Characterization of ENU-induced mutations in red blood cell structural proteins

Murine models with modified gene function as a result of N-ethyl-N-nitrosourea (ENU) mutagenesis have been used to study phenotypes resulting from genetic change. This study investigated genetic factors associated with red blood cell (RBC) physiology and structural integrity that may impact on blood component storage and transfusion outcome. Forward and reverse genetic approaches were employed with pedigrees of ENU-treated mice using a homozygous recessive breeding strategy. In a “forward genetic” approach, pedigree selection was based upon identification of an altered phenotype followed by exome sequencing to identify a causative mutation. In a second strategy, a “reverse genetic” approach based on selection of pedigrees with mutations in genes of interest was utilised and, following breeding to homozygosity, phenotype assessed. Thirty-three pedigrees were screened by the forward genetic approach. One pedigree demonstrated reticulocytosis, microcytic anaemia and thrombocytosis. Exome sequencing revealed a novel single nucleotide variation (SNV) in Ank1 encoding the RBC structural protein ankyrin-1 and the pedigree was designated Ank1EX34. The reticulocytosis and microcytic anaemia observed in the Ank1EX34 pedigree were similar to clinical features of hereditary spherocytosis in humans. For the reverse genetic approach three pedigrees with different point mutations in Spnb1 encoding RBC protein spectrin-1β, and one pedigree with a mutation in Epb4.1, encoding band 4.1 were selected for study. When bred to homozygosity two of the spectrin-1β pedigrees (a, b) demonstrated increased RBC count, haemoglobin (Hb) and haematocrit (HCT). The third Spnb1 mutation (spectrin-1β c) and mutation in Epb4.1 (band 4.1) did not significantly affect the haematological phenotype, despite these two mutations having a PolyPhen score predicting the mutation may be damaging. Exome sequencing allows rapid identification of causative mutations and development of databases of mutations predicted to be disruptive. These tools require further refinement but provide new approaches to the study of genetically defined changes that may impact on blood component storage and transfusion outcome.

A new plasmid display technology for the in vitro selection of functional phenotype–genotype linked proteins

Background: Display technologies which allow peptides or proteins to be physically associated with the encoding DNA are central to procedures which involve screening of protein libraries in vitro for new or altered function. Here we describe a new system designed specifically for the display of libraries of diverse, functional proteins which utilises the DNA binding protein nuclear factor κB (NF-κB) p50 to establish a phenotype–genotype link between the displayed protein and the encoding gene. Results: A range of model fusion proteins to either the amino- or carboxy-terminus of NF-κB p50 have been constructed and shown to retain the picomolar affinity and DNA specificity of wild-type NF-κB p50. Through use of an optimal combination of binding buffer and DNA target sequence, the half-life of p50–DNA complexes could be increased to over 47 h, enabling the competitive selection of a variety of protein–plasmid complexes with enrichment factors of up to 6000-fold per round. The p50-based plasmid display system was used to enrich a maltose binding protein complex to homogeneity in only three rounds from a binary mixture with a starting ratio of 1:108 and to enrich to near homogeneity a single functional protein from a phenotype–genotype linked Escherichia coli genomic library using in vitro functional selections. Conclusions: A new display technology is described which addresses the challenge of functional protein display. The results demonstrate that plasmid display is sufficiently sensitive to select a functional protein from large libraries and that it therefore represents a useful addition to the repertoire of display technologies.

A new plasmid display technology for the in vitro selection of functional phenotype-genotype linked proteins

Background Display technologies which allow peptides or proteins to be physically associated with the encoding DNA are central to procedures which involve screening of protein libraries in vitro for new or altered function. Here we describe a new system designed specifically for the display of libraries of diverse, functional proteins which utilises the DNA binding protein nuclear factor κB (NF-κB) p50 to establish a phenotype-genotype link between the displayed protein and the encoding gene. Results A range of model fusion proteins to either the amino- or carboxy-terminus of NF-κB p50 have been constructed and shown to retain the picomolar affinity and DNA specificity of wild-type NF-κB p50. Through use of an optimal combination of binding buffer and DNA target sequence, the half-life of p50-DNA complexes could be increased to over 47 h, enabling the competitive selection of a variety of protein-plasmid complexes with enrichment factors of up to 6000-fold per round. The p50-based plasmid display system was used to enrich a maltose binding protein complex to homogeneity in only three rounds from a binary mixture with a starting ratio of 1:108 and to enrich to near homogeneity a single functional protein from a phenotype-genotype linked Escherichia coli genomic library using in vitro functional selections. Conclusions A new display technology is described which addresses the challenge of functional protein display. The results demonstrate that plasmid display is sufficiently sensitive to select a functional protein from large libraries and that it therefore represents a useful addition to the repertoire of display technologies.

