Biomedical subjects

Abdelnaby Khalyfa

Publications and source records attributed to Abdelnaby Khalyfa.

8 recordsLinked to original sources

PubMed2026

Monocarboxylate Transporter 2 (MCT2) Reduction Is Associated with Increased Lung Tumor Growth and Alterations in the Immune Microenvironment in a Subcutaneous Tumor Model.

Monocarboxylate transporter 2 (MCT2; SLC16A7) is a high-affinity pyruvate transporter implicated in cancer metabolism. However, its role in lung cancer progression and the tumor microenvironment remains unclear. This study examined the effects of MCT2 reduction on tumor growth and cell-type-specific transcriptional changes within the tumor microenvironment. MCT2 loxP/loxP mice were crossed with mCre-Tg mice, and MCT2 deletion was induced by tamoxifen. Control (CO) mice received vehicle treatment. TC1 cells (100,000 cells/mouse) were injected subcutaneously, and tumors were harvested after 24 days. Single-nucleus RNA sequencing (snRNA-seq) was performed on isolated tumor nuclei (4000 nuclei/sample; n = 3 per group) using the 10x Genomics Chromium platform. Data were processed with Cell Ranger v3.0.2 and Seurat v5.2.1, followed by differential expression and pathway enrichment analyses integrated with macrophage bulk RNA-seq data. Tumors in mice with systemic MCT2 reduction grew significantly faster than those in control mice, demonstrating an association between host MCT2 reduction and increased tumor growth. Transcriptomic analysis generated high-quality profiles from 6864 CO and 10,055 KO nuclei. Clustering identified 12 cellular populations and cell types. MCT2 reduction altered pathways involved in glycolysis, the tricarboxylic acid cycle, oxidative phosphorylation, and fatty acid metabolism across multiple populations. Macrophages showed prominent transcriptional changes, including enrichment of MAPK, PI3K-Akt, IgSF-CAM, ECM, and cytokine-cytokine signaling pathways. These findings were supported by macrophage bulk RNA-seq data. Systemic MCT2 reduction was associated with increased tumor growth and broad transcriptional alterations within the tumor micro-environment. Differences in metabolic and immune-related transcriptional programs, particularly in macrophages, identify potential mechanisms associated with tumor progression that warrant further functional investigation.

Animals↗

PubMed2005

MPrime: efficient large scale multiple primer and oligonucleotide design for customized gene microarrays.

BACKGROUND: Enhancements in sequencing technology have recently yielded assemblies of large genomes including rat, mouse, human, fruit fly, and zebrafish. The availability of large-scale genomic and genic sequence data coupled with advances in microarray technology have made it possible to study the expression of large numbers of sequence products under several different conditions in days where traditional molecular biology techniques might have taken months, or even years. Therefore, to efficiently study a number of gene products associated with a disease, pathway, or other biological process, it is necessary to be able to design primer pairs or oligonucleotides en masse rather than using a time consuming and laborious gene-by-gene method. RESULTS: We have developed an integrated system, MPrime, in order to efficiently calculate primer pairs or specific oligonucleotides for multiple genic regions based on a keyword, gene name, accession number, or sequence fasta format within the rat, mouse, human, fruit fly, and zebrafish genomes. A set of products created for mouse housekeeping genes from MPrime-designed primer pairs has been validated using both PCR-amplification and DNA sequencing. CONCLUSION: These results indicate MPrime accurately incorporates standard PCR primer design characteristics to produce high scoring primer pairs for genes of interest. In addition, sequence similarity for a set of oligonucleotides constructed for the same set of genes indicates high specificity in oligo design.

Animals↗

PubMed2005

Human adult olfactory neuroepithelial derived progenitors retain telomerase activity and lack apoptotic activity.

