Development of pro-apoptotic peptides as potential therapy for peritoneal endometriosis.

Endometriosis is a common gynaecological disease associated with pelvic pain and infertility. Current treatments include oral contraceptives combined with nonsteroidal anti-inflammatory drugs or surgery to remove lesions, all of which provide a temporary but not complete cure. Here we identify an endometriosis-targeting peptide that is internalized by cells, designated z13, using phage display. As most endometriosis occurs on organ surfaces facing the peritoneum, we subtracted a phage display library with female mouse peritoneum tissue and selected phage clones by binding to human endometrial epithelial cells. Proteomics analysis revealed the z13 receptor as the cyclic nucleotide-gated channel ß3, a sorting pathway protein. We then linked z13 with an apoptosis-inducing peptide and with an endosome-escaping peptide. When these peptides were co-administered into the peritoneum of baboons with endometriosis, cells in lesions selectively underwent apoptosis with no effect on neighbouring organs. Thus, this study presents a strategy that could be useful to treat peritoneal endometriosis in humans.

Virus activation and immune function during intense training in rugby football players.

Epidemiological studies suggest that highly trained athletes are more susceptible to upper respiratory tract infections (URTI) compared with the general population. Upper respiratory symptoms (URS) often appear as either primary invasion of pathogenic organisms and/or reactivation of latent viruses such as Epstein-Barr virus (EBV). The purpose of this study was to examine the relationship between EBV reactivation and the appearance of URS during intensive training in collegiate rugby football players. We evaluated EBV-DNA expression in saliva and examined the relationship between onset of URS and daily changes in EBV-DNA as well as secretory immunoglobulin A (SIgA) levels among 32 male collegiate rugby football players during a 1-month training camp. The EBV-DNA expression tended to be higher in subjects who exhibited sore throat (p=0.07) and cough (p=0.18) than that of those who had no symptoms, although their differences were not significant. The SIgA level was significantly lower 1 day before the EBV-DNA expression (p<0.05). The number of URS increased along with the EBV-DNA expression and decrease of SIgA levels. These results suggest that the appearance of URS is associated with reactivation of EBV and reduction of SIgA during training.

Tryptophan aspartate-containing coat protein (CORO1A) suppresses Toll-like receptor signalling in Mycobacterium leprae infection.

Mycobacterium leprae is an intracellular pathogen that survives within the phagosome of host macrophages. Several host factors are involved in producing tolerance, while others are responsible for killing the mycobacterium. Tryptophan aspartate-containing coat protein (TACO; also known as CORO1A or coronin-1) inhibits the phagosome maturation that allows intracellular parasitization. In addition, the Toll-like receptor (TLR) activates the innate immune response. Both CORO1A and TLR-2 co-localize on the phagosomal membrane in the dermal lesions of patients with lepromatous leprosy. Therefore, we hypothesized that CORO1A and TLR-2 might interact functionally. This hypothesis was tested by investigating the effect of CORO1A in TLR-2-mediated signalling and, inversely, the effect of TLR-2-mediated signalling on CORO1A expression. We found that CORO1A suppresses TLR-mediated signal activation in human macrophages, and that TLR2-mediated activation of the innate immune response resulted in suppression of CORO1A expression. However, M. leprae infection inhibited the TLR-2-mediated CORO1A suppression and nuclear factor-kappaB activation. These results suggest that the balance between TLR-2-mediated signalling and CORO1A expression will be key in determining the fate of M. leprae following infection.

A rat model of saliva secretory immunoglobulin: a suppression caused by intense exercise.

