A selection system to study C5a–C5a-receptor interactions: Phage display of a novel C5a anaphylatoxin,Fos-C5aAla27

Binding and effector domains of the human anaphylatoxin C5a have been determined by either site directed mutagenesis or synthetic peptide studies. However, the lack of specific selection methods, which allow direct investigation of C5a–C5a-receptor interaction made these studies laborious. To overcome these limitations we have constructed a novel Fos-C5a expressed on the tip of a filamentous phage. To guarantee for a free C-terminus which is required for C5a activity C5a cDNA was cloned into the phagemid vector pJuFo. Helper phage infection of pJuFo-C5a transformed cells resulted in a mutant phage displaying Fos-C5a on its surface. However studies with Bt₂cAMP differentiated U937 cells revealed that phage displayed Fos-C5a is functional inactive. Subsequently we replaced a nonconserved cysteine residue at position 27 by alanine and obtained Fos-C5a^Ala27. Both the purified and the phage displayed Fos-C5a^Ala27 proteins were functional active and induced enzyme release from differentiated U937 cells. In addition, purified Fos-C5a^Ala27 exhibited the same binding profile as compared to rhC5a. Fos-C5a^Ala27 displaying phages were mixed with phage harboring only the pJuFo plasmid at a ratio of 10⁶. After four successive rounds of panning on differentiated U937 cells Fos-C5a^Ala27 phages were enriched to 100% as shown by C5a-specific ELISA. We expect this approach to prove helpful for studying C5a–C5a-receptor interactions, i.e. to screen C5a libraries for high affinity binders with agonistic or antagonistic properties directly on cells.     

Synthesis of c-5′-nor-Dideoxycarbanucleosides Structurally Related to Neplanocin C

Abstract

Purine carbanucleosides built on a 6-oxabicyclo[3.1.0]hexane template were synthesized from readily available 2-cyclopentenone employing a Mitsunobu reaction to incorporate the base onto the carbocyclic ring. Both adenosine and guanosine analogues exhibited moderate antiviral activity.     

Perfusion of rabbit kidneys with glycerol solutions at 5 °C

The long-term preservation of whole organs will almost certainly require the use of subzero temperatures and cryoprotectants. An essential part of such a technique is the ability to add a cryoprotectant in adequate concentration and subsequently to remove it without damage to the organ. In this study rabbit kidneys have been perfused with solutions containing 3% dextran and 2 m glycerol at 5 °C, and their function has been measured after removal of the glycerol. The assay technique involved the measurement of glomerular filtration rate, protein leakage, and tubular reabsorption of sodium and glucose. The results indicate that the inclusion in the perfusate of an impermeant solute (mannitol) and limitation of the rate of change of glycerol concentration (to 30 mm min^?1) permits rabbit kidneys to retain a degree of function similar to that found in perfused control kidneys, although somewhat reduced in comparison with freshly isolated kidneys.     

Variability of physiological parameters of unacclimatized males during a two—hour cold stress of 5°C

The metabolic and temperature responses of 11 male Caucasians to a 2-hr exposure to 5 ± 1°C, 70 ± 2% RH were compared with control data obtained in an ambient environment of 28 ± 1°C, 45 ± 2% RH. The heat production increased during the cold exposure attaining an approximately stable level during the final 30 min. The group variability in response to the cold was greatest during the first 30 min and declined for the remainder of the cold exposure. All skin temperatures approached a stable value during the final 30 min of cold exposure. The correlation between mean skin temperature and thigh temperature was significant (p < 0.001) and the use of thigh temperature as an approximate mean skin temperature was suggested. The calculation of tissue conductance with or without the inclusion of heat exchanges due to changes in body heat content and respiratory losses was in agreement only during the final 30 min of cold exposure, thus indicating a stage of physiological equilibrium. All measured parameters except the toe and finger temperatures approached minimum variability of response during the final 30 min of cold exposure.

