[Efficacy and safety of combination therapy with chemotherapy, programmed death-1 inhibitor and anlotinib in the treatment of advanced dedifferentiated liposarcoma].

Objective: To investigate the efficacy and safety of the combination therapy with chemotherapy, programmed death-1 (PD-1) inhibitor and anlotinib in the treatment of advanced dedifferentiated liposarcoma (DDLPS). Methods: The clinical data of patients with dedifferentiated liposarcoma who received chemotherapy combined with PD-1 inhibitor and anlotinib in the Department of Medical Oncology, Zhongshan Hospital Affiliated to Fudan University from January 1, 2020 to November 30, 2021 were retrospectively analyzed. A total of 24 patients were included in this study, including 12 males and 12 females, with a median age of onset of 56 years (range, 31-69 years). Efficacy and safety in those patients were assessed. Results: All patients had unresectable or metastatic dedifferentiated liposarcoma with G2 (moderate differentiation) or G3 (differential differentiation) in a concise three-grade grading scheme of tumor pathology. Twelve patients received the regimen as the first-line treatment, while the other 7 taken the regimen as second-line treatment and 5 as third-line or above. The median follow-up time for overall survival (OS) was 7.7 months. The overall response rate (ORR) was 20.8% (5/24) and disease control rate (DCR) was 83.3% (20/24) with 5 partial response (PR), 15 stable disease (SD) and 4 progressive disease (PD). Overall, the median progression-free survival (PFS) was 4.9 months (95%CI: 3.4-16.2 months). The ORR of anthracycline-based, eribulin-based or gemcitabine-based regimens was 1/12, 2/6 and 2/6, respectively; and the median PFS was 7.7, 7.3 and 4.4 months, respectively. Waterfall plots showed notable tumor shrinkage of any degree in eribulin and gemcitabine-based regimens(3/6 and 2/6, respectively), while there were more patients presented with SD in anthracycline-based group(9/12). Common adverse reactions included myelosuppression, fatigue, anorexia, rash, pruritus, palpitate, hypothyroidism and hypertension. Conclusions: The combination regimen with chemotherapy, PD-1 inhibitor and anlotinib in the treatment of advanced DDLPS is effective and well tolerable. There are more responders in eribulin or gemcitabine-based regimens.

[Mediating effect of hemoglobin and hematocrit on the association between alcohol consumption and blood pressure among middle-aged and elderly male residents in Guangzhou].

Objective: To explore the mediating effect of hemoglobin (Hb) and hematocrit (HCT) on the association between alcohol consumption and blood pressure, and provide evidence for the prevention and control of alcohol-attributed hypertension. Methods: 1 091 male (age >50 years old) participants with drinking habit were selected from the Guangzhou biobank cohort study (GBCS). Mediation analysis was used to evaluate the mediating effect of Hb and HCT on the association of alcohol consumption (unit/day) with systolic blood pressure (SBP), diastolic blood pressure(DBP), pulse pressure(PP) and mean arterial pressure (MAP). Results: After adjusting for age, body mass index, education level, personal annual income, smoking, occupation and physical activity, the associations of alcohol consumption with SBP, DBP, PP and MAP were partly mediated by Hb, the proportion of mediating effect was 11.8% (95%CI 4.8%-24.7%), 15.3% (95%CI 6.5%-32.0%), 8.4% (95%CI 2.2%-22.5%) and 13.5% (95%CI 5.9%-27.5%), respectively. The associations of alcohol consumption with SBP, DBP, and MAP were also partly mediated by HCT, the proportion of mediating effect was 6.3% (95%CI 1.0%-16.0%), 8.7% (95%CI 1.4%-21.4%), and 7.5% (95%CI 1.0%-18.6%), respectively. Conclusion: There is a significant mediating effect of Hb and HCT on the association between alcohol consumption and blood pressure. Besides efforts on alcohol control, the potential effects of alcohol-induced increase on Hb and HCT, which might also increase the blood pressure, need to be considered to achieve optimal monitoring and prevention of alcohol-related hypertension.

Hybrid Pixel-Waveform (HPWF) Enabled CdTe Detectors for Small Animal Gamma-Ray Imaging Applications.

