Multiomic Metabolic Enrichment Network Analysis Reveals Metabolite–Protein Physical Interaction Subnetworks Altered in Cancer

Metabolism is recognized as an important driver of cancer progression and other complex diseases, but global metabolite profiling remains a challenge. Protein expression profiling is often a poor proxy since existing pathway enrichment models provide an incomplete mapping between the proteome and metabolism. To overcome these gaps, we introduce multiomic metabolic enrichment network analysis (MOMENTA), an integrative multiomic data analysis framework for more accurately deducing metabolic pathway changes from proteomics data alone in a gene set analysis context by leveraging protein interaction networks to extend annotated metabolic models. We apply MOMENTA to proteomic data from diverse cancer cell lines and human tumors to demonstrate its utility at revealing variation in metabolic pathway activity across cancer types, which we verify using independent metabolomics measurements. The novel metabolic networks we uncover in breast cancer and other tumors are linked to clinical outcomes, underscoring the pathophysiological relevance of the findings.     

A comparative Proteomics Analysis Identified Differentially Expressed Proteins in Pancreatic Cancer–Associated Stellate Cell Small Extracellular Vesicles

Human pancreatic stellate cells (HPSCs) are an essential stromal component and mediators of pancreatic ductal adenocarcinoma (PDAC) progression. Small extracellular vesicles (sEVs) are membrane-enclosed nanoparticles involved in cell-to-cell communications and are released from stromal cells within PDAC. A detailed comparison of sEVs from normal pancreatic stellate cells (HPaStec) and from PDAC-associated stellate cells (HPSCs) remains a gap in our current knowledge regarding stellate cells and PDAC. We hypothesized there would be differences in sEVs secretion and protein expression that might contribute to PDAC biology. To test this hypothesis, we isolated sEVs using ultracentrifugation followed by characterization by electron microscopy and Nanoparticle Tracking Analysis. We report here our initial observations. First, HPSC cells derived from PDAC tumors secrete a higher volume of sEVs when compared to normal pancreatic stellate cells (HPaStec). Although our data revealed that both normal and tumor-derived sEVs demonstrated no significant biological effect on cancer cells, we observed efficient uptake of sEVs by both normal and cancer epithelial cells. Additionally, intact membrane-associated proteins on sEVs were essential for efficient uptake. We then compared sEV proteins isolated from HPSCs and HPaStecs cells using liquid chromatography–tandem mass spectrometry. Most of the 1481 protein groups identified were shared with the exosome database, ExoCarta. Eighty-seven protein groups were differentially expressed (selected by 2-fold difference and adjusted p value ≤0.05) between HPSC and HPaStec sEVs. Of note, HPSC sEVs contained dramatically more CSE1L (chromosome segregation 1–like protein), a described marker of poor prognosis in patients with pancreatic cancer. Based on our results, we have demonstrated unique populations of sEVs originating from stromal cells with PDAC and suggest that these are significant to cancer biology. Further studies should be undertaken to gain a deeper understanding that could drive novel therapy.     

Multi-lineage Lung Regeneration by Stem Cell Transplantation across Major Genetic Barriers

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The effect of L-2-amino-4-methoxy-trans-3-butenoic acid on serine hydroxymethyl transferase

The tumour growth inhibitor L-2-amino-4-methoxy-trans-3-butenoic acid (Ro07-7957) inhibits serine hydroxymethyltransferase in cytosolic extracts of Walker carcinoma non-competitively with respect to L-serine with an apparent inhibition constant similar to the K_m-value for L-serine. The kinetics of inactivation suggest that it reacts as an irreversible substrate analogue. Incubation of Walker cells with Ro07-7957 causes an increase in serine hydroxymethyltransferase activity which is most pronounced at concentration ≤LD₅₀. This increase in enzyme activity does not occur in the presence of cycloheximide. These results suggest that inhibition of serine hydroxymethyltransferase in intact cells is accompanied by an increase in enzyme biosynthesis and that the growth inhibitory property of Ro07-7957 does not involve interference with the conversion of serine to glycine.     

Induction of unscheduled DNA synthesis by the carcinogen 7-bromomethylbenz(a)anthracene and its removal from the DNA of normal and xeroderma pigmentosum lymphocytes

The carcinogen 7-bromomethylbenz(a)anthracene (BBA), which can bind strongly to DNA, induces unscheduled DNA synthesis (DNA repair) in normal lymphocytes but almost none in lymphocytes from patients with Xeroderma pigmentosum (XP), and inherited disease known to be defective in excision repair of ultraviolet-damaged DNA. We studied [³H]BBA's ability to bind to DNA of normal and XP lymphocytes, its influence on unscheduled DNA synthesis, and its removal from the DNA of both cell types. We found that 20–30% of the BBA is bound to macromolecules other than DNA and that its binding to DNA is essentially complete after 30 min. The induction of unscheduled DNA synthesis by the carcinogen in XP lymphocytes was approximately 10% of that induced in normal lymphocytes. While 15–20% of the BBA was removed from the DNA of normal cells 6 h after treatment, only 1–2% was removed from the DNA of XP cells. Thus, XP cells not only are defective in repairing ultraviolet-damaged DNA and excising thymine dimers but also fail to repair DNA damaged by certain carcinogens, and, most importantly, fail to remove the DNA-bound carcinogen, BBA.     