We aimed to develop a valid model of immunosuppression induced by intense exercise in rats. Rats were divided into three groups. In the rest (Rest) group, saliva was collected from resting rats on 4 consecutive days. In the exercise (Ex) group, rats ran on a treadmill until exhaustion (exercise time: 60.0 +/- 3.7 min), and their saliva was collected before and after exercise; the salivary glands were removed after exercise. In the control (Con) group, saliva collection and gland removal were also performed, but the rats did not exercise. Secretory immunoglobulin A (SIgA) concentrations in saliva and polymeric immunoglobulin receptor (pIgR) mRNA expression in the glands were measured. There was no significant change in SIgA concentration in the Rest group over 4 days. In the Ex group, SIgA concentration decreased significantly after exercise compared with before, whereas there was no significant change in the Con group. The expression of pIgR mRNA was significantly lower in the Ex group post-exercise than in the Con group. Our procedure for saliva collection appeared suitable, and the exercise-induced SIgA suppression was probably caused by a decline in pIgR mRNA expression. We propose to use this reproducible and reliable rat model of exercise-induced SIgA suppression in future studies.

The in vivo role of alpha-mannosidase IIx and its role in processing of N-glycans in spermatogenesis.

The surfaces of mammalian cells are covered by a variety of carbohydrates linked to proteins and lipids. N-glycans are commonly found carbohydrates in plasma membrane proteins. The structure and biosynthetic pathway of N-glycans have been analyzed extensively. However, functional analysis of cell surface N-glycans is just under way with recent studies of targeted disruption of genes involved in N-glycan synthesis. This review briefly introduces the potential role of processing alpha-mannosidases in N-glycan biosynthesis and recent findings derived from the alpha-mannosidase IIx (MX) gene knockout mouse, which shows male infertility. Thus, the MX gene knockout experiment unveiled a novel function of specific N-glycan, which is N-acetylglucosamine-terminated and fucosylated triantennary structure, in the adhesion between germ cells and Sertoli cells. Analysis of the MX gene knockout mouse is a good example of a multidisciplinary approach leading to a novel discovery in the emerging field of glycobiology.

Effects of 12 months of exercise training on salivary secretory IgA levels in elderly subjects.

BACKGROUND: The immune system declines in efficiency with advancing age, making the elderly less resistant to pathogenic microorganisms. Upper respiratory tract infection (URTI) is a common illness. Recent studies have shown that suppression of secretory immunoglobulin A (SIgA) is associated with increased incidence of URTI. OBJECTIVE: To assess the effect of exercise on salivary SIgA in elderly subjects. METHODS: Forty five elderly subjects (18 men, 27 women; mean (SD) age 64.9 (8.4) years) performed both 60 minute resistance and 60 minute moderate endurance training a week for 12 months. Saliva samples were obtained before training, and at four and 12 months during the training period. Salivary SIgA concentrations were measured by enzyme linked immunosorbent assay, and the SIgA secretion rate was calculated. RESULTS: SIgA concentrations before training, and at four and 12 months during training were 24.7 (14.4), 27.2 (14.2), and 33.8 (18.5) micro g/ml respectively. SIgA secretion rates were 29.5 (26.0), 33.8 (27.2) and 46.5 (35.1) micro g/min respectively. The results indicate that both the concentration and secretion rate of SIgA significantly (p<0.01) increased during 12 months of exercise in these elderly subjects. CONCLUSION: Regular moderate exercise seems to enhance mucosal immune function in elderly subjects.

The role of N-glycans in spermatogenesis.

Many proteins, in particular those in the plasma membranes, are glycosylated with carbohydrates, which are grouped into O-glycans and N-glycans. O-glycans are synthesized step by step by glycosyltransferases, whereas N-glycans are synthesized by en-bloc transfer of the so-called high-mannose-type oligosaccharide from lipid-linked precursor to polypeptide. The high-mannose-type N-glycans are then modified by processing alpha-mannosidases. Alpha-mannosidase IIx (MX) was identified as the gene product of processing alpha-mannosidase II (MII)-related gene. MX apparently plays subsidiary role for MII in many cell types, as N-glycan patterns of MX null mouse tissues are not altered significantly. Surprisingly MX null male mice are infertile due to a failure of spermatogenesis. This review provides a brief overview of the in vivo role of N-glycans which are revealed by the gene knockout mouse approach, and introduce our studies on the MX gene knockout mouse. The MX gene knockout experiments unveiled a novel function of a specific N-glycan, which is N-acetylglucosamine-terminated and has a fucosylated triantennary structure, in the adhesion between germ cells and Sertoli cells. The study of MX is a good example of how the in vivo roles of an apparently redundant gene product are determined by the gene knockout approach.

