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Escherichia albertii is an emerging zoonotic foodborne pathogen. Several outbreaks of E. albertii have occurred particularly in Japan. Although birds have been considered as one of the most important reservoirs of this bacterium, information regarding the prevalence in birds is still scanty. We performed a survey of E. albertii in wild birds in Japan, and examined characteristics of the isolates. E. albertii specific gene was detected in 5 cloacal swabs out of 156 birds by PCR. Four E. albertii were isolated from a swallow with 2 different E. albertii strains and 2 pigeons in a flock by XRM-MacConkey agar. These isolates were assigned to biogroup 3, shown no resistance to any antimicrobials tested, and classified into 2 EAO-genotypes (EAOg2 and EAOg33) and untypable. Similar to clinical E. albertii strains, these isolates carried virulence genes including eae (n=4), paa (n=4), Eccdt-I (n=2) and stx2f (n=1) in addition to Eacdt. Interestingly, stx2f genes in a strain were located on an inducible bacteriophage, which can confer the ability to produce Stx2f to E. coli. In conclusion, Japanese wild birds carried E. albertii at the similar levels to the reported prevalence in birds. These isolates may have a potential to cause gastroenteritis in humans.

An infant hamadryas baboon exhibiting an albino phenotype —white body hair and red eyes— was born to parents with wild-type body color. Pigmentation on some parts of its body surfaced during childhood and progressed with age. This baboon in adulthood has gray hair on parts of its body, such as the tail, distal portion of the legs, and face, with the remainder being white. This pigmentation pattern resembles that of the Siamese cat and the Himalayan variants of the mouse and the mink. The distinguishing phenotypes in these animals are known to be caused by a temperature-sensitive activity of tyrosinase, an enzyme essential for biosynthesis of melanin. We sequenced all the five exons of the tyrosinase (TYR) gene of this albino baboon, which were amplified by PCR, and found a base substitution leading to alteration of the 365th amino acid from Ala to Thr. Tyrosinase requires copper as a cofactor for its enzyme function. It has two copper-binding sites, the second of which contains His residues in positions 363 and 367 that are critical to its function. Thus, p.(Ala365Thr) due to a mutation in the TYR gene is a likely candidate for the cause of the albino phenotype in this baboon., シャム猫のような体色パターンをもつマントヒヒの遺伝子変異を同定 --メラニン合成を担う酵素の遺伝子が変化--. 京都大学プレスリリース. 2020-03-17.

Escherichia albertii is an emerging zoonotic foodborne pathogen. Several outbreaks of E. albertii have occurred, particularly in Japan. Although birds have been considered as one of the most important reservoirs of this bacterium, information regarding its prevalence in birds is still scarce. We performed a survey of E. albertii in wild birds in Japan and examined the characteristics of these isolates. E. albertii-specific genes were detected in five cloacal swabs from 156 birds by PCR. Four E. albertii strains were isolated from a swallow with two different E. albertii strains and two pigeons in a flock using XRM-MacConkey agar. These isolates were assigned to biogroup 3, showed no resistance to any tested antimicrobials, and were classified into two EAO-genotypes (EAOg2 and EAOg33) and were untypable. Similar to clinical E. albertii strains, these isolates carried virulence genes, including eae (n = 4), paa (n = 4), Eccdt-I (n = 2), and stx2f (n = 1), as well as Eacdt. Furthermore, stx2f genes in a strain were located on an inducible bacteriophage, which can confer the ability to produce Stx2f in E. coli. In conclusion, Japanese wild birds carried E. albertii at levels similar to the reported prevalence in birds. These isolates may have the potential to cause gastroenteritis in humans.

Lipocalin-type prostaglandin D synthase (L-PGDS) catalyses the formation of prostaglandin D(2) (PGD(2)) and also functions as a transporter for lipophilic ligands, including all-trans retinoic acid (RA). Here, we show that human epidermal melanocytes produce and secrete L-PGDS and PGD(2) in culture medium, whereas L-PGDS is not expressed in human melanoma cell lines, HMV-II, SK-MEL-28, 624 mel and G361. Treatment with RA (1 or 10 microM) for 4 days decreased the proliferation of melanocytes (30% decrease), but not melanoma cells. We therefore isolated L-PGDS-expressing cell lines from 624 mel cells. Treatment with RA decreased the proliferation of L-PGDS-expressing cells by 20%, but not mock-transfected cell lines lacking L-PGDS expression. RA induced expression of a cyclin-dependent kinase inhibitor p21(Cip1) in L-PGDS-expressing cells, but not mock-transfected cells. Moreover, RA increased the transient expression of a reporter gene carrying the RA-responsive elements in L-PGDS-expressing cell lines (at least 5-fold activation), compared to the 2-fold activation in mock-transfected cell lines, suggesting that L-PGDS may increase the sensitivity to RA. Lastly, the knockdown of L-PGDS expression by RNA interference was associated with the restoration of the RA-mediated decrease in proliferation of human and mouse melanocytes. In conclusion, L-PGDS may fine-tune the RA signalling in melanocytes.

