[Comparison of extracorporeal liver assist devices - albumin dialysis versus plasma exchange - in acute-on-chronic liver failure]. / Vergleich extrakorporaler Leberunterstützungsverfahren - Albumin-Dialyse versus Plasmaaustausch - bei akut-auf-chronischem Leberversagen.

BACKGROUND AND AIM: Extracorporeal liver assist devices are besides causal and symptomatic approaches important therapeutic options in acute-on-chronic (AOC) liver failure. In this retrospective analysis, albumin dialysis was compared to therapeutic plasma exchange (TPA) under various aspects. PATIENTS AND METHODS: Data from 20 patients per group (10 women, 10 men in each group, mean age 51 ±â€…12,6 years and 48,2 ±â€…15,2 years, respectively) treated over a period of 3 months were analyzed. During the first treatment, 5 sessions of dialysis were performed (week 1) for both procedures, 3 more sessions were completed in the second and in the third week each. Data were acquired on days 1, 8, 13, 20, 28 and 90. RESULTS: After 28 days, 13 out of 20 patients following albumin dialysis and 7 out of 20 patients following plasma exchange had survived (p = 0,11). After 90 days, 10 patients following albumin dialysis and 5 patients following plasma exchange were alive (p = 0,19). Degree of hepatic encephalopathy (HE) had not improved significantly. Rates of complication (infections, bleeding or system clotting) were similar under both procedures. CONCLUSION: Extracorporeal liver assist devices can be considered equally well as a therapeutic option in acute-on-chronic liver failure. Differences in 90-day survival were not observed in our study.

Low penetrance susceptibility to glioma is caused by the TP53 variant rs78378222.

BACKGROUND: Most of the heritable risk of glioma is presently unaccounted for by mutations in known genes. In addition to rare inactivating germline mutations in TP53 causing glioma in the context of the Li-Fraumeni syndrome, polymorphic variation in TP53 may also contribute to the risk of developing glioma. METHODS: To comprehensively evaluate the impact of variation in TP53 on risk, we analysed 23 tagSNPs and imputed 2377 unobserved genotypes in four series totaling 4147 glioma cases and 7435 controls. RESULTS: The strongest validated association signal was shown by the imputed single-nucleotide polymorphism (SNP) rs78378222 (P=6.86 × 10(-24), minor allele frequency ~0.013). Confirmatory genotyping confirmed the high quality of the imputation. The association between rs78378222 and risk was seen for both glioblastoma multiforme (GBM) and non-GBM tumours. We comprehensively examined the relationship between rs78378222 and overall survival in two of the case series totaling 1699 individuals. Despite employing statistical tests sensitive to the detection of differences in early survival, no association was shown. CONCLUSION: Our data provided strong validation of rs78378222 as a risk factor for glioma but do not support the tenet that the polymorphism being a clinically useful prognostic marker. Acquired TP53 inactivation is a common feature of glioma. As rs78378222 changes the polyadenylation signal of TP53 leading to impaired 3'-end processing of TP53 mRNA, the SNP has strong plausibility for being directly functional contributing to the aetiological basis of glioma.

[The Heinz Nixdorf Recall study]. / Die Heinz Nixdorf Recall Studie.

The Heinz Nixdorf Recall Study is a population-based study that aims to improve the prediction of cardiovascular events by integrating new imaging and non-imaging modalities in risk assessment. One focus of the study is the evaluation of the quantification of subclinical coronary artery calcifications (coronary artery calcification, CAC) as a prognostic factor in predicting cardiac events. Primary endpoints are myocardial infarction and sudden cardiac death. The study was initiated in the late 1990s and enrolled a total of 4,814 participants aged 45-75 years between December 2000 and August 2003. A 5-year follow-up examination took place between 2006 and 2008. Currently, the 10-year follow-up is under way and is estimated to be finished in July 2013. Extending the original aims of the study, serial CAC measurements will allow the characterization of the natural history of CAC dynamics, the identification of its determinants and an understanding of the impact of CAC progression on the primary endpoints. The Heinz Nixdorf Recall Study will significantly extend our knowledge about new modalities in the prediction of cardiac events.

Percentage of smudge cells determined on routine blood smears is a novel prognostic factor in chronic lymphocytic leukemia.

