAC của lợn với mục tiêu phân tích trình điều khiển của nó và xác định các bản sao của nó. Các gen và isoform đã được xác định bằng phương pháp PCR phiên mã ngược, phân tích Northern blot và RACE (Khuếch đại nhanh các đầu cDNA). Các trình điều khiển tiềm năng của gen MYL1 đã được đặc trưng bằng cách sử dụng thử nghiệm báo cáo luciferase và các thử nghiệm dịch chuyển di điện (EMSA). Các isoform MLC1f, MLC3f và ba isoform bổ sung của MYL1 lợn, MLC5f-A, -B và -C đã được phát hiện. Đến nay, ba isoform mới này chưa được báo cáo trong người hoặc chuột. Phân tích Northern blot cho thấy MLC1f, MLC3f và MLC5fs chỉ được biểu hiện trong các cơ dài lưng. Hai vị trí khởi đầu và kết thúc phiên mã đã được xác định bằng RACE. Phân tích trình điều khiển và EMSA chứng tỏ sự hiện diện của một vị trí liên kết MEF3 (tăng cường phiên mã chuyên biệt cho cơ vân) (+384 đến +481), có thể là thiết yếu cho phiên mã MYL1 ở lợn. Kết quả của chúng tôi cho thấy rằng năm biến thể phiên mã đã được tạo ra bằng cách sử dụng các trình điều khiển thay thế, hai vị trí bắt đầu phiên mã và các vị trí polyA, cũng như sự thay đổi splicing biến đổi của exon 5 gen MYL1 lợn. Việc xác định các trình điều khiển thay thế và các biến thể splicing, sự biểu hiện của các biến thể splicing trong các mô cơ khác nhau, và định nghĩa các yếu tố điều hòa cung cấp kiến thức di truyền phân tử quan trọng liên quan đến gen MYL1.

Ca2+ homeostasis plays a pivotal role in maintaining cell growth and function. Many heart diseases are related to the abnormalities in Ca2+ mobilization and extrusion. Ca2+-sensitive fluorescent dyes have been used successfully to estimate intracellular free Ca2+ ([Ca2+]i) level and the mechanisms of Ca2+ movements in living cells. This article is focused on the methodology involving the use of Fura-2/AM or free Fura-2 to measure agonist-induced Ca2+ mobilization as well as the mechanisms of changes in [Ca2+]i in cardiomyocytes. Methods involving Fura-2 technique for the measurement of Ca2+ extrusion from the cells and Ca2+ reuptake by sarcoplasmic reticulum (SR) are also described. The prevention of KCl-induced increase in the intracellular Ca2+ is shown by chelating the extracellular Ca2+ with EGTA or by the presence of Ca2+-channel inhibitors such as verapamil and diltiazem. The involvement of SR in the ATP-induced increase in intracellular Ca2+ is illustrated by the use of Ca2+-pump inhibitors, thapsigargin and cyclopiazonic acid as well as ryanodine which deplete the SR Ca2+ storage. The use of 2-nitro-4-carboxyphenyl N,N-diphenyl carbamate (NCDC), an inhibitor of inositol 1,4,5-trisphosphate (IP3) production, is described for the attenuation of phosphatidic acid (PA) induced increase in Ca2+-mobilization. The increase in intracellular Ca2+ in cardiomyocytes by PA, unlike that by KCl or ATP, was observed in diabetic myocardium. Thus, it appears that the Fura-2 method for the measurement of Ca2+ homeostasis in cardiomyocytes is useful in studying the pathophysiology and pharmacology of Ca2+ movements.

