A phase I study of binimetinib (MEK 162), a MEK inhibitor, plus carboplatin and pemetrexed chemotherapy in non-squamous non-small cell lung cancer.

INTRODUCTION: MEK inhibition is a potential therapeutic strategy in non-small cell lung cancer (NSCLC). This phase I study evaluates the MEK inhibitor binimetinib plus carboplatin and pemetrexed in stage IV non-squamous NSCLC patients (NCT02185690). METHODS: A standard 3 + 3 dose-escalation design was used. Binimetinib 30 mg BID (dose level 1 [DL1]) or 45 mg BID (dose level 2 [DL2]) was given with standard doses of carboplatin and pemetrexed using an intermittent dosing schedule. The primary outcome was determination of the recommended phase II dose (RP2D) and safety of binimetinib. Secondary outcomes included efficacy, pharmacokinetics, and an exploratory analysis of response based on mutation subtype. RESULTS: Thirteen patients (6 DL1, 7 DL2) were enrolled: 7 KRAS, 5 EGFR, and 1 NRAS mutation. The RP2D was binimetinib 30 mg BID. Eight patients (61.5%) had grade 3/4 adverse events, with dose limiting toxicities in 2 patients at DL2. Twelve patients were evaluated for response, with an investigator-assessed objective response rate (ORR) of 50% (95% CI 21.1%-78.9%; ORR 33.3% by independent-review, IR), and disease control rate 83.3% (95% CI 51.6%-97.9%). Median progression free survival (PFS) was 4.5 months (95% CI 2.6 months-NA), with a 6-month and 12-month PFS rate of 38.5% (95% CI 19.3%-76.5%) and 25.6% (95% CI 8.9%-73.6%), respectively. In an exploratory analysis, KRAS/NRAS-mutated patients had an ORR of 62.5% (ORR 37.5% by IR) vs. 25% in KRAS/NRAS wild-type patients. In MAP2K1-mutated patients, the ORR was 42.8%. CONCLUSION: The addition of binimetinib to carboplatin and pemetrexed appears to have manageable toxicity with evidence of activity in advanced non-squamous NSCLC.

Role of Coronary Angiography in Pre-Liver Transplantation Cardiac Evaluation: Experience From an Asian Transplant Institution.

BACKGROUND: Liver transplant (LT) patients with significant coronary artery disease (CAD) have poorer outcomes. Pre-LT coronary angiography (CA) is associated with significant complications in cirrhotic patients. METHODS: This study aimed to identify predictors of abnormal CA in pre-LT cardiac assessment and to develop a predictive model to reduce unnecessary CA. From January 2006 to June 2013, 122 patients underwent CA based on the current institutional protocol. RESULTS: Forty-one (33.6%) patients had abnormal CA. Univariate analysis showed age ≥65 years (P = .001), cryptogenic cirrhosis (P = .046), cardiac comorbidities (P = .027), ischemic heart disease (IHD; P = .002), left ventricular hypertrophy (LVH; P = .004), hypertension (P = .002), diabetes mellitus (P = .017), dyslipidemia (P < .001), metabolic syndrome (P = .003), ≥2 CAD risk factors (P = .001), and high Framingham risk score (hard CAD risk, P = .018; cardiovascular disease: lipids, P = .002; body mass index, P < .001) to be significant predictors of abnormal CA. A predictive model was developed with the use of multivariable logistic regression and included diabetes, dyslipidemia, IHD, age ≥65 years, and LVH, achieving a specificity of 55.1% and sensitivity of 90.0%. This would reduce unnecessary CA by up to one-half in our study population (from 81 to 35) while maintaining a false negative rate of only 8.5%. CONCLUSIONS: Diabetes, dyslipidemia, IHD, age ≥65 years, and LVH appear to be predictors of abnormal CA in pre-LT patients. Our predictive model may help to better select patients for CA, although further validation is required.

Palliative surgical intervention in metastatic colorectal carcinoma: a prospective analysis of quality of life.

