Survival and Repopulation of Irradiated 'Pre-Cfu-C' In Mice

ABSTRACT Supernatants of murine bone-marrow cultures contain a colony-promoting factor (CPF) which increases the number of granulocyte and macrophage colonies in semi-solid agar cultures in the presence of colony-stimulating factor (CSF). Incubation of bone-marrow cells with CPF results in an increase in the number of granulocyte/macrophage progenitor cells (CFU-c) and the CPF-responsive cells may be younger than the CFU-c. We have investigated the radiosensitivity and the pattern of the recovery after irradiation of CPF-responsive cells. We found that the radiosensitivity of CPF-responsive cells was significantly lower than those of CFU-c. burst-forming units-erythroid (BFU-e) and pluripotent stem cells in vivo (CFU-s) and in vitro (CFU-mix). the CPF-responsive cells remained subnormal even at 28 days after irradiation of the mice, a time when the CFU-s and CFU-c had recovered completely. Therefore the CPF-responsive cells may constitute a separate compartment, namely ‘pre-CFU-c’, in the maturation sequence of granulopoiesis, and this maturation of the ‘pre-CFU-c’ to CFU-c seems to be highly stimulated after irradiation to counterbalance the influx from CFU-s.     

Regulation of angiogenesis by the aging suppressor gene klotho

Advanced age is a major risk factor of peripheral artery disease. We examined the effects of the aging-suppressor gene klotho on angiogenesis in response to ischemia by introducing ischemic hindlimb model in mice heterozygously deficient for the klotho gene and in wild type mice. Blood flow recovery as assessed by laser doppler perfusion imaging and angiogenesis as assessed by density of PECAM-1/CD31-positive positive capillaries were markedly impaired in mice heterozygously deficient for the klotho gene (both <0.05). Our findings show that the aging-suppressor gene klotho affects angiogenesis and the possibility that age-related impairment of angiogenesis might be regulated by the klotho gene. Our results present a new possibility of therapeutic angiogenesis for patients of advanced age.     

Role of chlorogenic acid quinone and interaction of chlorogenic acid quinone and catechins in the enzymatic browning of apple

Chlorogenic acid (CQA) is one of the major polyphenols in apple and a good substrate for the polyphenol oxidase (PPO) in apple. Apple contains catechins as well as CQA, and the role of CQA quinone and its interaction with catechins in the enzymatic browning of apple were examined. Browning was repressed and 2-cysteinyl-CQA was formed when cysteine was added to apple juice. CQA quinone was essential for browning to occur. Although catechins and CQA were oxidized by PPO, some catechins seemed to be non-enzymatically oxidized by CQA quinone.     

Genetic variants of the IgA Fc receptor (FcαR, CD89) promoter in chronic hepatitis C patients

Fc receptor for IgA (FcαR, CD89) is capable of triggering IgA-mediated immune responses to pathogens and has been proposed to function in circulating IgA clearance. Because inheritable variations modifying individual immune responses or immunoglobulin catabolism may affect the chronicity of viral infection, we investigated whether promoter polymorphisms of the FcαR gene (FCAR) affect chronic hepatitis C virus (HCV) infection and its disease progression. The two −311T/C and −142T/C single-nucleotide polymorphisms (SNPs) were studied by direct DNA sequencing in 177 Japanese patients with chronic hepatitis C (CHC). Both −311CC and −142CC genotypes were more frequent in CHC patients (15.9 and 18.6%) compared with 210 healthy controls (5.7 and 10.0%) [p = 0.001, odds ratio (OR) = 3.10, 95% confidence interval CI) = 1.53–6.30 and p = 0.014, OR = 2.06, 95% CI = 1.14–3.72, respectively], and were associated with infection with HCV genotype 2a/2b (p = 0.019 and p = 0.005, respectively). Conversely, −311CC and −142CC were decreased in 59 patients at advanced stages of disease as assessed on the basis of hepatic fibrosis markers such as decreased platelet count (PLT) ( < 150,000/μl) (5.1 and 8.5%) compared with 91 patients with normal PLT ( ≥ 150,000/μl) (24.2 and 26.4%) (p = 0.006 and p = 0.005, respectively). Moreover, among the patients with normal PLT (but not with decreased PLT), −311CC or −142CC was significantly associated with decreased serum IgA levels (p = 0.023 or p = 0.007, respectively). These results suggest that the FCAR promoter SNPs may be related to chronic HCV infection and disease progression in Japanese CHC, which might be explained by altered FcαR expression affecting IgA-mediated immune responses and/or IgA catabolism.     

l‐DOPA Produced Nitric Oxide in the Vitreous and Caused Greater Vasodilation in the Choroid and the Ciliary Body of Melanotic Rats than in those of Amelanotic Rats

