Single-strand conformation polymorphism analysis on the delta-sarcoglycan gene in Japanese patients with hypertrophic cardiomyopathy.

To elucidate the etiology of hypertrophic cardiomyopathy (HC) in humans, we analyzed the delta-sarcoglycan gene (SG), which is reported to be the causal gene for HC in the Syrian hamster BIO14.6. We performed polymerase chain reaction (PCR) single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses on the delta-SG in 102 patients with HC. SSCP was detected in exon 2 of the gene, but not in the other exons. The direct sequencing analysis of exon 2 revealed a C-->T substitution at nucleotide residue 84 (TAC-->TAT) with no amino acid alteration (Tyr-->Tyr). There were no significant differences in allele frequencies of C/T between the patients with HC and the control group. Patients with HC were classified into 4 subgroups: obstructive HC, nonobstructive HC, apical HC, and familial HC. The allele frequency of C/T polymorphism in each of these groups was compared with that of the control group. The obstructive HC group showed a significantly greater frequency of the allele T than in the control group (31.6% vs 15.1%, RR = 2.6, p = 0.023). No other significant differences were observed. Thus, amino acid alteration in delta-SG may not be a common cause of HC in Japanese patients.

Bioartificial endocrine pancreas (Bio-AEP) for treatment of diabetes: effect of implantation of Bio-AEP on the pancreas.

Recently, we described a diffusion chamber for a bioartificial endocrine pancreas (Bio-AEP). Pancreatic islet cells in the Bio-AEP device were isolated from the immune system of the host by an artificial barrier, while nutrients, electrolytes, oxygen, and bioactive secretory products were exchanged across this barrier. This experiment was designed to evaluate whether the diffusion chamber could be useful as a Bio-AEP in the treatment of diabetes. Six streptozotocin (STZ)-induced diabetic rats each received a diffusion chamber containing 8 x 10(6) MIN6 cells as a xenograft Bio-AEP. In the STZ diabetic rats with Bio-AEPs, a return to normoglycemia was observed up to 30 weeks after implantation, without the use of any immunosuppressant. A gradual increase in the body weight of the rats was also observed. In three STZ diabetic rats, diffusion chambers without MIN6 cells were implanted as a sham operation. The fasting blood glucose levels in these three rats remained higher than 600 mg/dl, after implantation, and they lost weight. Thirty-five weeks after implantation, the pancreata were removed from the rats that underwent xenoimplantation, those that had the sham operation, and the normal control rats. In the sham-operated animals, the exocrine tissues of the pancreata were vacuolated and pancreatic B cells were not seen in the islets. In contrast, in the pancreata from the xenoimplantation, the exocrine tissues were normal, and a few pancreatic B cells were seen in the islets. These results indicated that xenoimplantation using the Bio-AEP might retard the progress of diabetes.

Membrane immunoisolation of a diffusion chamber for bioartificial pancreas.

Immunoisolation is a potentially important approach to transplanting islets without any immunosuppressive therapy. The concept of immunoisolation is outlined in systems in which the transplanted tissue is separated from the immune system of the host by an artificial barrier. We previously described a diffusion chamber as a bioartificial endocrine pancreas (Bio-AEP), which was constructed by placing pancreatic endocrine cells, trapped in a mixed matrix, in the center of a ring holder sandwiched between nucleopore membranes, which were shielded by silicone. This experiment was designed to evaluate a suitable pore size for the nucleopore membrane to ensure immunoisolation during xenoimplantation of the Bio-AEP in vitro and in vivo. A nucleopore membrane of pore size 0.1 microm or 0.2 microm was employed as the semipermeable membrane which provided a mechanical barrier between the endocrine pancreas graft and the host immune system. The protective effect of the Bio-AEP from humoral immunity was determined in vitro, using sensitized sheep erythrocytes (EAs). A complement protein did not destroy the cell membranes of the EAs in the diffusion chamber containing the mixed matrix with the nucleopore membrane of 0.1 microm pore size. In an in vivo experiment, 6 streptozotocin (STZ) induced diabetic rats were implanted with Bio-AEPs constructed with nucleopore membranes of pore size 0.1 microm and containing MIN6 cells in the mixed matrix. In the STZ diabetic rats with Bio-AEPs, a return to normoglycemia was observed up to 50 weeks after implantation without the use of any immunosuppressant. Also, the body weights of the rats gradually increased. During the observation, when the Bio-AEPs were removed from the STZ diabetic rats, the blood glucose immediately returned to preimplantation levels, and the body weights of the rats also decreased. The membranes of the Bio-AEPs removed from the STZ diabetic rats showed a very thin layer of fibroblastic cells on the outer surfaces. The results indicated that the Bio-AEP, in which pancreatic endocrine cells were trapped in a mixed matrix and with a 0.1 microm pore size membrane, should be useful for xenoimplantation into diabetic animals and may open a new field in the therapy of human diabetics.

