High-order oligomers of intrinsically disordered brain proteins BASP1 and GAP-43 preserve the structural disorder.

Brain acid-soluble protein-1 (BASP1) and growth-associated protein-43 (GAP-43) are presynaptic membrane proteins participating in axon guidance, neuroregeneration and synaptic plasticity. They are presumed to sequester phosphatidylinositol-4,5-bisphosphate (PIP2 ) in lipid rafts. Previously we have shown that the proteins form heterogeneously sized oligomers in the presence of anionic phospholipids or SDS at submicellar concentration. BASP1 and GAP-43 are intrinsically disordered proteins (IDPs). In light of this, we investigated the structure of their oligomers. Using partial cross-linking of the oligomers with glutaraldehyde, the aggregation numbers of BASP1 and GAP-43 were estimated as 10-14 and 6-7 monomer subunits, respectively. The cross-linking pattern indicated that the subunits are circularly arranged. The circular dichroism (CD) spectra of the monomers were characteristic of coil-like IDPs showing unordered structure with a high population of polyproline-II conformation. The oligomerization was accompanied by a minor CD spectral change attributable to formation of a small amount of α-helix. The number of residues in the α-helical conformation was estimated as 13 in BASP1 and 18 in GAP-43. However, the overall structure of the oligomers remained disordered, indicating a high degree of 'fuzziness'. This was confirmed by measuring the hydrodynamic dimensions of the oligomers using polyacrylamide gradient gel electrophoresis and size-exclusion chromatography, and by assaying their sensitivity to proteolytic digestion. There is evidence that the observed α-helical folding occurs within the basic effector domains, which are presumably tethered together via anionic molecules of SDS or PIP2 . We conclude that BASP1 and GAP-43 oligomers preserve a mostly disordered structure, which may be of great importance for their function in PIP2 signaling pathway.

Vitamin D receptor is highly expressed in precancerous lesions and esophageal adenocarcinoma with significant sex difference.

Bile acid reflux into the esophagus is important in the development of esophageal adenocarcinoma (EAC). Recently, vitamin D receptor (VDR) was recognized as a bile acid receptor as well as a vitamin receptor. Expression of VDR is reported to influence the development of various types of cancer, such as those of the breast, liver, and colon. However, little is known about the role of VDR in esophageal neoplasms. We investigated the clinicopathological role of VDR in esophageal tumors. We analyzed genomic DNA from 116 EACs for copy number aberrations. The VDR locus was amplified in 7% of EACs. Expression of the VDR protein was also detected by immunohistochemistry from tissue microarrays created from tissues of Barrett esophagus (BE), low-grade (LGD) and high-grade dysplasia (HGD), columnar cell metaplasia (CCM), squamous epithelium (SE), EAC, and esophageal squamous cell carcinoma (ESCC). The protein was highly expressed in 88% of CCM (58/66), 95% of BE (35/37), 100% of the 19 LGD, 94% of HGD (15/16), and 79% of EAC (86/109), but expression in SE and ESCC was rare. Female patients with EAC and CCM were significantly less likely to have high VDR expression than male patients. The overall survival rate was significantly different for patients with tumors exhibiting VDR amplification versus nonamplification. Our findings suggest that VDR plays a role in the early development of EAC through a bile acid ligand. The sex difference in VDR expression may help to explain why men have a high incidence of EAC.

MACC1 is related to colorectal cancer initiation and early-stage invasive growth.

OBJECTIVES: To investigate metastasis associated in colon cancer 1 (MACC1) and MET expression in colorectal adenoma, Tis, early-stage invasive (T1 and T2), and advanced adenocarcinoma with liver metastasis using immunohistochemistry. METHODS: Ninety-three paraffin-embedded colorectal tumor specimens were immunohistochemically analyzed for MACC1 and MET protein expression. RESULTS: MACC1 expression was upregulated in the transition from adenoma to Tis; its expression was further elevated during tumor progression from Tis to early invasive carcinoma. MET expression was constant from adenoma to Tis and to T1 but significantly increased as tumor progression to T2. Both MACC1 and MET expression were enhanced in advanced carcinoma with liver metastasis. CONCLUSIONS: Stepwise elevation of MACC1 expression in key points of colorectal cancer development suggests that MACC1 may contribute to cancer initiation and early invasive growth. High expression of both MACC1 and MET may relate to distant metastasis.

Diagnostic value of HMGAs, p53 and ß-catenin in discriminating adenocarcinoma from adenoma or reactive atypia in ampulla and common bile duct biopsies.

