A monoclonal antibody that induces neuronal apoptosis binds a metastasis marker.

The cell surface molecules controlling apoptosis in cortical neurons are largely unknown. A monoclonal antibody was derived that induces cultured neocortical neurons to undergo apoptosis. A Fab fragment of the antibody, however, lacked the ability to induce cell death. The antigen was purified, and characterized by compositional analysis, fast atom bombardment (FAB) mass spectrometry, sequential exoglycosidase treatments, methylation analysis, and (1)H-nuclear magnetic resonance spectroscopy, proving to be isoglobotetraosylceramide (IsoGb4). IsoGb4 has been shown previously to be a metastasis marker, antibodies against which block metastases in a mammary adenocarcinoma model (S. A. Carlsen et al., Cancer Res., 53: 2906-2911, 1993). Addition of the purified antigen to cells lacking this glycolipid demonstrated that it is capable of functioning as a portable apoptosis-transducing molecule. Intracellular ceramide levels were increased after the treatment with the apoptosis-inducing antibody, but the membrane sphingomyelin level remained unchanged. Fumonisin B1 inhibited both the ceramide increase and the apoptosis induced via IsoGb4, which indicated that the ceramide synthase pathway is likely to be involved in apoptosis induction by IsoGb4.

Magnetic, microplate-format plasmid isolation protocol for high-yield, sequencing-grade DNA.

We have developed a rapid, microplate-format plasmid isolation procedure to purify sequencing-grade DNA templates for high-throughput DNA sequencing operations. A modified lysozyme/boiling method is used to produce a plasmid-containing supernatant that is then purified by iron bead capture. After binding, the beads are pelleted in a magnetic field, washed and the DNA eluted in water. The method yields up to 10 micrograms plasmid DNA from a 1-mL overnight culture in a deep-well microplate. The procedure is suitable for large-scale experiments, amenable to automation and does not require expensive reagents or equipment. The entire protocol can be completed in as little as 2 h, and one technician with a 96-well pipetting station can process up to 48 plates per day. This protocol is ideal for any high-throughput operation in which template quantity, quality and reproducibility are of primary importance.

Structural organization of virulence-associated plasmids of Yersinia pestis.

The complete nucleotide sequence and gene organization of the three virulence plasmids from Yersinia pestis KIM5 were determined. Plasmid pPCP1 (9,610 bp) has a GC content of 45.3% and encodes two previously known virulence factors, an associated protein, and a single copy of IS100. Plasmid pCD1 (70,504 bp) has a GC content of 44.8%. It is known to encode a number of essential virulence determinants, regulatory functions, and a multiprotein secretory system comprising the low-calcium response stimulation that is shared with the other two Yersinia species pathogenic for humans (Y. pseudotuberculosis and Y. enterocolitica). A new pseudogene, which occurs as an intact gene in the Y. enterocolitica and Y. pseudotuberculosis-derived analogues, was found in pCD1. It corresponds to that encoding the lipoprotein YlpA. Several intact and partial insertion sequences and/or transposons were also found in pCD1, as well as six putative structural genes with high homology to proteins of unknown function in other yersiniae. The sequences of the genes involved in the replication of pCD1 are highly homologous to those of the cognate plasmids in Y. pseudotuberculosis and Y. enterocolitica, but their localization within the plasmid differs markedly from those of the latter. Plasmid pMT1 (100,984 bp) has a GC content of 50.2%. It possesses two copies of IS100, which are located 25 kb apart and in opposite orientations. Adjacent to one of these IS100 inserts is a partial copy of IS285. A single copy of an IS200-like element (recently named IS1541) was also located in pMT1. In addition to 5 previously described genes, such as murine toxin, capsule antigen, capsule anchoring protein, etc., 30 homologues to genes of several bacterial species were found in this plasmid, and another 44 open reading frames without homology to any known or hypothetical protein in the databases were predicted.

Anti-human class I MHC antibodies induce apoptosis by a pathway that is distinct from the Fas antigen-mediated pathway.

