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.

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.

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.

Insect lipid transfer particle catalyzes diacylglycerol exchange between high-density and very-high-density lipoproteins.

Facilitated diacylglycerol exchange between Manduca sexta [3H]diacylglycerol-labeled high-density lipophorin and Heliothis zea very-high-density chromolipoprotein was studied. M. sexta lipid transfer particle was employed in assays which measured exchange of [3H]diacylglycerol. Following incubations with lipid transfer particle, donor and acceptor lipoproteins were reisolated by density-gradient ultracentrifugation to determine facilitated exchange. The reaction was limited to diacylglycerol exchange, while donor or acceptor particle apoprotein exchange did not occur. Lipid analysis of donor and acceptor lipoproteins after the lipid-exchange reaction revealed that the labeled diacylglycerol remained unchanged. Lipid transfer particle-catalyzed diacylglycerol exchange was linear up to 0.3 micrograms lipid transfer particle protein in a standard assay and exchange occurred at a rate of 2.5 micrograms diacylglycerol min-1.micrograms-1 lipid transfer particle protein. The assay method was used to show that the hemolymph concentration of lipid transfer particle increased during development.

The nucleotide sequence of a microvitellogenin encoding gene from the tobacco hornworm, Manduca sexta.

A microvitellogenin (mVg)-coding gene (mvg) has been isolated from a lambda phage library prepared from the tobacco hornworm, Manduca sexta. One of the lambda clones had a 15-kb insert and contained the entire mvg gene. A DNA fragment (3.0 kb) containing this mvg gene has been sequenced. Southern blot analysis showed that there may be more than one mvg gene in M. sexta. The putative transcriptional start point (tsp) for the cloned mvg was determined by primer extension analysis. This gene contains a single intron in the 5'-noncoding region. The 5'-flanking sequence was compared to the 5'-conserved regions of yolk polypeptide-encoding genes (yp) of Drosophila melanogaster. Two regions were found in the 5'-flanking sequence of the mvg gene that have 66% similarity to the D. melanogaster yp consensus sequence that is believed to be involved in gene expression controlled by ecdysteroids. Furthermore, the sequences flanking these two regions are also similar to the ecdysone-responsive elements found in several genes of D. melanogaster. In fact, preliminary experiments showed that mVg mRNA synthesis is induced by the 20-hydroxyecdysone. Four regions of the mvg gene resemble the upstream conserved regions of the two vitellogenin-encoding genes of the locust, Locusta migratoria. The nucleotide sequence of mvg has 70% similarity to the sequence of one or more of the 30-kDa hemolymph proteins of Bombyx mori. This indicates a very close evolutionary relationship between these proteins.

Sequestration of insecticyanin, a blue hemolymph protein, into the egg of the hawkmoth Manduca sexta. Evidence for receptor-mediated endocytosis.

Sequestration of the blue biliprotein, insecticyanin, into developing oocytes of the hawkmoth, Manduca sexta was investigated. Immunodiffusion assays revealed that insecticyanin concentration in mature eggs (29.6 microM) is slightly higher than that in hemolymph (25.8 microM). The endocytotic uptake of insecticyanin was visualized at the light microscopic level using autoradiography. Uptake of 125I-insecticyanin by isolated oocytes was saturable. Analysis of in vitro uptake data estimated that the value of K(uptake) (insecticyanin concentration at half-maximal uptake rate) is 4.2 microM and that the Vmax (maximum rate of uptake) is 1 pmol follicle-1 h-1. Labeled insecticyanin was shown to bind to sonicated follicle membranes with high specificity and affinity. The KD (equilibrium dissociation constant) and the Bm (total number of binding sites per follicle), were estimated as 4 x 10(-8) M and 8 x 10(7) respectively. Competition studies showed that binding of labeled insecticyanin to oocyte membranes was blocked by excess amounts of unlabeled insecticyanin but not by lipophorin and vitellogenin of M. sexta. Additional membrane binding experiments demonstrated that receptors for insecticyanin are only present in the oocytes membranes, not in fat body or gut tissue.

Rapid and efficient isolation of transferrin and ferritin from Manduca sexta.

We report methods for the rapid purification of two iron-binding proteins from larval hemolymph of Manduca sexta. Ferritin was purified in two steps by density gradient ultracentrifugation. To accomplish this, we utilized the relatively high level of ferritin present in the hemolymph of this animal and augmented the density of the protein in vivo by injection of iron sulfate. Nitrocellulose blots analyzed by laser densitometry showed hemolymph from iron-injected insects contained about 0.4 mg of ferritin per ml (approximately 0.7% of total hemolymph protein); of this, 62% was found as pure ferritin in the pellet formed during ultracentrifugation. Following the density ultracentrifugation, we purified transferrin from the hemolymph subphase by immobilized metal ion affinity chromatography using a new gel, Novarose-SE1000/40 coupled to dipicolylamine (DPA) chelated with nickel. Higher capacity Ni2+DPA-gel permitted good resolution of transferrin in the first chromatography; a lower capacity of the same gel allowed purification of transferrin in a second step. Overall transferrin recovery was 52%. Larval hemolymph contained 0.770 mg transferrin/ml, representing about 1.3% of the total protein.

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.

Increased frequency of obstructive airway abnormalities with long-term tracheostomy.

Eighty-one patients with long-term tracheostomy tubes (mean duration, 4.9 months) were examined via fiberoptic bronchoscopy prior to decannulation. Obstructive airway lesions were observed in 54 patients (67 percent). All tracheal lesions were anatomically located proximal to the stoma. No cuff lesions were observed. The two most commonly observed lesions were tracheal granuloma (60 percent) and tracheomalacia (29 percent). Less frequently observed lesions were tracheostenosis (14 percent) and vocal cord and laryngeal dysfunction (8 percent). As a result of the high frequency of tracheal abnormalities, especially that for tracheal granuloma which has not been previously reported (to our knowledge), we recommend that all decannulation candidates undergo anatomic examination of the airways.

