Aberrant E-cadherin staining patterns in invasive mammary carcinoma

Background

Advances in our understanding of the molecular biology of colorectal cancer have fuelled the search for novel molecular prognostic markers to complement existing staging systems. Markers assessed in combination may perform better than those considered individually. Using high-throughput tissue microarray technology, we describe the prognostic value of combined p53 / Bcl-2 status in colorectal cancer.

Patients and methods

Tumour samples from 462 patients who underwent elective surgery to resect a primary colorectal cancer between 1994 and 2000 (mean follow-up of 75 months) were assembled in tissue microarray format. Clinico-pathological data including tumour grade, stage, vascular invasion status along with disease specific survival data has been collected prospectively. Immunohistochemical analysis of p53 and Bcl-2 expression was performed using antibodies DO-7 (p53) and 124 (Bcl-2), and results correlated with known clinico-pathological variables and outcomes.

Results

Abnormal nuclear p53 accumulation and Bcl-2 overexpression were detected in 221/445 (49.6%) and199/437 (45.5%) tumours respectively, with a significant inverse correlation between the two markers (p = 0.023). On univariate analysis no correlations were found between either marker and standard clinico-pathological variables, however nuclear p53 expression was associated with a significantly reduced survival (p = 0.024). Combined analysis of the two markers indicated that 112/432 (24.2%) cases displayed a p53(-)/Bcl-2(+) phenotype, this occurring more frequently in earlier stage tumours. Kaplan-Meier analysis revealed a significant survival advantage in these p53(-)/Bcl-2(+) tumours compared with the remaining cases (p = 0.0032). On multivariate analysis using the Cox proportional hazards model, neither p53 expression nor Bcl-2 expression alone were of independent prognostic significance, however the combined p53(-)/Bcl-2(+) phenotype was significantly associated with a good prognosis in this series (HR 0.659, 95%CI 0.452–0.959, p = 0.029).

Conclusion

Patient stratification by combined p53 / Bcl-2 phenotype provides stage-independent prognostic information in colorectal cancer. Specifically, that up to a quarter of patients display a good prognosis p53(-)/Bcl-2(+) phenotype. This may indicate a more clinically indolent phenotype and a subset of patients for whom less aggressive adjuvant treatment appropriate.     

Polypeptide substrate recognition by calnexin requires specific conformations of the calnexin protein

Calnexin is an endoplasmic reticulum chaperone that binds to substrates containing monoglucosylated oligosaccharides. Whether calnexin can also directly recognize polypeptide components of substrates is controversial. We found that calnexin displayed significant conformational lability for a chaperone and that heat treatment and calcium depletion induced the formation of calnexin dimers and higher order oligomers. These conditions enhanced the chaperone activity of calnexin toward glycosylated and non-glycosylated major histocompatibility complex (MHC) class I heavy chains, and enhanced calnexin binding to MHC class I heavy chains. In contrast to these observations, calnexin binding to oligosaccharide substrates has been reported to be impaired under calcium-depleting conditions. Calnexin dimers were induced in HeLa cells upon heat shock and under calcium-depleting conditions, and heat shock enhanced calnexin binding to MHC class I heavy chains in HeLa cells. Virus-induced endoplasmic reticulum stress also resulted in the appearance of calnexin dimers. Tunicamycin treatment of HeLa cells induced a slow accumulation of calnexin dimers, the appearance of which correlated with enhanced calnexin binding to deglycosylated MHC class I heavy chains. In vitro, the presence of calnexin-specific oligosaccharides inhibited the formation of calnexin dimers and higher order structures. Together, these data indicate that polypeptide binding is favored by conditions that induce partial unfolding of calnexin monomers, whereas oligosaccharide binding is favored by conditions that enhance the structural stability (folding) of calnexin monomers. Conditions that induce the calnexin "polypeptide-binding" conformation also induce self-association of calnexin if the concentration is sufficiently high; however, calnexin dimerization/oligomerization per se is not essential for polypeptide substrate binding.     

Impaired E-cadherin expression in human spermatozoa in a male factor infertility subset signifies E-cadherin-mediated adhesion mechanisms operative in sperm-oolemma interactions

Cadherins comprise a family of calcium-dependent glycoproteins that function in mediating cell-cell adhesion in virtually all solid tissues of multicellular organisms. We have examined the presence of a cadherin on spermatozoon and its possible involvement in sperm-oocyte interaction. Spermatozoa from fertile human subjects showed the presence of E-cadherin on its head domains, detectable only after permeabilization of the surface membranes. On the contrary, spermatozoa from oligozoospermic subjects did not possess E-cadherin on their principal acrosomal and equatorial domains. Immunoprecipitation and Western blot studies also showed the presence of E-cadherin in spermatozoa from fertile males and its absence in oligozoospermic males. Using RT-PCR, we detected E-cadherin message in the round cells of fertile males, which was absent in the cells from oligozoospermic males. The presence of anti-E-cadherin antibody brought about quantitative reduction in the sperm-oocyte binding in vitro. These observations indicate the possibility of the interplay of a cadherin-dependent homophilic and/or heterophilic adhesion interaction between spermatozoa and oocyte during fertilization. The absence of a key adhesion molecule in a human male infertility disorder points towards genetic defects causing failure in gamete interactions.     