The nitrogen cycle initiates direct reduction of N₂ to NH₃ by enzymatic reactions. We hypothesize that l ‐dihydroxyphenylalanine (l ‐DOPA), a catecholamine, could be a source of nitric oxide (NO). In order to determine whether l ‐DOPA generates NO and induces any biological change in the eye, we measured the generation of NO in vitro and in vivo, and investigated the histopathological changes caused by injection of l ‐DOPA into the vitreous of rats. We also hypothesized that melanin granules may affect the generation of NO during the metabolism of l ‐DOPA, since l ‐DOPA is a precursor of melanin in the brain and the eye. Therefore, we compared the effects of l ‐DOPA on the generation of NO between amelanotic and melanotic rats. NO was measured as diffusion currents by NO electrodes. In vitro, various concentrations of l ‐DOPA (5, 29.9, 79.4, 152.7, and 249 μM) were added to the medium. The inhibition of NO generation by 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazole‐1‐oxyl 3‐oxide (carboxy‐PTIO) was tested. In vivo, NO generation in the vitreous of rats was measured and the eyes were enucleated under anesthesia after l ‐DOPA injection. The ocular tissues were subjected to histological examination. NO was produced from l ‐DOPA in a dose‐dependent manner and was scavenged by carboxy‐PTIO in vitro. NO in the vitreous of melanotic rats was generated from l ‐DOPA. Histological examination with hematoxylin‐eosin staining revealed vasodilation in the ciliary vessels and the choroid after l ‐DOPA injection. Both effects were greater in melanotic rats than in amelanotic rats. The vasodilation may be attributable to NO as well as to superoxides, which can be regulated by the existence of melanin.     

L-DOPA produced nitric oxide in the vitreous and caused greater vasodilation in the choroid and the ciliary body of melanotic rats than in those of amelanotic rats

The nitrogen cycle initiates direct reduction of N2 to NH3 by enzymatic reactions. We hypothesize that L-dihydroxyphenylalanine (L-DOPA), a catecholamine, could be a source of nitric oxide (NO). In order to determine whether L-DOPA generates NO and induces any biological change in the eye, we measured the generation of NO in vitro and in vivo, and investigated the histopathological changes caused by injection of L-DOPA into the vitreous of rats. We also hypothesized that melanin granules may affect the generation of NO during the metabolism of L-DOPA, since L-DOPA is a precursor of melanin in the brain and the eye. Therefore, we compared the effects of L-DOPA on the generation of NO between amelanotic and melanotic rats. NO was measured as diffusion currents by NO electrodes. In vitro, various concentrations of L-DOPA (5, 29.9, 79.4, 152.7, and 249 microM) were added to the medium. The inhibition of NO generation by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (carboxy-PTIO) was tested. In vivo, NO generation in the vitreous of rats was measured and the eyes were enucleated under anesthesia after L-DOPA injection. The ocular tissues were subjected to histological examination. NO was produced from L-DOPA in a dose-dependent manner and was scavenged by carboxy-PTIO in vitro. NO in the vitreous of melanotic rats was generated from L-DOPA. Histological examination with hematoxylin-eosin staining revealed vasodilation in the ciliary vessels and the choroid after L-DOPA injection. Both effects were greater in melanotic rats than in amelanotic rats. The vasodilation may be attributable to NO as well as to superoxides, which can be regulated by the existence of melanin.     

Survival and repopulation of irradiated 'pre-CFU-c' in mice

Supernatants of murine bone-marrow cultures contain a colony-promoting factor (CPF) which increases the number of granulocyte and macrophage colonies in semi-solid agar cultures in the presence of colony-stimulating factor (CSF). Incubation of bone-marrow cells with CPF results in an increase in the number of granulocyte/macrophage progenitor cells (CFU-c) and the CPF-responsive cells may be younger than the CFU-c. We have investigated the radiosensitivity and the pattern of the recovery after irradiation of CPF-responsive cells. We found that the radiosensitivity of CPF-responsive cells was significantly lower than those of CFU-c, burst-forming units-erythroid (BFU-e) and pluripotent stem cells in vivo (CFU-s) and in vitro (CFU-mix). The CPF-responsive cells remained subnormal even at 28 days after irradiation of the mice, a time when the CFU-s and CFU-c had recovered completely. Therefore the CPF-responsive cells may constitute a separate compartment, namely 'pre-CFU-c', in the maturation sequence of granulopoiesis, and this maturation of the 'pre-CFU-c' to CFU-c seems to be highly stimulated after irradiation to counterbalance the influx from CFU-s.     

