Stability of subtilisins and related proteinases (subtilases)

The stability towards thermal and chemical (guanidine hydrochloride, GnHCl) denaturation of six inhibited subtilases (mesentericopeptidase, subtilisins BPN′, Carlsberg and DY, proteinase K and thermitase) has been investigated by kinetic and equilibrium studies. The unfolding processes were monitored by circular dichroic and fluorescence spectroscopy. Experiments in the absence and presence of extraneous calcium in the concentration range 2×10^?3-10^?1 M were performed. The presence of calcium in the weak calcium binding site changes the denaturation drastically. The heat- (or GnHCl-) induced unfolding curves obtained using CD spectroscopy show two independent transitions which seem not to have been resolved before. The presence of Ca²⁺ in the second (third in the case of thermitase) binding site increases the T_m, values by 11-21 °C and the δG_D(H₂O) values obtained from denaturation experiments in GnHCl by 6.7-7.2 kcal/mol when an extraneous Ca²⁺ concentration of 2 × 10^?2 M was used. One interpretation is that the initial step of denaturation in the presence of added calcium is the formation of a partially unfolded intermediate form, retaining a highly ordered structure with 60-85% of the a-helix structure of the native enzyme. This intermediate then unfolds at a temperature considerably higher than that of the same proteinases in the absence of added Ca²⁺. The free energy of stabilization of the intermediates is increased by 1.8-2.8 times in comparison with that for the unfolding reactions of the subtilases with empty Ca2/Ca3 binding sites. A second interpretation is that the two steps in the unfolding curves correspond to enzyme without and with calcium in the weak binding site. Fluorescence experiments confirm the mechanism involving the formation of intermediate states. The results are discussed in relation to the X-ray models of the six subtilases.     

用Sauvegrain法测定青春期骨龄准确性的研究

背景：Sauvegrain方法是通过分析肘部X线片来评估骨龄，常用于青春期生长发育最为迅速的两年间的骨龄测定。本文旨在研究该方法的准确性以及在小儿骨科中的应用价值。 方法：Sauvegrmn法主要评估肘部的4个解剖标志点：外髁、肱骨滑车、尺骨鹰嘴突以及桡骨近端骨骺。它是一个27分制的评分系统。对上述结构所得的评分进行合计而得出一个总分，然后使用标准图表确定骨龄。让三位观察者分别利用该方法进行骨龄评估。三位观察者通过分析60个男孩和60个女孩样本左肘部的前后位和侧位X线片来测定骨龄，并将该结果与通过分析左手和腕部后前位X线片的Greulich和Pyle图谱而得出的骨龄结果进行比较。间隔4周后每个观察者再测定骨龄一次。 结果：通过分析肘部X线片的方法测定骨龄更为精确，因为采用该方法测定骨龄可以精确到半岁。根据观察者的评定，Sauvegrain法显示出很好的观察者之间的相关性（r=0．93）和可重复性（r=0．96）。Sauvegrain法与Greulich和Pyle图谱之间有很好的相关性（r=0．85）。然而，一些肘部生长中心显示出一种中间的发育形态，这种形态学不适合Sauvegrain法的评分。这就导致了数据分析时出现误差。我们建议对于这些样本设定中间评分，并且通过修改原始图表而使评分更为精确。 结论：改良的Sauvegrain法简单、可靠而且可重复性高，它补充了Greulich和Pyle图谱的不足。在临床实践中，骨骼成熟度可以通过骨龄、年生长率以及第二性征而得以很准确的评估。因此，当青春期需要进行骨骺或脊柱关节融合术时，这种方法对于确定手术时间具有重要意义。 可信水平：诊断性研究，Ⅱ级，进一步可信度参见作者介绍。     

Clinical Experience with Expanded Polytetrafluoroethylene Gore-Tex® Surgical Membrane for Pericardial Closure: A Study of 110 Cases

Complete closure of the pericardium after cardiac operations has the advantage of avoiding injury of the heart and great vessels during reoperation. Between 1985 and 1987, the pericardium was closed with Gore-Tex Surgical Membrane (SM) in a selected series of 110 patients 1 month to 76 years of age. Fifty-three patients had congenital heart lesions and 57 patients had acquired heart disease. Overall hospital mortality was 3/110 cases. In no instance was there a relationship between occurrence of death and pericardial closure with SM. There was one episode of cardiac tamponade on the seventh postoperative day. One patient developed fever and leukocytosis due to a mediastinal hematoma. During a mean follow-up of 15 months, four patients had to be reoperated upon three, four, eight weeks, and eight months after primary operation. The anterior wall of the heart had no adhesion with the SM and the other parts of pericardium could be dissected easily. Scanning electron microscopic examination of the explanted SM patches showed neither cellular ingrowth nor immunocompetent cellular elements. The Gore-Tex Surgical Membrane has the advantages of easy availability and lack of reaction between its surface and the epicardium and pericardium. We believe its routine use should be encouraged in patients with high probability of reoperation after repair of complex cardiac anomalies, implantation of bioprostheses, coronary revascularization for one- or two-vessel disease, and repair of degenerative disease of the ascending aorta.     

