Body weight reduction in rats by oral treatment with zinc plus cyclo-(His-Pro).

BACKGROUND AND PURPOSE: We have previously shown that treatment with zinc plus cyclo-(His-Pro) (CHP) significantly stimulated synthesis of the insulin degrading enzyme and lowered plasma insulin and blood glucose levels, alongside improving oral glucose tolerance in genetically type 2 diabetic Goto-Kakizaki (G-K) rats and in aged obese Sprague-Dawley (S-D) rats. Thus, we postulated that zinc plus CHP (ZC) treatment might also improve body weight control in these rats. We therefore determined the effects of ZC treatment on body weights in both genetically diabetic, mature G-K rats and non-diabetic, obese S-D rats. EXPERIMENTAL APPROACH: G-K rats aged 1.5-10 months and non-diabetic overweight or obese S-D rats aged 6-18 months were treated with 0-6 mg CHP plus 0-10 mg zinc L(-1) drinking water for 2-4 weeks, and changes in weight, serum leptin and adiponectin levels, food and water intakes were measured. KEY RESULTS: The optimal dose of CHP (in combination with zinc) to reduce weight and plasma leptin levels and to increase plasma adiponectin levels was close to 0.1 mg kg(-1) day(-1), in either mature G-K rats and aged overweight or obese S-D rats. Food and water intake significantly decreased in ZC treated rats in both aged S-D rats and mature G-K rats, but not in young S-D and G-K rats. CONCLUSIONS AND IMPLICATIONS: ZC treatment improved weight control and may be a possible treatment for overweight and obesity.

Increase of oligodendrocyte progenitor cells after spinal cord injury.

The reaction of oligodendrocyte progenitor cells (OPCs) after spinal cord injury (SCI) is poorly understood. In this study, we examined oligodendroglial reactions after contusion SCI in adult rats by immunohistochemistry. OPCs were identified by staining with monoclonal antibodies (mAbs) A2B5 and O4. Each of the A2B5-, O4-positive OPCs and galactocerebroside-positive oligodendrocytes dramatically increased in the lesion of the dorsal posterior funiculus. Bromodeoxyuridine (BrdU) incorporation studies showed that most O4-positive cells in the lesion were labeled with BrdU, suggesting that these OPCs were proliferative. In contrast, the expression of myelin basic protein was decreased in the lesion compared with controls that received laminectomy only. From the injured cord, OPCs were isolated by immunopanning with mAb A2B5. We observed an increased number of OPCs from the injured spinal cords compared with those isolated from controls and unoperated animals. After several days in culture, the OPCs from the lesion expressed galactocerebroside. These results suggest that OPCs are induced and can differentiate following SCI in the adult rat.

Functional analysis for peripheral myelin protein PASII/PMP22: is it a member of claudin superfamily?

Two major glycoproteins, P0 and PASII/PMP22, are specifically expressed in peripheral myelin. Point mutations of these proteins and over or under expression of PASII/PMP22 cause various hereditary peripheral neuropathies. P0 is well characterized as a major adhesion molecule in PNS myelin, but the function of PASII/PMP22 is still unknown. Recently, an oligodendrocyte-specific protein (OSP) was identified as a member of the claudin family and as a component of tight junctions of central myelins. Since PASII/PMP22 shows similarity in structure to OSP, which is a tetraspan membrane protein, we speculated if PASII/PMP22 could be a member of claudin superfamily. The primary structure of PASII/PMP22 showed a significant homology of 48% and a 21% identity with the OSP sequence. Exogenous expression of PASII/PMP22 in C6 cells significantly inhibited BrdU incorporation to the cells. The C6 cells stably transfected with PASII/PMP22 cDNA showed no homophilic cell adhesive activity. When dorsal root ganglion (DRG) neurons were cocultured on PASII/PMP22 expressing cells, both neurite extension and branching of DRG neurons were significantly inhibited. These results indicate that PASII/PMP22 may play a role in a turning point of Schwann cell development from proliferation to differentiation. On the other hand, the cells expressing claudin family proteins are reported to show strong cell adhesive activity and an ability to form tight junctions with neighboring cells. For this reason, we currently do not have any functional data supporting that PASII/PMP22 is the member of claudin superfamily.

