Ultrasonic Nakagami Imaging of High-intensity Focused Ultrasound-induced Thermal Lesions in Porcine Livers: Ex Vivo Study.

High-intensity focused ultrasound (HIFU) has demonstrated the capacity to be used for local thermal ablation in clinical surgery; however, relying solely on conventional ultrasound B-mode imaging to monitor HIFU thermal ablation and determine ablation levels remains a challenge. Here, we experimentally demonstrate the ability to use Nakagami imaging to monitor HIFU-induced thermal lesions in porcine livers ex vivo. Ultrasonic Nakagami imaging has been proven to be able to characterize tissues with different scatterer concentrations and distributions. The pathological sections from HIFU thermally ablated porcine liver tissues reveal that normal and denatured tissues significantly differ in scatterer concentration and distribution. Therefore, we believe that Nakagami imaging can be used to monitor thermal ablation by tracing Nakagami parameter changes in liver tissues. The ex vivo porcine liver experiments were performed using a homemade HIFU device synchronized with a commercial diagnostic ultrasound scanner to obtain the ultrasound envelope data before and after thermal ablation. These data were used to evaluate the performance of thermal lesion characterization using Nakagami imaging and were compared with those derived from conventional B-mode imaging. Experimental results showed that Nakagami imaging can be used to identify thermal lesions, which are difficult to visualize using conventional B-mode imaging because there is no apparent bubble formation. In cases with apparent bubble formation, Nakagami imaging could provide a more accurate estimation of lesion size and position. In addition, the Nakagami imaging algorithm is characterized by low computational complexity, which means it can be easily integrated as postprocessing for existing array imaging systems.

Sulforaphane protects immature hippocampal neurons against death caused by exposure to hemin or to oxygen and glucose deprivation.

Oxidative stress is a mediator of cell death following cerebral ischemia/reperfusion and heme toxicity, which can be an important pathogenic factor in acute brain injury. Induced expression of phase II detoxification enzymes through activation of the antioxidant response element (ARE)/Nrf2 pathway has emerged as a promising approach for neuroprotection. Little is known, however, about the neuroprotective potential of this strategy against injury in immature brain cells. In this study, we tested the hypothesis that sulforaphane (SFP), a naturally occurring isothiocyanate that is also a known activator of the ARE/Nrf2 antioxidant pathway, can protect immature neurons from oxidative stress-induced death. The hypothesis was tested with primary mouse hippocampal neurons exposed to either O(2) and glucose deprivation (OGD) or hemin. Treatment of immature neurons with SFP immediately after the OGD during reoxygenation was effective in protecting immature neurons from delayed cell death. Exposure of immature hippocampal neurons to hemin induced significant cell death, and both pre- and cotreatment with SFP were remarkably effective in blocking cytotoxicity. RT-PCR analysis indicated that several Nrf2-dependent cytoprotective genes, including NAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO1), and glutamate-cysteine ligase modifier subunit (GCLM), which is involved in glutathione biosynthesis, were up-regulated following SFP treatment both in control neurons and following exposure to OGD and hemin. These results indicate that SFP activates the ARE/Nrf2 pathway of antioxidant defense and protects immature neurons from death caused by stress paradigms relevant to those associated with ischemic and traumatic injury to the immature brain.

Properties and expression of a multidrug efflux pump AcrAB-KocC from Klebsiella pneumoniae.

We previously reported that we had cloned genes responsible for multidrug resistance from the chromosomal DNA of Klebsiella pneumoniae MGH78578 using a drug-hypersusceptible Escherichia coli strain as a host. One of the recombinant plasmids pETV6 conferred resistance to host cells against a wide range of antimicrobial agents, dyes and detergents. It was revealed that this plasmid carried the acrBKp gene and a part of the acrAKp gene coding for a multidrug efflux pump belonging to the RND family. We cloned the whole acrAKpBKp operon of K. pneumoniae and characterized the pump. The AcrAB pump utilized TolC as an outer membrane component in cells of E. coli. Elevated energy-dependent efflux of ethidium was observed with cells possessing AcrAKp BKp-TolC. We cloned a gene coding for an ortholog of TolC from chromosomal DNA of K. pneumoniae, and designated it kocC. It seems that the AcrAKpBKp-KocC complex functions as a potent multidrug efflux pump in K. pneumoniae. We observed a higher level of expression of acrAKp in K. pneumoniae MGH78578, a multidrug resistant strain, compared with ATCC10031, a drug susceptible strain.

