Amplification of ssb-1 mutant single-stranded DNA-binding protein in Escherichia coli.

The ssb-1 gene encoding a mutant Escherichia coli single-stranded DNA-binding protein has been cloned into plasmid pACYC184. The amount of overproduction of the cloned ssb-1 gene is dependent upon its orientation in the plasmid. In the less efficient orientation, 25-fold more mutant protein is produced than in strains carrying only one (chromosomal) copy of the gene; the other orientation results in more than 60-fold overproduction of this protein. Analysis of the effects of overproduction of the ssb-1 encoded protein has shown that most of the deficiencies associated with the ssb-1 mutation when present in single gene copy, including temperature-sensitive conditional lethality and deficiencies in amplified synthesis of RecA protein and ultraviolet light-promoted induction of prophage lambda +, are reversed by increased production of ssb-1 mutant protein. These results provide evidence in vivo that SSB protein plays an active role in recA-dependent processes. Homogenotization of a nearby genetic locus (uvrA) was identified in the cloning of the ssb-1 mutant gene. This observation has implications in the analysis of uvrA- mutant strains and will provide a means of transferring ssb- mutations from plasmids to the chromosome. On a broader scale, the observation may provide the basis of a general strategy to transfer mutations between plasmids and chromosomes.

Noninvasive predictors of patency for infrapopliteal PTFE bypasses with combined arteriovenous fistula and vein interposition technique.

BACKGROUND: Despite recent increased indications for infrapopliteal prosthetic bypass grafts with complementary arteriovenous fistulas, objective documentation of improved perfusion to the foot is lacking. In addition, the value of post operative noninvasive testing in the prediction of bypass success remains unclear. PATIENTS AND METHODS: Over a 3-year period, 41 patients with limb-threatening ischemia were treated with 41 infrapopliteal 6-mm polytetrafluoroethylene (PTFE) bypasses with a complementary arteriovenous fistula at our institution. Twenty-four patients were men and 17 were women, with an average age 71.3 +/- 8.6 years. Thirty-one patients (76%) had undergone at least 1 previous failed ipsilateral arterial bypass. Preoperative and early postoperative (less than 1 month) pulse-volume recordings of transmetatarsal amplitude (TMA) were available for analysis in 28 patients. Postoperative duplex evaluations of graft velocity, fistula patency, and prograde distal arterial flow were performed in 26 of the original 41 patients. These data were correlated to early graft failure in an attempt to identify specific noninvasive predictors. RESULTS: Cumulative primary patency rates of the original 41 patients were 79.0%, 69.2%, and 63.8% at 1, 2, and 3 years, respectively. The early postoperative TMA values ranged from 3 to 50 mm with a mean of 21.6 +/- 14.8 mm (P < 0.001). Twenty-one patients (75%) had patent grafts on follow-up of 2 to 37 months (mean 18.6). The early postoperative TMA in this group of patients was 26.5 +/- 12.4 mm compared with 3.3 +/- 2.8 mm in the 6 patients whose grafts failed within 6 months (P < 0.001). A TMA of < 5 mm was 83% sensitive and 95% specific for the prediction of early graft failure. Of the graft examined by duplex ultrasonography, 21 (81%) remained patent during the follow-up period. The midgraft peak systolic velocity (PSV) of these grafts was 109 +/- 8.0 cm/s compared with 74.2 +/- 15.3 cm/s for the 5 initially patent bypasses that subsequently failed at any time during the follow-up period (P < 0.05). PSV of < 70 cm/s was 60% sensitive and 86% specific in predicting early graft failure. The combination of early postoperative TMA < 5 mm and early midgraft PSV < 70 cm/s was 100% sensitive and 100% specific for the prediction of early graft failure. CONCLUSIONS: These data show that infrapopliteal PTFE arterial bypasses with complementary arteriovenous fistulas significantly improve arterial perfusion at the level of the foot in the majority of patients. Also, both the postoperative TMA and midgraft PSV appear to be reliable predictors of graft outcome. Further experience with the noninvasive surveillance of these bypasses may become as rewarding as it is in standard vein bypasses.

The Appearance of New Active Forms of Trypsin Inhibitor in Germinating Mung Bean (Vigna radiata) Seeds.