A role for IL-12 receptor expression and signal transduction in host defense in leprosy.

The generation of cell-mediated immunity against intracellular infection involves the production of IL-12, a critical cytokine required for the development of Th1 responses. The biologic activities of IL-12 are mediated through a specific, high affinity IL-12R composed of an IL-12Rbeta1/IL-12Rbeta2 heterodimer, with the IL-12Rbeta2 chain involved in signaling via Stat4. We investigated IL-12R expression and function in human infectious disease, using the clinical/immunologic spectrum of leprosy as a model. T cells from tuberculoid patients, the resistant form of leprosy, are responsive to IL-12; however, T cells from lepromatous patients, the susceptible form of leprosy, do not respond to IL-12. We found that the IL-12Rbeta2 was more highly expressed in tuberculoid lesions compared with lepromatous lesions. In contrast, IL-12Rbeta1 expression was similar in both tuberculoid and lepromatous lesions. The expression of IL-12Rbeta2 on T cells was up-regulated by Mycobacterium leprae in tuberculoid but not in lepromatous patients. Furthermore, IL-12 induced Stat4 phosphorylation and DNA binding in M. leprae-activated T cells from tuberculoid but not from lepromatous patients. Interestingly, IL-12Rbeta2 in lepromatous patients could be up-regulated by stimulation with M. tuberculosis. These data suggest that Th response to M. leprae determines IL-12Rbeta2 expression and function in host defense in leprosy.

Functional repression of Islet-2 by disruption of complex with Ldb impairs peripheral axonal outgrowth in embryonic zebrafish.

Islet-2 is a LIM/homeodomain-type transcription factor of the Islet-1 family expressed in embryonic zebrafish. Two Islet-2 molecules bind to the LIM domain binding protein (Ldb) dimers. Overexpression of the LIM domains of Islet-2 or the LIM-interacting domain of Ldb proteins prevented binding of Islet-2 to Ldb proteins in vitro and caused similar in vivo defects in positioning, peripheral axonal outgrowth, and neurotransmitter expression by the Islet-2-positive primary sensory and motor neurons as the defects induced by injection of Islet-2-specific antisense morpholino oligonucleotide. These and other experiments, i.e., mosaic analysis, coexpression of full-length Islet-2, and overexpression of the chimeric LIM domains derived from two different Islet-1 family members, demonstrated that Islet-2 regulates neuronal differentiation by forming a complex with Ldb dimers and possibly with some other Islet-2-specific cofactors.

Expression of the neural RNA-binding protein Musashi1 in human gliomas.

Tumor cells arising from a particular tissue may exhibit the same gene expression patterns as their precursor cells. To test this proposition, we have analyzed the expression of a neural RNA-binding protein, Musashi1, in primary human central nervous system (CNS) tumors. In rodents, Musashi1 is expressed predominantly in proliferating multipotent neural precursor cells, but not in newly generated postmitotic neurons. The expression of Musashi1 is downregulated with the successive progression of neurogenesis. In normal adult human tissues, we detected low levels of Musashi1 expression in brain and testis by RT-PCR analysis. In an RNA panel of 32 cancer tissues and cell lines, elevated expression of Musashi1 was seen in all five malignant gliomas studied, in contrast to the slight expression seen in other tumor cells, including those in several melanomas and a prostate cancer. Western blot analysis showed strong Musashi1 expression in malignant gliomas compared with nonneoplastic brain tissue. Glioblastomas, the most malignant form of glioma, showed higher Musashi1 expression than less malignant gliomas by immunohistochemical analysis. Tumors with strong Musashi1 expression tended to have high proliferative activity. Thus, the expression of Musashi1 correlated with the grade of the malignancy and proliferative activity in gliomas. These results suggest that primary CNS tumors may share gene expression patterns with primitive, undifferentiated CNS cells and that Musashi1 may be a useful marker for the diagnosis of CNS tumors.

Treatment of human renal cell carcinoma by a conditionally replicating herpes vector G207.