Recently developed molecular prognostic tests in patients with early Binet stage chronic lymphocytic leukemia (B-CLL) are costly and often require a high level of technologic expertise. Recent data give evidence for the prognostic relevance of the percentage of smudge cells in B-CLL. In our study we analysed the prognostic potential of this novel marker in a cohort of 100 CLL patients. The percentage of smudge cells ranged from 0% to 70% (median 21%). Patients with 20% smudge cells. Multivariate Cox regression analysis identified percentages of smudge cells, stage according to Binet and CD38 expression as independent prognostic markers. The percentage of smudge cells was significantly lower in CD38+, ZAP-70+ and unmutated IgVH patients. Combined analysis of smudge cell percentages with CD38 expression provided complementary prognostic information identifying three patient subgroups with good, intermediate and poor prognosis. Comparing gene expression profiles in a subset of 12 patients we identified eight differentially expressed genes in groups with high vs. low percentage of smudge cells suggesting a role of these differentially expressed genes, especially for Tribbles homolog 2 (Trib2), in the disease progression of high risk CLL patients. In conclusion, our data confirm previous studies showing that the simple and inexpensive microscopic detection of smudge cells on blood smears prepared for routine diagnostic purposes is a novel independent factor predicting overall survival in CLL.

Differential expression of drug-resistance-related genes between sensitive and resistant blasts in acute myeloid leukemia.

Drug resistance constitutes a considerable problem in the therapy of acute myeloid leukemia (AML). In order to identify genes which might be related to drug resistance, we retrospectively studied gene expression patterns in blast populations of 14 patients with de novo AML, focusing on known or potential resistance mechanisms against cytosine arabinoside and anthracyclines. Following induction and postremission chemotherapy, 7 patients achieved a complete remission (CR) for more than 1 year, while 7 patients showed blast persistence (BP) after induction and salvage chemotherapy. Gene expression analysis was performed using RNA extracted from archived guanidine extracts and Affymetrix HGU133A gene chips. We utilized the Gene Ontology category Biological Process to select genes implicated in DNA metabolism, nucleoside and nucleotide metabolism and transport, reactive oxygen species metabolism, apoptosis and response to drugs and identified 32 differentially expressed genes. From this functional perspective, we found differences between the CR and BP groups with regard to nucleotide metabolism (PBEF1, G6PD; p = 0.048), apoptosis (TNFAIP3, TNFAIP8, MPO, BCL2A1, BAX, SON, BNIP3L; p = 0.039) and reactive oxygen species metabolism (SOD2, KIAA0179; p = 0.048). However, the attempt to construct a predictive model of chemoresistance failed. BP samples had a 2-fold higher expression of CD34 than CR samples. Thus, our findings are in line with reports describing differences in apoptosis resistance between CD34+ and CD34- blast populations. Taken together, our results suggest that drug resistance in AML is a heterogenous phenomenon that might be better defined by means of disturbed biological processes than by focusing on the alteration of the expression of distinct genes.

Anticancer versus antigrowth activities of three analogs of the growth-inhibitory peptide: relevance to physicochemical properties.

A 34-amino acid peptide has been synthesized based on an amino acid sequence from the third domain of native full-length alpha-fetoprotein, which has been shown to have both antigrowth and anticancer activities. This peptide, known as the growth-inhibitory peptide (GIP), has two cysteine residues and demonstrates reduced antigrowth activity after long-term storage, presumably due to disulfide bond formation. The disulfide bridge problem was addressed by replacing the two naturally occurring cysteines with either glycines, alanines, or serines (to produce the G-, A- and S-peptides, respectively). The non-hydrophobic G- and S-peptides were found to exist as dimers, while the more hydrophobic C- and A-peptides formed trimers in solution under certain conditions of pH and peptide concentration. The A-peptide was already known to display anticancer activity; however, the G- and S-serine analogs have not been studied in depth since they had demonstrated low antigrowth activities in rodent uterine assays. Using both in vivo and in vitro assays, the A-, G- and S-peptides were shown to exhibit various degrees of cancer growth suppression. An in vitro culture assay, using MCF-7 breast cancer cells, demonstrated that both the G- and S-peptides showed modest cancer growth suppression, while the A- analog showed strong inhibition at doses ranging from 10(-5) M to 10(-7) M. In contrast, an in vivo ascites tumor study of all four peptides showed them to have notable activity in the suppression of mouse mammary tumor growth. Overall, our data indicated that physicochemical properties, such as hydrophobicity, oligomeric state and secondary structure, contribute to the anticancer activity of both the active C- peptide and its analogs. In addition, the antigrowth rodent uterine assay was not always predictive of the anticancer potential of the peptide forms, suggesting a difference between the mechanism of peptide action in the antigrowth models and that in the anticancer assay. Notably, the antigrowth assay failed to predict the marked anticancer activity of the analogs against a mammary tumor, indicating that the growth bioassay cannot always be relied upon as a screening protocol.