Diabetes mellitus (DM) causes myocardial remodeling on the subcellular level and alterations in the function of the cell membranes ion transport systems resulting in contractile dysfunction. The present study was aimed to investigate the expression and activation of mitogen-activated protein kinases (MAPKs) and their possible role in the acute diabetic rat hearts. Rats were injected with single dose of streptozotocin (45 mg/kg, i.v.), and after 1 week the disease was manifested by hyperglycemia and cardiac dysfunction. The Langendorff-perfused hearts were subjected to ischemia (5 or 30 min occlusion of LAD coronary artery). The protein pattern in cytosolic fraction of the heart tissue was determined after electrophoretic separation. The levels and activation of MAPKs were determined by Western blot analysis using specific antibodies. No differences between the diabetics and controls in the level of ERKs were found at baseline. However, in DM samples ERKs phosphorylation was markedly increased, and further changes occurred during ischemia. Also content of phoshorylated c-Raf kinase (an upstream activator of ERKs) was slightly increased at baseline conditions in the diabetic samples. In contrast, no significant changes in the contents and phosphorylation of p38-MAPK were observed at baseline. But some differences in the p38-MAPK phosphorylation were found during ischemia. The results show that differential pattern of protein kinase cascades activation in the diabetic hearts might be account for the modulation of their response to ischemia.

Angiotensin II (Ang II) is an important regulator of the function of medullary thick ascending limb of loop of Henle (MTAL). Recent studies showed that changes in Ang II receptor expression occur and underlie changes in the function of proximal tubules during altered sodium intake. The present experiment was designed to determine (1) whether expression of the type 1 Ang II (AT1) receptor in the MTAL is regulated by altered sodium intake, and (2) the specific pathway(s) mediating sodium-induced AT1 expression in the MTAL. Wistar rats were fed a normal sodium (0.5%, NS), low sodium (0.07%, LS), or high sodium (4%, HS) diet for 2 weeks. Northern blot analysis and radioligand binding showed that in rats fed a normal sodium diet the rank of order for both AT1 mRNA expression and receptor density was outer medulla > cortex > inner medulla. Sodium restriction significantly increased both AT1 mRNA expression and receptor density in the outer medulla. In contrast, neither AT1 mRNA expression nor receptor density in the outer medulla was altered by sodium loading. Losartan treatment (3 mg/kg/per day by oral gavage for 2 weeks) prevented low sodium-induced upregulation of the AT1 receptor in the outer medulla, but it had no effect on AT1 expression in the outer medulla of rats fed a normal sodium diet. Highly purified suspensions of MTAL were isolated from rats fed a normal or low sodium diet. Low sodium intake significantly increased AT1 mRNA level by 184% and AT1 receptor density by 58% in MTALs. Primary cultures of MTAL cells were treated with PBS, Ang II (10-8 M), and Ang II + 17 octadecynoic (17 ODYA, 10 μM). Ang II caused about 2-fold increase in AT1 mRNA levels, and this increase was diminished by about 30% by the addition of 17 ODYA. We conclude that (1) sodium restriction but not sodium loading increases AT1 receptor expression in the MTAL, (2) low sodium-induced upregulation of the AT1 receptor in the MTAL is Ang II-dependent, and (3) Ang II-induced upregulation of the AT1 receptor in the MTAL is mediated, at least in part, by cytochrome P450 pathways.

The effect of diabetic status and insulin on adipocyte plasma membrane properties and fatty acid uptake was examined. Studies with inhibitors and isolated adipocyte ghost plasma membranes indicated 9Z, 11E, 13E, 15Z-octatetraenoic acid (cis-parinaric acid) uptake was protein mediated. Cis-parinaric acid uptake was inhibited by trypsin treatment or incubation with phloretin, and competed with stearic acid. The initial rate, but not maximal uptake, of cis-parinaric acid uptake was enhanced two-fold in adipocytes from diabetic rats. Concomitantly, the structure and lipid composition of adipocyte ghost membranes was dramatically altered. However, the increased initial rate of cis-parinaric acid uptake in the diabetic adipocytes was not explained by membrane alterations or by a two-fold decrease in cytosolic adipocyte fatty acid binding protein (ALBP), unless ALBP stimulated fatty acid efflux. Thus, diabetic status dramatically altered adipocyte fatty acid uptake, plasma membrane structu re, lipid composition, and cytosolic fatty acid binding protein. (Mol Cell Biochem 167: 51-60, 1997)