AIMS: Biopsies from the ampulla of Vater and the common bile duct often pose diagnostic challenges. The aim of this study was to investigate the expression patterns of HMGA1, HMGA2, ß-catenin and p53 in biopsy specimens, in order to evaluate the potential diagnostic value of these proteins in differentiating adenocarcinoma from reactive atypia or adenoma. METHODS AND RESULTS: Forty-eight biopsies (10 from the common bile duct and 38 from the ampulla) were selected for immunohistochemical studies; they included 14 cases of reactive atypia, 12 adenomas, and 22 adenocarcinomas. Expression of HMGA1 was seen in 21% of the reactive atypia cases, 42% of adenomas, and 91% of adenocarcinomas. HMGA2 was positive in 14% of reactive atypias, 42% of adenomas, and 86% of adenocarcinomas. The staining intensity of HMGA1 and HMGA2 was also significantly higher in adenocarcinomas than in adenomas or reactive atypias. Interestingly, coexpression of HMGA1 and HMGA2 was found in 86% of adenocarcinomas, 0% of reactive atypias, and 8% of adenomas. p53 and ß-catenin expression seemed not to provide additional value for discriminating adenocarcinoma from reactive atypia or adenoma. CONCLUSIONS: HMGA1 and HMGA2 might serve to discriminate between reactive atypia, adenoma and adenocarcinoma in ampulla and common bile duct biopsies.

Tissue pattern recognition error rates and tumor heterogeneity in gastric cancer.

The anatomic pathology discipline is slowly moving toward a digital workflow, where pathologists will evaluate whole-slide images on a computer monitor rather than glass slides through a microscope. One of the driving factors in this workflow is computer-assisted scoring, which depends on appropriate selection of regions of interest. With advances in tissue pattern recognition techniques, a more precise region of the tissue can be evaluated, no longer bound by the pathologist's patience in manually outlining target tissue areas. Pathologists use entire tissues from which to determine a score in a region of interest when making manual immunohistochemistry assessments. Tissue pattern recognition theoretically offers this same advantage; however, error rates exist in any tissue pattern recognition program, and these error rates contribute to errors in the overall score. To provide a real-world example of tissue pattern recognition, 11 HER2-stained upper gastrointestinal malignancies with high heterogeneity were evaluated. HER2 scoring of gastric cancer was chosen due to its increasing importance in gastrointestinal disease. A method is introduced for quantifying the error rates of tissue pattern recognition. The trade-off between fully sampling tumor with a given tissue pattern recognition error rate versus randomly sampling a limited number of fields of view with higher target accuracy was modeled with a Monte-Carlo simulation. Under most scenarios, stereological methods of sampling-limited fields of view outperformed whole-slide tissue pattern recognition approaches for accurate immunohistochemistry analysis. The importance of educating pathologists in the use of statistical sampling is discussed, along with the emerging role of hybrid whole-tissue imaging and stereological approaches.

Ion channel activity of brain abundant protein BASP1 in planar lipid bilayers.

BASP1 (also known as CAP-23 and NAP-22) is a brain abundant myristoylated protein localized at the inner surface of the presynaptic plasma membrane. Emerging evidence suggests that BASP1 is critically involved in various cellular processes, in particular, in the accumulation of phosphatidylinositol-4,5-diphosphate (PIP(2)) in lipid raft microdomains. We have recently shown that BASP1 forms heterogeneously-sized oligomers and higher aggregates with an outward similarity to oligomers and protofibrils of amyloid proteins. However, BASP1 is not known to be related to any amyloid disease. In the present study, we show that BASP1 induces single channel currents across negatively-charged planar lipid bilayers (containing phosphatidylserine or PIP(2)) bathed in 0.1-0.2 M KCl (pH 7.5). By their characteristics, BASP1 channels are similar to amyloid protein channels. BASP1 channels exhibit multiple conductance levels, in the range 10-3000 pS, with the most frequently observed conductance state of approximately 50 pS. The channels demonstrate a linear current-voltage relationship and voltage-independent kinetics of opening and closing. Their K(+) to Cl(-) permeability ratio is approximately 14, indicating that BASP1 channels are cation-selective. The ion channel activity of BASP1 is in accordance with the pore-like structure of BASP1 oligomers observed by electron microscopy on a lipid monolayer. Neuronal protein GAP-43, which is functionally related to BASP1 and also forms oligomers, elicited no ion channel currents under the conditions used in the present study. Elucidation of the physiological or pathological roles of ion channel activity of membrane-bound BASP1 oligomers will help to define the precise mechanism of amyloid protein toxicity.

Suppressed expression of type 2 3alpha/type 5 17beta-hydroxysteroid dehydrogenase (AKR1C3) in endometrial hyperplasia and carcinoma.

