Detrimental effects after dobutamine infusion on rat left ventricular function: mechanical work and energetics

We have previously reported that continuous infusion of dobutamine into the coronary artery induces positive inotropic effects but induces no detrimental effects in cross-circulated, excised normal rat hearts and even in Ca2+ overload-induced contractile failing rat hearts. However, we hypothesized that some detrimental effects on left ventricular (LV) function are induced after continuous dobutamine infusion and the following clearance of blood dobutamine, as is the case after beta-adrenergic receptor stimulation. To test this hypothesis, we investigated LV mechanical work and energetics in the same type of preparations that underwent continuous dobutamine infusion and clearance of blood dobutamine. We found that both mean end-systolic pressure and systolic pressure-volume area (PVA; a measure of total mechanical energy per beat) at midrange LV volume were significantly (P < 0.01) decreased. The mean myocardial oxygen consumption per beat intercept, which is composed of for the total Ca2+ handling in excitation-contraction coupling and basal metabolism, of the and PVA linear relation was also significantly (P < 0.05) decreased (n=8). The mean slope of the linear relation was unchanged in such hearts. Post-dobutamine basal metabolism was unchanged (n = 5 of the 8 hearts). The moderate proteolysis of a cytoskeleton protein, alpha-fodrin was identified (n = 7 of the 8 hearts with the decreased intercept), after clearance of blood dobutamine. In agreement with our hypothesis, the detrimental effect of the post-beta-adrenergic receptor stimulation was induced by a moderate concentration of dobutamine; we found systolic dysfunction due to the impairment of Ca2+ handling in excitation-contraction coupling in the rat LV and proteolysis of a cytoskeleton protein, alpha-fodrin.     

Hypoxia-induced renal epithelial cell death through caspase-dependent pathway: role of Bcl-2, Bcl-xL and Bax in tubular injury

Although injury of epithelial cells has been reported to be responsible for renal disease such as acute renal failure, its molecular mechanisms are largely unknown. As hypoxia has been postulated as the initial trigger of epithelial injury, we studied the molecular mechanisms of apoptosis induced by hypoxia in human renal epithelial cells. Severe hypoxia caused epithelial cell death, accompanied by a significant increase in LDH release (p<0.01). In addition, hypoxic treatment of epithelial cells resulted in a significant increase in apoptotic cells as assessed by cell morphology (p<0.01). The apoptotic change in epithelial cells under hypoxic condition was also confirmed by a significant increase in caspase-3-like activity and release of cytochrome c (p<0.01). The decrease in epithelial cell number was completely abolished by addition of a wide-spectrum caspase inhibitor, Z-VAD, rather than Z-DEVD, a specific caspase-3 inhibitor (p<0.01). Thus, we further studied the molecular mechanisms of apoptosis induced by hypoxia. Anti-apoptotic factors, Bcl-2 and Bcl-xL, were significantly decreased in epithelial cells under a hypoxic condition as assessed by Western blotting (p<0.01). In contrast, hypoxia did not alter their location. Of particular importance, translocation of a proapoptotic factor, Bax, from the cytoplasm to the mitochondrial membrane was observed in response to hypoxia, whereas total Bax protein was not changed by hypoxia. Overall, this study demonstrated that hypoxia caused epithelial cell death induced by caspase-3-like activity-dependent apoptosis. The pro-apoptotic mechanisms of hypoxia in epithelial cells largely depend on a significant decrease in Bcl-2 and Bcl-xL. In addition, the present results demonstrate that translocation of Bax from the cytosol to the mitochondrial membrane occurred under hypoxia, thereby leading to pathological tissue destruction.     

High-grade dysplasia associated with fundic gland polyposis in a familial adenomatous polyposis patient, with special reference to APC mutation profiles.

We report a patient with familial adenomatous polyposis who developed high-grade dysplasia against a background of fundic gland polyposis. Two large high-grade dysplasia lesions were found in the gastric body, where numerous fundic gland polyps were present. In both lesions, the dysplastic epithelium covered non-neoplastic oxyntic glands that occasionally exhibit cystic changes. A genetic analysis for APC (adenomatous polyposis coli) revealed a somatic 50-bp deletion involving codons 1502-1517 and 2-bp deletion at codon 1465 in each lesion of high-grade dysplasia. In contrast, six of the 18 fundic gland polyps were found to harbor an identical mutation: 1-bp insertion at codon 1556. Both lesions of high-grade dysplasia and the fundic gland polyps were similarly located in the fundic gland area and were caused by the inactivation of APC; however, their mutation profiles of APC were different. These results imply that fundic gland polyps and high-grade dysplasia of the stomach have distinct preferences for APC genotypes in their development.     