Biophysical studies and anti-growth activities of a peptide, a certain analog and a fragment peptide derived from alpha-fetoprotein.

A chemically synthesized 34-amino acid peptide, an analog, and a fragment of the peptide have been purified and studied. Biophysical studies were carried out to determine some of the metal ion binding properties of the original peptide and an analog of this parent peptide, in which the two histidine residues were replaced by alanines. As shown by visible absorption spectroscopy, Co (II) forms a complex with the parent peptide, but not with the analog peptide, and one or two histidines in the parent peptide are ligands for Co (II) ion binding. The effects on disulfide bond formation in the peptide by Zn (II) and Co (II) ions were also examined for this analog. Anti-growth assays were performed using the original cysteine-containing peptide with Zn (II) ion complexed to the peptide through the two cysteine residues. These rat uterine growth assays showed that the complexing of Zn (II) ion to the peptide maintained the anti-growth activity of the peptide, while gel-filtration experiments showed the zinc ions maintained the peptide in its anti-growth form indefinitely in solution. A saliently important part of this research was the discovery that a fragment of the peptide consisting of a middle sequence of 14 amino acids was found to have significant anti-growth activity in the rat uterine assay. Its activity suggested that this fragment might be considered a viable candidate for testing in anti-cancer protocols.

Interrelationships among biological activity, disulfide bonds, secondary structure, and metal ion binding for a chemically synthesized 34-amino-acid peptide derived from alpha-fetoprotein.

A 34-amino-acid peptide has been chemically synthesized based on a sequence from human alpha-fetoprotein. The purified peptide is active in anti-growth assays when freshly prepared in pH 7.4 buffer at 0.20 g/l, but this peptide slowly becomes inactive. This functional change is proven by mass spectrometry to be triggered by the formation of an intrapeptide disulfide bond between the two cysteine residues on the peptide. Interpeptide cross-linking does not occur. The active and inactive forms of the peptide have almost identical secondary structures as shown by circular dichroism (CD). Zinc ions bind to the active peptide and completely prevents formation of the inactive form. Cobalt(II) ions also bind to the peptide, and the UV-Vis absorption spectrum of the cobalt-peptide complex shows that: (1) a near-UV sulfur-to-metal-ion charge-transfer band had a molar extinction coefficient consistent with two thiolate bonds to Co(II); (2) the lowest-energy visible d-d transition maximum at 659 nm, also, demonstrated that the two cysteine residues are ligands for the metal ion; (3) the d-d molar extinction coefficient showed that the metal ion-ligand complex was in a distorted tetrahedral symmetry. The peptide has two cysteines, and it is speculated that the other two metal ion ligands might be the two histidines. The Zn(II)- and Co(II)-peptide complexes had similar peptide conformations as indicated by their ultraviolet CD spectra, which differed very slightly from that of the free peptide. Surprisingly, the cobalt ions acted in the reverse of the zinc ions in that, instead of stabilizing anti-growth form of the peptide, they catalyzed its loss. Metal ion control of peptide function is a saliently interesting concept. Calcium ions, in the conditions studied, apparently do not bind to the peptide. Trifluoroethanol and temperature (60 degrees C) affected the secondary structure of the peptide, and the peptide was found capable of assuming various conformations in solution. This conformational flexibility may possibly be related to the biological activity of the peptide.

Studies on analogs of a peptide derived from alpha-fetoprotein having antigrowth properties.

