The effect of dietary restriction on mouse T cell functions.

Forty percent dietary restriction on 9-weeks-old C3H/He mice caused decrease of the weight of central lymphoid organs in parallel with the reduction of body weight. However, the percentage of splenic T cells was dramatically increased in diet-restricted mouse spleen cells. Generally, normal mouse spleen cells contained about 30% of Thy 1.2+ T cells, but the restricted mouse spleen cells contained 80% Thy 1.2+ T cells. Ly 1+, L3T4+ T cells, but not Ly 2+ T cells, also increased in diet-restricted mouse compared with the unrestricted mice. In parallel with the dramatic changes of splenic T cells, spleen cells obtained from diet-restricted mice showed higher immunological responses against alloantigen and interleukin 2. It was also demonstrated than nylon-passed splenic T cells obtained from diet-restricted mice showed higher levels of T cell responses against r-IL-2 and alloantigen, indicating that dietary restriction modulates T cell functions themselves.

Action of serine carboxypeptidase from paecilomyces carneus on oligopeptides containing carboxy-terminally amidated peptides

Paecilomyces carneus carboxypeptidase sequentially liberated amino acids from the carboxy-terminus of neurotensin, angiotensin I, bradykinin, and delta sleep-inducing peptide, indicating that the sequential hydrolysis of peptides was limited by the occurrence of intermediates with the structure of -Gly-X (X = L-amino acid), -Pro-X, -X-Gly, and -X-Pro. The enzyme had carboxyamidase and/or amidase activities for the carboxy-terminally amidated peptides. The enzyme essentially acted as a carboxyamidase for the long carboxy-terminally amidated peptides; an amidase became dominant for the substrates in the presence of bulky amino acids such as Arg, Met, Leu, and Phe in the penultimate (P1) and P2 positions, corresponding with the S1 and S2 sites of the enzyme, and the P3 position of carboxy-terminally amidated peptides played a significant role in the action as a carboxyamidase or a amidase.

Dietary restriction induces microcytic change and shortened life span of erythrocytes without anemia in mice.

Dietary restriction is well known to increase mean and maximal life span in rodents and other species, although the mechanisms remain unclear. To investigate the effects of dietary restriction on erythrocyte cellular life span, mice were fed 40% restricted diet and erythrocyte properties were examined. At the end of the 8th week of a feeding period, erythrocytes in the restricted mice showed strikingly shorter life span than those in the unrestricted mice (restricted; T1/2 = 15.4 +/- 1.1 days, unrestricted; T1/2 = 18.9 +/- 1.2 days). However, the restricted mice never showed anemic conditions although they had microcytes, in addition. Significant increase in the erythrocyte count was observed in the restricted mice. These results strongly suggest that increased erythropoiesis should exist in the restricted mice, and that some unknown biophysical significance made shortened erythrocyte life span in mice fed restricted amount of diet.

Significant calcium accumulation in fragile erythrocytes by dietary restriction in mice.

Previously we reported that mice adapted to a 40% dietary restriction showed shortened erythrocyte life span (Internat. J. Vit. Nutr. Res. 1989, 59, 406-412). To clarify the mechanism of this phenomenon, influences of dietary restriction on erythrocyte fragility and on some factors associated with erythrocyte deformability were investigated. Forty percent dietary restriction caused an increase of erythrocyte osmotic fragility, a remarkable decrease of ATP levels and a striking accumulation of calcium in erythrocytes. In the diet restricted mice, it was further observed in the erythrocytes that the activity of rate limiting enzymes of glycolysis was significantly decreased. Remarkable decreases of the activities of calmodulin and membrane Ca2+/Mg2(+)-ATPase were also demonstrated in the erythrocytes from the diet restricted mice. These results strongly suggest that the fragile and short-lived erythrocytes in the diet restricted mice may be caused by the decreased activity of pumping out calcium from the cells. The fragile erythrocytes may be destroyed and removed from the blood flow earlier than flexible erythrocytes of the control mice. However, it is also suggested that the essential function of the erythrocytes, the oxygen supply to the peripheral tissues, may be maintained as 2,3-bisphosphoglyceric acid levels were not altered with erythrocyte aging in spite of the decreased glycolytic activity in the diet restricted mice.