5H7, an anti-human class I MHC mAb that recognizes a monomorphic determinant of the alpha3 domain, profoundly inhibits T lymphocyte activation. The present study was designed to determine the role of programmed cell death in 5H7-mediated immune suppression. Incubation of PBMC with 5H7 mAb induced a marked reduction in viable cell recovery (VCR) of T cells (<10% VCR), NK cells (<1% VCR), and B cells (<1% VCR). In addition, 5H7 inhibited proliferation and VCR of JY, DBS-521, and BevD tumor lines as well as cells transfected with HLA-B27. Morphologic changes characteristic of apoptosis were induced by 5H7, including cell membrane blebbing, cytoplasmic vacuolization, condensation of nuclear chromatin, and nuclear fragmentation. Furthermore, DNA fragmentation was demonstrated in 5H7-treated PBMC using a TdT-mediated end-labeling (TUNEL) technique. 5H7, but not anti-Fas mAb, induced apoptosis of cell lines derived from patients with Niemann-Pick disease that lack acidic sphingomyelinase activity. In addition, induction of cell death by 5H7 was not inhibited by IL-1beta-converting enzyme (ICE) inhibitors under conditions that fully suppressed Fas-mediated apoptosis. These findings suggest that signal transduction through "classical" human class I MHC molecules induces apoptosis by a Fas-independent pathway that does not require acidic sphingomyelinase, is independent of ICE protease, and may represent a unique pathway of cell death in human lymphocytes.

Fas-mediated apoptosis and sphingomyelinase signal transduction: the role of ceramide as a second messenger for apoptosis.

In this article, we review the role of sphingomyelinases and ceramide in the Fas-mediated apoptosis signal transduction cascade. Several stimuli, including ligation of Fas, have been shown to enhance either neutral and/or acidic sphingomyelinase activity and increase ceramide content in intact cells or cell membrane preparations. Ceramide seems to have different functions, including induction of apoptosis, growth arrest, and/or differentiation, depending on cell type or location of sphingomyelin hydrolysis within the cell. Several putative targets for ceramide activity, including a kinase and a phosphatase, have also been identified. While ceramide and acidic sphingomyelinase activity appear to be involved in apoptotic signalling for Fas and other members of the tumour necrosis factor receptor family, it is clear that other signals and mechanisms are necessary for Fas-mediated apoptosis.

Apoptosis induced in Jurkat cells by several agents is preceded by intracellular acidification.

We have previously shown that in neutrophils deprived of granulocyte colony-stimulating factor, apoptosis is preceded by acidification and that the protection against apoptosis conferred on neutrophils by granulocyte colony-stimulating factor is dependent upon delay of this acidification. To test the hypothesis that acidification could be a general feature of apoptosis, we examined intracellular pH changes in another cell line. Jurkat cells, a T-lymphoblastoid line, were induced to undergo apoptosis with anti-Fas IgM, cycloheximide, or exposure to short-wavelength UV light. We found that acidification occurred in response to treatment with these agents and that acidification preceded DNA fragmentation. Jurkat cells were also found to possess an acid endonuclease that is active below pH 6.8, compatible with a possible role for this enzyme in chromatin digestion during apoptosis. Incubation of the cells with the bases imidazole or chloroquine during treatment with anti-Fas antibody or cycloheximide or after UV exposure decreased apoptosis as assessed by nuclear morphology and DNA content. The alkalinizing effect of imidazole and chloroquine was shown by the demonstration that the percentage of cells with an intracellular pH below 6.8 after treatment with anti-Fas antibody, cycloheximide, or UV was diminished in the presence of base as compared with similarly treated cells incubated in the absence of base. We conclude that acidification is an early event in programmed cell death and may be essential for genome destruction.

Detection of cytomegalovirus in paraffin-embedded postmortem coronary artery specimens of heart transplant recipients by the polymerase chain reaction: implications of cytomegalovirus association with graft atherosclerosis.

To assess the presence of cytomegalovirus in various tissues and its relevance to the development of graft atherosclerosis, 54 postmortem paraffin-embedded tissue samples from 15 heart transplant recipients who survived more than 100 days were analyzed by the polymerase chain reaction for cytomegalovirus. Eight patients had known previous cytomegalovirus exposure; 7 patients did not. Of the eight with known previous exposure, three patients (38%) died of graft atherosclerosis versus four (57%) of the seven patients without previous exposure. Of the 54 specimens, 49 were positive for beta-globin (inclusive positive control), including 38 coronary artery, five lung, five gastrointestinal, and one kidney. Only two coronary artery specimens were cytomegalovirus positive in a single patient with known cytomegalovirus exposure who did not have evidence of graft atherosclerosis at autopsy. Other tissues tested showed positive lung and stomach specimens in the patient with cytomegalovirus-positive coronary artery specimens and positive kidney, lung, and gastrointestinal specimens in a second patient. No specimens were cytomegalovirus positive in the remaining patients, despite the presence of graft atherosclerosis or previous cytomegalovirus exposure. Our data do not support the hypothesis that graft atherosclerosis is associated with latent cytomegalovirus infection of the coronary arteries. The role of cytomegalovirus in the pathogenesis of graft atherosclerosis is unknown, but possibly it represents a modulation of the immune system by remote infection.

