Src tyrosine kinase inhibits apoptosis through the Erk1/2- dependent degradation of the death accelerator Bik.

Src, the canonical member of the non-receptor family of tyrosine kinases, is deregulated in numerous cancers, including colon and breast cancers. In addition to its effects on cell proliferation and motility, Src is often considered as an inhibitor of apoptosis, although this remains controversial. Thus, whether the ability of Src to generate malignancies relies on an intrinsic aptitude to inhibit apoptosis or requires preexistent resistance to apoptosis remains somewhat elusive. Here, using mouse fibroblasts transformed with v-Src as a model, we show that the observed Src-dependent resistance to cell death relies on Src ability to inhibit the mitochondrial pathway of apoptosis by specifically increasing the degradation rate of the BH3-only protein Bik. This effect relies on the activation of the Ras-Raf-Mek1/2-Erk1/2 pathway, and on the phosphorylation of Bik on Thr124, driving Bik ubiquitylation on Lys33 and subsequent degradation by the proteasome. Importantly, in a set of human cancer cells with Src-, Kras- or BRAF-dependent activation of Erk1/2, resistances to staurosporine or thapsigargin were also shown to depend on Bik degradation rate via a similar mechanism. These results suggest that Bik could be a rate-limiting factor for apoptosis induction of tumor cells exhibiting deregulated Erk1/2 signaling, which may provide new opportunities for cancer therapies.

A continuous assay for DNA cleavage: the application of "break lights" to enediynes, iron-dependent agents, and nucleases.

Although extensive effort has been applied toward understanding the mechanism by which enediynes cleave DNA, a continuous assay for this phenomenon is still lacking. In fact, with the exception of assays for DNase, continuous assays for most DNA cleavage events are unavailable. This article describes the application of "molecular break lights" (a single-stranded oligonucleotide that adopts a stem-and-loop structure and carries a 5'-fluorescent moiety, a 3'-nonfluorescent quenching moiety, and an appropriate cleavage site within the stem) to develop the first continuous assay for cleavage of DNA by enediynes. Furthermore, the generality of this approach is demonstrated by using the described assay to directly compare the DNA cleavage by naturally occurring enediynes [calicheamicin and esperamicin), non-enediyne small molecule agents (bleomycin, methidiumpropyl-EDTA-Fe(II), and EDTA-Fe(II]), as well as the restriction endonuclease BamHI. Given the simplicity, speed, and sensitivity of this approach, the described methodology could easily be extended to a high throughput format and become a new method of choice in modern drug discovery to screen for novel protein-based or small molecule-derived DNA cleavage agents.

A high voltage electrical burn of lung parenchyma.

High voltage electrical trauma may cause severe visceral injuries. We report a case of direct electrical injury to the lung parenchyma, without evidence of any thoracic wall contact injury, in an electrician who sustained a 20 kV-electrical shock while working in a substation cubicle. The diagnosis of a true electrical burn of the left lower lobe was suggested early on by imaging and then confirmed by surgical exploration, histological findings and the significant improvement of the patient's condition following resection of the infarcted lobe. All possible causes of bronchial and pulmonary pathologies in such a context were ruled out. The fatal outcome of two previous similar cases and the generally high mortality of any electrical visceral injury support early surgical management as the only rational life-saving treatment. Current pathophysiological knowledge substantiates the theory of an isolated visceral injury located far away from the contact wounds. However, the pathogenesis of such severe injuries is not entirely understood.

Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction.

The invasive signal amplification reaction has been previously developed for quantitative detection of nucleic acids and discrimination of single-nucleotide polymorphisms. Here we describe a method that couples two invasive reactions into a serial isothermal homogeneous assay using fluorescence resonance energy transfer detection. The serial version of the assay generates more than 10(7) reporter molecules for each molecule of target DNA in a 4-h reaction; this sensitivity, coupled with the exquisite specificity of the reaction, is sufficient for direct detection of less than 1,000 target molecules with no prior target amplification. Here we present a kinetic analysis of the parameters affecting signal and background generation in the serial invasive signal amplification reaction and describe a simple kinetic model of the assay. We demonstrate the ability of the assay to detect as few as 600 copies of the methylene tetrahydrofolate reductase gene in samples of human genomic DNA. We also demonstrate the ability of the assay to discriminate single base differences in this gene by using 20 ng of human genomic DNA.

Direct genetic analysis by matrix-assisted laser desorption/ionization mass spectrometry.

