A new method for post-translationally labeling proteins in live cells for fluorescence imaging and tracking.

We present a novel method to fluorescently label proteins, post-translationally, within live Saccharomycescerevisiae. The premise underlying this work is that fluorescent protein (FP) tags are less disruptive to normal processing and function when they are attached post-translationally, because target proteins are allowed to fold properly and reach their final subcellular location before being labeled. We accomplish this post-translational labeling by expressing the target protein fused to a short peptide tag (SpyTag), which is then covalently labeled in situ by controlled expression of an open isopeptide domain (SpyoIPD, a more stable derivative of the SpyCatcher protein) fused to an FP. The formation of a covalent bond between SpyTag and SpyoIPD attaches the FP to the target protein. We demonstrate the general applicability of this strategy by labeling several yeast proteins. Importantly, we show that labeling the membrane protein Pma1 in this manner avoids the mislocalization and growth impairment that occur when Pma1 is genetically fused to an FP. We also demonstrate that this strategy enables a novel approach to spatiotemporal tracking in single cells and we develop a Bayesian analysis to determine the protein's turnover time from such data.

Resistance to HSP90 inhibition involving loss of MCL1 addiction.

Inhibition of the chaperone heat-shock protein 90 (HSP90) induces apoptosis, and it is a promising anti-cancer strategy. The mechanisms underpinning apoptosis activation following HSP90 inhibition and how they are modified during acquired drug resistance are unknown. We show for the first time that, to induce apoptosis, HSP90 inhibition requires the cooperation of multi BH3-only proteins (BID, BIK, PUMA) and the reciprocal suppression of the pro-survival BCL-2 family member MCL1, which occurs via inhibition of STAT5A. A subset of tumour cell lines exhibit dependence on MCL1 expression for survival and this dependence is also associated with tumour response to HSP90 inhibition. In the acquired resistance setting, MCL1 suppression in response to HSP90 inhibitors is maintained; however, a switch in MCL1 dependence occurs. This can be exploited by the BH3 peptidomimetic ABT737, through non-BCL-2-dependent synthetic lethality.

Temozolomide: a new oral cytotoxic chemotherapeutic agent with promising activity against primary brain tumours.

Temozolomide, a new oral cytotoxic agent, has been given to 28 patients with primary brain tumours. Treatment was given at a dose of 150 mg/m2/day for 5 days (i.e. total dose 750 mg/m2) escalating, if no significant myelosuppression was noted on day 22, to 200 mg/m2/day for 5 days (i.e. total dose 1000 mg/m2) for subsequent courses at 4 week intervals. A major improvement in computer tomography (CT) scan was noted in 5/10 patients with astrocytomas recurrent after radiotherapy, with a major clinical improvement but minor improvement on CT scan in one further patient. Reduction in the size of the CT lesion was also observed in 4/7 patients with newly diagnosed high grade astrocytomas given 2-3 courses of temozolomide prior to irradiation. 1 patient with recurrent medulloblastoma had a clinical response in bone metastases. Temozolomide was well tolerated with little subjective toxicity and usually predictable myelosuppression and is a promising new drug in the treatment of primary brain tumours.

The Charing Cross Hospital experience with temozolomide in patients with gliomas.

Temozolomide, a new oral cytotoxic agent, was given to 75 patients with malignant gliomas. The schedule used was for the first course 150 mg/m2 per day for 5 days (i.e. total dose 750 mg/m2), escalating, if no significant myelosuppression was noted on day 22, to 200 mg/m2 per day for 5 days (i.e. total dose 1000 mg/m2) for subsequent courses at 4-week intervals. There were 27 patients with primary disease treated with two courses of temozolomide prior to their radiotherapy and 8 (30%) fulfilled the criteria for an objective response. There were 48 patients whose disease recurred after their initial surgery and radiotherapy and 12 (25%) fulfilled the criteria for an objective response. This gave an overall objective response rate of 20 (27%) out of 75 patients. Temozolomide was generally well tolerated, with little subjective toxicity and predictable myelosuppression. However, the responses induced with this schedule were of short duration and had relatively little impact on overall survival. In conclusion, temozolomide given in this schedule has activity against high grade glioma. However, studies evaluating chemotherapy in primary brain tumours should include a quality-of-life/performance status evaluation in addition to CT or MRI scanning assessment.

