β-Trichosanthin: a new abortifacient protein from the Chinese drug,Wangua, Trichosanthes cucumeroides

β-Trichosanthin was a new abortifacient protein purified from the Chinese drug, Wangua, root tubers of Trichosanthes cucumeroides (Cucurbitaceae). The purification procedure involved acetone fractionation, ammonium sulfate precipitation, ion-exchange chromatography on CM-Sepharose and preparative agarose electrophoresis. Homogeneity of β-trichosanthin was demonstrated in immunoelectrophoresis, agarose electrophoresis and SDS-polyacrylamide gel electrophoresis. It had a molecular weight of 28 000 and no cysteine in its molecule. It differed from trichosanthin, a known abortifacient protein isolated from a related Chinese drug, Tianhuafen, root tubers of Trichosanthes kirilowii (Cucurbitaceae), in molecular weight, carbohydrate content, charge and amino acid composition. β-Trichosanthin was, however, immunochemically identical to trichosanthin and was about twice as potent as trichosanthin in inducing mid-term abortion in mice.     

Analysis of γβ, βγ, γγ, ββ continuous turns in proteins

Abstract: We report the observation of continuous turns in proteins which comprise individual γ‐turns or β‐turns or both that are situated immediately one after the other along the polypeptide chain. The continuous turns were identified from a representative data set of three‐dimensional protein crystal structures. The γβ/βγ, γγ and ββ continuous turns represent peptides of varying amino acid residue lengths and conformations. The continuous turns frequently observed in proteins were: γβ, between a coil and a strand; βγ, between a helix and a strand; γγ, between coils; and ββ, either between a strand and a coil or between strands or coils. We determined the statistically significant amino acid residue preferences at individual positions in the turn, calculated amino acid positional potentials and analyzed main chain hydrogen bonds and side‐chain interactions likely to stabilize the continuous turns. The data on continuous turns have been integrated in the database of structural motifs in proteins (DSMP) on our web server at ( http://www.cdfd.org.in/dsmp.html ). This is useful to make queries on sequences compatible with different continuous turns.     

Analysis of γβ, βγ, γγ, ββ multiple turns in proteins

Abstract: The number of γ‐turns in a representative protein dataset selected from the current Protein Data Bank has increased almost seven times during the past decade. Eighty percent classic γ‐turns and 57% inverse γ‐turns are associated as multiple turns with either another γ‐turn or a β‐turn. We refer to these as multiple turns of the (γβ)_1,2,3 or (βγ)_1,2,3 type, depending upon whether the γ‐turn is before or after the β‐turn along the protein chain, respectively. However, for multiple turns involving only γ‐turns, we follow the nomenclature analogous to that proposed earlier for the multiple (or double) β‐turns. Fifty‐eight per cent β‐turns are associated as multiple turns with another β‐turn. We extracted multiple turns from the protein dataset and classified them on the basis of individual γ‐ or β‐turn types and the number of overlapping residues. Furthermore, we evaluated the amino acid positional potentials and determined the statistically significant amino acid preferences, hydrogen bond/side‐chain interaction preferences in the multiple turns and secondary structure preferences for residues immediately flanking these turns. The results of our analysis would be useful in the modeling, prediction or design of multiple turns in proteins. The amino acid sequence corresponding to the multiple turn, position in the protein chain, PDB Code/chain in which multiple turn is present and the individual turn types constituting the multiple turns are available from our website and this information would also be integrated in our Database of Structural Motifs in Proteins ( http://www.cdfd.org.in/dsmp.html ).     

Recent advances in trichosanthin, a ribosome-inactivating protein with multiple pharmacological properties.

