Scanning alanine mutagenesis of the CDP-alcohol phosphotransferase motif of Saccharomyces cerevisiae cholinephosphotransferase

Cholinephosphotransferase (EC 2.7.8.2) catalyzes the formation of a phosphoester bond via the transfer of a phosphocholine moiety from CDP-choline to diacylglycerol forming phosphatidylcholine and releasing CMP. A motif, Asp113-Gly114-(X)2-Ala117-Arg118-(X)8-Gly127+ ++-(X)3-Asp131-(X)3-Asp135, located within the CDP-choline binding region of Saccharomyces cerevisiae cholinephosphotransferase (CPT1 ?/Author: Please confirm that a gene is meant here.) is also found in several other phospholipid synthesizing enzymes that catalyze the formation of a phosphoester bond utilizing a CDP-alcohol and a second alcohol as substrates. To determine if this motif is diagnostic of the above reaction type scanning alanine mutagenesis of the conserved residues within S. cerevisiae cholinephosphotransferase was performed. Enzyme activity was assessed in vitro using a mixed micelle enzyme assay and in vivo by determining the ability of the mutant enzymes to restore phosphatidylcholine synthesis from radiolabeled choline in an S. cerevisiae strain devoid of endogenous cholinephosphotransferase activity. Alanine mutants of Gly114, Gly127, Asp131, and Asp135 were inactive; mutants, Ala117 and Arg118 displayed reduced enzyme activity, and Asp113 displayed wild type activity. The analysis described is the first molecular characterization of a CDP-alcohol phosphotransferase motif and results predict a catalytic role utilizing a general base reaction proceeding through Asp131 or Asp135 via a direct nucleophilic attack of the hydroxyl of diacylglyerol on the phosphoester bond of CDP-choline that does not proceed via an enzyme bound intermediate. Residues Ala117 and Arg118 do not participate directly in catalysis but are likely involved in substrate binding or positioning with Arg118 predicted to associate with a phosphate moiety of CDP-choline.     

The Tenet experience and the design of protocols for integrated-services internetworks

The research done by the Tenet Group in multimedia networking has reached a point where it may be useful to reflect on the significance of its results for the current debate on how integrated-services internetworks should be designed. Such reflections constitute the main subject of this paper. The principles of the work and the conclusions reached so far by the Tenet researchers are discussed in the light of the conflict between the two major technologies being proposed to build future information infrastructures: namely, the Internet and the ATM technologies. The Tenet approach suggests one feasible way for resolving the conflict to the advantage of all the users of those infrastructures. This paper discusses various fundamental aspects of integrated-services network design: the choice of the service model, the type of charging policy to be adopted, and the selection of a suitable architecture.     

Development of technologies aiding large-tissue engineering

Apoptosis associated oligonucleosomal fragmentation of DNA can result from the activation of endonucleases that exhibit different pH optima and are either sensitive or insensitive to divalent cations. DNA fragmentation due to activation of cation sensitive endonucleases occurs in the absence of a change in intracellular pH whereas intracellular acidification is a feature of apoptosis characterized by activation of cation insensitive acidic endonuclease. We have reported earlier that somatostatin (SST) induced DNA fragmentation and apoptosis is signaled in a receptor subtype selective manner uniquely via human somatostatin receptor subtype 3 (hSSTR3). In the present study we investigated the pH dependence and cation sensitivity of endonuclease induced in hSSTR3 expressing CHO-K1 cells by the SST agonist octreotide (OCT) and its effect on intracellular pH. We show that OCT induced apoptosis is associated with selective stimulation of a divalent cation insensitive acidic endonuclease. The intracellular pH of of cells undergoing OCT induced apoptosis was 0.9 pH units lower than that of control cells. The effect of OCT on endonuclease and pH was inhibited by orthovanadate as well as by pretreatment with pertussis toxin, suggesting that hSSTR3 initiated cytotoxic signaling is protein tyrosine phosphatase mediated and is G protein dependent. These findings suggest that intracellular acidification and activation of acidic endonuclease mediate wild type p53 associated apoptosis signaled by hormones acting via G protein coupled receptors.     