Olfactory epithelium (OE) contains a population of progenitors responsible for its life-long regenerative capacity. Procedures for the isolation of these progenitors have been established [F.J. Roisen, K.M. Klueber, C.L. Lu, L.M. Hatcher, A. Dozier, C.B. Shields, Adult human olfactory stem cells, Brain Res., 890 (2001) 11-12.] and over 40 patient-specific cell lines from adult postmortem OE and endoscopic biopsy from patients undergoing nasal sinus surgery have been obtained. As these cells emerged in primary cultures, they formed neurospheres (NSFCs). The purpose of the present study was to further characterize these adult human olfactory-derived progenitors. Subcultures of the NSFCs have been passaged nearly 200 times, with a mitotic cycle of 18-20 h. Telomerase activity remains in stem cells; therefore, ELISA was employed to determine the telomerase activity of different lines and passages. Since progenitors undergo low levels of apoptosis, the levels of apoptosis were also examined in these populations. The levels of telomerase and apoptotic activity in 12 NSFC lines remained relatively constant irrespective of donor age, culture duration, or sex. To further study the apoptotic characteristics of the NSFCs, nine different caspases (cysteine proteases) known to be critical in apoptosis were evaluated using gene-microarrays comparing cells from a single line at passages 14, 88, and 183. No increases were found in caspase activity in all passages studied. ELISA confirmed the absence of caspase activity over the entire range of passages. This study further suggests that NSFCs can be obtained and used from patients, irrespective of age, sex, or time in culture without altered viability expanding the potential utility of these cells for autologous transplantation and possible diagnostic testing.

Adult↗

PubMed2005

Serum deprivation induces apoptotic cell death of transformed rat retinal ganglion cells via mitochondrial signaling pathways.

PURPOSE: Apoptosis-related signaling pathways were investigated in a cultured rat retinal ganglion cell (RGC-5) line deprived of growth factors after serum withdrawal from the culture medium. METHODS: RGC-5 cells were subjected to serum deprivation for 2 to 6 days and compared with RGC-5 cells cultured in growth medium containing 10% fetal calf serum. Cell viability was determined by a neutral red dye uptake assay. Apoptosis of RGC-5 cells was established by DNA laddering. The expression of various apoptosis-related genes was investigated by immunoblot analysis, and or reverse transcription polymerase chain reaction (RT-PCR) analysis. The redox state of the cell was determined by biochemical methods, including NF-kappaB binding activity by electrophoretic mobility gel shift assays (EMSA) and mitochondrial damage by JC-1 (5,5', 6,6'-tetrachloro 1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide) staining, using live cell confocal microscopy and cytosolic release of cytochrome c. RESULTS: Fifty percent cell loss was evident after 2 days of serum deprivation, as demonstrated by neutral red dye uptake assay. This cell loss was due to apoptotic cell death, as established by DNA laddering. The oxidative state of serum-deprived RGC-5 cells was perturbed as suggested by the increase in malonyldialdehyde (MDA) and a decrease in reduced glutathione (GSH) levels in cell lysates. The apoptosis of the RGC-5 cells was associated with the activation of caspase-3, -8, and -9, and increased levels of Bax with corresponding decreases in Bcl-2 levels and NF-kappaB (NF-kappaB) binding activity. Serum deprivation was also associated with a loss of mitochondrial function, as revealed by cytosolic release of cytochrome c and JC-1 staining of mitochondria of dying RGC-5 cells. CONCLUSIONS: Taken together, these results indicate that serum withdrawal induces apoptotic cell death in RGC-5 cells via mitochondrial pathways. These studies lead to the speculation that growth factor deprivation arising from blockade of retrograde transport of neurotrophins may involve similar mechanism(s) of retinal ganglion cell death in glaucoma.

Animals↗

PubMed2003

Transcription profiling of estrogen target genes in young and old mouse uterus.

The goal of this study was to identify age-related changes in the expression of estrogen target genes in mouse uterus. We developed a novel 'estrogen response element (ERE) Chip' microarray bearing 297 genes including both known estrogen target genes and genes identified by searching the mouse genome database to have EREs, AP-1 sites, and Sp1 sites, all targets of estrogen receptor (ER) regulation. 400-500 bp PCR products of these 297 genes were printed onto nylon membranes creating the 'ERE Chip' microarray. This microarray is unique because it is the first estrogen-responsive gene-specific microarray to identify changes in uterine gene expression in young versus old mice. Using this ERE microarray we identified 10 uterine genes whose expression was up-regulated in old mice, e.g. beta-actin, calcium binding protein 45a, Sp1, and COUP-TFII. In contrast, the expression of only 4 uterine genes, i.e., complement C3, lactoferrin, Muc-1, and 17-beta-hydroxysteroid dehydrogenase 8 (H2-Ke6) was down-regulated in old mice. These changes may reflect an increase in stromal and a decrease in glandular epithelial gene expression, and may be associated with age-related changes in these tissue compartments within the uterus, possibly leading to the decline in reproductive function in C57Bl/6 mice.

