RalA GTPase Tethers Insulin Granules to L‐ and R‐Type Calcium Channels Through Binding α2δ‐1 Subunit

RalA GTPase has been implicated in the regulated delivery of exocytotic vesicles to the plasma membrane (PM) in mammalian cells. We had reported that RalA regulates biphasic insulin secretion, which we have now determined to be contributed by RalA direct interaction with voltage‐gated calcium (Ca_v) channels. RalA knockdown (KD) in INS‐1 cells and primary rat β‐cells resulted in a reduction in Ca²⁺ currents arising specifically from L‐(Ca_v1.2 and Ca_v1.3) and R‐type (Ca_v2.3) Ca²⁺ channels. Restoration of RalA expression in RalA KD cells rescued these defects in Ca²⁺ currents. RalA co‐immunoprecipitated with the Ca_vα₂δ‐1 auxiliary subunit known to bind the three Ca_vs. Moreover, the functional molecular interactions between Ca_vα₂δ‐1 and RalA on the PM shown by total internal reflection fluorescent microscopy/FRET analysis could be induced by glucose stimulation. KD of RalA inhibited trafficking of α₂δ‐1 to insulin granules without affecting the localization of the other Ca_v subunits. Furthermore, we confirmed that RalA and α₂δ‐1 functionally interact since RalA KD‐induced inhibition of Ca_v currents could not be recovered by RalA when α₂δ‐1 was simultaneously knocked down. These data provide a mechanism for RalA function in insulin secretion, whereby RalA binds α₂δ‐1 on insulin granules to tether these granules to PM Ca²⁺ channels. This acts as a chaperoning step prior to and in preparation for sequential assembly of exocyst and excitosome complexes that mediate biphasic insulin secretion.     

Dodecyl α‐L‐Rhamnopyranosyl‐(1→2)‐β‐D‐fucopyranoside Derivatives from the Glandular Trichome Exudate of Erodium pelargoniflorum

Chemical investigation of the glandular trichome exudate of Erodium pelargoniflorum (Geraniaceae) led to the isolation of two dodecyl disaccharide derivatives, named pelargoside A1 and pelargoside B1 ( 1 and 2 , resp.). The structures of 1 and 2 were determined as dodecyl 4‐O‐acetyl‐α‐L ‐rhamnopyranosyl‐(1→2)‐4‐O‐acetyl‐β‐D ‐fucopyranoside and dodecyl 3,4‐di‐O‐acetyl‐α‐L ‐rhamnopyranosyl‐(1→2)‐4‐O‐acetyl‐β‐D ‐fucopyranoside, respectively, by spectroscopic studies, including 2D‐NMR, and chemical transformations. In addition, undecyl, tridecyl, and tetradecyl homologs of 1 and 2 , named pelargosides A2–A4 and pelargosides B2–B4, were also characterized as minor constituents of the exudate.     

5α‐Androst‐16‐en‐3α‐ol β‐D‐Glucuronide,Precursor of 5α‐Androst‐16‐en‐3α‐ol in Human Sweat

5α‐Androst‐16‐en‐3α‐ol (α‐androstenol) is an important contributor to human axilla sweat odor. It is assumed that α‐andostenol is excreted from the apocrine glands via a H₂O‐soluble conjugate, and this precursor was formally characterized in this study for the first time in human sweat. The possible H₂O‐soluble precursors, sulfate and glucuronide derivatives, were synthesized as analytical standards, i.e., α‐androstenol, β‐androstenol sulfates, 5α‐androsta‐5,16‐dien‐3β‐ol (β‐androstadienol) sulfate, α‐androstenol β‐glucuronide, α‐androstenol α‐glucuronide, β‐androstadienol β‐glucuronide, and α‐androstenol β‐glucuronide furanose. The occurrence of α‐androstenol β‐glucuronide was established by ultra performance liquid chromatography (UPLC)/MS (heated electrospray ionization (HESI)) in negative‐ion mode in pooled human sweat, containing eccrine and apocrine secretions and collected from 25 female and 24 male underarms. Its concentration was of 79 ng/ml in female secretions and 241 ng/ml in male secretions. The release of α‐androstenol was observed after incubation of the sterile human sweat or α‐androstenol β‐glucuronide with a commercial glucuronidase enzyme, the urine‐isolated bacteria Streptococcus agalactiae, and the skin bacteria Staphylococcus warneri DSM 20316, Staphylococcus haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919, reported to have β‐glucuronidase activities. We demonstrated that if α‐ and β‐androstenols and androstadienol sulfates were present in human sweat, their concentrations would be too low to be considered as potential precursors of malodors; therefore, the H₂O‐soluble precursor of α‐androstenol in apocrine secretion should be a β‐glucuronide.     