Peripancreatic lymphoadenopathy and extrahepatic immunological manifestations in chronic hepatitis C

Abstract. The aim of the study was to determine the role of peripancreatic lymph node swelling in systemic immunological alterations during chronic hepatitis C (HC). The prospective study was carried out as a clinical study in a university hospital. Clinical, haematochemical and ultrasonographic findings in 182 patients were studied. Ultrasonography was performed by the same operator and the findings were evaluated blind without the operator knowing the clinical and haematochemical parameters. Hepatitis B virus (HBV) markers, anti-HCV antibodies. LKMl, cryoglobulinaemia, rheumatoid factor and anti-tissue antibodies were determined. Liver biopsy was carried out in 43 of the 182 patients. One or two pathological peripancreatic lymph nodes (PLNs) were present in 30 of the 182 patients and, of the 30, 28 were anti-HC positive. Only one patient in the non-PLN group was positive for anti-HCV, there being statistical significance ( P <0.0001) between the PLN and non-PLN groups. In HCV-positive patients, extrahepatic immunological manifestations were observed (cryoglobulinaemia; positivity to anti-smooth muscle, antinuclear and antimitochondrial antibodies; positivity to rheumatoid factor and LKMl). In five patients the presence of focal lymphocytic aggregates was detected by biopsy, whereas one patient presented typical ocular lesion of Mikulicz's syndrome. Our results may confirm the marked lymphotropism shown by the HC virus and indicate more complex immune system involvement, especially in view of the coexisting signs of immune system involvement related to the presence of intrahepatic cellular aggregates detected in our study. We believe that the peripancreatic adenopathy in chronic HCV hepatitis is an important diagnostic sign and may indicate an involvement of the C virus in the still unexplained extrahepatic immunological disorders.     

Antigenic variation in Giardia lamblia: cellular and humoral immune response in a mouse model

Neonatal mice (CR:NIH:S) were infected with a cloned human isolate of Giardia lamblia (GS/M-83-H7) and the surface antigens of the intestinal trophozoites, as well as the cellular and humoral immune responses, were analysed during the course of infection. Infections in mice peaked 2-3 weeks after inoculation and were self-cured by day 42 post-infection (p.i.). The proportion of trophozoites expressing the Mr 72,000 surface antigen of the initial inoculum had decreased by day 12 and approached zero by day 22 p.i., similar to infections in humans. The predominant parasite-specific humoral response was an IgM- and IgG-isotype directed to the original Mr 72,000 surface antigen as well as other antigens. T-lymphocytes (predominantly LY4(CD4)+) isolated from Peyer's patches 12 days p.i. and later showed a significant proliferative response to Giardia lamblia antigens. Spleen and lymph node cells showed no lymphoproliferative response. T-cell blot analysis revealed the presence of dominant T-cell epitopes in the areas of Mr 200,000-75,000 and less than 50,000 polypeptides. No response was demonstrated in the Mr 72,000 region (migration site of the major surface antigen), suggesting T-cell dependent mechanisms are most likely not responsible for the surface antigen switch which occurred during the course of infection. This model infection can be used to study the role of immunological mechanisms in Giardia lamblia variant antigen switching and in the control of infections.     

Molecular detection of t(8;21)/AML1-ETO in AML M1/M2: correlation with cytogenetics,morphology and immunophenotype

The t(8;21) identifies a subgroup of acute myeloid leukaemia (AML) with a relatively good prognosis which may merit different treatment. It is associated predominantly, but not exclusively, with AML M2, and corresponds to rearrangements involving the AML1 and ETO genes. AML1-ETO positive, t(8;21) negative cases are well recognized but their incidence is unknown. In order to determine optimal prospective AML1-ETO RT-PCR screening strategies, we analysed 64 unselected AML M1 and M2 cases and correlated the results with other biological parameters. Molecular screening increased the overall detection rate from 8% to 14%. AML1-ETO was found in 3% (1/32) of AML M1 and 25% (8/32) of M2, including three patients without a classic t(8;21) but with chromosome 8 abnormalities. It was more common in younger patients. Correlation with morphology enabled development of a scoring system which detected all nine AML1-ETO-positive cases with a false positive rate of 7% (4/55). Although certain AML1-ETO-positive cases demonstrated characteristic immunological features (CD19 and CD34 expression, CD33 negativity), each of these markers was insufficiently specific to permit prediction in an individual case. We conclude that initial routine prospective molecular screening for AML1-ETO in all AMLs, combined with standardized morphological and immunological analysis, is desirable in order to produce improved prognostic stratification and to determine whether screening can ultimately be restricted to appropriate subgroups.