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Objective: This study aims to show the usefulness of basic carbohydrate counting (BCC) in the glycemic control of patients with type 2 diabetes (T2DM) and to investigate the effects of BCC on body compositions. Subjects and Methods: We included T2DM 20 patients in no insulin therapy with stable dosage of antidiabetic medication 8 weeks before and during the study period. We provided the meal planning according to the indicated sugar mass, which was set to 50% of their energy amount per day. HbA1c levels, daily carbohydrate intake, and body compositions (measured with InBody 770) were collected before and 12 ± 4 weeks after BCC. Results: Before/after BCC of their daily carbohydrate intake and HbA1c levels were 210.7 ± 42.1/171.5 ± 34.9 g (p = 0.0018), and 8.1 ± 1.1/7.4 ± 0.9 % (p = 0.0002), respectively. To examine the effects of BCC on their body compositions, 16 patients with improved HbA1c levels were selected for further analysis. BCC improved their body weight (before/after BCC, 64.4 ± 17.6/63.0 ± 16.6 kg, p = 0.005), BMI (25.7 ± 5.3/25.2 ± 5.0 kg/m2, p = 0.004), body fat mass (21.8 ± 9.5/20.1 ± 8.7 kg, p = 0.014), and abdominal circumference (95.0 ± 13.6/92.9 ± 13.2 cm, p = 0.0004). There was no significant difference in overall SMI, but the muscle mass of right arm, left arm, and trunk significantly decreased while the muscle mass of legs was not affected by BCC. Discussion and Conclusion: Our findings demonstrate that BCC is an effective strategy for improving the glycemic control of T2DM patients. The patients with improved HbA1c levels also showed significant reduction in their body weight, BMI, body fat mass, and abdominal circumference. While preserving the muscle mass of their legs, the muscle mass reduction in their arms and trunks was observed. Combining with resistant training of arms and trunk, BCC may make more positive effects on T2DM patients. Disclosure M. Taniguchi: None. K. Maeda: None. R. Morita: None. R. Harada: None. F. Yoshino: None. Y. Mukai: None. M. Ikeda: None. K. Harada: None. M. Yoshiyama: None. H. Nakano: None.

Kunii et al. reveal that the SNARE protein SNAP23 plays distinct roles in the secretion of amylase in exocrine cells and of insulin in endocrine cells the pancreas and show that MF286, a novel inhibitor of SNAP23, may be a new drug candidate for diabetes., The membrane fusion of secretory granules with plasma membranes is crucial for the exocytosis of hormones and enzymes. Secretion disorders can cause various diseases such as diabetes or pancreatitis. Synaptosomal-associated protein 23 (SNAP23), a soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE) molecule, is essential for secretory granule fusion in several cell lines. However, the in vivo functions of SNAP23 in endocrine and exocrine tissues remain unclear. In this study, we show opposing roles for SNAP23 in secretion in pancreatic exocrine and endocrine cells. The loss of SNAP23 in the exocrine and endocrine pancreas resulted in decreased and increased fusion of granules to the plasma membrane after stimulation, respectively. Furthermore, we identified a low molecular weight compound, MF286, that binds specifically to SNAP23 and promotes insulin secretion in mice. Our results demonstrate opposing roles for SNAP23 in the secretion mechanisms of the endocrine and exocrine pancreas and reveal that the SNAP23-binding compound MF286 may be a promising drug for diabetes treatment.

p31, the mammalian orthologue of yeast Use1p, is an endoplasmic reticulum (ER)-localized soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) that forms a complex with other SNAREs, particularly syntaxin 18. However, the role of p31 in ER function remains unknown. To determine the role of p31 in vivo, we generated p31 conditional knockout mice. We found that homozygous deletion of the p31 gene led to early embryonic lethality before embryonic day 8.5. Conditional knockout of p31 in brains and mouse embryonic fibroblasts (MEFs) caused massive apoptosis accompanied by upregulation of ER stress-associated genes. Microscopic analysis showed vesiculation and subsequent enlargement of the ER membrane in p31-deficient cells. This type of drastic disorganization in the ER tubules has not been demonstrated to date. This marked change in ER structure preceded nuclear translocation of the ER stress-related transcription factor C/EBP homologous protein (CHOP), suggesting that ER stress-induced apoptosis resulted from disruption of the ER membrane structure. Taken together, these results suggest that p31 is an essential molecule involved in the maintenance of ER morphology and that its deficiency leads to ER stress-induced apoptosis.