AIM: Quality of life (QOL) was assessed after palliative surgery for incurable metastatic colorectal cancer (CRC). METHOD: Newly diagnosed patients with incurable metastatic CRC who were offered elective palliative surgical intervention were included. The European Organization for Research and Treatment of Cancer QLQ-C30 and QLQ-CR29 questionnaire was used for the assessment of QOL at baseline and at 3 and 6 months after surgery. Generalized estimating equations were used to estimate the mean change in the QOL score from baseline. RESULTS: Twenty-four patients formed the study group. Sixteen underwent resection of the primary tumour and eight had a proximal diversion or bypass. The Global Health (GH) score and Social Functioning (SF) score improved at 3 and 6 months after intervention respectively (GH +11, P = 0.021; SF +15, P = 0.005). Mean anxiety scores were markedly improved from the baseline of 51 to 71 (P = 0.004, 3 months) and 76 (P = 0.002, 6 months). Weight concerns also improved significantly when compared with baseline (3 months, +20, P < 0.001; 6 months, +14, P = 0.012). Symptoms of diarrhoea (3 months, --17, P = 0.007; 6 months,--16, P = 0.008) and nausea (--8, P = 0.032) improved. CONCLUSION: In patients with incurable metastatic CRC, surgery improved QOL.

Aedes aegypti transferrin. Gene structure, expression pattern, and regulation.

Mosquitoes and all other insects so far examined have an abundant haemolymph transferrin (Tsf). The exact function of these proteins has not been determined, but they may be involved in iron transport, in oogenesis and in innate immune defence against parasites and pathogens. The Tsf gene of Aedes aegypti has been cloned and sequenced. It contains a single small intron, which contrasts it to vertebrate Tsf genes that contain up to sixteen introns. The promoter region of the gene is rich in putative NF-kappaB binding sites, which is consistent with the postulated role of Tsf in insect innate immunity. Tsf message levels are very low in embryos and early larvae, but high in late larvae, pupae and adults. Western blotting experiments revealed high levels of Tsf protein in pupae and adults. Late larvae and ovaries of blood-fed mosquitoes have little intact protein, but two prominent proteolytic degradation products. These may represent biologically active peptides, as has been shown for other organisms. Tsf message is down-regulated by inorganic iron in the diet or environment, but up-regulated by a blood meal in the adult female. The up-regulation following a blood meal may, in part, be due to the decrease in juvenile hormone (JH) that is known to follow blood feeding. Treatment of blood-fed females with methoprene, an analogue of JH, resulted in decrease of the Tsf message.

Drosophila melanogaster ferritin: cDNA encoding a light chain homologue, temporal and tissue specific expression of both subunit types.

Drosophila melanogaster secreted ferritin like the cytosolic ferritins of other organisms is composed of two subunits, a heavy chain homologue (HCH) and a light chain homologue (LCH). We report the cloning of a cDNA encoding the ferritin LCH of this insect. As predicted from the gene sequence, it contains no iron responsive element (IRE). Northern blot analysis reveals two mRNAs that differ in length due to the choice of polyadenylation signals. Message levels vary through the life cycle of the fly and are markedly increased by high levels of dietary iron. The gut is the main site of increased message synthesis and iron preferentially increases the amount of shorter messages. Western blotting reveals that LCH is the predominant ferritin subunit in all life stages. The amount of LCH protein corresponds well with the message levels in control animals, while in iron-fed animals LCH does not increase proportionally with the message levels. In contrast, the amount of HCH is less than that would be predicted from message levels in control animals, but corresponds well in iron-fed animals. Ferritin is abundant in gut and hemolymph of larvae and adults and in ovaries of adult flies. At pupariation, ferritin becomes more abundant in hemolymph than in other tissues.

On the biosynthesis of Rhodnius prolixus heme-binding protein.

