Hemorrhagic typhlocolitis associated with attaching and effacing Escherichia coli in common marmosets.

We describe the necropsy results from three common marmosets presented during an outbreak of hemorrhagic diarrhea in a colony of 230 common marmosets (Callithrix jacchus). Necropsy revealed consistent hemorrhagic typhlocolitis and variable ileitis associated with gram-negative bacilli closely adherent to enterocytes. Electron microscopy revealed bacilli attached intimately to shallow cup-like projections of enterocyte apical membranes with loss of microvilli (attaching and effacing). Escherichia coli isolates from affected marmosets were serogroup O26 and were positive for the E. coli attaching and effacing locus and negative for shiga-like toxin I and II, heat-stable enterotoxins a and b, and heat-labile enterotoxin by DNA probe hybridization. Results of a Vero cell cytotoxicity assay confirmed that the E. coli isolates did not produce shiga-like toxin. The increased availability of diagnostic probes for specific virulence factors such as attaching and effacing E. coli should lead to a greater understanding of the frequency of E. coli as a cause of diarrhea in nonhuman primates.

Tryptophan micro-scale determinations by rapid hydrolysis.

A rapid acid-hydrolysis micro method was developed for the accurate determination of the tryptophan contents of proteins. Sample protein (5 micrograms) was hydrolysed in 30 microliters of 3M mercaptoethanesulfonic acid at 166 degrees C for 25 min, resulting in total hydrolysis of the peptide bonds. The subjection of the hydrolysate to an amino-acid analyser revealed the amino-acid composition, including the content of tryptophan with a high recovery of 92%.

A rapid method for acid hydrolysis of protein with a mixture of trifluoroacetic acid and hydrochloric acid.

Proteins have regions which resist hydrolysis with mineral acid. The presence of a strong organic acid was found to be efficient for hydrolysis of a hydrophobic peptide bond. The proposed condition, a 2:1 (by vol.) mixture of concentrated hydrochloric acid and trifluoroacetic acid at 166 degrees C for 25 min was observed to be equivalent to the conventional conditions (6 M HCl at 110 degrees C for more than 24 h) without significant decomposition of amino acids. The method was shown to be superior to the conventional conditions, especially for hydrophobic proteins. The present method destroys tryptophan, as the conventional acid hydrolysis does.

A new polyacrylamide gel electrophoresis system. Separation of small peptides and proteins in a volatile buffer system after modification with a strongly acidic fluorescent NH2 reagent.

Polyacrylamide gel electrophoresis is one of the most efficient methods for separation and identification of proteins. A new gel electrophoresis system has been developed in order to facilitate both separation and extraction of peptides and proteins ranging in molecular weight from approximately 200 up to 100 000. This system involves the use of a volatile buffer, triethylamine/formic acid pH 11.7, and a reaction with a covalently binding NH2 reagent, 1,3,6-trisulfonylpyrene 8-isothiocyanate. Under these conditions, the addition of strongly negative charges to proteins is achieved which allows migration according to molecular weight. The modified proteins being fluorescent require neither fixation nor staining for their detection. This is an absolute requirement especially when dealing with small peptides. An additional advantage of such a modification is the increased solubility of the proteins which makes their extraction easier and more efficient. THe extracted proteins are easily freed from salts and other small molecules simply by evaporation and they are readily available for sequencing analysis using Edman degradation, carboxypeptidase digestion or amino acid analysis.