The trophinin gene encodes a novel group of MAGE proteins, magphinins, and regulates cell proliferation during gametogenesis in the mouse.

Trophinin is a membrane protein that mediates apical cell adhesion between trophoblastic cells and luminal epithelial cells of the endometrium and is implicated in the initial attachment during the process of human embryo implantation. The present study identified novel trophinin gene transcripts, which encode proteins structurally distinct from trophinin protein in the mouse. We designated these proteins "magphinins," because they share consensus amino acid sequences with MAGE (melanoma-associated antigen) superfamily proteins. Among many MAGE proteins, magphinins are closely related to NRAGE, which mediates p75 neurotrophin receptor-dependent apoptosis, and necdin, which is a strong suppressor of cell proliferation in post-mitotic neurons. There are three major forms of magphinins, i.e. magphinin-alpha, -beta, and -gamma, in the mouse, which are formed due to alternative usage of different exons. Northern blot analysis revealed that magphinins are expressed in brain, ovary, testis, and epididymis. In addition, Western blot analysis and in vitro translation experiments showed that magphinins expressed in the mouse ovary and testis are translation products utilizing the second initiation AUG codon and contain an active nuclear localization signal. Ectopic expression of magphinins in mammalian cells resulted in nuclear localization of magphinin and suppressed cell proliferation. Immunohistochemistry of the mouse ovary and testis showed that magphinin proteins are distributed in the cytoplasm of the male and female germ cells, whereas these proteins are translocated to the nucleus at a specific stage of gametogenesis. These results strongly suggest that magphinins regulate cell proliferation during gametogenesis in the mouse.

Human ITCH is a coregulator of the hematopoietic transcription factor NF-E2.

We have cloned a new protein that interacts with the hematopoietic DNA-binding transcription factor, p45/NF-E2, by screening a human erythroleukemia cell cDNA library with the yeast two-hybrid approach. Predicted peptide sequence and chromosomal mapping identified the cloned molecule to be the product of the human ortholog of the mouse Itch gene, which has been implicated previously in the regulation of growth and differentiation of erythroid and lymphoid cells. Transfection experiments indicate that this human ITCH protein can act as a transcriptional corepressor of p45/NF-E2. Our data provide novel insights into the functional roles of the mammalian ITCH proteins in the development of hematopoietic cell lineages.

Overexpression of the Golgi-localized enzyme alpha-mannosidase IIx in Chinese hamster ovary cells results in the conversion of hexamannosyl-N-acetylchitobiose to tetramannosyl-N-acetylchitobiose in the N-glycan-processing pathway.

Golgi alpha-mannosidase II is an enzyme that processes the intermediate oligosaccharide Gn(1)M(5)Gn(2) to Gn(1)M(3)Gn(2) during biosynthesis of N-glycans. Previously, we isolated a cDNA encoding a protein homologous to alpha-mannosidase II and designated it alpha-mannosidase IIx. Here, we show by immunocytochemistry that alpha-mannosidase IIx resides in the Golgi in HeLa cells. When coexpressed with alpha-mannosidase II, alpha-mannosidase IIx colocalizes with alpha-mannosidase II in COS cells. A protein A fusion of the catalytic domain of alpha-mannosidase IIx hydrolyzes a synthetic substrate, 4-umbelliferyl-alpha-D-mannoside, and this activity is inhibited by swainsonine. [(3)H]glucosamine-labeled Chinese hamster ovary cells overexpressing alpha-mannosidase IIx show a reduction of M(6)Gn(2) and an accumulation of M(4)Gn(2). Structural analysis identified M(4)Gn(2) to be Man alpha 1-->6(Man alpha 1-->2Man alpha 1-->3)Man beta 1-->4GlcNAc beta 1-->4GlcNAc. The results suggest that alpha-mannosidase IIx hydrolyzes two peripheral Man alpha 1-->6 and Man alpha 1-->3 residues from [(Man alpha 1-->6)(Man alpha 1-->3)Man alpha 1-->6](Man alpha 1-->2Man alpha 1-->3)Man beta 1-->4GlcNAc beta 1-->4GlcNAc, during N-glycan processing.

