Highly resolving two-dimensional gels for protein sequencing.

Two-dimensional (2D) PAGE, using carrier ampholytes for the first-dimension separation, has provided a tool for the simultaneous analysis of cellular proteins. To extend the utility of 2D PAGE to the preparative level, we have investigated the use of immobilized pH gradients (IPG) for the first-dimension separation. The results we have obtained indicate that as much as 1 mg of cellular protein can be loaded onto a single IPG gel without loss of resolution. Mutant polypeptides previously detected in carrier ampholyte-based 2D gels were equally detectable in IPG-based 2D gels. With IPG gels several hundred cellular polypeptides can be isolated, from as few as 10 gels, in sufficient amount for sequencing with current sequencing technology. We therefore conclude that IPG greatly enhances the prospects for the large-scale sequencing of cellular proteins for the development of 2D gel-related protein data bases and for the identification of new polypeptide gene products, with the attendant implications for a genome sequencing effort.     

High levels of p19/nm23 protein in neuroblastoma are associated with advanced stage disease and with N-myc gene amplification.

The gene encoding a novel protein designated nm23-H1, which was recently identified as identical to the A subunit of nucleotide diphosphate kinase from human erythrocytes, has been proposed to play a role in tumor metastasis suppression. We report that untreated neuroblastoma tumors contain a cellular polypeptide (Mr = 19,000) designated p19, identified in two-dimensional electrophoretic gels, which occurs at significantly higher levels (P = 0.0001) in primary tumors containing amplified N-myc gene. The partial amino acid sequence obtained for p19 is identical to the sequence of the human nm23-H1 protein. An antibody to the A subunit of erythrocyte nucleotide diphosphate kinase reacted exclusively with p19. In this study, significantly higher levels of p19/nm23 occurred in primary neuroblastoma tumors from patients with advanced stages (III and IV) relative to tumors from patients with limited stages (I and II) of the disease. Even among patients with a single copy of the N-myc gene, tumors from patients with stages III and IV had statistically significantly higher levels of p19/nm23 than tumors from patients with stages I and II. Our findings indicate that, in contrast to a proposed role for nm23-H1 as a tumor metastasis suppressor, increased p19/nm23 protein in neuroblastoma is correlated with features of the disease that are associated with aggressive tumors. Therefore, nm23-H1 may have distinct if not opposite roles in different tumors.     

单克隆抗体与丝裂霉素交联物对人胃癌细胞的选择性杀伤作用

本文选用抗胃癌单克隆抗体(MoAb)3H 11与Mitomycin C(MMC)制备交联物。体外实验结果表明,3H 11-MMC对人胃癌细胞BGC 823有明显的杀伤作用。而且有明显的选择性:3H 11-MMC(相当于MMC 100 ng/ml)对靶细胞的杀伤率为71%,而对非靶细胞MCF-7仅为14%。MoAb 3H11与靶细胞预温后可使杀伤率(1 μg/ml)由85%下降为38%,而与MoAb 3G9预温却无明显影响。体内实验表明。3 H 11-MMC处理组,肿瘤形成的时间较各对照组明显延长,肿瘤生长速度明显减慢,具有显著性差异。     

Prevalence of hydatidosis in slaughtered animals from North Jordan

The prevalence of hydatid disease was investigated in 704 sheep, 391 goats, 280 cattle and 68 camels slaughtered at two abattoirs in North Jordan. The infection rates for these animals were 4.0, 3.6, 11.4 and 8.8%, respectively. In general, older animals had higher infection rates than younger ones. The percentage of infected animals that had fertile cysts was 66.7% in camels, 34.3% in cattle, 28.6% in goats and 7.1% in sheep. Out of 143 single cysts recovered from cattle, 52.4% were fertile; 29.2% of cysts in goats, 14.7% of cysts in camels and 8.0% of those in sheep were fertile. However, infected sheep had more fertile multiple cysts than other animals. The lung was the predominant location for hydatid cysts in camels. In cattle and goats more hydatid cysts were found in the lung than in the liver, while in sheep the reverse was true.     

Characteristics of the genetically determined allozymic forms of human serum paraoxonase/arylesterase.

Human serum paraoxonase/arylesterase is an esterase with broad substrate specificity. It occurs in two genetically determined allozymic forms, which we have designated types A and B. These allozymes are presumed to be the products of two allelic genes located at the paraoxonase locus on chromosome 7, which is closely linked to the gene for cystic fibrosis. Paraoxonase activity of the B-type isozyme is considerably higher and stimulated more by 1 M NaCl than A-type paraoxonase. The ratio of paraoxonase activity/arylesterase activity of the B-isozyme is about 8, and that of the A-isozyme about 1. Purified isozymes A or B are free of nearly all other serum proteins, and the broad substrate specificity of the serum esterase is preserved after purification. A variety of substrates are hydrolyzed; these include: diisopropylfluorophosphate, soman, sarin, 4-nitro-phenylacetate, 2-nitro-phenylacetate, 2-naphthylacetate, and phenylthioacetate. The isozymic distinctions in kinetic properties and substrate specificity are preserved during purification. It is likely that the allozymes have very similar turnover numbers with phenylacetate (arylesterase activity), but differ considerably in their turnover numbers with paraoxon. Isozymes A and B have about the same minimal molecular weight of 43,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Further detailed studies on the individual isozymic proteins (or the DNA coding for their amino acid sequence) will be required to detect the exact structural differences in the isozymes.     