Controlled non-heart-beating donor liver transplantation: a successful single center experience, with topic update.

BACKGROUND: The critical shortage of transplantable organs necessitates utilization of unconventional donors. We describe a successful experience of controlled non-heart-beating donor (NHBD) liver transplantation. METHODS: Controlled NHBDs had catastrophic head injury, prognosis for no meaningful recovery, decision to withdraw life support, and subsequent consent for donation. After stopping mechanical ventilation in the operating room, death determination by a nontransplant caregiver, and rapid aortic cannulation, liver and kidneys were recovered. RESULTS: Controlled NHBDs contributed 5% of hepatic allografts (8/164) from August 1996 through June 1999 (9% in 1998). Sixteen NHBDs afforded 8 livers and 24 kidneys. Liver donors (n=8) were 11-66 years old; half were >50 years old. Premortem alanine aminotransferase was 25-157 U/L. Arrest occurred 3-27 min after stopping ventilation. Perfusion started 3-5 min after incision, and <22 min after hypotension (mean arterial pressure: <50 mmHg). Patient and graft survivals are 100% at 18+/-12 months follow-up. There was no intraoperative complication, reperfusion syndrome, poor graft function, primary nonfunction, arterial thrombosis, biliary complication, or serious infection. Postoperative day 2 prothrombin time was 13+/-1 sec. Peak alanine aminotransferase was 980+/-601 U/L. Intensive care unit and posttransplant lengths of stay were 2+/-2 and 10+/-7 days, respectively. Soon after transplantation there was frequent temporary hyperbilirubinemia (five of eight recipients; bilirubin peak: 7-29 mg/dl, 2-3 weeks after transplantation) and rejection (4/8 recipients, <3 weeks after transplantation). CONCLUSIONS: NHBDs significantly and safely expanded our donor pool. NHBD surgeons must be capable of rapid procurement. Cautious liberalization of criteria for accepting livers from NHBDs with confounding risk factors is justified. Refined ethics guidelines would broaden approval of NHBDs.

Multiple vascular profiles in the umbilical cord; an indication of maternal smoking habits and intrauterine distress.

A total of 644 umbilical cords were examined over a period of 5 years, 40 cords (6.2 per cent) showed more than the normal three vascular profiles on cross-section. Both the umbilical arteries and vein showed multiple profiles, although arterial profiles were far more commonly multiple. Clinical data were correlated with the histological findings. Heavy smoking was significantly associated with multiple vascular profiles. Maternal age and rhesus group were not significantly related however, but there was some association with primiparity. Fetuses with a gestational age between 33 and 38 weeks were significantly more likely to have multiple vessels than either fetuses beyond 38 weeks or less than 33 weeks gestation. Multiple channels also occurred significantly more frequently in still birth than in live birth cords. We suggest that multiple vascular profiles represent a response to intrauterine hypoxia. Inflammation of the cord was associated with multiple channels and suggests linkage between these two indications of fetal distress.

EB virus-specific IgA in serum of patients with infectious mononucleosis and of healthy people of different ages.

The following sera were tested for EB virus-specific IgA: serial sera from 61 cases of infectious mononucleosis (IM) and from 195 EBV IgG positive healthy students; single sera from each of 1469 persons of different ages, 63 cases of untreated Hodgkin's disease, and 22 neonates. EBV specific IgA was found in the sera from 88% of cases of IM, from 18.5% of EBV IgG positive healthy students, and in 13.5% of repeat samples from the same students three years later. The incidence of EBV IgA varied from 5 to 30% at different ages in single sera from EBV IgG positive persons aged 2 to 70 years. The higher percentages occurred in the age groups where recent sero-conversion rates were high. Fifteen percent of sera from cases of Hodgkin's disease were positive for EBV IgA, an incidence similar to that for healthy adults in the age group 25-45 years. None of the EBV IgG positive sera from neonates gave a positive reaction for EBV IGA.

Screening tests for hepatitis B antigen and antibody in two colleges of education and studies on the relationship between nonspecific positive antibody tests and EB virus infection.