Trichosanthin (TCS), a ribosome-inactivating protein extracted from the root tuber of Chinese medicinal herb Trichosanthes kirilowii Maximowicz, has multiple pharmacological properties including abortifacient, anti-tumor and anti-HIV. It is traditionally used to induce abortion but its antigenicity and short plasma half-life have limited the repeated clinical administration. In this review, work to locating antigenic sites and prolonging plasma half-life are discussed. Studies on structure-function relationship and mechanism of cell entry are also covered. Recently, TCS has been found to induce apoptosis, enhance the action of chemokines and inhibit HIV-1 integrase. These findings give new insights on the pharmacological properties of TCS and other members of ribosome-inactivating proteins.     

Alpha and beta momorcharins

Two abortifacient proteins designated α and β momorcharins were isolated from seeds of the bitter gourd Momordica charantia by a procedure involving acetone fractionation, CM-Sepharose chromatography and gel filtration on Sephadex G-100. The two proteins were approximately equipotent in inducing midterm abortion in mice. Hemagglutinating (lectin) activity was lacking. Homogeneity of the preparations was attested by a single band in polyacrylamide gel electrophoresis and immunoelectrophoresis. Although the two proteins had similar molecular weights as judged by SDS-polyacrylamide gel electrophoresis, they were immunochemically distinct.     

Conformational studies on β-bend containing a cis peptide unit

Conformational studies have been carried out on the X-cis-Pro tripeptide system (a system of three linked peptide units, in the trans-cis-trans configuration) using energy minimization techniques. For X, residues Gly, L-Ala, D-Ala and L-Pro have been used. The energy minima have been classified into different groups based upon the conformational similarity. There are 15, 20, 18 and 6 minima that are possible for the four cases respectively arid these fall into 11 different groups. A study of these minima shows that, (i) some minima contain hydrogen bonds - either 4→1 or 1→2 type, (ii) the low energy minima qualify themselves as bend conformations, (iii) cis′ and trans′ conformations are possible for the prolyl residue as also the C^γ-endo and C^γ-exo puckerings, and (iv) for Pro-cis-Pro, cis′ at the first prolyl residue is ruled out, due to the high energy. The available crystal structure data on proteins and peptides, containing cis-Pro segment have been examined with a view to find the minima that occur in solid state. The data from protein show that they fall under two groups. The conformation at X in X-cis-Pro is near extended when it is a non-glycyl residue. In both peptides and proteins there exists a preference for trans′ conformation at prolyl residue over cis′ when X is a non-glycyl residue. The minima obtained can be useful in modelling studies.     

Thiophilic interaction chromatography of Alzheimer's β‐amyloid peptides

Abstract: Alzheimer's disease is characterized by a progressive formation of senile plaques in the brain, the major constituent of which is β‐amyloid (Aβ) peptide, a proteolytic product of the transmembrane β‐amyloid precursor protein (APP). Prior to the measurement of levels of the Aβ peptide for diagnostic purposes, this peptide must be isolated from the myriad of proteins resident in the human serum. Thiophilic interaction chromatography is an effective method for the isolation of proteins and peptides containing clusters of aromatic residues such as tryptophan, phenylalanine and tyrosine. The purpose of the present study was to develop a protocol for binding and recovery of Aβ peptides (1–38), (1–40) and (1–42) to T‐gels by varying T‐gel type and elution conditions such as the salt concentration and type of eluent. We established the minimal salt concentration necessary for the binding of the Aβ(1–40) peptide to the 3S‐gel; binding at that concentration was subsequently compared with that of model proteins, lysozyme and α‐chymotrypsin and this methodology was extended to 2S‐gels and PyS. β‐Amyloid peptide (1–40) showed a remarkably strong affinity for all three types of T‐gels in comparison to lysozyme and α‐chymotrypsin and was found to bind best to 2S‐gel.     

