Hepatic ABCG5/G8 overexpression reduces apoB-lipoproteins and atherosclerosis when cholesterol absorption is inhibited

We previously reported that liver-specific overexpression of ABCG5/G8 in mice is not atheroprotective, suggesting that increased biliary cholesterol secretion must be coupled with decreased intestinal cholesterol absorption to increase net sterol loss from the body and reduce atherosclerosis. To evaluate this hypothesis, we fed low density lipoprotein receptor-knockout (LDLr-KO) control and ABCG5/G8-transgenic (ABCG5/G8-Tg)xLDLr-KO mice, which overexpress ABCG5/G8 only in liver, a Western diet containing ezetimibe to reduce intestinal cholesterol absorption. On this dietary regimen, liver-specific ABCG5/G8 overexpression increased hepatobiliary cholesterol concentration and secretion rates (1.5-fold and 1.9-fold, respectively), resulting in 1.6-fold increased fecal cholesterol excretion, decreased hepatic cholesterol, and increased (4.4-fold) de novo hepatic cholesterol synthesis versus LDLr-KO mice. Plasma lipids decreased (total cholesterol, 32%; cholesteryl ester, 32%; free cholesterol, 30%), mostly as a result of reduced non-high density lipoprotein-cholesterol and apolipoprotein B (apoB; 36% and 25%, respectively). ApoB-containing lipoproteins were smaller and lipid-depleted in ABCG5/G8-TgxLDLr-KO mice. Kinetic studies revealed similar 125I-apoB intermediate density lipoprotein/LDL fractional catabolic rates, but apoB production rates were decreased 37% in ABCG5/G8-TgxLDLr-KO mice. Proximal aortic atherosclerosis decreased by 52% (male) and 59% (female) in ABCG5/G8-TgxLDLr-KO versus LDLr-KO mice fed the Western/ezetimibe diet. Thus, increased biliary secretion, resulting from hepatic ABCG5/G8 overexpression, reduces atherogenic risk in LDLr-KO mice fed a Western diet containing ezetimibe. These findings identify distinct roles for liver and intestinal ABCG5/G8 in modulating sterol metabolism and atherosclerosis.     

Development of dynamic knee stability after acute ACL injury.

Recently, a training program that includes perturbation of support surfaces has been shown to allow most active individuals with ACL injury who pass a screening examination to successfully return to high level activities. The purpose of this study was to identify the effect of this rehabilitation program on involved side muscle activation during walking in subjects with acute ACL rupture and to determine if the activation changes were coincident with improved function. Nine subjects with an acute, unilateral ACL injury or rupture of an ACL graft, who met the screening examination criteria, received ten sessions of rehabilitation that included perturbation training. Motion analysis of five self-paced walking trials were performed before and after training. Electromyographic (EMG) data were collected during stance. After training during walking, the vastus lateralis (VL) integral of activity increased, and relationships between muscles were significantly altered. During walking, VL activation variables were dependent on lateral hamstrings (LH) and/or the soleus (SOL) activation, while no relationships were found before training. Function improved after training, and all subjects returned to their pre-injury activities without experiencing instability. The relationships formed between muscles post-training suggests that perturbation training enhances dynamic knee stability by inducing a well-coordinated strategy among muscles that affect tibial translation.     

Sequence-specific adenylations and deadenylations accompany changes in the translation of maternal messenger RNA after fertilization of Spisula oocytes

A dramatic change in the pattern of protein synthesis occurs within ten minutes after fertilization of Spisula oocytes. This change is regulated entirely at the translational level. We have used DNA clones complementary to five translationally regulated messenger RNAs to follow shifts in mRNA utilization at fertilization and to characterize alterations in mRNA structure that accompany switches in translational activity in vivo. Four of the mRNAs studied are translationally inactive in the oocyte. After fertilization two of these mRNAs are completely recruited onto polysomes, and two are partially recruited. All four of these mRNAs have very short poly(A) tracts in the oocyte; after fertilization the poly(A) tails lengthen considerably. In contrast, a fifth mRNA, that encoding alpha-tubulin mRNA, is translated very efficiently in the oocyte and is rapidly lost from polysomes after fertilization. Essentially all alpha-tubulin mRNA in the oocyte is poly(A)+ and a large portion of this mRNA undergoes complete deadenylation after fertilization. These results reveal a striking relationship between changes in adenylation and translational activity in vivo. This correlation is not perfect, however. Evidence for and against a direct role for polyadenylation in regulating these translational changes is discussed. Changes in poly(A) tails are the only alterations in mRNA sizes that we have been able to detect. This indicates that, at least for the mRNAs studied here, translational activation is not due to extensive processing of larger translationally incompetent precursors. We have also isolated several complementary DNA clones to RNAs encoded by the mitochondrial genome. Surprisingly, the poly(A) tracts of at least two of the mitochondrial RNAs also lengthen in response to fertilization.     