Dietary restriction augments erythropoiesis in mice.

Previously, we have reported that a 40% dietary restriction caused microcytic and short-lived erythrocytes without anemia in mice (Internat. J. Vitr. Nutr. Res. (1989) 59, 406-412). To elucidate the mechanism of these phenomena, the effects of dietary restriction on the proliferation of erythroid precursor cells were investigated. In the diet restricted mice, a striking increase of erythropoietin (EP - responsive erythroid precursor cells without increment of EP was observed. Remarkable increases in the numbers of femoral nucleated cells and erythroid stem cells (CFU-S) were also observed in the diet restricted mice. Furthermore, the percentage of femoral CFU-S in DNA synthetic stage (S phase) revealed remarkably higher levels in the diet restricted mice than in the control mice. Our results strongly suggest that the microcytic change of erythrocytes in the diet restricted mice may be attributed to the increased number of erythroid precursor cells without an increment of hemoglobin synthesis and that the avoidance of becoming anemic from the short-lived erythrocytes in the diet restricted mice may be caused by a vigorous erythropoiesis.

Effects of intraperitoneally administered dietary fibers on superoxide generation from peritoneal exudate macrophages in mice.

To clarify whether edible polysaccharides (dietary fibers) have a host defense stimulating activity, the effects of intraperitoneally administered dietary fiber on the generation of microbicidal superoxide anion (O2-) in mouse peritoneal exudate macrophages (MP) were investigated. MP obtained from mice injected cellulose or pectin generated larger amount of O2- when stimulated by 12-o-tetradecanoylphorbol-13-acetate (TPA) or during phagocytosis of opsonized zymosan than those from mice injected proteose peptone, a widely used elicitor for MP. Especially, generated O2- from cellulose-elicited MP was as strikingly large as that from MP elicited by lentinan, a strong antitumor polysaccharide, when stimulated by TPA. Yeast mannan had no effect on O2(-)-generation. These results suggest the possibility that cellulose and pectin may act as host defense stimulators. The augmentation by cellulose or pectin of O2(-)-generation during phagocytosis was not inhibited by the calmodulin (CaM) antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). However, the augmentation was completely disappeared when the protein kinase C (PKC) inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) was added. These results strongly suggest that the augmentation of O2(-)-generation in phagocytosing MP by cellulose or pectin might be due to the increased activity of PKC in the MP, and that CaM may not be involved in the augmentation.

Dietary restriction augments superoxide generation in mouse peritoneal macrophages: an investigation using a chemiluminescence probe specific for superoxide anion.

It has not been clarified whether dietary restriction alters macrophage functions, although the augmentation of T cell functions by dietary restriction is well known. Forty percent dietary restriction on 9-week-old male C3H/He mice caused a decrease of body weight. However, one of the major macrophage functions, the generation of superoxide anion (O2), was augmented in proteose peptone-elicited peritoneal macrophages (MPs) from diet-restricted mice. This increase was more striking when the cells were stimulated by 12-o-tetradecanoylphorbol-13-acetate (TPA), which directly activated protein kinase C, than by opsonized zymosan which binded to receptors on the cells. These results strongly suggest that the augmentation of O2- generation in MPs by dietary restriction is due to the increased activity of protein kinase C which phosphorylate and activate O2-generating enzyme system NADPH oxidase. It is thought that one of the major factors for the reduced incidence of tumor and infection in diet-restricted animals is the augmentation of O2-generation in MPs.

Augmentation of superoxide generation in phagocytosing murine peritoneal macrophages by dietary restriction.

The effects of dietary restriction on the generation of superoxide anion (O2-) in phagocytosing mouse peritoneal macrophages (MPs) were investigated. MPs obtained from diet-restricted mice generated larger amount of O2- during phagocytosis of opsonized zymosan than those from unrestricted mice. This augmentation of the O2- generation was not inhibited by the protein kinase C (PKC) inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). However, the augmentation was clearly disappeared when the calmodulin (CaM) antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) was added. These results strongly suggest that the augmentation of O2- generation in phagocytosing MPs by dietary restriction is due to the increased activity of CaM in the MPs, and that PKC is not involved in the augmentation. It is thought that one of the major factors for the reduced incidence of tumor and infection in diet-restricted animals is the augmentation of O2- generation in MPs.

