RESUMO
The Mycobacterium xenopi gyrase A mini-intein has been engineered to yield a controllable N-terminal or C-terminal, single-splice-junction autocleavage element. When combined with an affinity tag, these modified mini-inteins can be used to purify target proteins after a single combined chromatography/cleavage step. Cleavage at the intein N terminus was induced with thiol reagents, while cleavage at the intein C terminus was induced by a temperature shift to 16 degrees-25 degrees C. Different preferences for the residue immediately preceding the intein were observed during thiol-induced, N-terminal splice-junction cleavage of the M. xenopi gyrase A mini-intein vs. the Saccharomyces cerevisiae vacuolar ATPase, subunit A (VMA) intein present in the IMPACT purification system. Furthermore, the M. xenopi gyrase A mini-intein C-terminal autocleavage vector allows isolation of polypeptides with N-terminal cysteine residues that are active in the Intein Mediated Protein Ligation method of protein semisynthesis.
Assuntos
Proteínas de Bactérias/isolamento & purificação , DNA Topoisomerases Tipo II/química , Mycobacterium xenopi/enzimologia , Processamento de Proteína , Proteínas de Bactérias/química , Proteínas de Transporte/genética , Clonagem Molecular , DNA Girase , DNA Topoisomerases Tipo II/genética , Escherichia coli , Lectinas de Ligação a Manose , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/enzimologia , Temperatura , Tiorredoxinas/química , Tropomiosina/químicaRESUMO
The gregarious braconid wasp Cotesia congregata parasitizes host larvae of Manduca sexta, and several other sphingid species. Parasitism induces host immunosuppression due to the disruptive action of the wasp's polydnavirus (PDV) on host blood cells. During the initial stages of parasitism, these cells undergo apoptosis followed by cell clumping, which clears the hemolymph of a large number of cells. In this study, the persistence and expression of Cotesia congregata PDV (CcPDV) were examined using Southern and Northern blots, respectively. Digoxygenin-labelled total polydnaviral DNA was used to probe genomic DNA isolated from fat body and brains of hosts with emerged wasps taken 6 days following egress of the parasitoids, and significant cross-hybridization between the host fat body genomic DNA with viral DNA was seen. Thus, the virus persists in the host for the duration of parasitism, even during the post-emergence period, and may even be integrated in the host caterpillar DNA. Viral gene expression was examined using Northern blots and probes to the Cotesia rubecula CrV1 homolog, and the CrV1-like mRNAs were expressed as early as 4 h post-parasitization for at least 72 h and faint hybrization is even seen at the time the wasps eclose. In contrast, in Pieris rapae larvae the CrV1 transcript is expressed only for a brief time, during which time hemocyte function is disrupted. The effect is transitory, and hemocytes regain their normal functions after the parasites emerge as first instars.The genome of CcPDV contains one copy of the CrV1-like homolog as shown on Southern blots of viral genomic DNA. In conjunction with our earlier studies of the PDV-encoded early protein 1, the current work suggests multiple viral transcripts are produced following parasitization of the host, and likely target host hemocytes to induce their apoptosis, thereby preventing encapsulation of the parasitoid's eggs. Whether viral DNAs are integrated in the host's genomic DNA remains to be proven, but our results provide preliminary evidence that viral DNAs are detected in the host's fat body cells examined at the time of wasp emergence and several days later.
Assuntos
Larva/virologia , Manduca/virologia , Polydnaviridae/isolamento & purificação , Vespas/virologia , Animais , Northern Blotting , Southern Blotting , DNA Viral/genética , Polydnaviridae/genéticaRESUMO
Four cases of multiple liver metastasis of colorectal cancer treated with hepatic resection after chemotherapy were investigated. The 4 patients consisted of 3 males and 1 female with a mean age of 67.8 years. Two patients underwent systemic chemotherapy, and the other 2 received hepatic arterial infusion chemotherapy. The effect was PR in all patients, and mean duration until resection was 8.5 months. One patient had lung metastasis and one had recurrence in a local lesion and residual liver after hepatic resection, but all patients remain alive. The longest survival periods in 4 patients who underwent hepatic resection and 13 who did not were 32 and 24 months, respectively. It is suggested that hepatic resection after chemotherapy for multiple liver metastasis of colorectal cancer may be useful for improvement of outcome.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Camptotecina/análogos & derivados , Neoplasias Colorretais/patologia , Hepatectomia , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas/cirurgia , Idoso , Camptotecina/administração & dosagem , Cisplatino/administração & dosagem , Esquema de Medicação , Feminino , Fluoruracila/administração & dosagem , Humanos , Irinotecano , Leucovorina/administração & dosagem , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Taxa de SobrevidaRESUMO
In the patient-specific vascular CFD, determination of the inlet and outlet boundary conditions (BCs) is an important issue for a valid diagnosis. The 3D cine phase-contrast MRI (4D Flow) velocimetry is promising for this issue; yet, its measured velocities contain relatively large error and are not admissible as the BCs without any correction. This paper proposes a novel correction method for determining the BCs accurately using the 4D Flow velocimetry. First, we reveal that the error of the velocity measured by the 4D Flow at each measurement voxel is large but is distributed symmetrically. Secondly, our method pays attention to the incompressibility of the blood and the fact that the volume flow rate (VFR) in each vessel is constant on any cross sections. We reveal that the average of the cross-sectional VFRs integrated from many measurement voxel in each vessel is accurate despite the large error. Finally, we propose the novel correction method, which applies a smoothing to the measured velocities on each inlet or outlet boundary with a low-pass filter and then corrects them with the VFR. The results of the several phantom studies are presented to validate the accuracy of our method. A demonstrative analysis for an actual aneurysm is also presented to show the feasibility and effectiveness of our method.
