Repair of infarcted myocardium mediated by transplanted bone marrow-derived CD34+ stem cells in a nonhuman primate model

Rodent and human clinical studies have shown that transplantation of bone marrow stem cells to the ischemic myocardium results in improved cardiac function. In this study, cynomolgus monkey acute myocardial infarction was generated by ligating the left anterior descending artery, and autologous CD34(+) cells were transplanted to the peri-ischemic zone. To track the in vivo fate of transplanted cells, CD34(+) cells were genetically marked with green fluorescent protein (GFP) using a lentivirus vector before transplantation (marking efficiency, 41% on average). The group receiving cells (n = 4) demonstrated improved regional blood flow and cardiac function compared with the saline-treated group (n =4) at 2 weeks after transplant. However, very few transplanted cell-derived, GFP-positive cells were found incorporated into the vascular structure, and GFP-positive cardiomyocytes were not detected in the repaired tissue. On the other hand, cultured CD34(+) cells were found to secrete vascular endothelial growth factor (VEGF), and the in vivo regional VEGF levels showed a significant increase after the transplantation. These results suggest that the improvement is not the result of generation of transplanted cell-derived endothelial cells or cardiomyocytes; and raise the possibility that angiogenic cytokines secreted from transplanted cells potentiate angiogenic activity of endogenous cells.     

ZZ domain is essentially required for the physiological binding of dystrophin and utrophin to beta-dystroglycan

An intracellular protein, dystrophin, plays an important role in keeping muscle fibers intact by binding at its N-terminal end to the subsarcolemmal cytoskeletal actin network and via its C-terminal end to the transmembraneous protein beta-dystroglycan. Duchenne muscular dystrophy is caused by the loss of dystrophin, which can result from the loss of this binding. The N-terminal part of the latter binding site of dystrophin has been well documented using overlay assay and X-ray diffraction assays. However, the binding site at the C-terminal region of dystrophin has not been examined in detail. In the present work, we report a detailed analysis of the C-terminal binding domain as follows. (1). The full binding activity corresponding to the effective binding in vivo is expressed by the dystrophin fragment spanning amino acids 3026-3345 containing the ZZ domain at the C-terminus. Determination of this binding range is important not only for understanding of the mechanism of dystrophy, but also useful for the design of truncated dystrophin constructs for gene therapy. (2). The ZZ domain binds to EF1 domain in the dystrophin fragment to reinforce the binding activity. (3). The cysteine 3340 in the ZZ domain is essential for the binding of dystrophin to beta-dystroglycan. A reported case of DMD due to missense mutation C3340Y may be caused by inability to fix dystrophin beneath the cell membrane. (4). The binding mode of utrophin is different from that of dystrophin. The difference is conspicuous concerning the cysteine residues present in the ZZ domain.     

Localization of alkaline phosphatase and osteopontin during matrix mineralization in the developing cartilage of coccygeal vertebrae

We observed the manner in which alkaline phosphatase (ALPase) and osteopontin were localized in the cartilage and intramembranous bone of coccygeal vertebrae during matrix mineralization, shedding considerable light on the manner in which they develop. In the cartilage matrix of coccygeal vertebrae, we observed the localization of ALPase activity in the boundary of the proliferative and the hypertrophic zones. Granular nodules of mineralization were consistently found in the boundary of both zones, and increased in size when close to the hypertrophic zone. While osteopontin was rarely present in the early stages of mineralization, its localization along the margins of mineralized matrices in the hypertrophic zone was prominent. In contrast to cartilage, mineralized nodules in the intramembranous bone in the mid-portion of the vertebra displayed osteopontin-immunoreactivity, indicating its early synthesis and subsequent accumulation to early-stage mineralized nodules. When blood vessels, accompanied by osteoblastic and osteoclastic cell populations, invaded the cartilage, osteopontin was localized in the lower region of the hypertrophic zone, despite its maintaining the localization of ALPase and early-stage mineralization. Thus, our investigation demonstrated ALPase activity consistent with early-stage mineralization in the cartilage matrix. However, the fact that osteopontin-localization could not be pinpointed might account for its multifunctionality as concerns both the regulation of mineralization and the attachment of migrating osteogenic and osteoclastic cells to the mineralized matrix.     

Spatiotemporal changes of coxsackievirus and adenovirus receptor in rat hearts during postnatal development and in cultured cardiomyocytes of neonatal rat

Coxsackievirus B is the most common cause of viral myocarditis and is particularly virulent in neonates and children. Adenovirus is also a leading cause of the disease. The determinant of tropism for both viruses is considered to be the expression of coxsackievirus and adenovirus receptor (CAR) in target organs. However, developmental change and physiological localization of CAR in the heart are unknown. We examined expression levels of CAR in rat hearts by quantitative real-time polymerase chain reaction and Western blot analysis and found that CAR decreased gradually during postnatal development, although CAR was detectable, even in adults. Immunohistochemistry revealed CAR on the whole surface of cardiomyocytes in immature rat hearts. In contrast, CAR was detected predominantly on intercalated disks in the adult heart and was accumulated especially at the contact point between the cultured cardiomyocytes, even though they were prepared from the neonatal rat heart. In conclusion, CAR was expressed abundantly on the whole surface of cardiomyocytes in immature rat hearts. Both the expression level and the localization of CAR are possible determinants of the susceptibility to viral myocarditis of neonates and children.     

