Activation of carbonic anhydrase II by active-site incorporation of histidine analogs

The hydration of CO2 catalyzed by human carbonic anhydrase II (HCA II) is accompanied by proton transfer from the zinc-bound water of the enzyme to solution. We have replaced the proton shuttling residue His 64 with Ala and placed cysteine residues within the active-site cavity by mutating sites Trp 5, Asn 62, Ile 91, and Phe 131. These mutants were modified at the single inserted cysteine with imidazole analogs to introduce new potential shuttle groups. Catalysis by these modified mutants was determined by stopped-flow and 18O-exchange methods. Specificity in proton transfer was demonstrated; only modifications of the Cys 131-containing mutant showed enhancement in the proton transfer step of catalysis compared with unmodified Cys 131-containing mutant. Modifications at other sites resulted in up to 3-fold enhancement in rates of CO2 hydration, with apparent second-order rate constants near 350 microM(-1) s(-1). These are among the largest values of kcat/Km observed for a carbonic anhydrase.     

Comparative efficacy of VIP and analogs on activation and internalization of the recombinant VPAC2 receptor expressed in CHO cells

Using a monoclonal antibody interacting with the extracellular amino-terminus of the human VPAC₂ receptor but that did not interfere with ligand binding, we measured by flow cytometry receptor internalization and trafficking induced by full agonists, partial agonists and an antagonist in Chinese hamster ovary cells expressing the recombinant receptor. The agonists, but not the antagonist, induced a rapid, dose-dependent receptor internalization blocked by hypertonic sucrose that was more pronounced for the VIP analog N-hexanoyl-VIP (80%) than for VIP and Ro 25-1553 (50%) and the [A¹¹]-VIP (20%). Re-expression of the receptors at the membrane was achieved within two hours after exposure to VIP and Ro 25-1553 was blocked by 25 μM monensin but not by 10 μg/ml cycloheximide. Re-expression was much slower after exposure to the acylated peptide and was blocked by preincubation with 25 μM monensin and 10 μg/ml cycloheximide.     

Pharmacological studies of proctolin receptors on foregut and hindgut of blaberus craniifer

Proctolin (Arg-Tyr-Leu-Pro-Thr) and proctolin analogs modified at position 1, 2, or 5 caused dose dependent contractions of Blaberus fore- and hindgut. The varying contractile effects between both tissues revealed the possible presence of receptor subtypes as identified by [GABA¹]-proctolin. A single population of binding sites (K_d ≈ 100 n ) was deduced from Scatchard analysis. In addition, nanomolar concentrations of proctolin induced a dose-dependent hydrolysis of phosphoinositides (PIns) augmented by GTPγS (1 μ ) on foregut membranes but no accumulation of cAMP. Proctolin induced contractions are likely mediated via a phospholipase C linked to a heptahelical receptor bound to heterotrimeric G-proteins.     

The use of Fmoc-Lys(Pac)-OH and penicillin G acylase in the preparation of novel semisynthetic insulin analogs.

In this paper, we present the detailed synthetic protocol and characterization of Fmoc-Lys(Pac)-OH, its use for the preparation of octapeptides H-Gly-Phe-Tyr-N-MePhe-Thr-Lys(Pac)-Pro-Thr-OH and H-Gly-Phe-Phe-His-Thr-Pro-Lys(Pac)-Thr-OH by solid-phase synthesis, trypsin-catalyzed condensation of these octapeptides with desoctapeptide(B23-B30)-insulin, and penicillin G acylase catalyzed cleavage of phenylacetyl (Pac) group from Nepsilon-amino group of lysine to give novel insulin analogs [TyrB25, N-MePheB26,LysB28,ProB29]-insulin and [HisB26]-insulin. These new analogs display 4 and 78% binding affinity respectively to insulin receptor in rat adipose membranes.     

Isolation and characterization of resistance and defense gene analogs in cotton (Gossypium barbadense L.)

Plant disease resistance gene (R gene) and defense response gene encode some conserved motifs. In the present work, a PCR strategy was used to clone resistance gene analogs (RGAs) and defense gene analogs (DGAs) from Sea-island cotton variety Hai7124 using oligonucleotide primers based on the nucleotide-binding site (NBS) and serine/threonine kinase (STK) in the R-gene and pathogenesis-related proteins of class 2 (PR2) of defense response gene. 79 NBS sequences, 21 STK sequences and 11 DGAs were cloned from disease-resistance cotton. Phylogenic analysis of 79 NBS-RGAs and NBS-RGAs nucleotide sequences of cotton already deposited in GenBank identified one new sub-cluster. The deduced amino acid sequences of NBS-RGAs and STK-RGAs were divided into two distinct groups respectively: Toll/Interleukin-1 receptor (TIR) group and non-TIR group, A group and B group. The expression of RGAs and DGAs having consecutive open reading frame (ORF) was also investigated and it was found that 6 NBS-RGAs and 1 STK-RGA were induced, and 1 DGA was up-regulated by infection of Verticillium dahliae strain VD8. 4 TIR-NBS-RGAs and 4 non-TIR-NBS-RGAs were arbitrarily used as probes for Southern-blotting. There existed 2–10 blotted bands. In addition, since three non-TIR-NBS-RGAs have the same hybridization pattern, we conjecture that these three RGAs form a cluster distribution in the genome.     