The diagnosis of endometrial hyperplasia and endometrial type adenocarcinoma arising within the uterine cavity has long been rested on morphologic criteria. Although distinction between normal endometrial epithelium from adenocarcinoma is usually straightforward, the separation between normal and hyperplastic endometrium, particularly those cases without atypia, can be a diagnostic challenge. The same is true in separation of hyperplastic endometrium with atypia from endometrial-type endometrial adenocarcinoma. Type 2 3alpha-/type 5 17beta-hydroxysteroid dehydrogenase (HSD) (AKR1C3) is a multifunctional enzyme involved in androgen, estrogen, progesterone, and pros-taglandin metabolism. Its expression has been shown in the epithelium of the renal tubules, urothelial epithelium, and endothelial cells in normal tissues as well as in prostatic adenocarcinoma. The proliferation and maintenance of endometrial epithelium is dependent on both estrogen and progesterone; and AKR1C3-mediated steroid metabolism may play a critical role in the maintenance of viable normal and abnormal endometrial epithelium. We studied the expression of AKR1C3 in 33 endometrial biopsy specimens including 13 cases of normal proliferative endometrium, 8 cases of hyperplastic endometrium with and without atypia, and 12 cases of primary endometrial adenocarcinoma of endometrial type. We demonstrated a uniform, diffuse, and strong expression of AKR1C3 in normal endometrial epithelium but not in endometrial stromal cells. In contrast, the expression of AKR1C3 is reduced in both hyperplastic and carcinomatous endometrial epithelium. These findings suggest that AKR1C3 may play important roles in the physiology of endometrial cells and that suppressed AKR1C3 expression may represent a feature that allows differentiation of hyperplastic and neoplastic endometrial epithelium from normal endometrial epithelium. However, reduced AKR1C3 expression cannot distinguish hyperplastic endometrium from endometrial adenocarcinoma of endometrial type. The biologic and pathological roles of AKR1C3 in endometrial epithelium require further investigation.

Oligomeric structure of brain abundant proteins GAP-43 and BASP1.

Brain abundant proteins GAP-43 and BASP1 participate in the regulation of actin cytoskeleton dynamics in neuronal axon terminals. The proposed mechanism suggests that the proteins sequester phosphatidylinositol-4,5-diphosphate (PIP(2)) in the inner leaflet of the plasma membrane. We found that model anionic phospholipid membranes in the form of liposomes induce rapid oligomerization of GAP-43 and BASP1 proteins. Multiply charged phosphoinositides produced the most potent effect. Anionic detergent sodium dodecyl sulfate (SDS) at submicellar concentration stimulated formation of similar oligomers in solution. BASP1, but not GAP-43, also formed oligomers at sufficiently high concentration in the absence of lipids and SDS. Electron microscopy study demonstrated that the oligomers have disk-shaped or annular structure of 10-30nm in diameter. BASP1 also formed higher aggregates of linear rod-like structure, with average length of about 100nm. In outward appearance, the oligomers and linear aggregates are reminiscent of oligomers and protofibrils of amyloid proteins. Both the synthetic N-terminal peptide GAP-43(1-40) and the brain-derived fragment GAP-43-3 preserved the ability to oligomerize under the action of acidic phospholipids and SDS. On the contrary, BASP1 fragment truncated by the short N-terminal myristoylated peptide was unable to form oligomers. GAP-43 and BASP1 oligomerization can be regulated by calmodulin, which disrupts the oligomers and displaces the proteins from the membrane. We suggest that in vivo, the role of membrane-bound GAP-43 and BASP1 oligomers consists in accumulation of PIP(2) in functional clusters, which become accessible for other PIP(2)-binding proteins after dissociation of the oligomers.

A mouse model of lethal synergism between influenza virus and Haemophilus influenzae.

Secondary bacterial infections that follow infection with influenza virus result in considerable morbidity and mortality in young children, the elderly, and immunocompromised individuals and may also significantly increase mortality in normal healthy adults during influenza pandemics. We herein describe a mouse model for investigating the interaction between influenza virus and the bacterium Haemophilus influenzae. Sequential infection with sublethal doses of influenza and H. influenzae resulted in synergy between the two pathogens and caused mortality in immunocompetent adult wild-type mice. Lethality was dependent on the interval between administration of the bacteria and virus, and bacterial growth was prolonged in the lungs of dual-infected mice, although influenza virus titers were unaffected. Dual infection induced severe damage to the airway epithelium and confluent pneumonia, similar to that observed in victims of the 1918 global influenza pandemic. Increased bronchial epithelial cell death was observed as early as 1 day after bacterial inoculation in the dual-infected mice. Studies using knockout mice indicated that lethality occurs via a mechanism that is not dependent on Fas, CCR2, CXCR3, interleukin-6, tumor necrosis factor, or Toll-like receptor-4 and does not require T or B cells. This model suggests that infection with virulent strains of influenza may predispose even immunocompetent individuals to severe illness on secondary infection with H. influenzae by a mechanism that involves innate immunity, but does not require tumor necrosis factor, interleukin-6, or signaling via Toll-like receptor-4.