A 34-amino acid portion of the third domain of alpha-fetoprotein possesses antigrowth and anticancer activities. Three analogs of this sequence were chemically synthesized, in which the two cysteines of the original sequence were replaced by alanines, glycines or serines. The original cysteine and alanine peptides formed trimers at 0.20 g/L in pH 7.4 phosphate buffer, and the glycine and serine peptides formed dimers. Trimer preparations were more potent in inhibiting estrogen-induced growth in the mouse uterine assays than the two dimeric oligomers. Of salient importance is that the alanine peptide retained its trimeric form in solution much longer than the cysteine peptide. Antigrowth assays were performed starting with stock solutions at a peptide concentration of 0.20 g/L, because at very high peptide concentration (8.0 g/L) the peptides aggregated extensively. All the peptides, although differing in biological activity, had almost identical secondary structures. Unlike alpha-fetoprotein, the three peptides have low amounts of alpha-helix. Trifluoroethanol has the ability to convert peptides into a helical conformation when they have a propensity for that structure. At trifluoroethanol concentrations of 20% and higher, the alanine and glycine peptides were changed into highly helical structures.

Studies on a growth-inhibitory peptide derived from alpha-fetoprotein and some analogs.

A 34-amino acid synthetic peptide was derived from the third domain of human alpha-fetoprotein, and the peptide was shown to inhibit estrogen-stimulated growth. Under certain conditions, however, the peptide lost growth-inhibitory activity. A biophysical study of the peptide was undertaken with a goal of obtaining completely reliable preparations. The peptide was studied using gel-filtration column chromatography as a function of peptide concentration and age of solution, and was found to exhibit complex aggregation behaviors. During the early period (0-3 h) after dissolving lyophilized peptide into pH 7.4 buffer, solutions were composed mostly of trimers. At higher peptide concentrations (> or = 3.0 g/L), the trimers aggregated extensively to a large aggregate (minimum size approximately 102 peptides). At 5.0-8.0 g/L, these large aggregates increased in size (up to approximately 146 peptides) until trimers were largely exhausted from solution. During the later times (>3 h) after sample preparation, the trimeric oligomer of the peptide dissociated slowly to form dimers for samples at 0.10-3.0 g/L. After their build-up, a very small number of dimers associated to form hexamers. Disulfide bonds stabilized the dimers as indicated by the conversion of dimers to trimers upon the addition of a reducing agent, and the failure of dimers to form in the presence of reducing agent. Reducing agent did not affect trimer or large aggregate formation. Trimers were found to be active in an assay monitoring inhibition of estrogen-stimulated growth, whereas dimers and large aggregates were inactive. The two cysteines in the peptide were modified to either S-methylcysteine or S-(2-aminoethyl)cysteine, and both derivatives showed significant growth-inhibition activity. A serine analog in which both cysteines were replaced had very different aggregation behavior than the cysteine peptide and lacked its growth inhibitory ability. Peptide aggregation is critically important in establishing the ability of the peptide to inhibit growth and have anticancer activity, but the state of its two cysteines is of little influence.

Studies on C-phycocyanin from Cyanidium caldarium, a eukaryote at the extremes of habitat.

C-Phycocyanin, a biliprotein, was purified from the red alga, Cyanidium caldarium. This alga grows at temperatures up to 57 degrees C, a very high temperature for a eukaryote, and at pH values down to 0.05. Using the chromophores on C-phycocyanin as naturally occurring reporter groups, the effects of temperature on the stability of the protein were studied by circular dichroism and absorption spectroscopy. The protein was unchanged from 10 to 50 degrees C, which indicates that higher temperatures are not required to cause the protein to be photosynthetically active. At 60 and 65 degrees C, which are above the temperatures at which the alga can survive, the protein undergoes irreversible denaturation. Gel-filtration column chromatography demonstrated that the irreversibility is caused by the dissociation of the trimeric protein to its constitutive polypeptides. Upon cooling, the alpha and beta polypeptides did not reassemble to the trimer. Unlike phycocyanins 645 and 612, the C-phycocyanin does not show a reversible conformational change at moderately high temperatures. At constant temperature, the C-phycocyanin was more stable than a mesophilic counterpart. It is designated a temperature-resistant protein.

