Transmission electron microscopy of the liver mitochondria of N-Nitrosodibutylamine-treated mice.

N-Nitrosodibutylamine (DBN) has been found in a wide variety of products because of the nitrosation of amines present in these products. An extensive series of synthesis and carcinogenicity studies of various compounds related to DBN and its metabolites have revealed that they have markedly different carcinogenic effects. The role of mitochondria in disease has been found to be expanded beyond the respiratory chain, as defects in additional mitochondrial functions and behaviors have been linked to cancer, metabolic disorders, and many neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Huntington's disease (Nunnari & Suomalainen (2012) Cell, 148, 1145-1159). The sample preparation was performed using the protocol standardized by Sophisticated Analytical Instrumentation Facility (SAIF) laboratory in NEHU. The prepared sample was then mounted and observed under the transmission electron microscope model JEM-100 CC II. The micrographs obtained from transmission electron microscopy (TEM) at various magnifications 2,000, 5,000, and 12,000× from DBN-treated mice showed significant changes. Mitochondria showed variability in number, size, and shape. This significant alteration in the morphology and population of liver mitochondria observed in DBN-treated animals in comparison to that of the age-matched normal control mice provide a unique potential for the use of mitochondria as markers for the early detection for the treatment of liver cancer.

Long-term outcomes of dual kidney transplantation-a single center experience.

The shortage of kidney donors has led to broadening of the acceptance criteria for deceased donor organs beyond the traditional use of young donors. We determined long-term post-transplant outcomes in recipients of dual expanded criteria donor kidneys (dECD, n = 44) and compared them to recipients of standard criteria donor kidneys (SCD, n = 194) and single expanded criteria donor kidneys (sECD, n = 62). We retrospectively reviewed these 300 deceased donor kidney transplants without primary non-function (PNF) or death in the first two wk, at our center from 1996 to 2003. The three groups were similar in baseline characteristics. Kidney allograft survival and patient survival (nine yr) were similar in the three respective donor groups, SCD, sECD and dECD (60% vs. 59% vs. 64% and 82% vs. 73% vs. 73%). Acute rejection in the first three months was 23.2%, 16.1%, and 22.7% in SCD, sECD and dECD, respectively (p = 0.49) and delayed graft function was 25.2%, 31.9% and 17.1% in the three groups, respectively (p = 0.28). When PNF and death within the first two wk was included, there was no significant difference in graft survival between the three groups. In our population, recipients of dECD transplants have acceptable patient and graft survival with kidneys that would have usually been discarded.

Admission hyperglycemia and length of hospital stay in patients with diabetes and heart failure: a prospective cohort study.

The authors assessed the relationship between glycemia and length of hospital stay (LOS) in a prospective cohort study of patients with diabetes mellitus and heart failure (HF). Of 212 patients with acute HF exacerbation, 119 (56%) also had diabetes. The mean age of the cohort was 63+/-0.87 years, and the mean body mass index was 29.3 kg/m2. Diabetic patients had significantly longer LOS compared with the nondiabetics (5.0+/-0.29 vs 3.4+/-0.19; P<.001). In patients with diabetes, the mean glycated hemoglobin A1c was 8.3%, admission blood glucose (BG) was 169+/-7.7 mg/dL, and average BG was 196+/-8.1 mg/dL. After adjusting for age, sex, weight, hypertension, renal function, and anemia, LOS was significantly correlated with admission BG (r=0.31; P<.001) and average BG (r=0.34; P=.001). In patients with acute HF exacerbation, diabetes significantly prolonged LOS. Hyperglycemia correlated with LOS.

Carcinogenesis response modulation induced by gelonin encapsulated in liposome.

The effectiveness of gelonin to arrest protein synthesis, thereby limiting the growth of cancer cells was studied by encapsulating it into liposomes. The protein was extracted from the seeds of Indian plant Gelonium multiflorum by ammonium sulfate precipitation and purified using cation-exchange and gel-filtration chromatography. Biological activity of purified gelonin was determined using a rabbit reticulocyte lysate assay in the cell-free translational experiments. Gelonin was encapsulated in conventional liposomes prepared by the dry film method in order to retain biological activity of the entrapped protein. Carcinogenesis was induced in Swiss albino mice by intravenous administration of DBN (10 mg kg(-1) body weight) at weekly intervals. Marker enzyme assays (GGT, AChE, and GST), GSH levels, cell proliferation assay, hepatocyte DNA analysis, histological examination of micro sections of liver tissues were parameters used to monitor carcinogenesis induction, and regression in mice. From the in vitro experiments conducted, it was observed that gelonin upon its encapsulation into liposome, resulted in significant destruction of the transformed liver cells by its cytotoxic effects that arrest protein synthesis. Various parameters studied to monitor regression also suggested mass cell destruction to liver upon administration of liposomal gelonin in mice exposed to DBN.