Heat shock proteins play a crucial role in tumor-specific apoptosis by REIC/Dkk-3.

We recently showed that overexpression of REIC/Dickkopf-3 (Dkk-3), a tumor suppressor gene, induced apoptosis in a tumor cell-specific manner. The aim of the present study was to determine the mechanisms underlying the selective induction of apoptosis. At first, we found a mouse renal carcinoma cell line, RENCA, to be extremely sensitive to an adenovirus carrying REIC/Dkk-3 (Ad-REIC), and we showed that activation of c-Jun N-terminal kinase (JNK) was a critical step in cell death, i.e. a process similar to that in human prostate and testicular cancer observed in our previous studies. Among the proteins interfering with the activation of JNK, heat shock protein (Hsp)70/72 was reduced in expression in RENCA cells compared with that in NIH3T3 cells. An Hsp70/72 inducer protected RENCA cells from Ad-REIC-induced apoptosis, while an Hsp70/72 inhibitor sensitized NIH3T3 cells for apoptosis induction. These results indicate that functionally active Hsp70/72 is a key factor in tumor cell-specific induction of apoptotic cell death and that analyses of the expression levels of Hsp70/72 may be essential in determining the significance of Ad-REIC-based gene therapy against human cancer.

Involvement of deterioration in S100C/A11-mediated pathway in resistance of human squamous cancer cell lines to TGFbeta-induced growth suppression.

Recently, we demonstrated that S100C/A11 comprises an essential pathway for growth suppression by TGFbeta in normal human keratinocytes. Nuclear transfer of S100C/A11 was a hallmark of the activation of the process. In the present study, we examined the possible deterioration in the pathway in human squamous cancer cell lines, focusing on intracellular localization of S100C/A11 and its functional partners Smad3 and Smad4. All four human squamous cancer cell lines examined (A431, BSCC-93, DJM-1, and HSC-5) were resistant to growth suppression by TGFbeta. In BSCC-93, DJM-1, and HSC-5 cells exposed to TGFbeta, S100C/A11 was not transferred to the nuclei, and p21(WAF1) was not induced. Overexpression of nucleus-targeted S100C/A11 partially recovered induction of p21(WAF1) and p15(INK4B) and growth suppression by TGFbeta1 in these cells. These results indicate that the deterioration in the S100C/A11-mediated pathway conferred upon the cancer cell lines resistance to TGFbeta. In A431 cells, S100C/A11, Smad3, and Smad4 were simultaneously transferred to the nuclei, and p21(WAF1) was induced upon exposure to TGFbeta. We provide evidence to indicate that refractoriness of A431 cells to TGFbeta was probably because the amount of p21(WAF1) induced by TGFbeta was insufficient to counteract cyclin A, which is highly overexpressed in A431 cells. Thus, the newly found S100C/A11-mediated pathway is at least partly involved in conferring upon human squamous cell cancers resistant to TGFbeta-induced growth suppression, which is considered to play a critical role for the initiation and progression of many human cancers.

Multidrug resistance in Klebsiella pneumoniae MGH78578 and cloning of genes responsible for the resistance.

Klebsiella pneumoniae MGH78578, a clinical isolate, showed high level of resistance to many antimicrobial agents. We cloned genes responsible for drug resistance from chromosomal DNA of K. pneumoniae MGH78578 by shotgun method using Escherichia coli KAM32, a drug hypersensitive strain, as host. We obtained 43 hybrid plasmids that made host cells resistant to several antimicrobial agents. We classified them into 17 groups based on growth properties in the presence of each one of 9 antimicrobial agents and on restriction patterns of each hybrid plasmid. Analysis of the cloned genes must be very useful for investigation of major parts of multidrug resistance systems including multidrug efflux pumps in K. pneumoniae MGH78578 in which genome sequence is available.