Time to face the fats : What can mass spectrometry reveal about the structure of lipids and their interactions with proteins?

Since the 1950s, X-ray crystallography has been the mainstay of structural biology, providing detailed atomic-level structures that continue to revolutionize our understanding of protein function. From recent advances in this discipline, a picture has emerged of intimate and specific interactions between lipids and proteins that has driven renewed interest in the structure of lipids themselves and raised intriguing questions as to the specificity and stoichiometry in lipid-protein complexes. Herein we demonstrate some of the limitations of crystallography in resolving critical structural features of ligated lipids and thus determining how these motifs impact protein binding. As a consequence, mass spectrometry must play an important and complementary role in unraveling the complexities of lipid-protein interactions. We evaluate recent advances and highlight ongoing challenges towards the twin goals of (1) complete structure elucidation of low, abundant, and structurally diverse lipids by mass spectrometry alone, and (2) assignment of stoichiometry and specificity of lipid interactions within protein complexes.

International hermelin brain tumor symposium on matricellular proteins in normal and cancer cell-matrix interactions

The second of the Hermelin Brain Tumor Center Symposia was held once again at Henry Ford Hospital in Detroit, Michigan on October 24th and 25th, 2003. A public conference was held on the 24th while a closed-door session took place on the 25th. The purpose of these symposia is to bring together experts in a particular field of study with the aim to share information with each other and the public, but then to meet privately to present novel data, hold discussions, and share concepts. While the interaction is intended to benefit all involved, the incentive is the expectation that the shared information will aid researchers at the Hermelin Brain Tumor Center in their quest to identify potential therapeutic targets and explore translational therapeutic strategies for the treatment of patients suffering nervous system tumors...

Localization of fibronectin in the rat testis : effects of serum and dibutyryl cyclic AMP on fibronectin deposition by peritubular myoid cells in culture

The peritubular zone of the rat testis has an extensive extracellular matrix (ECM). Fibronectin (FN) is distributed primarily in the basal lamina of the seminiferous tubule boundary tissue and is synthesized by peritubular myoid cells. Several extracellular changes are mediated by growth factors and these changes occur at the time of hormone mediated testicular development, particularly in the peritubular zone. The effects of serum or dibutyryl cyclic AMP (cAMP) on FN production by the mesenchymal peritubular myoid cells were evaluated. Rats of various ages (10, 15, 20, 40 and 80 days) were employed for immunofluorescent localization of rat testicular FN in frozen sections. In all age groups tested, FN was primarily present in a broad layer around each seminiferous tubule, and blood vessel, and in variable distribution throughout the interstitial stroma. By day 20 there was no clear distinction in FN staining between the peritubular zone and the interstitial tissue. This indicates an involvement of FN in the ECM developments which occur in the peritubular zone of the testis at this time. The peritubular myoid cells were isolated from 20-22 day old rat testis and cultured on glass coverslips. These cells were grown to confluence with 10% fetal calf serum (FCS) in medium until day 4 and then subcultured to have secondary monocultures maintained with or without serum. By means of immunofluorescence and cytochemistry using avidin-biotin peroxidase complex it was observed that peritubular myoid cells were positive for FN and most of the FN was localized in the perinuclear region. Subcultured peritubular myoid cells maintained for 4 days in medium containing FCS developed an extensive interconnecting FN matrix. In the presence of 0.5 mM cAMP in culture, FN became localized along the filamentous process of peritubular myoid cells and more prominently in the areas of triangulated multi-cell aggregates as well as on the surface of the contracted small spherical cells. The addition of cAMP in the presence of FCS, also caused a noticeable change in the staining pattern; FN was detected along the filamentous process developing into a complex network of cells encased in an extensive matrix. It would appear that the translocation of FN in the cytoplasmic extensions of peritubular myoid cells may be a direct consequence of morphological changes associated with metabolic regulation of cAMP. This may also be related to the puberty associated development of in vivo changes in the ECM produced by peritubular myoid cells.

Proteins, drug targets and the mechanisms they control : the simple truth about complex networks

Realizing the promise of molecularly targeted inhibitors for cancer therapy will require a new level of knowledge about how a drug target is wired into the control circuitry of a complex cellular network. Here we review general homeostatic principles of cellular networks that enable the cell to be resilient in the face of molecular perturbations, while at the same time being sensitive to subtle input signals. Insights into such mechanisms may facilitate the development of combination therapies that take advantage of the cellular control circuitry, with the aim of achieving higher efficacy at a lower drug dosage and with a reduced probability of drug-resistance development.

