Downregulation of Max dimerization protein 3 is involved in decreased visceral adipose tissue by inhibiting adipocyte differentiation in zebrafish and mice.

BACKGROUND: The diet-induced obesity model of zebrafish (DIO-zebrafish) share a common pathophysiological pathway with mammalian obesity. OBJECTIVES: We aimed to investigate the role of Max dimerization protein 3 (MXD3) in visceral fat accumulation and adipocyte differentiation, by conducting knockdown experiments using zebrafish and mouse preadipocytes. METHODS: To identify genes related to visceral adiposity, we conducted transcriptome analyses of human and zebrafish obese populations using the Gene Expression Omnibus and DNA microarray. We then intraperitoneally injected morpholino antisense oligonucleotides (MO-mxd3) to knockdown mxd3 gene expression in DIO-zebrafish and measured several parameters, which reflected human obesity and associated metabolic diseases. Finally, lentiviral Mxd3 shRNA knockdown in mouse 3T3-L1 preadipocytes was conducted. Quantitative PCR analyses of several differentiation markers were conducted during these gene knockdown experiments. RESULTS: We found that MXD3 expression was increased in the obese population in humans and zebrafish. Intraperitoneal MO-mxd3 administration to DIO-zebrafish suppressed the increase in body weight, visceral fat accumulation and the size of mature adipocytes. Subsequently, dyslipidemia and liver steatosis were also ameliorated by MO-mxd3. In mouse adipocytes, Mxd3 expression was drastically increased in the early differentiation stage. Mxd3 shRNA inhibited preadipocyte proliferation and adipocyte maturation. Quantitative PCR analyses showed that the early differentiation marker, CCAAT/enhancer-binding protein delta (Cebpd) and late differentiation markers (CCAAT/enhancer-binding protein, alpha and peroxisome proliferator-activated receptor gamma) were downregulated by Mxd3 knockdown in 3T3-L1 cells and DIO-zebrafish. Subsequently, mature adipocyte markers (adiponectin and caveolin 1 for zebrafish, and fatty acid binding protein 4 and stearoyl-coenzyme A desaturase 1 for mouse adipocytes) were also decreased. CONCLUSION: Mxd3 regulates preadipocyte proliferation and early adipocyte differentiation via Cebpd downregulation in vitro and in vivo. Integrated analysis of human and zebrafish transcriptomes allows identification of a novel therapeutic target against human obesity and further associated metabolic disease.

Vasoactive intestinal peptide and inflammatory cytokines enhance vascular endothelial growth factor production from epidermal keratinocytes.

BACKGROUND: Overexpression of vascular endothelial growth factor (VEGF) in epidermal lesions of psoriasis is well documented; however, its underlying mechanisms are largely unknown. We have recently demonstrated that vasoactive intestinal peptide (VIP) induces the production of cytokines such as interleukin-6 and stem cell factor from keratinocytes, thereby contributing to the development of inflammatory dermatoses such as psoriasis. OBJECTIVES: In this study, we attempted to determine whether VIP could increase the production of VEGF in human keratinocytes. METHODS: We examined the expression of VEGF using reverse transcription-polymerase chain reaction, immunocytochemistry, enzyme-linked immunosorbent assay and immunoblotting in normal human epidermal keratinocytes and human epidermal keratinocyte cell line DJM-1 cultured in the absence or presence of VIP and/or inflammatory cytokines. RESULTS: We demonstrate that human keratinocytes produced VEGF in a steady state at both mRNA and protein levels. VIP significantly upregulated the production of VEGF in keratinocytes in a dose- and time-dependent manner. The VIP-mediated production of VEGF was further enhanced by inflammatory cytokines such as interferon-gamma, tumour necrosis factor-alpha and interleukin-4, with maximum enhancement being observed with the combination of VIP and interferon-gamma. CONCLUSIONS: VIP and other cytokines from nerve endings, mast cells and local inflammatory cells are capable of enhancing VEGF production from epidermal keratinocytes, which may underlie excessive angiogenesis and vasodilation in skin lesions of psoriasis.

An anti-alpha-fetoprotein antibody-daunorubicin conjugate with a novel poly-L-glutamic acid derivative as intermediate drug carrier.

