To examine whether there is a circadian variation in skin blood flow response to passive heat stress and maximal skin blood flow, which was measured by local warming to 42 degrees C for 45 min, we studied six men at an ambient temperature of 28 degrees C at four different times of day [0400-0700 (morning), 1000-1300 (daytime), 1600-1900 (evening), and 2200-0100 hours (night)], each time of day being examined on separate days. Heat stress at rest was performed by immersing the legs below the knee in hot water (42 degrees C) for 60 min. The esophageal temperature (Tes) at rest was significantly higher in the evening than in the morning. The maximal skin blood flow (SkBFmax) on both sites, back and forearm, did not show a significant difference among the four times of day. The variation in Tes thresholds for cutaneous vasodilation to heat stress was similar to the circadian rhythm in resting Tes. The relationship of the percentage of SkBFmax (%SkBF) with Tes was significantly lower in the morning than in the evening. The results suggest that the maximal skin blood flow during local warming does not show variation over the day, but the sensitivity of vasodilation to passive heat stress shows a circadian variation.

Adult↗

PubMed1997

Expression of multiple alpha1-antitrypsin-like genes in hibernating species of the squirrel family.

In the chipmunk, a mammalian hibernator, a 140 kDa protein complex found in the blood, drastically decreases in concentration during hibernation. This complex contains four species of proteins, HP-20, -25, -27 and -55. In the present study, cDNA clones coding for the chipmunk HP-55 were isolated from a liver cDNA library. Sequence analysis revealed that HP-55 is produced as a precursor protein of 413 amino acids (aa), that it has a signal peptide of 24 aa, and that it contains four potential N-glycosylation sites. The deduced aa sequence shows 63% identity with that of rat alpha1-antitrypsin (alpha1-AT); however, the sequence corresponding to the reactive center P1-P1' residues was found to be Met-Leu, whereas it is Met-Ser in the rat alpha1-AT. During screening of the chipmunk liver cDNA library, four other related classes of cDNA clones were obtained, each also coding for an alpha1-AT-like protein. In spite of more than 86% overall aa sequence identity among the five chipmunk alpha1-AT-like proteins, they are highly divergent in the putative reactive center region; the putative P1-P1' sequences are Met-Leu (HP-55 or CM55-ML), Met-Met (CM55-MM), Met-Ser (CM55-MS), Ser-Ile (CM55-SI) and Ser-Thr (CM55-ST). Each of the alpha1-AT-like protein mRNAs was expressed in chipmunk liver, and the HP-55 mRNA level was greatly reduced during hibernation. Genomic Southern blot analysis and screening of a liver cDNA library from another hibernating squirrel species, the ground squirrel, also revealed expression of multiple members of the alpha1-AT gene family, whereas analysis of a cDNA library from a non-hibernating species, the tree squirrel, found only a single alpha1-AT gene.

Amino Acid Sequence↗

PubMed1997

BLM (the causative gene of Bloom syndrome) protein translocation into the nucleus by a nuclear localization signal.

Bloom syndrome (BS) is a rare genetic disorder characterized by small body size, sun sensitivity, immunodeficiency and a high predisposition to various types of cancer. BLM was identified as the causative gene for BS, and BLM protein is homologous to DNA helicase. There are two putative nuclear localization signals (NLSs) within amino acid residues 1334-1349 in the C-terminus of the BLM protein, which has the distinctive structure of two basic residue arms separated by a spacer. The entire coding or deleted BLM sequences of various sizes were ligated into an enhanced green fluorescent protein (EGFP) vector and transfected into HeLa cells. The EGFP vector harboring the entire BLM coding sequence was transported to the nucleus. The BLM protein truncated at 1341 amino acid, containing an intact helicase domain and only one proximal arm, was not transported to the nucleus. The BLM protein truncated at 1357 amino acid, containing an intact helicase domain and two arms, was transported to the nucleus. The EGFP vector harboring DNA fragments encoding a protein having only the distal arms of basic amino acids in the C-terminus was also transported to the nucleus. The truncated BLM proteins corresponding to previously reported mutated BLM proteins were retained in the cytoplasm or both the cytoplasm and the nucleus as was the EGFP vector with no insert. These results show that the BLM protein translocates into the nucleus and that the distal arm of the bipartite basic residues in the C-terminus of the BLM protein is essential for targeting the nucleus.

Adenosine Triphosphatases↗

PubMed1997

Mass screening test for mucopolysaccharidoses using the 1,9-dimethylmethylene blue method: positive interference from paper diapers.

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Glycosaminoglycans↗

PubMed1997

An alternative form of nucleolysin binds to a T-cluster DNA in the silencer element of platelet factor 4 gene.

