Radio-frequency-induced coagulation necrosis in rabbits: immediate detection at US with a synthetic microsphere contrast agent.

PURPOSE: To determine whether a synthetic ultrasonographic (US) contrast agent can be used to differentiate coagulation necrosis from untreated tumor immediately after radio-frequency ablative therapy. MATERIALS AND METHODS: VX2 (adenocarcinoma) tumors (0.8-1.5-cm diameter) were implanted into 12 rabbits. Gray-scale and color Doppler US were performed with or without intravenous injection of a US contrast agent composed of poly-lactide-co-glycolic acid polymeric (PLGA) microspheres (2-micron diameter) filled with perfluorocarbon gas. Radio frequency was applied to each nodule for 6 minutes at 127 mA +/- 33 (mean +/- SD) (tip temperature, 92 degrees C +/- 2). Repeat US with a second dose of the contrast agent was performed immediately after ablation. In four animals, a third dose was administered 30-120 minutes after ablation. Radiologic-histopathologic correlation was performed and included in vivo staining and studies of mitochondrial function. RESULTS: Intense contrast agent enhancement was seen throughout the tumor prior to ablation. At gray-scale US, ablation produced hyperechoic foci, which were within 1 mm of the foci identified at histopathologic examination in seven of 12 animals (58%). After the administration of contrast material, foci devoid of previously visualized enhancement, which measured 7.3-15.0 mm, were identified. These were within 1 mm of the size of the foci identified at histopathologic examination in 11 of 12 animals (92%, P < .01). In two animals, enhancement depicted viable tumor, which appeared hyperechoic, on nonenhanced images. On delayed images, hyperechoic areas decreased in size, whereas the nonenhanced region remained unchanged. CONCLUSION: A PLGA microspherical US contrast agent enabled the immediate detection of coagulation necrosis as a region devoid of contrast enhancement after radio-frequency ablation in rabbit hepatic tumors. Therefore, this agent could provide real-time guidance during complex ablative procedures and may provide an efficient technique for postprocedural assessment.

A 23S rDNA-targeted polymerase chain reaction-based system for detection of Staphylococcus aureus in meat starter cultures and dairy products.

A polymerase chain reaction-based system for detection of Staphylococcus aureus was developed. The system consisted of the following components: (i) selective enrichment, (ii) DNA isolation, (iii) amplification of DNA with primers targeted against the 23S rRNA gene, and (iv) evaluation of the specificity of the polymerase chain reaction by Southern hybridization and nested polymerase chain reaction. The method achieved a high degree of sensitivity and unambiguity as required for the detection of contaminants in food starter preparations. The method permitted detection of Staphylococcus aureus in preparations of meat starter cultures containing Staphylococcus carnosus either alone or in combination with lactobacilli, pediococci, and/or Kocuria varians. Detection limits were sufficiently low to show within 12 h the presence of 10(0) CFU of S. aureus in starter preparations containing 10(10) CFU of S. carnosus. The system was also applied to dried skim milk and cream. For detection without selective enrichment, a protocol was developed and permitted detection of 120 CFU of S. aureus in 1 ml of cream within 6 h. With nested polymerase chain reaction, the detection limit was decreased by one order of magnitude.

Calpain as a novel target for treating acute neurodegenerative disorders.

Calpains are cytosolic, neutral proteases that normally exist in an inactive or quiescent state. They require higher than normal levels of calcium for activation which, once accomplished, lead to irreversible proteolysis of numerous cytoskeletal, membrane-associated and regulatory proteins. Because of these characteristics, calpain is gaining attention as a potentially important pathogenic variable in ischemic neuronal death. This manuscript explores this hypothesis by briefly reviewing current support for the role played by calpain in ischemic neurodegeneration, and then discussing a series of recently published studies which: 1. offer further evidence for the hypothesis, and 2. provide direct support for the idea that selective inhibition of calpain can greatly limit the neuronal damage that would normally occur following both global as well as focal brain ischemia. Thus, the data reviewed in this manuscript support the ideas that unregulated activation and proteolysis of intraneuronal calpain plays a significant role in the brain damage that occurs following an ischemic event and that delivering selective and membrane permeant calpain inhibitors to ischemic tissue may provide a powerfully effective therapeutic means of limiting neuronal damage.

Calpain inhibitor AK295 protects neurons from focal brain ischemia. Effects of postocclusion intra-arterial administration.