Semi-synthetic salinomycin analogs exert cytotoxic activity against human colorectal cancer stem cells

Salinomycin, a polyether antibiotic, is a well-known inhibitor of human cancer stem cells. Chemical modification of the allylic C20 hydroxyl of salinomycin has enabled access to synthetic analogs that display increased cytotoxic activity compared to the native structure. The aim of this study was to investigate the activity of a cohort of C20-O-acyl analogs of salinomycin on human colorectal cancer cell lines in vitro. Two human colorectal cancer cell lines (SW480 and SW620) were exposed to three C20-O-acylated analogs and salinomycin. The impact of salinomycin and its analogs on tumor cell number, migration, cell death, and cancer stem cell specifity was analyzed. Exposure of human colorectal cancer cells to the C20-O-acylated analogs of salinomycin resulted in reduced tumor cell number and impaired tumor cell migration at lower concentrations than salinomycin. When used at higher (micromolar) concentrations, these effects were accompanied by induction of apoptotic cell death. Salinomycin analogs further expose improved activity against cancer stem cells compared to salinomycin.     

Analogs of the autoinducer of bioluminescence inVibrio fischer

The enzymes for luminescence inVibrio fischeri are induced only when a sufficient concentration of a metabolic product (autoinducer) specifically produced by this species accumulates. It has previously been shown that the autoinducer is 3-oxohexanoyl homoserine lactone and that it enters the cells by simple diffusion. To further study the mechanism of induction, we have synthesized several analogs of the autoinducer. The analogs were tested withV. fischeri for their inducing activity and for their ability to inhibit the action of the natural autoinducer. The compounds were found to display various combinations of inducing and inhibiting abilities. None of the compounds tested appeared to have any effect on cells ofV. harveyi strain MAV orPhotobacterium leiognathi strain 721, but several of the compounds decreased light output byP. phosphoreum strain 8265. These studies show that 1) the site of action of the autoinducer is not highly sterically constrained 2) the autoinducers of other species of luminous bacteria are likely to be quite different from that ofV. fischeri and 3) a simple mode in which one autoinducer molecule binds to a single receptor protein site and thus initiates luciferase synthesis is inadequate. The analogs should prove useful in the study of the binding site and mode of action of the autoinducer.Abbreviations SWC sea water complete     

Binding of ethylene analogs and cyclic olefins to a Triton X-100 extract from plants: Comparison with in vivo activities

The binding constants of various olefins were determined with a Triton X-100 extract of mung bean sprouts. The olefins tested included compounds that have been reported to induce an ethylene response in vivo as well as olefins reported to block the ethylene response. Both types of compounds were bound by the Triton X-100 extract, and the binding constants in vitro were usually considerably lower than those obtained in in vivo studies as measured by gas phase concentrations. Increased solubility due to Triton X-100 solution appears to be partly responsible. The in vitro binding order of compounds reported to induce an ethylene response was similar to their order of in vivo activity. Also, the compounds which gave an anti-ethylene response in vivo bound to the extract in approximately the same order as their in vivo effectiveness. These results suggest that binding of olefins is not the only factor necessary for an ethylene response. Although binding is necessary for activity, another factor must be involved after binding, and this may be -acceptance.     

Amiloride and its analogs as tools in the study of ion transport

Amiloride inhibits most plasma membrane Na+ transport systems. We have reviewed the pharmacology of inhibition of these transporters by amiloride and its analogs. Thorough studies of the Na+ channel, the Na+/H+ exchanger, and the Na+/Ca2+ exchanger, clearly show that appropriate modification of the structure of amiloride will generate analogs with increased affinity and specificity for a particular transport system. Introduction of hydrophobic substituents on the terminal nitrogen of the guanidino moiety enhances activity against the Na+ channel; whereas addition of hydrophobic (or hydrophilic) groups on the 5-amino moiety enhances activity against the Na+/H+ exchanger. Activity against the Na+/Ca2+ exchanger and Ca2+ channel is increased with hydrophobic substituents at either of these sites. Appropriate modification of amiloride has produced analogs that are several hundred-fold more active than amiloride against specific transporters. The availability of radioactive and photoactive amiloride analogs, anti-amiloride antibodies, and analogs coupled to support matrices should prove useful in future studies of amiloride-sensitive transport systems. The use of amiloride and its analogs in the study of ion transport requires a knowledge of the pharmacology of inhibition of transport proteins, as well as effects on enzymes, receptors, and other cellular processes, such as DNA, RNA, and protein synthesis, and cellular metabolism. One must consider whether the effects seen on various cellular processes are direct or due to a cascade of events triggered by an effect on an ion transport system.