PURPOSE: Surgical removal remains the only potentially curative therapy for renal cell carcinoma. In this study we evaluated the inhibitory effect of the replication competent engineered herpes simplex virus type 1, G207, for renal cell carcinoma in vitro and in vivo. MATERIALS AND METHODS: The nature of G207 enables it to replicate within cancer cells, thus, causing cytolysis, but replication is restricted within normal cells. The susceptibility of the human renal cancer cell lines ACHN and A498 to G207 at a multiplicity of infection of 0.1 was examined. In addition, the growth characteristics of G207 was assessed. In vivo athymic mice bearing subcutaneous tumors were inoculated with 1 x 10(7) plaque forming units of G207 intra-neoplastically. For pathological analysis subcutaneous tumors were stained with X-gal. RESULTS: Two cell lines were efficiently destroyed by G207 within 1 week. The viral yields of G207 increased in a time dependent manner. In vivo the intra-neoplastic inoculation of G207 caused significantly decreased tumor growth in athymic mice harboring subcutaneous human renal cancer cells. On day 14 the mean growth ratio of ACHN and A498 lesions was significantly inhibited in G207 treated compared to control tumors (p <0.005 and <0.0001, respectively). CONCLUSIONS: These results suggest that G207 should be considered another potential therapeutic agent for renal cell carcinoma.

Expression of peripheral myelin protein zero in sural nerve of patients with Charcot-Marie-Tooth disease 1B.

A Charcot-Marie-Tooth disease 1B (CMT1B) family with a mutation of the Po gene is presented. A to G substitution of nucleotide 389 in exon 3 resulted in Lys 131 Arg substitution. Immunostaining for Po in biopsied sural nerve from one family member with CMT1B was expressed in a small number of myelinated fibers. Immunoblot analysis for Po revealed that it was of normal molecular weight (29 kDa) although significantly reduced in amount. This heterozygous mutation could lead to a reduction in the total amount of normal protein in peripheral nerves through a mechanism of loss of function.

A novel monoclonal antibody K1 recognises early neurones in the rat cortex.

The aim of this study was to produce monoclonal antibodies specific for neurones that are generated earliest in the rat neocortex. One of the established clones, K1, showed a strong immunoreactivity in the marginal zone at the 19th day of gestation (E19). The immunoreactivity of K1 initially appeared in the primordial plexiform layer at E15, in the subplate at E16, and in the marginal zone by E17. It became undetectable in the first postnatal week. The immunoreactivity was not detected in the neocortex of adults or elderly. Western blotting analysis revealed reactive bands at positions corresponding to proteins of 290 and 280 kDa for the neocortical membrane fractions prepared from E16 to E21 embryos. In these stages, smears of bands were also found at positions corresponding to higher molecular weights. A single band of protein of 280 kDa was detected for the soluble fractions prepared from the embryos at E19 and E21. These reactivities were susceptible to protease, but not to enzymatic or chemical destruction of carbohydrate residues. Electron microscopic analysis showed that the K1 immunoreactivity was detected primarily on the cellular membranes of neurites. In the marginal zone at E19, the K1 immunoreactivity was localised where neurites make contact with other neurites or somata. A portion of these contact points had typical features of synapses. In the cortical plate of the same stage, arrays of tiny K1-immunoreactive puncta were observed on a subset of radial processes. These results suggest that monoclonal antibody K1 is a marker recognising neurites of subplate neurones that extend radially and make neuronal contacts in the marginal zone.

Conservation of K1 immunoreactivity against early cortical neurones in the vertebrate telencephalon.

A monoclonal antibody, K1, immunostains neurones generated earliest in the rat neocortex. The K1 immunoreactivity was found in both mouse and human embryos. In the human marginal zone, the subpial granular layer and the inner sublayer were stained at the 19th and 20th week of gestation, respectively. Western blot analysis revealed that the K1 immunoreactivity was conserved in a variety of vertebrates. While a protein of low molecular weight (200 kDa) reacted dominantly in an amphibian (Xenopus laevis) and a reptile (Agkistrodon blomhoffii), proteins of higher molecular weights (280 and 290 kDa) reacted dominantly in mammals (mouse, rat and macaque). In the brain of the reptile (Lacerta triliniata) embryo, K1 stained a marginal part of the superficial molecular layer in the dorsal cortex that is probably homologous to the mammalian marginal zone in the neocortex. In the chick embryo at the 8th day of incubation, the immunoreactivity was observed on neurones generated earliest in the dorsal cortex but not in the superficial molecular layer. The dorsal ventricular ridge and pallial thickening in either the reptile or chick were not stained. The K1 antigen could be a good marker for evolutional study of the mammalian neocortex.

