Immunogenomic profiles associated with response to neoadjuvant chemoradiotherapy in patients with rectal cancer.

BACKGROUND: Accumulating evidence suggests that radiotherapy success has an immune-associated component. The immunogenomic profiles associated with responses to chemoradiotherapy (CRT) were assessed in patients with locally advanced rectal cancer in this study. METHODS: CD8+ tumour-infiltrating lymphocyte (TIL) and stromal lymphocyte densities were assessed by immunohistochemistry using pretreatment biopsies from patients with advanced rectal cancer who had preoperative CRT. Whole-exome sequencing and gene expression microarray analysis were conducted to investigate the genomic properties associated with the response to CRT and CD8+ TIL density. Response to CRT was determined based on Dworak tumour regression grade (TRG); tumours with complete (TRG 4) or near-complete (TRG 3) regression were grouped as good responders, and those with TRG 1 as non-responders. RESULTS: Immunohistochemical examinations (275 patients) showed that pre-CRT CD8+ TIL density was associated with better response to CRT and improved recurrence-free survival, whereas pre-CRT stromal CD8+ cell density was not associated with either response to CRT or recurrence-free survival. Whole-exome sequencing (74 patients) showed that the numbers of single-nucleotide variations (SNVs) and neoantigens predicted from SNVs were higher in good responders than in non-responders, and these correlated positively with CD8+ TIL density (rS = 0·315 and rS = 0·334 respectively). Gene expression microarray (90 patients) showed that CD8A expression correlated positively with the expression of programmed cell death 1 (PDCD1) (rS = 0·264) and lymphocyte-activation gene 3 (LAG3) (rS = 0·507). CONCLUSION: Pre-CRT neoantigen-specific CD8+ T cell priming may be a key event in CRT responses where immune checkpoint molecules could be useful targets to enhance tumour regression.

ANTECEDENTES: Las evidencias existentes sugieren que el éxito de la radioterapia tiene un componente asociado con el sistema inmunitario. En este estudio se evaluaron los perfiles inmunogenómicos asociados con la respuesta a la quimiorradioterapia (chemoradiotherapy, CRT) en pacientes con cáncer de recto localmente avanzado. MÉTODOS: Las densidades de los linfocitos infiltrantes de tumor CD8+ (tumour-infiltrating lymphocyte, TIL) y de los linfocitos del estroma se evaluaron por inmunohistoquímicas en las biopsias antes del tratamiento de pacientes con cáncer de recto localmente avanzado que recibieron CRT preoperatoria. Se realizó secuenciación de todo el exoma, así como microarrays de expresión génica, para investigar las propiedades genómicas asociadas con la respuesta a la CRT y a la densidad de los TIL CD8+. La respuesta a la CRT se determinó según el grado de regresión del tumor de Dworak (tumour regression grade, TRG), agrupándose como buenos respondedores los casos de regresión tumoral completa (TRG4) o casi completa (TRG3) y como no respondedores, los casos de grado TRG1. RESULTADOS: Los exámenes inmunohistoquímicos (n = 275) mostraron que la densidad pre-CRT de TIL CD8+ se asoció con una mejor respuesta a la CRT y una mejor supervivencia libre de recidiva, aunque la densidad de células CD8+ del estroma previa a la CRT no se asoció con la respuesta a la CRT ni con la supervivencia libre de recidiva. La secuenciación de todo el exoma (n = 74) mostró que el número de variaciones de nucleótidos únicos (single nucleotide variations, SNVs) y los neoantígenos predichos a partir de los SNVs fueron mayores en los que respondieron bien que en los que no respondieron, y éstos se correlacionaron positivamente con la densidad de los TIL CD8+ (Spearman r = 0,315 y r = 0,334 respectivamente). Los microarrays de expresión génica (n = 90) mostraron que la expresión CD8A se correlacionó positivamente con la expresión del ligando de muerte programada-1 (r = 0,264) y con el antígeno linfocitario del gen 3 (r = 0,507). CONCLUSIÓN: La activación de células T CD8+ específicas para neoantígenos previa a la CRT puede ser un evento clave en la respuesta a la misma donde las moléculas del punto de control inmunitario podrían ser dianas útiles para intensificar la regresión del tumor.

Reconstruction of transcription-translation dynamics with a model of gene networks.