Sera from 627 students entering Colleges of Education between 1969 and 1972 were tested for hepatitis B surface antigen and antibody. One was found positive for antigen, none for antibody. Six for 15 positive Hepanosticon results and two positive Hepatest results occurred in sera which also gave positive heterophil antibody tests indicative of current or recent EB virus infection. One of these six sera was still positive in the Hepanosticon test after one absorption, and one of two Hepatests gave no positive reaction with the control cells. Eleven of 14 sera from cases of infectious mononucleosis gave positive Hepanosticon results and two were still positive after one absorption. Seven were positive in the Hepatest and only three of these were positive with the control cells. The control tests in the Hepanosticon and Hepatest do not clearly identify all false positives due to Paul Bunnell antibody. It is suggested that when a positive result in a passive haemagglutination test can be removed by absorption or if positive after absorption cannot be confirmed by other tests for hepatitis Bs antigen, the patient from whom the serum specimen was taken should be investigated for indications of current EB virus infection.

Studies on the diagnostic value of an immunofluorescence test for EB virus-specific IgM.

A modification of the test for EB virus/IgM introduced by Schmitz and Scherer (1972) is described. It is simple and gives reproducible results.EB virus/IgM was demonstrated in all but one case of infectious mononucleosis and in students with minor illness shown to have acquired EB virus/IgG recently. Unlike the EB virus/IgG, the IgM disappears within a few months. Although the Paul-Bunnell-Davidsohn test is still the test of choice for the diagnosis of infectious mononucleosis, the EB virus/IgM test could be useful to establish a diagnosis of current or recent EB virus infection where the Paul-Bunnell-Davidsohn test was negative or equivocal.

Demonstration of Chlamydia trachomatis in colposcopic cervical biopsy specimens by an immunoperoxidase method.

A total of 31 cervical biopsy specimens were taken from 29 women attending a genitourinary medicine clinic, nine women (11 biopsy specimens) were known to have Chlamydia trachomatis cervicitis and 20 women were known to be free of chlamydial infection. The specimens were routinely processed to paraffin wax and stained by an anti-Chlamydia immunoperoxidase technique to localise the organisms. Of the 11 positive biopsy specimens three showed positive staining of elementary/reticulate bodies. In one case the surface endocervical cells showed large inclusions which were packed with chlamydial bodies. The diagnosis of chlamydial infection is difficult to make clinically and in routine cytological and histological specimens but immunoperoxidase staining can clearly identify C trachomatis inclusions in cervical biopsy specimens provided infection is severe.

Incidence of anti-intermediate filament antibody in serum samples of students with suspected glandular fever.

Serum samples from 40 students with suspected infectious mononucleosis were tested for the presence of antibodies to intermediate filaments (AIFA) of the cytoskeleton. Twenty had antibodies to the Epstein-Barr virus capsid antigen before their illness, and during it their sera remained negative by the Paul-Bunnell test. The other 20 patients did not have antibodies to the Epstein-Barr virus capsid antigen before their illness and seroconverted during the illness. These patients (true infectious mononucleosis group) developed positive Paul-Bunnell tests. Sera from normal subjects (blood donors) were also tested for AIFA. AIFA was present in titres greater than 1/10 in 80% of the infectious mononucleosis group (mean titre 1/40-1/80), 10% of the Paul-Bunnell negative glandular fever group, and 8.5% of the normal blood donors.

Comparison of the indirect fluorescent antibody test with agglutination, complement-fixation, and Coombs test for Brucella antibody.

A comparison is made of a Brucella fluorescent antibody test with four standard tests on 342 normal, 86 diagnostic, and 41 known positive human sera. Where positive titres of accepted significance were obtained in standard tests, a titre of 1/10 or more was achieved in the fluorescence test.

Laboratory diagnosis of EB virus infection in some cases presenting as hepatitis.

In 1975 and 1976, 7580 serum specimens were tested for HBsAg by the passive haemagglutination test, Hepanosticon. Serum from 38 people gave a positive result after absorption with the absorbent provided, but the presence of HBsAg could not be confirmed by other tests. Tests for current infection with EB virus, namely, the presence of heterophil antibody and EB virus specific IgM, were performed and were positive in 11 of the 38. In six of these the clinical picture was of hepatitis rather than infectious mononucleosis.

Measurement of heterophil antibody and antibodies to EB viral capsid antigen IgG and IgM in suspected cases of infectious mononucleosis.

