Identification of five phytosterols from Aloe vera gel as anti-diabetic compounds.

The genus Aloe in the family Liliaceae is a group of plants including Aloe vera (Aloe barbadensis MILLER) and Aloe arborescens (Aloe arborescens MILLER var. natalensis BERGER) that are empirically known to have various medical efficacies. In the present study, we evaluated the anti-hyperglycemic effect of Aloe vera gel and isolated a number of compounds from the gel. On the basis of spectroscopic data, these compounds were identified as lophenol, 24-methyl-lophenol, 24-ethyl-lophenol, cycloartanol, and 24-methylene-cycloartanol. These five phytosterols were evaluated for their anti-hyperglycemic effects in type 2 diabetic BKS.Cg-m(+/+)Lepr(db/J) (db/db) mice. In comparison with the hemoglobin A1c (HbA1c) levels of vehicle-treated mice, statistically significant decreases of 15 to 18% in HbA1c levels were observed in mice treated with 1 mug of the five phytosterols. Considering the ability to reduce blood glucose in vivo, there were no differences between the five phytosterols. Administration of beta-sitosterol did not reduce the blood glucose levels in db/db mice. After administration of the five phytosterols for 28 d, fasting blood glucose levels decreased to approximately 64%, 28%, 47%, 51%, and 55% of control levels, respectively. Severe diabetic mice treated with phytosterols derived from Aloe vera gel did not suffer weight reduction due to glucose loss in the urine. These findings suggest that Aloe vera gel and phytosterols derived from Aloe vera gel have a long-term blood glucose level control effect and would be useful for the treatment of type 2 diabetes mellitus.

Effects of bovine alpha-lactalbumin on gastric defense mechanisms in naive rats.

We recently investigated the effects of the major proteins in cow's milk on gastric mucosal injuries in rat ulcer models. We found that alpha-lactalbumin (alpha-LA) has marked preventive effects against gastric mucosal injuries and that prostaglandin (PG) synthesis may contribute to these effects [Matsumoto et al., Biosci. Biotechnol. Biochem., 65, 1104-1111, 2001]. In this study, we investigated the effects of alpha-LA on several defense mechanisms of gastric mucosa by evaluating gastric PGE2 content, gastric mucin content, gastric luminal pH, gastric fluid volume, and gastric emptying in naive rats. Oral administration of alpha-LA (200, 500, and 1000 mg/kg) elevated endogenous PGE2 levels in gastric tissue and increased the gastric mucin contents of both the gastric fluid and the adherent mucus gel layer. In addition to these PG-related responses, alpha-LA also caused PG-independent responses such as elevation of gastric luminal pH, increase in gastric fluid volume, and delay in gastric emptying. These responses were observed to be dose-dependent (200-1000 mg/kg of alpha-LA). Thus, we demonstrated that alpha-LA enhances both PG-dependent and PG-independent gastric defense mechanisms in naive rats. Both of these mechanisms are probably involved in its gastroprotective action.

Booster effect of interleukin-2 on natural killer 1.1+ cells stimulated by administration of macrophage colony-stimulating factor in mice.

The authors studied the combined effects of macrophage colony-stimulating factor (M-CSF) and interleukin (IL)-2 on the functions and antitumor activity of natural killer (NK) 1.1+ cells in vitro and in vivo. NK1.1+ cells were isolated from the spleen of mice treated with saline or M-CSF, and their functions (proliferation, production of IFN-gamma, and cytotoxicity) evaluated in vitro. Although the proliferation of and production by NK1.1+ cells was stimulated by the addition of IL-2, the cells from the M-CSF-treated mice responded better. Furthermore, the cytotoxicity against Yac-1 cells and B16 melanoma cells was stimulated by M-CSF administration and enhanced by the addition of IL-2 and IL-12. These results demonstrated that M-CSF treatment augmented the functions of NK1.1 cells, and IL-2 and IL-12 boosted these activities in vitro. The authors then examined the effects of co-administration of M-CSF and IL-2 in vivo. The clearance of B16 cells in lung was augmented by the administration of M-CSF but not IL-2. However, M-CSF + IL-2 treatment further enhanced the clearance activity. The anti-metastatic activity was also enhanced by the M-CSF + IL-2 treatment. Furthermore, the survival of B16-bearing mice was prolonged by M-CSF + IL-2. These results suggested that administration of IL-2 boosts the functions of NK1.1+ cells, which are augmented preliminarily by the administration of M-CSF.

Molecular cloning of a novel immunoglobulin superfamily gene preferentially expressed by brain and testis.

We have cloned and characterized a novel gene from both human and mouse that encodes a new member of the immunoglobulin superfamily. The gene is preferentially expressed in both brain and testis, and hence, termed BT-IgSF (brain- and testis-specific immunoglobulin superfamily). The predicted protein consists of V-type and C2-type immunoglobulin domains as well as a hydrophobic signal sequence, a single transmembrane region, and a cytoplasmic domain. Human BT-IgSF protein (431 amino acids) is 88% identical to the mouse protein (428 amino acids) and both show significant homology to coxsackie and adenovirus receptor (CAR) and endothelial cell-selective adhesion molecule (ESAM). We examined the expression of BT-IgSF with various cultured cells and found that the gene was expressed in both neurons and glial cells in vitro. Furthermore, the expression was preferentially detected in pyramidal cell layers of the dentate gyrus and hippocampus and in commissure fibers of the corpus callosum, in brain tissue sections examined. These findings suggest that BT-IgSF plays a role in the development or function of the central nervous system.