The roles of cell adhesion molecules on the formation of peripheral myelin.

Several cell adhesion molecules of the immunoglobulin superfamily (IGSF) are expressed differently during the development and myelination of the peripheral nervous system. To examine the relationship between the expression of IGSF molecules and Schwann cell differentiation, we established a useful system for myelin formation in vitro on collagen gel using primary neuron/Schwann cell co-cultures from neonatal dorsal root ganglions (DRG). At 10 days in vitro (DIV), many Schwann cells were found in the areas surrounding aggregates of DRG neurons. After 20 DIV, Schwann cells positioned next to axons and elongated their processes along the axons. Some of them started loosely elaborating a large axon. Under electron microscopy, compact myelin was shown to be formed at 30 DIV. Thus the speed of myelination was much slower in vitro than in vivo. In co-cultures, L1 and neural cell adhesion molecule (NCAM) were detected at the premyelinating stage, L1 was precisely expressed earlier than NCAM. Expression of myelin associated glycoprotein (MAG) was transiently up-regulated at the early stage of myelination, and then P0 expression was finally increased as myelination proceeded. The change of expression pattern of these molecules in co-cultures was quite similar to that observed in the development in vivo. When Schwann cell proliferation was blocked by low serum culture condition, L1 and NCAM expressions were up-regulated. In contrast, the presence of cholera toxin in low serum media markedly increased expressions of P0 and MAG, but decreased the levels of both L1 and NCAM. These results suggest that both L1 and NCAM play roles in the contact and/or recognition between axons and Schwann cells at an early stage of myelination. On the other hand, MAG and P0 are important for axon ensheathment and myelin compaction.

Oncolytic viral therapy for human prostate cancer by conditionally replicating herpes simplex virus 1 vector G207.

Over the last few years, a conditionally replicating herpes simplex virus 1 (HSV-1) vector, G207 has been used for the treatment of several malignant tumors. In this article we evaluate the anti-tumoral effect of G207 against prostate cancer in vitro and in vivo. The susceptibility of the human prostate cancer cell lines, DU145 and PC3 to G207 at a multiplicity of infection (MOI) of 0.1 was examined. In addition, the growth characteristics of G207 were assessed. Athymic mice with s.c. tumors were inoculated in vivo intraneoplastically with 1 x 10(7) plaque-forming units (PFU) of G207. For the pathological analyses, s.c. tumors were stained with X-gal. DU145 and PC3 were efficiently destroyed by G207 within 7 days. The viral yields of G207 increased time-dependently. In vivo, the intraneoplastic inoculation of G207 induced a significant inhibition of the tumor growth. The mean tumor growth ratio was significantly inhibited in the G207-treated tumors (DU145, P < 0.0001; PC3, P < 0.001 versus controls). In a pathological study, many lacZ-positive cells were diffusely present in the G207-treated tumors. G207 showed a significant antitumoral effect against human prostate cancer cell lines, and thus may be considered a useful agent for the treatment of prostate cancer.

Lysenin-sphingomyelin binding at the surface of oligodendrocyte lineage cells increases during differentiation in vitro.

We have investigated the relationship between the developmental expression of sphingomyelin, a major component of myelin, and oligodendrocyte lineage. Using lysenin as a cytochemical probe for membrane sphingomyelin, we have now determined the distribution pattern of sphingomyelin on the plasma membrane of rat cultured oligodendrocytes. Although lysenin does not bind to A2B5(+)/NG2(+) bipolar oligodendrocyte progenitors, lysenin recognizes sphingomyelin on the cell bodies of multipolar A2B5(+) cells, but not on their processes. O4(+) and O1(+) immature and MBP(+) mature oligodendrocytes are strongly labeled by lysenin from cell bodies to the tips of processes. The content of sphingomyelin in immature and mature oligodendrocytes is approximately 2-fold higher than that in oligodendrocyte progenitors. These findings show that sphingomyelin increases during differentiation of cells in the oligodendrocyte lineage. In multipolar oligodendrocyte progenitors exposed to Triton X-100 at 4 degrees C, lysenin labels cell processes in addition to cell bodies. In contrast, Triton X-100 extraction does not alter the distribution of lysenin binding on O4(+), O1(+) and MBP(+) cells, although the immunocytochemical intensities of the lysenin bindings increase. Our data suggest that the alteration in sphingomyelin content and distribution in the oligodendrocyte lineage cells could have important consequences for cell recognition and downstream signaling events through sphingomyelin-rich domains.

