Objectives-To describe the frequency of pepsin-positive tracheal secretions (a proxy for the aspiration of gastric contents), outcomes associated with aspiration (including a positive Clinical Pulmonary Infection Score [a proxy for pneumonia] and use of hospital resources), and risk factors associated with aspiration and pneumonia in a population of critically ill tube-fed patients.Design-Prospective descriptive study conducted over a 2-yr period. Setting-Five intensive care units in a university-affiliated medical center with level I trauma status.Patients-Each of the 360 adult patients participated for 4 days. Among the inclusion criteria were mechanical ventilation and tube feedings. An exclusion criterion was physician-diagnosed pneumonia at the time of enrollment. Intervention-None.Measurements and Major Results-Almost 6,000 tracheal secretions collected during routine suctioning were assayed for pepsin; of these, 31.3% were positive. At least one aspiration event was identified in 88.9% (n = 320) of the participants. The incidence of pneumonia (as determined by the Clinical Pulmonary Infection Score) increased from 24% on day 1 to 48% on day 4. Patients with pneumonia on day 4 had a significantly higher percentage of pepsin-positive tracheal secretions than did those without pneumonia (42.2% vs. 21.1%, respectively; p < .001). Length of stay in the intensive care unit and need for ventilator support were significantly greater for patients with pneumonia (p < .01). A low backrest elevation was a risk factor for aspiration (p = .024) and pneumonia (p = .018). Other risk factors for aspiration included vomiting (p = .007), gastric feedings (p = .009), a Glasgow Coma Scale score <9 (p = .021), and gastroesophageal reflux disease (p = .033). The most significant independent risk factors for pneumonia were aspiration (p < .001), use of paralytic agents (p = .002), and a high sedation level (p = .039).Conclusions-Aspiration of gastric contents is common in critically ill tube-fed patients and is a major risk factor for pneumonia. Furthermore, it leads to greater use of hospital resources. Modifiable risk factors for aspiration need to be addressed. Keywords critical illness; enteral nutrition; aspiration pneumonia; risk factors; gastroesophageal reflux; gastric emptyingThe authors have no financial interests to disclose.The study was performed at Saint Louis University Hospital, in St. Louis, MO. NIH Public Access Author ManuscriptCrit Care Med. Author manuscript; available in PMC 2008 May 23. Published in final edited form as:Crit Care Med. 2006 April ; 34(4): 1007-1015. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptTracheobronchial aspiration can be defined as the inhalation of oropharyngeal or gastric contents into the respiratory tract (1). Although aspiration from either source is important, the type of greatest concern in critically ill tube-fed patients is tracheobronchial aspiration of gastric contents. The extent to which aspiration of gastric contents occurs is difficu...

We previously characterized a methionine aminopeptidase (EC 3.4.11.18; Met-AP1; also called peptidase M) in Saccharomyces cerevisiae, which differs from its prokaryotic homologues in that it (i) In all living cells, protein synthesis is initiated with either methionine (in the cytosol of eukaryotes), or formylmethionine (in prokaryotes, mitochondria, and chloroplasts). Where formylmethionine is used, the formyl group is usually removed cotranslationally by a deformylase, leaving methionine bearing a free NH2 group. The methionine NH2 is then removed by one or more methionine aminopeptidases (Met-APs), if the penultimate residue is small and uncharged-e.g

Angiogenesis inhibitors are a novel class of promising therapeutic agents for treating cancer and other human diseases. Fumagillin and ovalicin compose a class of structurally related natural products that potently inhibit angiogenesis by blocking endothelial cell proliferation. A synthetic analog of fumagillin, TNP-470, is currently undergoing clinical trials for treatment of a variety of cancers. A common target for fumagillin and ovalicin recently was identified as the type 2 methionine aminopeptidase (MetAP2). These natural products bind MetAP2 covalently, inhibiting its enzymatic activity. The specificity of this binding is underscored by the lack of inhibition of the closely related type 1 enzyme, MetAP1. The molecular basis of the high affinity and specificity of these inhibitors for MetAP2 has remained undiscovered. To determine the structural elements of these inhibitors and MetAP2 that are involved in this interaction, we synthesized fumagillin analogs in which each of the potentially reactive epoxide groups was removed either individually or in combination. We found that the ring epoxide in fumagillin is involved in the covalent modification of MetAP2, whereas the side chain epoxide group is dispensable. By using a fumagillin analog tagged with fluorescein, His-231 in MetAP2 was identified as the residue that is covalently modified by fumagillin. Site-directed mutagenesis of His-231 demonstrated its importance for the catalytic activity of MetAP2 and confirmed that the same residue is covalently modified by fumagillin. These results, in agreement with a recent structural study, suggest that fumagillin and ovalicin inhibit MetAP2 by irreversible blockage of the active site.

