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PubMed2002

Protein microarrays to detect protein-protein interactions using red and green fluorescent proteins.

Proteomics, the study of protein function on a global scale, will play an important role in furthering our understanding of gene functions, complex biological pathways, and discovery of novel drug targets. A number of techniques have been developed for proteomic studies to identify and analyze proteins, compare protein expression levels, and study protein-protein interactions. Recent developments have applied a DNA array-type approach to immobilize proteins on a surface for high-throughput analysis. Here we report the development and construction of protein chips using derivatized glass and nitrocellulose-coated slides and the employment of recombinant proteins fused with green and red fluorescent proteins for detection. Fluorescent signals were found to be proportional to the amount of arrayed proteins and could be readily detected with a conventional fluorescence slide scanner. This technique allows the investigation of protein-protein interactions without the need for additional labeling steps of probe proteins.

Antibodies↗

PubMed2000

Active site modulation in the N-acetylneuraminate lyase sub-family as revealed by the structure of the inhibitor-complexed Haemophilus influenzae enzyme.

The N-acetylneuraminate lyase (NAL) sub-family of (beta/alpha)(8) enzymes share a common catalytic step but catalyse reactions in different biological pathways. Known examples include NAL, dihydrodipicolinate synthetase (DHDPS), d-5-keto-4-deoxyglucarate dehydratase, 2-keto-3-deoxygluconate aldolase, trans-o-hydroxybenzylidenepyruvate hydrolase-aldolase and trans-2'-carboxybenzalpyruvate hydratase-aldolase. Little is known about the way in which the three-dimensional structure of the respective active sites are modulated across the sub-family to achieve cognate substrate recognition. We present here the structure of Haemophilus influenzae NAL determined by X-ray crystallography to a maximum resolution of 1.60 A, in native form and in complex with three substrate analogues (sialic acid alditol, 4-deoxy-sialic acid and 4-oxo-sialic acid). These structures reveal for the first time the mode of binding of the complete substrate in the NAL active site. On the basis of the above structures, that of substrate-complexed DHDPS and sequence comparison across the sub-family we are able to propose a unified model for active site modulation. The model is one of economy, allowing wherever appropriate the retention or relocation of residues associated with binding common substrate substituent groups. Our structures also suggest a role for the strictly conserved tyrosine residue found in all active sites of the sub-family, namely that it mediates proton abstraction by the alpha-keto acid carboxylate in a substrate-assisted catalytic reaction pathway.

Amino Acid Sequence↗

PubMed2002

The role of nitric oxide in ocular surface diseases.

For the first time, the current series of studies provide a possible pathophysiologic mechanism of NO-induced ocular surface disease. NO is present in tear and aqueous humor and is suspected of having an important physiological role in maintaining normal homeostasis of the ocular surface. NO concentrations are higher in aqueous humor compared to tears, though some variability exists between different species. When inflammation was induced by PTK wounding or LPS, three forms of NOS expression were seen in corneal cells. Each isoform of NOS was expressed uniquely according to the specific location of inflammation. When concentrations of NO peaked, the levels of iNOS were markedly increased in fibroblasts and inflammatory cells. The correlation between NO and inflammation was confirmed by treatment with NOS inhibitor, which abrogated the amount of both NO and inflammation. The tissue damage by NO was measured by nitrotyrosine formation. Damage was detected mainly in inflammatory cells, especially those localized in and around the limbal vessel. It is likely that expression of iNOS in limbal fibroblasts has other roles related to survival of limbal stem cells and fibroblasts as well. Because the main source of NO are fibroblasts, we were able to determine the effect of various concentrations of NO on cell viability using a fibroblast culture system. Cell viability increased in dose dependent manner from 10 microM to 500 microM of the NO generator SNAP, but decreased at concentrations above 1000 microM, suggesting that the in vivo mechanism of cell death was indirect, through specific biologic pathways. Therefore, the pathophysiological mechanism of NO action is bimodal with a toxicological component in ocular surface diseases. Furthermore, its concentration and interaction with other oxygen mediators appear to vary depending on the degree of inflammation.

Animals↗

PubMedDate not supplied

Chromium in plants. Comparison between the concentration of chromium in Brazilian nonhypo and hypoglycemic plants.