Intravesical and intravenous therapy of human bladder cancer by the herpes vector G207.

G207, a conditionally replicating herpes vector, efficiently kills human bladder cancer cells in vitro. To evaluate the therapeutic potential of G207, we have established three in vivo models similar to the clinical situation. In vivo, G207 was intraneoplastically, intravesically, or intravenously inoculated in nude mice. Intraneoplastic inoculation into subcutaneous tumor caused significant tumor growth inhibition. Intravesical inoculation of G207 also caused decreased tumor growth in an orthotopic human bladder cancer model. Furthermore, multiple intravenous inoculation markedly inhibited subcutaneous tumor growth. These results suggest that intravesical therapy with G207 is effective for localized bladder tumor, especially for carcinoma in situ (CIS), and intravenous therapy with G207 is promising for invasive or metastasized bladder tumor.

Structure of a major oligosaccharide of PASII/PMP22 glycoprotein in bovine peripheral nerve myelin.

The amino acid sequence of the glycopeptide obtained from bovine PASII/PMP22 protein in the PNS myelin was determined to be Gln-Asn-Cys-Ser-Thr, where the asparagine was glycosylated. To eliminate all the contaminated P(o) glycopeptides from the PASII/PMP22 glycopeptide preparation, we used a fluorescent probe, N-[2-(2-pyridylamino)ethyl]maleimide, which reacts with the cysteine of the PASII/PMP22 glycopeptides. The labeled PASII/PMP22 glycopeptides were isolated by HPLC and were digested further with glycopeptidase A. The resultant oligosaccharides were conjugated with 2-aminopyridine (PA) as a fluorescent tag. One major PA-oligosaccharide, OPPE1, was purified by HPLC. The structure of OPPE1 was elucidated by fast atom bombardment mass spectrometry and (1)H-NMR studies and comparing the derivatives of PA-OPPE1 and PA-oligosaccharides of gamma-globulin on HPLC. The structure, SO(4)-3GlcAbeta1-3Galbeta1-4GlcNAcbeta1-2Manalpha1+ ++-6(GlcNAcbeta1-4) (GlcNAcbeta1-2Manalpha1-3)Manbeta1-4GlcNAcbeta1- 4(Fucalpha1-6)GlcNAc- PA, was identical to the pyridylaminated form of the major oligosaccharide D8 of bovine P(o) previously reported.

A role for CD40-CD40 ligand interactions in the generation of type 1 cytokine responses in human leprosy.

The interaction of CD40 ligand (CD40L) expressed by activated T cells with CD40 on macrophages has been shown to be a potent stimulus for the production of IL-12, an obligate signal for generation of Th1 cytokine responses. The expression and interaction of CD40 and CD40L were investigated in human infectious disease using leprosy as a model. CD40 and CD40L mRNA and surface protein expression were predominant in skin lesions of resistant tuberculoid patients compared with the highly susceptible lepromatous group. IL-12 release from PBMC of tuberculoid patients stimulated with Mycobacterium leprae was partially inhibited by mAbs to CD40 or CD40L, correlating with Ag-induced up-regulation of CD40L on T cells. Cognate recognition of M. leprae Ag by a T cell clone derived from a tuberculoid lesion in the context of monocyte APC resulted in CD40L-CD40-dependent production of IL-12. In contrast, M. leprae-induced IL-12 production by PBMC from lepromatous patients was not dependent on CD40L-CD40 ligation, nor was CD40L up-regulated by M. leprae. Furthermore, IL-10, a cytokine predominant in lepromatous lesions, blocked the IFN-gamma up-regulation of CD40 on monocytes. These data suggest that T cell activation in situ by M. leprae in tuberculoid leprosy leads to local up-regulation of CD40L, which stimulates CD40-dependent induction of IL-12 in monocytes. The CD40-CD40L interaction, which is not evident in lepromatous leprosy, probably participates in the cell-mediated immune response to microbial pathogens.