Human corneal GlcNac 6-O-sulfotransferase and mouse intestinal GlcNac 6-O-sulfotransferase both produce keratan sulfate.

Human corneal N-acetylglucosamine 6-O-sulfotransferase (hCGn6ST) has been identified by the positional candidate approach as the gene responsible for macular corneal dystrophy (MCD). Because of its high homology to carbohydrate sulfotransferases and the presence of mutations of this gene in MCD patients who lack sulfated keratan sulfate in the cornea and serum, hCGn6ST protein is thought to be a sulfotransferase that catalyzes sulfation of GlcNAc in keratan sulfate. In this report, we analyzed the enzymatic activity of hCGn6ST by expressing it in cultured cells. A lysate prepared from HeLa cells transfected with an intact form of hCGn6ST cDNA or culture medium from cells transfected with a secreted form of hCGn6ST cDNA showed an activity of transferring sulfate to C-6 of GlcNAc of synthetic oligosaccharide substrates in vitro. When hCGn6ST was expressed together with human keratan sulfate Gal-6-sulfotransferase (hKSG6ST), HeLa cells produced highly sulfated carbohydrate detected by an anti-keratan sulfate antibody 5D4. These results indicate that hCGn6ST transfers sulfate to C-6 of GlcNAc in keratan sulfate. Amino acid substitutions in hCGn6ST identical to changes resulting from missense mutations found in MCD patients abolished enzymatic activity. Moreover, mouse intestinal GlcNAc 6-O-sulfotransferase had the same activity as hCGn6ST. This observation suggests that mouse intestinal GlcNAc 6-O-sulfotransferase is the orthologue of hCGn6ST and functions as a sulfotransferase to produce keratan sulfate in the cornea.

Antiprothrombin autoantibodies in severe preeclampsia and abortion.

PURPOSE: We examined the levels of autoantibodies against prethrombin-1 and fragment-1 in pregnant women to determine the type of autoantibodies that were associated with severe preeclampsia or spontaneous abortion. SUBJECTS AND METHODS: We measured autoantibodies bound to prothrombin, prethrombin-1, and fragment-1 by using an enzyme-linked immunosorbent assay (ELISA) in 12 healthy nonpregnant women, 36 women with normal pregnancies, 28 pregnant women with severe preeclampsia, and 19 pregnant women who subsequently had spontaneous abortion. RESULTS: Plasma samples in 10 (36%) of the 28 women with severe preeclampsia and 11 (58%) of the 19 women with spontaneous abortion were positive for antiprothrombin antibodies as compared with 3 (9%) of the 36 women with normal pregancies. All 11 of the positive samples from women who had spontaneous abortions were positive for antiprethrombin-1 antibody, but only 1 was positive for antifragment-1 antibody. The mean (+/- SD) titer of antiprethrombin-1 antibodies in patients with spontaneous abortion (36 +/- 9 U) was higher than that in women with normal pregnancies (10 +/- 4 U; P < 0.01). Antiprethrombin-1 antibody was detected in only 2 women with severe preeclampsia, whereas all 10 women with antiprethrombin antibodies were positive for antifragment-1 antibody. The antifragment-1 antibody titer in patients with severe preeclampsia (49 +/- 15 U) was higher than in women with normal pregnancies (13 +/- 6 U, P < 0.01). CONCLUSIONS: There is a strong and specific association between various types of antiprothrombin antibodies with severe preeclampsia and spontaneous abortion.