Chromium is an essential trace element and is associated with some biological pathways, especially with glucose tolerance. For these reasons, we decided to determine the concentration of chromium in two sets of Brazilian medicinal plants. The first group consisted of plants that are considered as antidiabetic, whereas the second included plants that do not have this therapeutic property. The concentration of chromium was determined by flameless atomic absorption. All the plants analyzed contain chromium in the normal range for this element, but the hypoglycemic plants contain more chromium than the others (1-4 micrograms/g compared to 0.5-1.5 micrograms/g).

Brazil↗

PubMed2002

Differential display analysis of gene expression in mammals: a p53 story.

Differential display is used worldwide as a method to identify changes in gene expression and to discover novel genes that are involved in important biological pathways. The principle of differential display is the systematic amplification of the 3' termini of messenger RNAs by using anchored oligo-dT primers in combination with upstream arbitrary primers. The separation of the polymerase chain reaction products by gel electrophoresis and their direct comparison allows the identification of differentially regulated genes. Recently, fluorescent differential display was established as the first nonradioactive differential display system with equivalent sensitivity to originally 33P isotopic labeling method. Because of its simplicity, sensitivity, reproducibility and automation, which increase the throughput and accuracy, differential display has become one of the most widely used gene-screening methods in biomedical research involving mammals. This chapter provides a glimpse of the application of differential display in search of target genes of the p53 tumor suppressor gene.

Animals↗

PubMed2005

A genetic perspective on myopia.

Myopia is a refractive error of the eye that has a significant socioeconomic impact due to its increasing prevalence and the fact that it causes visual impairment. Its aetiology is complex and is likely to involve the interaction of environmental and genetic influences. Tight environmental influence is exemplified by defocus-induced myopia produced in animal models, while genetic factors predominate in familial occurrence of myopia with a Mendelian inheritance pattern. The involvement of numerous mediators, such as cytokines, neurotransmitters and transcription factors, in myopia development has been indicated through various lines of investigation, particular interest focussing on scleral extracellular matrix proteins and developmental genes of the eye. As high-throughput technology for large-scale genotyping and RNA expression analysis enters the field of myopia research, a productive avenue will open up for deciphering the aetiological heterogeneity of myopia and the biological pathways underlying its development.

Animals↗

PubMed2004

Day/night rhythms in gene expression of the normal murine heart.

Molecular circadian oscillators have recently been identified in heart and many other peripheral organs; however, little is known about the physiologic significance of circadian gene cycling in the periphery. While general temporal profiles of gene expression in the heart have been described under constant lighting conditions, patterns under normal day/night conditions may be distinctly different. To understand how gene expression contributes to cardiac function, especially in human beings, it is crucial to examine these patterns in 24-h light and dark environments. High-density oligonucleotide microarrays were used to assess myocardial expression of 12,488 murine genes at 3-h intervals under the normal conditions of light and dark cycling. Variation in genetic activity was considerable, as 1,634 genes (approximately 13% of genes analyzed) exhibited statistically significant changes across the 24-h cycle. Some genes exhibited rhythmic expression, others showed abrupt change at light-to-dark and dark-to-light transitions. Importantly, genes that exhibited significant cycling rhythms mapped to key biological pathways, including for example cardiac cellular growth and remodeling, as well as transcription, translation, mitochondrial respiration, and signaling pathways. Gene expression in the heart is remarkably different in the day versus the night. Some gene cycling may be driven by the central circadian pacemaker, while other changes appear to be responses to light and dark. This has important implications regarding our understanding of how the molecular physiology of the heart is controlled, including temporal patterns of organ growth, renewal, and disease, comparative gene expression, and the most appropriate times for administration of therapy.

Animals↗

PubMed2006

A perfusion protocol for highly efficient transduction of intact pancreatic islets of Langerhans.

