The mitochondrial genome of Saccharomyces cerevisiae codes for 24 tRNAs. The nucleotide sequences of the tRNA genes suggest a unique set of rules that govern the decoding of the mitochondrial genetic code. The four codons of unmixed families are recognized by single tRNAs that always have a U in the wobble position of the anticodon. The codons of the mixed families are read by two different tRNAs. Codons terminating in a C or U are recognized by tRNAs with a G and codons terminating in a G or A are recognized by tRNAs with a U in the corresponding positions of the anticodons. There are two exceptions to these rules. In the AUN family for isoleucine and methionine, the isoleucine tRNA has a G and the methionine tRNA has a C in the wobble position. The tRNA for the arginine CGN family also has an A in the wobble position of the anticodon. It is of interest that the CGN codons have not been found in the mitochondrial genes sequenced to date. The simplified decoding system of yeast mitochondria allows all the codons to be recognized by only 24 tRNAs. Yeast mitochondria contain a complete set of tRNAs that function in the translation of a discrete number of mitochondrial messengers. During the past year, the sequences of most of the mitochondrial tRNA genes have been determined and virtually all of their anticodons are now known (1-4). Together with the codon usage in five mitochondrial genes, this information has permitted us to formulate the coding properties of this self-contained translational system.In this communication we present evidence that yeast mitochondria are capable of interpreting their genetic code with less than the 32 tRNAs required by the wobble hypothesis (5). The reduction in the number of tRNAs is achieved by the use of a single tRNA to recognize the four codons in each of the eight unmixed families.* These families are read by the "two out of three" method first proposed by Lagerkvist (6). Furthermore, because only two tRNAs are used to discriminate among the codons of the mixed families, the entire system including the formylmethionine initiator consists of 24 tRNAs. The 24 tRNAs represent the minimum number needed to interpret a degenerate code whose amino acid assignments follow the general outline of the universal code. Number of mitochondrial tRNAs in Saccharomyces cerevisiae Several approaches have been used to estimate the number of mitochondrial tRNAs in yeast. Separation of the aminoacylated tRNAs by reverse phase chromatography on RPC-5 has provided information about the number of isoaccepting species for each of the 20 amino acids. Such analyses indicate [31][32][33][34][35] chromatographically distinct tRNA species (7-9). This number includes species that arise from posttranscriptional modification and therefore is an overestimate of the actual number of tRNA genes. For example, Berlani et al. (3) have recently shown that a point mutation in mitochondrial DNA abolishes charging of the four histidine tRNAs, suggesting that all four isoacceptors are products of a single gen...

The aim of this study was to determine the effect of adoptive transfer of regulatory natural killer T (NKT) lymphocytes on the metabolic disorder in leptin-deficient ob/ob mice, which feature depletion and defective function of NKT and CD4 lymphocytes. Leptin-deficient ob/ob mice were subjected to transplantation of 1 x 10(6) of either ob/ob or wild-type-derived NKT lymphocytes, or to transplantation of either ob/ob or wild-type-derived splenocytes. The effect on hepatic fat content was measured by magnetic resonance imaging (signal intensity index) and histology, using the steatohepatitis grading scale. The degree of glucose intolerance was measured by an oral glucose tolerance test (GTT). Adoptive transfer of wild-type or ob/ob-derived regulatory NKT cells led to a 12% decrease in hepatic fat content. A significant histological shift from macrosteatosis to microsteatosis was observed. Marked improvement in the GTT was noted in wild-type or ob/ob-derived NKT recipients. Metabolic effects were associated with a significant decrease in peripheral and intrahepatic CD4/CD8 lymphocyte ratios. Intrahepatic CD8 trapping was observed in all responders. Serum interleukin 10 levels decreased significantly. In conclusion, adoptive transfer of a relatively small number of regulatory NKT lymphocytes into ob/ob mice results in a significant reduction in hepatic fat content, a shift from macro to microsteatosis, and significant improvement in glucose intolerance. These effects were associated with decreased peripheral and intrahepatic CD4/CD8 ratios and decreased interleukin 10 levels. The results further support a role for regulatory NKT lymphocytes in the pathogenesis of non-alcoholic steatohepatitis in the leptin-deficient murine model.

Concanavalin A (ConA) induces natural killer T (NKT) cell-mediated liver damage. Glucocerebroside (GC) is a naturally occurring glycolipid. Our aims were to determine the effect of GC in a murine model of ConA-induced hepatitis. Mice in groups A and B were treated with GC 2 h before and 2 h following administration of ConA, respectively; group C mice were treated with ConA; group D mice was treated with GC; group E mice did not receive any treatment. Liver damage was evaluated by serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and liver histology. The immune effect of GC was determined by fluorescence-activated cell sorter analysis of intrahepatic and intrasplenic NKT lymphocytes, measurement of cytokine levels, and Western blot analysis for STAT 1, 4, 6, and NF-kappaB expression. The effect of GC on NKT cell proliferation was assessed in vitro. Serum AST and ALT levels were markedly reduced in GC-treated group A mice compared with nontreated group C animals, and histological damage was markedly attenuated in group A. The beneficial effect of GC was associated with a 20% decrease of intrahepatic NKT lymphocytes, significant lowering of serum IFN-gamma levels, and decreased STAT1 and STAT6 expression. In vitro administration of GC led to a 42% decrease of NKT cell proliferation in the presence of dendritic cells but not in their absence. Intraperitoneally administered radioactive GC was detected in the liver and bowel. Administration of GC led to amelioration of ConA hepatitis associated with an inhibitory effect on NKT lymphocytes. GC holds promise as a new immune-modulatory agent.