The biosynthesis of Rhodnius prolixus heme-binding protein (RHBP), which is present in the hemolymph and oocytes of Rhodnius prolixus, was investigated. Fat bodies of female insects incubated in vitro with 14C-leucine were able to synthesize and secrete 14C-RHBP to the culture medium. Titrtion of synthesized RHBP with hemin showed that the protein secreted by the fat bodies is bound to heme, despite the presence of apo-RHBP in the hemolymph. The sequence of the RHBP cDNA encodes a pre-protein of 128 amino acids with no significant homology to any known protein. Northern-blot assays revealed that RHBP expression was limited to fat bodies. The levels of both RHBP mRNA and secreted protein increased in response to blood meal. In addition, the time-course of RHBP secretion in vitro paralleled mRNA accumulation observed in vivo. The inhibition of the de novo heme biosynthesis by treatment of fat bodies with succinyl acetone (SA), an irreversible inhibitor of delta-aminolevulinic acid-dehydratase, led to a significant decrease of heme-RHBP secretion. Nevertheless, the levels of RHBP mRNA were not modified by SA treatment, suggesting that the heme availability is involved in a post-transcriptional control of the RHBP synthesis.

Transcriptional control is relevant in the modulation of mosquito ferritin synthesis by iron.

In yellow fever mosquito cells (Aag2 clone), iron treatment induces a threefold increase in ferritin message (fer mRNA) and protein (ferritin) by 16 h. These data contrast with work in mammalian hepatocytes and fibroblasts in which fer mRNA levels do not change with iron stimulation, but ferritin levels increase 50-fold. Pretreatment of the Aag2 cells with actinomycin D blocks induction of fer mRNA and reduces the ferritin subunit synthesis, suggesting that iron induction of ferritin subunit synthesis is subjected to transcriptional control. A putative iron-regulatory protein has also been identified in cytoplasmic extracts from Aag2 cells.

A novel lipoprotein from the hemolymph of the cochineal insect, Dactylopius confusus.

A new type of insect lipoprotein was isolated from the hemolymph of the female cochineal insect Dactylopius confusus. The lipoprotein from the cochineal insect hemolymph was found to have a relative molecular mass of 450 000. It contains 48% lipid, mostly diacylglycerol, phospholipids and hydrocarbons. The protein moiety of the lipoprotein consists of two apoproteins of approximately 25 and 22 kDa, both of which are glycosylated. Both apolipoproteins are also found free in the hemolymph, unassociated with any lipid. Purified cochineal apolipoproteins can combine with Manduca sexta lipophorin, if injected together with adipokinetic hormone into M. sexta. This could indicate that the cochineal lipoprotein can function as a lipid shuttle similar to lipophorins of other insects, and that the cochineal insect apolipoproteins have an overall structure similar to insect apolipophorin-III.

Iron availability dramatically alters the distribution of ferritin subunit messages in Drosophila melanogaster.

Insect ferritins have subunits homologous to the heavy and light chains of vertebrate ferritins. Cloning and sequence of the heavy chain homologue (HCH) of Drosophila melanogaster ferritin subunit have been reported earlier. When Northern blots of D. melanogaster RNA were probed with a cDNA for this HCH, three bands were observed. It was shown that these represented at least four classes of mRNA of various lengths. The polymorphism results from alternative splicing of an intron in the 5' untranslated region (UTR) that contains the iron-responsive element (IRE) and from two alternative polyadenylation sites in the 3' UTR. This has also been reported by others [Lind, M. I., Ekengren, S., Melefors, O. & Söderhäll, K. (1998) FEBS Lett. 436, 476-482]. By hybridizing Northern blots with specific probes, it has been shown that the relative proportions of the messages vary with the life stage and especially with iron supplementation of the diet. Iron significantly increases the amount of ferritin HCH messages and dramatically shifts the balance toward those messages that lack an IRE and/or have a short 3' UTR. In the larvae this change takes place in the gut, but not in the fat body. We speculate that this dramatic shift in message distribution may result from an effect of iron on the rate of transcription or message degradation, or from an effect on the splicing process itself. Synthesis of ferritin HCH subunit mRNAs that lack an IRE may be important under conditions of iron overload.