Fluorescence polarization studies on four biliproteins and a bilin model for phycoerythrin 545.

Fluorescence (excitation) polarization spectroscopy in the wavelength region of the bilin chromophores was applied to phycoerythrocyanin (CV-phycocyanin), phycocyanins 645 and 612, and phycoerythrin 545. The cryptomonad biliproteins - phycoerythrin 545 and phycocyanins 612 and 645 - were studied as both protein dimers having an alpha(2)beta(2) polypeptide structure and as alphabeta monomers. The cyanobacterial phycoerythrocyanin (CV-phycocyanin) was a trimeric oligomer. The changes in polarization across the spectrum were attributed to transfers of energy between bilins. Cryptomonad biliproteins are isolated as dimers. The similarities between their steady-state fluorescence polarization spectra and those of the corresponding monomers suggested that the monomers' conformations were analogous to the dimers. This supports the use of monomers in the study of dimer bilin organization. The unusual polarization spectrum of phycoerythrin 545 was explained using a model for the topography of its bilins. Obtaining the emission spectra of phycoerythrin 545 at several temperatures and a deconvolution of the dimer circular dichroism spectrum also successfully tested the bilin model. Circular dichroism spectroscopy was used to determine which polarization changes are formed by Förster resonance energy transfers and which may be produced by internal conversions between high- and low-energy states of pairs of exciton-coupled bilins. Attempts were made to assign energy transfer events to the corresponding changes in fluorescence polarization for each of the four biliproteins.

Bilin organization in cryptomonad biliproteins.

The bilin organization of three cryptomonad biliproteins (phycocyanins 612 and 645 and phycoerythrin 545) was examined in detail. Two others (phycocyanin 630 and phycoerythrin 566) were studied less extensively. Phycocyanin 645 and phycoerythrin 545 were suggested to have one bilin in each monomeric (alphabeta) unit of the dimer (alpha2beta2) isolated from the others, and the remaining six bilins may be in pairs. One pair was found across the monomer-monomer interface of the protein dimer, and two identical pairs were proposed to be within the monomer protein units. For phycocyanin 612, a major surprise was that a pair of bilins was apparently not found across the monomer-monomer interface, but the remaining bilins were distributed as in the other two cryptomonad proteins. The effect of temperature on the CD spectra of phycocyainin 612 demonstrated that two of the bands (one positive and one negative) behaved identically, which is required if they are coupled. The two lowest-energy CD bands of phycocyanin 612 originated from paired bilins, and the two higher-energy bands were from more isolated bilins. The paired bilins within the protein monomers contained the lowest-energy transition for these biliproteins. Using the bilins as naturally occurring reporter groups, phycocyanin 612 was shown to undergo a reversible change in tertiary structure at 40 degrees C. Protein monomers were shown to be functioning biliproteins. A hypothesis is that the coupled pair of bilins within the monomeric units offers important advantages for efficient energy migration, and other bilins transfer energy to this pair, extending the wavelength range or efficiency of light absorption.

Phycoerythrin 545: monomers, energy migration, bilin topography, and monomer/dimer equilibrium.

Phycoerythrin 545 was isolated having an alpha2beta2 (dimer) protein structure at pH 6.0 and 2 g/L protein concentration with eight bilin chromophores. Monomers (alphabeta) were produced by lowering the protein concentration to 0.15 g/L and the pH to 4.5. Dimer dissociation was monitored by dynamic light scattering and gel-filtration column chromatography. Monomers were stable and had bilin optical spectra different from the alpha2beta2 dimers, although they have very similar protein secondary structures. The optical spectra of phycoerythrin 545 showed four types of behavior with temperature: 10-20 degrees C, dimers; 40-50 degrees C, dimers/monomers; 60 degrees C, nearly fully disordered; 70 degrees C, disordered alpha and beta polypeptides. At 40 degrees C, the protein dissociated partially to monomer, which could be totally reversed to dimers at 20-25 degrees C. The visible circular dichroism difference spectrum for the protein dimers minus monomers exhibited positive and negative bands--such spectra may indicate exciton splitting between closely-spaced bilins. Circular dichroism also revealed a spectrum suggesting exciton coupling for the second excited state of the bilins. Ultrafast fluorescence using a two-photon method showed the fastest time for protein dimers to be 2. 4 ps and monomers had a 39-ps lifetime. Phycocyanin 645 was found to have a 550-fs lifetime.