This paper presents the design and preliminary evaluation of small-pixel CdTe gamma ray detectors equipped with a hybrid pixel-waveform (HPWF) readout system for gamma ray imaging applications with additional discussion on CZT due to its similarity. The HPWF readout system utilizes a pixelated anode readout circuitry which is designed to only provide the pixel address. This readout circuitry works in coincidence with a high-speed digitizer to sample the cathode waveform which provides the energy, timing, and depth-of-interaction (DOI) information. This work focuses on the developed and experimentally evaluated prototype HPWF-CdTe detectors with a custom CMOS pixel-ASIC to readout small anode pixels of 350 µm in size, and a discrete waveform sampling circuitry to digitize the signal waveform induced on the large cathode. The intrinsic timing, energy, and spatial resolution were experimentally evaluated in this paper in conjunction with methods for depth of interaction (DOI) partitioning of the CdTe crystal. While the experimental studies discussed in this paper are primarily for evaluating HPWF detectors for small animal PET imaging, these detectors could find their applications for ultrahigh-resolution SPECT and other imaging modalities.

[Association between body weight change during early and middle adulthood and the risk of type 2 diabetes in middle aged and elderly population].

Objective: To examine the association between weight changes during early and middle adulthood and the risk of type 2 diabetes mellitus in middle aged and elderly population. Methods: Based on the Guangzhou Biobank Cohort Study (GBCS), 28 736 residents aged ≥50 years were included in Guangzhou. Multivariate logistic regression was used to analyze the association between body weight changes during early or middle adulthood and age when the heaviest weight reaching the threshold on the risk of type 2 diabetes mellitus in middle age or elderly population. Adjustments on age, smoking, alcohol consumption, physical activity, education level, occupation, district of residence and body mass index etc., were made. Results: The mean age was 64.3 (standard deviation=6.7) years in men and 61.0 (standard deviation=7.0) years in women, with the prevalence rates of diabetes as 13.1% and 13.7% in men and women, respectively. Compared to those with stable body weight, the risk of diabetes increased with weight gain during early and middle adulthood in both men and women (both P values for trend<0.01). Participants who gained more than 20 kg during early and middle adulthood were associated with the highest risk of diabetes in men (OR=2.83, 95%CI:1.99-4.02) and women (OR=3.13, 95%CI: 2.47-3.96). Compared to those who reached the highest weight at age 20, those who reaching the highest weight at 40 to 49 years were associated with the highest risk of diabetes, with OR being 5.32 (95%CI: 1.92-14.8) in men and 3.41 (95%CI: 2.49-4.67) in women, respectively. Weight loss in adulthood was associated with self-reported but not newly diagnosed diabetic cases in both middle and older aged men and women. Conclusion: Weight gain during early and middle adulthood may increase the risk of diabetes in middle and older aged population. The detrimental effect of obesity on diabetes might become significantly visible in the next decades.

Effects of alfalfa and cereal straw as a forage source on nutrient digestibility and lactation performance in lactating dairy cows.

This study was conducted to investigate the nutrient digestibility and lactation performance when alfalfa was replaced with rice straw or corn stover in the diet of lactating cows. Forty-five multiparous Holstein dairy cows were blocked based on days in milk (164 ± 24.8 d; mean ± standard deviation) and milk yield (29.7 ± 4.7 kg; mean ± standard deviation) and were randomly assigned to 1 of 3 treatments. Diets were isonitrogenous, with a forage-to-concentrate ratio of 45:55 [dry matter (DM) basis] and contained identical concentrate mixtures and 15% corn silage, with different forage sources (on a DM basis): 23% alfalfa hay and 7% Chinese wild rye hay (AH), 30% corn stover (CS), and 30% rice straw (RS). The experiment was conducted over a 14-wk period, with the first 2 wk for adaptation. The DM intake of the cows was not affected by forage source. Yield of milk, milk fat, protein, lactose, and total solids was higher in cows fed diets of AH than diets of RS or CS, with no difference between RS and CS. Contents of milk protein and total solids were higher in AH than in RS, with no difference between CS and AH or RS. Feed efficiency (milk yield/DM intake) was highest for cows fed AH, followed by RS and CS. Cows fed AH excreted more urinary purine derivatives, indicating that the microbial crude protein yield may be higher for the AH diet than for RS and CS, which may be attributed to the higher content of fermentable carbohydrates in AH than in RS and CS. Total-tract apparent digestibilities of all the nutrients were higher in cows fed the AH diet than those fed CS and RS. The concentration of rumen volatile fatty acids was higher in the AH diet than in CS or RS diets, with no difference between CS and RS diets. When the cereal straw was used to replace alfalfa as a main forage source for lactating cows, the shortage of fermented energy may have reduced the rumen microbial protein synthesis, resulting in lower milk protein yield, and lower nutrient digestibility may have restricted milk production.