Effect of coadministration of M-CSF and IFN-alpha on NK1.1+ cells in mice.

The purpose of this study was to evaluate the effect of coadministration of macrophage colony-stimulating factor (M-CSF) and interferon-alpha (IFN-alpha) on NK1.1(+) cells in mice. Administration of M-CSF, but not IFN-alpha, increased the number of NK1.1(+) cells and CD11b(+) cells in spleen and blood. Coadministration of the two agents induced a greater increase in NK1.1(+) cells than did administration of M-CSF alone. Administration of M-CSF or IFN-alpha augmented the clearance activity of Yac-1 cells in lung, and coadministration of these agents further augmented this effect. The combination of M-CSF and IFN-alpha effectively reduced the formation of tumor nodules in lung and liver in an experimental metastasis model using B16 melanoma. The combination of M-CSF and IFN-alpha induced the increase and activation of NK1.1(+) cells more than either agent alone. These effects may contribute to the antimetastatic reaction by NK1.1(+) cells in vivo.

Leukemic cell-surface CD13/aminopeptidase N and resistance to apoptosis mediated by endothelial cells.

BACKGROUND: Attachment of leukemic cells to vascular endothelial cells induces the vascular endothelial cells to release endothelial cell-derived interleukin 8 (endothelial IL-8), which then induces leukemic cells to undergo apoptosis. NB4, a human promyelocytic leukemic cell line that expresses high levels of cell-surface CD13/aminopeptidase N, does not undergo endothelial IL-8-induced apoptosis. Consequently, we investigated the relationship between cell-surface aminopeptidase activity and endothelial IL-8 induction of apoptosis in various leukemic cell lines. METHODS: CD13/aminopeptidase N activity and IL-8-induced apoptosis were examined in leukemic cell lines. Endothelial IL-8-induced apoptosis was examined further in NB4 cells, K562 cells (human chronic myelogenous leukemic cells expressing low levels of CD13/aminopeptidase N), CD13/aminopeptidase N-transfected K562 (K562/CD13) cells that overexpress aminopeptidase, and mock-transfected K562 cells (vector only). These cells were also cocultured with a vascular endothelial cell layer to investigate the association between aminopeptidase activity and apoptosis in this system. All statistical tests were two-sided. RESULTS: Endothelial IL-8 induced apoptosis in K562 cells but not in K562/CD13 cells. A combination of an aminopeptidase inhibitor (such as bestatin) and endothelial IL-8 induced apoptosis in NB4 cells and K562/CD13 cells (2.88-fold difference [95% confidence interval [CI] = 1.82-fold to 3.94-fold], P =.004 for bestatin-treated NB4 cells and 4.31-fold difference [95% CI = 3.52-fold to 5.10-fold], P<.001 for bestatin-treated K562/CD13 cells). When aminopeptidase activity in NB4 cells was modulated by aminopeptidase inhibitors, a statistically significant correlation was found between aminopeptidase activity and the proportion of apoptotic cells induced by endothelial IL-8 (r = -.837, P<.001 by Pearson's correlation coefficient; r = -.697, P =.013 by Spearman's correlation analysis by ranks). K562/CD13 cells cocultured with vascular endothelial cells did not undergo apoptosis, but the addition of bestatin resulted in the induction of apoptosis in K562/CD13 cells (2.70-fold difference [95% CI = 1.77-fold to 3.63-fold], P<.001). Bestatin treatment increased the level of IL-8 mRNA in and the amount of IL-8 secreted by vascular endothelial cells. CONCLUSIONS: High levels of cell-surface CD13/aminopeptidase N appear to allow leukemic cells to resist endothelial IL-8-induced apoptosis. The combination of endothelial IL-8 and bestatin induce leukemic cells expressing high levels of CD13/aminopeptidase N to undergo apoptosis. Bestatin may be useful for treating patients with leukemia.

New human myelodysplastic cell line, TER-3: G-CSF specific downregulation of Ca2+/calmodulin-dependent protein kinase IV.

We have established a new hematopoietic cell line from a patient with myelodysplastic syndrome (MDS), which was refractory anemia with excess blasts (RAEB). This cell line, designated TER-3, depends on several cytokines for long-term survival and growth, and requires interleukin-3 (IL-3) for continuous growth. Cytochemical analysis revealed that TER-3 cells are weakly dianisidine positive and nonspecific esterase positive, but peroxidase negative. The surface marker profile shows that the TER-3 cells are strongly positive for myeloid, lymphoid, and megakaryocytic antigens such as CD15, CD19, and CD61, and negative for some common multilineage antigens such as CD13, CD33, and CD34. Thus, this cell line has a multilineage phenotype, suggesting that the transformation event occurred in multipotent stem cells. Dianisidine- and nonspecific esterase-positive TER-3 cells increase with granulocyte-colony stimulating factor (G-CSF) rather than with IL-3. These results suggest that the cell line is useful for understanding the mechanism underlying G-CSF-associated hematopoietic cell differentiation and activation in the patient with MDS.