The cDNA of a T-cluster binding protein (TCBP) has been cloned using the Southwestern method. The cDNA sequence of TCBP reveals that it has 78% homology to that of nucleolysin, a factor involved in apoptosis in cytolytic lymphocyte target cells. In particular, the 0.8kb sequences of the 5'-half of both cDNAs were identical. However, nucleolysin has a lysosome-targeting motif at the carboxy terminus, while TCBP has a hydrophobic sequence instead. Southern blot experiments have revealed that both cDNA sequences existed in the same YAC clone derived from chromosome 10. This strongly suggests that the TCBP cDNA is an alternatively spliced product of the nucleolysin/TCBP gene. The TCBP mRNA is ubiquitously expressed, not only in megakaryocytic cells but also in other hematopoietic cells. However, when HEL cells were induced to differentiate to megakaryocytes by DMSO, TCBP mRNA was reduced, while PF4 gene expression increased simultaneously. Gel mobility shift analysis demonstrated that recombinant TCBP specifically bound to the T-cluster and the proximal T-rich region of the PF4 promoter. Co-transfection experiments showed that TCBP reduced the gene expression from the PF4 promoter. On the other hand, TCBP did not affect expression from the PF4 promoter in which the T-cluster and the T-rich region had been removed. These results indicate that TCBP may participate in the regulation of PF4 gene expression by binding to the T-cluster and the T-rich sequence.

Amino Acid Sequence↗

PubMed1997

Succinyl-CoA:3-ketoacid coenzyme A transferase (SCOT): development of an antibody to human SCOT and diagnostic use in hereditary SCOT deficiency.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) is a key enzyme for ketone body utilization. Hereditary SCOT deficiency in humans (McKusick catalogue number 245050) is characterized by intermittent ketoacidotic attacks and permanent hyperketonemia. Since previously-available antibody to rat SCOT did not crossreact with human SCOT, we developed an antibody against recombinant human SCOT expressed in a bacterial system. The recombinant SCOT was insoluble except under denaturing conditions. Antibody raised to this polypeptide recognized denatured SCOT and proved useful for immunoblot analysis. On immunoblots, SCOT was easily detectable in control fibroblasts and lymphocytes but was detected neither in fibroblast extracts from four SCOT-deficient patients, nor in lymphocytes from two SCOT-deficient patients. These data indicate that immunoblot analysis is useful for diagnosis of SCOT deficiency in combination with enzyme assay.

Animals↗

PubMed1997

Studies on selectin blockers. 4. Structure-function relationships of sulfated sialyl Lewis X hexasaccharide ceramides toward E-, P-, and L-selectin binding.

In order to clarify the real ligand structure of L-selectin proposed by Rosen et al., we first synthesized 6-sulfated sLe(x) hexasaccharide ceramide 1, 6'-sulfated sLe(x) hexasaccharide ceramide 2, and 6,6'-disulfated sLe(x) hexasaccharide ceramide 3 and examined their binding avidities for L-selectin. As a result, we found that the 6'-sulfated sLe(x) hexasaccharide ceramides 1-3 have similar binding avidities to L-selectin and their binding to L-selectin appeared somewhat stronger than that of sLe(x). For P-selectin, the sulfated sLe(x) derivatives 1-3 showed a similar avidity to sLe(x). On the other hand, 6-sulfated sLe(x) 2 was recognized to E-selectin and the binding avidity was apparently weak as compared to that of sLe(x) hexasaccharide ceramide. Surprisingly, 6'-sulfated and 6,6'-disulfated sLe(x)s 1 and 3 did not bind to E-selectin at all. We constructed the E-selectin-sLe(x) complex model and investigated the binding mode. Namely, the galactose 6'-position was directed toward the negatively charged residues, Glu80 and Asp100. Our results with E-selectin indicate that the replacement of 6'-OH position from anionic charged group to cationic charged one, e.g., amino groups, could have a marked affect on E-selectin recognition. These results could provide useful information for the drug design of selectin blockers.

Animals↗

PubMed1997

Selectin-ligand interactions revealed by molecular dynamics simulation in solution.