Aging↗

PubMed2003

Changes in protein levels of elongation factors, eEF1A-1 and eEF1A-2/S1, in long-term denervated rat muscle.

PURPOSE: This study was designed to determine whether the quantitative relationship between the levels of the eEF1A-1(developmental) and eEF1A-2/S1 (adult) isoforms of peptide elongation factor remains stable after denervation of skeletal muscle or whether in response to denervation the relative amount of the developmental form would increase. In normal postnatal rat muscle, eEF1A-2/S1 is the dominant form represented, and levels of eEF1A-1 are extremely low. METHODS: One hind limb in young adult rats was permanently denervated. Denervated and corresponding contralateral control muscles were removed for biochemical and morphological analysis from 2 days to 25 months after denervation. RESULTS: By one month after denervation, relative levels of eEF1A-1 rose dramatically in relation to those of eEF1A-2/S1, and they remained high throughout the remainder of the 25-month denervation period. Ultrastructural analysis showed a complex mix of muscle fiber atrophy, dying muscle nuclei and muscle fibers, and newly forming muscle fibers in the same tissue. CONCLUSIONS: As during muscle regeneration, levels of the developmental eEF1A-1 isoform of peptide elongation factor greatly increased relative to those of the adult eEF1A-2/S1 adult isoform following denervation in rat muscles. However, in contrast to regeneration, the elevated level of eEF1A-1 did not return to the basal minimal level. Since switching from eEF1A-1 to eEF1A-2/S1 is an indicator that terminal differentiated is completed, the failure of eEF1A-1 to return to basal level may be indicative of the persistence of an unstable tissue environment that includes muscle fiber atrophy, degeneration and neomyogenesis. The specific cellular basis for the increase in eEF1A-1 could not be determined from this study.

Animals↗

PubMed2002

Development-dependent disappearance of caspase-3 in skeletal muscle is post-transcriptionally regulated.

Caspase-3, a major player in apoptosis, engages apoptosis-activated cells into an irreversible pathway leading to cell death. In this article, we report that caspase-3 protein is absent from rat and mouse adult skeletal muscles, despite the abundant presence of its mRNA. During skeletal muscle development, caspase-3 protein is present in neonatal animals, but its expression gradually decreases, and disappears completely by 1 month of age, when there is still abundant caspase-3 mRNA. This discordance between caspase-3 message and protein expression is unique to skeletal muscle, as in all other analyzed tissues the protein presence correlates with the presence of the mRNA. The only circumstance in which caspase-3 protein appears in adults is in regenerating muscles; once regeneration is complete, however, it again becomes undetectable in repaired muscles. We conclude that caspase-3 protein in skeletal muscle is uniquely regulated at the post-transcriptional level, unseen in other tissues such as brain, heart, lung, kidney, thymus, spleen, liver, or testis. The post-transcriptional regulation of caspase-3 might serve as a fail-safe mechanism to avoid accidental cell death.

Aging↗

PubMed2002

Effects of long-term denervation on skeletal muscle in old rats.

We compared the reactions to denervation of limb muscles between young adult and old rats. After denervation for up to 4 months in 24-month-old rats, limb muscles were removed and analyzed for contractile properties, morphology, and levels of several key molecules, including the peptide elongation factors eEF1A-1 and eEF1A-2/S1, myogenin, gamma-subunit of the acetylcholine receptor, and cyclin D3. The principal difference between denervated old and young muscle is a somewhat slower rate of atrophy in denervated older muscle, especially among the type II fibers. Expression levels of certain molecules were higher in old than in young control muscle, but after denervation, levels of these molecules increased to the same absolute values in both young and old rats. Although many aspects of postdenervation reactions do not differ greatly between young and old animals, the lesser degree of atrophy in the old rats may reflect significant age-based mechanisms.

Age Factors↗