Quantitative studies on the neutralization reaction between African horse-sickness virus and antiserum

Summary Kinetic neutralization curves formed with African horse-sickness virus were studied by the plaque reduction technique. There was no recovery phase in surviving virus titer during a 60-minute incubation period at 37°C when antiserum was diluted 1100 or more.Factors affecting the accuracy of the serum neutralization test in tube cultures of monkey kidney stable (MS) cells were studied to ascertain optimal conditions. Dose-response patterns, variations within and between tests, and the relationship between the amount of virus and serum titers were examined to develop a test procedure showing accuracy of less than a 3-fold dilution deviation under ordinary circumstances.With antisera against 8 different types of AHS virus, it was demonstrated that there is linear relationship between the amount of serum antibody and amount of virus neutralized. When tested in MS cell tube cultures, neutralization slopes of all AHS virus types ranged between 1.33 and 1.59. A decrease in the slope was noticed when tested in mice.This work was undertaken at the Near East Animal Health Institute, a project established by the United Nations Development Program Special Fund through the Food and Agriculture Organization in cooperation with the Ministry of Agriculture of Iran.     

Effects of intra- and extracellular H+ and Na+ concentrations on Na+-H+ antiport activity in the lacrimal gland acinar cells

Kinetic properties of the Na⁺-H⁺ antiport in the acinar cells of the isolated, superfused mouse lacrimal gland were studied by measuring intracellular pH (pH_i) and Na⁺ activity (aNa_i) with the aid of double-barreled H⁺- and Na⁺-selective microelectrodes, respectively. Bicarbonate-free solutions were used throughout. Under untreated control conditions, pH_i was 7.12±0.01 and aNa_i was 6.7±0.6 mmol/l. The cells were acid-loaded by exposure to an NH ₄ ⁺ solution followed by an Na⁺-free N-methyl-d-glucamine (NMDG⁺) solution. Intracellular Na⁺ and H⁺ concentrations were manipulated by changing the duration of exposure to the above solutions. Subsequent addition of the standard Na⁺ solution rapidly increased pH_i. This Na⁺-induced increase in pH_i was almost completely inhibited by 0.5 mmol/l amiloride and was associated with a rapid, amiloride-sensitive increase in aNa_i. The rate of pH_i recovery induced by the standard Na⁺ solution increased in a saturable manner as pH_i decreased, and was negligible at pH_i 7.2–7.3, indicating an inactivation of the Na⁺-H⁺ antiport. The apparent K _m for intracellular H⁺ concentration was 105 nmol/l (pH 6.98). The rate of acid extrusion from the acid-loaded cells increased proportionally to the increase in extracellular pH. Depletion of aNa_i to less than 1 mmol/l by prolonged exposure to NMDG⁺ solution significantly increased the rate of Na⁺-dependent acid extrusion. The rate of acid extrusion increased as the extracellular Na⁺ concentration increased following Michaelis-Menten kinetics (V _max was 0.55 pH/min and the apparent K _m was 75 mmol/l at pH_i 6.88). The results clearly showed that the Na⁺-H⁺ antiport activity is dependent on the chemical potential gradient of both Na⁺ and H⁺ ions across the basolateral membrane, and that the antiporter is asymmetric with respect to the substrate affinity of the transport site. The data agree with the current model of activation and inactivation of the antiporter by an intracellular site through changes in the intracellular Na⁺ and H⁺ concentrations.     

Mechanisms of blood coagulation induced by latex particles and the roles of blood cells

Latex particles with highly negative or positive charges shortened the clotting time of whole blood and platelet-rich plasma and activated platelet factor 3. Platelet-poor plasma was clotted by the particles with a highly negative charge, but not by those with a positive charge, except hydrophobic particles. Blood coagulation by positively-charged particles was attributed to platelet activation. An enhancement of blood coagulation was also observed in the presence of erythrocytes, leucocytes, their cell membranes or negatively charged phospholipids, and phosphatidylserine instead of platelets. Hydrophilic and low-charged particles suppressed blood coagulation.     

Ultrastructural, cytochemical, and biophysical aspects of mechanisms of bone matrix calcification

Primary calcification in embryonic ossification occurs as follows: crystallization within matrix vesicles, formation of calcified nodules, and finally the establishment of expansive calcified matrix. However, the participation of the matrix vesicles in other types of bone calcification, such as bone formation during bone remodeling in adults has not been examined sufficiently. We introduce our recent observations on the presence of matrix vesicles in aged bones. In addition, although it is well known that the extracellular fluid supersaturates the calcification crystal, hydroxyapatite, the specific mechanisms by which bone matrix calcify remain unclear. In order to further approach the mechanisms of bone matrix calcification, we also review ultrastructural and localizational alterations of the matrix organics according to the progression of calcification, and an evaluation of mineral micro-environment in the calcifying sites by energy-filter transmission electron microscopy.