Igor Zusman

αMUPA mice: a transgenic model for longevity induced by caloric restriction

Caloric restriction (CR) is currently the only therapeutic intervention known to attenuate aging in mammals, but the underlying mechanisms of this phenomenon are still poorly understood. To get more insight into these mechanisms, we took advantage of the alphaMUPA transgenic mice that previously were reported to spontaneously eat less and live longer compared with their wild-type control mice. Currently, two transgenic lines that eat less are available, thus implicating the transgenic enzyme, i.e. the urokinase-type plasminogen activator (uPA), in causing the reduced appetite. This phenotypic change could have resulted from the ectopic transgenic expression that we detected in the adult alphaMUPA brain, or alternatively, from a transgenic interference in brain development. Here, we have summarized similarities and differences so far found between alphaMUPA and calorically restricted mice. Recently, we noted several changes in the alphaMUPA liver, at the mitochondrial and cellular level, which consistently pointed to an enhanced capacity to induce apoptosis. In addition, alphaMUPA mice showed a reduced level of serum IGF-1 and a reduced incidence of spontaneously occurring or carcinogen-induced tumors in several tissues. In contrast, alphaMUPA did not differ from wild type mice in the levels of low molecular weight antioxidants when compared in several tissues at a young or an old age. Overall, the alphaMUPA model suggests that fine-tuning of the threshold for apoptosis, possibly linked in part to modulation of serum IGF-1 and mitochondrial functions, could play a role in the attenuation of aging in calorically restricted mice.

Mitochondrion-mediated apoptosis is enhanced in long-lived αMUPA transgenic mice and calorically restricted wild-type mice

Caloric restriction (CR) can extend the life-span of multiple species and is the only intervention known to attenuate aging in mammals. Mechanisms mediating the CR influence are as yet unclear. To get insight into these mechanisms we took advantage of alphaMUPA transgenic mice that have previously been reported to spontaneously eat less and live longer compared with their wild-type (WT) control. Here we report that mitochondria isolated from young adult alphaMUPA livers showed increased susceptibility to calcium-induced high-amplitude swelling, increased cytochrome c release and enhanced glutathione levels. Furthermore, young adult alphaMUPA mice showed significantly enhanced caspase-3 activity in liver homogenates, increased fraction of apoptotic hepatocytes, and a lower level of serum IGF-1. In addition, alphaMUPA mice showed a decreased rate of spontaneously occurring lung tumors at an old age. Short-term (8 weeks) calorically restricted WT mice also showed an increase of mitochondrial swelling and caspase-3 activity compared with ad libitum (AL) fed WT mice. These results provide the first indication that CR can enhance mitochondrion-mediated apoptotic capacity. Collectively, the results are consistent with the possibility that long lasting, moderately increased apoptotic capacity, possibly linked in part to IGF-1 and GSH modulation, could play a role in the CR-induced anti-aging influence in mice.

The effects of high-sucrose diets and of maternal diabetes on the ultrastructure of the visceral yolk sac endoderm in rat embryos developing in vivo and in vitro

The ultrastructure of the visceral yolk sac endoderm of in vivo developing 9- to 13-day-old embryos from 2 diabetic rat models (streptozotocin diabetes and Cohen--genetically determined--diabetes) and from nondiabetic rats fed high sucrose diets have been studied. This was compared to yolk sacs from 9.5-day-old embryos cultured for 48 h in sera from diabetic and nondiabetic rats fed a high-sucrose diet. Light-microscopic, TEM and SEM studies showed that the pathological cellular changes in the visceral yolk sac endoderm from diabetic rats were first observed on day 9 and were most severe among 11-day-old embryos. In vitro culture of control rat embryos in serum from experimental animals induced a reduction in the number of microvilli, of vacuolar intracellular inclusions and an increase in the number of degenerated endodermal cells. SEM studies showed that in addition to disappearance of microvilli, the majority of cells were collapsed and had degenerated cell membranes. Culture of embryos from diabetic animals in control serum only slightly reversed the pathological changes in the visceral yolk sac endoderm. A good correlation exists between the rate of embryonic malformations in diabetic rats and an index of endodermal-cell damage in the visceral yolk sac.

Purification of sheep immunoglobin G using protein A trapped in sol-gel glass.