First imaging result with an ultrahigh resolution stationary MR compatible SPECT system.

In this paper, we will present the design and preliminary performance of an ultrahigh resolution stationary MR compatible SPECT (MRC-SPECT) system that is developed in our lab. The MRC-SPECT system is based on the second-generation energy-resolved photon-counting (ERPC) CdTe detectors and there are several key features associated with this system. Firstly, up to a total of twenty ERPC detectors will be assembled as a very compact ring, which provides an adequate angular sampling capability and a relatively high detection efficiency. The detectors are supported on a gantry made of high strength polyamide structure constructed using 3-D printing. This compact system can be directly operated inside an MR scanner. The detector module used in this system offers an intrinsic resolution of 350µm and an excellent energy resolution of around 3~4kev. Each ERPC detector module consists of four pixelated CdTe detectors with a total dimension of 4.5cm×2.25cm. Secondly, a die-cast platinum pinhole inserts and cast lead apertures are developed for this stationary SPECT system. Four 300/500µm diameter pinholes are used for each detector and all pinholes are mounted around a casted cylinder lead aperture tube. The inner diameter of the lead aperture tube is 6cm and the lead tube thickness is 16mm. The opposite detectors are placed 15.6cm apart and the magnification factor of this SPECT system is about 1.2. Thirdly, a comprehensive charge collection model inside strong magnetic field has been developed to account for the magnetic field induced distortion in the SPECT image. This model can accurately predict the detector's energy and spatial response to gamma ray incident events and then help to compensate for the event position recording error due to the strong magnetic field. In this development, we have made an effort to minimize the amount of magnetic materials in the system to alleviate potential interference to magnetic field inhomogeneity.

Methods to improve electrochemical treatment effect of dye wastewater.

Several methods, including changing electrolyte concentration, temperature, stirring, and voltage were studied to improve the degradation effect of electrochemical treatment in dye wastewater. In addition, nanophase TiO2 catalyst and Co-Bi-PbO2/Ti anode have been prepared to expedite color removal. Enhancement of temperature leads to proportional increase of color removal. As for voltage, at low levels its increase could greatly improves color removal. After voltage reaches about 3.0 V, its improvement effect declines quickly. The influence of electrolyte concentration and aerating on color removal are similar to that of voltage. So it favored to degrade organic pollutants using high salt concentration, high voltages and large electric current to improve treatment effect. However, the efficiency of energy supplied during electrolysis decreases. A nonlinear model is established to evaluate the influence of electrolyte concentration and voltage on color removal. The model agrees with the experiment data very well. It is suggested by the simulation result of this model that electrolysis degradation should better be carried out at about 3.0 V, in 0.01 M Na2SO4 concentration for high energy efficiency. Additionally, either catalyst or Co-Bi-PbO2/Ti anode brings about 0.15 times more color removal without increasing electric current. Together, they could bring forth some 0.22 times higher color removal.

PEGylated recombinant human tumor necrosis factor alpha: preparation and anti-tumor potency.

