Effects of introduced aspartic and glutamic acid residues on the P'1 substrate specificity, pH dependence and stability of carboxypeptidase Y

Carboxypeptidase Y is a serine carboxypeptidase isolated from Saccharomyces cerevisiae with a preference for C-terminal hydrophobic amino acid residues. In order to alter the inherent substrate specificity of CPD-Y into one for basic amino acid residues in P'1, we have introduced Asp and/or Glu residues at a number of selected positions within the S'1 binding site. The effects of these substitutions on the substrate specificity, pH dependence and protein stability have been evaluated. The results presented here demonstrate that it is possible to obtain significant changes in the substrate preference by introducing charged amino acids into the framework provided by an enzyme with a quite different specificity. The introduced acidic amino acid residues provide a marked pH dependence of the (kcat/Km)FA-A-R-OH/(kcat/Km)FA-A-L-OH ratio. The change in stability upon introduction of Asp/Glu residues can be correlated to the difference in the mean buried surface area between the substituted and the substituting amino acid. Thus, the effects of acidic amino acid residues on the protein stability depend upon whether the introduced amino acid protrudes from the solvent accessible surface as defined by the surrounding residues in the wild type enzyme or is submerged below.     

Muscle transformation of the sartorius muscle in a canine model: clinical impact for electrodynamic graciloplasty as a "neosphincter"

PURPOSE: Transformation of fast-twitching skeletal muscles to slow-twitching, slowly fatigable muscles has become of clinical interest in the recent past. Transposition and transformation of the gracilis muscle to use it as a substitute for a resected or defected anal sphincter (graciloplasty) have been reported as achieving promising results in the treatment of fecal incontinence caused by sphincter defects or following abdominoperineal anorectal excision for cancer. METHOD: This experimental study used a canine model and the sartorius muscle to evaluate the functional efficiency of two different configurations of the muscle loop to compare the presently applied transformation program (8 weeks) with a shorter (5 weeks) protocol. In six beagle dogs, both sartorius muscles were wrapped around two stomas, either in an alpha fashion or in the so-called split-sling technique. Muscle transformation was achieved by controlled neuromuscular stimulation either during eight (Program A) or five weeks (Program B). After completion of the transformation period, the function of the muscle slings was evaluated by manometry, and histomorphologic evaluation of the sartorius muscles was performed. RESULTS: It was shown that muscle transformation led to a slowly fatigable muscle that made it possible to perform continuos (tetanic) contraction, regardless of the configuration or the duration of the transformation. Median pressures created by these muscles also did not differ significantly. In accordance with these functional findings, the histologic evaluation showed the typical, significant increase of Type I fibers in both muscle slings and following both transformation protocols. Although the decrease of fast-twitching Type II fibers was more pronounced following the conventional (8 weeks) program, this finding did not influence the functional results. CONCLUSIONS: Results of our experiment indicate the possibility for using a shorter transformation protocol for transformation of the gracilis muscle during graciloplasty in the clinical setting. Furthermore, the efficacy and safety of the modified (split-sling) wrap technique was demonstrated.     

Role of maternal depression in perceptions of child maladjustment.

We investigated (a) the relation of maternal depression to perceptions of externalizing and internalizing disorders in children and (b) quality of communication in a mother–child interaction task as a function of maternal depression and perceptions of the child. 64 children of unipolar or bipolar mothers, chronic medically ill or normal mothers were studied; clinical diagnoses, children's reports, and teachers' ratings served as objective criteria of children's maladjustment. Maternal depression defined by current symptomatology on the Beck Depression Inventory and by psychiatric status was not associated with misperceptions of psychopathology. Maternal depression interacted with children's actual behaviors to predict mothers' perceptions: nondepressed mothers were less accurate reporters of problems in children than were depressed mothers. Depressed mothers who perceived maladjustment in their disturbed children made more negative comments in interactions than did nondepressed mothers of disturbed children. The apparent mutual impact of mothers' and children's maladjustment requires fuller exploration in offspring studies. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Evidence for superantigen involvement in insulin-dependent diabetes mellitus aetiology

Insulin-dependent diabetes mellitus (IDDM) is a T-cell-mediated autoimmune disease whose onset is believed to be triggered by unknown environmental factors acting on a predisposing genetic background. Islet-infiltrating T (IIT) cells from two IDDM patients, who had died at the onset of the disease from brain swelling as a complication of ketoacidosis, were analysed. The results provided evidence for the involvement of a pancreatic islet cell membrane-bound superantigen as a diabetes aetiopathogenetic factor. There was a selective expansion of a T-cell receptor (TCR) variable segment of the beta-chain (V beta 7) in these IIT cells in association with unselected V alpha-chain segments; extensive junctional diversity of the TCR V beta 7 chains; and evidence of positive selection, after exposure to diabetic islet cell membrane preparations, of V beta 7+ T-cell clones among peripheral blood lymphocytes from non-diabetic individuals.     