Macular corneal dystrophy type I and type II are caused by distinct mutations in a new sulphotransferase gene.

Macular corneal dystrophy (MCD; MIM 217800) is an autosomal recessive hereditary disease in which progressive punctate opacities in the cornea result in bilateral loss of vision, eventually necessitating corneal transplantation. MCD is classified into two subtypes, type I and type II, defined by the respective absence and presence of sulphated keratan sulphate in the patient serum, although both types have clinically indistinguishable phenotypes. The gene responsible for MCD type I has been mapped to chromosome 16q22, and that responsible for MCD type II may involve the same locus. Here we identify a new carbohydrate sulphotransferase gene (CHST6), encoding an enzyme designated corneal N-acetylglucosamine-6-sulphotransferase (C-GlcNAc6ST), within the critical region of MCD type I. In MCD type I, we identified several mutations that may lead to inactivation of C-GlcNAc6ST within the coding region of CHST6. In MCD type II, we found large deletions and/or replacements caused by homologous recombination in the upstream region of CHST6. In situ hybridization analysis did not detect CHST6 transcripts in corneal epithelium in an MCD type II patient, suggesting that the mutations found in type II lead to loss of cornea-specific expression of CHST6.

Palmar fasciitis and polyarthritis associated with gastric carcinoma: complete resolution after total gastrectomy.

Palmar fasciitis and polyarthritis (PFA) is a rare paraneoplastic rheumatic syndrome characterized by flexion contractures of both hands and thickening of palmar fascia. Several reports have suggested that this syndrome is a tumor-associated autoimmune disorder. We report a 44-year-old Japanese man who presented with flexion contractures of both hands associated with thickening of palmar fascia and polyarthritis. These clinical pictures were suggestive of PFA associated with occult neoplasm. Upper gastrointestinal endoscopic examination revealed advanced gastric cancer. Resection of the cancer resulted in a gradual resolution of palmar fasciitis and polyarthritis. This clinical course suggests an underlying tumor-related immunologic process in this syndrome.

A genetic linkage map of the MSM Japanese wild mouse strain with restriction landmark genomic scanning (RLGS).

A high-resolution genetic map of the Mus musculus molossinus (MSM) Japanese wild mouse strain was constructed with restriction landmark genomic scanning (RLGS) and compared with that of the laboratory strain C3H. MSM is phylogenetically 1 million years apart from common laboratory mouse strains and is distinctly resistant to chemical carcinogenesis. Since it exhibits frequent genetic polymorphisms with laboratory mice but can still be easily crossed with laboratory strains, hybrids between MSM and carcinogen-sensitive laboratory mouse strains provide excellent materials for analysis of modifier genes and genetic changes during carcinogenesis. We have generated MSM backcross progeny with the C3H strain, which is extremely sensitive to hepatocarcinogenesis, to construct the present map. RLGS profiles with two combinations of restriction enzymes (NotI-PvuII-PstI, NotI-PstI-PvuII) yielded more than 2000 spots each. The polymorphism rate was about 39.2%, and of a total of 1732 polymorphic spot loci identified, 1371 could be assigned to specific chromosomes by comparison with 79 microsatellite marker loci. Thus, 1450 loci, on all chromosomes except for Y, effectively mapped 90% of the genome (1431.7 cM length). Although some spots might be derived from the same NotI site, each NotI site potentially generating two fragments, the presence of at least 515 loci groups with different progeny distribution patterns dispersed through the genome with an average spacing of 3 cM, means that this genetic map should be useful for analysis of various biological phenomena, including carcinogenesis and ontogenesis, at the gene level.

The relationship between serum immunoglobulin levels and pulmonary involvement in systemic sclerosis.