Most known organisms encode proteases that are crucial for constitutive proteolytic events. In the present paper, we describe a method to define these events in proteomes from Escherichia coli to humans. The method takes advantage of specific N-terminal biotinylation of protein samples, followed by affinity enrichment and conventional LC (liquid chromatography)-MS/MS (tandem mass spectrometry) analysis. The method is simple, uses conventional and easily obtainable reagents, and is applicable to most proteomics facilities. As proof of principle, we demonstrate profiles of proteolytic events that reveal exquisite in vivo specificity of methionine aminopeptidase in E. coli and unexpected processing of mitochondrial transit peptides in yeast, mouse and human samples. Taken together, our results demonstrate how to rapidly distinguish real proteolysis that occurs in vivo from the predictions based on in vitro experiments.

We describe here a new homogeneous antibody-based protein sensor design (molecular pincers) that allows rapid and sensitive detection of a specific protein in solution. In the presence of the target protein these sensors produce fluorescence signal derived from target-dependent annealing of short complementary fluorochrome-labeled oligonucleotides attached to a pair of target-specific antibodies via nanometer-scale flexible linkers. The sensors allow near-instantaneous detection of the target with sensitivity and specificity approaching that of enzyme-linked immunosorbent assay (ELISA) but requiring no sample manipulation other then the addition of the sample to the sensor mix. We used cardiac troponin I and C-reactive protein as the targets to validate these desirable properties of the sensors. Due to the availability of antibodies to thousands of interesting targets and the straightforward design blueprint of the sensors we expect their wide-ranging applications in research and medical diagnosis, especially when simplicity, high throughput, and short detection time are essential.

Limited proteolysis of intact yeast methionine aminopeptidase (MAP1) with trypsin releases a 34 kDa fragment whose NH2-terminal sequence begins at Asp70, immediately following Lys69. These results suggest that yeast MAP may have a two-domain structure consisting of an NH2-terminal zinc finger domain and a C-terminal catalytic domain. To test this, a mutant MAP lacking residues 2-69 was generated, overexpressed, purified and analyzed. Metal ion analyses indicate that 1 mol of wild-type yeast MAP contains 2 mol of zinc ions and at least 1 mol of cobalt ion, whereas 1 mol of the truncated MAP lacking the putative zinc fingers contains only a trace amount of zinc ions but still contains one mole of cobalt ion. These results suggest that the two zinc ions observed in the native yeast MAP are located at the Cys/His rich region and the cobalt ion is located in the catalytic domain. The kcat and Km values of the purified truncated MAP are similar to those of the wild-type MAP when measured with peptide substrates in vitro and it appears to be as active as the wild-type MAP in vivo. However, the truncated MAP is significantly less effective in rescuing the slow growth phenotype of map mutant than the wild-type MAP. These findings suggest that the zinc fingers are essential for normal MAP function in vivo, even though the in vitro enzyme assays indicate that they are not involved in catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Human serum albumin is the most abundant protein in the body and is an important biomarker used for disease-related diagnosis. Although the traditional enzyme-linked immunosorbent assay (ELISA) approach can precisely measure the concentration of human serum albumin, the multi-step procedure and time-consuming preparations of ELISA limit its diagnostic applications, preventing accurate point-of-care testing, for example. Herein, we report the recent development of an antibody-based albumin sensor that allows for a homogeneous measurement of albumin concentrations in saliva, urine and serum, in which this type of sensor is validated for the first time. The assay only requires simple mixing, and relies on time-resolved (TR) fluorescence resonance energy transfer (FRET) to produce robust, sensitive signals. The whole process, from sample preparation to final read-out, is expected to take less than one hour and requires only a standard plate-reader, thus making the sensor a convenient and cost-effective tool for albumin analysis.

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