Characterisation of Trypanosoma cruzi populations by dna polymorphism of the cruzipain gene detected by single-stranded dna conformation polymorphism (SSCP) and direct sequencing

Fifty fresh isolates of Trypanosoma cruzi from Triatoma dimidiata vectors and 31 from patients with Chagas disease were analysed for DNA polymorphisms within the 432-bp core region of the cruzipain gene which encodes the active site of cathepsin L-like cystein proteinase. The cruzipain gene showed signs of polymorphism consisting of four different DNA sequences in Central and South American isolates of T. cruzi. The PCR fragments of Guatemalan isolates could be divided into three groups, Groups 1, 2 and 3, based on different patterns of single-stranded DNA conformation polymorphism. All of the strains isolated from Brazil, Chile, and Paraguay, except for the CL strain, showed a Group 4 pattern. Two to four isolates from each group were analysed by cloning and sequencing. A silent mutation occurred between Groups 1 and 2, and five nucleotides and two aa substitutions were detected between Groups 1 and 3. The DNA sequence of Group 4 contained five nucleotides and one aa substitution from Group 1. All of the DNA sequences corresponded well with the single-stranded DNA conformation polymorphism. The Group 1 isolates, the majority in the Guatemalan population (70/81, 86.4%), were isolated from both triatomines and humans, but Group 3 were isolated only from humans. Moreover, the Group 2 isolates were detected only in triatomine vectors (9/50; 18%), but never in humans (0/32, P<0.05) suggesting that this group has an independent life-cycle in sylvatic animals and is maintained by reservoir hosts other than humans.     

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A previous paper has demonstrated that enhanced tumor-specific immunity could be induced by priming mice with Bacillus Calmette Guerin (BCG) and subsequently immunizing them with syngeneic tumor cells modified with BCG-cross-reactive muramyl dipeptide (MDP) hapten [15]. The present study establishes a tumorspecific immunotherapy protocol for a murine chronic leukemia based on the above T-T cell collaboration between antitumor effector T cells and anti-MDP hapten helper T cells induced by BCG priming. BALB/c mice which had been primed to BCG were injected intravenously (i.v.) with viable, syngeneic BCL1 leukemia cells. One week later, these mice were immunized intraperitoneally (i.p.) with unmodified or MDP hapten-modified, 10,000 R X-irradiated BCL1 cells, followed by 4 booster immunizations at 5-day intervals. The administration of unmodified BCL1 tumor cells into BCG-primed mice failed to prevent them from tumor death due to the persistent growth of preinjected BCL1 cells. In contrast, the immunization of BCG-primed, BCL1 leukemia-cell-bearing mice with MDP-modified BCL1 cells resulted in a high growth inhibition of leukemia cells and protection of these mice from death by leukemia. It was also revealed that potent tumorspecific, T-cell-mediated immunity was generated in mice which survived in this immunotherapy model. Thus, these results indicate that administration of MDP hapten-modified, syngeneic leukemia cells into leukemia-bearing mice which have been primed with BCG results in potent tumor-specific, T-cell-mediated immunity attributable to preventing the growth of disseminated leukemic cells.This work was supported by a Grant-in-Aid for the Special Project Cancer-Bioscience from the Ministry of Education, Science, and Culture, Japan Abbreviations used: TATA, tumor-associated transplantation antigens; MDP, muramyl dipeptide; MTP, muramyl tripeptide; BCG, Bacillus Calmette Guerin     

Deafferentation pain and stimulation of the thalamic sensory relay nucleus: clinical and experimental study

This report summarizes our clinical experience in which the effects of both thalamic sensory relay nucleus (TSRN) and periaqueductal gray (PAG) stimulation were tested in the same series of patients with various forms of pain. The clinical data indicated that neurogenic pain due to deafferentation at the level of the peripheral nerves or the spinal cord was often controlled by TSRN stimulation but not by PAG stimulation. We also review the results of our experimental investigations in cats which were undertaken in an attempt to clarify the neurophysiologic basis of such differential clinical effects of TSRN and PAG stimulation. It appeared that abnormal hyperactivity within the trigeminal medullary dorsal horn following retrogasserian rhizotomy was far more frequently inhibited by TSRN stimulation than by PAG stimulation.     