Thermal denaturation of tryptophan synthase α-subunit

Thermal denaturation for the wild-type of tryptophan synthase α-subunit from E. coli and one of its mutant proteins was followed by CD measurements at various pHs in the alkaline region and the results from van't Hoff analyses of the thermal denaturation curves were compared with those from calorimetry. Although the far-u.v. CD spectra of the thermally denatured proteins differed from those of the completely denatured states in 3.2 M guanidine hydrochloride, the titration curves by denaturants at higher temperatures were not sigmoidal but straight lines, indicating that the cooperative structure of the proteins has been completely destroyed by heating. The ratio of calorimetric enthalpy change to van't Hoff enthalpy change obtained from calorimetric study was unity, indicating that the thermal denaturation of the proteins was a two-state system. The unfolding heat capacity change (ΔCp) of the wild-type protein from van't Hoff analysis of the thermal denaturation curves by CD measurement was estimated to be 2.45 kcal/mol ± deg, which was similar to that from calorimetry. The values of unfolding enthalpy change at denaturation temperatures were lower by about 15 kcal/mol compared to those from calorimetry.     

Prediction of γ‐turns from amino acid sequences

Abstract: We predicted γ‐turns from amino acid sequences using the first‐order Markov chain theory and enlarged representative data sets corresponding to protein chains selected from the Protein Data Bank (PDB). The following data sets were used for training and deriving the probability values: (1) an initial data set containing 315 protein chains comprising 904 γ‐turns and (2) a later data set in order to include new entries in the PDB, containing 434 protein chains and comprising 1053 γ‐turns. By excluding 93 protein chains that were common to these two training data sets, we generated two mutually exclusive data sets containing 222 and 341 protein chains for testing our predictions. Applying amino acid probability values derived from training data sets on to testing data sets yielded overall prediction accuracies in the range 54–57%. We recommend the use of probability values derived from the data set comprising 315 protein chains that represents more γ‐turns and also provides better predictions.     

Effect of Serum, α-1 Acid Glycoprotein,Lipoproteins and Albumin on Human Mononuclear Leucocyte β-Adrenoceptors

Abstract: The effects of serum, α-1 acid glycoprotein (AAG), serum lipoproteins (SLP) and human serum albumin (HSA) on ³H-(-)-dihydroalprenolol (³H-(-)-DHA) binding and (-)-isoproterenol ((-)-IPR) induced cyclic AMP (cAMP) elevation in human peripheral blood mononuclear leucocytes (MNL) were investigated. The saturable binding of ³H-(-)-DHA was decomposed into two classes of binding sites with maximum binding capacity of approximately 1400 and 30000 sites/cell and with dissociation constants (K_d) of approximately 0.7 and 65 nM. Stimulation of the MNL β-adrenoceptors by (-)-IPR caused a concentration dependent cAMP accumulation (EC50 ～0.2 μM) with maximum level approximately 250% above basal. For all single leucocyte preparations, 30–35 min. exposure to serum, AAG and SLP increased the number of β-adrenoceptors with 100–200% and the maximal responsiveness to (-)-IPR with 30–90%. The presence of proteins did not change the K_d or the EC50. (-)-Alprenolol inhibited concentration dependently the serum induced increment in (-)-IPR-responsiveness. Serum, AAG and SLP did also increase the number of low affinity binding sites with 25–40% without effect on the K_d. HSA had no consistent effect on β-adrenergic binding or stimulation. The present study shows that serum, AAG and SLP influence the number and function of MNL β-adrenoceptors in vitro.     

Synthesis of uniformly deuterated n‐dodecyl‐β‐d‐maltoside (d39‐DDM) for solubilization of membrane proteins in TROSY NMR experiments