Drosophila melanogaster transferrin. Cloning, deduced protein sequence, expression during the life cycle, gene localization and up-regulation on bacterial infection.

Drosophila melanogaster transferrin cDNA was cloned from an ovarian cDNA library by using a PCR fragment amplified by two primers designed from other dipteran transferrin sequences. The clone (2035 bp) encodes a protein of 641 amino acids containing a signal peptide of 29 amino acids. Like other insect transferrins, Drosophila transferrin appears to have a functional iron-binding site only in the N-terminal lobe. The C-terminal lobe lacks iron-binding residues found in other transferrins, and has large deletions which make it much smaller than functional C-terminal lobes in other transferrins. In-situ hybridization using a digoxigenin labeled transferrin cDNA probe revealed that the gene is located at position 17B1-2 on the X chromosome. Northern blot analysis showed that transferrin mRNA was present in the larval, pupal and adult stages, but was not detectable in the embryo. Iron supplementation of the diet resulted in lower levels of transferrin mRNA. When adult flies were inoculated with bacteria (Escherichia coli), transferrin mRNA synthesis was markedly increased relative to controls.

Mosquito transferrin, an acute-phase protein that is up-regulated upon infection.

When treated with heat-killed bacterial cells, mosquito cells in culture respond by up-regulating several proteins. Among these is a 66-kDa protein (p66) that is secreted from cells derived from both Aedes aegypti and Aedes albopictus. p66 was degraded by proteolysis and gave a virtually identical pattern of peptide products for each mosquito species. The sequence of one peptide (31 amino acids) was determined and found to have similarity to insect transferrins. By using conserved regions of insect transferrin sequences, degenerate oligonucleotide PCR primers were designed and used to isolate a cDNA clone encoding an A. aegypti transferrin. The encoded protein contained a signal sequence that, when cleaved, would yield a mature protein of 68 kDa. It contained the 31-amino acid peptide, and the 3' end exactly matched a cDNA encoding a polypeptide that is up-regulated when A. aegypti encapsulates filarial worms [Beerntsen, B. T., Severson, D. W. & Christensen, B. M. (1994) Exp. Parasitol. 79, 312-321]. This transferrin, like those of two other insect species, has conserved iron-binding residues in the N-terminal lobe but not in the C-terminal lobe, which also has large deletions in the polypeptide chain, compared with transferrins with functional C-terminal lobes. The hypothesis is developed that this transferrin plays a role similar to vertebrate lactoferrin in sequestering iron from invading organisms and that degradation of the structure of the C-terminal lobe might be a mechanism for evading pathogens that elaborate transferrin receptors to tap sequestered iron.

Isolation and properties of Drosophila melanogaster ferritin--molecular cloning of a cDNA that encodes one subunit, and localization of the gene on the third chromosome.

Ferritin was purified from iron-fed Drosophila melanogaster extracts by centrifugation in a gradient of potassium bromide. On polyacrylamide gel electrophoresis, the product showed two protein bands corresponding to the ferritin monomer and dimer. Electrophoresis following dissociation with SDS and 2-mercaptoethanol revealed three strong bands of approximately 25, 26, and 28 kDa. N-terminal amino acid sequences were identical for the 25-kDa and 26-kDa subunits, but different for the 28-kDa subunit. Conserved ferritin PCR primers were used to amplify a 360-bp cDNA product, which was used to isolate a clone from a D. melanogaster cDNA library that contained the complete coding sequence for a ferritin subunit. Additional 5' sequence obtained by the RACE method revealed the presence of a putative iron regulatory element. The PCR product was also used to locate the position of the ferritin subunit gene at region 99F on the right arm of the third chromosome. The deduced amino acid sequence of the D. melanogaster ferritin subunit contained a signal sequence and resembled most closely ferritin of the mosquito Aedes aegypti. The evolution of ferritin sequences is discussed.

Characterization of the solubilized oocyte membrane receptor for insecticyanin, a biliprotein of the hawkmoth, Manduca sexta.