A transcription-translation model of gene networks and a method to reconstruct it from gene expression data are proposed. The model is a hybrid system based on the Glass network with continuous-time dynamics and logical interactions. Transcription-translation dynamics is introduced into the Glass network. The reconstruction of gene networks is reduced to the problem of estimating logical functions from binary representations of quantities of mRNAs and proteins. The reconstruction method is applied to the gene expression data of circadian rhythms. The response characteristics of the reconstructed gene network to periodic stimuli are analyzed. The results suggest the existence of a receiver gene that responds to an external signal, consistently with biological knowledge.

cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS factor (Arnt2) with close sequence similarity to the aryl hydrocarbon receptor nuclear translocator (Arnt).

We isolated mouse cDNA clones (Arnt2) that are highly similar to but distinct from the aryl hydrocarbon receptor (AhR) nuclear translocator (Arnt). The composite cDNA covered a 2,443-bp sequence consisting of a putative 2,136-bp open reading frame encoding a polypeptide of 712 amino acids. The predicted Arnt2 polypeptide carries a characteristic basic helix-loop-helix (bHLH)/PAS motif in its N-terminal region with close similarity (81% identity) to that of mouse Arnt and has an overall sequence identity of 57% with Arnt. Biochemical properties and interaction of Arnt2 with other bHLH/PAS proteins were investigated by coimmunoprecipitation assays, gel mobility shift assays, and the yeast two-hybrid system. Arnt2 interacted with AhR and mouse Sim as efficiently as Arnt, and the Arnt2-AhR complex recognized and bound specifically the xenobiotic responsive element (XRE) sequence. Expression of Arnt2 successfully rescued XRE-driven reporter gene activity in the Arnt-defective c4 mutant of Hepa-1 cells. RNA blot analysis revealed that expression of Arnt2 mRNA was restricted to the brains and kidneys of adult mice, while Arnt mRNA was expressed ubiquitously. In addition, whole-mount in situ hybridization of 9.5-day mouse embryos showed that Arnt2 mRNA was expressed in the dorsal neural tube and branchial arch 1, while Arnt transcripts were detected broadly in various tissues of mesodermal and endodermal origins. These results suggest that Arnt2 may play different roles from Arnt both in adult mice and in developing embryos. Finally, sequence comparison of the currently known bHLH/PAS proteins indicates a division into two phylogenetic groups: the Arnt group, containing Arnt, Arnt2, and Per, and the AhR group, consisting of AhR, Sim, and Hif-1alpha.

Two new members of the murine Sim gene family are transcriptional repressors and show different expression patterns during mouse embryogenesis.

From a cDNA library of mouse skeletal muscle, we have isolated mouse Sim1 (mSim1) cDNA encoding a polypeptide of 765 amino acids with striking amino acid identify in basic helix-loop-helix (89% identify) and PAS (89 % identify) domains to previously identified mSim2, although the carboxy-terminal third of the molecule did not show any similarity to mSim2 or Drosophila Sim (dSim). Yeast two-hybrid analysis and coimmunoprecipitation experiments demonstrated that both of the mSim gene products interacted with Arnt even more efficiently than AhR, a natural partner of Arnt, suggesting a functional cooperativity with Arnt. In sharp contrast with dSim having transcriptional-enhancing activity in the carboxy-terminal region, the two mSims possessed a repressive activity toward Arnt in the heterodimer complex. This is the first example of bHLH-PAS proteins with transrepressor activity, although some genetic data suggest that dSim plays a repressive role in gene expression (Z. Chang, D. Price, S. Bockheim, M. J. Boedigheimer, R. Smith, and A. Laughon, Dev. Biol. 160:315-322, 1993; D. M. Mellerick and M. Nirenberg, Dev. Biol. 171:306-316, 1995). Whole-mount in situ hybridization showed restricted and characteristic expression patterns of the two mSim mRNAs in various tissues and organs during embryogenesis, such as those for the somite, the nephrogenic cord, and the mesencephalon (for mSim1) and those for the diencephalon, branchial arches, and limbs (for mSim2). From sequence similarity and chromosomal localization, it is concluded that mSim2 is an ortholog of hSim2, which is proposed to be a candidate gene responsible for Down's syndrome. The sites of mSim2 expression showed an overlap with the affected regions of the syndrome, further strengthening involvement of mSim2 in Down's syndrome.