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Zusammenfassung Die Reaktionen des Stoffwechsels und der Hauttemperatur von 11 männlichen Angehörigen der weissen Rasse während einer 2-stündigen Exponierung bei 5 ± 1°C, 50–70 % RF wurden mit den Kontrollwerten bei 28 ± 1°C und 45 ± 2% RF verglichen. Während der Kälteexponierung stieg die Wärmebildung an und erreichte wie die Hauttemperatur in den letzten 30 Min ein ungefähr konstantes Niveau. Die Gruppenvariabilität war in den ersten 30 Min am grössten und liess dann nach. Die Korrelation zwischen mittlerer Hauttemperatur und Oberschenkeltemperatur war hochsignifikant (p < 0,001). Es wird vorgeschlagen letztere als mittlere Hauttemperatur zu verwenden. Die Berechnung der Gewebeleitfähigkeit mit oder ohne Einbeziehung des Wärmeaustausches als Folge von Änderungen des Wärmegehaltes des Körpers und Wärmeverlustes bei der Atmung stimmte nur während der letzten 30 Min. Alle gemessenen Parameter ausser der Zehen- und Fingertemperatur näherten sich während der letzten 30 Min der Kälteexponierung einer minimalen Variabilität. Dies weist auf ein physiologisches Gleichgewicht hin.

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Resume On a comparé le métabolisme et la température cutanée de 11 personnes de la race blanche caucasienne exposées durant 2 heures à une température de 5 ± 1°C et à une humidité relative de 70 ± 2% aux mêmes valeurs obtenues par 28 ± 1°C et 45 ± 2%. La production de chaleur a augmenté durant l'exposition au froid pour atteindre un niveau relativement stable durant les 30 dernières minutes. La variabilité du groupe quant à la réaction au froid fut très importante durant les 30 premières minutes. Elle a notablement diminué le reste du temps. Toutes les températures cutanées se sont stabilisées durant les 30 dernières minutes de l'exposition au froid. La corrélation entre la température de la peau et celle de la cuisse fut hautement significative (p < 0,001) et l'on propose d'utiliser cette dernière température comme température cutanée moyenne. Le calcul de la conductibilité des tissus en y incluant ou excluant les échanges de chaleur dus aux variations thermiques du corps ou les pertes imputables à la respiration n'est exact que pour les 30 dernières minutes. Tous les paramètres mesurés, à l'exception des températures des doigts et des orteils tendent vers un minimum de variabilité durant ce même laps de temps. Ceci indique qu'un état d'équilibre physiologique est alors atteint.

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Supported in part by United States Public Health Service Grant No. HD-00235; and the Air Force Office of Scientific Research, Office of Aerospace Research, United States Air Force, under AFOSR Grant No. 69-1653. Data analysis (on IBM System 360 Model 75 computer) was made possible by funds from the Special Research Resources Branch, Division of Research Facilities and Resources, National Institutes of Health.     

Evidence for the phosphorylation of serine259 of histone deacetylase 5 by protein kinase Cδ

Signaling via pro-growth G protein coupled receptors triggers phosphorylation of HDAC5 on two serine residues (Ser₂₅₉ and Ser₄₉₈), resulting in nuclear export of HDAC5 and de-repression of downstream target genes. In the previous paper we reported the important role of PKD isozymes in the regulation of HDAC5 by phosphorylating Ser₄₉₈ of HDAC5 [Q.K. Huynh, T.A. Mckinsey, Arch. Biochem. Biophys. 450 (2006) 141–148]. In the present paper, we provide evidence that PKCδ can directly phosphorylate Ser₂₅₉ of HDAC5. The evidence is based on the following facts (a) isolated kinase fraction from human failing heart tissues contained PKCδ that phosphorylated HDAC5 Ser₂₅₉ peptide and no significant activity was found for the unbound fraction after they were immunoprecipitated with PKCδ specific antibody; (b) specific inhibitors for PKCδ inhibited kinase activity from isolated fraction and recombinant human PKCδ with similar IC₅₀ values; (c) recombinant human PKCδ can directly phosphorylate full length Ser₂₅₉ HDAC5 protein and HDAC5 Ser₂₅₉ peptide. The results suggest that in addition to activation of protein kinase D isozymes by phosphorylating Ser₇₄₄ and Ser₇₄₈ at their activation sites, PKCδ may also play a role in the regulation of HDAC5 by phosphorylation of Ser₂₅₉.     