The US market for SO2 permits: Policy implications of the low price and trading volume

The price of SO₂ permits and the volume of trading under the US Acid Rain Program have been lower than expected. This can be explained by the creation and distribution of more permits than were initially authorized, by the sale of permits by high cost abaters who are subject to more stringent local emission constraints or who have irreversibly invested in high cost abatement technologies and by the deregulation of the natural gas and railroad industries which, in combination with incentives for cost-cutting under the new market approach to SO₂ control, has lowered marginal costs of abatement curves and made them more uniform across powerplants. The low price of permits and low trading volume are evidence that market approaches to pollution control can be more cost-effective than command and control regulations. The effect of public policies and technological changes on the allowance market are usefully examined in the context of an ideal market, in which the equilibrium price of allowances equals the marginal cost of abatement of individual powerplants. Using recent data from Coggins and Swinton (1996), we are able to explain the current price of permits with some accuracy.     

Prostaglandin synthase-1 and prostaglandin synthase-2 are coupled to distinct phospholipases for the generation of prostaglandin D2 in activated mast cells

Aggregation of IgE cell surface receptors on MMC-34 cells, a murine mast cell line, induces the synthesis and secretion of prostaglandin D2 (PGD2). Synthesis and secretion of PGD2 in activated MMC-34 cells occurs in two stages, an early phase that is complete within 30 min after activation and a late phase that reaches a maximum about 6 h after activation. The early and late phases of PGD2 generation are mediated by prostaglandin synthase 1 (PGS1) and prostaglandin synthase 2 (PGS2), respectively. Arachidonic acid, the substrate for both PGS1 and PGS2, is released from membrane phospholipids by the activation of phospholipases. We now demonstrate that in activated mast cells (i) secretory phospholipase A2 (PLA2) mediates the release of arachidonic acid for early, PGS1-dependent synthesis of PGD2; (ii) secretory PLA2 does not play a role in the late, PGS2-dependent synthesis of PGD2; (iii) cytoplasmic PLA2 mediates the release of arachidonic acid for late, PGS2-dependent synthesis of PGD2; and (iv) a cytoplasmic PLA2-dependent step precedes secretory PLA2 activation and is necessary for optimal PGD2 production by the secretory PLA2/PGS1-dependent early pathway.     

Properties of permease dimer, a fusion protein containing two lactose permease molecules from Escherichia coli

An engineered fusion protein containing two tandem lactose permease molecules (permease dimer) exhibits high transport activity and is used to test the phenomenon of negative dominance. Introduction of the mutation Glu-325-->Cys into either the first or the second half of the dimer results in a 50% decrease in activity, whereas introduction of the mutation into both halves of the dimer abolishes transport. Lactose transport by permease dimer is completely inactivated by N-ethylmaleimide; however, 40-45% activity is retained after N-ethylmaleimide treatment when either the first or the second half of the dimer is replaced with a mutant devoid of cysteine residues. The observations demonstrate that both halves of the fusion protein are equally active and suggest that each half may function independently. To test the possibility that oligomerization between dimers might account for the findings, a permease dimer was constructed that contains two different deletion mutants that complement functionally when expressed as untethered molecules. Because this construct does not catalyze lactose transport to any extent whatsoever, it is unlikely that the two halves of the dimer interact or that there is an oligomeric interaction between dimers. The approach is consistent with the contention that the functional unit of lactose permease is a monomer.     

Safe and effective navigation of autonomous robots in hazardous environments

The development of autonomous mobile machines to perform useful tasks in real work environments is currently being impeded by concerns over effectiveness, commercial viability and, above all, safety. This paper introduces a case study of a robotic excavator to explore a series of issues around system development, navigation in unstructured environments, autonomous decision making and changing the behaviour of autonomous machines to suit the prevailing demands of users. The adoption of the Real-Time Control Systems (RCS) architecture (Albus, 1991) is proposed as a universal framework for the development of intelligent systems. In addition it is explained how the use of Partially Observable Markov Decision Processes (POMDP) (Kaelbling et al., 1998) can form the basis of decision making in the face of uncertainty and how the technique can be effectively incorporated into the RCS architecture. Particular emphasis is placed on ensuring that the resulting behaviour is both task effective and adequately safe, and it is recognised that these two objectives may be in opposition and that the desired relative balance between them may change. The concept of an autonomous system having “values” is introduced through the use of utility theory. Limited simulation results of experiments are reported which demonstrate that these techniques can create intelligent systems capable of modifying their behaviour to exhibit either ‘safety conscious’ or ‘task achieving’ personalities.