Isolation of a cDNA probe for a human jejunal brush-border hydrolase,sucrase-isomaltase,and assignment of the gene locus to chromosome 3

We report the nucleotide sequence and derived amino acid sequence of a cDNA clone encoding most of the N-terminal, isomaltase region of human sucrase-isomaltase (SI). A plasmid containing this cDNA, pS 12, identifies a 6-kb mRNA found in human jejunum and the human colon carcinoma cell Une Caco-2. This human SI cDNA shows extensive overall homology with recently published rabbit SI cDNA. Using pSI2 to probe DNA from a panel of somatic cell hybrids, we have assigned the gene encoding human SI to chromosome 3.     

Chitin Accelerates Activation of a Novel Haloarchaeal Serine Protease That Deproteinizes Chitin-Containing Biomass

The haloarchaeon Natrinema sp. strain J7-2 has the ability to degrade chitin, and its genome harbors a chitin metabolism-related gene cluster that contains a halolysin gene, sptC. The sptC gene encodes a precursor composed of a signal peptide, an N-terminal propeptide consisting of a core domain (N*) and a linker peptide, a subtilisin-like catalytic domain, a polycystic kidney disease domain (PkdD), and a chitin-binding domain (ChBD). Here we report that the autocatalytic maturation of SptC is initiated by cis-processing of N* to yield an autoprocessed complex (N*-I^WT), followed by trans-processing/degradation of the linker peptide, the ChBD, and N*. The resulting mature form (M^WT) containing the catalytic domain and the PkdD showed optimum azocaseinolytic activity at 3 to 3.5 M NaCl, demonstrating salt-dependent stability. Deletion analysis revealed that the PkdD did not confer extra stability on the enzyme but did contribute to enzymatic activity. The ChBD exhibited salt-dependent chitin-binding capacity and mediated the binding of N*-I^WT to chitin. ChBD-mediated chitin binding enhances SptC maturation by promoting activation of the autoprocessed complex. Our results also demonstrate that SptC is capable of removing proteins from shrimp shell powder (SSP) at high salt concentrations. Interestingly, N*-I^WT released soluble peptides from SSP faster than did M^WT. Most likely, ChBD-mediated binding of the autoprocessed complex to chitin in SSP not only accelerates enzyme activation but also facilitates the deproteinization process by increasing the local protease concentration around the substrate. By virtue of these properties, SptC is highly attractive for use in preparation of chitin from chitin-containing biomass.     

C-2 (E)-4-(Styryl)aniline substituted diphenylpyrimidine derivatives (Sty-DPPYs) as specific kinase inhibitors targeting clinical resistance related EGFRT790M mutant

With the aim to overcome the drug resistance induced by the EGFR T790M mutation (EGFR^T790M), herein, a family of diphenylpyrimidine derivatives (Sty-DPPYs) bearing a C-2 (E)-4-(styryl)aniline functionality were designed and synthesized as potential EGFR^T790M inhibitors. Among them, the compound 10e displayed strong potency against the EGFR^T790M enzyme, with the IC₅₀ of 11.0 nM. Compound 10e also showed a higher SI value (SI = 49.0) than rociletinib (SI = 21.4), indicating its less side effect. In addition, compound 10e could effectively inhibit the proliferation of H1975 cells harboring the EGFR^T790M mutation, within the concentration of 2.91 μM. Significantly, compound 10e has low toxicity against the normal HBE cell (IC₅₀ = 22.48 μM). This work provided new insights into the discovery of potent and selective inhibitor against EGFR^T790M over wild-type (EGFR^WT).     

Combination of Pre-Treatment DWI-Signal Intensity and S-1 Treatment: A Predictor of Survival in Patients with Locally Advanced Pancreatic Cancer Receiving Stereotactic Body Radiation Therapy and Sequential S-1