Effects of Nigella sativa Extracts on Antibody Response of Rats Vaccinated with Brucella Vaccine (Rev-1)

Volatile oil (VO), polysaccharide (PS) and ethanol extracts (EE) of N. sativa were evaluated for their effects on the antibody (Ab) titer of rats vaccinated with Brucella melitensis vaccine (Rev-1). Thirty rats were divided into five equal groups. Group 1(control) animals were injected with PBS, group 2 with Rev-1 alone, group 3 with Rev-1 and VO, group 4 with Rev-1 and PS and group 5 with Rev-1 and EE. The Ab titer was measured by Rose Bengal (RB) test, complement fixation (CF) test and tube agglutination (TA) test. All of the tests were negative in group 1. RB test occurred significantly faster in groups 4 and 5 than group 2 (P < 0.05 and 0.01, respectively). When the mean values of Ab titers of CF and TA in the groups was compared, significant differences (P < 0.05 or 0.01) were found between each of 2 and 3, 4, 5 and also between groups 3 and 4. When the percentages of lymphocytes and neutrophils were compared between the groups, significant differences were found between groups 1 and 3, and between 3 and 5. Histopathological examination of lymph nodes revealed a remarkably consistent picture of reactive lymphoid hyperplasia with prominence of germinal centers being evident in group 5. Infiltration of the medullary sinuses with plasma cells, lymphocytes, and macrophages was also seen. Our results suggest that all fractions of N. sativa had a significant adjuvant effect on the response of Br. melitensis vaccine, and that PS was the most effective fraction.     

Pathological,Immunological and Molecular Diagnosis of Rabies in Clinically Suspected Animals of Different Species Using Four Detection Techniques in Jordan

A total of 29 brain tissue samples (BTS) were examined for rabies infection by different diagnostic techniques. None of the examined brain tissues were presented as a whole intact brain. Twenty‐seven brain tissue samples from various animal species – dog (13 cases), cat (one case), fox (one case), pig (one case), cow (three cases), sheep (two cases), goat (one case), camel (one case), horse (one case) and donkey (three cases) – were provided by the Vaccine and Sera Department/Al‐Bashir Central Hospital in Amman/Jordan from July 2009 up to May 2010. All these samples were frozen at −20°C, for a period of time and then fixed in 10% formalin after being tested for rabies virus by fluorescence antibody test (FAT). The results showed that 21 (77.77%) of 27 BTS were positive for rabies by FAT. Seventeen samples (58.62%) of 29 were positive by histopathology, 2 (6.90%) were positive by histopathology, immunohistochemistry (IHC) and of those which were fixed for 24 h only, and 21 (72.42%) were positive using RT‐PCR assay. Five of 29 BTS had no pathological lesions, 17 had Negri bodies and the remaining had non‐suppurative encephalitis and necrosis. Thirteen BTS that were diagnosed positive by FAT were also positive by RT‐PCR and histopathology, but negative by IHC. Four BTS that were positive by FAT were negative by histopathology, IHC and RT‐PCR. Also, 3 BTS (cases 19, 22, and 25) that were negative by FAT were positive by RT‐PCR and negative by IHC. One of these was negative, while two were positive by histopathology. Therefore, definitive diagnosis of rabies under these conditions in Jordan needs one or more other diagnostic tests in addition to FAT. Also, freezing and prolonged formalin fixation of BTS is not suitable for the detection of rabies virus antigen using IHC.     

普萘洛尔或血管紧张素Ⅱ结合胃癌单克隆抗体与丝裂霉素交联物导向治疗的实验研究

观察了普萘洛尔和血管紧张素Ⅱ(AT-Ⅱ)对胃癌单克隆抗体3H11和丝裂霉素(MMC)交联物导向治疗的增强作用。在荷瘤裸鼠体内观察,交联物的抑瘤率达50%,联合应用普萘洛尔或AT-Ⅱ,可使抑瘤率分别提高到79%和60%。从¹³¹Ⅰ-3H11,在荷瘤裸鼠体内的生物学分布实验中也看到,普萘洛尔或AT-Ⅱ均能提高肿瘤组织对¹³¹Ⅰ-3H11的摄取量。此结果表明,血管活性物质确可通过改善肿瘤血管的血流灌注,增加肿瘤内交联物含量,增强导向治疗的疗效。