Dendritic cells (DCs) are antigen presenting cells that play a role in T-cell activation. Liver-associated natural killer T lymphocytes (NKTs) are a unique subset of lymphocytes that may be important in antitumor immunity. Hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) expresses hepatitis B virus surface antigen (HBsAg) on its cell surface and may serve as a tumor-associated antigen. The aim of the study was to evaluate the antitumor effect of DC pulsed with tumor or viral-associated antigens in HBV-expressing HCC in mice and to determine the role of NKT lymphocytes in this process. Balb/c mice were sublethally irradiated and transplanted with Hep3b HCC cell line, followed by transplantation of naive splenocytes. DCs were separated using CD11c beads and pulsed with HBV-enveloped proteins (group A), HCC cell lysate (group B), or BSA (control group C). Mice were followed for survival and tumor size. To determine the mechanism of the antitumor effect, intrasplenic and intrahepatic lymphocyte subpopulations were analyzed by FACS for NKT, CD4 and CD8 markers. Tumor-associated antigens-specific IFN␥ ELISPOT, T-cell proliferation assays and serum cytokine analysis were performed. Treatment with tumor-associated antigen-pulsed DC significantly improved survival (40% and 50% as compared with 0% in groups A, B, and control group C, respectively; p < 0.005). Tumor size decreased to 12.8 ؎ 0.4 and 0 from 60.4 ؎ 0.9 mm 3 in groups A, B, and control group C, respectively (p < 0.005). Adoptive transfer of HBV or Hep3b-associated antigens-pulsed DC induced a 6-fold increase in peripheral CD8 ؉ lymphocytes (from 1% in control mice to 6% and 5.5% in groups A and B, respectively), along with a decrease in CD4 ؉ lymphocytes (from 3.5% in controls to 1.4% and 2.3% in A and B, respectively; p < 0.005). The CD8 ؉ / CD4 ؉ ratio increased from 0.28 in controls to 4.28 and 2.39 in groups A and B, respectively (p < 0.005). Intrasplenic NKT cells increased from 7% in control mice to 7.98% and 14.6% in groups A and B, respectively. In contrast, an opposite shift was observed inside the liver. Intrahepatic lymphocyte analysis showed a marked increase in CD4 ؉ and a decrease in CD8 ؉ lymphocytes in treated groups. The intrahepatic CD4 ؉ number increased from 0.5% in controls to 2.15% and 25.8% in groups A and B, respectively (p < 0.005). In contrast, a significant decrease in the intrahepatic CD8 ؉ numbers was observed (from 7% in controls to 1.0% and 2.4% in groups A and B, respectively; p < 0.005). A significant increase was noted in HBV-specific IFN␥ spot-forming T-cell colonies from 0.0 to 8.8 ؎ 1.7 and 1.8 ؎ 2.9 in groups C, A, and B, respectively (p < 0.005). Similarly, a significant increase in the HBV-specific T-cell stimulation index, from 0. Key words: hepatocellular carcinoma; dendritic cells; immunotherapy; natural killer T lymphocytesDendritic cells (DCs) are specialized antigen presenting cells (APCs) capable of taking up a diverse array of antigens and modulating T-cell immune response driving naive T-cells ...

Non-alcoholic steatohepatitis (NASH) is a common cause of cryptogenic cirrhosis in the Western world. In an animal model of NASH, leptin-deficient ob/ob mice present with alterations in number and function of hepatic NKT and peripheral CD4 lymphocytes. Oral immune regulation is a method to alter the immune response towards orally administered antigens. To determine the effect of oral immune regulation towards liver-extracted proteins on the metabolic disorders in ob/ob mice, ob/ob mice and their lean littermates were orally administered liver extracts from wild-type or ob/ob mice or bovine serum albumin for 1 month. The effect of treatment on hepatic fat content was measured by magnetic resonance imaging (MRI) and using a histological steatohepatitis grading scale. Glucose tolerance was measured by an oral glucose tolerance test (GTT). T lymphocyte subpopulations were assessed by flow cytometry analysis. Induction of immune regulation by oral presentation of liver-extracted proteins resulted in a significant 18% reduction of the hepatic fat content in ob/ob mice fed with either wild-type or ob/ob liver extracts for 1 month. The MRI signal intensity index in treated mice decreased to 0.48 and 0.51, respectively, compared with 0.62 in BSA-fed controls (p = 0.037 and p = 0.019, respectively), while the histological steatohepatitis score decreased in both treated groups to 2.0, compared with 2.4 in BSA-fed controls (p = 0.05). A significant improvement in GTT was noted in treated ob/ob mice. These changes were accompanied by a marked increase in the intrahepatic NKT lymphocyte population in mice fed with proteins extracted from both wild-type and ob/ob mice (46.96% and 56.72%, respectively, compared with 26.21% in BSA-fed controls; p < 0.05) and a significant elevation in serum IL-10 levels. Oral immune regulation towards liver extracted proteins in leptin-deficient mice resulted in a marked reduction in hepatic fat content and improved glucose tolerance. This effect was associated with a significant increase in the intrahepatic NKT lymphocyte population and serum IL-10 levels, suggesting a Th1 to Th2 immune shift. Immune regulation towards disease-associated antigens holds promise as a new mode of therapy for NASH.