A number of proteins are known to be involved in apical/basolateral transport of proteins in polarized epithelial cells. The small GTP-binding protein Rab8 was thought to regulate basolateral transport in polarized kidney epithelial cells through the AP1B-complex-mediated pathway. However, the role of Rab8 (Rab8A) in cell polarity in vivo remains unknown. Here we show that Rab8 is responsible for the localization of apical proteins in intestinal epithelial cells. We found that apical peptidases and transporters localized to lysosomes in the small intestine of Rab8-deficient mice. Their mislocalization and degradation in lysosomes led to a marked reduction in the absorption rate of nutrients in the small intestine, and ultimately to death. Ultrastructurally, a shortening of apical microvilli, an increased number of enlarged lysosomes, and microvillus inclusions in the enterocytes were also observed. One microvillus inclusion disease patient who shows an identical phenotype to Rab8-deficient mice expresses a reduced amount of RAB8 (RAB8A; NM_005370). Our results demonstrate that Rab8 is necessary for the proper localization of apical proteins and the absorption and digestion of various nutrients in the small intestine.

Patients with pemphigus vulgaris (PV) have circulating anti-desmoglein (Dsg) 3 immunoglobulin G (IgG) autoantibodies that induce blister formation. We developed an in vitro quantitative assay to evaluate the pathogenic strength of anti-Dsg3 IgG autoantibodies in blister formation. To obtain intercellular adhesion mediated dominantly by Dsg3, we used primary cultured normal human keratinocytes expressing low level of Dsg2 in the presence of exfoliative toxin A that specifically digests Dsg1. After incubation with various antibodies, monolayers released by dispase were subjected to mechanical stress by pipetting, and the number of cell fragments were counted. When anti-Dsg3 monoclonal antibodies (mAb) obtained from pemphigus model mice were tested, pathogenic AK23 mAb yielded significantly higher number of cell fragments than AK7 or AK20 non-pathogenic mAb. Dissociation scores, defined with AK23 mAb as the positive control, were significantly higher with active stage PV sera (n=10, 77.4 ± 21.4) than controls (n=11, 16.0 ± 9.6; p=0.003). When pair sera obtained from 6 PV patients in active stage and in remission were compared, the dissociation scores reflected well the disease activity as those in active stage were four to 17 times higher than those in remission. When sera from different patients showing similar ELISA scores but different clinical severity were tested (n=6), the dissociation scores with sera from severe disease activity were significantly higher than those with sera in remission. These findings indicate that this dissociation assay will provide a simple and objective biological method to measure the pathogenic strength of pemphigus autoantibodies.

Gene therapies for recessive genetic diseases may provoke unwanted immune responses against the introduced gene product because patients, especially those with null mutation of a certain protein, have no tolerance for the protein of interest. This study used desmoglein 3 knockout (Dsg3-/-) mice as a disease model for a genetic defect in DSG3, to investigate whether nonviral gene therapy induces an immune response against Dsg3 and whether the reaction against Dsg3 can be prevented. When mouse Dsg3 cDNA was injected in the skin of Dsg3-/- mice, 50% of treated Dsg3-/- mice developed anti-Dsg3 IgG, which can bind native Dsg3 in vivo. To prevent this response, we used an anti-CD40L monoclonal antibody, MR1, which blocks the costimulatory interaction between CD40 and CD40L. To evaluate the effect of MR1, we grafted Dsg3+/+skin on Dsg3-/- mice, to mimic stable gene transfer of Dsg3. After skin grafting, all the recipient Dsg3-/- mice were treated with either MR1 (n=8) or control hamster IgG (n=8). All of the control IgG-treated mice developed circulating anti-Dsg3 IgG about 2 wk after grafting, and IgG deposition was observed on the surfaces of keratinocytes in the grafted Dsg3+/+skin. Such anti-Dsg3 IgG production was significantly prevented, however, when the recipient mice were treated with MR1. These findings suggested that gene therapies for recessive diseases may provoke an immune response against the transgene product, and that the CD40-CD40L interaction might be a reasonable target for effective prevention of such undesirable immune responses, leading, in turn, to a successful gene therapy.

Mammalian protein kinase D (PKD) isoforms have been proposed to regulate diverse biological processes, including the establishment and maintenance of neuronal polarity. To investigate the function of PKD in neuronal polarization in vivo, we generated PKD knockout (KO) mice. Here, we show that the brain, particularly the hippocampus, of both PKD1 KO and PKD2 KO mice was similar to that of control animals. Neurite length in cultured PKD1 KO and PKD2 KO hippocampal neurons was similar to that of wild-type neurons. However, hippocampal neurons deficient in both PKD1 and PKD2 genes showed a reduction in axonal elongation and an increase in the percentage of neurons with multiple axons relative to control neurons. These results reveal that whereas PKD1 and PKD2 are essential for neuronal polarity, there exists a functional redundancy between the two proteins.