We report the solubilization and characterization of the oocyte membrane receptor for insecticyanin, a blue biliprotein of the hawkmoth Manduca sexta. The insecticyanin receptor was solubilized using 40 mM CHAPS. Strong binding affinity of [125I]insecticyanin to its solubilized receptor was demonstrated to be heat-labile, pH-dependent, Ca2+-dependent, and saturable. The binding was inhibited by excess unlabeled insecticyanin, but not by two other major hemolymph and oocyte proteins, vitellogenin and lipophorin. The receptor for insecticyanin showed tissue specificity: it was present only in oocyte membranes, not in membranes of fat body, midgut or ovariole sheath. The equilibrium data for the solubilized receptor, K(d) and B(max), were estimated to be 17 nM and 11.4 pmol/mg solubilized proteins, respectively. The results from co-immunoprecipitation showed that the apparent molecular mass for the insecticyanin receptor is approximately 185 kDa while chemical crosslinking of the insecticyanin-receptor complex revealed a product with a molecular mass near 10(3) kDa. This suggests that the insecticyanin receptor has a multimeric structure, or that four receptor molecules can bind to one insecticyanin tetramer.

Comparative nutrition of iron and copper.

The suggestion from nutritional studies with mammals of a link between iron and copper metabolism has been reinforced by recent investigations with yeast cells. Iron must be in the reduced ferrous (FeII) state for uptake by yeast cells, and reoxidation to ferric (FeIII) by a copper oxidase is part of the transport process. Thus, yeast cells deficient in copper are unable to absorb iron. In an analogous way, animals deficient in copper appear to be unable to move FeII out of cells, probably because it cannot be oxidized to FeIII. Invertebrate animals use copper and iron in ways very similar to vertebrates, with some notable exceptions. In the cases where vertebrates and invertebrates are similar, the latter may be useful models for vertebrate metabolism. In cases where they differ (e.g. predominance of serum ferritin in insects, oxygen transport by a copper protein in many arthropods, central importance of phenoloxidase, a copper enzyme in arthropods), the differences may represent processes that are exaggerated in invertebrates and thus more amenable to study in these organisms. On the other hand, they may represent processes unique to invertebrates, thus providing novel information on species diversity.

Cockroach transferrin closely resembles vertebrate transferrins in its metal ion-binding properties: a spectroscopic study.

The optical and electron paramagnetic resonance (EPR) spectroscopic properties of a transferrin from the cockroach Blaberus discoidalis have been investigated to determine the relation of this protein to vertebrate transferrins. Difference spectrophotometry substantiates the involvement of tyrosyl residues in iron binding, and confirms the specific binding of two equivalents of iron per molecule. The far-UV CD spectrum also indicates a secondary structure with marked similarity to those of vertebrate transferrins. EPR studies show a dependence of iron binding on (bi)carbonate, consistent with the absolute requirement of transferrins for a synergistic anion in binding iron. Continuous wave (CW) and pulsed EPR studies of the cupric complex of the protein implicate a histidyl nitrogen ligand in metal coordination, as in human transferrin. Additional studies establish that the pH-dependent release of iron is similar to that of human serum transferrin. The present data confirm cockroach transferrin as an authentic member of the transferrin superfamily, thereby suggesting an ancestral relationship of insect to vertebrate transferrins.

Purification of an expressed insect transferrin from cell culture media using high-capacity Ni(2+)-dipicolylamine gel.

Vertebrate transferrin is a well characterized iron transport protein. In contrast, little is known concerning the role of transferrin in insects. Yet, study of iron metabolism in insects could give insights into strategies for insect control, particularly for insects that transmit disease.

Purification of recombinant insect transferrin from large volumes of cell culture medium using high capacity Ni(2+)-dipicolylamine gel.