Formation and dynamic alterations of horizontal microdomains in sperm membranes during progesterone-induced acrosome reaction

A peptidomics approach was applied to determine the peptides in the larval central nervous system of the grey flesh fly, Neobellieria bullata. Fractions obtained by high performance liquid chromatography were analysed by MALDI-TOF and ESI-Q-TOF mass spectrometry. This provided biochemical evidence for the presence of 18 neuropeptides, 11 of which were novel Neobellieria peptides. Most prominently present were the FMRFamide-related peptides: 7 FMRFamides, 1 FIRFamide, and Neb-myosuppressin. The three putative capa-gene products Neb-pyrokinin and the periviscerokinins Neb-PVK-1 and -2 were detected, as well as another pyrokinin. This Neb-PK-2 was also present in the ring gland along with corazonin, Neb-myosuppressin, and Neb-AKH-GK, an intermediate processing product of the adipokinetic hormone. Furthermore, the central nervous system contained Neb-LFamide, proctolin, and FDFHTVamide, designated as Neb-TVamide. With this study, we considerably increased our knowledge of the neuropeptidome of the pest fly N. bullata, which is an important insect model for physiological research.     

Mycobacterium tuberculosis cells growing in macrophages are filamentous and deficient in FtsZ rings

FtsZ, a bacterial homolog of tubulin, forms a structural element called the FtsZ ring (Z ring) at the predivisional midcell site and sets up a scaffold for the assembly of other cell division proteins. The genetic aspects of FtsZ-catalyzed cell division and its assembly dynamics in Mycobacterium tuberculosis are unknown. Here, with an M. tuberculosis strain containing FtsZ(TB) tagged with green fluorescent protein as the sole source of FtsZ, we examined FtsZ structures under various growth conditions. We found that midcell Z rings are present in approximately 11% of actively growing cells, suggesting that the low frequency of Z rings is reflective of their slow growth rate. Next, we showed that SRI-3072, a reported FtsZ(TB) inhibitor, disrupted Z-ring assembly and inhibited cell division and growth of M. tuberculosis. We also showed that M. tuberculosis cells grown in macrophages are filamentous and that only a small fraction had midcell Z rings. The majority of filamentous cells contained nonring, spiral-like FtsZ structures along their entire length. The levels of FtsZ in bacteria grown in macrophages or in broth were comparable, suggesting that Z-ring formation at midcell sites was compromised during intracellular growth. Our results suggest that the intraphagosomal milieu alters the expression of M. tuberculosis genes affecting Z-ring formation and thereby cell division.     

The intracellular Ca2+ channels of membrane traffic

Regulation of organellar fusion and fission by Ca²⁺ has emerged as a central paradigm in intracellular membrane traffic. Originally formulated for Ca²⁺-driven SNARE-mediated exocytosis in the presynaptic terminals, it was later expanded to explain membrane traffic in other exocytic events within the endo-lysosomal system. The list of processes and conditions that depend on the intracellular membrane traffic includes aging, antigen and lipid processing, growth factor signaling and enzyme secretion. Characterization of the ion channels that regulate intracellular membrane fusion and fission promises novel pharmacological approaches in these processes when their function becomes aberrant. The recent identification of Ca²⁺ permeability through the intracellular ion channels comprising the mucolipin (TRPMLs) and the two-pore channels (TPCs) families pinpoints the candidates for the Ca²⁺ channel that drive intracellular membrane traffic. The present review summarizes the recent developments and the current questions relevant to this topic.     

Recognition pliability is coupled to structural heterogeneity: a calmodulin intrinsically disordered binding region complex

Protein interactions within regulatory networks should adapt in a spatiotemporal-dependent dynamic environment, in order to process and respond to diverse and versatile cellular signals. However, the principles governing recognition pliability in protein complexes are not well understood. We have investigated a region of the intrinsically disordered protein myelin basic protein (MBP(145-165)) that interacts with calmodulin, but that also promiscuously binds other biomolecules (membranes, modifying enzymes). To characterize this interaction, we implemented an NMR spectroscopic approach that calculates, for each conformation of the complex, the maximum occurrence based on recorded pseudocontact shifts and residual dipolar couplings. We found that the MBP(145-165)-calmodulin interaction is characterized by structural heterogeneity. Quantitative comparative analysis indicated that distinct conformational landscapes of structural heterogeneity are sampled for different calmodulin-target complexes. Such structural heterogeneity in protein complexes could potentially explain the way that transient and promiscuous protein interactions are optimized and tuned in complex regulatory networks.     

Orally efficacious thrombin inhibitors with cyanofluorophenylacetamide as the P2 motif

2-Cyano-6-fluorophenylacetamide was explored as a novel P2 scaffold in the design of thrombin inhibitors. Optimization around this structural motif culminated in 14, which is a potent thrombin inhibitor (K_i = 1.2 nM) that exhibits robust efficacy in canine anticoagulation and thrombosis models upon oral administration.