Ungerminated seeds of mung bean contain a single major species (F) of trypsin inhibitor with five minor species (A-E) separable on diethylaminoethyl-cellulose. During germination the level of trypsin inhibitory activity decreases from 1.8 units/grams dry weight in ungerminated cotyledons to 1.2 units/grams in cotyledons from seeds germinated 5 days. This decrease is accompanied by major changes in the distribution of inhibitory activity among the inhibitor species. By 48 hours of germination, inhibitor F has largely disappeared with an accompanying rapid increase in inhibitor C. Similarly, though less rapidly, inhibitor E decreases while inhibitor A increases. A similar sequence of changes is found in vitro when purified inhibitor F is incubated with extracts from seeds germinated 96 hours. The combined in vivo and in vitro data suggest a conversion sequence of: F --> E --> C --> A. The in vitro conversion is inhibited by phenylmethyl sulfonyl fluoride but not by iodoacetamide, indicating that at least the initial phases of inhibitor conversion are not catalyzed by the mung bean vicilin peptidohydrolase.

Preliminary results of a nonoperative approach to saphenofemoral junction thrombophlebitis.

PURPOSE: A prospective, nonrandomized study was conducted to evaluate the efficacy of a nonoperative approach of anticoagulation therapy to manage saphenofemoral junction thrombophlebitis (SFJT). METHODS: Over a 2-year period between January 1993 and January 1995, 20 consecutive patients with SFJT were entered into the study. They were hospitalized and given a full course of heparin. Duplex ultrasonography was performed before admission, both to establish the diagnosis and to evaluate the deep venous system. Two to 4 days after admission a follow-up scan was obtained to assess resolution of SFJT and to reexamine the deep venous system. Patients with SFJT alone and resolution of SFJT by duplex scanning were maintained on warfarin (Coumadin) for 6 weeks. Those with SFJT and deep venous thrombosis (DVT) were maintained on Coumadin for 6 months. Incidence of concurrent DVT and its location were noted. The efficacy of anticoagulation therapy was evaluated by measuring SFJT resolution, recurrent episodes of SFJT, and occurrence of pulmonary embolism (PE). RESULTS: There was a 40% incidence (eight of 20 patients) of concurrent DVT with SFJT. Of the eight patients, four had unilateral DVT, two had bilateral DVT, and two had development of DVT with anticoagulation. DVT was contiguous with SFJT in five patients and noncontiguous in three patients. Of 13 duplex scans obtained at 2 to 8 months follow-up, seven demonstrated partial resolution of SFJT, five had complete resolution, and one did not demonstrate resolution. There were no episodes of PE, no recurrences, and no complications of anticoagulation at maximum follow-up of 14 months. CONCLUSIONS: Anticoagulation therapy to manage SFJT was effective in achieving resolution, preventing recurrence and preventing PE within our follow-up period. There is a high incidence of DVT associated with SFJT that merits careful evaluation of the deep venous system during the course of management.

Isolation of uvrA mutation on a multicopy plasmid: preliminary characterization of the mutant protein.

A new uvrA mutation (uvrA276) has been isolated on a multicopy plasmid and shown to reside within the region of the uvrA gene defined by the KpnI to SalI endonuclease sites. The protein produced by the uvrA276 mutant gene is identical in size to the wild-type protein and binds to single-stranded DNA under the same conditions as the wild-type protein. However, extracts prepared from strains containing this mutant are deficient at incision of DNA that has been irradiated with UV light.

Superior vena caval Greenfield filters: indications, techniques, and results.

PURPOSE: This study assessed the clinical feasibility of placement of the titanium Greenfield filter within the superior vena cava (SVC) in patients with upper extremity deep venous thrombosis and evaluated the short-term results. METHODS: During the past 14 months, we have encountered 6 patients who had upper extremity central venous thrombosis in whom anticoagulation was deemed either contraindicated or ineffective in preventing recurrent pulmonary embolism. All patients underwent percutaneous insertion of a Greenfield filter into the SVC for prophylaxis for pulmonary embolism. Two of these patients had already undergone insertion of an inferior vena cava filter and two other patients had simultaneous insertion of SVC and inferior vena caval filters. RESULTS: No complications such as filter migration, misplacement, excessive hemorrhage, or pneumothorax occurred. Two patients subsequently underwent uncomplicated insertion of a Swan-Ganz catheter despite the presence of the filter. Two patients died of unrelated causes without clinical evidence of complications related to the filter. During the follow-up period from 4 to 14 months, there was no clinical evidence of pulmonary embolism in the remaining four patients. CONCLUSIONS: This procedure can be performed safely and is effective for the prevention of pulmonary embolism in patients with upper extremity venous thrombosis.