Assuntos
Hemodinâmica/fisiologia , Imageamento Tridimensional/métodos , Imageamento por Ressonância Magnética/métodos , Humanos , Modelos Teóricos , ReologiaRESUMO
Maternal undernutrition (MUN) results in growth-restricted newborns with reduced nephron numbers that is associated with increased risk of hypertension and renal disease. The total adult complement of nephrons is set during nephrogenesis suggesting that MUN affects the staged development of nephrons in as yet unknown manner. A possible cause may be the increased renal apoptosis; therefore, we investigated whether apoptotic signaling and cell death were increased in MUN rat kidneys. Pregnant rat dams were fed an ad libitum diet [control] or were 50% food restricted (MUN) starting at embryonic day (E) 10. Male offspring kidneys (n = 5 each, MUN and control) were analyzed for mRNA using quantitative PCR (E20) and for protein expression using Western blotting and immunohistochemistry (E20 and postnatal day 1, P1). Apoptosis was measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Upregulation of pro-apoptotic protein expression was detected at E20 (Fas receptor, caspase 9) and at P1 (caspase 3, Bax). The anti-apoptotic factor Bcl2 was significantly decreased in P1 kidneys. Kidney TUNEL showed apoptotic nuclei significantly increased in the P1 nephrogenic zone (MUN 3.3 + 0.3 v. C 1.6 + 0.5, P = 0.002). The majority of apoptotic nuclei co-localized to mesenchyme and pretubular aggregates in the nephrogenic zone. Differential regulation of apoptosis in mesenchyme and pretubular aggregates following parturition suggests a mechanism for nephropenia in gestational programming of the kidney.
RESUMO
Ferromagnetism and superconductivity are thought to compete in conventional superconductors, although in principle it is possible for any metal to become a superconductor in its non-magnetic state at a sufficiently low temperature. At pressures above 10 GPa, iron is known to transform to a non-magnetic structure and the possibility of superconductivity in this state has been predicted. Here we report that iron does indeed become superconducting at temperatures below 2 K at pressures between 15 and 30 GPa. The transition to the superconducting state is confirmed by both a drop in resistivity and observation of the Meissner effect.
RESUMO
Direct electrical transport measurements in a diamond anvil cell provide evidence for the metallization of cesium iodide (CsI) at a pressure of 115 gigapascals. A drop in the temperature dependence of the resistance was found at pressures above 180 gigapascals, indicating that the CsI was superconductive. The superconductivity changed under the influence of a magnetic field to a lower critical temperature and disappeared above 0.3 tesla. The highest critical temperature at which superconductivity was observed was 2 kelvin, and the critical temperature decreased with increasing pressure.
RESUMO
Fractional flow reserve was measured in three patients with coronary artery dissection occurring after percutaneous coronary intervention. In Case 1, fractional flow reserve decreased from 0.88 to 0.73 and angiography showed coronary artery dissection 20 min after balloon angioplasty. In Case 2, angiography showed good results, but the fractional flow reserve decreased to a low value(0.69). Intravascular ultrasonography revealed dissection. In Case 3, angiography clearly showed dissection, but fractional flow reserve remained high(0.91). Stent implantation was performed in all three patients, but might not have been necessary in Case 3. Dissection with low or diminished fractional flow reserve value may cause a pressure gradient in the true lumen. Stent implantation is necessary in such cases. On the other hand, cases of dissection in which the fractional flow reserve value is maintained may also cause a pressure gradient in true lumen, but stent implantation may not be necessary. Fractional flow reserve measurements may be useful for the assessment of coronary artery dissection and evaluating the indications for stent implantation.
Assuntos
Angioplastia Coronária com Balão/efeitos adversos , Dissecção Aórtica/diagnóstico , Aneurisma Coronário/diagnóstico , Circulação Coronária , Dissecção Aórtica/etiologia , Dissecção Aórtica/fisiopatologia , Aneurisma Coronário/etiologia , Aneurisma Coronário/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , StentsRESUMO
A 64-year-old woman was admitted to our hospital with acute myocardial infarction. She underwent emergent percutaneous transluminal coronary angioplasty. Transthoracic echocardiography revealed mild pericardial effusion on the third day. Pericarditis or cardiac rupture were suspected, so transthoracic echocardiography was repeated serially. On the sixth day, transthoracic echocardiography showed increasing pericardial effusion and abrupt interruption of the apical myocardium of the left ventricle and intact epicardial imaging with systolic expansion. The diagnosis was oozing type cardiac rupture of a subepicardial aneurysm. Surgical treatment was successful and the accuracy of the echocardiographic diagnosis was established.