Protein Kinase D (PKD) 1, 2, and 3 are members of the PKD family. PKDs influence many cellular processes, including cell polarity, structure of the Golgi, polarized transport from the Golgi to the basolateral plasma membrane, and actin polymerization. However, the role of the PKD family in cell polarity has not yet been elucidated in vivo. Here, we show that KO mice displayed similar localization of the apical and basolateral proteins, transport of VSV-G and a GPI-anchored protein, and similar localization of actin filaments. As DKO mice were embryonic lethal, we generated MEFs that lacked all PKD isoforms from the PKD1 and PKD2 double floxed mice using Cre recombinase and PKD3 siRNA. We observed a similar localization of various organelles, a similar time course in the transport of VSV-G and a GPI-anchored protein, and a similar distribution of F-actin in the PKD-null MEFs. Collectively, our results demonstrate that the complete deletion of PKDs does not affect the transport of VSV-G or a GPI-anchored protein, and the distribution of F-actin. However, simultaneous deletion of PKD1 and PKD2 affect embryonic development, demonstrating their functional redundancy during development.

Background Pityriasis rosea (PR) is a common papulosquamous skin disorder that is suspected to have an infectious aetiology. Objectives We aimed to study the role of human herpesvirus (HHV)-7 and HHV-6 in the pathogenesis of PR. Methods We performed seroepidemiological studies (indirect immunofluorescence test) and polymerase chain reaction (PCR) analysis for HHV-6 and HHV-7 in patients with PR. Seventy-two serum samples and 37 samples of peripheral blood mononuclear cells (PBMC) from 44 patients with PR were obtained. Twenty-five patients with other skin disorders such as drug eruption, urticaria or herpes zoster were studied as controls in the PCR analysis. Results HHV-7 DNA was detected in 13 of 30 (43%) samples of PBMC of the patients with PR and 14 of 25 (56%) samples of PBMC of controls. HHV-6 DNA was detected in six of 29 (21%) patients with PR and nine of 23 (39%) controls. Thus there was no difference in the prevalence of HHV-6 or HHV-7 in PBMC between patients with PR and those with other skin disorders. In the seroepidemiological study, two cases of at least a fourfold rise in titre and five cases of a fourfold decrease in titre to HHV-7 antibody, and two cases of a fourfold rise in titre and two cases of a fourfold decrease in titre to HHV-6 antibody, were observed in 24 patients with PR. This seroepidemiological study revealed antibody responses consistent with active infection in several PR patients, but the greater proportion of the patients had no definite increase in the antibody titres. Conclusions We conclude that HHV-7 and HHV-6 may play a part in some patients with PR, but that other causative agents may exist. Further analyses are needed to determine the causative agents of PR.

We report two cases of Trichophyton mentagrophytes infection. Case 1: A 10-year-old girl visited Tokyo Electric Power Hospital in June 1994 for evaluation of an erythematous lesion on her head. Three months of topical steroid therapy exacerbated the lesion with pustular formation. Histopathological and mycological examination revealed that the patient had tinea capitis caused by T. mentagrophytes. T. mentagrophytes was also isolated from her pet, a hamster. Case 2: A-14-year-old girl was referred to Shonan Clinic in January 1996 with scaly erythema on her face. She had been treated with neticonazole hydrochloride at another clinic, but the lesion became worse. Direct microscopic examination of the scale was negative at that time, so treatment with topical steroid was started. After 10 days, the lesion was almost cured, but one month later it recurred with an annular distribution. KOH preparation of the scale revealed mycelia and T. mentagrophytes was isolated on culture. T. mentagrophytes was also isolated from her pet, a chinchilla. In both cases, the oral administration of itraconazole at 50 mg/day was effective. The isolated pathogen was identified as Arthroderma vanbreuseghemii with species-specific primers of chitin synthase 1 gene. T. mentagrophytes is one of the most common dermatophytes isolated from man and animals. Rodents like the hamster and the chinchilla have recently become popular as pets in Japan. We should be aware that rodents may carry this kind of fungal pathogen as they become even more popular as pets.