AIMS/HYPOTHESIS: Successful gene transfer to pancreatic islets might be a powerful tool for dissecting the biological pathways involved in the functional impairment and destruction of beta cells in type 1 diabetes. In the long run, such an approach may also prove useful for promoting islet graft survival after transplantation in diabetic patients. However, efficient genetic modification of primary insulin-producing cells is limited by the specific compact structure of the pancreatic islet. We present here a whole-pancreas perfusion-based transduction procedure for genetic modification of intact pancreatic islets. MATERIALS AND METHODS: We used flow cytometry analysis and confocal microscopy to evaluate the efficiency of in vitro and perfusion-based transduction protocols that use adenoviral and lentiviral vectors expressing green fluorescent protein. Islet cell viability was assessed by fluorescence microscopy and beta cell function was determined via glucose-stimulated insulin secretion. RESULTS: In intact rat and human pancreatic islets, adenoviral and lentiviral vectors mediated gene transfer to about 30% of cells, but they did not reach the inner cellular mass within the islet core. Using the whole-pancreas perfusion protocol, we demonstrate that at least in rodent models the centrally located insulin-producing cells can be transduced with high efficiency, while preserving the structural integrity of the islet. Moreover, islet cell viability and function are not impaired by this procedure. CONCLUSIONS/INTERPRETATION: These results support the view that perfusion-based transduction protocols may significantly improve the yield of successfully engineered primary insulin-producing cells for diabetes research.

Adenoviridae↗

PubMed1999

The budding yeast cohesin gene SCC1/MCD1/RHC21 genetically interacts with PKA, CDK and APC.

Cohesin is a protein that plays a key role in the cohesion and separation of sister chromatids. During the duplication of chromatids, cohesin holds sister chromatids together until the onset of anaphase, and thereby prevents the premature separation of sister chromatids which would otherwise jeopardize the faithful segregation of chromosomes. To investigate the molecular mechanisms of sister chromatid cohesion, we have isolated multicopy suppressors of a temperature-sensitive (ts) mutation in the SCC1/MCD1/RHC21 gene which encodes a component of the cohesin complex in budding yeast. Isolation of multicopy suppressors of rhc21-sk16 and further genetic analyses revealed that several distinct biological pathways are involved in the regulation of SCC1/MCD1/RHC21 function. Firstly, PDE2 and BCY1, each of which inhibits the activity of protein kinase A (PKA), suppressed the temperature sensitivity of the rhc21-sk16 mutant. Secondly, PDE2 suppressed the temperature sensitivity of the cdc16-1 mutant. These results suggest that SCC1/MCD1/RHC21 is negatively regulated by the PKA pathway via the anaphase promoting complex (APC). Thirdly, ZDS1, a multicopy suppressor of cdc28-1N, and its homologue ZDS2 were isolated as multicopy suppressors of rhc21-sk16. Furthermore, the rhc21-sk16 mutant did not grow in the presence of the cdc28-1N mutation. Hence, SCC1/MCD1/RHC21 is positively regulated by the mitotic CDK, CDC28. Finally, SCC1/MCD1/RHC21 was found to interact genetically with CDC20, an activator of APC. Overexpression of CDC20 suppressed the temperature sensitivity of rhc21-sk16, and rhc21-sk16 was shown to be synthetically lethal with cdc20-1. In addition, the growth of the rhc21-sk16 mutant was inhibited by overproduction of the anaphase inhibitor Pds1p, whose degradation is mediated by Cdc20p in APC-dependent proteolysis. The functional relationships between SCC1/MCD1/RHC21 and PKA, CDK or APC are discussed.

Cdc20 Proteins↗

PubMed2004

Comparative physical maps of the human and mouse Meckel syndrome critical regions.

Meckel syndrome (MKS-OMIM 24900) is an autosomal recessive disease characterized by cystic kidneys, occipital encephalocele, polydactyly, and fibrotic changes of the liver, typically resulting in postnatal death. A Meckel syndrome critical region (MKS1) maps to human Chromosome (Chr) 17, in a region of homology to mouse Chr 11. Here we report the comparison of human Chr 17q23 with mouse Chr 11. We have generated physical maps of the human and mouse MKS1 critical regions. Additionally, we have created a transcript map of the MKS1 critical region in both species. By comparing these physical maps, we observe a high degree of similarity in gene order in the human and mouse Meckel syndrome critical regions. We have also examined the expression patterns of genes in the MKS1 region to assess their potential as MKS1 candidates. Finally, we have analyzed genes present in the other Meckel syndrome critical regions, MKS2 and MKS3, to determine whether any of the candidate genes for the three MKS loci have similar gene functions or are members of a common biological pathway.