Real-time imaging of individual fluorescent-protein color-coded metastatic colonies in vivo

We have established stable, bright green fluorescent protein (GFP)- or red fluorescent protein (RFP)-expressing HT-1080 human fibrosarcoma clones. These cell lines showed similar cell proliferation rates and high-frequency experimental lung metastasis. The HT-1080-GFP and -RFP clones enable simultaneous real-time dual-color imaging in the live animal. HT-1080 cells were transduced with retroviral vectors containing GFP or RFP and the neomycin resistance gene. Stable transformants were selected stepwise with G418 up to 800 μl/ml. Subsequently, high GFP- or RFP-expressing clones, HT-1080-GFP or HT-1080-RFP, respectively, were selected. 3×10⁶ cells from each clone were mixed and injected into the tail vein of SCID mice. The cells seeded the lung at high frequency with subsequent formation of pure green and pure red colonies as well as mixed yellow colonies with different patterns visualized directly on excised lungs. The lung metastases were also visualized by external fluorescence imaging in live animals through skin-flap windows over the chest wall. Lung metastases were observed on the lung surface of all mice. SCID mice well tolerated multiple surgical procedures for direct-view imaging via skin-flap windows. Real-time metastatic growth of the two different colored clones in the same lung was externally imaged with resolution and quantification of green, red, or yellow colonies in live animals. The color coding enabled determination of whether the colonies grew clonally or were seeded as a mixture with one cell type eventually dominating, or whether the colonies grew as a mixture. The simultaneous real-time dual-color imaging of metastatic colonies described in this report gives rise to the possibility of color-coded imaging of clones of cancer cells carrying various forms of gene of interest. This revised version was published online in July 2006 with corrections to the Cover Date.     

Neural changes in cat auditory cortex after a transient pure-tone trauma

Here we present the changes in cortical activity occurring within a few hours after a 1-h exposure to a 120-dB SPL pure tone (5 or 6 kHz). The changes in primary auditory cortex of 16 ketamine-anesthetized cats were assessed by recording, with two 8-microelectrode arrays, from the same multiunit clusters before and after the trauma. The exposure resulted in a peripheral threshold increase that stabilized after a few hours to on average 40 dB in the frequency range of 6-32 kHz, as measured by the auditory brain stem response. The trauma induced a shift in characteristic frequency toward lower frequencies, an emergence of new responses, a broadening of the tuning curve, and an increase in the maximum of driven discharges. In addition, the onset response after the trauma was of shorter duration than before the trauma. The results suggest the involvement of both a decrease and an increase in inhibition. They are discussed in terms of changes in central inhibition and its implications for tonotopic map plasticity.     

Clonality analysis of different histological components in combined small cell and non-small cell carcinoma of the lung

Combined small cell and non-small cell carcinoma is relatively rare in the lung. Examination of the clonal relationship of different components in this type of tumor may give a clue to the rarity. We retrieved 6 such tumors; all 6 had small cell carcinoma and adenocarcinoma components, and 3 had an additional squamous cell carcinoma component. We examined the point mutations in the p53 gene and allelic loss (ie, the loss of heterozygosity [LOH] pattern) of chromosome 3p in each component. p53 mutations were detected in the small cell carcinoma component of 5 tumors and in the non-small cell carcinoma components of 2 tumors. In 1 case, the squamous cell carcinoma component had a p53 mutation locus identical to that in the small cell carcinoma component, but in the other case, the adenocarcinoma component had a different mutation than that in the small cell carcinoma component. Chromosome 3p LOH loci in the squamous cell carcinoma component were present in the small cell carcinoma component in all 3 cases, but some LOH loci were not identical in the small cell carcinoma and adenocarcinoma components in 3 cases. These results suggest that the small cell and squamous cell carcinoma components of combined small cell lung carcinomas have an intimate clonal relationship. On the other hand, the adenocarcinoma component often may be derived from a separate clone or, more likely, undergo a progressive process separate from the squamous cell-small cell carcinoma beginning in a very early stage, that is, before the appearance of p53 and chromosome 3p abnormalities. This tumorigenesis process may explain the relative rarity of combined small cell and non-small cell carcinoma, which occurs primarily in the peripheral lung, an infrequent site of squamous cell carcinoma.     

