MscL is a mechanosensitive channel in bacteria that responds directly to membrane tension by opening a large conductance pore. To determine functionally important residues within this molecule, we have randomly mutagenized mscL, expressed the genes in living bacteria, and screened for gain-of-function mutants with hampered growth. Expression of these genes caused leakage of cytoplasmic solutes on little or no hypo-osmotic stress. In excised patches, the mutant channels gated at membrane tensions that are less than that required for the gating of the wild-type MscL. Hence, the data suggest that the slowed or no-growth phenotype is caused by solute loss because of inappropriate gating of the channel. Most of the mutations mapped to the first transmembrane domain. When this domain is modeled as an ␣-helix, the most severe mutations are substitutions of smaller amino acids (three glycines and one valine) on one facet, suggesting an important role for this structure in MS channel gating.

Hemolytic uremic syndrome (HUS) usually occurs after infection with Shiga toxin-producing bacteria. Thrombotic thrombocytopenic purpura, a disorder with similar clinical manifestations, is associated with deficient activity of a circulating metalloprotease that cleaves von Willebrand factor at the Tyr842-Met843 peptide bond in a shear stress-dependent manner. We analyzed von Willebrand factor-cleaving metalloprotease activity and the status of von Willebrand factor in 16 children who developed HUS after Escherichia coli O157:H7 infection and in 29 infected children who did not develop this complication. Von Willebrand factor-cleaving metalloprotease activity was normal in all subjects, but von Willebrand factor size was decreased in the plasma of each of 16 patients with HUS. The decrease in circulating von Willebrand factor size correlated with the severity of thrombocytopenia and was proportional to an increase in von Willebrand factor proteolytic fragments in plasma. Immunohistochemical studies of the kidneys in four additional patients who died of HUS demonstrated glomerular thrombi in three patients, and arterial and arteriolar thrombi in one patient. The glomerular thrombi contained fibrin but little or no von Willebrand factor. A decrease in large von Willebrand factor multimers, presumably caused by enhanced proteolysis from abnormal shear stress in the microcirculation, is common in HUS. HUS, characterized by acute renal failure, hemolysis with schistocytes on blood smears, and thrombocytopenia, is accompanied by thrombotic microangiopathy of the kidneys and of other organs (1). The syndrome covers a diverse spectrum of microangiopathic disorders (2-4), but most cases occur after infection with Shiga toxin-producing bacteria, such as Escherichia coli O157:H7 (5) or Shigella dysenteriae serotype 1 (6).TTP, a disorder with some clinical, laboratory, and histopathologic similarities to HUS, has been associated with abnormal homeostasis of von Willebrand factor, a protein that is secreted from endothelial cells as a disulfide-linked polymer of a polypeptide with 2050 amino acid residues. Circulating von Willebrand factor is normally cleaved between Tyr842 and Met843 (7) in a shear stress-dependent manner (8) by a plasma metalloprotease (9, 10), generating a series of multimers. Without this metalloprotease activity, von Willebrand factor, when unfolded by shear stress (11), has increased platelet-aggregating activity (12). It is postulated that this increased activity facilitates the formation of arteriolar and capillary platelet thrombi in TTP. Indeed, acquired TTP has been associated with deficient von Willebrand factor-cleaving metalloprotease activity caused by inhibitory antibodies (12, 13).

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We report a case of human clenbuterol toxicity confirmed and correlated with qualitative and quantitative serum clenbuterol assays. This poisoned patient, a 28-year-old woman, developed sustained sinus tachycardia at 140/min, hypokalemia (2.4 mEq/L, 2.4 mmol/L), hypophosphatemia (0.9 mg/dL, 0.29 mmol/L), and hypomagnesemia (1.52 mg/dL, 0.76 mmol/L) after ingesting a reportedly small quantity of clenbuterol. The patient received repeated doses of metoprolol to treat her cardiovascular stimulation and potassium chloride to treat her hypokalemia. She remained symptomatic for more than 20 hours after the ingestion. Analysis by enzyme-linked immunosorbent assay and liquid chromatography/mass spectrometry revealed a serum clenbuterol concentration of 2.93 mcg/L 3 hours after the ingestion and an undetectable serum concentration 20 hours after ingestion. It is noteworthy that at a serum concentration below the limit of detection by liquid chromatography/mass spectrometry, the patient remained symptomatic. Acute clenbuterol toxicity is rarely reported following illicit use in humans, and this is the first such case to provide confirmatory toxicological analysis.

Participants were unable to inflate endotracheal tube cuff to safe pressures and were unable to identify endotracheal tube cuffs with excessive intracuff pressure by palpation. Clinicians should consider using devices such as manometers to facilitate safe inflation and accurate measurement of endotracheal tube cuff pressure.

The mtrB gene from Bacillus pumilus encodes a 76-amino-acid polypeptide with 77% identity to the trp RNA-binding attenuation protein (TRAP) from Bacillus subtilis. B. pumilus TRAP binds trp leader RNA from either B. subtilis or B. pumilus in a tryptophan-dependent manner. Altering threonine 52 to alanine eliminated RNA-binding activity of B. pumilus TRAP.The Bacillus subtilis trp operon is regulated by transcription attenuation involving the RNA-binding protein TRAP (trp RNA-binding attenuation protein) (7,8,11,17). TRAP, which is the product of the mtrB gene (8), is activated by L-tryptophan to bind to a target site in the 204-nucleotide trp leader RNA transcript upstream of the start codon of the first structural gene (5,15). This binding influences the secondary structure of the leader RNA to form a transcription terminator, thereby halting transcription in the leader and preventing expression of the operon (6). In the absence of tryptophan, the operon is expressed because TRAP does not bind, and an alternative antiterminator structure forms in the leader transcript. Kuroda et al. (12) showed that the cis-acting features of the Bacillus pumilus trp operon are similar to those in B. subtilis and that expression of the cloned B. pumilus trp leader transcript in B. subtilis resulted in deregulated overexpression of the trp operon, presumably by titrating the trp regulatory factor. They therefore proposed that the B. pumilus trp operon is also regulated by an attenuation mechanism similar to that which controls the B. subtilis operon (12).We have cloned and sequenced the B. pumilus mtrB gene and found it to encode a 76-amino-acid polypeptide (molecular weight, 8,301) with 77% identity to B. subtilis TRAP. Purified B. pumilus TRAP binds to both B. pumilus and B. subtilis trp leader RNAs in a tryptophan-dependent manner. We also show that a mutation which changes threonine 52 to alanine in B. pumilus TRAP eliminates the ability of this protein to bind RNA, apparently by disrupting tryptophan binding.Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1. B. subtilis was transformed by using natural competence (1) as modified in reference 12. Resistance to 5-fluorotryptophan (Ftrp) was tested as described elsewhere (12).Cloning and sequencing the mtrB gene from B. pumilus. PCR using oligonucleotide primers based on nucleotides 800 to 823 within mtrA and nucleotides 1390 to 1410 downstream of the mtrB gene in B. subtilis (8) resulted in amplification of a 580-bp fragment from B. pumilus genomic DNA which contained the mtrB gene.Purification of TRAP. Purification of B. subtilis TRAP has been described previously (15). B. pumilus TRAP was purified similarly, from overexpressing Escherichia coli SG62052/pGP1-2/pTZ18BpmtrB, except that the immunoaffinity column contained antibodies prepared against B. pumilus TRAP.Gel mobility shift assays. Uniformly 32 P-labelled trp leader RNA transcripts were prepared as described previously (15) by using T7 RNA polymerase to trans...