Abnormalities, Multiple↗

PubMed2025

High-fat and low-fat fermented milk and cheese intake, proteomic signatures, and risk of all-cause and cause-specific mortality.

PURPOSE: This study aimed to examine the associations between the intake of high- and low-fat fermented dairy (cheese and fermented milk), their proteomic profiles, and mortality risk. METHODS: This cohort study included 25,187 participants (mean age 57.7 years, 60.9% females). Fermented dairy intake was assessed by a modified diet history method. In a random subset of this cohort (n = 4359), we constructed proteomic signatures for fermented dairy intake using 136 candidate plasma proteins. RESULTS: During 23.5 years of follow-up, 9742 participants died. High-fat cheese (> 20% fat) intake was inversely associated with risk of all-cause mortality (HR for an increment of 20 g/day, 0.97; 95% CI, 0.96-0.99, P < 0.001) and cardiovascular disease mortality (HR, 0.96; 95% CI, 0.93-0.99, P = 0.006). Low-fat cheese intake showed an inverse association with all-cause mortality (HR, 0.98; 95% CI, 0.96-1.00, P = 0.047). Low-fat fermented milk intake was inversely associated with all-cause mortality (HR for an increment of 250 g/day, 0.91; 95% CI, 0.85-0.97, P = 0.006), while high-fat fermented milk (> 2.5% fat) showed null association. A total of 42, 26, 0, and 39 proteins were identified for the signature of high-fat cheese, low-fat cheese, high-fat fermented milk, and low-fat fermented milk, respectively. Inverse associations with all-cause mortality were observed for all three signatures with identified proteins. The identified proteins were involved in biological pathways related to immune response and inflammation. CONCLUSION: Our study indicated that consuming high-fat cheese, low-fat cheese, and low-fat fermented milk was linked to survival benefits. Plasma proteins improve our understanding of the health effects of fermented dairy.

Humans↗

PubMed2003

Targeting enzymes involved in spermidine metabolism of parasitic protozoa--a possible new strategy for anti-parasitic treatment.

Sequencing data obtained from the Plasmodium, Anopheles gambiae and human genome projects provide a new basis for drug and vaccine development. One of the most characteristic features in the process of drug development against parasitic protozoa is target identification in a biological pathway. The next step must be a structure-based rational drug design if the target is not only present in the parasite. In mouse models of malaria, such drugs should be tested for efficacy of the new therapies. Here, we present data that pinpoint the existence of two enzymes of the polyamine pathway involved in spermidine metabolism in P. falciparum, i.e. deoxyhypusine synthase (DHS; EC 1.1.1.249) and homospermidine synthase (HSS; EC 2.5.1.45). Recent data obtained from the malaria genome databases showed that at least a putative gene encoding DHS is present in the parasite. Sequencing data from the P. falciparum genome project prove that the eukaryotic initiation factor eIF5A (the substrate for DHS) exists in P. falciparum. Here, we present the amino acid sequence of eIF5A from P. vivax, which causes tertiary malaria. EIF5A from P. vivax shows 82% nucleic acid and 97% amino acid identity to its homologue from P. falciparum. GC/MS data and inhibitor studies with agmatine prove that the triamine homospermidine occurs in the parasite. These data suggest a separate locus encoding HSS in P. falciparum. The hss gene recruits from the dhs gene in eukaryotes. Here, we present genomic DNA fragments obtained by amplification with primers of a conserved region (amino acid positions 550-1,043) between the putative P. falciparum DHS gene ( dhs) and the HSS gene ( hss) from the plant Senecio vulgaris (Asteraceae). The amplification product from different P. falciparum strains reveals differences in sequence identity, compared with the putative dhs gene from P. falciparum strain 3D7. Expression of the full-length clone and determination of HSS-specific activity will finally prove whether a separate region encoding HSS exists.

Alkyl and Aryl Transferases↗

PubMed2001

The maize WD-repeat gene ZmRbAp1 encodes a member of the MSI/RbAp sub-family and is differentially expressed during endosperm development.