An effective method for purifying immunoglobulins has been developed utilizing a sol-gel glass support system. Sol-gel glass is an effective support for chemically active ligands entrapped in this medium at room temperature. There are two problems associated with the utilization of such sol-gel glasses for entrapping macromolecules. One is the phenomenon of nonspecific absorption of proteins onto the glass. The second is that only a portion of the entrapped molecules may retain their biological activity. In the present study a sol-gel glass was treated with gamma-aminopropyltriethoxysilane to provide a matrix that eliminated nonspecific absorption of proteins. The method of entrapping molecules was modified to increase the proportion of entrapped molecules that retained their reactivity. Protein A was entrapped in the modified sol-gel glass column and used to purify IgG from sheep sera by affinity chromatography. The purity of the IgG, as determined by SDS-PAGE, was comparable to that obtained from commercially available protein A columns and, if the capacity of the column was not exceeded, the yield approached 100%. Although the quality and quantity of the yields were comparable, the methodology described herein can be accomplished more rapidly and with greater ease. Furthermore, the sol-gel glass ligand preparation is extremely stable and can be reused for isolation of gamma-globulin. The technique has great potential for isolating macromolecules utilizing various ligands.

Pathology of lymphoid organs in low birth weight infants subjected to antigen-related diseases: A morphological and morphometric study

&NA; The pathology of lymphoid organs in 38 low birth weight (LBW) human infants has been evaluated by morphological and morphometric features. The gestational ages of infants ranged from 22 to 32 wks and their age at death varied from 1 hr to 153 days post partum. Infants were divided into 3 groups: 1) without antigenic effects, 2) with mild (bronchopneumonia), and 3) with severe antigenic effects, mainly sepsis. In mildly affected LBW infants, the fetal type of the immune reaction was found. It continued during the period studied (till 5 mths) and was manifested in the reaction of macrophages and the transformation of lymphocytes to lymphoblasts. Reactive centres of follicles and mature plasmocytes were not found. During the first months of postnatal development, an increase in the amount of lymphocytes in the lymphoid organs and in the rate of proliferation of reticular epithelium and a decrease in the area of the cortex in the thymus were found in all infants. In severely affected infants, the number and the size of follicles in the spleen decreased significantly and the total number of cells decreased more than 3 times. Similar changes were found in lymph nodes. These changes as well as the weak reaction of the thymus are the main features of the insufficiency and fast devastation of the lymphoid system. A compensatory increase in the number of neutrophils and eosinophils in the red pulp of the spleen and lymph nodes was found after the second week.Abbreviations: LBW, low birth weight; HMD, hyaline membrane disease; RDS, respiratory distress syndrome; LN, lymph node; AI, accidental involution of thymus; TC, thymic corpuscles.

A high degree of aneuploidy, loss of p53 gene, and low soluble p53 protein serum levels are detected in ulcerative colitis patients.

PURPOSEThe causes for the increased risk of colorectal cancer associated with ulcerative colitis have not been fully defined. Colonic tissue of ulcerative colitis patients was examined for changes in chromosome-17-centromere copy number, loss of the p53 gene, and alterations in serum levels of the 53-kDa protein. This study was performed under the assumption that these molecular events correlate with ulcerative colitis status and duration.METHODSUlcerative colitis patients (n = 42) and healthy controls (n = 37) participated in the study. All participants were histopathologically and medically diagnosed. The stage of ulcerative colitis patients was stratified according to increasing risk factors for the development of colorectal cancer: left-sided colitis, pancolitis, sclerosing cholangitis, and dysplasia-associated lesions or masses. Changes in centromere number of chromosome 17 alone or in association with changes in copy number of the p53 gene were analyzed in colon tissue biopsies by fluorescence in situ hybridization. Serum p53 level was determined in blood samples by immunoprecipitation followed by separation using high-pressure liquid chromatography.RESULTSChanges in chromosome 17 and p53 copy number and lower levels of serum p53 protein in ulcerative colitis patients directly correlated with colorectal cancer risk factors. All values significantly differed from controls. Significant direct correlations were obtained for ulcerative colitis disease duration, levels of p53 in the serum, and extent of aneuploidy.CONCLUSIONSWe demonstrate that in the colonic mucosa of ulcerative colitis patients, high levels of genomic instability, changes in p53 gene copy number, and lower levels of p53 in the serum directly correlate with the extent of disease duration and increased risk factors for colorectal cancer. Any of the measurements described herein can provide an acceptable prognostic tool in the assessment of colorectal cancer risk in ulcerative colitis patients.

Effects of High‐fiber diets on pathological changes in DMH‐induced rat colon cancer

The protective role of dietary fibers on tumorigenic effects of 1,2-dimethylhydrazine (DMH) on the rat colon was studied using histochemical, immunohistochemical, and biochemical analyses. Rats were injected with DMH (20 mg/kg sc) for five weeks, once a week, and were fed laboratory chow (Control I), semisynthetic fiber-free diet (Control II), or 18% or 25% assorted fiber (cellulose, beans, corn) diet. The rats were sacrificed 12, 16, and 24 weeks after the carcinogenic injections. Adenomatous tumors developed in 90-100% of the animals fed chow or fiber-free diets. A diet with high concentrations (25%) of corn dietary fiber significantly decreased the tumor incidence (40% and 42%) and tumor yield (p < 0.01). Diets with lower concentrations of fibers (18%) did not protect against tumorigenic effects of DMH. No differences were found in pathological parameters of tumors obtained from different dietary groups: the damage index (percentage of damaged cells per section) was very similar in all groups studied. Diets enriched with dietary fibers (25%) had a significant protective effect in DMH-induced rat colon cancer.