Antitumoral effects of defective herpes simplex virus-mediated transfer of tissue inhibitor of metalloproteinases-2 gene in malignant glioma U87 in vitro: consequences for anti-cancer gene therapy.

We set up experiments to evaluate the effects of defective herpes simplex virus (HSV)-mediated in vitro gene transfer of tissue inhibitor of metalloproteinases-2 (TIMP-2) in malignant glioma cells. Intrinsic TIMPs are known to be inhibitors of the strong invasive activities of matrix metalloproteinases in malignant gliomas. The defective HSV vectors dvSRaTIMP2 was engineered to express human TIMP-2 (hTIMP-2) with a combination of replication-competent HSV mutant, temperature-sensitive HSV-tsK, and amplicon plasmid-containing hTIMP-2. The hTIMP-2 gene was driven by the simian virus 40 promoter. The helper virus (HSV-tsK) was thermosensitive; consequently, this vector could proliferate only at 31.5 degrees C. After infection of U87 human glioblastoma cells with the vector in vitro, expression of TIMP-2 was confirmed by reverse zymography. The U87 cells infected in vitro either with dvSRaTIMP2 or HSV-tsK were efficiently destroyed under replication-permissive conditions (at 31.5 degrees C) and significantly lowered under replication-nonpermissive conditions (at 37 degrees C). The invasive activity of U87 was clearly inhibited by dvSRaTIMP2 infection at both 31.5 degrees C and 37 degrees C. Our studies suggest that TIMP-2 expressing the defective HSV vector is possibly useful for the treatment of malignant brain tumors.

Expression of L1 and TAG-1 in the corticospinal, callosal, and hippocampal commissural neurons in the developing rat telencephalon as revealed by retrograde and in situ hybridization double labeling.

In the telencephalon, the corticospinal (CS), callosal, and hippocampal commissural neurons are the major types of neurons that have axons crossing the midline of the brain. To understand the mechanisms involved in crossing the midline structure and to examine whether the expression patterns of L1 and TAG-1 in the commissural neurons are similar to those in the spinal cord, we investigated L1 and TAG-1 expression in these neurons in rats by using a double-labeling technique involving retrograde labeling and in situ hybridization. Expression of L1 messenger RNA was detected in the retrogradely labeled CS projection neurons by 1,1;-dioctadecyl-3,3, 3;,3;-tetramethylindocarbocyanine perchlorate (DiI) injection into the pons at embryonic day (E) 19, but expression of TAG-1 messenger RNA was not detected in these neurons. Also, after their axons crossed the pyramidal decussation, continued expression of L1 but no expression of TAG-1 in the CS projection neurons was shown by an additional double-labeling experiment involving DiI injection into the spinal cord at postnatal day (P) 1. An immunohistochemical study showed that L1 was continuously present in each level of the CS tract at E21 and P3, but TAG-1 immunoreactivity was not found in any level at any stage. Finally, we examined the expression of L1 and TAG-1 messenger RNAs in the callosal and hippocampal commissure neurons after their axons had crossed the midline by using the double-labeling technique. In both cases, hybridization signals of the L1 and TAG-1 messenger RNAs were observed in the retrogradely labeled neurons at P3. These results suggest that the roles of L1 and TAG-1 in the formation of the commissures in the forebrain are different from their roles in the spinal cord.

Application of conditionally replicating herpes vector for gene therapy treatment of urologic neoplasms.

Herpes vector has been widely used for experimental gene therapy. We herein review the strategies of such therapy for the treatment of urologic neoplasms. Most experimental studies of genetically altered viruses have employed replication-incompetent vectors. However, such viruses are unable to infect additional cells subsequent to the initial infection event. Therefore, this strategy has relied heavily on the bystander effect because a large number of noninfected tumor cells remain. Conditionally replicating herpes vector G207 has been developed in order to overcome potential problems of safety and tumor specificity for human use. It has been used to treat malignant brain tumors because of its neural tropism. In the last few years, applications of G207 for non-neural tumors have been reported. Because G207 may be useful for the treatment of urologic malignant tumors, we evaluated the antitumor effect against several types of tumor cells both in vitro and in vivo. Our data suggest that G207 may be applicable for the treatment of urologic malignant tumors.