The amplified peptidome : the new treasure chest of candidate biomarkers

Mass spectrometric analysis of the low-molecular weight (LMW) range of the serum/plasma proteome is revealing the existence of large numbers of previously unknown peptides and protein fragments predicted to be derived from low- abundance proteins. This raises the question of why such low abundance molecules would be retained at detectable levels in the circulation, instead of being rapidly cleared and excreted. Theoretical models of biomarker production and association with serum carrier proteins have been developed to elucidate the mechanisms governing biomarker half-life in the bloodstream. These models predict that the vast majority of LMW biomarkers exist in association with circulating high molecular mass carrier proteins. Moreover, the total serum/ plasma concentration of the biomarker is largely determined by the clearance rate of the carrier protein, not the free-phase biomarker clearance itself. These predictions have been verified experimentally using molecular mass fractionation of human serum before mass spectrometry sequence analysis. These principles have profound implications for biomarker discovery and measurement.

Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing

Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2), plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI). Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops), and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

Epithelial-mesenchymal transition and mesenchymal-epithelial transition are essential for the acquisition of stem cell properties in hTERT-immortalised oral epithelial cells

BACKGROUND INFORMATION: Evidence has shown that mesenchymal-epithelial transition (MET) and epithelial-mesenchymal transition (EMT) are linked to stem cell properties. We currently lack a model showing how the occurrence of MET and EMT in immortalised cells influences the maintenance of stem cell properties. Thus, we established a project aiming to investigate the roles of EMT and MET in the acquisition of stem cell properties in immortalised oral epithelial cells. RESULTS: In this study, a retroviral transfection vector (pLXSN-hTERT) was used to immortalise oral epithelial cells by insertion of the hTERT gene (hTERT(+)-oral mucosal epithelial cell line [OME]). The protein and RNA expression of EMT transcriptional factors (Snail, Slug and Twist), their downstream markers (E-cadherin and N-cadherin) and embryonic stem cell markers (OCT4, Nanog and Sox2) were studied by reverse transcription PCR and Western blots in these cells. Some EMT markers were detected at both mRNA and protein levels. Adipocytes and bone cells were noted in the multi-differentiation assay, showing that the immortal cells underwent EMT. The differentiation assay for hTERT(+)-OME cells revealed the recovery of epithelial phenotypes, implicating the presence of MET. The stem cell properties were confirmed by the detection of appropriate markers. Altered expression of alpha-tubulin and gamma-tubulin in both two-dimensional-cultured (without serum) and three-dimensional-cultured hTERT(+)-OME spheroids indicated the re-programming of cytoskeleton proteins which is attributed to MET processes in hTERT(+)-OME cells. CONCLUSIONS: EMT and MET are essential for hTERT-immortalised cells to maintain their epithelial stem cell properties.

Serum 25-hydroxy vitamin D concentrations are more deficient/insufficient in peritoneal dialysis than haemodialysis patients in a sunny climate

Background Research has identified associations between serum 25(OH)D and a range of clinical outcomes in chronic kidney disease and wider populations. The present study aimed to investigate vitamin D deficiency/insufficiency in dialysis patients and the relationship with vitamin D intake and sun exposure. Methods A cross-sectional study was used. Participants included 30 peritoneal dialysis (PD) (43.3% male; 56.87 ± 16.16 years) and 26 haemodialysis (HD) (80.8% male; 63.58 ± 15.09 years) patients attending a department of renal medicine. Explanatory variables were usual vitamin D intake from diet/supplements (IU day−1) and sun exposure (min day−1). Vitamin D intake, sun exposure and ethnic background were assessed by questionnaire. Weight, malnutrition status and routine biochemistry were also assessed. Data were collected during usual department visits. The main outcome measure was serum 25(OH)D (nm). Results Prevalence of inadequate/insufficient vitamin D intake differed between dialysis modality, with 31% and 43% found to be insufficient (<50 nm) and 4% and 33% found to be deficient (<25 nm) in HD and PD patients, respectively (P < 0.001). In HD patients, there was a correlation between diet and supplemental vitamin D intake and 25(OH)D (ρ = 0.84, P < 0.001) and average sun exposure and 25(OH)D (ρ = 0.50, P < 0.02). There were no associations in PD patients. The results remained significant for vitamin D intake after multiple regression, adjusting for age, gender and sun exposure. Conclusions The results highlight a strong association between vitamin D intake and 25(OH)D in HD but not PD patients, with implications for replacement recommendations. The findings indicate that, even in a sunny climate, many dialysis patients are vitamin D deficient, highlighting the need for exploration of determinants and consequences.