OBJECTIVE: To identify whether the combination of pre-treatment radiological and clinical factors can predict the overall survival (OS) in patients with locally advanced pancreatic cancer (LAPC) treated with stereotactic body radiation and sequential S-1 (a prodrug of 5-FU combined with two modulators) therapy with improved accuracy compared with that of established clinical and radiologic risk models. METHODS: Patients admitted with LAPC underwent diffusion weighted imaging (DWI) scan at 3.0-T (b = 600 s/mm²). The mean signal intensity (SI_b = 600) of region-of-interest (ROI) was measured. The Log-rank test was done for tumor location, biliary stent, S-1, and other treatments and the Cox regression analysis was done to identify independent prognostic factors for OS. Prediction error curves (PEC) were used to assess potential errors in prediction of survival. The accuracy of prediction was evaluated by Integrated Brier Score (IBS) and C index. RESULTS: 41 patients were included in this study. The median OS was 11.7 months (2.8-23.23 months). The 1-year OS was 46%. Multivariate analysis showed that pre-treatment SI_b = 600 value and administration of S-1 were independent predictors for OS. The performance of pre-treatment SI_b = 600 and S-1 treatment in combination was better than that of SI_b = 600 or S-1 treatment alone. CONCLUSION: The combination of pre-treatment SI_b = 600 and S-1 treatment could predict the OS in patients with LAPC undergoing SBRT and sequential S-1 therapy with improved accuracy compared with that of established clinical and radiologic risk models.     

In vitro phenotypic screening of 7-chloro-4-amino(oxy)quinoline derivatives as putative anti-Trypanosoma cruzi agents

In this study, a series of 22 pre-synthesized 7-chloro-4-amino(oxy)quinoline derivatives was assayed in vitro as potential antichagasic agents. A primary screening against Trypanosoma cruzi epimastigotes and a non-specific cytotoxicity assay on murine fibroblasts were simultaneously performed, resulting quinolines 3, 7 and 12 with great selectivity (SI) on the extracellular parasite (SI₇, SI₃, SI₁₂ and SI_BZ >9.44). Therefore, the activity of these derivatives was evaluated on intracellular amastigotes, achieving derivative 7 the best SI (SI = 12.73). These results, supported by the in silico prediction of a good oral bioavailability and a suitable risk profile, propose the 4-amino-7-chloroquinoline scaffold as a potential template for designing trypanocidal prototypes.     

A major mutation of KIF21A associated with congenital fibrosis of the extraocular muscles type 1 (CFEOM1) enhances translocation of Kank1 to the membrane

Congenital fibrosis of the extraocular muscles type 1 (CFEOM1) is associated with heterozygous mutations in the KIF21A gene, including a major (R954W) and a minor (M947T) mutation. Kank1, which regulates actin polymerization, cell migration and neurite outgrowth, interacted with the third and fourth coiled-coil domains of KIF21A protein at its ankyrin-repeat domain. While both KIF21A^R954W and KIF21A^M947T enhanced the formation of a heterodimer with the wild type, KIF21A^WT, these mutants also enhanced the interaction with Kank1. Knockdown of KIF21A resulted in Kank1 predominantly occurring in the cytosolic fraction, while KIF21A^WT slightly enhanced the translocation of Kank1 to the membrane fraction. Moreover, KIF21A^R954W significantly enhanced the translocation of Kank1 to the membrane fraction. These results suggest that KIF21A regulates the distribution of Kank1 and that KIF21A mutations associated with CFEOM1 enhanced the accumulation of Kank1 in the membrane fraction. This might cause an abrogation of neuronal development in cases of CFEOM1 through over-regulation of actin polymerization by Kank1.     

ArPIKfyve Regulates Sac3 Protein Abundance and Turnover: DISRUPTION OF THE MECHANISM BY Sac3I41T MUTATION CAUSING CHARCOT-MARIE-TOOTH 4J DISORDER*

The mammalian phosphatidylinositol (3,5)-bisphosphate (PtdIns(3,5)P₂) phosphatase Sac3 and ArPIKfyve, the associated regulator of the PtdIns3P-5 kinase PIKfyve, form a stable binary complex that associates with PIKfyve in a ternary complex to increase PtdIns(3,5)P₂ production. Whether the ArPIKfyve-Sac3 subcomplex functions outside the PIKfyve context is unknown. Here we show that stable or transient expression of ArPIKfyve^WT in mammalian cells elevates steady-state protein levels and the PtdIns(3,5)P₂-hydrolyzing activity of Sac3, whereas knockdown of ArPIKfyve has the opposite effect. These manipulations do not alter the Sac3 mRNA levels, suggesting that ArPIKfyve might control Sac3 protein degradation. Inhibition of protein synthesis in COS cells by cycloheximide reveals remarkably rapid turnover of expressed Sac3^WT (t_½ = 18.8 min), resulting from a proteasome-dependent clearance as evidenced by the extended Sac3^WT half-life upon inhibiting proteasome activity. Coexpression of ArPIKfyve^WT, but not the N- or C-terminal halves, prolongs the Sac3^WT half-life consistent with enhanced Sac3 protein stability through association with full-length ArPIKfyve. We further demonstrate that mutant Sac3, harboring the pathogenic Ile-to-Thr substitution at position 41 found in patients with CMT4J disorder, is similar to Sac3^WT with regard to PtdIns(3,5)P₂-hydrolyzing activity, association with ArPIKfyve, or rapid proteasome-dependent clearance. Remarkably, however, neither is the steady-state Sac3^I41T elevated nor is the Sac3^I41T half-life extended by coexpressed ArPIKfyve^WT, indicating that unlike with Sac3^WT, ArPIKfyve fails to prevent Sac3^I41T rapid loss. Together, our data indentify a novel regulatory mechanism whereby ArPIKfyve enhances Sac3 abundance by attenuating Sac3 proteasome-dependent degradation and suggest that a failure of this mechanism could be the primary molecular defect in the pathogenesis of CMT4J.     

