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.     

Unfavorable mechanical effects of heat and moisture exchangers in ventilated patients

OBJECTIVE: To investigate the mechanical effects of artificial noses. SETTING: A general intensive care unit of a university hospital. PATIENTS: 10 patients in pressure support ventilation for acute respiratory failure. INTERVENTIONS: The following three conditions were randomly tested on each patient: the use of a heated humidifier (control condition), the use of a heat and moisture exchanger without filtering function (HME), and the use of a combined heat and moisture exchanger and mechanical filter (HMEF). The pressure support level was automatically adapted by means of a closed-loop control in order to obtain constancy, throughout the study, of patient inspiratory effort as evaluated from airway occlusion pressure at 0.1 s (P0.1). Patient's ventilatory pattern, P0.1, work of breathing, and blood gases were recorded. MEASUREMENTS AND MAIN RESULTS: The artificial noses increased different components of the inspiratory load: inspiratory resistance, ventilation requirements (due to increased dead space ventilation), and dynamic intrinsic positive end-expiratory pressure (PEEP). The additional load imposed by the artificial noses was entirely undertaken by the ventilator, being the closed-loop control of P0.1 effective to maintain constancy of patient inspiratory work by means of adequate increases in pressure support level. CONCLUSIONS: The artificial noses cause unfavorable mechanical effects by increasing inspiratory resistance, ventilation requirements, and dynamic intrinsic PEEP. Clinicians should consider these effects when setting mechanical ventilation and when assessing patients' ability to breathe spontaneously.     

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.     

Evidence for disturbed S-adenosylmethionine : S-adenosylhomocysteine ratio in patients with end-stage renal failure: a cause for disturbed methylation reactions?

BACKGROUND: Elevated homocysteine concentrations have been associated with premature arteriosclerosis and with impairment of key methylation reactions through accumulation of the homocysteine metabolite S-adenosylhomocysteine. In end-stage renal failure high homocysteine concentrations are commonly found but thus far the concentrations of related adenosylated metabolites in plasma have not been assessed. METHODS: In this prospective study we determined plasma homocysteine and related metabolites in 25 patients on regular haemodialysis, and in 40 healthy volunteers. Blood samples from patients were drawn immediately before and in 10 patients additionally after the dialysis session. RESULTS: Folic acid and vitamin B12 in plasma were similar in patients (mean +/- SEM 25+/-2 nmol/l and 400+/-41 pmol/l respectively) and controls (24+/-3 and 324+/-23 respectively). In patients plasma homocysteine, S-adenosylmethionine and S-adenosylhomocysteine were markedly elevated (36.6+/-3.6 micromol/l, 381+/-32nmol/l and 1074+/-55 nmol/l respectively) compared to the control values (6.8+/-0.4 micromol/l, 60+/-3 nmol/l and 24.4+/-1.1 nmol/l respectively) whereas the molar ratio of plasma S-adenosylmethionine and S-adenosylhomocysteine was significantly decreased (0.36+/-0.02 and 2.7+/-0.2 in patients and controls respectively). Haemodialysis failed to normalize the abnormal levels of these metabolites. CONCLUSION: Since the ratio of S-adenosylmethionine : S-adenosylhomocysteine is closely linked to the activity of numerous enzymatic methylation reactions, these results suggest that methylation may be impaired in these patients.     

The protease-protected 30 kDa domain of SecA is largely inaccessible to the membrane lipid phase

SecA binds to the inner membrane of Escherichia coli through low affinity lipid interactions or with high affinity at SecYEG, the integral domain of preprotein translocase. Upon addition of preprotein and nucleotide, a 30 kDa domain of SecYEG-bound SecA is protected from proteolysis via membrane insertion. Such protection could result from some combination of insertion into the lipid phase, into a proteinaceous environment or across the membrane. To assess the exposure of SecYEG-bound SecA to membrane lipids, a radiolabeled, photoactivatable and lipid-partitioning crosslinker, 3-trifluoromethyl-3-(m[125I]iodophenyl) diazirine benzoic acid ester, was incorporated into inner membrane vesicles. The 30 kDa domain of SecYEG-bound SecA, inserted into the membrane in response to translocation ligands, is 18-fold less labeled than SecY, which is labeled effectively. In contrast, incorporation of the purified 30 kDa SecA fragment into crosslinker-containing detergent micelles or addition of detergent to crosslinker-containing membranes bearing the protease-protected SecA domain readily allows for labeling of this domain. We propose that the protease-inaccessible 30 kDa SecA domain is shielded from the fatty acyl membrane phase by membrane-spanning SecYEG helices and/or is largely exposed to the periplasm.     

Generation of chromosome fragment specific bovine DNA sequences by microdissection and DOP-PCR

Trends in causative organisms and sources of infection were studied in a series of 288 episodes of bacteremia in neutropenic cancer patients observed in a single institution from 1986 to 1993. The incidence of bacteremia increased significantly from 20 episodes per 1000 admissions in 1986 to 50 episodes per 1000 admissions in 1993 (p = 0.00001). Over the study period, a continuous increment in gram-positive bacteremia, which reached 81% of episodes in 1993 (p = 0.000001), was observed. Conversely, the incidence of gram-negative bacteremia remained stable. Coagulase-negative staphylococci and viridans group streptococci were the most commonly isolated pathogens. Bacteremia caused by coagulase-negative staphylococci increased from 3 episodes per 1000 admissions to 19 episodes per 1000 admissions (p = 0.0001), and viridans group streptococci bacteremia increased from 0 episodes per 1000 admissions to 19 episodes per 1000 admissions (p = 0.000001). The upward trend in gram-positive bacteremia appeared to be related to a significant increase in both intravascular catheters (p = 0.003) and oral mucositis (p = 0.003) as sources of infection. Specific strategies to prevent chemotherapy-induced mucositis and catheter-related bacteremia merit further investigations.     

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.     

Hierarchical Self‐Assembly of Peptide‐Coated Carbon Nanotubes

Numerous applications, from molecular electronics to super‐strong composites, have been suggested for carbon nanotubes. Despite this promise, difficulty in assembling raw carbon nanotubes into functional structures is a deterrent for applications. In contrast, biological materials have evolved to self‐assemble, and the lessons of their self‐assembly can be applied to synthetic materials such as carbon nanotubes. Here we show that single‐walled carbon nanotubes, coated with a designed amphiphilic peptide, can be assembled into ordered hierarchical structures. This novel methodology offers a new route for controlling the physical properties of nanotube systems at all length scales from the nano‐ to the macroscale. Moreover, this technique is not limited to assembling carbon nanotubes, and could be modified to serve as a general procedure for controllably assembling other nanostructures into functional materials.