We report a case of lymphomatoid papulosis (LyP) followed by Ki-1 positive anaplastic large cell lymphoma (LCL). A 33-year-old man developed subcutaneous nodules in the left inguinal region and the left thigh after a seven-year-history of self-healing papulonecrotic lesions of LyP. Histological and immunohistochemical examination of the subcutaneous nodules revealed LCL. DNA was isolated from a nodule of the initial stage of LyP in 1988, a subcutaneous nodule of LCL in 1993, and a papule of LyP in 1993 which appeared after chemotherapy for LCL. T cell receptor gene rearrangement analysis demonstrated an identical rearranged pattern in all the three specimens, indicating that a common T cell clone proliferated throughout the course in both the LyP and LCL lesions.

The molecular mechanisms of neuronal morphology and synaptic vesicle transport have been largely elusive, and only a few of the molecules involved in these processes have been identified. Here, we developed a novel morphology-based gene trap method, which is theoretically applicable to all cell lines, to easily and rapidly identify the responsible genes. Using this method, we selected several gene-trapped clones of rat pheochromocytoma PC12 cells, which displayed abnormal morphology and distribution of synaptic vesicle-like microvesicles (SLMVs). We identified several genes responsible for the phenotypes and analyzed three genes in more detail. The first gene was BTB/POZ domain-containing protein 9 (Btbd9), which is associated with restless legs syndrome. The second gene was cytokine receptor-like factor 3 (Crlf3), whose involvement in the nervous system remains unknown. The third gene was single-stranded DNA-binding protein 3 (Ssbp3), a gene known to regulate head morphogenesis. These results suggest that Btbd9, Crlf3, and Ssbp3 regulate neuronal morphology and the biogenesis/transport of synaptic vesicles. Because our novel morphology-based gene trap method is generally applicable, this method is promising for uncovering novel genes involved in the function of interest in any cell lines.—Hashimoto, Y., Muramatsu, K., Kunii, M., Yoshimura, S., Yamada, M., Sato, T., Ishida, Y., Harada, R., Harada, A. Uncovering genes required for neuronal morphology by morphology-based gene trap screening with a revertible retrovirus vector.

Background: Parapsoriasis en plaques (large-plaque type) is a premalignant condition capable of developing into cutaneous T-cell lymphoma (CTCL). However, it is not known whether the early stage of CTCL can be distinguished from parapsoriasis en plaques. Objective: Our purpose was to determine whether parapsoriasis en plaques can develop into CTCL. Methods: The clinical appearance, histopathologic features, immunophenotype, DNA rearrangements, and clinical course were analyzed in 20 cases. Results: T-cell receptor β-chain gene rearrangement was detected in four of the 20 cases. No clinical, histopathologic, or immunohistochemical differences were found between patients with and without gene rearrangement. Conclusion: The early stage of CTCL cannot be differentiated from parapsoriasis en plaques by clinical features, histopathologic characteristics, or immunophenotype. Patients with parapsoriasis en plaques exhibit heterogeneous findings, which may include monoclonal proliferation. Patients with long-standing parapsoriasis-like lesions resistant to conventional treatment require careful monitoring for the possible development of cutaneous lymphoma.

To investigate the neuronal function of genes in vivo, a neuron-specific and inducible gene targeting system is desirable. In this study, we generated a knockin mouse line that expresses a fusion protein consisting of the Cre recombinase and the progesterone receptor (CrePR) in neurons. The neuron-specific expression of CrePR was attained by inserting CrePR gene into the tau locus, because tau is expressed strongly in neurons but scarcely in glias and other tissues. By crossing this knockin mouse line (tau(CrePR)) with ROSA26 lacZ reporter mouse line (R26R), we observed that the antiprogesterone RU486 could induce recombinase activity of the CrePR specifically in neurons. Thus, tau (CrePR) knockin line is a useful tool for studying neuronal gene functions.

To determine the neuronal function of genes in vivo, the neuron-specific deletion of a target gene in animals is required. Tau, a microtubule-associated protein, is expressed abundantly in neurons but scarcely in glias and other tissues. Therefore, to generate mice that express Cre recombinase in neurons, we inserted Cre recombinase into the tau locus. By crossing these tau-Cre mice with ROSA26 lacZ reporter mice, we observed Cre recombinase activity in the neurons from most of the central nervous system, but not in glias nor in non-neuronal tissues. This neuronal-specific activity appeared during embryogenesis. We further crossed tau-Cre mice with rab8 'floxed' mice, and showed that the recombination was nearly complete in the brain, but incomplete or non-detectable in other tissues. Thus, tau-Cre knockin mouse is a useful tool for studying the neuronal function of a gene in vivo.