Complement permeability in bio-artificial endocrine pancreas using a diffusion chamber.

Immunoisolation is a potentially important approach to transplanting islets without any immunosuppressive therapy. This study was designed to investigate whether complement penetrates into the diffusion chamber that we developed for a bioartificial endocrine pancreas (Bio-AEP). The results showed that within 12-24 h, when a Nuclepore membrane (Nuclepore Corp., California) was the sole barrier, complement permeated 100%. However, the Nuclepore membrane used together with a mixed matrix prevented complement penetration. Also, the complement become inactivated during penetration through both the mixed matrix and the Nuclepore membrane with a pore size of 0.1 microm. In conclusion, immunoisolation of cells by a mixed matrix and a Nuclepore membrane of 0.1 microm pore size is a particularly effective method for transplanting xenogeneic cells. Thus, the clinical application of our Bio-AEP shows promise for long-term xenotransplantation without an immunosuppressant.

[Reaction of 3-aryl-2-nitroacrylates with titanium tetrachloride. Formation of 4H-1,2-oxazines and hydroxy-arylaldehydes].

The reaction of ethyl 3-aryl-2-nitroacrylate (1a: aryl = 3-methoxyphenyl) with toluene in the presence of titanium tetrachloride gave 4-(4'-methyl-phenyl)-4H-1,2-benzoxazine (3) in a 44.2% yield. The acrylate 1a reacted with dichloromethane in the presence of titanium tetrachloride to give 5-methoxy-salicylaldehyde (2a) in a 61.8% yield. Therefore, 2a was presumed to be formed via an intermediate 4H-1,2-benzoxazine (3'), followed by ring opening to quinone methide and cyano formate. In a similar reaction using both toluene and dichloromethane, 1 bearing a 2-naphthyl group gave a dimer of quinone methide and 1-hydroxy-2-naphthaldehyde via 4H-naphth[2,1-e]-1,2-oxazine, respectively.

[Flow cytometric analysis of immunophagocytosis using sensitized fluorescent microspheres bearing C3b].

We analyzed the phagocytic activity of purified human monocytes using fluorescent latex beads sensitized with IgG or IgG.C3 by flow cytometry. To prepare IgG-sensitized latex beads (BA), BSA-coated latex beads (B) were incubated with diluted rabbit IgG anti-BSA. To bind complement components, BA were incubated with whole serum pretreated with K-76 monocarboxylic acid (K-76COOH). K-76COOH inhibits the activity of factor I and C5, resulting in deposition of C1, C4b, C2a, C3b on BA (BAC). Phagocytic activity was assessed by percent phagocytosis and phagocytic index (PI). To eliminate the effects of non-phagocytosed latex beads, subtraction of the data at 4 degrees C from 37 degrees C was performed. Percent phagocytosis for 60 min. was B 5.0%, BA 18.3%, and BAC 57.5%, and PI (ingested latex beads/100 cells) was B 7.9, BA 36.8, and BAC 152.7, respectively. In addition, K-76COOH caused dose dependent inhibition on IgG.C3 mediated phagocytosis. Comparison of inhibition pattern on BAC and BA indicated that K-76COOH directly inhibited C3.C3-receptor binding.

Endoscopic diagnosis of catheter trouble in CAPD patients.

In case of CAPD catheter trouble, we applied a new diagnostic approach using industrial fiberoptics. This method was safe and useful to specify the causes of the catheter trouble.