Analysis of gene expression in wild‐type and Notch1 mutant retinal cells by single cell profiling

Results: To examine the molecular underpinnings of this observation, microarray analysis of single retinal cells from wild‐type or Notch1 conditional knockout retinas was performed. In situ hybridization was carried out to validate some of the findings. 相似文献   

Association of the ScaI atrial natriuretic peptide gene polymorphism with nonfatal myocardial infarction and extent of coronary artery disease

Background There is growing evidence from recent studies that atrial natriuretic peptide (ANP) plays an important part in coronary blood flow regulation and in atherosclerosis. Transition T2238→C in the atrial natriuretic peptide (ANP) precursor gene, which leads potentially to the translation of ANP with 2 additional arginines, has been suggested to be associated with salt-sensitive hypertension. According to our knowledge, this study is the first to look for the potential association of the ScaI ANP gene polymorphism with the history of nonfatal myocardial infarction and the extent of coronary artery disease (CAD).Methods The study was performed in 847 consecutive, white patients (719 men and 128 women) with significant coronary artery stenosis confirmed by means of elective coronary angiography (at least 1 coronary artery with ≥50% lumen narrowing). Screening for the T2238→C substitution was performed by means of polymerase chain reaction of genomic DNA, followed by ScaI digestion and agarose gel electrophoresis.Results We found a significant association of the A2A2 ScaI ANP genotype with a higher incidence of positive history of nonfatal myocardial infarction (odds ratio 1.85, 95% CI 1.33-2.58) and multiple-vessel CAD (odds ratio 1.45, 95% CI 1.02-2.06). The ScaI ANP genotype distribution did not differ with age, sex, body mass index, plasma lipids, hypertension, diabetes mellitus, and family history of CAD in studied groups.Conclusions Our results suggest that the ScaI ANP polymorphism may be associated with nonfatal myocardial infarction and the extent of CAD. However, the precise mechanism of this association remains to be determined. (Am Heart J 2003;145:125-31.)     

The –553 T/A polymorphism in the promoter region of the FGF2 gene is associated with increased breast cancer risk in Polish women

Introduction

Fibroblast growth factor-2 (FGF2) is an important signalling molecule contributing to angiogenesis, tumour growth and progression and its expression is implicated in breast cancer (BC) development. We investigated whether –553 T/A FGF2 gene polymorphism is associated with the risk and progression of BC in Polish women.

Material and methods

The –553 T/A polymorphism was genotyped in 230 breast cancer patients and 245 control subjects, using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach. Moreover, FastQuant human angiogenesis array was used to measure FGF2 levels in tumour (n = 127) and serum (n = 76) samples.

Results

The T/A genotypes (OR = 2.12, 95% CI: 1.20–3.74) (p = 0.08) and the combined heterozygotes T/A and homozygote A/A (OR = 2.18, 95% CI: 1.24–3.83) (p = 0.006) had an increased risk of BC. The median FGF2 levels in the tumours of A allele carriers were significantly increased compared to T/T patients, whereas in serum FGF2 levels were hardly altered among different genotype carriers. Significantly higher frequency of A allele was found in patients with lymph node metastases (OR = 2.53; 95% CI: 1.23–5.17) (p = 0.009) and human epidermal growth factor receptor 2 positive tumour (OR = 3.22, 95% CI: 1.49–6.99) (p = 0.002). Furthermore, Kaplan-Meier survival analysis showed that the A allele predicted worse disease-free survival (DFS) in BC patients.

Conclusions

Our study shows for the first time that the –553 T/A FGF2 gene polymorphism may be associated with a risk of BC developing and progression in Polish women and may have prognostic value for the assessment of BC high-risk groups.     

Biological Activation of Inert Ceramics: Recent Advances Using Tailored Self-Assembled Monolayers on Implant Ceramic Surfaces

High-strength ceramics as materials for medical implants have a long, research-intensive history. Yet, especially on applications where the ceramic components are in direct contact with the surrounding tissue, an unresolved issue is its inherent property of biological inertness. To combat this, several strategies have been investigated over the last couple of years. One promising approach investigates the technique of Self-Assembled Monolayers (SAM) and subsequent chemical functionalization to create a biologically active tissue-facing surface layer. Implementation of this would have a beneficial impact on several fields in modern implant medicine such as hip and knee arthroplasty, dental applications and related fields. This review aims to give a summarizing overview of the latest advances in this recently emerging field, along with thorough introductions of the underlying mechanism of SAMs and surface cell attachment mechanics on the cell side.     