AIM: To assess the merits of polyethylene glycol-modified recombinant human tumor necrosis factor alpha (PEG-rHuTNF-alpha). METHODS: The rHuTNF-alpha was modified with N-succinimidyl succinate monomethoxy polyethylene glycol (SS-PEG) of three different molecular weights. The PEG-rHuTNF-alpha was separated into fractions of various molecular weights by gel filtration chromatography. In vitro activities of various fractions were determined with L929 cell assay and in vivo anti-tumor potencies of main fractions were studied with respect to necrosis of S-180 solid tumor. RESULTS: The rHuTNF-alpha could be modified using SS-PEG under mild conditions. The main fraction of PEG5000-rHuTNF-alpha contained four PEG molecules, and PEG12000-rHuTNF-alpha and PEG20000-rHuTNF-alpha contained two PEG molecules, respectively. There was a higher activity when rHuTNF-alpha was coupled to less numbers of the same molecular weight PEG molecules. When PEG-rHuTNF-alpha was of the same molecular weight, rHuTNF-alpha modified with bigger molecular weight PEG molecules had a higher activity. PEG-rHuTNF-alpha was resistant to proteolysis, and over 70 % activity remained after 8 h, but the activity of rHuTNF-alpha was time-dependently diminished by incubation with bovine trypsin. PEG5000-rHuTNF-alpha (1500 IU per mouse) had a similar anti-tumor potency compared with rHuTNF-alpha (3000 IU per mouse). PEG12000-rHuT NF-alpha (1500 IU per mouse) had an increased anti-tumor potency compared with rHuTNF-alpha (3000 IU per mouse). In particular, PEG20000-rHuTNF-alpha at a dose of 1500 IU per mouse had a higher anti-tumor potency than rHuTNF-alpha at a dose of 6000 IU per mouse. CONCLUSION: PEG-modified rHuTNF-alpha could be more suitable for therapeutic use

Environmentally induced reversible conformational switching in the yeast cell adhesion protein alpha-agglutinin.

The yeast cell adhesion protein alpha-agglutinin is expressed on the surface of a free-living organism and is subjected to a variety of environmental conditions. Circular dichroism (CD) spectroscopy shows that the binding region of alpha-agglutinin has a beta-sheet-rich structure, with only approximately 2% alpha-helix under native conditions (15-40 degrees C at pH 5.5). This region is predicted to fold into three immunoglobulin-like domains, and models are consistent with the CD spectra as well as with peptide mapping and site-specific mutagenesis. However, secondary structure prediction algorithms show that segments comprising approximately 17% of the residues have high alpha-helical and low beta-sheet potential. Two model peptides of such segments had helical tendencies, and one of these peptides showed pH-dependent conformational switching. Similarly, CD spectroscopy of the binding region of alpha-agglutinin showed reversible conversion from beta-rich to mixed alpha/beta structure at elevated temperatures or when the pH was changed. The reversibility of these changes implied that there is a small energy difference between the all-beta and the alpha/beta states. Similar changes followed cleavage of peptide or disulfide bonds. Together, these observations imply that short sequences of high helical propensity are constrained to a beta-rich state by covalent and local charge interactions under native conditions, but form helices under non-native conditions.

Interaction of alpha-agglutinin and a-agglutinin, Saccharomyces cerevisiae sexual cell adhesion molecules.

alpha-Agglutinin and a-agglutinin are complementary cell adhesion glycoproteins active during mating in the yeast Saccharomyces cerevisiae. They bind with high affinity and high specificity: cells of opposite mating types are irreversibly bound by a few pairs of agglutinins. Equilibrium and surface plasmon resonance kinetic analyses showed that the purified binding region of alpha-agglutinin interacted similarly with purified a-agglutinin and with a-agglutinin expressed on cell surfaces. At 20 degrees C, the K(D) for the interaction was 2 x 10(-9) to 5 x 10(-9) M. This high affinity was a result of a very low dissociation rate ( approximately 2.6 x 10(-4) s(-1)) coupled with a low association rate (= 5 x 10(4) M(-1) s(-1)). Circular-dichroism spectroscopy showed that binding of the proteins was accompanied by measurable changes in secondary structure. Furthermore, when binding was assessed at 10 degrees C, the association kinetics were sigmoidal, with a very low initial rate. An induced-fit model of binding with substantial apposition of hydrophobic surfaces on the two ligands can explain the observed affinity, kinetics, and specificity and the conformational effects of the binding reaction.