Altered folding, turnover, and polarized sorting act in concert to define a novel pathomechanism of congenital sucrase-isomaltase deficiency

Naturally occurring mutants of membrane and secretory proteins are often associated with the pathogenesis of human diseases. Here, we describe the molecular basis of a novel phenotype of congenital sucrase-isomaltase deficiency (CSID), a disaccharide malabsorption disorder of the human intestine in which several structural features and functional capacities of the brush-border enzyme complex sucrase-isomaltase (SI) are affected. The cDNA encoding SI from a patient with CSID reveals a mutation in the isomaltase subunit of SI that results in the substitution of a cysteine by an arginine at amino acid residue 635 (C635R). When this mutation is introduced into the wild type cDNA of SI a mutant enzyme, SI(C635R), is generated that shows a predominant localization in the endoplasmic reticulum. Nevertheless, a definite localization of SI(C635R) in the Golgi apparatus and at the cell surface could be also observed. Epitope mapping with conformation-specific mAbs protease sensitivity assays, and enzymatic activity measurements demonstrate an altered folding pattern of SI(C635R) that is responsible for a substantially increased turnover rate and an aberrant sorting profile. Thus, SI(C635R) becomes distributed also at the basolateral membrane in contrast to wild type SI. Concomitant with the altered sorting pattern, the partial detergent extractability of wild type SI shifts to a complete detergent solubility with Triton X-100. The mutation has therefore affected an epitope responsible for the apical targeting fidelity of SI. Altogether, the combined effects of the C635R mutation on the turnover rate, function, polarized sorting, and detergent solubility of SI constitute a unique and novel pathomechanism of CSID.     

Selective activity of 2,4-diaryl-1,2,3,4-tetrahydroquinolines on Trypanosoma cruzi epimastigotes and amastigotes expressing β-galactosidase

The growth inhibitory effect on Trypanosoma cruzi epimastigotes and the unspecific cytotoxicity over NCTC-929 fibroblasts of two series of previously synthesized 2,4-diaryl-1,2,3,4-tetrahydroquinolines (THQ), have been studied in vitro and compared with those of benznidazole (BZ). Derivatives AR39, AR40, AR41, AR91 and DM15 achieved outstanding selectivity indexes (SI) on the extracellular form (SI_THQ > SI_BZ > 9.44) and thus, were tested in a more specific in vitro assay against amastigotes, showing less effectiveness than the reference drug (SI_BZ > 320) but also accomplishing great selectivity on the intracellular stage (SI_THQ > 25). These promising results, supported by the in silico prediction of high bioavailability and less potential risk than benznidazole, reveal several tetrahydroquinolines as prototypes of potential antichagasic drugs.     

Cross-species Binding Analyses of Mouse and Human Neonatal Fc Receptor Show Dramatic Differences in Immunoglobulin G and Albumin Binding

The neonatal Fc receptor (FcRn) regulates the serum half-life of both IgG and albumin through a pH-dependent mechanism that involves salvage from intracellular degradation. Therapeutics and diagnostics built on IgG, Fc, and albumin fusions are frequently evaluated in rodents regarding biodistribution and pharmacokinetics. Thus, it is important to address cross-species ligand reactivity with FcRn, because in vivo testing of such molecules is done in the presence of competing murine ligands, both in wild type (WT) and human FcRn (hFcRn) transgenic mice. Here, binding studies were performed in vitro using enzyme-linked immunosorbent assay and surface plasmon resonance with recombinant soluble forms of human (shFcRn^WT) and mouse (smFcRn^WT) receptors. No binding of albumin from either species was observed at physiological pH to either receptor. At acidic pH, a 100-fold difference in binding affinity was observed. Specifically, smFcRn^WT bound human serum albumin with a K_D of ∼90 μm, whereas shFcRn^WT bound mouse serum albumin with a K_D of 0.8 μm. shFcRn^WT ignored mouse IgG1, and smFcRn^WT bound strongly to human IgG1. The latter pair also interacted at physiological pH with calculated affinity in the micromolar range. In all cases, binding of albumin and IgG from either species to both receptors were additive. Cross-species albumin binding differences could partly be explained by non-conserved amino acids found within the α2-domain of the receptor. Such distinct cross-species FcRn binding differences must be taken into consideration when IgG- and albumin-based therapeutics and diagnostics are evaluated in rodents for their pharmacokinetics.     