Bilin chromophores as reporters of unique protein conformations of phycocyanin 645.

At 45 degrees C, phycocyanin 645 maximally undergoes a reversible and stable conformational change. The change is observed in the visible (chromophore) region of the absorption and circular dichroism (CD) spectra. In the absorption spectrum, the absorbance is lower at 45 degrees C but remains much closer to the normal spectrum than to a strongly denatured spectrum. In the CD, a similar situation exists except that a negative band on the blue edge of the spectrum is much more strongly affected at 45 degrees C than the other bands. On returning to 20 degrees C, all these changes are restored to the original states. The protein is an alpha 2 beta 2 dimer at both 20 and 45 degrees C, and CD in the far-UV shows the identical protein secondary structures at both 20 and 45 degrees C. Fluorescence studies show that energy transfer occurs at both temperatures. At 50 degrees C the results are saliently different as the secondary structure changes and the spectral changes are mostly irreversible. At 50 degrees C, some monomers (alpha beta) are produced, and these monomers are very unstable at that temperature, resulting in the formation of some fully denatured polypeptides. Stable monomers can be produced at 20 degrees C and have visible absorption and CD spectra identical to the dimer at 45 degrees C. Therefore, the chromophores are reporting a tertiary conformational change at 45 degrees C, in which the two halves of the dimer each assume a monomer-like conformation prior to dissociating. These results are compared with a hypothesis for the chromophore topography, and the CD change at the blue edge of the spectra may result from the separation at 45 degrees C of a chromophore pair.

R-phycoerythrins having two conformations for the same aggregate.

The visible circular dichroism (CD) spectrum of an R-phycoerythrin (Porphyra tenera) is composed of several positive bands. The protein in aqueous buffer very slowly exhibits changes in the CD spectrum of its chromophores, a band at 489 nm undergoes an increase in intensity and a red shift. When the band reached a 493 nm maximum, the spectrum became very stable. The aggregation state of the protein did not change during this spectral conversion. The chromophore CD spectrum was also obtained in the presence of a low concentration of urea or sodium thiocyanate, and the identical change in the CD was noted, but the change was much faster. The visible absorption and CD in the far UV spectra were unaffected by urea. Unchanged visible absorption and protein secondary structure (61% alpha helix) contradicted by comparatively salient alterations in the visible CD spectra suggested very subtle structural changes are influencing some of the chromophores. For a second R-phycoerythrin (Gastroclonium coulteri), the CD of the chromophores had a negative band on the blue edge of the spectrum. This is the first negative CD band observed for any R-phycoerythrin. Treatment of this protein with low concentrations of urea produced a change in the visible CD with the negative band being completely converted to a positive band. Fluorescence studies showed that the treatment by urea did not affect energy migration. Deconvolution of the CD spectra were used to monitor the chromophores. The results demonstrated that the same aggregate of each R-phycoerythrin could exist in two conformations, and this is a novel finding for any red algal or cyanobacterial biliprotein. The two forms of each protein would differ in tertiary structure, but retain the same secondary structures.

The discovery of a novel R-phycoerythrin from an antarctic red alga.

A novel biliprotein, named R-phycoerythrin IV, has been discovered. It absorbs blue light better than any other known red algal biliprotein. The protein was found in Phyllophora antarctica, a benthic macroalga, which grows beneath the coastal waters of McMurdo Sound, Antarctica. Fluorescence emission and fluorescence excitation polarization spectroscopy demonstrated that R-phycoerythrin IV behaved as a typical R-phycoerythrin in the functioning of energy migration and has an emission maximum at 577 nm. The circular dichroism (CD) spectrum of the chromophores was compared with visible absorption spectrum, and both were deconvoluted. This process showed the energy states of various individual chromophores. The molecular weight of the protein suggested a alpha6beta6gamma polypeptide structure, and far UV CD studies revealed polypeptides with highly alpha-helical secondary structures. Dynamic light scattering indicated that the protein had a 5.54 nm radius, and its shape was nonspherical. R-phycoerythrin was also purified from a second benthic Antarctic red alga, Iridaea cordata. Its spectroscopic properties were similar to those of some R-phycoerythrins from nonpolar regions. The unique spectroscopic properties of R-phycocerythrin IV may help enable the alga to occupy its niche deeper in the water column than the red alga that has the typical R-phycoerythrin.