An approach to analyzing single-nucleotide polymorphisms (SNPs) found in the human genome has been developed that couples a recently developed invasive cleavage assay for nucleic acids with detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The invasive cleavage assay is a signal amplification method that enables the analysis of SNPs by MALDI-TOF MS directly from human genomic DNA without the need for initial target amplification by PCR. The results presented here show the successful genotyping by this approach of twelve SNPs located randomly throughout the human genome. Conventional Sanger sequencing of these SNP positions confirmed the accuracy of the MALDI-TOF MS analysis results. The ability to unambiguously detect both homozygous and heterozygous genotypes is clearly demonstrated. The elimination of the need for target amplification by PCR, combined with the inherently rapid and accurate nature of detection by MALDI-TOF MS, gives this approach unique and significant advantages in the high-throughput genotyping of large numbers of SNPs, useful for locating, identifying, and characterizing the function of specific genes.

Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes.

Flap endonucleases (FENs) isolated from archaea are shown to recognize and cleave a structure formed when two overlapping oligonucleotides hybridize to a target DNA strand. The downstream oligonucleotide probe is cleaved, and the precise site of cleavage is dependent on the amount of overlap with the upstream oligonucleotide. We have demonstrated that use of thermostable archaeal FENs allows the reaction to be performed at temperatures that promote probe turnover without the need for temperature cycling. The resulting amplification of the cleavage signal enables the detection of specific DNA targets at sub-attomole levels within complex mixtures. Moreover, we provide evidence that this cleavage is sufficiently specific to enable discrimination of single-base differences and can differentiate homozygotes from heterozygotes in single-copy genes in genomic DNA.

Blood vessel and lymphatic vessel invasion in resected nonsmall cell lung carcinoma. Correlation with TNM stage and disease free and overall survival.

BACKGROUND: The objective of this prospective study was to assess in 96 patients with resected nonsmall cell lung carcinoma (NSCLC) the prevalence of both blood and lymphatic vessel invasion (BVI and LVI) according to stage, as well as their prognostic value for disease free and overall survival. METHODS: BVI and LVI were evaluated by hematoxylin and eosin stains on surgical specimens after resection. Associations among variables were tested by Fisher's exact test or the chi-square test; prognostic values on time-failure data were analyzed by the log rank test and the multivariate Cox model. RESULTS: BVI was present in 52% of NSCLC cases and LVI in 59%. Venous but not arterial vascular invasion correlated with the T factor and pTNM, whereas LVI correlated with the N factor and pTNM. In univariate analysis, LVI but not BVI was associated with a short disease free interval (P = 0.0007) and poor survival (P = 0.0001). The estimated relative risk of death in patients with LVI was 3.2 compared with patients without LVI. In multivariate analysis, LVI and pTNM were additional predictors for poor disease free and overall survival. In this series, BVI had no prognostic value. CONCLUSIONS: The prevalence of BVI and LVI appeared high in patients with NSCLC, especially those with advanced pTNM stages. LVI was predictive of poor outcome, both time to recurrence and death.

Expanding the scope of RNA catalysis.

The basic notions of transition state theory have been exploited in the past to generate highly selective catalysts from the vast library of antibody molecules in the immune system. These same ideas were used to isolate an RNA molecule, from a large library of RNAs, that catalyzes the isomerization of a bridged biphenyl. The RNA-catalyzed reaction displays Michaelis-Menten kinetics with a catalytic rate constant (kcat) of 2.8 x 10(-5) per minute and a Michaelis constant (Km) of 542 microM; the reaction is competitively inhibited by the planar transition state analog with an inhibition constant (Ki) value of approximately 7 microM. This approach may provide a general strategy for expanding the scope of RNA catalysis beyond those reactions in which the substrates are nucleic acids or nucleic acid derivatives.

Increasing the chemical potential of the germ-line antibody repertoire.

To augment the chemical potential of the immunological repertoire, a metal ion-binding light chain has been introduced into the murine genome. Mice containing the transgene were subsequently immunized with a fluorescein conjugate. The transgenic light chain was found at a high frequency in the anti-fluorescein memory B-cell compartment. This general method should be applicable to other cofactors and small molecules and should lead to generation of antibodies with unique catalytic activities.

Acquired von Willebrand disease: correction of hemostatic defect by high-dose intravenous immunoglobulins.

High-dose intravenous immunoglobulins (ivIG) were used in a 57-year-old patient with acquired von Willebrand disease in order to correct a hemostatic defect before pneumonectomy for lung carcinoma. IvIG induced a rapid and complete correction of factor VIII (F VIII) and von Willebrand factor (vWF) and allowed surgery without additional factor coverage. F VIII and vWF returned to baseline values within 10 days after ivIG.