Expression of AKR1C3 in renal cell carcinoma, papillary urothelial carcinoma, and Wilms' tumor.

Human aldo-keto reductase (AKR) 1C3 is a monomeric cytoplasmic multifunctional enzyme that reduces ketosteroids, ketoprostaglandins, and lipid aldehydes. AKR1C3 was initially identified as an enzyme involved in steroid metabolism. However, immunohistochemistry has demonstrated AKR1C3 in normal adult kidneys with expression in Bowman' capsule, the mesangial cells, proximal and distal tubules, as well as mature urothelial epithelium. The significance of its spatial distribution and metabolic activities in the kidney remains undefined. In addition to its ability to catalyze steroid hormones (including androgen, desoxycorticosterone, and progesterone) and involvement in prostaglandins metabolism, we suspect that AKR1C3 may function as a chemical barrier in the renal tubules for normal function in mature kidneys. Moreover, AKR1C3 may represent a developmental marker for some urological epithelial tissues. In this study, we demonstrate widespread expression of AKR1C3 in renal neoplasms with a phenotype recapitulating mature kidney (i.e., renal cell carcinoma) and urothelium also known as transitional epithelium (i.e., papillary urothelial carcinoma), but noted limited AKR1C3 expression in renal neoplasms with a phenotype recapitulating embryonic kidneys (i.e., Wilms' tumor). Our results suggest that AKR1C3 may represent a developmental marker that is related to renal epithelium maturity.

M-calpain-mediated cleavage of GAP-43 near Ser41 is negatively regulated by protein kinase C, calmodulin and calpain-inhibiting fragment GAP-43-3.

Neuronal protein GAP-43 performs multiple functions in axon guidance, synaptic plasticity and regulation of neuronal death and survival. However, the molecular mechanisms of its action in these processes are poorly understood. We have shown that in axon terminals GAP-43 is a substrate for calcium-activated cysteine protease m-calpain, which participates in repulsion of axonal growth cones and induction of neuronal death. In pre-synaptic terminals in vivo, in synaptosomes, and in vitro, m-calpain cleaved GAP-43 in a small region near Ser41, on either side of this residue. In contrast, micro-calpain cleaved GAP-43 in vitro at several other sites, besides Ser41. Phosphorylation of Ser41 by protein kinase C or GAP-43 binding to calmodulin strongly suppressed GAP-43 proteolysis by m-calpain. A GAP-43 fragment, lacking about forty N-terminal residues (named GAP-43-3), was produced by m-calpain-mediated cleavage of GAP-43 and inhibited m-calpain, but not micro-calpain. This fragment prevented complete cleavage of intact GAP-43 by m-calpain as a negative feedback. GAP-43-3 also blocked m-calpain activity against casein, a model calpain substrate. This implies that GAP-43-3, which is present in axon terminals in high amount, can play important role in regulation of m-calpain activity in neurons. We suggest that GAP-43-3 and another (N-terminal) GAP-43 fragment produced by m-calpain participate in modulation of neuronal response to repulsive and apoptotic signals.

Natural N-terminal fragments of brain abundant myristoylated protein BASP1.

BASP1 (also known as CAP-23 and NAP-22) is a novel myristoylated calmodulin-binding protein, abundant in nerve terminals. It is considered as a signal protein participating in neurite outgrowth and synaptic plasticity. BASP1 is also present in significant amounts in kidney, testis, and lymphoid tissues. In this study, we show that BASP1 is accompanied by at least six BASP1 immunologically related proteins (BIRPs), which are present in all animal species studied (rat, bovine, human, chicken). BIRPs have lower molecular masses than that of BASP1. Similarly to BASP1, they are myristoylated. Peptide mapping and partial sequencing have shown that BIRPs represent a set of BASP1 N-terminal fragments devoid of C-terminal parts of different length. In a definite species, the same set of BASP1 fragments is present in both brain and other tissues. The sum amount of the fragments is about 50% of the BASP1 amount in a tissue. Obligatory accompanying of BASP1 by a set of specific fragments indicates that these fragments are of physiological significance.