Members of the MSI/RbAp sub-family of WD-repeat proteins are widespread in eukaryotic organisms and form part of multiprotein complexes that are involved in various biological pathways, including chromatin assembly, regulation of gene transcription, and cell division. In this study we report the isolation and characterization of a cDNA sequence from Zea mays, which encodes an RbAp-like protein (ZmRbAp1) that binds acetylated histones H3 and H4 and suppresses mutations that have a negative effect on the Ras/cAMP pathway in yeast. The ZmRbAp genes form a gene family and are expressed in different tissues of Z. mays L. plants. Determination of its expression pattern during maize seed development revealed that ZmRbAp transcripts are abundant during the initial stages of endosperm formation. In addition, the transcripts are specifically localized in shoot apical meristem and leaf primordia of the embryo. A possible role for the ZmRbAp genes in early endosperm differentiation and plant development is discussed.

Acetylation↗

PubMed2026

CRISPR-Enabled functional genomics in hPSCs-derived neural models for autism spectrum disorder.

Autism Spectrum Disorder (ASD) is a genetically heterogeneous neurodevelopmental condition in which hundreds of individually rare risk variants converge on a small number of shared biological pathways, including synaptic scaffolding, chromatin remodeling, excitation-inhibition balance, and cellular energy metabolism. Translating this genetic heterogeneity into mechanistic insight requires experimental systems capable of interrogating individual gene functions in human-relevant neural contexts at scale. CRISPR-enabled functional genomics in human pluripotent stem cell (hPSC)-derived neural models, spanning neural progenitors, cortical and inhibitory neurons, astrocytes, microglia, and brain organoids, provides precisely this capability. By integrating pooled perturbation screens with multimodal readouts including single-cell and spatial transcriptomics, chromatin accessibility profiling, proximity labeling proteomics, multi-electrode array electrophysiology, and metabolic flux analysis, these platforms enable systematic, causal mapping of ASD gene function at system resolution. Early applications have already revealed convergent mechanisms: BAF complex disruption expands the ventral progenitor pool and biases its fate toward oligodendrocyte and interneuron lineages; ADNP loss impairs microglial synaptic pruning through altered endocytic trafficking; and mTOR pathway dysregulation in PTEN- and TSC2-perturbed models links genetic risk directly to metabolic and mitochondrial dysfunction. Computational frameworks including MIMOSCA and SCEPTRE enable causal network reconstruction and pseudotime inference from these datasets, moving the field from gene lists toward pathway-level models of ASD pathobiology. Translational applications leverage isogenic iPSC panels and variant-level base and prime editing to stratify ASD variants by functional impact, informing gene therapy design for haploinsufficient targets such as CHD8 and SCN2A via AAV or antisense oligonucleotide delivery. Remaining challenges, including model developmental immaturity, batch variability, and the difficulty of modeling polygenic risk, are addressed by a roadmap integrating spatial perturbomics, AI-driven causal inference, and population-scale standardized biobanks. This review synthesizes the current state of CRISPR-based functional genomics in human stem cell neural models as a coherent experimental framework for converting ASD genetic associations into mechanistic understanding and therapeutic opportunity.

Humans↗

PubMed2004

Advances in the genetics of inflammatory bowel disease.

Research efforts in the inflammatory bowel diseases have been uniquely successful in identifying genetic linkage regions likely containing susceptibility genes for Crohn's disease and ulcerative colitis. In two of these regions, definitive gene associations have been established, namely for the NOD2/CARD15 gene on chromosome 16 (IBD1) and the OCTN1/SLC22A4-OCT/SLC22A5 genes on chromosome 5q (IBD5), both conferring increased risk for developing Crohn's disease. Recently, significant gene associations have been reported for additional genes, including DLG5, MDR1, and TLR4 as well. The NOD2/CARD15 gene mutations are associated with ileal disease location and a modestly earlier age of onset compared with NOD2/CARD15 wild-type Crohn's disease patients. Future progress in the genetics of inflammatory bowel disease will likely involve systematic phenotyping, including the incorporation of clinical subtypes and novel biomarkers. The ultimate goal of genetic research in inflammatory bowel disease is to identify the earliest biologic pathways that are altered, resulting in disease pathogenesis. Identification of these key pathways will potentially highlight novel therapeutic targets.