An efficient antibody-catalyzed aminoacylation reaction.

An antibody generated against a neutral phosphonate diester transition-state analog was found to catalyze the aminoacylation of the 3'-hydroxyl group of thymidine with an alanyl ester. A comparison of the apparent second-order rate constant of the antibody-catalyzed reaction [5.4 x 10(4) molar-1 minute-1 (M-1 min-1)] with that of the uncatalyzed reaction (2.6 x 10(-4) M-1 min-1) revealed this to be a remarkably efficient catalyst. Moreover, although the concentration of water (55 M) greatly exceeds that of the secondary alcohol, the antibody selectively catalyzes acyl transfer to thymidine. The antibody exhibits sequential binding, with Michaelis constants of 770 microM and 260 microM for acyl acceptor and donor, respectively, and a dissociation constant of 240 pM for hapten. This antibody-catalyzed reaction provides increased insight into the requirements for efficient aminoacylation catalysts and may represent a first step toward the generation of "aminoacyl transfer RNA synthetases" with novel specificities.

A mutagenesis study of a catalytic antibody.

We have generated seven site-specific mutations in the genes encoding the variable region of the heavy chain domain (VH) of the phosphocholine-binding antibody S107. S107 is a member of a family of well-characterized highly homologous antibodies that bind phosphorylcholine mono- and diesters. Two of these antibodies, MOPC-167 and T15, have previously been shown to catalyze the hydrolysis of 4-nitrophenyl N-trimethylammonioethyl carbonate. Two conserved heavy-chain residues, Tyr-33 and Arg-52, were postulated to be involved in binding and hydrolysis of 4-nitrophenylcholine carbonate esters. To more precisely define the catalytic roles of these residues, three Arg-52 mutants (R52K, R52Q, R52C) and four Tyr-33 mutants (Y33H, Y33F, Y33E, Y33D) of antibody S107 were generated. The genes encoding the VH binding domain of S107 were inserted into plasmid pUC-fl, and in vitro mutagenesis was performed. The wild-type and mutant S107 antibodies were expressed in P-3X63-Ag8.653 (P-3) myeloma cells by using a modified SV2 shuttle vector. The catalytic properties of wild-type antibody S107 are similar to those of the phosphocholine-specific antibody T15, which has the same VH protein sequence. In general, mutations at Tyr-33 had little effect on catalytic activity, whereas mutations at Arg-52 that result in loss of the positively charged side chain significantly lower the catalytic activity of S107. One mutant, Y33H, catalyzed the hydrolysis of 4-nitrophenyl N-trimethylammonioethyl carbonate with a kcat of 5.7 min-1 and a Km of 1.6 mM at pH 7.5. These results not only demonstrate the importance of electrostatic interactions in catalysis by antibody S107 but also show that catalytic side chains can be introduced into antibodies to enhance their catalytic efficiency.

[Wegener's granulomatosis. Therapeutic indications and follow-up course. Apropos of 5 cases]. / La granulomatose de Wegener. Indications thérapeutiques et suivi évolutif. A propos de 5 observations.

Five cases of Wegener's granulomatosis treated with cyclophosphamide (CPM) and prednisolone are reported. Four of these patients received intermittent intravenous boluses of cyclophosphamide with the aim of improving the prognosis and renal function and at the same time to attenuate any haematological or vesical toxicity of CPM. The initial response to treatment by boluses of CPM was favourable in all cases but three patients presented with relapses, which were sometimes repeated and boluses of CPM did not enable a remission to be maintained at the time. Recourse to continuous oral therapy in place of bolus therapy proved viable for the maintenance of remission in two cases. The bladder and haematological tolerance to the bolus was satisfactory but an episode of severe neutropenia led to an adaptation of the dose of CPM. The intermittent administration above all the low cumulative dose of CPM obtained with the boluses will explain the better vesical and haematological tolerance observed depending on the capacity to maintain a prolonged remission. The indications for boluses of CPM in the treatment of Wegener's granulomatosis remain uncertain and do not seem to be totally without inconvenience.

[Fatal hemoptysis during bronchial aspergillosis with multiple pulmonary artery aneurysms]. / Hémoptysie fatale au cours d'une aspergillose bronchique par anévrysmes artériels pulmonaires multiples.