Through a computer modeling and simulation technique, we investigated the binding mode of a complex of E-selectin-GSC-150, which is a novel selectin blocker. GSC-150 is the 3'-sulfated Lewis X derivative with a long, branched alkyl chain. Initial attempts to construct a model for E-selectin-GSC-150 complex were performed based on a previously reported model of E-selectin-sialyl Lewis X (sLex) complex [Kogan, T.P.; Revelle, B.M.; Tapp, S.; Scott, D.; Beck, P.J.J. Biol. Chem. 1995, 270, 14047-14055]. In our model, the carbohydrate portion of GSC-150 interacted with the protein in a similar manner as that of sLex reported previously. Interestingly, each of the branched alkyl chains extended on the surface of E-selectin and interacted with two different hydrophobic portions. One of these hydrophobic portions consists of Tyr44, Pro46, and Tyr48. Another portion forms a shallow cavity, and it consists of Ala9, Leu114, and the alkyl moieties of the side chains of Lys111, Lys112, and Lys113. A subsequent 200-ps molecular dynamics simulation in solution revealed that the interactions involved in the sugar portion of the ligand were relatively weak, whereas the hydrophobic interactions involved in the branched alkyl chains were fairly stable in solution. These results suggest that the branched alkyl chain serves as an "anchor" for the tight binding of GSC-150 on the surface of E-Selectin. This is the first attempt to evaluate the dynamics of E-Selectin-ligand interactions in solution, and it sheds light on the nature of ligand recognition by selectins.

Carbohydrate Sequence↗

PubMed1997

Isolation of a new peroxisome-deficient CHO cell mutant defective in peroxisome targeting signal-1 receptor.

For the study of mechanism of peroxisome biogenesis, we attempted to isolate CHO cell mutants deficient in peroxisome biogenesis. We used as the parent strain a stable CHO transformant of rat PEX2 (formerly named peroxisome assembly factor-1) cDNA, to avoid unusually frequent isolation of Pex2 mutants. Among the three peroxisome-deficient mutants obtained, ZP102 was a new CHO mutant of complementation group 2, and was restored for peroxisome assembly by the transfection of human PEX5 (formerly called PXR1 or PTS1R) cDNA. This approach would facilitate the isolation of new complementation gorups of peroxisome-deficient CHO mutants and the identification of essential genes for peroxisome biogenesis.

Animals↗

PubMed1997

Hunter disease in a girl caused by R468Q mutation in the iduronate-2-sulfatase gene and skewed inactivation of the X chromosome carrying the normal allele.

Hunter disease is an X-linked recessive mucopolysaccharide storage disorder caused by iduronate-2-sulfatase deficiency and is rare in females. We describe here findings in a girl with Hunter disease of the severe type. She had a normal karyotype but a marked deficiency of iduronate-2-sulfatase activity in lymphocytes and cultured fibroblasts. In a sequence analysis of the iduronate-2-sulfatase gene, evidence was obtained for the R468Q (G1403 to A) mutation, a common one in Hunter disease. RT-PCR showed her cDNA to represent only the R468Q allele, although at the genomic level she was a heterozygote with one normal allele. Her brother had the R468Q mutation, and their mother was a carrier of this mutation. The fusion products of CHO (TG(R),Neo(R)) with patient's fibroblasts cultured in HAT/G418 selective medium, carried only the maternal allele. However, in genomic DNA from the patient's fibroblasts, only the paternal allele of the androgen receptor gene, a gene subjected to differential methylation of the inactive X-chromosome, was methylated. These findings strongly suggest that the severe form of Hunter disease in this girl was the result of selective expression of the maternal allele carrying the missense mutation R468Q, which in turn resulted from skewed X inactivation of the paternal nonmutant X chromosome.

Alleles↗

PubMed1997

Fourteen novel mucopolysaccharidosis IVA producing mutations in GALNS gene.

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by a deficiency of the lysosomal N-acetylgalactosamine-6-sulfate sulfatase. Here, we report our analysis of data on 21 patients of diverse ethnic and geographic origins studied by SSCP and sequencing analysis. Sixteen mutations were detected, including 14 new mutations (11 missense, one premature termination, one splice site alteration, and one cryptic site alteration). The donor splice site mutation (IVS4 + 1G-->A) predicts that normal splicing will be abolished and that translation would lead to an immediate premature termination (W141X). Another novel nucleotide change outside the coding sequence is an intronic alteration (IVS9-42C-->T:ggtcggtgcggttggtgc) creating a potential cryptic donor site. The nucleotide sequence surrounding this alteration is highly suggestive of a consensus donor splice site. All 12 missense and nonsense mutations were shown by transient expression to abolish or greatly reduce GALNS activity, thereby providing an explanation as to why they produce MPS IVA. All mutations were readily confirmed by restriction enzyme or by allelic specific oligonucleotide analysis (ASO). These findings, coupled with previously reported mutations, bring the total of different mutations to 41 among independent families with MPS IVA, illustrating the extensive allelic heterogeneity among mutations producing MPS IVA.

Chondroitinsulfatases↗