This work reports the first synthesis of uniformly deuterated n‐dodecyl‐β‐d ‐maltoside (d₃₉‐DDM). DDM is a mild non‐ionic detergent often used in the extraction and purification of membrane proteins and for solubilizing them in experimental studies of their structure, dynamics and binding of ligands. We required d₃₉‐DDM for solubilizing large α‐helical membrane proteins in samples for [¹⁵N–¹H]TROSY (transverse relaxation‐optimized spectroscopy) NMR experiments to achieve the highest sensitivity and best resolved spectra possible. Our synthesis of d₃₉‐DDM used d₇‐d ‐glucose and d₂₅‐n‐dodecanol to introduce deuterium labelling into both the maltoside and dodecyl moieties, respectively. Two glucose molecules, one converted to a glycosyl acceptor with a free C4 hydroxyl group and one converted to a glycosyl donor substituted at C1 with a bromine in the α‐configuration, were coupled together with an α(1 → 4) glycosidic bond to give maltose, which was then coupled with n‐dodecanol by its substitution of a C1 bromine in the α‐configuration to give DDM. ¹H NMR spectra were used to confirm a high level of deuteration in the synthesized d₃₉‐DDM and to demonstrate its use in eliminating interfering signals from TROSY NMR spectra of a 52‐kDa sugar transport protein solubilized in DDM.     

Synthetic peptides derived from the β2−β3 loop of Raphanus sativus antifungal protein 2 that mimic the active site

Abstract: Rs‐AFPs are antifungal proteins, isolated from radish (Raphanus sativus) seed or leaves, which consist of 50 or 51 amino acids and belong to the plant defensin family of proteins. Four highly homologous Rs‐AFPs have been isolated (Rs‐AFP1–4). The structure of Rs‐AFP1 consists of three β‐strands and an α‐helix, and is stabilized by four cystine bridges. Small peptides deduced from the native sequence, still having biological activity, are not only important tools to study structure?function relationships, but may also constitute a commercially interesting target. In an earlier study, we showed that the antifungal activity of Rs‐AFP2 is concentrated mainly in the β2?β3 loop. In this study, we synthesized linear 19‐mer peptides, spanning the entire β2?β3 loop, that were found to be almost as potent as Rs‐AFP2. Cysteines, highly conserved in the native protein, are essential for maintaining the secondary structure of the protein. Surprisingly, in the 19‐mer loop peptides, cysteines can be replaced by α‐aminobutyric acid, which even improves the antifungal potency of the peptides. Analogous cyclic 19‐mer peptides, forced to adopt a hairpin structure by the introduction of one or two non‐native disulfide bridges, were also found to possess high antifungal activity. The synthetic 19‐mer peptides, like Rs‐AFP2 itself, cause increased Ca²⁺ influx in pregerminated fungal hyphae.     

Synthese von γ,γ'-Dihydroxysulfonen und γ-Hydroxy-γ'-ketosulfonen

Syntheses of γ,γ'-Dihydroxysulfones and γ-Hydroxy-γ'-ketosulfones Reduction of γ,γ'-diketosulfones 1 with dimethylaminoborane leads to γ,γ'-dihydroxysulfones 3 via γ-hydroxy-γ'-ketosulfones 2 . The influence of substituents on the ratio of the yields of 2 and 3 is investigated.     

Binding of calcium to synthetic peptides containing γ-carboxyglutamic acid

The Ca²⁺ binding properties of various γ-carboxyglutamic acid (gla)-containing synthetic peptides with counterpart sequences in human protein C were investigated employing potentiometry with a Ca²⁺-selective electrode and titration calorimetric techniques. The shortest peptides, FL(gla)(gla)LR, DF(gla)(gla)AK, and the oxidized form of the cyclic hexapeptide CI(gla)(gla)IC, each of which contains one pair of gla residues, have a weak affinity for Ca²⁺, with some peptides probably involved in intermolecular bridging of the Ca²⁺. The best example of this is the oxidized form of the peptide, CI(gla)(gla)IC, where one g-atom of Ca²⁺ interacts with 2 mol of peptide (n= 0.5) with a K_d value of 1.6 mM. A second g-atom of Ca²⁺ interacts with 2 mol of this same peptide (n= 0.5) and is characterized by a K_d of 8.8 mM. A longer peptide containing this same sequence, viz. L(gla)R(gla)CI(gla)(gla)IC, possesses two binding sites (n= 2.0)for Ca²⁺ of K_d=16.1 mM, as well as a tighter site (n= 1), Of K_d= 0.4 mM. An increase in stoichiometry of tight binding sites as the peptide is elongated is observed from binding data obtained on a 38-residue peptide that possesses all nine of the gla-residues of protein C in their proper sequence positions. The strongest Ca²⁺ binding sites (n= 3–4) possess an average K_d of 0.4 mM, followed by another class of sites (n= 5–10, average K_d= 1.5–3.0 mM). The affinity and stoichiometry of these stronger sites mimic those observed for binding of Ca²⁺ to the gla region of prothrombin fragment 1. By selective [¹³C]-labeling of the essential gla 16 residue of the 38-mer peptide, we demonstrate that this particular gla residue participates as a donor for a high-affinity Ca²⁺ site. These similarities in binding properties between the synthetic peptide containing the entire gla domain and the gla domain as it exists in proteins and protein fragments indicate suitably designed peptides of this type may constitute appropriate models for investigation of the binding of Ca²⁺ to intact gla-containing proteins.     

