A Report on the Positive Response to an Outdoor Nature Challenge of a Snow Camp for Young Liver Transplant Patients.

OBJECTIVES: More than two decades have passed since the first living donor liver transplantation was performed in Japan in 1989. There are many reports about problems in adherence to taking medication and medical follow-ups in children who received liver transplants, because there is no transition strategy for those children and parents or guardians. The objective of this study is to measure the effect of nature and outdoor activity to improve children's medical adherence. METHODS: We recruited participants from 9-year-old children who are attending the outpatient liver transplant clinic in a stable condition (no event such as rejection or surgical procedure within 6 months). We took participants to a snow camp and measured its effect by using the IKIRU CHIKARA (IKR) tool, which contain 28 items divided into 3 categories: psychosocial ability, moral fitness, and physical ability. Children were tested on three occasions, before, just after, and 1 month after the camp. RESULTS: Eight patients participated in the snow camp and 7 patients were eligible for the study. The average age was 12.6 with a range 10 to 17 years. There were 3 girls and 4 boys. The average IKR scores before, just after, and 1 month after the camp were 127.9, 131.5, and 126.6, respectively. CONCLUSION: An outdoor activity such as a snow camp can be safely conducted, and it is an acceptable option to incorporate within a pediatric liver transplant program. There were no significant changes in IKR scores during this short observation. Longer observation is needed to measure the effect of nature and outdoor activities.

Unique structure of murine interleukin-2 as deduced from cloned cDNAs.

Interleukin-2 (IL-2) is a lymphokine originally described as a humoral factor required for the continued proliferation of activated T-cell clones. It also seems to be involved in the mitogenic response of thymocytes, in augmenting natural killer cell activity, in the generation of cytotoxic T cells and in the induction of other lymphokines such as gamma-interferon and a B-cell growth factor (BCGF-1). More recently, there has been evidence for the involvement of IL-2 per se in the stimulation of B-cell growth (ref. 10 and T. Kishimoto and J. Vilcek, personal communications). We have reported previously the cloning and expression of a human IL-2 complementary DNA. The cDNA encodes biologically active IL-2 which would consist of 153 amino acids, including a signal sequence. Because so much of the work on IL-2 has been done in the human and mouse, we sought to obtain cDNA encoding murine IL-2, and we now report the cloning, expression and sequence analysis of murine IL-2 cDNAs. The longest cDNA insert encodes a polypeptide of 169 amino acids, containing unique repeats of a CAG sequence which would encode 12 consecutive glutamine residues within the active IL-2 molecule.

Cloning and expression of a novel variant of human interferon-gamma cDNA.

A cDNA library was prepared from the poly(A) mRNA isolated from human peripheral blood lymphocytes which were induced by combined treatment with phytohemagglutinin and a phorbol ester. Recombinant plasmids containing human interferon-gamma (HuIFN-gamma) cDNAs were identified by the oligonucleotide-hybridization method. Nucleotide sequence analysis showed that the nucleotide and amino-acid sequences of HuIFN-gamma cDNA in plasmid pIFN gamma-G4 differed from the published data at amino acid position 9 (CAA for glutamine versus AAA for lysine). The cDNA in plasmid pIFN gamma-G4 was expressed under control of the simian virus 40 early promoter in monkey COS cells and a biologically active HuIFN-gamma was secreted from the cells. The cDNA was also inserted into an expression vector carrying an E. coli tryptophan promoter and was expressed in E. coli. The results suggest that the conversion from lysine to glutamine at amino acid position 9 might not affect the specific activity of HuIFN-gamma.

Efficient expression in Escherichia coli of a mature and a modified human interferon-beta 1.

Ten recombinant plasmids were constructed which direct the synthesis of a mature human interferon-beta 1 (IFN-beta 1) under the control of the Escherichia coli tryptophan (trp) promoter. The spacing between the Shine-Dalgarno sequence of the trpL and the ATG initiation codon of the interferon gene was varied from 6 to 23 nucleotides by utilizing a Cla I site located within the spacer region of the plasmids. The optimal spacing for expression of IFN-beta 1 was determined to be 8-13 nucleotides from the results of interferon assay. The E. coli lipoprotein (lpp) promoter was also used for expression of IFN-beta 1 in E. coli. The results with an expression vector carrying a lpp-lac promoter showed that a modified IFN-beta 1 containing an additional 7 amino acids at the amino-terminus might be less active than the mature molecule.