We report the purification of secreted recombinant Manduca sexta transferrin from Spodoptera frugiperda (Sf9) cell culture medium in a single step using high capacity Ni(2+)-dipicolylamine (DPA)-Novarose gel. Although the original sample was highly diluted (approximately 10 micrograms transferrin/ml medium) and the cell culture medium contained 10% surfactant (Pluronic F68) and a lipid emulsion, we were able to recover the recombinant transferrin (1 mg protein/100 ml) under gentle elution conditions with 70% yield at > 90% homogeneity. This work demonstrates the versatility of immobilized metal ion affinity chromatography using a high metal ion capacity gel to purify a recombinant protein and illustrates the potential of this affinity technique for protein separations from large volumes of cell culture media that contain surfactants.

Purification and cDNA cloning of ferritin from the hepatopancreas of the freshwater crayfish Pacifastacus leniusculus.

Ferritin was purified from the hepatopancreas of the freshwater crayfish Pacifastacus leniusculus after injection of iron. It has the same size as horse spleen ferritin (440 kDa) and migrates as two bands, 19 kDa and 20 kDa, respectively, in SDS/PAGE under reducing conditions. Crayfish ferritin (20 kDa) was cloned from a hepatopancreas cDNA library. The deduced amino acid sequence of the crayfish ferritin shows a closer relationship to vertebrate ferritin heavy chains than to insect ferritin and contains the conserved H-specific residues for the ferroxidase centre found in vertebrate ferritin heavy chain. An IRE(iron-responsive element)-like sequence with a predicted stem-loop structure was present in the 5' untranslated region of the crayfish ferritin mRNA. Crayfish ferritin does not share the atypical properties of insect ferritins, such as high molecular mass of intact protein, abundance in hemolymph, and export into vacuoles. We suggest that there are two different types of ferritins distributed in different species: insect-type or secretory ferritins which are predominant in the snail oocyte and insects, and vertebrate (crustacean)-type or cytosolic ferritins which are predominant in vertebrates and crustacea.

Manduca sexta lipid transfer particle: synthesis by fat body and occurrence in hemolymph.

Lipid transfer particle (LTP) is present in hemolymph of the tobacco hornworm Manduca sexta. Biosynthesis of LTP, occurrence in hemolymph, and the role of LTP-apoproteins in the lipid transfer reaction were investigated using antibodies specific for LTP or for each of the apoproteins. In vitro protein synthesis followed by immunoprecipitation demonstrated that LTP is synthesized by the fat body and secreted into the medium. In contrast to apolipophorin III, an exchangeable apoprotein of lipophorin (the major lipid transport protein in hemolymph), apoLTP-III could not be detected free in hemolymph. LTP concentrations in the hemolymph were measured by a sandwich ELISA using a mouse monoclonal antibody against apoLTP-III as capturing antibody and rabbit polyclonal antibody against apoLTP-I as detecting antibody. LTP concentration increased during the late fifth instar larval stage, followed by a decrease in the wandering stage. Subsequently, LTP concentrations were strongly increased in hemolymph of adult moths. The role of the three apoproteins of LTP in the lipid transfer reaction was analyzed using apoprotein-specific antibodies. All three, apoLTP-I, -II, and -III, appeared to be important for lipid transfer activity, as shown by inhibition of lipid transfer by antibodies specific for each of the three apoproteins.

Cloning and sequence analysis of the cDNA for a protein from Coccidioides immitis with immunogenic potential.

We have cloned and sequenced the cDNA encoding an immunoreactive protein from the pathogenic fungus Coccidioides immitis which stimulates human T cells and has been associated with protective vaccines in mice. The transcript contained an open reading frame encoding 194 amino acids with a calculated molecular weight of 19.5 kDa, a 151 base 5' untranslated region (UTR), and a 468 base 3'UTR. A four member repeat motif, usually thr-ala-glu-pro, exists for amino acids 98 through 141. Deduced amino acid sequence derived from the cDNA was identical with previously determined internal amino acid sequence from the native protein, and goat antiserum raised against the purified fungal protein reacted with an inducible fusion protein translated from this cDNA. Using this cDNA to produce recombinant protein will allow direct testing of its role in human immunity to coccidioidomycosis and may lead to new diagnostic tests.