Secretion in yeast of human lysozymes with different specific activities created by replacing valine-110 with proline by site-directed mutagenesis.

Computer graphics indicate that a steric hindrance exists between valine-110 side chain of human lysozyme (EC 3.2.1.17) and an acetyl group of a modified substrate that contains N6,O-diacetylmuramic acid. To alter the substrate specificity of human lysozyme to be effective on the modified substrate, we replaced the valine-110 residue with various amino acids by site-directed mutagenesis. One of the mutant proteins (valine residue replaced with proline:P110) was secreted in Saccharomyces cerevisiae as at least four components (P110-A, P110-B, P110-C, and P110-D) with different specific activities. Two components, P110-B and P110-D, were isolated in a pure form and structurally characterized. The results suggest that this mutation lowered the lytic activity against Micrococcus lysodeikticus by changing a local conformation of the catalytic site while keeping almost the same substrate binding sites. Our results also indicate that cis/trans isomerization of prolyl peptide bonds probably occurs in vivo and that the conformational change of protein as well as point mutations in genes might influence the molecular evolution of the protein.     

Osteogenesis associated with bone gla protein gene expression in diffusion chambers by bone marrow cells with demineralized bone matrix.

Diffusion chambers with rat bone marrow cells and demineralized bone matrix (DBM) were implanted subcutaneously to syngeneic 8-week-old rats and were harvested every week 3-7 weeks after implantation, and histochemical examination, determination of alkaline phosphatase activity, total calcium and phosphorus, the bone-specific vitamin K-dependent gla-containing protein (BGP) content, and detection of BGP mRNA relative to mineralization were performed. Alkaline phosphatase in diffusion chamber implants reached the highest activity at 4 weeks and then decreased. Calcium and phosphorus deposits occurred at 4 weeks after implantation and were followed by marked increases until 7 weeks, which was comparable to the accumulation of BGP. The BGP gene within the diffusion chambers began to be expressed at 5 weeks, and its expression increased markedly at 7 weeks after implantation. At 4-5 weeks after implantation, new bone adjacent to the membrane filters and cartilage toward the center of the diffusion chamber were observed histochemically. Light microscopic and immunohistologic examinations of chambers with marrow cells and DBM revealed production of mineralized matrices, typical of bone characterized by the appearance of BGP and mineralized nodules. In contrast, bone marrow cells alone did not show extensive bone formation and yielded very low values for these biochemical parameters. The present experiments demonstrate the potential of bone marrow cells and DBM to produce not only cartilage formation but also membranous bone formation associated with increasing expression of BGP mRNA during the later stages of bone formation, as well as a marked accumulation of BGP.     

A report on two siblings with vitamin D resistant rickets treated with 1 alpha-hydroxy-vitamin D3 until completion of the growth]

The treatment of vitamin D resistant rickets still raises much controversy. This report describes out experiences on 1 alpha OHD3 treatment in two siblings from childhood till completion of the growth. The treatment began from age 3 and 8 in these two patients respectively. The daily doses were necessary to maintain the healing and increased during the growth period so that the doses per body weight were virtually constant at 1 microgram/kg/day. Under this regimen, the improvement of rickets was recognized in the radiogram, and the lower leg deformity corrected. In one case the bone biopsy confirmed the improvement of disturbed calcification. The final height was 152.5 cm (-0.8 SD) and 149 cm (-1.4 SD) respectively suggesting the effectiveness of 1 alpha OHD3 on the growth promotion. Around the closure of growth plate, the regimen induced hypercalcemia. Thereafter the doses were reduced to one fourth or one fifth of those in the growth period.     

Relationships of site of infarction and history of previous infarction with short- and long-term prognosis after acute myocardial infarction in Japan.

We studied the outcome in 308 patients with acute myocardial infarction (MI) admitted to the coronary care unit of Kobe General Hospital. Short-term outcome (within 28 days after MI) and long-term outcome (more than 28 days) were examined with survival curves to find any relationship with a history of previous MI and with the site of the MI. In the short term, 38 of the 308 patients died of cardiac causes. The group with anterior MI tended to have higher mortality than the group of patients with inferior MI, and among patients without a previous MI, patients with anterior MI had significantly higher mortality (p = 0.01). In multivariate analysis by the logistic regression model, the site of the MI was found to be independently associated with the short-term outcome. In the long term, with a mean follow-up of 3.4 years, 23 of the 308 patients died of cardiac causes. Different sites of the MI did not result in different outcomes in patients with or without a previous MI. Of patients with anterior or inferior MI, those with a previous MI tended to have higher mortality, and of patients with an inferior MI, the difference was significant (p = 0.001). In multivariate analysis by the proportional hazards model, a history of MI was more predictive than the site of the MI. In conclusion, the site of the MI was associated more with the short-term outcome than with the long-term outcome, and a history of MI was associated more closely with the long-term outcome.     