BACKGROUND AND PURPOSE: This research was performed to determine whether a selective inhibitor of the calcium-dependent protease, calpain, could reduce ischemia-associated brain damage when peripherally administered after a vascular occlusion. METHODS: A variation of the rat middle cerebral artery occlusion model was used. A range of doses of AK295 (a novel calpain inhibitor synthesized for this purpose) was continuously infused through the internal carotid artery, beginning 1.25 hours from the initiation of the occlusion. Rats were killed at 21 hours, and the infarct volume was quantified. RESULTS: Postocclusion (1.25-hour) infusion of the calpain inhibitor AK295 elicited a dose-dependent neuroprotective effect after focal ischemia. The highest dose tested (3 mg/kg per hour) afforded the maximum effect, illustrated by a 32% reduction in infarct volume 21 hours after the ischemia (vehicle, 81.7 +/- 4.7 mm3; AK295, 54.9 +/- 6.9 mm3; P < .007). CONCLUSIONS: These data provide the first evidence that a peripherally administered calpain inhibitor can protect against ischemic brain damage. They offer further support for an important role of calpain proteolysis in the brain degeneration associated with cerebral ischemic events and suggest that selective calpain inhibitors provide a rational, novel, and viable means of treating such neurodegenerative problems.

Stereospecific synthesis of peptidyl alpha-keto amides as inhibitors of calpain.

Peptidyl alpha-keto amides have been synthesized and tested as inhibitors of the cysteine protease calpain. A stereospecific synthesis was devised in which Cbz-dipeptidyl-alpha-hydroxy amides were oxidized with TEMPO/hypochlorite to the corresponding alpha-keto amides. This oxidation was accomplished in good yields and without epimerization of the chiral center adjacent to the ketone. The potent inhibition of porcine calpain I by the L,L diastereomers, combined with the poor inhibition by the L,D diastereomers, established the requirement for the all-L stereochemistry of the active inhibitor. The early lead inhibitors were very hydrophobic and, therefore, poorly soluble in aqueous solutions. Using the stereospecific route, new compounds were prepared with polar groups at the C- and N-termini. These modifications resulted in more soluble inhibitors that were still potent inhibitors of calpain. Studies of the stability of these alpha-keto amides showed that absolute stereochemistry can be maintained in acidic and unbuffered environments but general base-catalyzed epimerization of the chiral center adjacent to the ketone occurred rapidly. The alpha-hydroxy precursors were inactive as inhibitors of calpain, which supports the hypothesis that the alpha-keto compounds reversibly form an enzyme-bound tetrahedral species that results from the nucleophilic addition of the catalytic thiol of calpain to the electrophilic ketone of the inhibitor.

Limited enzymatic digestion for the determination of the quantities of minor diastereomeric impurities in preparations of RMP-7, a peptide containing a reduced peptide bond.

RMP-7 is a bradykinin analogue containing all "L" amino acids and a reduced dipeptide bond between amino acids eight and nine. This reduced dipeptide bond [4-Me-Tyr-psi(CH2NH)-Arg] is created under synthetic conditions which could result in inversions of the chiral centers of either 4-Me-Tyr or Arg. Stereoisomers of RMP-7 would be expected to have altered biological specificity. Current chromatographic methods are not sufficiently sensitive to distinguish the anticipated stereoisomeric variants of the intact molecule. Therefore we have devised an analytical method based on limited enzymatic digestion of the compound followed by reversed-phase HPLC analysis of the peptide fragments. Using this method we have been able to carry out precise and reliable quantitative analysis of the stereoisomeric content of different batches of peptide prepared for biological testing.

Postischemic administration of AK275, a calpain inhibitor, provides substantial protection against focal ischemic brain damage.

Experiments were conducted to determine whether a potent, reversible calpain inhibitor could reduce the cortical ischemic brain damage associated with focal ischemia in the rat. AK275 (Z-Leu-Abu-CONH-CH2CH3), the active isomer of the diastereomeric mixture, CX275, was employed in conjunction with a novel method of perfusing drug directly onto the infarcted cortical surface. This protocol reduced or eliminated numerous, nonspecific pharmacokinetic, hemodynamic, and other potentially confounding variables that might complicate interpretation of any drug effect. Focal ischemia was induced using a variation of the middle cerebral artery occlusion method. These studies demonstrated a reliable and robust neuroprotective effect of AK275 over the concentration range of 10 to 200 microM (perfused supracortically at 4 microliters/h for 21 h). Moreover, a 75% reduction in infarct volume was observed when initiation of drug treatment was delayed for 3 h postocclusion. Our data further support an important role of calpain in ischemia-induced neuropathology and suggest that calpain inhibitors may provide a unique and potentially powerful means of treating stroke and other ischemic brain incidents.