Extraction of serologic and delayed hypersensitivity antigens from spherules of Coccidioides immitis.

We have used an aqueous toluene extraction procedure to obtain antigens from mature spherules of Coccidioides immitis. This extract contained many antigens as determined by immunoblotting and two-dimensional immunoelectrophoretic studies. These included antigens with specificity for tube precipitin-type antibodies having molecular weights greater than or equal to 100 KDa. The extract also displayed lymphocyte-transforming activity when tested on human peripheral blood mononuclear leukocytes from donors who react to coccidioidal skin tests but elicited no such stimulation of cells from persons whose coccidioidal skin tests were nonreactive. At high concentrations of the extract, lymphocyte transformation did not occur, a finding that could not be explained by nonspecific toxicity. When gel filtration was employed to separate antigens by size, tube precipitin-like activity and specific coccidioidal delayed-type hypersensitivity displayed overlap, although only the latter activity was apparent in lower molecular weight pools.

Adipokinetic hormone controls lipid metabolism in adults and carbohydrate metabolism in larvae of Manduca sexta.

The peptide hormone which controls activation of fat body glycogen phosphorylase in starving larvae of Manduca sexta was isolated from larval corpora cardiaca and sequenced by FAB tandem mass spectrometry. It was found to be identical with Manduca AKH. This, together with earlier observations, demonstrates that in M. sexta AKH controls glycogen phosphorylase activation in starving larvae while in adults it controls lipid mobilization during flight. Larval corpora cardiaca contain about 10 times less AKH than the corpora cardiaca of adults. The corpora cardiaca of M. sexta appear to contain only one AKH.

Preliminary biological characterization of a melanization stimulating factor (MSF) from the dorsal skin of the channel catfish, Ictalurus punctatus.

A two step fractionation of conditioned media made from the darkly pigmented dorsal skin of the channel catfish, Ictalurus punctatus, has produced fractions that contain a melanization stimulating factor (MSF). Isolated neural tubes of Xenopus laevis embryos exposed to conditioned media and to specific fractions exhibit greater melanization (increased numbers of melanized cells and elevated percentages of melanized cells), a greater number of dendrites per melanized cell, and a greater number of emigrated neural crest cells than control neural tubes. The presence of MSF activity in the darkly pigmented dorsal integument suggests a role for a molecule or molecules in the development and maintenance of the dorsal/ventral pigment pattern of this piscine species and possibly of other vertebrates.

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.

Ring location in cyclopropane fatty acid esters by a mass spectrometric method.

Ring location in cyclopropane fatty acid esters is accomplished simply and unequivocally with submilligram samples. The technique involves reductive ring opening with platinum catalyst and hydrogen in glacial acetic acid, to give a mixture of branched-chain and straight-chain acid esters. The sample is analyzed with a combination gas chrmatograph-mass spectrometer. Examination of the spectra obtained from the mixture of branched-chain acid esters permits assignment of the position of the methyl groups, and hence of the ring in the parent compound.

Novel fatty acids from the royal jelly of honeybees (Apis mellifera, L.).

Three new compounds isolated from the royal jelly of honeybees (Apis mellifera, L.) have been identified as 8-hydroxyoctanoic acid, 3-hydroxydecanoic acid and a dextrorotatory isomer of 3,10-dihydroxydecanoic acid.

Immunocytochemical localization of a follicle specific protein of the hawkmoth Manduca sexta.

A follicle specific protein (FSP-I) from the hawkmoth Manduca sexta, has been localized in developing follicles by immuno-fluorescence and immuno-gold labeling techniques. At the light microscopical level, the protein was demonstrated to be present in both the basolateral and apical parts of follicular epithelial cells, as well as in clearly defined, spherical compartments in the cortex of the developing oocyte. Immuno-gold labeling at the electron microscopical level revealed the localization of FSP-I in endoplasmic compartments of the follicular epithelial cells, in the extracellular matrix of the follicle and in endocytic compartments of the oocyte. Our results indicate that M. sexta FSP-I is synthetized and secreted by the follicular epithelial cells, after which it is taken up by the developing oocyte through endocytic routes.

Protein and lipoprotein uptake by developing oocytes of the hawkmoth Manduca sexta. An ultrastructural and immunocytochemical study.

The ultrastructure of developing Manduca sexta oocytes is described with respect to the endocytic pathway for protein incorporation. Three major (lipo) protein components of mature M. sexta eggs, lipophorin, vitellogenin and microvitellogenin, were localized along this pathway by immuno-fluorescence and immuno-gold labeling techniques. Labeling of the antigens was observed in the extracellular spaces of the follicle. In those cases where fixation and en bloc staining procedures did not destroy antigenicity, antigens were detected in coated pits and coated vesicles near the plasma membrane of the oocyte. All three antigens were demonstrated to be present in endosomes in the cortex of the oocyte. Both the morphology and the labeling pattern of the endosomes indicate that this organelle is a compartment of uncoupling of receptor and ligand. Tubular elements at the surface of the endosome, interpreted to be involved in the recycling of receptors and membrane to the oocyte surface, were not labeled. Strong labeling of lipophorin, vitellogenin and microvitellogenin was observed in the developing yolk bodies, the main protein storage compartment of the oocyte. The uptake and storage of hemolymph proteins and lipoproteins by M. sexta oocytes is discussed in comparison with other insect and vertebrate endocytic systems.