The inclusion of an omental flap in pancreatoduodenectomy

A technique for reducing the morbidity and mortality of pancreatoduodenectomy by using an omental flap to protect the anastomoses and splanchnic vessels exposed during dissection is described herein.     

Effect of FK-506 on xenografted human Graves' thyroid tissue in severe combined immunodeficient mice

OBJECTIVE We studied the macrolide antibiotic FK-506, an immunosuppressive agent, in an attempt to ameliorate the lesion of autoimmune thyroid disease in human thyroid tissue xenografted into severe combined immunodeficient (SCID) mice. It was not felt appropriate to employ this agent directly in patients with autoimmune thyroid disease because adequate therapeutic modalities are available and the introduction of new, experimental agents could not be justified. Moreover, the study of the tissue before and after treatment could not have been undertaken directly in patients. DESIGN Human thyroid xenografts from four patients with Graves' disease and two normal persons were xenografted into SCID mice. Two weeks after xenograft-ing, human immunoglobulin G (IgG) was detectable in all SCID mice xenografted with Graves' thyroid tissue. Mice were divided into two groups with human IgG levels similar to each other. Mice in the first group were treated with FK-506 daily for 6 weeks; mice in the second (similar) group were given phosphate-buffered saline (PBS) only (control group). MEASUREMENTS Blood samples were taken every 2 weeks from the tail veins for human IgG, thyroid stimulating antibody, thyroperoxidase antibodies, thyroglobulin antibodies, and interferon-gamma (IFN-7). After 8 weeks treatment, animals were sacrificed; thyroid tissue was examined histologically and for thyrocyte HLA-DR expression. FK-506 was also added to thyrocytes in in-vitro tissue culture conditions. RESULTS After 4–6 weeks of FK-506 therapy, human IgG, all thyroid antibodies and IFN-7 were suppressed, while the levels remained elevated in the control group. Lymphocytic infiltration virtually disappeared in the human thyroid tissue of the FK-506-treated mice and thyrocyte HLA-DR expression markedly declined; in the control mice, lymphocytic infiltration remained heavy and HLA-DR expression remained high. On the other hand, FK-506 added directly to thyrocytes in vitro (without lymphocytes) did not reduce thyrocyte HLA-DR expression. CONCLUSIONS FK-506 appears to suppress the activation of intrathyroidal lymphocytes, but not thyrocytes. From these observations, it is concluded that this agent, by its action on intrathyroidal lymphocytes, is able to ameliorate the immunologically mediated histological and serological disturbance in human autoimmune thyroid disease, at least under these circumstances.     

Independent association of antibodies against human papillomavirus type 16 and E7 proteins with cervical cancer

The E4 open reading frame (ORF) of human papillomaviruses (HPVs) is transcribed in abundant mRNAs encoding an fusion gene during the productive infection, and the HPV 16 E7 ORF encodes an oncoprotein detectable in the cell lines derived from cervical carcinoma. We examined 421 human sera, which included 108 samples from the patients with cervical carcinoma, for the presence of IgG antibodies against the HPV 16 E4 and E7 proteins by enzyme-linked immunosorbent assay. Bacterially expressed fusion protein lac- and nonfusion protein E7 were purified and used as antigens. All of the 22 serum samples positive for anti-E7 antibody and the 11 out of 15 samples positive for anti- antibody were from the patients with cervical carcinoma, but only one sample was found to contain both anti- and anti-E7 antibodies. These findings show specific and independent association of these antibodies with cervical carcinoma.     

Maintenance of Androgen-, Glucocorticoid- or Estrogen-responsive Growth in Shionogi Carcinoma 115 Subline Sustained in Castrated Mice with High Dose of Estrogen for 30 Generations (3 Years)

Shionogi carcinoma 115 (SC115), an androgen-dependent mouse mammary tumor, rapidly loses its androgen responsiveness after androgen withdrawal. The growth of this tumor can also be stimulated by high doses of estrogen or glucocorticoid. In the present study, the maintenance of hormone-responsive growth of SC115 tumors with a high dose of estrogen was examined in castrated male mice using an SCI 15 subline obtained by serial transplantations of SCI 15 tumors in estrogen-treated castrated mice for 3 years (30 generations) (subline E₂). Seed tumors from both SC115 and subline E₂ could rapidly grow in castrated mice given daily injections of testosterone propionate (TP), 17β-estradiol (E₂), or dexamethasone (Dex) (100 μg/mouse/day) but not in those given vehicle alone. Although SCI 15 and subline-E₂ tumors grown with TP or Dex showed temporary regression after steroid withdrawal, the tumors grown with E₂ did not show such temporary regression. The TP-, E₂-, or Dex-induced growth of subline-E₂ tumors was almost the same as that of the original SCI 15 tumors. However, responsiveness to androgen, estrogen or glucocorticoid of both tumors disappeared within one passage in steroid-depleted castrated mice. The present findings demonstrate that the loss of responsiveness to androgen as well as to high doses of estrogen or glucocorticoid of SCI 15 tumors can be prevented in castrated mice not only with androgen but also with high doses of estrogen.