The small GTP-binding protein Rab8 is known to play an essential role in intracellular transport and cilia formation. We have previously demonstrated that Rab8a is required for localising apical markers in various organisms. Rab8a has a closely related isoform, Rab8b. To determine whether Rab8b can compensate for Rab8a, we generated Rab8b-knockout mice. Though the Rab8b-knockout mice did not display an overt phenotype, the Rab8a and Rab8b double-knockout mice exhibited mislocalisation of apical markers and died earlier than the Rab8a-knockout mice. The apical markers accumulated in three intracellular patterns in the double-knockout mice. However, the localisations of basolateral/dendritic markers of the double-knockout mice were apparently normal. The morphology and the lengths of various primary/motile cilia and the frequency of ciliated cells appeared to be identical between the control and double-knockout mice. However, an additional knockdown of Rab10 in the double-knockout cells greatly reduced the percentage of ciliated cells. Our results highlight the compensatory effect of Rab8a and Rab8b in apical transport and the complexity of the apical transport process. In addition, neither Rab8a nor Rab8b are required for basolateral/dendritic transport. In the meantime, the simultaneous loss of Rab8a and Rab8b has little effect on ciliogenesis, while the additional loss of Rab10 greatly affects ciliogenesis. (203 words)

In order to clarify the effects of tissue processing on the ultrastructure and immunological properties of human papilloma virus (HPV), HPV particles were studied comparatively by negative staining and immunofluorescence (IF) technique before and after processing with various fixatives, such as formalin (F), ethanol (E), formalin and ethanol (FE), and formalin, ethanol, xylol and ethanol (FEXE). No significant ultrastructural alterations were demonstrated after processing with F, but considerable alterations were seen with E and FE. IF studies using antiserum against raw HPV revealed a remarkable decrease in specific fluorescence (SF) after processing with F or FEXE, and the disappearance of SF with E alone. However, IF studies using antiserum against formalin-processed HPV revealed identical SF before and after processing with F or FEXE. From these findings, it is concluded that the ultrastructure of HPV may to some degree be altered by usual tissue processing, and that the reactivity of HPV to the antiserum is most seriously affected by formalin fixation of the tissue processing methods employed in preparing deparaffinized sections.

Multicentric pigmented Bowen’s disease (MPBD) is a bowenoid atypia in the genitocrural region with peculiar clinical appearances. A 36-year-old Japanese female patient showed a variety of lesions. Clinically the lesions on the external genitalia consisted of brown-black papillomatous eruptions, black discrete or confluent papules, and whitish macerated papules. Histologically only black papules showed bowenoid atypia, but whitish papules also showed transient bowenoid atypia. Electron microscopically, in all three kinds of the lesions, spherical particles with a diameter of about 50 nm were scattered or gathered together in the nuclei of the keratinocytes beneath the horny layer. These particles were morphologically similar to human papilloma virus. Based upon clinical, histologic and electron microscopic observations, MPBD may be regarded as a new entity, and a term such as multicentric pigmented viral papulosis may be rather preferable than MPBD.

A microsurgical dissection technique for residual strawberry marks applied to patients 6 years of age or older (a total of 7 cases) resulted in minimal bleeding during the operations. Therefore, the same technique was used for patients aged 4 months through 3 years and 4 months. The latter 8 patients had a minimal amount of intraoperative bleeding (no tourniquet was applied to the upper arm case), direct skin closure of the wound, and no postoperative complications. The procedure is tedious but makes dissection of the hemangioma easier because of the watery subcutaneous structure in the infant; it also is less traumatic than the usual surgical dissection technique. We found this procedure to be valuable for complete removal of the unwanted strawberry mark with subcutaneous hemangioma in infants.

Pigmented cosmetic dermatitis1–4 is a new name for melanosis faciei feminase designated after the mechanisim and causative allergens of this pigmentary disorder were greatly clarified. In Japan, a large number of pigmented cosmetic dermatitis patients were noted in the 1960s and 1970s; however, the number of patients has decreased remarkably since 1978, when major cosmetic companies began to eliminate strong contact sensitizers from their products. This review is concerned with the way this pigmentary disorder was overcome through the use of patch testing and allergen control.

A new method of osmotic fragility test with the coil planet centrifuge is presented. The method is highly reproducible and accurate with a minute amount (3μl) of blood sample and most suitable for medical investigations, routine laboratory tests, and screening tests of blood at blood banks.