Lysine 73 is a conserved active-site residue in the class A beta-lactamases, as well as other members of the serine penicillin-sensitive enzyme family; its role in catalysis remains controversial and uncertain. Mutation of Lys73 to alanine in the beta-lactamase from Bacillus licheniformis resulted in a substantial reduction in both turnover rate (k(cat)) and catalytic efficiency (k(cat)/K(m)), and a very significant shift in pK(1) to higher pH in the bell-shaped pH-rate profiles (k(cat)/K(m)) for several penicillin and cephalosporin substrates. The increase in pK(1) is consistent with the removal of the positive ammonium group of the lysine from the proximity of Glu166, to which the acid limb has been ascribed. The alkaline limb of the k(cat)/K(m) vs profiles is not shifted appreciably, as might have been expected if this limb reflected the ionization of Lys73 in the wild-type enzyme. The k(cat)/K(m) at the pH optimum for the mutant was down about 200-fold for penicillins and around 10(4) for cephalosporins, compared to the wild-type, suggesting significant differences in the mechanisms for catalysis of penicillins compared to cephalosporins. Burst kinetics were observed with several substrates assayed with K73A beta-lactamase, indicating an underlying branched-pathway kinetic scheme, and rate-limiting deacylation. FTIR analysis was used to determine whether acylation or deacylation was rate-limiting. In general, acylation was the rate-limiting step for cephalosporin substrates, whereas deacylation was rate-limiting for penicillin substrates. The results indicate that Lys73 plays an important role in both the acylation and deacylation steps of the catalytic mechanism. The effects of this mutation (K73A) indicate that Lys73 does not function as a general base in the catalytic mechanism of beta-lactamase. The existence of bell-shaped pH-rate profiles for the K73A variant suggests that Lys73 is not directly responsible for either limb in such plots. It is likely that both Glu166 and Lys73 are important to each other in terms of maintaining the optimum electrostatic environment for fully efficient catalytic activity to occur.

Prior studies have established that the expression of the human apolipoprotein B (apoB) gene in the intestine is dependent on DNA sequences located a great distance from the structural gene. To identify the location of those sequences, we used recA-assisted restriction endonuclease (RARE) cleavage to truncate the 5 -or 3 -flanking sequences from a 145-kilobase (kb) bacterial artificial chromosome spanning the entire human apoB gene. Seven RARE cleavage-modified bacterial artificial chromosomes with different lengths of flanking sequences were used to generate transgenic mice. An analysis of those mice revealed that as little as 1.5 kb of 3 sequences or 5 kb of 5 sequences were sufficient to confer apoB expression in the liver. In contrast, apoB gene expression in the intestine required DNA sequences 54 -62 kb 5 to the structural gene. Those sequences retained their ability to direct apoB expression in the intestine when they were moved closer to the gene. These studies demonstrate that the intestinal expression of the apoB gene is dependent on DNA sequences located an extraordinary distance from the structural gene and that the RARE cleavage/transgenic expression strategy is a powerful approach for analyzing distant gene-regulatory sequences.The B apolipoproteins, apoB48 1 and apoB100, play important roles in the formation of the triglyceride-rich lipoproteins (1, 2). ApoB48 is essential for the assembly of chylomicrons in the intestine, and apoB100 is essential for the formation of VLDL in the liver. When apoB gene mutations prevent the synthesis of apoB48 and apoB100, neither chylomicrons nor VLDL can be detected in the plasma, and the plasma concentrations of triglycerides and cholesterol are extremely low (3,4).Although the basic function of apoB (the assembly of triglyceride-rich lipoproteins) is the same in the liver and intestine, the genetic control of apoB gene expression in these two tissues is strikingly different. In transgenic mouse expression studies, an ϳ80-kb P1 bacteriophage (p158) spanning the entire human apoB gene (and containing 19 kb of 5Ј sequences and 17.5 kb of 3Ј sequences) yielded human apoB expression in the liver of transgenic mice, but expression was completely absent in the intestine (5, 6). Similarly, a P1 bacteriophage clone spanning the mouse apoB gene (and containing 33 kb of 5Ј-sequences and 11 kb of 3Ј sequences) did not direct transgene expression in the intestine of transgenic mice (7). More recently, we identified a 145-kb bacterial artificial chromosome (BAC) clone spanning the human apoB gene and used it to generate human apoB transgenic mice. That clone, which contained 70 kb of 5Ј sequences and 22 kb of 3Ј sequences, conferred completely appropriate levels of apoB gene expression in the absorptive enterocytes of the intestine (8).In the current study, we have used transgenic mouse expression studies with BACs to further define the sequences that are important for the expression of the apoB gene in the intestine. Our approach involved the use of recA-assisted restrictio...

The MMSE was a useful predictor of capacity determination by PSM personnel, but using MMSE alone results in a number of erroneous determinations. Cognitive disorders were the most common primary psychiatric diagnoses in decisional capacity cases. Primary teams tended to be more accurate when they found patients to lack capacity.

Delirium is commonly encountered in the hospital setting, particularly in the intensive care unit. However, the diagnosis is often missed, due in part to the nature of the illness, fluctuating levels of consciousness, and varied presentation. Even when it is recognized, delirium can be hard to manage, with multiple factors contributing to its course. In this article, we review the latest information regarding the underlying mechanisms of the syndrome and treatment options available. This is accomplished by examining two complex cases encountered at a university medical center-based psychosomatic service.

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