Radiomodulatory effect of liposome encapsulated AK-2123 on tumor in mice exposed to hepatocarcinogen.

An attempt was made to evaluate the whole body gamma-radiation effect on tumor in the presence of free and liposome encapsulated AK-2123, a hypoxic cell radiosensitizer that has widely been used in combination with a number of cancer therapies such as thermotherapy, chemotherapy and radiotherapy. Entrapment efficiency of AK-2123 into liposome was determined by LASER Raman spectroscopy. Cancer induction in mice was carried out by repeated exposure of N-nitrosodiethylamine (DEN) in combination with partial hepatectomy. Parameters such as marker enzymes activities (GGT and AChE), rates of nucleic acid synthesis, viability modification factor and the histology of liver tissues monitored, supported the induction of cancer in liver. In addition, the effect of free as well as liposome encapsulated AK-2123 on haemopoietic parameters were also studied. It was observed that AK-2123 after incorporation into liposome afforded more efficient radiomodulatory effects than that of free AK-2123 as determined by the above-mentioned parameters. Neither free AK-2123 nor liposome encapsulated AK-2123 showed any detectable toxic effects on the mice. Thus, it is seen that treatment of cancer with a combination of radiation, a radiomodifier and a drug delivery system, opens a wide scope for exploitation for the improvement of existing cancer therapies.

Molecular characterization of tumor associated antigen in mice exposed to a hepatocarcinogen.

The present investigation is aimed to identify and characterize tumor-associated antigen (TAA) in animals exposed to hepatocarcinogen. Swiss albino mice showed an enhanced expression of an approximately 58 kDa glycoprotein in liver cells upon exposure to a potential hepatocarcinogen N-nitrosodibutylamine (DBN). Carcinogenesis induction in mice was monitored by assays of y-glutamyl transpeptidase (GGT), acetylcholine esterase (AChE), glutathione-S-transferase (GST) activities and the level of glutathione (GSH) in liver. DBN treated animals showed cell distortion and extensive necrosis as observed by histological examination. The over-expressed TAA was purified by ion-exchange chromatography and further characterized by SDS-PAGE. The carbohydrate contents and glycan linkage to the polypeptide backbone was analyzed by using the DIG glycan differentiation and de-glycosylation kits. The glycoprotein has glycan chains that are N-linked via asparagines to the polypeptide backbone. It was also observed that the molecule is rich in sialic acid residues with a significantly high carbohydrate to protein ratio (> 2:1). The over-expressed high molecular weight glycoprotein TAA was found to be highly immunogenic and could eventually be used to induce immune response in order to counter tumor regression.

Purification,characterization and chemical modification studies on a translation inhibitor protein from Luffa cylindrica.

A ribosome-inactivating protein (RIP), luffin has been isolated from the seeds of Luffa cylindrica of Cucurbitaceae family by ammonium sulfate fractionation followed by cation exchange and gel-filtration chromatography. Extensive physico-chemical, immunological and biological characterizations were carried out on luffin and compared with that of gelonin. The molecular mass of luffin was -28 kDa as determined by gel-filtration chromatography and SDS-PAGE. The epsilon-NH2 group(s) of luffin were sequentially modified by N-succinimidyl 6-[3-(2-pyridyldithio) propionamido] hexanoate (LC-SPDP), N-succinimidyl-3-(2-pyridylthio)propionate (SPDP) and 2-iminothiolane (2IT) and their effect on immunoreactivity and ribosome inactivating property was evaluated. Modification of single amino group resulted in about 80% inhibition of immunoreactivity and more than 90% loss of protein synthesis inhibition activity. Modification of 2-3 amino groups further hampered both immunoreactivity and protein-synthesis inhibition property LC-SPDP modification played more pronounced effects on immunoreactivity and RIP activity than that of SPDP. However, 2IT modification retained both the immunoreactivity and RIP activity of luffin-LC-SPDP substantially. SPDP showed more pronounced effect on immunoreactivity and RIP activity as compared to 2IT. Therefore, it seems that the positive charge on lysine residues plays an important role in immunological as well as protein synthesis inhibitory effect of luffin.