Heme oxygenase consists of two structurally related isozymes, heme oxygenase-1 and and heme oxygenase-2, each of which cleaves heme to form biliverdin, iron and carbon monoxide. Expression of heme oxygenase-1 is increased or decreased depending on cellular microenvironments, whereas little is known about the regulation of heme oxygenase-2 expression. Here we show that hypoxia (1% oxygen) reduces the expression levels of heme oxygenase-2 mRNA and protein after 48 h of incubation in human cell lines, including Jurkat T-lymphocytes, YN-1 and K562 erythroleukemia, HeLa cervical cancer, and HepG2 hepatoma, as judged by northern blot and western blot analyses. In contrast, the expression level of heme oxygenase-1 mRNA varies under hypoxia, depending on the cell line; it was increased in YN-1 cells, decreased in HeLa and HepG2 cells, and remained undetectable in Jurkat and K562 cells. Moreover, heme oxygenase-1 protein was decreased in YN-1 cells under the conditions used, despite the induction of heme oxygenase-1 mRNA under hypoxia. The heme oxygenase activity was significantly decreased in YN-1, K562 and HepG2 cells after 48 h of hypoxia. To explore the mechanism for the hypoxia-mediated reduction of heme oxygenase-2 expression, we showed that hypoxia shortened the half-life of heme oxygenase-2 mRNA (from 12 h to 6 h) in YN-1 cells, without affecting the half-life of heme oxygenase-1 mRNA (9.5 h). Importantly, the heme contents were increased in YN-1, HepG2 and HeLa cells after 48 h of incubation under hypoxia. Thus, the reduced expression of heme oxygenase-2 may represent an important adaptation to hypoxia in certain cell types, which may contribute to the maintenance of the intracellular heme level.

Microphthalmia-associated transcription factor (MITF) is responsible for differentiation of melanocytes. A recessive MITF mutant, black-eyed white Mitfmi-bw mouse, is characterized by white coat color and deafness, due to the lack of melanocytes in the skin and inner ears. By cDNA microarray analysis, we have identified lipocalin-type prostaglandin D synthase (L-PGDS), whose mRNA is undetectable in the homozygous Mitfmi-bw skin. Immunohistochemical analysis of wild-type mice identified the specific expression of L-PGDS in follicular melanocytes. L-PGDS mRNA is expressed in B16 mouse melanoma cells, but undetectable in human melanoma cell lines. RNA interference analysis against MITF suggests that L-PGDS expression is dependent on MITF in B16 melanoma cells. Furthermore, we have provided evidence that MITF is involved in the melanocyte lineage-specific transcription of the mouse L-PGDS gene. Thus, L-PGDS represents a newly identified melanocyte marker. MITF may modulate the production of prostaglandin D2 by activating the L-PGDS gene in melanocytes.

Dietary intake of cadmium is inevitable, causing age-related increase in cadmium accumulation in many organs, including hair, choroid and retinal pigment epithelium (RPE). Cadmium has been implicated in the pathogenesis of hearing loss and macular degeneration. The functions of cochlea and retina are maintained by melanocytes and RPE, respectively, and the differentiation of these pigment cells is regulated by microphthalmia-associated transcription factor (MITF). In the present study, we explored the potential toxicity of cadmium in the cochlea and retina by using cultured human melanocytes and human RPE cell lines. MITF consists of multiple isoforms, including melanocyte-specific MITF-M and widely expressed MITF-H. Levels of MITF-M protein and its mRNA in human epidermal melanocytes and HMV-II melanoma cells were decreased significantly by cadmium. In parallel with the MITF reduction, mRNA levels of tyrosinase, the key enzyme of melanin biosynthesis that is regulated by MITF-M, were also decreased. In RPE cells, however, the levels of total MITF protein, constituting mainly MITF-H, were not decreased by cadmium. We thus identify MITF-M as the molecular target of cadmium toxicity in melanocytes, thereby accounting for the increased risk of disability from melanocyte malfunction, such as hearing and vision loss among people with elevated cadmium exposure.

Microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix-leucine zipper protein, and plays an important role in the development of various cell types, such as neural-crest-derived melanocytes and optic-cup-derived retinal pigment epithelium. Three isoforms of MITF with distinct amino-termini have been described. These include melanocyte lineage-specific MITF-M, heart-type MITF-H, and the recently identified MITF-A. Here we identify a fourth isoform, MITF-C, with a unique amino-terminus of 34 amino acid residues, which shares about 43% sequence identity with putative transactivation segments of two previously identified leukemogenic factors, ENL and AF-9. Reverse transcription-polymerase chain reaction analysis revealed that MITF-C mRNA is expressed in many cell types, including retinal pigment epithelium, but is undetectable in melanocyte-lineage cells. In contrast, MITF-A and MITF-H mRNAs are coexpressed in all cell types examined. Transient cotransfection assays suggested that MITF-C, like other MITF isoforms, functions as a transcriptional activator of certain target genes, but its transactivation specificity for the target promoters is different from those of other MITF isoforms. Therefore, isoform multiplicity provides MITF with differential expression patterns as well as functional diversity.

Microphthalmia-associated transcription factor (Mitf) is required for the differentiation of melanoblasts of the neural crest origin. The mouse homozygous for the black-eyed white (Mitf(mi-bw) ) allele is characterized by white-coat color and deafness with black eye, due to the loss of melanoblasts during embryonic development. The Mitf(mi-bw) allele carries an insertion of long interspersed element-1 (L1) in intron 3 of the Mitf gene, which may cause the deficiency of melanocyte-specific Mitf-M. Here, we show that the L1 insertion results in the generation of alternatively spliced Mitf-M mRNA species, such as Mitf-M mRNA lacking exon 3, exon 4 or both exons 3 and 4, each of which encodes Mitf-M protein with an internal deletion. Transient expression assays showed the loss of or reduction in function of each aberrant Mitf-M protein and the dominant negative effect of Mitf-M lacking exon 4 that encodes an activation domain. Thus, the L1 insertion may decrease the expression level of functional Mitf-M. Importantly, Mitf-M mRNA is expressed in the wild-type mouse brain, with the highest expression level in the hypothalamus. Likewise, aberrant Mitf-M mRNAs are expressed in the bw mouse brain. The bw mice show the altered neurobehavior under a stressful environment, suggesting the role of Mitf-M in sensory perception.

Heme oxygenase 1 is an essential enzyme in heme catabolism that cleaves heme to form biliverdin, iron, and carbon monoxide. The human heme-oxygenase-1 gene is transcriptionally activated through the cis-regulatory element (MTE), GTCATATGAC (positions -156 to -147), during 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced differentiation of myelomonocytic cell lines, such as THP-1, to macrophages. MTE is responsible for the myelomonocytic-specific induction of heme-oxygenase-1 gene expression, and is bound by ubiquitous and myelomonocytic cell-line-restricted proteins. In this study, we cloned the cDNA segments coding for a portion of a protein that binds to MTE by a Southwestern procedure from a THP-1 cDNA expression library; we subsequently isolated putative full-length cDNAs by a conventional hybridization procedure. The deduced protein, termed MTB-Zf, consists of 1482 amino acid residues, has a molecular mass of about 162 kDa, and contains the two widely separated zinc-finger domains located near the N- and C-termini. MTB-Zf possesses other structural features characteristic of transcription factors, including a long stretch of acidic amino acids (amino acids 67 - 95), a proline-rich region (positions 733-849), a region rich in basic amino acids (positions 1161-1247), and a leucine repeat-like region (positions 486-514). We show that a portion of MTB-Zf, including an N-terminal zinc-finger domain, binds in vitro to MTE and that the transient coexpression of MTB-Zf cDNA leads to transactivation of the heme-oxygenase-1 gene promoter. Since the 6.5 kb MTB-Zf is expressed in various human cell lines of different lineages, MTB-Zf may represent a ubiquitous MTE-binding protein. Furthermore, the MTB-Zf gene has been mapped to human chromosome 1p35-36.1 by fluorescence in situ hybridization, a region which is frequently deleted in various solid tumors, including neurogenic tumors. We found remarkable differences in the expression patterns of MTB-Zf mRNA and two other hybridizable mRNAs of 5kb and 8.5 kb when human brain and primary brain tumors were compared. Both MTB-Zf and the 8.5-kb mRNAs were abundantly expressed in the five primary brain tumors examined, but only the 5-kb mRNA was detectable in the human brain. These results suggest that MTB-Zf is a transcription factor and may also play an important role in cell growth or differentiation.