Adenosine 5′-triphosphate-mediated activation of sucrose-phosphate synthase in bundle sheath cells of C4 plants

We report the ATP-mediated activation of sucrose-phosphate synthase in bundle sheath cells prepared from C₄ species. Sucrose synthesis was followed by measuring the incorporation of [¹⁴C]fructose 6-phosphate into sucrose in bundle sheath cells also provided with uridine 5′-diphosphoglucose (UDPGlc). Studies with Panicum miliaceum L. cells showed that activation was largely due to an increase in the affinity for UDPGlc and was therefore only evident at limiting UDPGlc concentrations. The apparent K _m UDPGlc for sucrose synthesis by cells pretreated and assayed with ATP was about 0.7 mM compared with 7–8 mM for control cells without ATP. The γ-thio derivative of ATP had a similar effect to ATP. The effect was also evident when ATP was rapidly removed from cells prior to assay. Sucrose-phosphate synthase activity in extracts from cells pretreated with or without ATP showed similar differences in K _m UDPGlc. These observations support the view that ATP is inducing a covalent modification of the enzyme. However, several protein kinase inhibitors did not prevent activation. Changes of more than threefold were observed for the K _m UDPGlc with sucrose-phosphate synthase extracted from bundle sheath cells rapidly isolated from attached leaves that were subjected to dark/light treatments. The possible relationship between these changes and those induced by ATP with isolated cells is discussed. Received: 22 October 1996 / Accepted: 7 January 1997     

Structural chemistry of lanthanidedicyclopentadienidehalides. Part 1. Two modifications of gadoliniumdicyclopentadienidebromide, [Gd(C5H5)2Br]2 and 1∞[Gd(C5H5)2Br]

The crystal structures of two modifications of gadoliniumdicyclopentadienidebromide, [Gd(C₅H₅)₂Br]₂ (I) and ¹_∞[Gd(C₅H₅)₂Br] (II) have been determined from X-ray diffraction data. I crystallizes in the [Sc(C₅H₅)₂Cl]₂-type structure, space group P2₁/c, with a=14.110(3), b=16.488(3), c= 13.765(3) Å, β=93.25(2)°, V=3197(2) ų, and D_c= 2.289 g cm⁻³ for Z=6 molecules. II crystallizes in space group P2₁/c with a=5.946(7), b=8.447(5), c=20.239(9) Å, β=90.11(4)°, V=1020(2) ų, D_c=2.392 g cm⁻³ for Z=4 formula units. The structures have been refined by full matrix least-squares techniques to conventional R factors of 0.034 for 3014 (I) and 1964 (II) reflections (with I>2σ(I)). I consists of dimers with two bromine bridges (mean GdBr 2.872 Å). II has a double chain structure with alternating juxtaposition of gadolinium and bromine atoms (GdBr 2.912 Å (once) and 3.133 Å (twice)). The arrangement of the C₅H₅ groups with regard to the metal η⁵ fashion) is nearly identical in I and II (mean GdC 2.63(1) Å (I) and 2.62(1) Å (II)). Single crystals of I and II are obtained by sublimation at different temperatures. The formation of both modifications is discussed as to its dependence on the state of the gaseous phase equilibrium [Gd(C₅H₅)₂Br]₂ ⇄ 2Gd(C₅H₅)₂Br. Obviously, I crystallizes from gaseous phase dimers while II forms from the monomers.     

Treatment of Colorectal Cancer Using a Combination of Liposomal Irinotecan (Irinophore C?) and 5-Fluorouracil

Purpose

To investigate the use of liposomal irinotecan (Irinophore C™) plus or minus 5-fluorouracil (5-FU) for the treatment of colorectal cancer.