In studies on antitumor antibody-cytotoxic drug conjugates as potential antitumor agents with improved tumor specificity, daunorubicin [(DM) daunomycin] was conjugated with an affinity-purified horse antibody to rat alpha-fetoprotein (AFP) with a novel derivative of poly-L-glutamic acid (PLGA) as the intermediate drug carrier. A single masked thiol group first was introduced by PLGA, and the thiol group was generated from it after the linking of DM to PLGA at the carboxyl groups of PLGA. The thiol group was used selectively for binding PLGA-DM to antibody that had been modified so as to have the maleimide groups. The conjugates (DM:PLGA:immunoglobulin molar ratio, 19.6:2.8:1 or 11.8:1.1:1) were more potent than DM in in vitro cytotoxicity against the AFP-producing rat ascites hepatoma cell line AH66. In therapeutic experiments, the conjugates were more efficacious in prolonging the lives of AH66 hepatoma-bearing DONRYU rats than DM, antibody, a mixture of DM and antibody, or a conjugate similarly prepared with normal horse immunoglobulin.

In vitro cytotoxicity of a human serum albumin-mediated conjugate of methotrexate with anti-MM46 monoclonal antibody.

In studies on antitumor antibody:drug conjugates as potential antitumor agents, methotrexate (MTX) was conjugated with a murine monoclonal antibody (aMM46) to an antigen on ascitic mouse mammary tumor MM46 cells (MM antigen) with human serum albumin (HSA) as an intermediary. MTX was linked to HSA which had been conditioned to have about 1 mol of thiol group per mol of HSA by dithiothreitol treatment followed by oxidation on standing at 4 degrees C. The MTX linking was performed, without protection of the thiol group of HSA, by using MTX N-succinimidyl ester prepared via MTX intramolecular anhydride. The resulting HSA:MTX was reacted with the immunoglobulin with the maleimide group introduced. The aMM46:HSA:MTX obtained retained both antibody binding and drug activities. The cytotoxicity of aMM46:HSA:MTX against MM antigen-positive MM46 cells was greater than that of control 96.5 (anti-human melanoma-associated antigen, p97):HSA:MTX and was inhibited by unconjugated aMM46. No different cytotoxicity of aMM46:HSA:MTX compared with that of 96.5:HSA:MTX was observed against MM antigen-negative mouse mammary tumor MM48 cells. The presence of ammonium chloride or leupeptin abrogated the selective cytotoxicity against MM46 cells of aMM46 conjugate but did not affect the nonspecific cytotoxicity of 96.5:HSA:MTX. These results support the idea that the selective cytotoxicity of aMM46:HSA:MTX is antibody directed and exhibited through lysosomal degradation of the conjugate.

Nature of linkage and mode of action of methotrexate conjugated with antitumor antibodies: implications for future preparation of conjugates.

The binding of methotrexate (MTX) to IgG in conjugates was examined by studies on a direct MTX conjugate with a monoclonal antibody (aMM46) to mouse mammary tumor MM46 cells and corresponding irrelevant antibody and normal gamma-globulin conjugates, all prepared by the active ester method with MTX N-succinimidyl ester (MTX-OSu). The binding was examined in terms of effects on the potency and selectivity of the cytotoxic activity of the aMM46 conjugate. The results obtained supported the following conclusions: (a) MTX-OSu reacts not only with the amino group of IgG to give an amide bond, but also with another group(s) to give a less stable bond(s) such as an ester bond; (b) in contrast to the amide bond-linked MTX, which is taken up by the cells by endocytic internalization, a substantial portion of the MTX linked by an ester or other less stable bond(s) is released from the conjugates extracellularly and enters the cells by the MTX active transport system, as revealed by the inhibitory effect of thiamine pyrophosphate on the cytotoxicity; (c) this MTX linked by a less stable bond(s) that causes nonspecific cytotoxicity can be removed by treatment with hydroxylamine; (d) the overall cytotoxicity of aMM46-MTX decreased on removal of this less stably linked MTX, suggesting that the lysosomal degradation of the conjugate carrying amide bond-linked MTX to liberate MTX derivatives of low molecular weight is insufficient; (e) the liberation of low-molecular-weight substances in the lysosomes is probably more important for efficient entry of active substances into the cytosol, than for inhibition of the activity of dihydrofolate reductase, because after hydroxylamine treatment, the amide bond-linked MTX showed greater decrease in drug cytotoxicity than in inhibitory activity against dihydrofolate reductase; (f) in combination with hydroxylamine treatment, insertion between MTX and IgG of a linkage capable of ready cleavage in lysosomes deserves exploitation as a method for making potent conjugates with less nonspecific activity.