Single-particle EM reveals plasticity of interactions between the adenovirus penton base and integrin αVβ3

Human adenoviruses are double-stranded DNA viruses responsible for numerous infections, some of which can be fatal. Furthermore, adenoviruses are currently used in clinical trials as vectors for gene therapy applications. Although initial binding of adenoviruses to host attachment receptors has been extensively characterized, the interactions with the entry receptor (integrins) remain poorly understood at the structural level. We characterized the interactions between the adenovirus 9 penton base subunit and α_Vβ₃ integrin using fluorescence correlation spectroscopy and single-particle electron microscopy to understand the mechanisms underlying virus internalization and infection. Our results indicate that the penton base subunit can bind integrins with high affinity and in several different orientations. These outcomes correlate with the requirement of the pentameric penton base to simultaneously bind several integrins to enable their clustering and promote virus entry into the host cell.Human adenoviruses are classified into more than 60 different serotypes and are associated with acute respiratory, gastrointestinal, and ocular infections, as well as fulminant infections among children and immunocompromised patients. Replication-defective or conditionally replicating human adenoviruses are also used in a large number of clinical trials for vaccine and therapeutic gene transfer (1). Adenoviruses are large, nonenveloped double-stranded DNA virions sharing a common capsid architecture based on T = 25 icosahedral symmetry (2–4). The hexon, penton base, and fiber proteins are the main capsid building blocks, with several cement proteins further contributing to capsid stability. The structural conservation of hexon- and penton-like proteins among several virus families infecting organisms of the three domains of life suggested that these virions are evolutionarily related and resulted in their classification in the PRD1-adenovirus lineage (2, 3, 5–11).The trimeric fiber protein and the pentameric penton base are the major players in adenovirus attachment and cell entry. Initial attachment of the virus occurs through the binding of the fiber C-terminal distal knob domain to attachment receptors such as coxsackie and adenovirus receptor (CAR), the complement receptor CD46, the GD1a glycan, or desmoglein-2 (DSG-2) (12). After initial cell attachment, a highly conserved arginine glycine aspartic acid (RGD) motif in a long loop of the penton base subunits engages α_vβ₃ or α_vβ₅ integrins, which serve as entry receptors. Binding to integrins promotes integrin clustering and in turn rapid receptor-mediated internalization of the virion (13). Partially disassembled capsids escape from the endosome and are transported along microtubules to the perinuclear envelope, where further disintegration of the capsid allows import of the viral genome into the nucleus (14).Although binding of the adenovirus trimeric fiber protein to the attachment receptors CAR, CD46, and GD1a has been extensively characterized, the interactions between the penton base and heterodimeric integrin remain less well understood at the structural level. Integrins are heterodimeric cell-surface transmembrane proteins involved in the regulation of adhesion, migration, growth, and differentiation (15). They form a stalk-like structure composed of a large “headpiece” that engages ligands and slender “legs” that anchor the heterodimer into the membrane. Integrins can assume a bent, inactive conformation, in which the legs are wrapped around the headpiece, as well as more extended, active conformations (16–18). The large multidomain extracellular moiety formed by the two integrin chains is able to bind a variety of ligands, including several viruses (e.g., adenoviruses), whereas the smaller intracellular domains interact with cytoskeletal proteins to carry out bidirectional signaling (19–23). Some integrins, such as α_vβ₃, are able to engage RGD sequences, which are found in various physiological ligands, and a crystal structure of a complex between α_vβ₃ integrin and a small peptide harboring this sequence has shown that the RGD motif binds to an exposed region in the headpiece (17). Located at the interface of the α_v and β₃ chains, the RGD sequence inserts into a cleft formed by the β-propeller domain of the α_v chain and the βA domain of the β₃ chain. All three amino acids—RGD—make specific contacts with headpiece residues. Previous studies have attempted to characterize the interactions between adenovirus and integrin using electron cryo-microscopy and biophysical techniques (24, 25). The results suggested that four integrins could simultaneously bind onto one penton base with varying orientations (due to steric hindrance). However, these interactions could not be analyzed in detail due to the large conformational flexibility of the integrin as well as the symmetry mismatch between the two partners.We set out to describe the interactions between the adenovirus penton base and integrin to understand the mechanisms underlying virus internalization and infection. Our approach differs from previous studies as we used a “minimalist” system, which is composed of the monomeric RGD loop-containing insertion domain of the adenovirus 9 penton base subunit (pb9) and the extracellular region of the α_Vβ₃ integrin. The adenovirus 9 serotype was chosen because the penton loop harboring the RGD sequence is among the shortest, and perhaps least flexible, in the adenovirus family. We characterized the binding affinity between α_Vβ₃ integrin and pb9 using fluorescence correlation spectroscopy (FCS) and demonstrated that the complex has a dissociation constant in the nanomolar range. We also analyzed the extracellular region of the α_Vβ₃ integrin, either unliganded or in complex with the penton base domain, using single-particle electron microscopy. The results unexpectedly indicate that the penton base can bind integrins in several orientations and suggest a putative mechanism for integrin clustering and virion internalization at the onset of infection.