Synthesis of [3α‐3H] 17α‐Hydroxy pregnenolone and [3α‐3H] Pregnenolone

For the first time, [3α‐³H] 17α‐hydroxy pregnenolone (1) was synthesized through a multiple step sequence. The presence of [3β‐³H] isomer in RP‐HPLC purified product was identified by tritium NMR. The [3β‐³H] isomer was then separated from [3α‐³H] 17α‐hydroxy pregnenolone with chiralPAK AD‐H column. [3α‐³H] pregnenolone (2) was synthesized from commercial available 5‐pregnen‐3,20‐dione in one step with an improved procedure.     

Calorimetric study of tryptophan synthase α-subunit and two mutant proteins

Heat-denaturation of tryptophan synthase α-subunit from E. coli and two mutant proteins (Glu 49 ± Gln or Ser; called Gln 49 or Ser 49, respectively) has been studied by the scanning microcalorimetric method at various pH, in an attempt to elucidate the role of individual amino acid residues in the conformational stability of a protein. The partial specific heat capacity in the native state at 20°, C_p²⁰, has been found to be (0.43 ± 0.02) cal ± K^-1 ± g^-1, the unfolding heat capacity change, Δ_dC_p, (0.10 ± 0.01) cal ± K^-1 ± g^-1, and the unfolding enthalpy value extrapolated to 110°, Δ_dh¹¹⁰, (9.3 ± 0.5) cal ± g^-1 for the three proteins. The value of C_p²⁰ was larger than those found for fully compact protein and that of Δ_dh¹¹⁰ was smaller. Unfolding Gibbs energy, Δ_dG at 25° for Wild-type, Gln 49, and Ser 49 were 5.8, 8.4, and 7.1 kcal ± mol^-1 at pH 9.3, respectively. Unfolding enthalpy, Δ_dH, of the three proteins seemed to be the same and equal to (23.2 ± 1.2) kcal ± mol^-1 at 25°. As a consequence of the same value of Δ_dH and the different value in Δ_dG, substantial differences in unfolding entropy, Δ_dS, were found for the three proteins. The values of Δ_dG for the three proteins at 25° coincided with those from equilibrium methods of denaturation by guanidine hydrochloride.     

Properties of a Resiniferatoxin-stimulated,Calcium Inhibited but Phosphatidylserine-dependent Kinase,which is Distinct from Protein Kinase C Isotypes α, β1γ, δ, ε and η

