Inhibition of TEM-2 beta-lactamase from Escherichia coli by clavulanic acid: observation of intermediates by electrospray ionization mass spectrometry

Clavulanic acid, the therapeutically important inhibitor of beta-lactamases containing a nucleophilic serine residue at their active sites, inhibits Escherichia coli TEM-2 beta-lactamase via a complex mechanism. Electrospray ionization mass spectrometry (ESIMS) studies revealed that a minimum of four different modified proteins are formed upon incubation of clavulanate with the TEM-2 enzyme. These exhibit mass increments relative to the unmodified TEM-2 beta-lactamase of 52, 70, 88, and 155 Da. Time course studies implied that no long-lived forms of clavulanate-inhibited TEM-2 beta-lactamase retain the carbons of the oxazolidine ring of clavulanate. The absence of a 199 Da increment to unmodified TEM-2 suggests rapid decarboxylation of clavulanate upon binding to the enzyme. Proteolytic digestions of purified forms of clavulanate inhibited TEM-2 beta-lactamase followed by analyses using high-performance liquid chromatography coupled to ESIMS (HPLC-ESIMS) and chemical sequencing were used to provide positional information on the modifications to the enzyme. Increments of 70 and 80 Da increments were shown to be located in a peptide containing Ser-70. A further 70 Da mass increment, assigned as a beta-linked acrylate, was localized to a peptide containing Ser-130. A mechanistic scheme for the reaction of clavulanate with TEM-2 beta-lactamase is proposed in which acylation at Ser-70 and subsequent decarboxylation is followed either by cross-linking with Ser-130 to form a vinyl ether or by reformation of unmodified enzyme via a Ser-70 linked (hydrated) aldehyde. Purified cross-linked vinyl ether was observed to slowly convert under acidic conditions to a Ser-70 linked (hydrated) aldehyde with concomitant conversion of Ser-130 to a dehydroalanyl residue.     

Anesthesia case of the month. Dystocia, cesarean section and acupuncture resuscitation of newborn kittens

OBJECTIVE: To assess the results of laparoscopic splenectomy as a treatment for immune thrombocytopenic purpura (ITP). MATERIAL AND METHODS: We conducted a retrospective study of all patients who underwent laparoscopic splenectomy for ITP at our institution between August 1992 and May 1997. RESULTS: Of 27 patients who underwent attempted laparoscopic splenectomy for ITP at our institution during the study period, 26 had completion of the procedure without conversion to an open splenectomy. The median postoperative hospital stay was 1.5 days, and no postoperative deaths occurred. In one patient, pancreatitis developed postoperatively. In four patients, splenectomy failed--two initially and two subsequently--and reinstitution of medical therapy was necessary. The other patients have remained free of medication, and 19 patients have platelet counts greater than 100 x 10(9)/L. The 3-year actuarial success rate was 81.5%. Response to corticosteroid therapy preoperatively may be an indicator of success of splenectomy. CONCLUSION: Laparoscopic splenectomy is safe and allows prompt recovery. Long-term response rates are similar to those achieved with open splenectomy.     

Novel antitumor 2-cyanoaziridine-1-carboxamides

A set of 20 2-cyanoaziridine-1-carboxamides was synthesized from 2-cyanoaziridine and appropriate isocyanates. These compounds were active against a variety of solid and hematological tumor cells in culture, including strains resistant to doxorubicin and mitoxantrone. Their potencies in these assays correlated with the lipophilicity of substituents. The N-phenyl derivative was more potent and equally effective to imexon, a cyclized 2-cyanoaziridine-1-carboxamide of clinical interest, against cloned fresh human tumors.     

The role of state policies and programs in buffering the effects of poverty on children's immunization receipt

OBJECTIVES: This study assessed the influence of public policies on the immunization status of 2-year old children in the United States. METHODS: Up-to-dateness for the primary immunization series was assessed in a national sample of 8100 children from the 1988 National Maternal and Infant Health Survey and its 1991 Longitudinal Follow-Up. RESULTS: Documented immunization rates of this sample were 33% for poor children and 44% for others. More widespread Medicated coverage was associated with greater likelihood of up-to-dateness among poor children. Up-to-dateness was more likely for poor children with public rather than private sources of routine pediatric care, but all children living in states where most immunizations were delivered in the public sector were less likely to be up to date. Poor children in state with partial vaccine replacement programs were less likely to be up to date than those in free-market purchase states. CONCLUSIONS: While state policies can enhance immunization delivery for poor children, heavy reliance on public sector immunization does not ensure timely receipt of vaccines. Public- and private-sector collaboration is necessary to protect children from vaccine-preventable diseases.     

Can duplex scan arterial mapping replace contrast arteriography as the test of choice before infrainguinal revascularization?

PURPOSE: Arteriography is the diagnostic test of choice before lower extremity revascularization, because it is a means of pinpointing stenotic or occluded arteries and defining optimal sites for the origin and termination of bypass grafts. We evaluated whether a duplex ultrasound scan, used as an alternative to arteriography, could be used as a means of accurately predicting the proximal and distal anastomotic sites in patients requiring peripheral bypass grafts and, therefore, replace standard preoperative arteriography. METHODS: Forty-one patients who required infrainguinal bypass grafts underwent preoperative duplex arterial mapping (DAM). Based on these studies, an observer blinded to the operation performed predicted what operation the patient required and the best site for the proximal and distal anastomoses. These predictions were compared with the actual anastomotic sites chosen by the surgeon. RESULTS: Whether a femoropopliteal or an infrapopliteal bypass graft was required was predicted correctly by means of DAM in 37 patients (90%). In addition, both anastomotic sites in 18 of 20 patients (90%) who had femoropopliteal bypass grafts and 5 of 21 patients (24%) who had infrapopliteal procedures were correctly predicted by means of DAM. CONCLUSION: DAM is a reliable means of predicting whether patients will require femoropopliteal or infrapopliteal bypass grafts, and, when a patient requires a femoropopliteal bypass graft, the actual location of both anastomoses can also be accurately predicted. Therefore, DAM appears able to replace conventional preoperative arteriography in most patients found to require femoropopliteal reconstruction. Patients who are predicted by means of DAM to require crural or pedal bypass grafts should still undergo preoperative contrast studies to confirm these results and to more precisely locate the anastomotic sites.