Study of properties of cholinephosphotransferase from fetal guinea pig lung mitochondria and microsomes

Summary We have reported earlier that cholinephosphotransferase (EC 2.7.8.2) is present in both mitochondria and microsomes of fetal guinea pig lung. This study was designed to compare the properties of mitochondrial and microsomal cholinephosphotransferase in fetal guinea pig lung. Various parameters, such as substrate specificity, K_m values, sensitivity to N-ethylmaleimide, dithiothreitol and trypsin were measured. Both showed significant preference for unsaturated diacylglycerols over saturated diacylglycerols. Data on K_m and V_max indicate that the affinity of this enzyme for different diacylglycerols varies between the two forms. The ID₅₀ values for N-ethylmaleimide were 20 mM and 12.5 mM for the mitochondrial and microsomal form of the enzyme, respectively. Dithiothreitol showed an inhibitory effect on both; however, the mitochondrial form was inhibited less than the microsomal form. The effects of N-ethylmaleimide and dithiothreitol on both forms of enzyme indicated that the microsomal cholinephosphotransferase requires a higher concentration of -SH for its activity than the mitochondrial enzyme does. The enzyme was inhibited by trypsin in both mitochondria and microsome under isotonic condition suggesting that this enzyme is on the outside of the membrane in both endoplasmic reticulum and mitochondria.     

High-affinity [3H]inositol uptake by dissociated brain cells and cultured fibroblasts from fetal mice

The accumulation of [³H]inositol by mechanically dissociated brain cells and cultured skin fibroblasts from fetal mice was examined. Uptake by both tissues was strongly dependent on temperature and the presence of sodium ions. Brain and fibroblast uptake also responded similarly to inhibition by inositol isomers and phloridzin. At lower concentrations of inositol, both tissues exhibited high-affinity uptake kinetics with apparent K_m values near 30 M, similar to values observed previously in human fibroblasts and other cultured cells. The activity of brain high-affinity uptake was nearly an order of magnitude lower than that of fibroblasts, however, and was in part confounded by the presence of a low-affinity or simple diffusion system operating at inositol concentrations above 100M. Brain preparation from adult mice also showed evidence of high-affinity, Na⁺ dependent uptake, but its activity was significantly diminished relative to that of fetal brain preparations. Our results demonstrate that a high-affinity inositol transport system closely resembling that found in cultured cells is expressed in the developing mouse brain.     

Mesenchymal control of branching pattern in the fetal mouse lung

The effect of mesenchyme on specialization of respiratory epithelium in the fetal mouse was tested in organ cultures. Heterologous combinations were made between respiratory and non-respiratory lung epithelia and the corresponding mesenchymes. Isolated terminal respiratory buds of fetal mouse lungs were recombined with mesenchyme from chick lung parabronchi, mouse trachea or from the avascular, non-respiratory air sacs of chick lungs. Isolated non-branching chick air sacs were combined with mouse terminal bud mesenchyme or mesenchyme from the respiratory branches of chick lungs. Air sac epithelia branched in a pattern characteristic of the chick lung when combined with chick respiratory mesenchyme and in a pattern characteristic of mouse lung when combined with mouse terminal bud mesenchyme. Mouse terminal bud epithelia did not branch with either mouse tracheal mesenchyme or chick air sac mesenchyme but branched in a chick pattern with chick parabronchial mesenchyme. Electron microscopic examination of the cultures showed that all chick air sac epithelial cultures failed to produce surfactant (lamellar bodies) even when they branched. Control cultures of mouse terminal buds contained large numbers of lamellar bodies; mesenchyme which suppressed branching reduced the number of lamellar bodies to only a few in a small proportion of the cells. Culture medium supplemented with growth factors and hormones increased the number of lamellar bodies in heterologous mouse combinations but did not bring the number to control levels. Supplemented medium had no effect on lamellar body production by chick air sac epithelium. The results indicate that branching pattern is determined by the mesenchyme surrounding the epithelial primordium. However, the capacity to synthesize surfactant is determined by the source of the epithelium; mesenchyme may control the degree of expression but not the absolute presence or absence of the differentiated condition.     

Duration of retained fetal membranes and subsequent fertility in dairy cows

Lactations from 1,111 heifers and 2,493 cows were evaluated for the effects of the duration of retained fetal membranes on subsequent fertility. Cows or heifers with metritis, ovarian cyst, both metritis and cyst, or neither were evaluated in separate strata in order to control for the effects of parity, metritis, and cyst on fertility. Duration of retention had no effect on fertility if an animal had metritis or cyst. There was a suggestion that retained fetal membranes in heifers free of metritis and cyst decreased conception rate at first service. In multiparous cows free of metritis and cyst there was a significant decrease in conception rate at first service when retention exceeded 5 days, and delays of +18 days to 1st service and +57 days to conception when retention exceeded 7 days.     

Molecular cloning and sequence analysis of cDNA for human transferrin

A cDNA clone for human transferrin was identified from a human liver cDNA library by pre-screening with different ss-cDNA probes against length-fractionated liver mRNAs, positive hybridization-selection and nucleotide sequence analysis. The insert was of 1 kb, encoding human transferrin from aminoacid 403 through the COOH terminus, with a 3' non coding region of 166 nucleotides. This insert hybridized with a single major mRNA species of about 2.4 kb and several genomic DNA restriction fragments. Hybridization of the Southern blots with different parts of the transferrin insert and at different stringences suggest that the various bands observed correspond to splice sites inside one gene rather than to hybridization to several related genes. Finally, a single or a low number of transferrin gene copies seem to exist in the human genome.