Unsharp masking technique using multiresolution analysis for computed radiography image enhancement.

An unsharp masking technique (USM) is one of the image processing methods used in the computed radiography (CR) system. To further promote the performance of the USM, we developed the NEW-USM processing that can control the frequency enhancement characteristics flexibly and accommodate an extensive range of diagnostic targets. The NEW-USM and USM were performed on femur images acquired by computed radiography (model FCR9000; Fuji Medical Systems USA Inc, Stamford, CT), and the resulting images were compared. In the NEW-USM image, bone structures are enhanced as sharply as in the USM image, whereas the surrounding soft tissue structures, such as muscle are enhanced more strongly than in the USM image. Furthermore, the absence of the bone structure that may suggest pathological change is more obvious in the NEW-USM image. The newly developed NEW-USM can appropriately enhance diagnostic information over the whole range of image frequencies, thereby expanding utility of the USM.

Production, purification, and properties of serine carboxypeptidase from Paecilomyces carneus.

Seventeen strains of the genus Paecilomyces were examined for their ability to produce serine carboxypeptidase. Paecilomyces carneus IFO 7012 exhibited the highest potency for serine carboxypeptidase production. A maximum yield of serine carboxypeptidase was obtained by koji culture of the strain at 22 degrees C for 7 days. The serine carboxypeptidase was purified to homogeneity from an extract of the koji culture. The molecular weight of the enzyme was estimated to be 47,000 by HPLC. The isoelectric point of the enzyme was determined to be 4.0, and the optimum pH was 4.0 toward benzyloxycarbonyl-L-glutamyl-L-tyrosine (Z-Glu-Tyr) and benzyloxycarbonyl-L-phenylalanyl-L-alanine (Z-Phe-Ala), respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and p-chloromercurybenzoate. Relative hydrolysis rates of N-acylpeptides and kinetic studies indicated that the enzyme preferred substrates having bulky amino acids in the penultimate position from their carboxy-termini.

Dietary restriction reduces the incidence of 3-methylcholanthrene-induced tumors in mice: close correlation with its potentiating effect on host T cell functions.

All mice treated with 3-methylcholanthrene (MC) suffered with tumor 114 days after treatment. However, 40% dietary restriction caused a great inhibition of tumor incidence. In order to understand the mechanisms by which dietary restriction decreased the occurrence of tumor in mice, we investigated the correlation between tumor incidence and host T cell immune responses. At 114 days after MC administration, the mice were sacrificed and their T cell immune responses were assessed. Flow cytometry studies demonstrated that dietary restriction caused a marked increase of the proportion of Thy 1.2+, L3T4+ T cells in MC-treated diet-restricted mice. Consistent with this result, T cell responses against concanavalin A and interleukin-2 were also potentiated in spleen cells obtained from MC-treated diet-restricted mice, while spleen cells obtained from MC-treated unrestricted mice showed decreased T cell responses because of their tumor burden. Such potentiation of T cell functions by dietary restriction was also observed at earlier stages of MC-induced tumorigenesis. During the course of carcinogenesis, spleen cells obtained from diet-restricted mice showed decreased natural killer activity in vivo. However, in vitro induction of cytotoxic T cells was markedly augmented in MC-treated diet-restricted mice compared with unrestricted mice. These results strongly suggest that the increase of host T cell immune responses might be one of the major causes for the reduction of tumor occurrence by dietary restriction.

Augmentation of mouse immune functions by dietary restriction: an investigation up to 1 year of age.

Mice fed a 40% restricted diet until 1 year of age showed a 35% drop in body weight and markedly lower weights in the central lymphoid organs such as spleen and thymus than those of unrestricted mice. In contrast, the percentage of splenic Thy 1.2+ T cells was dramatically increased by dietary restriction. Splenic Ly 1+ T cells were also increased in the restricted mice. Spleen cells of the restricted mice revealed significantly higher responses not only in macrophage (MP)-dependent responses such as concanavalin A response and mixed-lymphocyte reaction but also in MP-independent T cell responses to recombinant interleukin 2 even at 1 year of age. These results strongly suggest that dietary restriction causes an enrichment of Thy 1.2+ T cells in spleen and augments the functions of T cells in mice.