Properties of monoclonal antibodies to nicotinic acetylcholine receptor from chick muscle

Four stable, hybrid-cell lines secreting monoclonal antibodies to distinct determinants on the nicotinic acetylcholine receptor from chick muscle have been established. These were characterised by the following criteria: immunoglobulin isotype, ability to produce experimental autoimmune myasthenia gravis in mice and reactivity towards homologous and heterologous acetylcholine receptor proteins. Two monoclonal antibodies were found to inhibit the reaction of alpha-bungarotoxin with homologous acetylcholine receptor; in addition one of these, on binding to receptor-toxin, induced a rapid dissociation of the complex (t1/2 = 0.5 h at 23 degrees C). Three of the antibody preparations recognised epitopes on this receptor from muscle of other species and two of these caused experimental autoimmune myasthenia gravis in BALB/c mice following passive transfer. The latter two recognised to significant extents the alpha-bungarotoxin binding component purified from chick optic lobe and brain cortex. Sedimentation analysis demonstrated that two of the monoclonal antibodies form a distinct size (s20, w = 12S) of complex with the receptor of chick muscle which most probably corresponds to a 1:1 attachment of antibody and receptor; this may involve cross-linking of two determinants within the same oligomer. A similar observation was made with the alpha-bungarotoxin binding component from optic lobe using one of the cross-reacting antibodies. Another monoclonal antibody was found to be capable of forming much heavier complexes with the receptor from chick muscle, these are thought to involve inter-molecular cross-linking of oligomers. The observed properties of these antibodies are discussed in relation to their myasthenogenicity and with reference to the extent of structural similarities between the peripheral nicotinic acetylcholine receptor and the alpha-bungarotoxin binding protein from brain.     

Activation of the cAMP-generating system by vasoactive intestinal polypeptide (VIP) in the human laryngeal malignant cell line HEp-2

In the presence of 3-isobutyi-l-methylxanthine, VIP produced a dose-related (3×10^–9–10^–7 M) increase (g-fold) in cAMP production in isolated HEp-2 cells incubated at 15°C in KRP buffer. Among the peptides structurally related to VIP, including secretin (10^–7 M), pancreatic glucagon (10^–6 M), PHI, somatostatin-14 (10^–6 M), hpGRF (10^–8–4×10^–M), GIP (2×10^–7 M), only PHI (3×10^–7 M and above) is able to activate the cAMP-generating system in HEp-2 cells, but at 102 times lower potency. Under the same conditions, histamine (10^–3 M) was also ineffective, while PGE 2 (10^–7–10^–4 M) increased (0-fold) basal cAMP levels in HEp-2 cells. The VIP effect is related to the interaction os the peptide on VIP recognition sites (12SI-VIP-binding capacity ), coupled to the membrane-bound adenylate cyclase . The results indicate that the transformed laryngeal cell line HEp-2 possessesa receptor-cAMP system preferentially activated by VIP (relative potencies: VIP > PHI other peptides of the secretin family), and suggest that this neuropeptide could modulate biological functions in normal laryngeal epithelia in man.     

Association between SIRT1 Gene Polymorphisms and Breast Cancer in Egyptians

Background

Breast cancer is reported to cause the highest mortality among female cancer patients. Previous studies have explored the association of silent mating-type information regulator 2 homolog 1 (SIRT1) gene expression with prognosis in breast cancer. However, no studies exist, so far, on the role of SIRT1 gene polymorphism in breast cancer risk or prognosis. The present study aimed to assess the association between SIRT1 gene polymorphisms and breast cancer in Egyptians.

Methods

The study comprised 980 Egyptian females divided into a breast cancer group (541 patients) and a healthy control group (439 subjects). SIRT1 gene single nucleotide polymorphisms (SNPs) rs3758391, rs3740051 and rs12778366 were genotyped using real-time polymerase chain reaction (RT-PCR). Allelic and genotypic frequencies were determined in both groups and association with breast cancer and clinicopathological characteristics was assessed.

Results

Breast cancer patients exhibited elevated serum SIRT1 levels which varied among different tumor grades. SIRT1 rs3758391 and rs12778366 TT genotypes were more frequent, exhibited higher SIRT1 levels than CC and CT genotypes and were associated with histologic grade and lymph node status. SIRT1 rs12778366 TT genotype also correlated with negative estrogen receptor (ER) and progesterone receptor (PR) statuses. The T allele frequency for both SNPs was higher in breast cancer patients than in normal subjects. Combined GG and AG genotypes of rs3740051 were more frequent, showed higher serum SIRT1 levels than the AA genotype, and were associated with ER and PR expression. Furthermore, inheritance of the G allele was associated with breast cancer.

Conclusions

Our findings reveal that rs3758391 and rs12778366 polymorphisms of SIRT1 gene are associated with breast cancer risk and prognosis in the Egyptian population.     