Suppression of the Double-Strand-Break-Repair Defect of the Saccharomyces cerevisiae rad57 Mutant

The Rad51 paralogs Rad55 and Rad57 form a heterodimer required to mediate the formation and/or stabilization of the Rad51 filament. To further characterize the function of Rad55-Rad57, we used a combination of rad57 partial suppressors to determine whether the DNA repair and recombination defects of the rad57 mutant could be completely suppressed. The combination of all suppressors, elevated temperature, srs2, rad51-I345T, and mating-type (MAT) heterozygosity resulted in almost complete suppression of the rad57 mutant defect in the recruitment of Rad51 to DNA-damaged sites, as well as survival in response to ionizing radiation and camptothecin. In a physical assay to monitor the kinetics of double-strand-break (DSB)-induced gene conversion, the rad57 mutant defect was effectively suppressed by srs2 and MAT heterozygosity, but these same suppressors failed to suppress the spontaneous recombination defect. Thus the Rad55-Rad57 heterodimer appears to have a unique function in spontaneous recombination that is not essential for DSB repair. Furthermore, we investigated the currently unknown mechanism of rad57 suppression by MAT heterozygosity and found that it is independent of DNL4.     

Betulin and ursolic acid synthetic derivatives as inhibitors of Papilloma virus

The synthesis of new betulin and ursolic acid derivatives and evaluation of their antiviral activity in vitro is reported. Betulin was modified at positions C-3, C-20 and C-28 to afford the derivatives with nicotinoyl-, methoxycynnamoyl-, alkyne and aminopropoxy-2-cyanoethyl-moieties. The two stage conversion of betulin to the new ursane-type triterpenoid by treatment of allobetulin with Ac₂O–HClO₄ is suggested. Cyanoethylation of ursonic acid oxime led to cyanoethyloximinoderivative. According to the results of antiviral screening against human papillomavirus type 11 the selectivity index for tested triterpenoids has a range from 10 to 35 with no cellular cytotoxicite, the most remarkable activity was found for 3β,28-di-O-nicotinoylbetulin. 3β,28-Dihydroxy-29-norlup-20(30)-yne was also active against HCV replicon (EC₅₀ 1.32; EC₉₀ 16.82; IC₅₀ 12.41; IC₉₀ >20; SI₅₀ 9.4; SI₉₀ >1.19). 28-O-Methoxycynnamoylbetulin was active against influenza type A virus (H1N1) (ЕС₅₀ 2; IC₅₀ >200; SI >100).     

Generation of a Functionally Distinct Rhizopus oryzae Lipase through Protein Folding Memory

Rhizopus oryzae lipase (ROL) has a propeptide at its N-terminus that functions as an intramolecular chaperone and facilitates the folding of mature ROL (mROL). In this study, we successfully generated a functionally distinct imprinted mROL (mROL^imp) through protein folding memory using a mutated propeptide. The mutated propeptide left its structural memory on mROL and produced mROL^imp that exhibited different substrate specificities compared with mROL^WT (prepared from the wild type propeptide), although the amino acid sequences of both mROLs were the same. mROL^imp showed a preference for substrates with medium chain-length acyl groups and, noticeably, recognized a peptidase-specific substrate. In addition, ROL^imp was more stable than mROL^WT. These results strongly suggest that proteins with identical amino acid sequences can fold into different conformations and that mutations in intramolecular chaperones can dynamically induce changes in enzymatic activity.     

