BJ-48, a novel thrombin-like enzyme from the Bothrops jararacussu venom with high selectivity for Arg over Lys in P1: Role of N-glycosylation in thermostability and active site accessibility.

BJ-48, a serine protease from the venom of Bothrops jararacussu, was purified to homogeneity using affinity chromatography on p-aminobenzamidine-agarose followed by HPLC gel filtration. BJ-48 presented 52kDa by SDS-PAGE analysis and 48,036Da by electron spray mass spectrometry. The enzyme was shown to be highly glycosylated with 42% of N-linked carbohydrates composed of Fuc(1):GalN(4):GlcN(5):Gal(1):Man(2) and a high content of sialic acid residues (8-12%). BJ-48 had optimal esterase activity at pH 7.5 and displayed maximum catalytic rate at 50 degrees C. Its hydrolytic activity was strongly inhibited by aprotinin and dithiothreitol while N-tosyl-l-phenylalanine chloromethyl ketone, 6-aminocaproic acid, E-64 and soybean trypsin inhibitor (SBTI) were ineffective. The kinetics of BJ-48 with chromogenic substrates revealed an unprecedented selectivity (10(4)-fold) for Arg over Lys in P1. BJ-48 proved to be a thrombin-like enzyme (TLE) with a specific fibrinogen-clotting activity of 73.4NIH units/mg. The TLE rapidly digested human fibrinogen Bbeta chain, but the Aalpha chain was cleaved specifically to release fibrinopeptide A with k(cat)/K(m)=2.1muM(-1)s(-1). The TLE showed no activity toward other thrombin substrates like protein C, protease-activated receptor-1 or inhibitors such as hirudin and antithrombin. A non-denaturing procedure using PNGase F and neuraminidase followed by hydrophobic interaction chromatography was employed to obtain active BJ-48 forms with variable carbohydrate content. Compared to the native enzyme, total or partially deglycosylated BJ-48 forms presented up to 2-fold reduction in their specific activities upon heating at 55/65 degrees C or treatment with SBTI. These results point out a role for BJ-48 glycosylation in thermostability and controlling the access of some canonical protein inhibitors to the active site.     

Comparison of glycaemic control over 1 year with pioglitazone or gliclazide in patients with Type 2 diabetes.

AIMS: To compare long-term (1 year) efficacy and safety of pioglitazone and gliclazide in patients with Type 2 diabetes. METHODS: This was a double-blind, multicentre, comparative, parallel group trial in 283 patients with Type 2 diabetes, who were randomized to receive 1-year treatment with pioglitazone 30-45 mg/day or gliclazide 80-320 mg/day. Drug dose was titrated on the basis of self-monitored blood glucose (SMBG) measurements and HbA1c values. The 1-year changes in HbA1c, fasting blood glucose (FBG), insulin, HOMA-S (HOmeostatic Model Assessment) and SMBG were compared. In a subgroup of patients (n = 10), systemic glucose production and utilization were determined by a combination of isotopic (deuterated glucose) and clamp techniques. RESULTS: In both groups, there were similar decreases in HbA1c (pioglitazone: -0.79%; gliclazide: -0.79%) and FBG (pioglitazone: -1.0 mmol/l; gliclazide: -0.7 mmol/l), whereas the slope of the reduction of fasting blood glucose was different between groups (P = 0.004). Insulin levels as well as insulin resistance assessed using HOMA-S decreased significantly only after pioglitazone treatment (-11.94 pmol/l and -1.03, respectively, both P = 0.002 vs. baseline). A significantly greater reduction in systemic glucose production was observed in the pioglitazone group (-2.48 micromol/kg/min, P = 0.042) than in the gliclazide group (-1.02 micromol/kg/min). A few, mild adverse events occurred in both groups. CONCLUSIONS: A comparable decrease in HbA1c and FBG was observed with pioglitazone and gliclazide. However, with pioglitazone there was a continuous decrease in FBG over 1 year, whereas gliclazide failed to maintain a similar trend. This favourable effect of pioglitazone was due to its insulin-sensitizing effect and ability to decrease systemic glucose production.     

In vitro/in vivo correlation of prolonged release dosage forms containing diltiazem HCl

Six preparations were considered: three multiple unit dosage forms (micropellets in capsules) (D, E and G) and one matrix tablet (B) were experimental prolonged release formulations, two non-disintegrating tablets (A and C) were commercial products. The in vitro dissolution behaviour of the differing formulations was investigated using the USP XXII paddle apparatus. The in vivo study was effected on a panel of 12 healthy volunteers. The two commercial tablets (A and C) showed mean dissolution time (MDT) of 1.34 and 1.44 h and t_d of 91 and 92 min, respectively; for prolonged release formulations (B, E, D, and G) MDT ranged between 2.28 and 4.23 h and t_d between 149 and 291 min. The mean residence time (MRT) was 8.68 and 6.47 h for tablets A and C, respectively; it ranged between 9.62 and 10.24 h for the multiple unit formulations E, D, and G and was 11.27 h for matrix B. Formulation B also showed the higher apparent elimination half-life t_1/2 (7.12 h), while apparent t_1/2 for all the other formulations were very similar, ranging between 5.04 and 5.28 h. High variability between the various formulations was found for C_max and AUC values, and no relationships could be established with the type of formulation. An in vitro/in vivo correlation was found for all the formulations examined on the basis of analogous parameters (MDT and MRT); (r = 0.83, p <0.05). In a few cases the Wagner-Nelson deconvolution method was applied to individual plasma level versus time curves and the corresponding absorption curves were obtained. In these cases the in vitro/in vivo correlation was tested on the basis of the comparison of the in vivo absorption curves with the in vitro dissolution profiles. This was accomplished using the ‘Levy's plot’ (per cent released versus per cent absorbed) approach and provided further support for the correlation found.     