3 alpha-hydroxysteroid dehydrogenase in the cytosol of rat submandibular gland

We examined the in vitro shuttle metabolism between dihydrotestosterone (DHT) and 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) by 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD, E.C. 1.1.1.50) in rat submandibular gland (SMG) and ventral prostate (VP). The protein having molecular weight of 30 kDa, which was revealed by Sephacryl S-200 column chromatography, had 3 alpha-HSD activity to produce 3 alpha-diol from DHT, and also showed an oxidative 3 alpha-HSD (3 alpha-HSDO) ability to produce DHT from 3 alpha-diol. From the kinetic studies, the apparent Km and Vmax values of 3 alpha-HSD for DHT and NADPH were 6.4 microM, 1429 pmol/mg protein per min and 33.0 microM, 1205 pmol in SMG, and 9.3 microM, 377 pmol and 34.0 microM, 192 pmol in VP. The corresponding values of 3 alpha-HSDO for 3 alpha-diol and NADP+ were 18.0 microM, 714 pmol and 14.0 microM, 445 pmol in SMG, and 14.0 microM, 417 pmol and 36.0 microM, 77 pmol in VP. The affinities for DHT and 3 alpha-diol and the cosubstrate requirements of this enzyme in SMG were similar to those in VP. However, higher capacities of 3 alpha-HSD and 3 alpha-HSDO in SMG than in VP were shown. This suggests that there may be more 3 alpha-HSD in the SMG.     

A randomized trial of chemoimmunotherapy of acute nonlymphocytic leukemia in adults using a protein-bound polysaccharide preparation

Summary The effect of immunotherapy with a protein-bound polysaccharide preparation termed PSK on remission duration and survival of adults with acute nonlymphocytic leukemia (ANLL) was studied in a prospective randomized cooperative trial. After having achieved complete remission and receiving a consolidation therapy, 73 patients were randomized either to maintenance chemotherapy or to maintenance chemotherapy plus immunotherapy with PSK. Ultimately 36 patients in the chemotherapy group and 31 in the chemoimmunotherapy group were evaluable. Six months after the last entry, immunotherapy with PSK showed a borderline beneficial effect on remission duration (P=0.089) and on duration of survival (P=0.062). When the data were analyzed 12, 18, and 24 months after the last entry there were no significant differences in duration of remission and survival between the two groups. However, analysis of the data of patients who had maintained complete remission for more than 270 days revealed that immunotherapy had a suggestive beneficial effect (P=0.105), prolonging the 50% remission period by 418 days (885 vs 467 days). Thus, immunotherapy with PSK seems to be active in the treatment of adult ANLL when used for maintenance therapy in combination with chemotherapy, especially in patients with a good prognosis.     

Melanin granules prevent the cytotoxic effects of L-DOPA on retinal pigment epithelial cells in vitro by regulation of NO and superoxide radicals

Inasmuch as the nitrogen cycle elicits the direct reduction of N2 to NH3 through enzymatic reactions, and inasmuch as L-DOPA (L-dihydroxyphentlalamine), a catecholamine, can be a source of nitric oxide (NO), it is possible that melanin granules in the eye affect the generation of NO, which causes damage to the retinal pigment epithelial (RPE) cells during the oxidation of L-DOPA. In order to confirm this possibility, we analyzed the correlations of NO generation, cell growth, and superoxide dismutase (SOD) activities in two types (melanotic and amelanotic) of bovine RPE cells following exposure to L-DOPA. NO generation from L-DOPA was determined using an NO detector that is reliant on redox currents. The concentration of NO was measured in terms of diffusion currents run between a working electrode and a counter electrode, both being set in culture medium placed in a Petri dish. For the assays, L-DOPA was added to the medium at various concentrations (5, 29.9, 79.4, 152.7 or 249 microM), and 6 min after addition, an NO-trapping agent 2,4-carboxyphenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (carboxy-PTIO) was also added. The melanotic and amelanotic types of RPE cells were cultured separately in medium with L-DOPA under an atmosphere containing 20, 10 or 5% oxygen. Cell numbers were counted using a Coulter counter, and SOD activities were determined following incubation for 24, 48 or 72 hr using a modification of the luminol assay. The results obtained indicated that: (a) NO was produced from L-DOPA in a concentration-dependent manner and was trapped quantitatively by carboxy-PTIO; (b) the generation of NO was inhibited more markedly in the melanotic cell line than in the amelanotic one, suggesting an increased tolerance to L-DOPA-derived cytotoxicity in the former; and (c) the SOD activities were more affected by oxygen concentration in the melanotic cells than in the amelanotic ones. From these results, it is concluded that melanin granules in RPE cells have a role in preventing the cytotoxicity derived from L-DOPA and in regulating the generation of NO and superoxide radicals.