Alleles↗

PubMed2024

Cohort Studies and Multi-omics Approaches to Low-Dose Ionizing Radiation-Induced Cardiovascular Disease: A Comprehensive Review.

The effect of low-dose ionizing radiation exposure on the risk of cardiovascular disease (CVD) represents a significant concern in the field of radiation protection. The prevailing approach to mitigating the adverse effects of low-dose or low-dose-rate radiation does not currently incorporate the potential risk of CVD, despite the possibility that such risk may be a substantial contributor to overall health hazards. Current evidence suggests a potential association between radiation exposure and CVD; however, the overall findings remain inconclusive. This is particularly due to the uncertainty surrounding the influence of significant non-radiation risk factors on the associations reported in epidemiological studies. It is difficult to discern the underlying connection in observational epidemiology when there is substantial variation in baseline risk factors. The paucity of epidemiological research in this domain is being partially offset by the advancement of multi-omics approaches. These methods assist in identifying radiosensitive targets, comprehending underlying biological processes, and pinpointing biomarkers. This, in turn, fortifies the evidence gleaned from epidemiological studies. In this review, we delve into the body of epidemiological research pertaining to CVD induced by low-dose ionizing radiation and the application of multi-omics techniques. The integration of these two methodologies holds the promise of identifying specific molecules or biological pathways that can be employed to validate endpoints related to radiation risk assessment.

Humans↗

PubMed1984

Abnormal abdominal computerized tomography with amiodarone therapy and clinical significance.

Although the antiarrhythmic agent, amiodarone, is known to cause elevation of liver function tests, the effect of this drug on abdominal computerized tomography (CT) scans in patients on amiodarone therapy is unknown. Since iodine is in its molecular structure, the presence of amiodarone or its metabolites might be expected to produce higher CT numbers similar to the effect of contrast agents. To test this hypothesis, CT scans were performed in a series of 25 patients receiving amiodarone, 14 receiving short-term (mean 2.5 +/- 1.3 days) and 19 receiving long-term therapy (mean 130 +/- 75 days), as well as in a control group not receiving amiodarone. Gastrointestinal symptoms (if any) and liver function tests (LFT) prior to instituting amiodarone therapy and at the time of CT scan were also documented. CT scans showed a modestly increased density in multiple organs in the short-term group, but a markedly increased hepatic density in all but one patient in the long-term group. Presence of gastrointestinal symptoms or abnormal LFT did not appear to correlate with the CT number. Such findings would suggest that increased hepatic density on CT scan is to be expected in patients receiving long-term amiodarone therapy, and although the exact biologic pathways of amiodarone metabolism are uncertain, the liver appears to be a major site of drug storage and/or metabolism.

Adult↗

PubMed1994

Sulfotransferase gene expression in rat hepatic and extrahepatic tissues.

Enzymatic sulfation has been implicated to play a key role in a number of essential biological pathways including xenobiotic detoxication, carcinogen activation, and the regulation of intra-tissue hormone activity. In order to increase our understanding of the critical determinants governing the regulation of sulfotransferase gene expression, we investigated age-, gender-, and xenobiotic-related alterations in hydroxysteroid sulfotransferase-a or aryl sulfotransferase-IV gene expression. Northern blot and slot blot analyses showed that rat hepatic hydroxysteroid sulfotransferase-a mRNA expression was responsive to age- and gender-related signals. The results also suggested that the rat hepatic aryl sulfotransferase-IV and hydroxysteroid sulfotransferase-a genes are differentially regulated. Northern blot and reverse transcriptase polymerase chain reaction analyses demonstrated that hydroxysteroid sulfotransferase-a mRNA was expressed to a greater extent in female rat liver than in lung or kidney tissue. In addition, rat hepatic hydroxysteroid sulfotransferase-a gene expression in mature female rats, although not substantially altered in response to short-term fasting or high-dose dexamethasone treatment, was suppressed after treatment with the polycyclic aromatic hydrocarbon, 3-methylcholanthrene.

Aging↗