Fine structures in denaturation curves of bacteriophage lambda DNA. Their relation to the intramolecular heterogeneity in base compositon.

Precise recording of polyphasic optical melting curves was carried out for three kinds of bacteriophage lambda DNA differing in length (lambdac1857s7, lambdacIb2 and lambdacIb2b5). Each of denaturation steps in melting profiles was characterized by two parameters, the melting temperature and the relative size. Any difference in fine structures in melting profiles was not recognized between the intact lambdacI857s7DNA and the DNA fragmented into halves. The change in fine structures in melting profiles caused by the deletions of the b2 and b5 region agreed qualitatively well with the prediction based on the physical and the genetical maps of phage lambda chromosome. The combined results indicate that, first, the well-known linear relationship between melting temperature and G+C content may apply also to each of denaturation steps in polyphasic melting curves due to heterogeneity of nucleotide distribution in a single DNA species, and, second, the effect of molecular ends on melting fine structures can be neglected at moderate salt concentration (0.01 M less than or equal to Na+ less than or equal to 0.2 M) for such a high molecular weight DNA. The heterogeneous distribution of nucleotides was derived for lambdaDNA and for its b2 and b5 regions.

Location of the cooperative melting regions in bacteriophage fd DNA.

Differential melting profiles of the linear replicative form (RF-III) DNA of bacteriophage fd, of the fragments obtained by the restriction endonuclease R.HinHI and of those obtained by R.Hga were investigated. With these results a physical map which locates the cooperative melting regions on the DNA was constructed, and compared with the genetic map.

Positioning of the A . T-rich regions in rat mitochondrial DNA by electron microscopy and analysis of the hysteresis of denaturation.

Intramolecular heterogeneity in the base composition of rat mitochondrial DNA (mtDNA) was shown by a combination of an improved denaturation mapping technique using electron microscopy and analysis of high-resolution optical melting-renaturation profiles. Circular mtDNA starts to melt in one specific region and then forms loops in four other regions in random order. These five early melting regions are all located in one half of the molecule. The arrangement of the early melting regions in rat mtDNA bears a remarkable resemblance not only to those of mtDNAs from several species of Drosophila but also to those of several species of Drosophila but also to those of several plasmid DNAs and phage DNA.

Membrane-bound transferrin-like protein (MTf): structure, evolution and selective expression during chondrogenic differentiation of mouse embryonic cells.

Mouse membrane-bound transferrin-like protein (MTf) cDNA was cloned to examine its expression during chondrogenic differentiation in the mouse embryonic cell line ATDC5, and to analyze the phylogenetic relationships among the MTfs of four animal species and 23 other transferrin members. Phylogenetic analysis indicated that the MTf gene diverged from the common ancestor gene earlier than the genes of the other transferrins such as serum transferrin, lactoferrin and ovotransferrin, and that the divergence occurred after the divergence of vertebrates and invertebrates. MTf, as well as the other transferrins, consists of two repeated domains. The similarity between the N-terminal and the C-terminal domains of MTf is much higher than that of the other transferrins, although the five amino acid residues required for iron binding were not conserved in the C-terminal domain of MTf in contrast to the conservation of these residues in both domains of the other transferrins. Among various adult mouse tissues, MTf mRNA was expressed at the highest level in cartilage and at a moderate level in the testis. MTf mRNA was expressed only at very low levels in the brain, spleen, thymus, muscle, lung, skin and intestine, and hardly detected in the heart, kidney, stomach and liver. In cultures of the mouse ATDC5 cell line, MTf is developmentally expressed in parallel with the expression of type II collagen and aggrecan, in the pattern commensurate with the onset of chondrogenesis to form cartilage nodules. The structural characteristics and the expression pattern suggest that during development and in adult tissues, MTf has some functions that are different from those of other transferrins.

Evolutionary aspects of variant types of rat mitochondrial DNA'S.

Mitochondrial DNA's (mtDNAs) were prepared from various kinds of individual Norway rats, Rattus norvegicus, and from three types of individual black rats, Rattus rattus, (Asian type, Ceylon type, and Oceanian type). Intra- and interspecies divergence of their mtDNA sequences were calculated based on changes in restriction endonuclease cleavage sites. The extent of intraspecies divergence of black rats (about 8%) is much larger than that of Norway rats (1%) and the mtDNA of Asian-type black rats resembles the mtDNA of Norway rats more closely than it resembles the mtDNA of other types of black rats. These results strongly suggest that during the course of intraspecies differentiation of black rats, probably long after the separation of the three types of black rats, some Asian-type black rats were isolated sexually and formed a new species, Norway rats. On the basis of our observations we propose a hypothetical process to explain the evolution of animal mtDNA.