U.K. Prospective Diabetes Study. XV: Relationship of renin-angiotensin system gene polymorphisms with microalbuminuria in NIDDM

We performed a case-control study to determine whether molecular variants of genes of the renin-angiotensin system were associated with the presence of albuminuria in non-insulin dependent diabetes mellitus (NIDDM). A total of 180 diabetic patients with persistent microalbuminuria [median urinary albumin (interquartile range) of 74 (54 to 126 mg/liter)] were matched with two control groups of diabetic patients without microalbuminuria [median urinary albumin 7 (5 to 10) mg/liter] for variables known to be associated with raised urinary albumin concentration including hemoglobin A1c and triglyceride. One control group was also matched for blood pressure and the other group was not, to allow assessment of interactions with hypertension. Association with the I/D polymorphism of the ACE gene and M235T variant of the angiotensinogen gene (AGT) with microalbuminuria and retinopathy was examined. There were no significant differences in genotype frequency between cases and controls for ACE or AGT irrespective of blood pressure matching. However, among subjects with microalbuminuria, those with the ACE DD genotype had a significantly greater urinary albumin excretion than individuals with a non-DD genotype [median 88 (68 to 170) mg/liter vs. 67 (53 to 113) mg/liter, P < 0.001]. More subjects with the DD than non-DD genotype had persistent albuminuria > 100 mg/liter, twice the upper normal range (60% vs. 38%, P = 0.006). When increased albumin excretion occurs, the presence of the ACE DD genotype appears to be associated with higher urinary albumin levels. No association with retinopathy was observed.     

The association of intra-abdominal infection and abdominal wound dehiscence

Concurrent infection is a risk factor for abdominal wound dehiscence. We reviewed our experience with fascial dehiscence to determine the incidence and to identify prognostic factors for associated intra-abdominal infection. Over a 7-year period, 107 patients with abdominal wound dehiscence were identified. Seventeen were managed nonoperatively, and 90 underwent exploratory laparotomy, 43 of whom had no intra-abdominal pathology and 47 of whom had intra-abdominal infections. Demographic factors, comorbid diseases, and potential indicators of systemic infection did not distinguish patients with intra-abdominal infection from those without. Patients with an intra-abdominal infection were more likely to have undergone an emergency operation (74% vs 48%; P < 0.02), an operation on the colon (55% vs 25%; P < 0.005), or an operation with a higher wound classification (P < 0.02). Mortality was higher in patients with intra-abdominal infection than in those without (44% vs 20%; P < 0.02). Wound dehiscence after emergent operations, and operations with a higher wound classification, especially those involving the colon, should raise concern for intra-abdominal infection. Thorough abdominal exploration should be performed at the time of dehiscence repair. Before nonoperative management is chosen, intra-abdominal infection should be excluded.     

Recursive algorithms for implementing digital image filters

The B-spline functions are used to develop recursive algorithms for the efficient implementation of two-dimensional linear digital image filters. These filters may be spatially varying. The B-splines are used in a representation of the desired point spread function. We show that this leads to recursive algorithms and hardware implementations which are more efficient than either direct spatial domain filter realizations or FFT implementations. The Z-transform is used to develop a discrete version of Duhamel's theorem. A computer architecture for B-spline image filters is proposed and a complexity analysis and comparison to other approaches is provided.     

A low-cost real time virtual system for postural stability assessment at home

Background and objective

The degeneration of the balance control system in the elderly and in many pathologies requires measuring the equilibrium conditions very often. In clinical practice, equilibrium control is commonly evaluated by using a force platform (stabilometric platform) in a clinical environment.In this paper, we demonstrate how a simple movement analysis system, based on a 3D video camera and a 3D real time model reconstruction of the human body, can be used to collect information usually recorded by a physical stabilometric platform.

Methods

The algorithm used to reconstruct the human body model as a set of spheres is described and discussed. Moreover, experimental measurements and comparisons with data collected by a physical stabilometric platform are also reported. The measurements were collected on a set of 6 healthy subjects to whom a change in equilibrium condition was stimulated by performing an equilibrium task.

Results

The experimental results showed that more than 95% of data collected by the proposed method were not significantly different from those collected by the classic platform, thus confirming the usefulness of the proposed system.

Conclusions

The proposed virtual balance assessment system can be implemented at low cost (about 500$) and, for this reason, can be considered a home use medical device. On the contrary, astabilometric platform has a cost of about 10,000$ and requires periodical calibration. The proposed system does not require periodical calibration, as is necessary for stabilometric force platforms, and it is easy to use. In future, the proposed system with little integration can be used, besides being an emulator of a stabilometric platform, also to recognize and track, in real time, head, legs, arms and trunk, that is to collect information actually obtained by sophisticated optoelectronic systems.