Mi protein encoded at the mouse microphthalmia (mi) locus is a transcription factor with a basic helix-loop-helix/leucine zipper structure. To assess the function of the human homolog of Mi protein, termed microphthalmia-associated transcription factor (MITF), we analyzed the effects of MITF on the promoter function of the mouse tyrosinase and tyrosinase-related protein 1 (TRP-1) genes. These two gene promoters are able to direct transcription preferentially in melanin-producing cells, and an enhancer element M box of 11 bp, containing a CATGTG motif, is conserved in both promoters. By transient expression assays, we have localized the cis-acting element of the tyrosinase gene responsible for pigment cell-specific expression to the proximal 82-bp region, which contains a CATGTG motif (positions -12 to -7) but lacks the M box (positions -107 to -97). We also provide evidence that the 82-bp region and the M box are involved in the transactivation of the tyrosinase promoter by MITF and that the M box is bound by MITF in vitro. Furthermore, MITF activated the TRP-1 gene promoter possibly through the M box (positions -44 to -34). These results suggest that MITF is a common factor regulating transcription of the pigment cell-specific genes.

Heme is an essential requirement for cell survival. Heme oxygenase (HO) is the rate-limiting enzyme in heme catabolism and consists of two isozymes, HO-1 and HO-2. To identify the protein that regulates the expression or function of HO-1 or HO-2, we searched for proteins that interact with both isozymes, using protein microarrays. We thus identified 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) that synthesizes or degrades fructose-2,6-bisphosphate, a key activator of glycolysis, depending on cellular microenvironments. Importantly, HO-2 and PFKFB4 are predominantly expressed in haploid spermatids. Here, we show a drastic reduction in expression levels of PFKFB4 mRNA and protein and HO-2 mRNA in HepG2 human hepatoma cells in responses to glucose deprivation (≤ 2.5 mM), which occurred concurrently with remarkable induction of HO-1 mRNA and protein. Knockdown of HO-2 expression in HepG2 cells, using small interfering RNA, caused PFKFB4 mRNA levels to decrease with a concurrent increase in HO-1 expression. Thus, in HepG2 cells, HO-1 expression was increased, when expression levels of HO-2 and PFKFB4 mRNAs were decreased. Conversely, overexpression of HO-2 in HepG2 cells caused the level of co-expressed PFKFB4 protein to increase. These results suggest a potential regulatory role for HO-2 in ensuring PFKFB4 expression. Moreover, in D407 human retinal pigment epithelial cells, glucose deprivation decreased the expression levels of PFKFB4, HO-1, and HO-2 mRNAs. Thus, glucose deprivation consistently down-regulated the expression of PFKFB4 and HO-2 mRNAs in both HepG2 cells and RPE cells. We therefore postulate that PFKFB4 and HO-2 are expressed in a coordinated manner to maintain glucose homeostasis.

Granulated metrial gland (GMG) cells are a natural killer (NK) cell-like population present in large numbers in the pregnant rodent uterus. By day 8 of gestation GMG cells are large and granulated and localized to the mesometrial side of each implantation site. GMG cells appear to be highly migratory both in vivo and in vitro; however, little is known regarding their functions. Using indirect fluorescence immunohistochemistry, murine uteri and implantation sites were studied on successive days of gestation to characterize the extracellular matrix receptors of the VLA-integrin family displayed by GMG cells. On days 3 and 6 of gestation, double immunostaining using the monoclonal antibody LGL-1 was employed to recognize GMG cells because their morphology early in pregnancy resembles that of other lymphocytes. Between days 8-15 of gestation, GMG cells can be recognized by their unique morphology. The day 3 and day 6 LGL-1+ cells were positive for all antigens examined; that is, beta 1 plus alpha 1, alpha 3, alpha 4, alpha 5 and alpha 6. From days 8-15 of gestation, GMG cells were beta 1+, alpha 4+, alpha 5+ but alpha 1-, alpha 3-, alpha 6-. Thus, between days 6-8 of gestation, major changes occur in the uterine NK/GMG cell population which include the loss of the surface molecules VLA alpha 1, alpha 3 and alpha 6 or the rapid expansion of NK cells not expressing these proteins. It is postulated that major changes in the functions of uterine NK cells are likely to be associated with these alterations in cell surface phenotype and that functional studies of uterine NK cells should focus upon this relatively early gestational time point.