The EBV IgG titres in acute and convalescent specimens from 97 cases of infectious mononucleosis were compared with titres from acute and convalescent sera from 96 students with illnesses resembling infectious mononucleosis but without heterophil antibody, EB IgM or EB IgG seroconversion; and also with titres from 91 healthy students known to have had EB IgG antibody for at least six months. These titres were related to the titre of the Research Standard A.66/235 for infectious mononucleosis serum prepared by the National Institute for Biological Standards and Control. Serial sera were tested for heterophil antibody and EBVCA specific IgG and IgM from 61 university students with infectious mononucleosis. The period of persistence of heterophil antibody and EBV IgM after illness was outlined from the results of the tests. Single sera from 406 patients in hospital or general practice sent to the diagnostic laboratory for heterophil antibody tests were also tested for EBV antibodies without prior knowledge of the heterophil antibody result. The close agreement between heterophil antibody and EBV IgM results is shown. False positive EB IgM results were correlated with the presence of rheumatoid factor.

EB virus antibody and infectious mononucleosis in a boarding school for boys.

A study of EB virus antibody was undertaken in a boarding school for boys between 11 and 18 years of age; 35% of boys had EBV antibody when first sampled and a small number acquired antibody each year. Some of these boys had had no recorded illness during the period before the first positive sample and some developed classic infectious mononucleosis which was accompainied by a positive heterophil antibody test and detection of EB virus specific IgM. In a school of 800 boys there were 13 cases of infectious mononucleosis in four years.

Correlation between serological and immunofluorescence results in the investigation of brucellosis in veterinary surgeons.

Four serological tests and three immunofluorescence tests for IgG, IgM, and IgA were compared for value in the investigation of brucellosis in veterinary surgeons. No one serological test stood out over the others, and the immunofluorescence tests did not appear to have advantages over the serological tests. If a laboratory is limited in time and resources then the saline agglutination or the complement fixation test would be reasonably satisfactory. The 2-mercaptoethanol test and the antihuman globulin (Coombs' test) have no advantages over the other two and could be dropped. Immunofluorescence tests are not recommended for routine testing of brucellosis sera. The results and these recommendations apply to the 'vet' sera tested; it is reasonable to suppose that what applies to 'vet' sera will also apply to sera of those who work with or are in repeated contact with cattle and who will have had previous experience of brucella antigen, that is, dairy farmers, herdsmen, or slaughter house employees.

Limb salvage in end-stage renal disease (ESRD). Comparison of modern results in patients with and without ESRD.

We reviewed our experience with femoral-popliteal-tibial reversed vein bypasses performed for limb salvage in 226 patients without and 19 patients with end-stage renal disease (ESRD). While 18-month primary patency rates were comparable (85% and 89%), limb salvage was significantly lower (76% vs 95%) in patients with ESRD. Five amputations in the ESRD group were required for nonhealing, large foot ulcers in diabetic patients despite patent arterial bypass while only five of 13 amputations in patients without ESRD were required in the presence of patent grafts. The need for major amputation despite patent bypass in diabetic patients with ESRD who have extensive foot gangrene or ischemic ulceration occurs sufficiently often that we recommend primary amputation be considered in these patients without regard to possible vascular reconstruction.

Reducing perioperative myocardial infarction following vascular surgery. The potential role of beta-blockade.

OBJECTIVE: To determine which perioperative variables may influence the occurrence of perioperative myocardial infarction (PMI) following vascular surgery. DESIGN: Case-control study. SETTING: Combined Veterans Affairs Medical Center-university hospital vascular service. PATIENTS: During a 4-year period, all major vascular surgical operations (N = 2088) were evaluated with serial postoperative electrocardiography and cardiac enzyme measurements. Patients with PMI following nonemergent vascular surgery (N = 53) were matched with randomly selected control patients without PMI (N = 106) for age, gender, type of operation, hypertension, and symptoms of coronary artery disease. MAIN OUTCOME MEASURES: The two groups were compared for operative blood loss, blood pressure, and heart rate as well as length of operation, type of anesthetic, and use of perioperative beta-blockers, nitroglycerine, calcium channel blockers, vasopressors, and angiotensin-converting enzyme inhibitors. RESULTS: beta-Blockers were used less frequently in patients with PMI than in control patients without PMI (30% vs 50%; P = .01). Overall beta-blockade was associated with a 50% reduction in PMI (P = .03). Perioperative myocardial infarction was not associated with length of operation, type of anesthetic, blood pressure, or use of other medications. CONCLUSIONS: beta-Blockade is associated with a decreased incidence of PMI in patients undergoing vascular surgery. Prophylactic perioperative use of beta-blockers may decrease PMI in patients requiring major vascular surgery. A prospective randomized trial of beta-blockers in these patients appears to be warranted.