Complementary distal arteriovenous fistula and deep vein interposition: a five-year experience with a new technique to improve infrapopliteal prosthetic bypass patency.

PURPOSE: In an attempt to improve graft patency results of prosthetic bypasses to infrapopliteal arteries, we used a new type of adjunctive technique that combines an arteriovenous fistula and vein interposition (AVF/VI). METHODS: Over the past 5 years, 68 such reconstructions were performed in 62 patients with critical ischemia in whom a totally autogenous vein bypass was not feasible. Forty-seven patients (76%) had one or more failed ipsilateral infrainguinal bypasses. The AVF/VI was performed by transposing the central portion of the adjacent deep vein onto the side of the recipient artery. The distal end of a 6-mm polytetrafluoroethylene (PTFE) ringed graft then was anastomosed to the hood of the AVF. The segment of vein interposed between the PTFE graft and the recipient artery widened the anastomosis and improved the compliance mismatch. Simultaneous pressure measurements of the radial artery and the distal portion of the graft were obtained in all cases. RESULTS: Significant pressure gradients ranging from 35 to 70 mm Hg were detected in 26 bypasses (38%), which led to banding of the venous outflow that decreased the gradient to within 20 mm Hg. A gradient < or = 30 mm Hg was found in 28 bypasses (41%), and no banding was required if the absolute intragraft systolic pressure was > or = 100 mm Hg. Only 14 bypasses (21%) had no detectable pressure gradients. Twenty-six bypasses originated from femoral arteries, 34 from iliac arteries, and 8 from patent proximal grafts. The recipient arteries were the anterior tibial artery in 33 cases, posterior tibial in 17, peroneal in 15, dorsalis pedis in 2, and lateral plantar in 1. All patients began a regimen of heparin 6 to 8 hours after surgery and continued to receive chronic anticoagulation. Cumulative, 3-year assisted primary graft patency rates were 78%, 70%, and 62%, respectively. Cumulative 3-year AVF patency rates were 65%, 57%, and 46%, respectively. The 3-year limb salvage rate was approximately 78%. CONCLUSION: Adjunctive AVF/VI significantly improves infrapopliteal PTFE graft patency and limb salvage rates. The combination of a decreased compliance mismatch at the distal anastomosis and the abolishment of a large pressure gradient at the distal anastomosis while maintaining higher graft flow rates may have contributed to the improved results.

Saphenous vein thrombophlebitis (SVT): a deceptively benign disease.

PURPOSE: The association between deep vein thrombosis (DVT) and the hypercoagulable state is a well-established entity. However, the association between saphenous vein thrombophlebitis and coagulation abnormalities has not been investigated. Although thrombosis of varicose veins typically runs a benign course, phlebitis of the saphenous system may propagate to the deep system or saphenofemoral junction that requires more aggressive therapy. Given the potential similarity in clinical outcome between saphenous vein thrombophlebitis (SVT) and DVT, we have investigated the coagulation profile of patients presenting with isolated SVT. METHODS: Seventeen consecutive patients who presented to our vascular laboratory with isolated SVT had a coagulation profile performed that included antithrombin III (AT III), protein C (PC), protein S (PS) antigen and activity levels, activated protein C (APC) resistance, factor V DNA mutation, and coagulation factors II and X. All patients had duplex scans performed on both the superficial and deep venous systems. Patients with SVT only were treated with nonsteroidal antiinflammatory drugs (NSAIDs) and warm soaks as outpatients, whereas those patients found to have DVT or a clot at the saphenofemoral junction were fully anticoagulated with heparin and coumadin therapy. All 17 patients had at least one repeat coagulation profile performed up to 5 months after their SVT occurrence to ensure that the results of hypercoagulability were not transient. RESULTS: Ten (59%) of the 17 patients with SVT had abnormal coagulation profiles on initial presentation. All 10 patients who were hypercoagulable had repeat tests and 6 (35%) remained abnormal. Four patients who had abnormal results converted to normal values. Seven patients with normal coagulation profiles on initial presentation had repeat tests and all remained normal. CONCLUSION: The incidence of the hypercoagulable state in patients with SVT is high. Thirty-five percent of patients with isolated SVT had consistently abnormal coagulation profiles. Patients with SVT may be prone to the development of DVT or saphenofemoral junction thrombophlebitis and should be closely followed after the initial diagnosis of hypercoagulability.