Differential subcellular and extracellular localisations of proteins required for insulin-like growth factor- and extracellular matrix-induced signalling events in breast cancer progression

Background: Cancer metastasis is the main contributor to breast cancer fatalities as women with the metastatic disease have poorer survival outcomes than women with localised breast cancers. There is an urgent need to develop appropriate prognostic methods to stratify patients based on the propensities of their cancers to metastasise. The insulin-like growth factor (IGF)-I:IGF binding protein (IGFBP):vitronectin complexes have been shown to stimulate changes in gene expression favouring increased breast cancer cell survival and a migratory phenotype. We therefore investigated the prognostic potential of these IGF- and extracellular matrix (ECM) interaction-induced proteins in the early identification of breast cancers with a propensity to metastasise using patient-derived tissue microarrays. Methods: Semiquantitative immunohistochemistry analyses were performed to compare the extracellular and subcellular distribution of IGF- and ECM-induced signalling proteins among matched normal, primary cancer and metastatic cancer formalin-fixed paraffin-embedded breast tissue samples. Results: The IGF- and ECM-induced signalling proteins were differentially expressed between subcellular and extracellular localisations. Vitronectin and IGFBP-5 immunoreactivity was lower while β1 integrin immunoreactivity was higher in the stroma surrounding metastatic cancer tissues, as compared to normal breast and primary cancer stromal tissues. Similarly, immunoreactive stratifin was found to be increased in the stroma of primary as well as metastatic breast tissues. Immunoreactive fibronectin and β1 integrin was found to be highly expressed at the leading edge of tumours. Based on the immunoreactivity it was apparent that the cell signalling proteins AKT1 and ERK1/2 shuffled from the nucleus to the cytoplasm with tumour progression. Conclusion: This is the first in-depth, compartmentalised analysis of the distribution of IGF- and ECM-induced signalling proteins in metastatic breast cancers. This study has provided insights into the changing pattern of cellular localisation and expression of IGF- and ECM-induced signalling proteins in different stages of breast cancer. The differential distribution of these biomarkers could provide important prognostic and predictive indicators that may assist the clinical management of breast disease, namely in the early identification of cancers with a propensity to metastasise, and/or recur following adjuvant therapy.

Identification of optimal epitopes for Plasmodium falciparum rapid diagnostic tests that target histidine-rich proteins 2 and 3

Rapid diagnostic tests (RDTs) represent important tools to diagnose malaria infection. To improve understanding of the variable performance of RDTs that detect the major target in Plasmodium falciparum, namely, histidine-rich protein 2 (HRP2), and to inform the design of better tests, we undertook detailed mapping of the epitopes recognized by eight HRP-specific monoclonal antibodies (MAbs). To investigate the geographic skewing of this polymorphic protein, we analyzed the distribution of these epitopes in parasites from geographically diverse areas. To identify an ideal amino acid motif for a MAb to target in HRP2 and in the related protein HRP3, we used a purpose-designed script to perform bioinformatic analysis of 448 distinct gene sequences from pfhrp2 and from 99 sequences from the closely related gene pfhrp3. The frequency and distribution of these motifs were also compared to the MAb epitopes. Heat stability testing of MAbs immobilized on nitrocellulose membranes was also performed. Results of these experiments enabled the identification of MAbs with the most desirable characteristics for inclusion in RDTs, including copy number and coverage of target epitopes, geographic skewing, heat stability, and match with the most abundant amino acid motifs identified. This study therefore informs the selection of MAbs to include in malaria RDTs as well as in the generation of improved MAbs that should improve the performance of HRP-detecting malaria RDTs.

Global sequence variation in the histidine-rich proteins 2 and 3 of Plasmodium falciparum: implications for the performance of malaria rapid diagnostic tests

Background Accurate diagnosis is essential for prompt and appropriate treatment of malaria. While rapid diagnostic tests (RDTs) offer great potential to improve malaria diagnosis, the sensitivity of RDTs has been reported to be highly variable. One possible factor contributing to variable test performance is the diversity of parasite antigens. This is of particular concern for Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-detecting RDTs since PfHRP2 has been reported to be highly variable in isolates of the Asia-Pacific region. Methods The pfhrp2 exon 2 fragment from 458 isolates of P. falciparum collected from 38 countries was amplified and sequenced. For a subset of 80 isolates, the exon 2 fragment of histidine-rich protein 3 (pfhrp3) was also amplified and sequenced. DNA sequence and statistical analysis of the variation observed in these genes was conducted. The potential impact of the pfhrp2 variation on RDT detection rates was examined by analysing the relationship between sequence characteristics of this gene and the results of the WHO product testing of malaria RDTs: Round 1 (2008), for 34 PfHRP2-detecting RDTs. Results Sequence analysis revealed extensive variations in the number and arrangement of various repeats encoded by the genes in parasite populations world-wide. However, no statistically robust correlation between gene structure and RDT detection rate for P. falciparum parasites at 200 parasites per microlitre was identified. Conclusions The results suggest that despite extreme sequence variation, diversity of PfHRP2 does not appear to be a major cause of RDT sensitivity variation.