Role of PAT proteins in lipid metabolism

One of the central reactions in bodily energy metabolism is lipolysis in adipocytes, the reaction that governs the release of stored fatty acids from the adipocyte triacylglycerol pool, which constitutes the major energy reserve in animals. These fatty acids are then transported by serum albumin to various tissues to supply their energy requirements. This reaction was previously thought to result from phosphorylation and activation of hormone-sensitive lipase by protein kinase A (PKA) but is now known to be governed by a translocation of the lipase from the cytosol to the surface of the intracellular lipid droplet that houses the reservoir of TAG. This droplet is coated with perilipin A, which is also phosphorylated by PKA in response to lipolytic stimuli, and phosphorylation of perilipin A is essential for HSL translocation and stimulated lipolysis.     

Structure and function of carbonic anhydrases from Mycobacterium tuberculosis

Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to form bicarbonate. This activity is universally required for fatty acid biosynthesis as well as for the production of a number of small molecules, pH homeostasis, and other functions. At least three different carbonic anhydrase families are known to exist, of which the alpha-class found in humans has been studied in most detail. In the present work, we describe the structures of two of the three beta-class carbonic anhydrases that have been identified in Mycobacterium tuberculosis, i.e. Rv1284 and Rv3588c. Both structures were solved by molecular replacement and then refined to resolutions of 2.0 and 1.75 A, respectively. The active site of Rv1284 is small and almost completely shielded from solvent, whereas that of Rv3588c is larger and quite open to solution. Differences in coordination of the active site metal are also observed. In Rv3588c, an aspartic acid side chain displaces a water molecule and coordinates directly to the zinc ion, thereby closing the zinc coordination sphere and breaking the salt link to a nearby arginine that is a feature of Rv1284. The two carbonic anhydrases thus exhibit both of the metal coordination geometries that have previously been observed for structures in this family. Activity studies demonstrate that Rv3588c is a completely functional carbonic anhydrase. The apparent lack of activity of Rv1284 in the present assay system is likely exacerbated by the observed depletion of zinc in the preparation.     

Crystal structure of Thermobifida fusca endoglucanase Cel6A in complex with substrate and inhibitor: the role of tyrosine Y73 in substrate ring distortion

Endoglucanase Cel6A from Thermobifida fusca hydrolyzes the beta-1,4 linkages in cellulose at accessible points along the polymer. The structure of the catalytic domain of Cel6A from T. fusca in complex with a nonhydrolysable substrate analogue that acts as an inhibitor, methylcellobiosyl-4-thio-beta-cellobioside (Glc(2)-S-Glc(2)), has been determined to 1.5 A resolution. The glycosyl unit in subsite -1 was sterically hindered by Tyr73 and forced into a distorted (2)S(o) conformation. In the enzyme where Tyr73 was mutated to a serine residue, the hindrance was removed and the glycosyl unit in subsite -1 had a relaxed (4)C(1) chair conformation. The relaxed conformation was seen in two complex structures of the mutated enzyme, with cellotetrose (Glc(4)) at 1.64 A and Glc(2)-S-Glc(2) at 1.04 A resolution.     

Mycobacterium tuberculosis ribose-5-phosphate isomerase has a known fold, but a novel active site

Ribose-5-phosphate isomerases (EC 5.3.1.6) inter-convert ribose-5-phosphate and ribulose-5-phosphate. This reaction allows the synthesis of ribose from other sugars, as well a means for salvage of carbohydrates after nucleotide breakdown. Two unrelated types of enzyme are known to catalyze the isomerization. The most common one, RpiA, is present in almost all organisms. The second type, RpiB, is found in many bacterial species.Here, we demonstrate that the RpiB from Mycobacterium tuberculosis (Rv2465c) has catalytic properties very similar to those previously reported for the Escherichia coli RpiB enzyme. Further, we report the structure of the mycobacterial enzyme, solved by molecular replacement and refined to 1.88A resolution. Comparison with the E.coli structure shows that there are important differences in the two active sites, including a change in the position and nature of the catalytic base. Sequence comparisons reveal that the M.tuberculosis and E.coli RpiB enzymes are in fact representative of two distinct sub-families. The mycobacterial enzyme represents a type found only in actinobacteria, while the enzyme from E.coli is typical of that seen in many other bacterial proteomes. Both RpiBs are very different from RpiA in structure as well as in the construction of the active site. Docking studies allow additional insights into the reactions of all three enzymes, and show that many features of the mechanism are preserved despite the different catalytic components.