Potential of the bean α‐amylase inhibitor αAI‐1 to inhibit α‐amylase activity in true bugs (Hemiptera)

True bugs (Hemiptera) are an important pest complex not controlled by Bt‐transgenic crops. An alternative source of resistance includes inhibitors of digestive enzymes, such as protease or amylase inhibitors. αAI‐1, an α‐amylase inhibitor from the common bean, inhibits gut‐associated α‐amylases of bruchid pests of grain legumes. Here we quantify the in vitro activity of α‐amylases of 12 hemipteran species from different taxonomic and functional groups and the in vitro inhibition of those α‐amylases by αAI‐1. α‐Amylase activity was detected in all species tested. However, susceptibility to αAI‐1 varied among the different groups. α‐Amylases of species in the Lygaeidae, Miridae and Nabidae were highly susceptible, whereas those in the Auchenorrhyncha (Cicadellidae, Membracidae) had a moderate susceptibility, and those in the Pentatomidae seemed to be tolerant to αAI‐1. The species with αAI‐1 susceptible α‐amylases represented families which include both important pest species but also predatory species. These findings suggest that αAI‐1‐expressing crops have potential to control true bugs in vivo.     

Roles of the three L‐domains in β‐retrovirus budding

Retroviral Gag protein plays a critical role during the late stage of virus budding and possesses a so‐called L‐domain containing PT/SAP, PPxY, YxxL or FPIV motifs that are critical for efficient budding. Mason–Pfizer monkey virus (M‐PMV) contains PSAP, PPPY, and YADL sequences in Gag. This study was performed to investigate the roles of these three L‐domain‐like sequences in virus replication in three different cell lines, 293T, COS‐7 and HeLa cells. It was found that the PPxY motif plays an essential role in progeny virus production as a major L‐domain in all three cell lines. The PSAP sequence was shown to function as an additional L‐domain in HeLa cells and to promote efficient release of M‐PMV; however, this sequence was dispensable for M‐PMV production in 293T and COS‐7 cells, suggesting that the role of the PSAP motif as an L‐domain in M‐PMV budding is cell type‐dependent. Viruses possessing multiple L‐domains appear to change the L‐domain usage to replicate in various cells. On the other hand, the YADL motif was required for M‐PMV production as a transport signal of Gag to the plasma membrane, but not as an L‐domain.     

Synthesis of the kyotorphin precursor benzoyl‐L‐tyrosine‐L‐argininamide with immobilized α‐chymotrypsin in sequential batch with enzyme reactivation

α‐Chymotrypsin was immobilized in activated agarose support and the stability of the biocatalyst was assessed in three polar organic solvents, namely, ethanol, diglyme, and acetonitrile. Ethanol was the solvent in which the stability of the enzyme was higher and was then selected to perform the synthesis of the kyotorphin derivative benzoyl‐tyrosine argininamide, evaluating enzyme reactivation after synthesis. Substrates for reaction were benzoyl tyrosine ethyl ester and argininamide, the reaction being performed under kinetic control. High conversion yield (85%) was obtained and the immobilized enzyme was successfully used in sequential batch reactor operation with enzyme reactivation after three batches. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:54–59, 2016     

An efficient and enantioselective Michael addition of aromatic oximes to α,β‐unsaturated aldehydes promoted by a chiral diamine catalyst derived from α,α‐diphenyl prolinol

Chiral diamine catalysts 11a–e derived from α ,α ‐diphenyl prolinol were prepared and successfully applied to the Michael addition of aromatic oximes to α ,β ‐unsaturated aldehydes in mediocre to good yields (up to 78%) and good to high enantioselectivities (up to 93% ee ).     