Aspergillosis with fatal outcome are usually pulmonary invasive aspergillosis with or without dissemination, developed in patients with severe immunosuppression. We report a fatal case of bronchial necrotizing aspergillosis in a young woman with diabetes mellitus, who developed similar lesions to "Semi-invasive Aspergillosis", so-called "Chronic Necrotizing Pulmonary Aspergillosis". This aspergillosis was complicated by large pulmonary artery aneurysms requiring an hemostatic lobectomy. These aneurysms, secondary to the bronchial lesions, contrast with infectious aneurysms (so-called mycotic) secondary to septic embols. They differ from Rasmussen's aneurysms, due to tuberculosis, by their size, fusiform shape and extent. Lesions of vessels' walls and parietal fungal invasion in the vicinity of an endo-bronchial aspergilloma explain the vascular rupture. The multiplicity of these aneurysms, showed on C T Scan, is responsible for death by post-surgical recurrence of hemoptysis.

[Castleman's disease (giant lymph node hyperplasia): clinical, biological and developing polymorphism. Apropos of 4 cases]. / Maladie de Castleman (hyperplasie lymphoïde angiofolliculaire): polymorphisme clinique, biologique et évolutif. A propos de 4 observations.

Four cases of plasma cell type Castleman's disease (CD) are described. Two patients had localized forms (one mediastinal and the other mesenteric) and presented systemic manifestations associated with hypergammaglobulinemia and severe anemia. In both cases, the lesions were revealed by computerized tomography scans and cures were obtained by the complete surgical removal of the masses, which led to the rapid disappearance of the systemic manifestations. The other 2 patients had the multicentric form of CD and presented more extensive clinical and biological symptoms. One of these developed severe peripheral neuropathy and endocrine anomalies during the late phase of his disease, which led us to discuss the relationship between multicentric CD and the POEMS (polyneuropathy, organomegaly, endocrinopathy, M-protein, skin change) syndrome first described in Japan. Three of our patients presented with hypochromic microcytic anemia too severe to be explained by an inflammatory syndrome alone, and was likely due to several mechanisms. The etiology of CD remains unknown. The histological characteristics of angiofollicular lymph node hyperplasia are among the most important criteria for the diagnosis of localized and multicentric forms of CD, which can easily be made on a lymph node biopsy. However, it must be noted that this lesion can also be observed (but only rarely) in HIV (human immunodeficiency virus) - infected patients. The localized form is always considered to be benign, but, to date, there is no formal argument definitively supporting the malignancy of the multicentric one, in spite of its clinical similarity to a lymphoproliferative syndrome.

Observations of colloidal gold labelled platelet microtubules: high voltage electron microscopy and low voltage-high resolution scanning electron microscopy.

18 nm colloidal gold-antitubulin and 4 nm colloidal gold-antitubulin were used to label microtubules in adherent, fully spread platelets. Both sizes of marker effectively labelled microtubules in the partially extracted platelets. However only the 4 nm gold penetrated the dense microfilament matrix of the inner filamentous zone so that portions of microtubules within this cytoskeletal zone could be tracked. The gold marker could be visualized well with 1 MeV high voltage transmission EM and with 5 kV or greater secondary imaging or 20 kV backscattered imaging of carbon only coated samples. 1 kV secondary imaging permitted high resolution imaging of the surface of tubules and the microfilaments with their respective associated material. Individual gold-antibody complexes were difficult to identify by shape alone due to the tendency of the antibody coats to blend together when in very close approximation and due to the presence of other molecules or molecular aggregates similar in size to the gold-antibody labels. Microtubules were seen to wind in and out of the inner and outer filamentous zones as they encircled the granulomere. Some tubules were seen to "dead end" at the peripheral web. Numerous smaller microtubule loops were present principally in the outer filamentous zone and tubules could be followed as they went from the outer filamentous zone through the inner filamentous zone and into the granulomere.

Computed tomography of mediastinal lymph nodes in nonsmall cell lung cancer. A new approach based on the lymphatic pathway of tumor spread.

Computed tomography was used to evaluate mediastinal lymph nodes in 97 patients with nonsmall cell lung cancer. All patients had thorough surgical-pathological determination of mediastinal node status. Twenty-three patients were found to have metastatic lymph nodes. The usual lymphatic pathways of tumor spread into the mediastinum were defined using the node mapping scheme suggested by the American Thoracic Society. We considered mediastinal nodes abnormal when the short axis of the largest mediastinal node in the lymphatic drainage territory of the cancer was greater than or equal to 10 mm and the difference between this node and the largest node in the other territories is greater than 5 mm. The sensitivity was 78%, the specificity 99%, the positive predictive value 95%, the negative predictive value 94%, and the accuracy 94%. Comparing our method to those that used the size criterion alone, the number of false positives was reduced.