Delineation of functional regions within the subunits of the Saccharomyces cerevisiae cell adhesion molecule a-agglutinin.

a-Agglutinin from Saccharomyces cerevisiae is a cell adhesion glycoprotein expressed on the surface of cells of a mating type and consists of an anchorage subunit Aga1p and a receptor binding subunit Aga2p. Cell wall attachment of Aga2p is mediated through two disulfide bonds to Aga1p (Cappellaro, C., Baldermann, C., Rachel, R., and Tanner, W. (1994) EMBO J. 13, 4737-4744). We report here that purified Aga2p was unstable and had low molar specific activity relative to its receptor alpha-agglutinin. Aga2p co-expressed with a 149-residue fragment of Aga1p formed a disulfide-linked complex with specific activity 43-fold higher than Aga2p expressed alone. Circular dichroism of the complex revealed a mixed alpha/beta structure, whereas Aga2p alone had no periodic secondary structure. A 30-residue Cys-rich Aga1p fragment was partially active in stabilization of Aga2p activity. Mutation of either or both Aga2p cysteine residues eliminated stabilization of Aga2p. Thus the roles of Aga1p include both cell wall anchorage and cysteine-dependent conformational restriction of the binding subunit Aga2p. Mutagenesis of AGA2 identified only C-terminal residues of Aga2p as being essential for binding activity. Aga2p residues 45-72 are similar to sequences in soybean Nod genes, and include residues implicated in interactions with both Aga1p (including Cys(68)) and alpha-agglutinin.

Structure of Saccharomyces cerevisiae alpha-agglutinin. Evidence for a yeast cell wall protein with multiple immunoglobulin-like domains with atypical disulfides.

alpha-Agglutinin of Saccharomyces cerevisiae is a cell wall-associated protein that mediates cell interaction in mating. Although the mature protein includes about 610 residues, the NH2-terminal half of the protein is sufficient for binding to its ligand a-agglutinin. alpha-Agglutinin20-351, a fully active fragment of the protein, has been purified and analyzed. Circular dichroism spectroscopy, together with sequence alignments, suggest that alpha-agglutinin20-351 consists of three immunoglobulin variable-like domains: domain I, residues 20-104; domain II, residues 105-199; and domain III, residues 200-326. Peptide sequencing data established the arrangement of the disulfide bonds in alpha-agglutinin20-351. Cys97 is disulfide-bonded to Cys114, forming an interdomain bond between domains I and II. Cys202 is bonded to Cys300, in an atypical intradomain disulfide bond between the A and F strands of domain III. Cys227 and Cys256 have free sulfhydryls. Sequencing also showed that at least two of three potential N-glycosylation sites with sequence Asn-Xaa-Thr are glycosylated. At least one of three Asn-Xaa-Ser sequences is not glycosylated. No residues NH2-terminal to Ser282 were O-glycosylated, whereas Ser282, and all hydroxy amino acid residues COOH-terminal to this position were modified. Therefore O-glycosylated Ser and Thr residues cluster in the COOH-terminal region of domain III, and the O-glycosylation continues into a Ser/Thr-rich sequence that extends from domain III to the COOH-terminal of the full-length protein.

A mannose-specific lectin from Vicia villosa seeds.

A lectin specific to mannose has been purified from Vicia villosa seed by (NH4)2SO4 fractionation, GalNAc-Sepharose and Man-Sepharose affinity chromatography. It was defined as VVLM, which showed a single band on an acidic-PAGE stained with Coomassie brilliant blue. The molecular weight of VVLM was 50 kDa as determined by gel filtration on Biogel P-100 column. The VVLM molecule consists of 2 distinct subunits with apparent molecular weight of 30 kDa and 22 kDa determined by SDS-PAGE. VVLM has at least four isolectins with similar haemagglutinating activity. Its extinction coefficient is calculated as A1(1cm) = 16.4 at 280 nm. Sugars could not be detected by phenol-sulfuric acid method. The circular dichroism analysis at far UV indicated that VVLM was a beta-sheet-rich protein, and gave no alpha-helix, 69% beta-sheet, 14% beta-turn by Provencher and Glockner method. The lectin was inhibited by alpha-methyl-D-mannose at 12.5 mM and glucose or GlcNAc at 50 mM. The carbohydrate binding specificity of VVLM was investigated by using affinity chromatography on a VVLM-Sepharose column. Among various Asn-linked oligosaccharides, core structure Man alpha 1-->3(Man alpha 1-->6)Man beta 1-->4GlcNAc beta 1-->4GlcNAcOT were found to have high affinity for VVLM-Sepharose. The antisera of VVLM did not produce precipitin line with VVLG in agar double diffusion plate indicating no serological relationship between VVLM and VVLG. However VVLM showed similar immunodeterminants of some other lectins of mannose specificity such as Con A, PSL, LCA and VFL.