Long-lived αMUPA transgenic mice exhibit increased mitochondrion-mediated apoptotic capacity

Abstract: Caloric restriction (CR) is currently the only therapeutic intervention known to attenuate aging in mammals, but the mechanisms underlying this phenomenon are still poorly understood. To study this issue, the transgenic model of αMUPA mice, which previously were reported to spontaneously eat less and live longer compared with their wild‐type (WT) control mice, were used. Currently, two transgenic lines that eat less are available, thus implicating the transgenic enzyme, that is, the urokinase‐type plasminogen activator (uPA), in causing the reduced appetite. Recently, several changes in the αMUPA liver were noted, at the mitochondrial and cellular level, which consistently pointed to an enhanced capacity to induce apoptosis. In addition, αMUPA mice showed a reduced level of serum IGF‐1 and a reduced incidence of spontaneously occurring or carcinogen‐induced tumors in several tissues. Overall, the αMUPA model suggests that long‐lasting, moderately increased apoptotic capacity, possibly linked in part to modulation of serum IGF‐1 and mitochondrial functions, could play a role in the attenuation of aging in calorically restricted mice.

Immunoglobulin A in the epithelium of the respiratory tract and intrahepatic bile ducts of fetuses and newborns with pneumonia and sepsis

The level of different immunoglobulins (IgA, IgG, IgM) in the tissues of 28 late fetuses and newborns was studied with peroxidase-labeled monoclonal antibodies. IgA+ and IgM+ lymphocytes were found in the spleen, lymph nodes and sometimes in the liver. IgG+ lymphocytes were not found. A high level of IgA+ material was found in the epithelium of the trachea, the epithelium and submucosal glands of the bronchi, but not the bronchioles, and in the epithelium of hepatic bile ducts and in their lumina. Such IgA is considered to be secretory--sIgA. Secretory IgA-containing epithelial cells appeared at 20 to 21 weeks of gestation; their number increased from 2.5 cells/10,000 microns2 in 23- to 26-week-old fetuses, to 8 cells/10,000 microns2 in 36- to 40-week-old fetuses. Secretory IgG and IgM were not detected. In fetuses with pneumonia or sepsis, the number of IgM+ and IgA+ lymphocytes increased significantly. IgM+ lymphocytes appeared not only in the spleen and lymph nodes, but also in the lungs. In such cases, the number of sIgA-containing epithelial cells in the trachea, bronchi and intrahepatic bile ducts decreased, sometimes completely disappearing. The amount of IgA+ material in the lumina of these organs increased, reflecting an intensification of sIgA secretion during infections. The presence of a marked amount of sIgA in fetuses from week 20 of gestation is considered to reflect the high importance of this immunoglobulin against normal contamination by microbes after birth, and to evidence the early maturation of the immune system.

Comparative study of the role of serum levels of p53 antigen and its tumor cell concentration in colon cancer detection

The role of serum levels of p53 antigen in detection of colon cancer was studied in different groups of cancer and noncancer patients and was compared with the results of immunohistochemical analyses. The p53 antigen was isolated from the human serum as a cytoplasmic fraction using the recently described new type of columns for affinity chromatography, gel fiberglass columns (Zusman and Zusman, 1995). Its concentration was detected by high performance liquid chromatography. The serum level of the p53 antigen significantly increased in cancer patients (3.6 mg ml(-1)) as compared to its concentration in patients with benign tumors (1.7 mg ml(-1)) or in patients with noncancer disorders (0.49 mg ml(-1)), and this was found to be a result of higher concentration of p53 protein in tumor cells. Coefficient of correlation between cellular concentration of p53 protein and its serum level was 0.44 in noncancer lesions and 0.48 in cancer patients. Serum levels of p53 antigen was shown to be highly active either in patients with noncancer lesions or in patients with cancer (r = 0.46 and 0.51 respectively), whereas the cell determination of p53 protein was effective only among noncancer patients (r = 0.61) but not in cancer patients (r = 0.22). The findings suggests that serum determination of p53 antigen can perhaps reveal this oncoprotein already in the early stages of cancer or even predict the putative development of cancer. The possibility to use the serum-levels of p53 antigen in the follow up patients with chronic diseases and to detect transformation of these diseases into cancer, or monitoring former cancer patients in order to detect as early as possible the incidence of recurrent cancer is discussed.