Preoperative Chemotherapy Alone and Combined with Preoperative Radiotherapy in Small-Size Breast Cancer

Abstract: Breast conservation surgery is an effective and safe treatment for many breast carcinomas. It may be possible to further limit the extent of resection (or expand the indication for breast conservation) by the application of preoperative chemotherapy and radiotherapy. We explored the feasibility of this in a pilot study.
Seventy-three patients (mean age 48, 63% premenopausal) with confirmed breast cancer, less than 2.5 cm, received chemotherapy (Group A) or chemotherapy plus radiotherapy (Group B) prior to limited resection (tumorectomy). Axillary dissection was always performed. Results: In 6/31 (19%) Group A and 17/42 (40%) Group B patients the tumor was not palpable after preoperative treatment, with complete pathological remission in 1 and 3 cases respectively. Histologic grading, mitosis, cellular alteration, and cellularity evaluations indicated a consistently greater therapeutic effect with chemoradiotherapy than with chemotherapy alone.
In conclusion, radiotherapy appears useful in the preoperative treatment of breast cancer and its use in association with various drug combinations should be further explored.     

Incidental prostatic carcinoma: four-year follow-up after treatment with cyproterone acetate

This report is a retrospective clinical randomized study carried out on 114 cases of incidental prostatic carcinoma aged 55-87 years, 58 untreated and 56 treated with cyproterone acetate (CPA 200 mg/day) for 6 months, immediately after surgery. 78 cases were staged A1 and the remaining 36 A2. In stage A1, 75 cases were histologically graded G1, and 3 G2, whereas in stage A2, 7 cases were G1, 19 G2 and 10 G3. Moreover, flow cytometric DNA analysis showed in A1 20 G1 carcinomas with nuclear diploidy and 3 G2 with nuclear aneuploidy, in stage A2, 4 G3 tumors with nuclear aneuploidy. During the 4-year follow-up, 25/28 patients of the untreated group and 15/56 of the CPA-treated group were found in progression. In A1, progression was found in 6/37 untreated patients and 5/41 CPA-treated, whilst in A2 progression was observed in 19/21 untreated patients and in 10/15 treated with CPA. The critical period for progression was between the 2nd and 3rd year of follow-up. In A1, therefore, 6 months of therapy with CPA does not modify the progression rate, which is significantly improved in A2 (66% in the treated and 90% in the untreated group) during the first 30 months of follow-up. The prognosis may probably be further improved by continuing endocrine therapy.     

Benign intracranial hypertension and recombinant growth hormone therapy in Australia and New Zealand

Benign intracranial hypertension (BIH) is reported in three children from Australia and one from New Zealand, who were being treated with recombinant human growth hormone (rhGH). Three males and one female, aged between 10.5 and 14.2 y, developed intracranial hypertension within 2 weeks to 3 months of starting treatment. A national database, OZGROW, has been prospectively collecting data on all 3332 children treated with rhGH in Australia and New Zealand from January 1986 to 1996. The incidence of BIH in children treated with growth hormone (GH) is small, 1.2 per 1000 cases overall, but appears to be greater with biochemical GHD (<10IUml ^-1), i.e. 6.5/1000 (3 in 465 cases), relative risk 18.4, 95% confidence interval 1.9-176.1, than in all other children on the database. The incidence in patients with Turner's syndrome was 2.3/1000 (1 in 428 cases). No cases in patients with partial GHD (10–20 IUml ^-1) or chronic renal failure were identified. Possible causative mechanisms are discussed. The authors'practice is now to start GH replacement at less than the usual recommended dose of 14IUm-² week^-1 in those children considered to be at high risk of developing BIH. Ophthalmological evaluation is recommended for children before and during the first few months following commencement of rhGH therapy and is mandatory in the event of peripheral or facial oedema, persistent headaches, vomiting or visual symptoms. The absence of papilledema does not exclude the diagnosis.     

Carcinoma of the male breast. Our experience

A case series of 5 male patients afflicted with breast carcinoma is reported. The Authors describe symptomatology, diagnostic iter, principles of surgical treatment and results. Underlined is the wider surgical demolition in male breast cancer in comparison with females, even if natural history and biological conduct are comparable. This is because in male breast carcinoma the local and remote infiltration is more rapid than female for the less development of breast gland. Finally, indications for radiochemotherapy are comparable to female breast cancer.     

大鼠肝细胞Ⅰ，Ⅲ型前胶原基因表达及PDGF的影响

目的观察大鼠肝细胞Ⅰ，Ⅲ型前胶原基因的表达及ＰＤＧＦ对其表达的影响．方法应用原位杂交技术检测分离培养的ＳＤ大鼠肝细胞（ｎ＝３０）内Ⅰ，Ⅲ型前胶原基因的表达．同时观察１０μｇ／Ｌ（ｎ＝３０）和３０μｇ／Ｌ（ｎ＝３０）ＰＤＧＦ促进前胶原基因表达的作用．测定基因表达颗粒总面积占细胞总面积的百分比，并作比较分析．结果无论正常肝细胞或是在两种浓度的ＰＤＧＦ存在时，肝细胞内均可见到Ⅰ，Ⅲ型前胶原基因的表达．正常肝细胞Ⅰ，Ⅲ型前胶原基因表达面积的百分比（％）为７７±１９和７５±２１；加１０μｇ／ＬＰＤＧＦ后为１１５±１９和１１２±１０，而加３０μｇ／Ｌ后为１５２±３４及１８１±２８，且在后者中表达明显增强（Ｐ＜００５及Ｐ＜００１）．结论ＰＤＧＦ在转录水平上促进肝细胞胶原的合成．