Differential physiologic responses of α7 nicotinic acetylcholine receptors to β‐amyloid1–40 and β‐amyloid1–42

The β‐amyloid peptides (Aβ), Aβ_1–40 and Aβ_1–42, have been implicated in Alzheimer's disease (AD) pathology. Although Aβ_1–42 is generally considered to be the pathological peptide in AD, both Aβ_1–40 and Aβ_1–42 have been used in a variety of experimental models without discrimination. Here we show that monomeric or oligomeric forms of the two Aβ peptides, when interact with the neuronal cation channel, α7 nicotinic acetylcholine receptors (α7nAChR), would result in distinct physiologic responses as measured by acetylcholine release and calcium influx experiments. While Aβ_1–42 effectively attenuated these α7nAChR‐dependent physiology to an extent that was apparently irreversible, Aβ_1–40 showed a lower inhibitory activity that could be restored upon washings with physiologic buffers or treatment with α7nAChR antagonists. Our data suggest a clear pharmacological distinction between Aβ_1–40 and Aβ_1–42. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 25–30, 2003     

Integrated Genome and Protein Editing Swaps α‐2,6 Sialylation for α‐2,3 Sialic Acid on Recombinant Antibodies from CHO

Immunoglobin G with α‐2,6 sialylation has been reported to have an impact on antibody‐dependent cellular cytotoxicity and anti‐inflammatory efficacy. However, production of antibodies with α‐2,6 sialylation from Chinese hamster ovary cells is challenging due to the inaccessibility of sialyltransferases for the heavy chain N‐glycan site and the presence of exclusively α‐2,3 sialyltransferases. In this study, combining mutations on the Fc regions to allow sialyltransferase accessibility with overexpression of α‐2,6 sialyltransferase produced IgG with significant levels of both α‐2,6 and α‐2,3 sialylation. Therefore, ST3GAL4 and ST3GAL6 genes were disrupted by CRISPR/Cas9 to minimize the α‐2,3 sialylation. Sialidase treatment and SNA lectin blot indicated greatly increased α‐2,6 sialylation level relative to α‐2,3 sialylation for the α‐2,3 sialyltransferase knockouts when combined with α‐2,6 sialyltransferase overexpression. Indeed, α‐2,3 linked sialic acids were not detected on IgG produced from the α‐2,3 sialyltransferase knockout‐α‐2,6 sialyltransferase overexpression pools. Finally, glycoprofiling of IgG with four amino acid substitutions expressed from an α‐2,3 sialyltransferase knockout‐α‐2,6 sialyltransferase stable clone resulted in more than 77% sialylated glycans and more than 62% biantennary disialylated glycans as indicated by both MALDI‐TOF and LC‐ESI‐MS. Engineered antibodies from these modified Chinese hamster ovary cell lines will provide biotechnologists with IgGs containing N‐glycans with different structural variations for examining the role of glycosylation on protein performance.     

Asymmetric Allylation of α‐Ketoester‐Derived N‐Benzoylhydrazones Promoted by Chiral Sulfoxides/N‐Oxides Lewis Bases: Highly Enantioselective Synthesis of Quaternary α‐Substituted α‐Allyl‐α‐Amino Acids

Chiral sulfoxides/N‐oxides (R)‐ 1 and (R,R)‐ 2 are effective chiral promoters in the enantioselective allylation of α‐keto ester N‐benzoylhydrazone derivatives 3a , 3b , 3c , 3d , 3e , 3f , 3g to generate the corresponding N‐benzoylhydrazine derivatives 4a , 4b , 4c , 4d , 4e , 4f , 4g , with enantiomeric excesses as high as 98%. Representative hydrazine derivatives 4a , 4b were subsequently treated with SmI₂, and the resulting amino esters 5a , 5b with LiOH to obtain quaternary α‐substituted α‐allyl α‐amino acids 6a , 6b , whose absolute configuration was assigned as (S), with fundament on chemical correlation and electronic circular dichroism (ECD) data. Chirality 25:529–540, 2013. © 2013 Wiley Periodicals, Inc.     

A quantitative anharmonic analysis of the amide A band in α‐helical poly(L‐alanine)

Polarized ir spectra of oriented films of α‐helical poly(l ‐alanine) (α‐PLA) have been obtained as a function of residual solvent dichloroacetic acid (DCA). The amide A, B, II, and V regions exhibit multiple bands whose structure depends on the residual DCA content, and those associated with the α_I‐PLA structure have been identified. A calculation of the relevant cubic anharmonic force constants indicates that, contrary to previous assignments, the overtone of amide II(A) is in Fermi resonance with the NH stretch fundamental, whose unperturbed frequency we now find to be at 3314 cm⁻¹, significantly higher than the previously suggested 3279 cm⁻¹. The presence of a structure in addition to the standard α_I‐PLA is indicated by our analysis. © 1999 John Wiley & Sons, Inc. Biopoly 49: 195–207, 1999