Structural and phylogenetic analyses of RGD-CAP/beta ig-h3, a fasciclin-like adhesion protein expressed in chick chondrocytes.

A cDNA for RGD-CAP/beta ig-h3 was cloned from a chick embryo chondrocyte cDNA library. The deduced amino acid sequence showed that the chick RGD-CAP/beta ig-h3 is 76-77% identical with human, mouse and pig forms of the protein, and 43% identical with human and mouse osteoblast specific factor 2 (OSF2). RGD-CAP/beta ig-h3 contained four internal repeat domains and two highly conserved sequences (H1 and H2) in each repeat. Chick RGD-CAP/beta ig-h3, as well as the mammalian RGD-CAP/beta ig-h3, contained an RGD sequence, which may serve as a recognition sequence for integrins, in the fourth repeat. Database searches revealed that the H1 and H2 sequences are conserved in some secreted or membrane proteins of several species including mammals, insects, sea urchins, plants, yeast and bacteria. Phylogenetic analysis showed that a portion of the common ancestor gene for RGD-CAP/beta ig-h3 and OSF2 was duplicated to form four repeat domains before the separation of the genes followed by the divergence of vertebrate species.

Significant improvement in accuracy of multiple protein sequence alignments by iterative refinement as assessed by reference to structural alignments.

The relative performances of four strategies for aligning a large number of protein sequences were assessed by referring to corresponding structural alignments of 54 independent families. Multiple sequence alignment of a family was constructed by a given method from the sequences of known structures and their homologues, and the subset consisting of the sequences of known structures was extracted from the whole alignment and compared with the structural counterpart in a residue-to-residue fashion. Gap-opening and -extension penalties were optimized for each family and method. Each of the four multiple alignment methods gave significantly more accurate alignments than the conventional pairwise method. In addition, a clear difference in performance was detected among three of the four multiple alignment methods examined. The currently most popular progressive method ranked worst among the four, and the randomized iterative strategy that optimizes the sum-of-pairs score ranked next worst. The two best-performing strategies, one of which was newly developed, both pursue an optimal weighted sum-of-pairs score, where the pair weights were introduced to correct for uneven representations of subgroups in a family. The new method uses doubly nested iterations to make alignment, phylogenetic tree and pair weights mutually consistent. Most importantly, the improvement in accuracy of alignments obtained by these iterative methods over pairwise or progressive method tends to increase with decreasing average sequence identity, implying that iterative refinement is more effective for the generally difficult alignment of remotely related sequences. Four well-known amino acid substitution matrices were also tested in combination with the various methods. However, the effects of substitution matrices were found to be minor in the framework of multiple alignment, and the same order of relative performance of the alignment methods was observed with any of the matrices.

Evolutionary relationships among five subspecies of Mus musculus based on restriction enzyme cleavage patterns of mitochondrial DNA.

The intra- and intersubspecific genetic distances between five subspecies of Mus musculus were estimated from restriction enzyme cleavage patterns of maps of mitochondrial DNA (mtDNA). The European subspecies, M. m. domesticus and Asian subspecies, M. m. bactrianus, M. m. castaneus, M. m. molossinus and M. m. urbanus were examined. For each subspecies, except M. m. urbanus, at least two local races from widely separated localities were examined. Intrasubspecific heterogeneity was found in the mtDNA cleavage patterns of M. m. bactrianus and M. m. castaneus. M. m. molossinus and M. m. domesticus, however, revealed no intrasubspecific heterogeneity. Four of the subspecies had distinct cleavage patterns. The fifth, M. m. urbanus, had cleavage patterns identical to those of M. m. castaneus with several enzymes. Estimates of genetic distances between the various races and subspecies were obtained by comparing cleavage maps of the mtDNAs with various restriction enzymes. Nucleotide sequence divergences of mtDNA between local races were estimated to be less than 0.4% in M. m. bactrianus and less than 0.3% in M. m. castaneus. The times of divergence of both subspecies were calculated to be 0.1--0.2 x 10(6) years. These values suggest that the intrasubspecific divergence began some 0.1--0.2 x 10(6) years ago. On the other hand, nucleotide sequence divergences between European subspecies M. m. domesticus and Asian subspecies M. m. bactrianus and M. m. castaneus were 7.1% ane 5.8%, respectively. The times of divergence were calculated to be 2.1--2.6 x 10(6) years. Further, the nucleotide sequence divergence and time of divergence between M. m. molossinus and the other two Asian subspecies were comparable to those between M. m. molossinus and M. m. domesticus (about 3% and 1 x 10(6) years, respectively). These results suggest that M. m. molossinus is situated in a unique evolutionary position among Asian subspecies.