Studies on the dissociation of cryptomonad biliproteins.

The spectroscopic properties of two biliproteins, phycocyanin 645 and phycoerythrin 566, have been studied by treating the proteins with two different agents, NaSCN at pH 6.0, or pH 4.0 without NaSCN. For phycoerythrin 566, treatment with NaSCN revealed that the visible CD spectrum of its chromophores was separated into a pair of different spectra, and each of these spectra was observed as a negative and one or more positive bands. For phycocyanin 645, two negative CD bands have been observed previously, together with two or more positive bands, in the dimer (alpha 2 beta 2) state, and NaSCN treatment caused elimination of one of these negative bands. The dimer was stable at pH 6.0, but at pH 4.0 the spectra of phycocyanin 645 had one less negative band than those at pH 6.0. Chromatography demonstrated that phycocyanin 645 was a monomer (alpha beta) at pH 4.0. Monomers of cryptomonad biliproteins have never been observed before. Excitation at 514 nm, in picosecond time-resolved fluorescence studies, produced lifetimes of 11.0 and 45.2 ps for dimers and monomers, respectively. Excitation at 566 nm yielded times of 1.38 and 1.24 ps, for dimers and monomers, respectively. CD in the far UV showed that monomers and dimers had very similar secondary structures. These results have been used to test an hypothesis that proposed two types of exciton splitting among the chromophores of phycocyanin 645, and perhaps phycoerythrin 566 could also have this chromophore organization.

Chromophore topography and exciton splitting in phycocyanin 645.

The biliprotein phycocyanin 645 has been purified from a photosynthetic cryptomonad Chroomonas species. It is composed of two copies each of two polypeptides (alpha and beta); each alpha polypeptide has one chromophore, and each beta polypeptide has three. There are one cryptoviolin and two phycocyanobilins on each beta polypeptide and one 697-nm bilin on each alpha polypeptide for a total of eight chromophores on the protein. Circular dichroism (CD) spectroscopy has been used to investigate the arrangement of these chromophores. Comparisons among the intact protein (alpha 2 beta 2) and various urea-treated products have yielded a tentative model for chromophore topography. The six chromophores on the two beta polypeptides are segregated into three pairs. The chromophores of each pair are close enough to experience electronic interactions. One pair, consisting of the two cryptoviolins, produces exciton splitting on the blue edge of the visible CD spectrum, and the two pairs of phycocyanobilins cause exciton splitting on the red edge of this spectrum. Deconvolution shows that the CD spectrum of each pair has a positive and a negative band, which are nearly conservative as expected for exciton coupling. The two chromophores on the alpha polypeptides are more isolated. The pairing of cryptoviolin chromophores occurs across two beta polypeptides, but the more likely position of each of the two pairs of phycocyanobilins is probably not across a beta-beta interface but within a single beta polypeptide. The exciton splitting events both increase the range of visible light absorption for the protein and establish the routes of exciton migration through the protein.

The binding domain structure of retinoblastoma-binding proteins.

The retinoblastoma gene product (Rb), a cellular growth suppressor, complexes with viral and cellular proteins that contain a specific binding domain incorporating three invariant residues: Leu-X-Cys-X-Glu, where X denotes a nonconserved residue. Hydrophobic and electrostatic properties are strongly conserved in this segment even though the nonconserved amino acids vary considerably from one Rb-binding protein to another. In this report, we present a diagnostic computer pattern for a high-affinity Rb-binding domain featuring the three conserved residues as well as the conserved physico-chemical properties. Although the pattern encompasses only 10 residues (with only 4 of these explicitly defined), it exhibits 100% sensitivity and 99.95% specificity in database searches. This implies that a certain pattern of structural and physico-chemical properties encoded by this short sequence is sufficient to govern specific Rb binding. We also present evidence that the secondary structural conformation through this region is important for effective Rb binding.