Study of Al3+ binding and conformational properties of the alanine-substituted C-terminal domain of the NF-M protein and its relevance to Alzheimer's disease.

NF-M13 [H-(Lys-Ser-Pro-Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly)-OH], NF-M17 [H-(Glu-Glu-Lys-Gly-Lys-Ser-Pro-Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly) -OH], and their phosphorylated derivatives, representing the C-terminal phosphorylation domain of the neurofilament protein midsize subunit, have four possible binding sites for metal ions: the COO- group of glutamate, the OH group of the serine residue, the PO3H- group of phosphoserine (when present), and the COO- at the terminus of the peptide chain. The CD titration of the phosphorylated neurofilament fragments with Al3+ and Ca2+ yielded a significant conformational change that resulted in conformations containing high beta-pleated-sheet contents, which precipitate on standing (intermolecular complex). Al3+ binding to the unphosphorylated NF-M13 and NF-M17 did not exhibit this behavior. Several alanine analogues of the parent NF-M17 peptide were synthesized in order to determine the relationship between metal ions and possible binding sites. CD titration of analogues with Ca2+ indicated that the critical residues of NF-M17 for Ca(2+)-induced conformational changes, from random to beta-pleated sheet, are the N-terminal serine or both phosphorylated serines. Al(3+)-induced conformational changes suggest that the critical sites of NF-M17 yielding the beta-pleated-sheet structure are the four glutamates or phosphorylated serines, especially the C-terminal SerP. On the basis of the titration data, it is very likely that analogues with a serine in position 11 form a stable intramolecular complex with Al3+ that, however, does not result in the adoption of the beta-conformation. Back-titration with citric acid fails to reverse the Al(3+)-induced conformational changes of the phosphorylated peptides. The above results, especially the possible formation of intramolecular and intermolecular Al3+ complexes, may have relevance to the molecular mechanism, through which the neurotoxin Al3+ gives rise to the formation of neurofilament tangles.

Stable intrachain and interchain complexes of neurofilament peptides: a putative link between Al3+ and Alzheimer disease.

The etiologic role of Al3+ in Alzheimer disease has been controversial. Circular dichroism (CD) spectroscopic studies on two synthetic fragments of human neurofilament protein mid-sized subunit (NF-M), NF-M13 (KSPVPKSPVEEKG) and NF-M17 (EEKGKSPVPKSPVEEKG), and their alanine-substituted and/or serine-phosphorylated derivatives were carried out in an attempt to find a molecular mechanism for the effect of Al3+ to induce aggregation of neuronal proteins or their catabolic fragments. Al3+ and Ca2+ ions were found to induce beta-pleated sheet formation in the phosphorylated fragments. The cation sensitivity depended on the length and charge distribution of the sequence and site of phosphorylation. Al3+-induced conformational changes were irreversible to citric acid chelation, whereas Ca(2+)-induced conformational changes were reversible with citric acid. Studies of the alanine derivatives demonstrated which residues affected Al3+ or Ca2+ binding. Peptides containing at least one free (nonphosphorylated) serine residue were shown to form an intramolecular Al3+ complex, rather than an intermolecular one. In the intramolecular (intrachain) complex, the ligand function of the deprotonated serine hydroxyl was delineated [(Al.pepH-1)-type complex]. Ca2+ ions did not show a tendency for intramolecular complexing. The potential role of Al3+ in Alzheimer disease tangle and plaque formation is strongly suggested.

Circular dichroic analysis of denatured proteins: inclusion of denatured proteins in the reference set.

We hypothesize that inclusion of denatured proteins in the set of reference native proteins may better represent the unordered form in the current circular dichroism (CD) analyses of proteins involving unfolding ones. Adding three denatured-protein spectra and one oligopeptide spectrum to 16 reference protein spectra markedly improved the correlation coefficients (r) between CD calculations and X-ray determinations for the unordered form and, to a lesser extent, for beta-turn, but the r-values for alpha-helix and beta-sheet decreased slightly. With 20 reference proteins the estimates of the unordered form of denatured proteins were significantly improved. Thus, we suggest that as a compromise the new set of reference proteins be used for estimating the changes in conformation for unfolding proteins. However, the current use of 16 reference native proteins appears to be adequate for CD analysis of native proteins and the expansion to 20 reference proteins including denatured ones may not enhance the analysis of native proteins.