Aberrant porphyrin metabolism in hepatocellular carcinoma

In 15 patients with hepatocellular carcinoma (HCC) and 14 patients with liver cirrhosis (LC), urinary excretions of delta-aminolevulinic acid (ALA), porphobilinogen (PBG), uroporphyrin (UP), coproporphyrin (CP), and erythrocyte contents of CP and protoporphyrin (PP) were examined. In patients with HCC, urinary excretions of ALA and PBG and erythrocyte contents of CP and PP were not increased, but urinary excretions of UP and CP were significantly increased more than those of LC patients. Urinary excretions of UP and CP had no correlations with liver function tests and excretion of UP correlated slightly with blood hemoglobin level. After administration of ALA intravenously, urinary excretions of UP and CP were clearly increased in patients with HCC compared to normal controls. A Red fluorescent area was present at the cancerous area but not in the noncancerous cirrhotic area in a patient with HCC. These results suggest that aberrant porphyrin metabolism occurred in patients with HCC compared to other liver diseases.     

Inactivation of T Antigen-Forming Capacities of Simian Virus 40 and Adenovirus 12 by Ultraviolet Irradiation

Methods to measure T antigen-forming capacities of simian virus 40 (SV40) and adenovirus 12 (Ad12) were investigated, and a method to measure the capacity in terms of T antigen-forming units was employed by the use of cytosine arabinoside. Plaque-forming units and T antigen-forming units of SV40, SV40 deoxyribonucleic acid, or Ad12 were inactivated by ultraviolet (UV) irradiation at the same rate, roughly following a single-hit curve. T-antigen formation by UV-irradiated SV40 and Ad12 was enhanced in cells multiply infected and in cells in a growing state. These observations showed that it was difficult or impossible to estimate the size of the gene for T antigen by UV inactivation.     

Novel ATP-binding heat-inducible protein of Mr = 37,000 that is sensitive to transformation in BALB/3T3 cells

Using affinity chromatography on ATP-agarose, we have identified a major ATP-binding protein in Nonidet P-40 extracts of avian and mammalian cells labeled with [35S]methionine. After washing ATP-agarose beads with high-ionic-strength buffer (0.4 M NaCl), the 37-kD protein was shown to be one of the major ATP-binding proteins while p72 and grp78, which are members of the hsp70 family, also bound to ATP-agarose. This protein consisted of several spots on two-dimensional gel electrophoresis. The isoelectric point of the most basic spot was approximately 9.2 in chick embryo fibroblasts, whereas it was about 8.8 in mouse 3T3 cells. The identities of these proteins in mouse and chick cells were confirmed by peptide mapping. After heat-shock treatment of BALB/3T3 cells, the major heat-shock protein, hsp70, was shown to be induced very rapidly after heat shock and was recovered in the ATP-binding fraction. Besides hsp70, a 37-kD protein was also found to be induced by heat shock. This protein was drastically induced by treating the cells with alpha,alpha'-dipyridyl, an iron chelating reagent, but not with sodium arsenite, calcium ionophore, or tunicamycin. The synthesis and the total amount of this ATP-binding protein increased in mouse 3T3 cells transformed by simian virus 40, methylcholanthrene, or activated c-Ha-ras oncogene compared to their normal counterparts. The incorporation of [32P]orthophosphate was not detected in either normal or transformed cells. These studies established that a major ATP-binding protein of Mr = 37,000 is a heat-inducible protein and that the synthesis of this protein is regulated by malignant transformation.     

Nucleotide sequence of the gene for cholesterol oxidase from a Streptomyces sp.

The nucleotide sequence of a 2.1-kilobase-pair fragment containing the Streptomyces choA gene, which codes a secreted cholesterol oxidase, was determined. A single open reading frame encodes a mature cholesterol oxidase of 504 amino acids, with a calculated Mr of 54,913. The leader peptides extend over 42 amino acids and have the characteristics of a signal sequence, including basic amino acids near the amino terminus and a hydrophobic core near the signal cleavage site. Analyses of the total amino acid composition and amino acid sequencing of the first 21 amino acids from the N terminus of the purified extracellular enzyme agree with the values deduced from nucleotide sequencing data.     

Mapping of the human gene encoding the mutual signal-transducing subunit (β-chain) of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and interleukin-5 (IL-5) receptor complexes to chromosome 22q13.1

The human gene encoding the mutual signal-transducing subunit (-chain) of granulocyte-macrophage colony-stimulating factor, interleukin-3, and interleukin-5 receptor complexes has been mapped to chromosome 22q13.1 by the fluorescence in situ hybridization method.     

Human high-affinity FcγRI (CD64) gene mapped to chromosome 1q21.2-q21.3 by fluorescence in situ hybridization

The human FcRI gene encodes for a highaffinity Fc receptor that plays pivotal roles in the immune response. We have used fluorescence in situ hybridization analysis to localize the FcRI gene to human chromosome 1. The human FcRI (CD64) gene has been assigned to human chromosome 1q21.2-q21.3 using R-banded human (pro)metaphase chromosomes.