Association of CYP1B1 genetic polymorphism with incidence to breast and lung cancer.

Cytochrome P450 1B1 (CYP1B1) participates in the metabolic activation of a number of procarcinogens including benzo[a]pyrene and the hydroxylation of 17beta-estradiol at the C-4 position. In this study, we investigated the association between CYP1B1 genetic polymorphism and breast or lung cancer incidence. The Ala-Ser polymorphism at codon 119 in presumed substrate recognition site 1 was significantly associated with the incidence of breast or squamous cell carcinoma of the lung. On the other hand, Leu-Val polymorphism at codon 432 did not show any association to the cancers. An allele containing both Ala and Leu simultaneously, comprised 75% of alleles among 315 Japanese healthy controls, was significantly inversely associated with breast cancer incidence. When expressed in a recombinant system, this CYP1B1 cDNA showed the lowest 17beta-estradiol 4-hydroxylase activity among four different variant forms of CYP1B1. Thus, inter-individual differences in activation of procarcinogens or metabolism of oestrogen originating from genetic polymorphisms of the human CYP1B1 gene may contribute to the susceptibility of human cancers.

P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature.

We provide here a list of 481 P450 genes and 22 pseudogenes, plus all accession numbers that have been reported as of October 18, 1995. These genes have been described in 85 eukaryote (including vertebrates, invertebrates, fungi, and plants) and 20 prokaryote species. Of 74 gene families so far described, 14 families exist in all mammals examined to date. These 14 families comprise 26 mammalian subfamilies, of which 20 and 15 have been mapped in the human genome and the mouse genome, respectively. Each subfamily usually represents a cluster of tightly linked genes widely scattered throughout the genome, but there are exceptions. Interestingly, the CYP51 family has been found in mammals, filamentous fungi and yeast, and plants-attesting to the fact that this P450 gene family is very ancient. One functional CYP51 gene and two processed pseudogenes, which are the first examples of intronless pseudogenes within the P450 superfamily, have been mapped to three different human chromosomes. This revision supersedes the four previous updates in which a nomenclature system, based on divergent evolution of the superfamily, has been described. For the gene, we recommend that the italicized root symbol "CYP' for human ("Cyp' for mouse and Drosophila), representing "cytochrome P450', be followed by an Arabic number denoting the family, a letter designating the subfamily (when two or more exist), and an Arabic numeral representing the individual gene within the subfamily. A hyphen is no longer recommended in mouse gene nomenclature. "P' ("ps' in mouse and Drosophila) after the gene number denotes a pseudogene; "X' after the gene number means its use has been discontinued. If a gene is the sole member of a family, the subfamily letter and gene number would be helpful but need not be included. The human nomenclature system should be used for all species other than mouse and Drosophila. The cDNAs, mRNAs and enzymes in all species (including mouse) should include all capital letters, and without italics or hyphens. This nomenclature system is similar to that proposed in our previous updates.

Nimodipine and the haemodynamic and histopathological consequences of middle cerebral artery occlusion in the rat.

The effect of the administration of nimodipine (1 microgram kg-1 min-1), initiated 5 min after occlusion of a middle cerebral artery (MCA), upon cerebral haemodynamics ([14C]iodoantipyrine autoradiography) and neuropathological outcome (volume of histologically ischaemic tissue) was investigated in anaesthetized rats. Measurements were made of the level of local CBF (LCBF) in a total of 37 neuroanatomically defined areas, either ipsilateral or contralateral to the occluded vessel, and the autoradiograms were examined using a new approach to quantitative densitometry that employed a frequency distribution analysis of the CBF in sections of the brain at different coronal planes. Both methods of analysis showed that nimodipine, administered after the ischemic event, did not modify the pattern of CBF distribution after MCA occlusion. The extent of ischaemic brain damage was determined by histological examination. There was no evidence that the extent of ischaemic damage, either in sections at eight different coronal planes or in overall volume, was significantly different in animals that received nimodipine from that observed in animals that received only the vehicle used to dissolve the drug. The lack of cerebral circulatory and neuropathological alterations when nimodipine administration is initiated after occlusion of the MCA is contrasted with the higher levels of LCBF and the reductions in the volume of ischaemic tissue that were found when nimodipine was administered before occlusion of the artery.