OBJECTIVE: To examine the relationship between serum immunoglobulin (Ig) levels and pulmonary function in patients with systemic sclerosis (SSc). METHODS: Twenty-four patients with SSc who had at least 2 sets of pulmonary function tests (PFT) at intervals of more than one year were eligible. Multiple linear regression models were constructed for prediction of the annualized rates of change of forced vital capacity (FVC), carbon monoxide diffusing capacity (DL(CO)), and DL(CO) per unit alveolar volume (K(CO)). RESULTS: The rates of change of FVC and K(CO) correlated with the annualized rate of change of IgG (p < 0.001 and p = 0.005, respectively), and the rate of change of DL(CO) correlated with the serum IgM level at the first PFT (p = 0.020) and with the annualized rate of change of IgG (p = 0.007). CONCLUSION: The rates of change of serum Ig levels are associated with those of pulmonary function in SSc. Use of this model may assist investigation of pulmonary involvement.

Molecular cloning and expression of two distinct human chondroitin 4-O-sulfotransferases that belong to the HNK-1 sulfotransferase gene family.

Using an expression cloning strategy, the cDNA encoding the human HNK-1 sulfotransferase (HNK-1ST) has been cloned. During this cloning we found that HNK-1ST and other Golgi-associated sulfotransferases cloned before share homologous sequences including the RDP motif (Ong, E., Yeh, J.-C., Ding, Y., Hindsgaul, O., and Fukuda, M. (1998) J. Biol. Chem. 223, 5190-5195). Using this conserved sequence in HNK-1ST as a probe, we identified two expressed sequence tags in EST data base which have 31.6 and 30.7% identity with HNK-1ST at the amino acid levels. Expression of these two full-length cDNAs failed to form HNK-1 glycan nor to add sulfate to CD34 or NCAM. Surprisingly, proteins expressed by these cDNAs transferred sulfate to the C-4 position of N-acetylgalactosamine in chondroitin and desulfated dermatan sulfate, thus we named these two enzymes, chondroitin 4-O-sulfotransferase 1 and -2 (C4ST-1 and C4ST-2). Both C4ST-1 and C4ST-2, however, did not form 4, 6-di-O-sulfated N-acetylgalactosamine when chondroitin sulfate C was used as an acceptor. Moreover, analysis of (35)S-labeled dermatan sulfate formed by C4ST-1 indicate that sulfation preferentially took place in GlcA-->GalNAc unit than in IdoA-->GalNAc unit, suggesting that 4-O-sulfation at N-acetylgalactosamine may precede epimerization of glucuronic acid to iduronic acid during dermatan sulfate biosynthesis. Northern analysis demonstrated that the transcript for C4ST-1 is predominantly expressed in peripheral leukocytes and hematopoietic tissues while the C4ST-2 transcript is more widely expressed in various tissues. These results indicate C4ST-1 and C4ST-2 play complementary roles in chondroitin and dermatan sulfate synthesis in different tissues.

Effect of brief maximal exercise on circulating levels of interleukin-12.

Interleukin-12 (IL-12) is a cytokine that was originally identified as natural killer cell stimulatory factor. It induces the activity of T-helper 1 (Th1) cells and exhibits strong anti-tumor activity. In this study, we studied the effects of brief anaerobic maximal exercise on circulating levels of IL-12. Six healthy males [mean (SD) 25.2 (2.6) years] performed a modified Wingate test exercise (resistance 0.075 kg/kg of body mass). The exercise consisted of five bouts of maximal cycling for 10 s, with rest intervals of 50 s between them. Blood samples were taken before, immediately after, 30 min after, 60 min after and 120 min after the exercise. Plasma concentrations of IL-12 were measured using an enzyme-linked immunosorbent assay. Data were corrected for hemoglobin and hematocrit measurements. Plasma concentrations of IL-12 averaged [mean (SD)] 234.2 (40.9) pg/ml before, 305.2 (62.1) pg/ml immediately after, 202.8 (24.2) pg/ml 30 min after, 239.7 (35.1) pg/ml 60 min after, and 199.6 (49.2) pg/ml 120 min after the exercise. We showed that plasma concentrations of IL-12 increased significantly immediately after brief anaerobic maximal cycle ergometer exercise (P < 0.01).