The cloning of eggplant seedling cDNAs encoding proteins from a novel cytochrome P-450 family (CYP76).

Using a CYP75 cDNA as a probe, we have cloned and sequenced two closely related cDNAs from eggplant seedlings, which encode typical cytochrome P-450 (P450) proteins. A database search revealed that the predicted proteins share less than 40% amino acid homology with other P450 proteins, which suggests that they form a novel P450 gene family (CYP76).

cDNAs sequences encoding cytochrome P450 (CYP71 family) from eggplant seedlings.

Three cytochrome P450 (P450) cDNAs were isolated from an eggplant hypocotyl cDNA library using eggplant CYP75 cDNA as a probe. These cDNAs have greater than 65% identity in their amino acid sequences, indicating that they belong to the same family. Comparison of these with P450 proteins from other sources showed that the protein with the greatest degree of homology is CYP71, isolated from avocado fruits (approximately 48%). We concluded that they are novel members of the CYP71 gene family (CYP71A2, 3 and 4). We have examined the level of mRNA transcripts from the CYP71 family in eggplant hypocotyl tissues and petunia flower buds, and found that the level of transcripts is developmentally regulated in the flower bud.

A novel covalent modification of antibodies at their amino groups with retention of antigen-binding activity.

A novel method of covalent modification of antibodies at their amino groups with retention of antigen-binding activity is described. The procedure is as follows: (a) blockade of those amino groups of antibodies whose integrity is essential to their antigen-binding activity with 2,3-dimethylmaleic anhydride, a reversible amino group-blocking reagent; (b) modification of residual amino groups with reagents reactive with the amino groups; and (c) removal of dimethylmaleyl groups by hydrolysis. This procedure was used for covalent conjugation of methotrexate (MTX) with two monoclonal antibodies against human melanoma-associated antigens using MTX N-succinimidyl ester. MTX attached to the antibodies at sites other than the amino groups via less stable bond(s) was removed by treatment with hydroxylamine.

A novel method of conjugation of daunomycin with antibody with a poly-L-glutamic acid derivative as intermediate drug carrier. An anti-alpha-fetoprotein antibody-daunomycin conjugate.

In studies on antitumor antibody-cytotoxic drug conjugates as potential antitumor agents with improved tumor specificity, daunomycin (DM) was first linked to a poly-L-glutamic acid (PLGA) derivative having a single masked thiol group. At the thiol group, DM-linked PLGA was bound to horse anti-rat alpha-fetoprotein (AFP) antibody. The anti-AFP antibody-PLGA-DM conjugate (anti-AFP conjugate, DM/PLGA/Ig molar binding ratio, 7.5/1.2/1.0) retained most of the antigen-binding activity of the parent antibody and was more potent than either unconjugated DM, a conjugate similarity prepared with normal horse immunoglobulin (normal conjugate), or an unconjugated mixture of anti-AFP antibody and DM in an in vitro cytotoxicity assay against the AFP-producing rat ascites hepatoma cell line AH66. Anti-AFP conjugate tended to be less cytotoxic than DM against the AFP-nonproducing rat ascites hepatoma AH272 cells, and in this case there was no difference between the cytotoxicities of anti-AFP conjugate and of normal conjugate.

Importance of the antigen-binding valency and the nature of the cross-linking bond in ricin A-chain conjugates with antibody.