Structure of the human interleukin 2 gene.

We have cloned two species of EcoRI-cleaved DNA segments that together cover the entire sequence for the human interleukin 2 gene and have determined the nucleotide sequence of the gene and its flanking regions. The gene contains three introns and the exon sequences can be aligned with the previously reported cDNA sequence almost perfectly except for a few nucleotides in the 3' nontranslated region. The promoter region contains a prototype "TATA" sequence as well as a notable palindromic sequence. Particularly interesting is the presence of sequences in this region that are homologous to the promoter region of the human interferon-gamma gene. In addition, a sequence that closely resembles the core sequence for the viral enhancer elements has been found in the second intron. Such sequences may play a role in the expression of the interleukin 2 gene in lectin- or antigen-stimulated T lymphocytes.

Structure and expression of a cloned cDNA for mouse interferon-beta.

A unique sequence in the mouse genome which cross-hybridized to a cloned human interferon-beta 1 gene was detected by DNA blot analysis. Taking advantage of this, a cDNA library prepared from partially purified mRNA for mouse interferon-beta was screened using human interferon-beta 1 DNA as a probe. One of the positive clones, pM beta-3, contained a 680-base pair cDNA insert, whose base sequence contained a single large open reading frame for 182 amino acids. The coding sequences of the cDNA showed homologies of 63% at the nucleotide and 48% at the amino acid level with respect to human interferon-beta 1 cDNA (Taniguchi, T., Ohno, S., Fujii-Kuriyama, Y., and Muramatsu, M. (1980) Gene 10, 11-15). The first 21 amino acids, considered to be the signal peptide, were followed by 24 amino acids, whose sequence was identical with the NH2-terminal sequence that had been reported for mouse interferon-beta from Ehrlich ascites tumor cells (Taira, H., Broeze, R. J., Jayaram, B. M., Lengyel, P., Hunkapiller, M. W., and Hood, L. E. (1980) Science (Wash. D.C.) 207, 528-530). The complete primary sequence of mature interferon-beta polypeptide consisting of 161 amino acids (Mr = 19,700) was deduced. There are three N-glycosylation sites, and this offers an explanation for the larger molecular size (Mr = 26,000-40,000) of natural mouse interferon-beta in comparison to the deduced interferon polypeptide. The cDNA, when fused to a SV40 promoter sequence and then introduced into COS-7 cells, directed the synthesis and secretion of a protein product indistinguishable from the authentic mouse interferon-beta.

Structure and expression of a cloned cDNA for human interleukin-2.

A cDNA coding for human interleukin-2 (IL-2) has been cloned from a cDNA library prepared from partially purified IL-2 mRNA. The DNA sequence codes for a polypeptide which consists of 153 amino acids including a putative signal sequence. A biologically active polypeptide, characteristic of human IL-2, was produced when the cDNA was fused to a simian virus 40 promoter sequence and used to transfect cultured monkey COS cells.

Construction and application of a novel plasmid "ATG vector" for direct expression of foreign genes in Escherichia coli.

A new type of plasmid expression vector was developed for direct expression of foreign genes in Escherichia coli. The plasmid vector, designated pTrS3, carries the E. coli tryptophan (trp) promoter and the Shine-Dalgarno (SD) sequence for the trp leader peptide as well as an ATG sequence located 13 bp downstream from the SD sequence. The dG residue of this ATG overlaps with the first dG residue of the single Sph I recognition sequence (GCATGC) of the vector DNA. After cleaving pTrS3 DNA by Sph I, the 3' protruding Sph I ends were converted into blunt ends using the Klenow fragment of E. coli DNA polymerase I. Subsequently, the DNA fragments coding for mature human interferon-beta or for the interferon lacking several aminoterminal amino acids, were ligated to this vector DNA and cloned in E. coli. Interferon activity was detected in the extracts of bacterial strains harboring the recombinant plasmids and the results indicated that the interferon-beta polypeptides without the five aminoterminal amino acids might be less active than the mature form.