Conformation of the abortifacient protein pinellin: a circular dichroic study.

The conformation of pinellin was studied by circular dichroism, which showed a minimum at 223 nm and a double maximum at 198-200 nm. The protein was rich in beta-sheet (about 40%) with little alpha-helix, based on current CD analyses. It was stable between pH4 and 10 beyond which it unfolded reversibly, but in alkaline solution, prolongly stored at, say, pH 12, it became irreversibly denatured. Thermal denaturation indicated a transition between 55 degrees and 68 degrees C; the solution at 80 degrees C was partially renatured upon air-cooling back to room temperature. Addition of sodium dodecyl sulfate caused a sharp increase in alpha-helix, which leveled off at 0.25 mM surfactant.

Conformation and activity of Phaseolus coccineus var. rubronanus lectin.

The conformation of native and denatured Phaseolus coccineus var. rubronanus lectin was studied by circular dichroism (CD) and correlated to the hemagglutinating activity. The far-UV CD spectrum at 25 degrees C showed a broad, negative band around 223 nm and a positive one at 196 nm. CD data analysis of the lectin indicated a beta-sheet-rich protein. At high temperatures, the spectrum was blue-shifted with increasing magnitude; these changes correlated well with the loss of the activity. The conformation of lectin between pH 2 and 10 remained essentially unchanged. At pH 13 the CD spectrum resembled that of unordered form with a negative band near 200 nm and the activity was completely lost. The denatured lectin in 6 M guanidine hydrochloride would be renatured upon diluting the denaturant to 0.75 M; the changes in CD spectrum again correlated well with the loss of the activity. The effect of sodium dodecyl sulfate on the lectin was drastic; it sharply increased the alpha-helix at the expense of the beta-sheet and reduced the activity; the changes reached a plateau above 20 mM surfactant.

Conformation and activity of mannose- and N-acetylgalactosamine-specific lectins from Vicia villosa seeds.

The conformation of two Vicia villosa lectins specific for mannose and N-acetylgalactosamine, respectively, was studied by circular dichroism. Both showed a broad negative CD band around 220 nm and a positive one above 190 nm. CD data analysis indicated that they were rich in beta-sheet. However, they differed in conformational stability against extreme pH, at elevated temperature, and in guanidine hydrochloride and sodium dodecyl sulfate solutions. The unusual feature was that the conformation of N-acetylgalactosamine-specific lectin was virtually unaltered in 6 M guanidine hydrochloride and 7.5 mM surfactant.

Conformational stability of porcine serum transferrin.

The conformation of porcine serum ferric transferrin (Tf) and its stability against denaturation were studied by circular dichroism. Tf was estimated to have 19-24% alpha-helix and 50-55% beta-sheet based on the methods of Chang et al. (Chang, C.T., Wu, C.-S.C., & Yang, J.T., 1978, Anal. Biochem. 91, 13-31) and Provencher and Glöckner (Provencher, S.W. & Glöckner, J., 1981, Biochemistry 20, 33-37). Removal of the bound ferric ions (apo-Tf) did not alter the overall conformation, but there were subtle changes in local conformation based on its near-UV CD spectrum. The Tfs were stable between pH 3.5 and 11. Denaturation by guanidine hydrochloride (Gu-HCl) showed two transitions at 1.6 and 3.4 M denaturant. The process of denaturation by acid and base was reversible, whereas that by Gu-HCl was partially reversible. The irreversible thermal unfolding of Tfs began at temperatures above 60 degrees C and was not complete even at 80 degrees C. The bound irons (based on absorbance at 460 nm) were completely released at pH < 4 or in Gu-HCl solution above 1.7 M, when the protein began to unfold, but they remained intact in neutral solution even at 85 degrees C. The NH2- and COOH-terminal halves of the Tf molecule obtained by limited trypsin digestion had CD spectra similar to the spectrum of native Tf, and the COOH-terminal fragment had more stable secondary structure than the NH2-terminal fragment.