Outcome of urgent percutaneous transluminal coronary angioplasty in acute myocardial infarction: comparison of single-vessel versus multivessel coronary artery disease.

Despite recent clinical trials of percutaneous transluminal coronary angioplasty (PTCA) in acute myocardial infarction, specific groups of patients that may benefit from adjunctive or alternative therapy have yet to be adequately characterized. The in-hospital outcome of 151 consecutive patients treated for acute myocardial infarction with urgent PTCA of the infarct-related artery was studied to identify a subgroup of patients at high risk. Patients were divided into two groups based on the angiographic presence of either single-vessel (n = 86) or multivessel (n = 65) coronary artery disease. Despite PTCA of only the infarct-related artery and similar baseline clinical characteristics such as age, peak serum creatine kinase concentration, left ventricular ejection fraction, and time from the onset of chest pain to arrival at the hospital, the group with multivessel disease had a lower rate of successful angioplasty (75% vs 92%, p < 0.005), with higher incidences of persistent total occlusion of the infarct-related artery (14% vs 3%, p < 0.02) and procedural complications during PTCA (28% vs 13%, p < or = 0.02), and were more likely to have multiple complications (12% vs 1%, p < 0.004). In addition, the group with multivessel disease had a higher rate of urgent (< or = 24 hours) coronary artery bypass graft surgery (13% vs 2%, p < 0.05) and a trend toward a higher in-hospital mortality rate (6% vs 1%, p < or = 0.17). By stepwise logistic regression, only the presence of single-vessel versus multivessel disease was predictive of PTCA success (p < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Right and left ventricular function after cardiac transplantation. Changes during and after rejection.

BACKGROUND: Attempts to identify noninvasive markers of ventricular dysfunction accompanying acute rejection have been hampered by a lack of detailed simultaneous hemodynamic data. Therefore, we prospectively performed serial monitoring of detailed left and right heart hemodynamic parameters in cardiac transplant recipients at the time of routine endomyocardial biopsy to better define the physiology of the allograft heart during and after acute rejection. METHODS AND RESULTS: To better assess the pathophysiology of the rejection process, 18 cardiac transplant patients were prospectively studied by serial right heart micromanometer catheterization and digital image processing at the time of routine endomyocardial biopsy. Eleven patients had 18 episodes of rejection. Studies of baseline (negative biopsy preceding rejection), rejection (acute moderate rejection), and resolved (first negative biopsy after rejection) states were compared. Seven patients who did not experience an episode of rejection served as the control group. Right ventricular minimum and end-diastolic pressures increased from baseline values of 0.9 +/- 3.2 and 6.9 +/- 3.7 mm Hg, respectively, to 3.2 +/- 5.5 and 9.9 +/- 6.6 mm Hg, respectively, with rejection (both variables, p less than 0.05) and remained elevated despite histological resolution of rejection (4.3 +/- 5.5 and 10.0 +/- 7.1 mm Hg, respectively; p less than 0.05 for both variables compared with baseline values). Concurrently, right ventricular end-diastolic volumes (133 +/- 29, 119 +/- 27, and 114 +/- 30 ml; baseline, rejection, and resolved, respectively) and left ventricular end-diastolic volumes (133 +/- 24, 117 +/- 20, and 113 +/- 30 ml; baseline, rejection, and resolved, respectively) significantly decreased during rejection and remained decreased after resolution of rejection (rejection and resolved compared with baseline values, p less than 0.05). Right ventricular chamber stiffness (0.055 +/- 0.035, 0.085 +/- 0.057, and 0.092 +/- 0.076 mm Hg/ml; baseline, rejection, and resolution, respectively; rejection and resolved compared with baseline values, p less than 0.05) increased with rejection and remained elevated after resolution of rejection. Right ventricular peak filling rate also increased from a baseline value of 2.48 +/- 0.45 to 2.76 +/- 0.63 ml end-diastolic volumes per second with rejection (p less than 0.05). Elevation of right ventricular filling pressures, peak filling rate, and chamber stiffness with a concomitant decrease in end-diastolic volume is consistent with a restrictive/constrictive physiology. Mean arterial blood pressure and systemic vascular resistance were elevated after the resolution of rejection (compared with either rejection or baseline values, p less than 0.05) associated with a higher mean daily dose of prednisone (resolved compared with either baseline or rejection values, p less than 0.05). The control group experienced a time-dependent increase in mean and diastolic systemic arterial pressures (both comparisons, p less than 0.05) without detectable diastolic dysfunction. CONCLUSIONS: Persistence of biventricular diastolic dysfunction may be due to an irreversible effect of rejection, although multifactorial changes in left ventricular afterload occur that may complicate serial assessment of ventricular function.