We cloned a cDNA coding for a putative human heme oxygenase isozyme, designated type 2 (HO-2), and analyzed its function by transient expression assays. HeLa cells transfected with either HO-2 cDNAs or a cDNA coding for authentic heme oxygenase (HO-1) expressed the activity of heme oxygenase, although no activity was detected in the mock transfected cells. Using specific anti-HO-1 antibody, we showed that expression of a HO-1 cDNA resulted in the increase in its protein levels, but HO-1 protein was not detectable in the cells expressing HO-2 cDNAs. We thus confirmed the functional identity of HO-1 and HO-2. Then, we analyzed their expression in an erythroid cell line, YN-1-0-A. Treatment with hemin or by heat shock (42 degrees C) led to a remarkable increase in the HO-1 mRNA levels, while HO-2 mRNA expression was not induced at all, suggesting that they are under separate regulation.

Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai, Japan, First Department of Internal Medicine, Fukushima Medical University, Fukushima, Japan, Division of Pathology, School of Health Sciences, Tohoku University, Sendai, Japan, Laboratory of Molecular Pharmacology, Tohoku University School of Medicine, Sendai, Japan, Department of Cardiovascular Medicine, Tohoku University School of Medicine, Sendai

Tyrosinase-related protein 2 (TRP-2), also known as DOPAchrome tautomerase, is an enzyme in melanin biosynthesis and may play an important role in detoxification of a metabolite derived from DOPA. TRP-2 is expressed in melanocytes of neural crest origin and retinal pigment epithelium (RPE), derived from the optic cup. TRP-2 has been established as an early differentiation marker for melanoblasts and RPE. It is therefore of significance to study the regulation of TRP-2/DOPAchrome tautomerase expression. Here we show that TRP-2 mRNA is expressed in Y79 human retinoblastoma cell line, derived from a primitive multipotential retinal cell. Retinoblastoma is the common primary intraocular tumor of childhood. Basal expression levels in Y79 retinoblastoma cells of TRP-2 mRNA and protein are comparable to those in melanoma cells, whereas mRNA for tyrosinase, the rate-limiting enzyme in melanogenesis, is undetectable in retinoblastoma cells. Transient transfection assays showed that the TRP-2 gene promoter efficiently directs the reporter gene expression in retinoblastoma cells as it does in melanoma cells. Moreover, the expression of TRP-2 mRNA was induced by retinoic acid in retinoblastoma cells but not noticeably affected by forskolin, a cAMP-elevating reagent, whereas in melanoma cells its expression was induced by forskolin but not by retinoic acid. These results suggest a difference in the regulation of TRP-2 expression between retinoblastoma and melanoma cells. Moreover, TRP-2 mRNA is expressed in the excised retinoblastoma specimens, as assessed by RT-PCR. The present study shows unexpected features of TRP-2 and may enhance our understanding of the pathophysiology of retinoblastoma.

Tyrosinase-related protein 2 (TRP-2)/DOPAchrome tautomerase is an enzyme involved in melanin biosynthesis and plays an important role in cytoprotection by preventing the production of a toxic melanin precursor, 5,6-dihydroxyindole. Neurofibromin is the protein product of a gene linked to neurofibromatosis type 1 (NF1), which is characterized by multiple neurofibromas and abnormalities in skin pigmentation. To explore the pathogenesis of NF1, we studied the role of neurofibromin in the regulation of TRP-2 gene expression. By means of transient cotransfection assays, we show that the expression of a reporter gene under the control of the TRP-2 gene promoter was increased by a neurofibromin-dependent signal through the 71-bp region (positions -415 to -345). A Lys-to-Glu substitution at position 1425 in neurofibromin abrogated this activating function. A dominant negative Ki-ras inhibitor mimics neurofibromin's function, and additively increases TRP-2 promoter activity when coexpressed with neurofibromin. Therefore, we suggest that neurofibromin is involved in the regulation of TRP-2 gene expression. Moreover, we found a single case of a glioblastoma multiforme that expresses TRP-2 mRNA but not tyrosinase mRNA, suggesting that TRP-2 may function in human neural tissues under certain conditions.