Characterization of uropathogenic E. coli O25b-B2-ST131, O15:K52:H1, and CGA: Neutrophils apoptosis,serum bactericidal assay,biofilm formation,and virulence typing

Pathogenic and drug-resistant strains of Escherichia coli (E. coli) O25b-B2-ST131, O15:H1-D-ST393, and CGA (clonal group A) clonal groups have spread worldwide. This study aimed at determining E. coli epidemic clonal groups, their virulence factors, biofilm formation, neutrophils apoptosis, and antimicrobial resistance pattern of uropathogenic E. coli. A total of 95 CTX-M-1-producing E. coli clinical isolates were enrolled. E. coli O25b-B2-ST131, CGA, and O15:K52:H1 were identified by serotyping and phylogrouping and allele-specific polymerase chain reaction-based assay. Antibiotic susceptibility, biofilm formation, hemolysis, and human serum bactericidal assay were performed. Neutrophil apoptosis was assayed by flow cytometry. Nine E. coli clonal groups including six O25b-B2-ST131 strains, two CGA, and one O15:K52:H1-D-ST393 strains were detected. One O25b-B2-ST131 isolate was a strong biofilm-producer. Three ST131 isolates had type I fimbriae. Furthermore, all the CGA and O15:K52:H1 and three of ST131 isolates harbored the P fimbriae. The virulence genes ompT, fimH, and traT were detected among all the clonal groups. The apoptosis was induced by O25b-B2-ST131, CGA, and O15:K52:H1 E. coli. There was no significant difference regarding apoptosis induction among clonal groups. Furthermore, the presence of the cdt, usp, and vat genes was significantly associated with the apoptosis of neutrophils by O25b-B2-ST131, CGA, and O15:K52:H1-D-ST393 clonal groups.     

Endothelial Cells Provide a Notch-Dependent Pro-Tumoral Niche for Enhancing Breast Cancer Survival,Stemness and Pro-Metastatic Properties

Treating metastasis has been challenging due to tumors complexity and heterogeneity. This complexity is partly related to the crosstalk between tumor and its microenvironment. Endothelial cells -the building blocks of tumor vasculature- have been shown to have additional roles in cancer progression than angiogenesis and supplying oxygen and nutrients. Here, we show an alternative role for endothelial cells in supporting breast cancer growth and spreading independent of their vascular functions. Using endothelial cells and breast cancer cell lines MDA-MB231 and MCF-7, we developed co-culture systems to study the influence of tumor endothelium on breast tumor development by both in vitro and in vivo approaches. Our results demonstrated that endothelial cells conferred survival advantage to tumor cells under complete starvation and enriched the CD44^HighCD24^Low/- stem cell population in tumor cells. Moreover, endothelial cells enhanced the pro-metastatic potential of breast cancer cells. The in vitro and in vivo results concordantly confirmed a role for endothelial Jagged1 to promote breast tumor through notch activation. Here, we propose a role for endothelial cells in enhancing breast cancer progression, stemness, and pro-metastatic traits through a perfusion-independent manner. Our findings may be beneficial in developing novel therapeutic approaches.     

Polyelectrolyte Effects on 9-Aminoacridine-DNA Binding

Abstract

The 9-aminoacridine-DNA binding curve is analyzed in two ways: with polyelectrolyte effects neglected and with polyelectrolyte effects included. It is found that the analysis which includes polyelectrolyte effects is consistent with the violation of neighbor exclusion displayed by diacridine complexes as observed by Atwell et al. and by Zimmerman and coworkers. However the analysis which neglects polyelectrolyte effects is inconsistent with the diacridine results. This comparison supports the necessity of including polyelectrolyte effects in the analysis of drug-DNA binding curves.     

Trichome micromorphology of Iranian Stachys (Lamiaceae) with emphasis on its systematic implication

Trichomes of 37 taxa of the genus Stachys and one species of Sideritis (S. montana) were examined using light and scanning electron microscopy. The indumentum shows considerable variability among different species, but is constant among different populations of one species, and therefore, affords valuable characters in delimitation of sections and species. The characters of taxonomic interest were presence of glandular and non-glandular trichomes, thickness of the cell walls, number of cells (unicellular or multi-cellular), presence of branched (dendroid) trichomes, presence of vermiform trichomes, orientation of trichomes in relation to the epidermal surface, curviness of trichomes, and presence of papillae on trichome surface. Two basic types of trichomes can be distinguished: glandular and non-glandular trichomes. The glandular trichomes can in turn be subdivided into subtypes: stalked, subsessile, or sessile. The stalks of the glandular trichomes can be uni- or multi-cellular. Simple unbranched and branched trichomes constitute two subtypes of non-glandular trichomes. Our data do not provide any support for separation of Sideritis from Stachys. The following evolutionary trends are suggested here for Stachys: vermiform trichomes with stellate base are primitive against vermiform trichomes with tuberculate base, long vermiform trichomes are primitive against the short simple trichomes, appressed trichomes are advanced against spreading ones, and loss of glandular trichomes is advanced against their presence. Overall, trichome micromorphology is more useful in separation of species within sections rather than characterizing large natural groups known as sections, except for few cases.