In vivo study of the effects of cryopreservation on heart valve xenotransplantation.

BACKGROUND: Recent reports have suggested that cryopreservation reduces the immunogenicity of donor tissue. The immunomodulation by cryopreservation might influence on the tissue durability after xenotransplantation. We investigated the in vivo morphologic changes in cryopreserved xenograft (CXG) heart valves. MATERIAL AND METHOD: We transplanted a fresh (fresh xenograft; FXG) and a cryopreserved (CXG) porcine aortic root and a cryopreserved canine (cryopreserved allograft; CAG) aortic root into the abdominal aorta of a dog without any immunosuppressive agents. Explanted grafts on the 21st to 49th days after implantation were analyzed morphologically with light microscopy using some special stains, immunohistochemical analysis, and scanning electron microscopy (SEM). RESULT: Light microscopy showed the absence of smooth muscle cells in the media of the aorta in any group after transplantation. FXG valves did not maintain any cellularity after transplantation. CXG valves contained cellular infiltration in themselves. CAG valves contained numerous fibroblasts, which showed the maintenance of tissue integrity without allowing cellular infiltration. The structure of elastic fibers was well maintained, even in the part of CXG valve with cellular infiltration. Immunohistochemical studies documented the infiltration of T lymphocytes in CXG valves that were labeled by anti-CD3 antibodies. SEM demonstrated that no endothelia were seen on the surface of the valves in any group after transplantation. CONCLUSION: We concluded that the cryopreservation method might provide an immunomodulation of xenogeneic heart valves for transplantation.     

Melanin pigmentation in mammalian skin and its hormonal regulation

Cutaneous melanin pigment plays a critical role in camouflage, mimicry, social communication, and protection against harmful effects of solar radiation. Melanogenesis is under complex regulatory control by multiple agents interacting via pathways activated by receptor-dependent and -independent mechanisms, in hormonal, auto-, para-, or intracrine fashion. Because of the multidirectional nature and heterogeneous character of the melanogenesis modifying agents, its controlling factors are not organized into simple linear sequences, but they interphase instead in a multidimensional network, with extensive functional overlapping with connections arranged both in series and in parallel. The most important positive regulator of melanogenesis is the MC1 receptor with its ligands melanocortins and ACTH, whereas among the negative regulators agouti protein stands out, determining intensity of melanogenesis and also the type of melanin synthesized. Within the context of the skin as a stress organ, melanogenic activity serves as a unique molecular sensor and transducer of noxious signals and as regulator of local homeostasis. In keeping with these multiple roles, melanogenesis is controlled by a highly structured system, active since early embryogenesis and capable of superselective functional regulation that may reach down to the cellular level represented by single melanocytes. Indeed, the significance of melanogenesis extends beyond the mere assignment of a color trait.     

Culture of chondrocytes in fibroin-hydrogel sponge

Fibroin-hydrogel sponge and collagen gel were used as scaffold for in vitro cartilage regeneration. Fibroin-hydrogel sponge was formed by phase separation from freezed fibroin solution. Chondrocytes were harvested from proximal humerus, distal femur and proximal tibia of 4-week-old Japanese white rabbits and inoculated in the fibroin-hydrogel sponge and collagen gel. Those constructs were cultured in DMEM supplemented with 10% FCS and 50 ml L-ascorbate at 37 degrees C. Histological observation, measurement of sulfated glycosaminoglycan and cell density were carried out at 3, 7, and 14 days after the cultivation. Well-defined cartilage tissue can be seen both in the fibroin-hydrogel sponge and in the collagen gel. The matrix was intensely stained by safranin-O and showed a metachromatic reaction in both group. However, the quantity of sulfated glycosaminoglycan and cell density of the fibroin-hydrogel sponge group were increased more rapidly than these of the collagen gel group. Thus, the chondrocytes proliferated in the fibroin sponge without losing their differentiated phenotype. It is possible that culture environment in the fibroin sponge was suitable for chondrocytes regeneration.