Heme oxygenase-1 is an essential enzyme in heme catabolism, and its human gene promoter contains a putative heat shock element (HHO-HSE). This study was designed to analyze the regulation of human heme oxygenase-1 gene expression under thermal stress. The amounts of heme oxygenase-1 protein were not increased by heat shock (incubation at 42 degrees C) in human alveolar macrophages and in a human erythroblastic cell line, YN-1–0-A, whereas heat shock protein 70 (HSP70) was noticeably induced. However, heat shock factor does bind in vitro to HHO-HSE and the synthetic HHO-HSE by itself is sufficient to confer the increase in the transient expression of a reporter gene upon heat shock. The deletion of the sequence, located downstream from HHO-HSE, resulted in the activation of a reporter gene by heat shock. These results suggest that HHO-HSE is potentially functional but is repressed in vivo. Interestingly, heat shock abolished the remarkable increase in the levels of heme oxygenase-1 mRNA in YN-1–0-A cells treated with hemin or cadmium, in which HSP70 mRNA was noticeably induced. Furthermore, transient expression assays showed that heat shock inhibits the cadmium-mediated activation of the heme oxygenase-1 promoter, whereas the HSP70 gene promoter was activated upon heat shock. Such regulation of heme oxygenase-1 under thermal stress may be of physiologic significance in erythroid cells.

Heme oxygenase-1 is an essential enzyme in heme catabolism, releasing carbon monoxide, iron, and biliverdin. By transient expression analysis of the fusion genes, containing the firefly luciferase gene as a reporter under the human heme oxygenase-1 gene promoter, we have identified a 10-bp sequence, TGCTAGATTT, that confers the cadmium-mediated induction on the fusion gene, located about 4 kb upstream from the transcriptional initiation site (J. Biol. Chem. 269: 22858–67, 1994). This cadmium-responsive element (CdRE) is different from the consensus sequence of the metal-regulatory element of the metallothionein genes. Both heme oxygenase-1 and metallothionein are considered to be involved in the defense system against metal toxicity. Here we showed that the CdRE of the human heme oxygenase-1 gene is not responsive to zinc, whereas a metal-regulatory element (MRE) of the human metallothionein IIA gene is able to respond to both cadmium and zinc. These results suggest that the metal-selective activation of each gene promoter is mediated by a separate mechanism.

Morphology of granulated metrial gland (GMG) cell, a uterine natural killer (NK) cell, was reported to be normal in pregnant uteri of NK cell-deficient mice and T-cell- and B-cell-deficient mice, but little is known about the number of GMG cells. To determine whether the number of GMG cells is influenced by such mutations, their number in the bg mice (genotype C57BL/6J-bg/bg) and SCID mice (genotype C.B-17/Icr-scid/scid) was compared to that of control mice on day 12 of gestation. GMG cells in these mutant mice were normal in number. Thus, the present results support the previous reports that GMG cells can differentiate normally in bg mice and that the GMG cell differentiation is not affected by functional T- nor B-cells.

The pigment cell-specific gene, located at the brown (b)-locus in mouse, encodes the protein that determines the type of melanin synthesized. This protein is known as tyrosinase-related protein, but here we tentatively term it b-locus protein to avoid confusions with the related sequence cross-hybridizing to the tyrosinase gene. In order to identify the mutation at the b-locus, we have cloned and characterized the b-locus protein gene of BALB/c mouse (b/b, c/c). The gene is about 18 kb long and organized into 8 exons and 7 introns. Sequence analysis of the b-locus protein gene reveals four base changes within the protein-coding regions: two missense mutations and two silent mutations. Two missense mutations result in the Cys to Tyr substitution at position 86 (codon 110) and the Arg to His substitution at position 302 (codon 326) of a b-locus protein molecule. Using allele-specific amplification, we confirmed that these missense mutations are actually present in the genomic DNA of two b-mutant strains examined, BALB/c and DBA/2 (b/b, C/C) mice, suggesting that these mutations are specific for the mutant mice at the b-locus. Moreover, we are able to show that the b-locus protein containing Tyr 86 is not reactive with the anti-b-locus protein monoclonal antibody, TMH-1, in transient expression assays.

The non-dialyzable portion of the water extract of the Oriental crude drug "karokon", the roots of Trichosanthes kirilowii, was found to reduce the plasma glucose level in mice. Activity-guided fractionation of this non-dialyzable portion led to the isolation of five glycans termed as trichosans A, B, C, D and E, showing hypoglycemic actions in normal mice. The main glycan, trichosan A, also exhibited activity in alloxan-induced hyperglycemic mice.