As a continuation of our work on toxin A-chain conjugates with antitumor antibodies for selective delivery of the toxin to the target cells, four ricin A-chain conjugates were prepared by linking A-chain to Fab' or F(ab')2 of rabbit IgG against L1210 with or without employing a cross-linking agent, N,N'-o-phenylenedimaleimide (PDM), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or N-succinimidyl m-(N-maleimido)benzoate (SMB), and the effects of antigen-binding valency and of the nature of the cross-linking bond on their in vitro cytotoxicity were studied. The relative potencies of the conjugates in terms of IC94's were as follows: F(ab')2-SPDP-A-chain, 100; Fab'-S-S-A-chain, 21; F(ab')2-SMB-A-chain, 1.3; Fab'-PDM-A-chain 0.38. Among the four conjugates, F(ab')2-SPDP-A-chain and Fab'-S-S-A-chain can be cleaved into the homing and the cytotoxic components with 2 mM 2-mercaptoethanol. These results suggest that divalency in antigen-binding and susceptibility of the cross-linking bond to cleavage by mercapto reagent are desirable for high potency. Protein synthesis in a cell-free system of rabbit reticulocyte lysate was inhibited by Fab'-S-S-A-chain and by Fab'-PDM-A-chain as effectively as by free A-chain, indicating that the liberation of A-chain is not important, at least on ribosomes, but it is important for the A-chain to reach a ribosome after binding of the conjugates to the cell-surface.

Ultraviolet light is an environmental factor aggravating facial lesions of adult atopic dermatitis.

An increasing number of adult patients with atopic dermatitis (AD) in Japan are distressed by persistent eczematous lesions of the face (so-called atopic red face). Phototests were carried out in 28 patients with the atopic red face to test a possibility that ultraviolet (UV) light could be an aggravating factor. Contact and photocontact dermatitis had been ruled out by repeated patch and photopatch tests. All of the patients had a normal response to a screening dose of UVA (10 J/sq cm) and a normal minimal erythema dose (MED) of UVB. Ten of these patients, however, showed an abnormal papular response to a single or 3-times consecutive UVB radiation above the MED (90 mJ/sq cm).

[Anti-tumor activity of an anti-human alpha-fetoprotein antibody-human serum albumin-mitomycin C conjugate against human yolk sac tumor maintained in vitro and in nude mice in vivo].

The anti-cancer drug, mitomycin C (MMC), was conjugated to an affinity-purified horse antibody to human alpha-fetoprotein (aAFP) via purified human serum albumin (HSA) as an intermediate carrier. The conjugate (aAFP immunoglobulin (IgG): HSA: MMC, molar ratio 1: 1.10:29.8) was 21 or 38 times as cytotoxic as free MMC or PBS at the MMC concentration of 100 ng/ml, respectively, against alpha-fetoprotein-producing human yolk sac tumor in vitro. The in vivo effects of the conjugate and various controls were also tested against human yolk sac tumor growing in nude mice. The conjugate over a total of 6 injections retarded the initial tumor growth at the concentration of MMC, containing equivalent amounts of 2 micrograms/mouse (0.1 mg/kg)/injection in the conjugate, whereas free MMC and normal horse IgG-HSA-MMC showed only slight inhibitory effects alone at non-toxic levels. These results suggest that the specific antibody-conjugate was considerably more effective than free MMC against the tumor maintained in nude mice as well as in vitro cultures.

[Involvement of substance P in gastric mucosal lesions of patients with rheumatoid arthritis].

We investigated the relation between gastric mucosal lesions and substance P (SP) in 64 patients with rheumatoid arthritis (RA) taking nonsteroidal antiinflammatory drugs (NSAIDs). In these patients, the incidence of gastric mucosal lesions was as high as 53.1%. Serum SP levels were significantly higher in patients with gastric mucosal lesions than in those without gastric lesions. Erythrocyte sedimentation rate, serum C-reactive protein and rheumatoid factor (RF) levels were also higher in patients with gastric mucosal lesions. A positive correlation between serum SP and RF levels was found in patients with RA. Experimental gastric mucosal lesions induced by an oral administration of indomethacin in rats were significantly enhanced by an additional intraperitoneal injection of SP. From these observations, it is suggested that, in addition to the effect of NSAIDs, SP elevation in blood has a role in the development of gastric mucosal lesion in patients with RA.