Possible steroid binding site common to adrenal cytochrome P-450scc and prostatic steroid binding protein.

By searching the entire PIR-protein-sequence data base, we have found that a dodecapeptide sequence in bovine adrenal cytochrome P-450scc is closely related to that in rat prostatic steroid binding protein. The two proteins belong to unrelated protein families, but both have steroids as substrates or ligands. Thus, the dodecapeptides may be important for substrate/ligand recognition in the individual proteins.

The differences between the primary structures of mitochondrial DNAs from rat liver and ascites hepatoma (AH-130).

Rat mitochondrial DNAs (mtDNAs) of ascites hepatoma (AH-130) and normal liver cells (Donryu strain) were digested by various restriction endonucleases and the cleavage patterns compared by agarose gel electrophoresis. Different cleavage patterns were observed between AH-130 and liver mtDNAs when they were digested by HindII and EcoRI. The mtDNA of AH-130 lost one clevage site of HindII and one clevage site of EcoRI. The cleavage patterns of mtDNAs from other organs and strains tested were the same as that of liver mtDNA. From these observations we concluded that the molecular clone of AH-130 mtDNA was different from that of other mtDNAs.

Computer analysis of the sequence relationships among 4.5S RNA molecular species from various sources.

Nucleotide sequence homology among 4.5S RNAs from various organisms was examined by computer analysis to evaluate their sequence relationships. Chloroplast 4.5S rRNAs of wheat and tobacco were not significantly related to Escherichia coli 4.5S RNA, but were closely related to the 3'-terminus of bacterial 23S rRNA. Significant sequence homology was found between rat Novikoff hepatoma 4.5S RNAI and mouse and hamster 4.5S RNAs, suggesting that these RNAs are products of a family of genes with diverged sequences. E. coli 4.5S RNA had no significant sequence homology with any rodent 4.5S RNAs as a whole sequence. The E. coli, mouse and hamster 4.5S RNAs, however, were found to share a homologous 14-nucleotide sequence at the center of the molecules, which is known to exist as a conserved sequence in both Alu and Alu-equivalent sequences of mammalian DNAs.

Structure of the gene encoding the human differentiation-stimulating factor/leukemia inhibitory factor receptor.

The human gene encoding the differentiation-stimulating factor (D-factor)/leukemia inhibitory factor (LIF) receptor was cloned and its structure was analyzed. The gene spans more than 70 kilobases and contains 20 exons. The D-factor/LIF receptor can be subdivided into several regions; cytokine receptor homologous domain 1, an Ig-like domain, cytokine receptor homologous domain 2, three fibronectin type III domains, a transmembrane domain and a cytoplasmic region. Each domain of the receptor is encoded by a set of exons. There is a TATA sequence upstream of the transcription initiation site. One unit of the Alu sequence is present in the 5' flanking region. An NF-IL6 site is located 31 bases downstream of the transcription initiation site.

Salt-concentration dependence of thermal denaturation of restriction fragment DNAs from phi X174.

High-resolution differential melting curves of phi X174 Y1 and Y2 restriction fragment DNAs, for which the base sequences were known, were measured at various sodium ion concentrations ranging from 195 to 2.3 mM. The curves were resolved into component peaks, and the change in the melting temperature, the change in the area, and the change in the breadth of each peak with change in salt concentration were examined. The locations of the melting regions corresponding to the peaks in the melting curves were assigned based on theoretical calculations of melting curves and stability maps. It was found that as the salt concentration was decreased from the high to the intermediate range, the breadths of the peaks on the low-temperature side decreased whereas those on the high-temperature side remained almost constant, and also the separation between the peaks along the temperature axis increased. Changes in the positions of peaks relative to one another were interpreted in terms of the difference in the free energy increase between a loop state and an end-coil state as the salt concentration decreased.