NK cells and monocytes modulate primary HTLV-1 infection

We investigated the impact of monocytes, NK cells, and CD8⁺ T-cells in primary HTLV-1 infection by depleting cell subsets and exposing macaques to either HTLV-1 wild type (HTLV-1_WT) or to the HTLV-1_p12KO mutant unable to infect replete animals due to a single point mutation in orf-I that inhibits its expression. The orf-I encoded p8/p12 proteins counteract cytotoxic NK and CD8⁺ T-cells and favor viral DNA persistence in monocytes. Double NK and CD8⁺ T-cells or CD8 depletion alone accelerated seroconversion in all animals exposed to HTLV-1_WT. In contrast, HTLV-1_p12KO infectivity was fully restored only when NK cells were also depleted, demonstrating a critical role of NK cells in primary infection. Monocyte/macrophage depletion resulted in accelerated seroconversion in all animals exposed to HTLV-1_WT, but antibody titers to the virus were low and not sustained. Seroconversion did not occur in most animals exposed to HTLV-1_p12KO. In vitro experiments in human primary monocytes or THP-1 cells comparing HTLV-1_WT and HTLV-1_p12KO demonstrated that orf-I expression is associated with inhibition of inflammasome activation in primary cells, with increased CD47 “don’t-eat-me” signal surface expression in virus infected cells and decreased monocyte engulfment of infected cells. Collectively, our data demonstrate a critical role for innate NK cells in primary infection and suggest a dual role of monocytes in primary infection. On one hand, orf-I expression increases the chances of viral transmission by sparing infected cells from efferocytosis, and on the other may protect the engulfed infected cells by modulating inflammasome activation. These data also suggest that, once infection is established, the stoichiometry of orf-I expression may contribute to the chronic inflammation observed in HTLV-1 infection by modulating monocyte efferocytosis.     

Gestational hypertension and the developmental origins of cardiac hypertrophy and diastolic dysfunction

The developmental origins of health and disease refer to the theory that adverse maternal environments influence fetal development and the risk of cardiovascular disease in adulthood. We used the chronically hypertensive atrial natriuretic peptide knockout (ANP?/?) mouse as a model of gestational hypertension, and attempted to determine the effect of gestational hypertension on left ventricular (LV) structure and function in adult offspring. We crossed normotensive ANP+/+ females with ANP?/? males (yielding ANP+/?^WT offspring) and hypertensive ANP?/? females with ANP+/+ males (yielding ANP+/?^KO offspring). Cardiac gene expression was measured using real-time quantitative PCR. Cardiac function was assessed using echocardiography. Daily injections of isoproterenol (ISO) were used to induce cardiac stress. Collagen deposition was assessed using picrosirius red staining. All mice were 10 weeks of age. Gestational hypertension resulted in significant LV hypertrophy in offspring, with no change in LV function. Treatment with ISO resulted in significant LV diastolic dysfunction with a restrictive filling pattern (increased E/A ratio and E/e′) and interstitial myocardial fibrosis only in ANP+/?^KO and not ANP+/?^WT offspring. Gestational hypertension programs adverse LV structural and functional remodeling in offspring. These data suggest that adverse maternal environments may increase the risk of heart failure in offspring later in life.     

Design,synthesis and anticancer evaluation of 1H-pyrazolo[3,4-d]pyrimidine derivatives as potent EGFRWT and EGFRT790M inhibitors and apoptosis inducers

In our attempt to develop effective EGFR-TKIs, two series of 1H-pyrazolo[3,4-d]pyrimidine derivatives were designed and synthesized. All the newly synthesized compounds were evaluated in vitro for their inhibitory activities against EGFR^WT. Compounds 15_b, 15_j, and 18_d potently inhibited EGFR^WT at sub-micro molar IC₅₀ values comparable to that of erlotinib. Moreover, thirteen compounds that showed promising IC₅₀ values against EGFR^WT were tested in vitro for their inhibitory activities against mutant EGFR^T790M. Compounds 17_d and 17_f exhibited potent inhibitory activities towards EGFR^T790M comparable to osimertinib. Compounds that showed promising IC₅₀ values against EGFR^WT were further tested for their anti-proliferative activities against three cancer cell lines bearing EGFR^WT (MCF-7, HepG2, A549), and two cancer cell lines bearing EGFR^T790M (H1975 and HCC827). Compounds 15_g, 15_j, 15_n, 18_d and 18_e were the most potent anticancer agents against the EGFR^WT containing cells, while compounds 15_e, 17_d and 17_f showed promising anti-proliferative activities against EGFR^T790M containing cells. Furthermore, the most active compound 18_d was selected for further studies regarding to its effects on cell cycle progression and induction of apoptosis in the HepG2 cell line. The results indicated that this compound is good apoptotic agent and arrests G₀/G₁and G₂/M phases of cell cycle. Finally, molecular docking studies were performed to investigate binding pattern of the synthesized compounds with the prospective targets, EGFR^WT (PDB: 4HJO) and EGFR^T790M (PDB: 3W2O).