Anticoagulation followed by elective carotid surgery in patients with repetitive transient ischemic attacks and high-grade carotid stenosis.

OBJECTIVE: To evaluate the results of preoperative heparin therapy followed by carotid surgery for patients with repetitive transient ischemic attacks (TIAs) and high-grade carotid stenoses. DESIGN: A 4-year prospective study. SETTING: Oregon Health Science University Hospital and Portland (Ore) Veterans Affairs Hospital. PATIENTS: Twenty-nine consecutive patients with repetitive TIAs referable to 30 high-grade (> or = 70%) ipsilateral carotid stenoses were treated with short-term heparin anticoagulation, followed by cerebral angiography, routine preoperative evaluation, and subsequent carotid reconstruction. INTERVENTIONS: Heparin sodium anticoagulation was maintained for a mean of 5 days. Surgical management consisted of 24 standard endarterectomies, five bypasses to the internal carotid artery, and one external carotid endarterectomy. MAIN OUTCOME MEASURES: Primary outcome variables included perioperative hemorrhage, thrombocytopenia, stroke, and death. Secondary outcome variables included carotid occlusion and recurrent TIAs with heparin therapy. RESULTS: One symptomatic common carotid occlusion and one asymptomatic internal carotid occlusion occurred during preoperative heparin therapy. Thirteen patients had additional sporadic TIAs while receiving heparin. There were no preoperative cerebral infarcts, thrombocytopenia, or clinical bleeding associated with heparin therapy. There was one postoperative stroke and one death due to myocardial infarction. CONCLUSION: When necessary, heparin anticoagulation and delayed carotid reconstruction would appear to be an acceptable alternative to emergency carotid surgery in patients with high-grade carotid stenosis and acute repetitive TIAs.

Extrathoracic arterial grafts performed for carotid artery occlusive disease not amenable to endarterectomy.

HYPOTHESIS: Extrathoracic cervical grafts are safe and provide long-lasting stroke prevention in patients with disease not amenable to standard carotid bifurcation endarterectomy. DESIGN: Review of a prospectively maintained vascular surgical registry. SETTING: Combined university and Department of Veterans Affairs vascular surgical service. PARTICIPANTS: Patients requiring surgery for carotid atherosclerotic occlusive disease not amenable to endarterectomy from January 1988 to March 1998. INTERVENTIONS: Carotid interposition grafting, subclavian-carotid bypass, or carotid-carotid bypass. MAIN OUTCOME MEASURES: Perioperative stroke and death, and life-table determination of freedom from stroke, stroke-free survival, and graft patency. RESULTS: Sixty patients (mean age, 65.8 years; range, 36-83) underwent cervically based carotid grafting. All had greater than 70% stenosis or occlusion of the innominate, common carotid, or internal carotid arteries, and 30 (50%) had undergone at least 1 previous ipsilateral carotid endarterectomy. Indication for operation was stroke or transient ischemic attack in 46 (77%) and asymptomatic high-grade stenosis in 14 (23%). Operative procedures included 31 (52%) carotid interposition grafts, 18 (30%) subclavian-carotid grafts, and 11 (18%) carotid-carotid grafts. Mean follow-up was 29 months (range, 1-117 months). Perioperative stroke rate was 5% (3/60) all in symptomatic patients, and there were no perioperative deaths. By life-table analysis, freedom from stroke was 92% at 1 and 5 years. Stroke-free survival was 90% at 1 year and 61% at 5 years. Primary graft patency was 94% at 1 year and 84% at 5 years, with assisted primary patency of 90% at 5 years. CONCLUSION: Cervical carotid artery grafts for complicated or recurrent carotid atherosclerosis not amenable to endarterectomy are durable and provide excellent freedom from stroke with low perioperative morbidity and mortality.