We have separated a resiniferatoxin-stimulated histone-kinase activity from human neutrophils, elicited mouse macrophages and murine alveolar macrophages by hydroxyapatite chromatography. The assay conditions for resiniferatoxin kinase were optimized as part of this study and in the presence of phosphatidylserine but absence of Ca²⁺ the K_a for histone IIIs phosphorylation by resiniferatoxin was calculated as 16 nm . Using a phosphate gradient of 20–500 mm , peaks of protein kinase C activity could be washed from the hydroxyapatite column in 300 nm phosphate and resiniferatoxin kinase recovered in 500 mm phosphate. At the optimum concentration of 160 nm , the ability of resiniferatoxin to induce enzyme activity was compared with a range of phorbol esters all at the same concentration. These related compounds failed to activate resiniferatoxin kinase although they have previously been shown to activate protein kinase C isotypes. Similarly sn-1,2,-dioleoylglycerol and the potent irritant capsaicin at 30 μm failed to activate the kinase. A Scatchard analysis of [³H] phorbol dibutyrate binding produced a linear plot (K_d 41·6 nm ; B_max 11·6 fmol unit^?1) and binding was inhibited by resiniferatoxin and 12-O-tetradecanoylphorbol-13-acetate (TPA), with resiniferatoxin 700 times more potent than TPA in this respect. A radiolabeled resiniferatoxin binding assay was also used to demonstrate specific binding of [³H]resiniferatoxin which could be inhibited by unlabelled compound. Resiniferatoxin kinase activity was shown to be distinct from the protein kinase C isotypes α, β₁, γ δ and ε by means of immunological analysis and from the η isotype, because that isotype was not stimulated by resiniferatoxin but was stimulated by TPA when a pseudosubstrate was used. In addition the resiniferatoxin-stimulated activity was inhibited in-vitro by the addition of Ca²⁺ (K_i 0·1-0·5 nm free Ca²⁺). Further purification of resiniferatoxin kinase by Superose chromatography indicated a major activity fraction of about 70–90 kDa. Thus resiniferatoxin kinase, isolated from human and mouse inflammatory cells is distinct from the known isotypes of protein kinase C and is a major resiniferatoxin receptor.     

N-terminal sequence of some ribosome-inactivating proteins

The N-terminal portion of some type 1 ribosome-inactivating proteins (RIPs) isolated from the seeds of Gelonium multifiorum, Momordica charantia, Bryonia dioica, Saponaria oficinalis and from the leaves of Saponaria oficinalis are reported in the present paper. Their relationship with other RIPS is discussed.     

The role of Krüppel‐like factor 4 in transforming growth factor‐β–induced inflammatory and fibrotic responses in human proximal tubule cells

Krüppel‐like factor 4 (KLF4) is known to mitigate inflammation in several cell types. Using human proximal tubule cells, the present study aimed to investigate the role of KLF4 in regulating transforming growth factor (TGF)‐β₁ induced inflammatory and fibrotic responses. Human kidney proximal tubular cells were exposed to high glucose, or TGF‐β₁ and KLF4 expressions were determined. Cells were then transfected with empty vector or KLF4 and exposed to 2‐ng/mL TGF‐β₁ for up to 72 h. Inflammatory proteins (macrophage migration inhibitory factor and monocyte chemoattractant protein‐1) and pro‐fibrotic proteins (fibronectin and collagen IV) were measured after 72 h by enzyme‐linked immunosorbent assay and western blot, respectively. To determine the relevance to in vivo models of chronic kidney disease, KLF4 protein expression in streptozotocin‐induced diabetic mice was determined. Krüppel‐like factor 4 messenger RNA (mRNA) levels were significantly reduced in high glucose‐treated human kidney proximal tubular cells. High glucose increased TGF‐β₁ mRNA expression, which significantly increased migration inhibitory factor and monocyte chemoattractant protein‐1 protein secretion. Transforming growth factor‐β₁ significantly increased fibronectin and collagen IV protein expression. The overexpression of KLF4 significantly reduced TGF‐β–mediated increases in migration inhibitory factor and monocyte chemoattractant protein‐1 but had no effect on TGF‐β–mediated fibronectin and collagen IV mRNA and protein expression. The levels of KLF4 mRNA were significantly reduced in the diabetic kidney, and diabetic animals had a significant reduction in renal tubular expression of KLF4 proteins. This data suggest that KLF4 reduces inflammation induced by TGF‐β₁, suggesting a therapeutic role for KLF4 in diabetic nephropathy.