Experimental Design

The effect of irinotecan (IRI) and/or 5-FU exposure times on cytotoxicity was assessed in vitro against HT-29 or LS174T human colon carcinoma cells. The pharmacokinetics and biodistribution of Irinophore C™ (IrC™) and 5-FU, administered alone or in combination, were compared in vivo. A subcutaneous model of HT-29 human colorectal cancer in Rag2-M mice was utilized to assess the efficacy of IrC™ alone, and in combination with 5-FU.

Results

The cytotoxicity of IRI and 5-FU were strongly dependent on exposure time. Synergistic interactions were observed following prolonged exposure to IRI/5-FU combinations. Pharmacokinetics/biodistribution studies demonstrated that the 5-FU elimination rate was decreased significantly when 5-FU was co-administered intravenously with IrC™, versus alone. Significant decreases in 5-FU elimination were also observed in plasma, with an associated increase of 5-FU in some tissues when 5-FU was given by intraperitoneal injection and IrC™ was given intravenously. The elimination of IrC™ was not significantly different when administered alone or in combination with 5-FU. Therapeutic studies demonstrated that single agent IrC™ was significantly more effective than the combination of IRI/5-FU; surprisingly, IrC™/5-FU combinations were no more effective than IrC™ alone. The administration of combinations of 5-FU (16 mg/kg) and IrC™ (60 mg IRI/kg) showed increased toxicity when compared to IrC™ alone. Treatment with IrC™ alone (60 mg IRI/kg) delayed the time required for a 5-fold increase in initial tumor volume to day 49, compared to day 23 for controls. When IrC™ (40 mg IRI/kg) was used in combination with 5-FU (16 mg/kg), the time to increase tumor volume 5-fold was 43 days, which was comparable to that achieved when using IrC™ alone (40 mg IRI/kg).

Conclusions

Single agent IrC™ was well tolerated and has significant therapeutic potential. IrC™ may be a suitable replacement for IRI treatment, but its use with free 5-FU is complicated by IrC™-engendered changes in 5-FU pharmacokinetics/biodistribution which are associated with increased toxicity when using the combination.     

Synthesis and SAR of geminal substitutions at the C5′ carbosugar position of pyrimidine-derived HCV inhibitors

The installation of geminal substitution at the C5′ position of the carbosugar in our pyrimidine-derived hepatitis C inhibitor series is reported. SAR studies around the C5′ position led to the installation of the dimethyl group as the optimal functionality. An improved route was subsequently designed to access these substitutions. Expanded SAR at the C2 amino position led to the utilization of C2 ethers. These compounds exhibited good potency, high selectivity, and excellent plasma exposure and bioavailability in rodent as well as in higher species.     

Purification of complement components by hydrophobic affinity chromatography on phenyl—Sepharose: Purification of human C5

Human complement components C5 and C3 were purified with 41% and 20% yields, respectively, by euglobulin precipitation, DEAE—Sephacel ion-exchange chromatography and gel filtration. Phenyl—Sepharose chromatography allowed the complete separation of C3 and C5. C3 bound loosely on the resin whereas C5 bound firmly and was eluted with 50% glycerin solution. Gel filtration on Sephacryl S-300 allowed the depletion of C4bp and H that contaminated C5 preparations. Homogeneity of C5 and C3 preparations was demonstrated by SDS—PAGE and immunochemical analysis. C5 and C3 consisted of two chains (α, 110000; β, 75000) linked by disulfide bridges.     

Influence of Inorganic Phosphate on Photosynthesis of Wheat Chloroplasts: I. PHOTOSYNTHESIS AND ASSIMILATE EXPORT AT 5 ?C AND 25 ?C

Chloroplasts were isolated from 10 d old wheat seedlings andilluminated at 5 ?C or 25 ?C in various concentrations of . Photosynthetic oxygen evolution, ATP content, andexport of triose phosphates and 3-phosphoglycerate were measured.Incorporation of ¹⁴C from NaH¹⁴CO₃ into pentose monophosphates,fructose monophosphate, and glucose monophosphates was determined. The ATP content in illuminated chloroplasts decreased when the concentration in the medium was low. The ATP content increased when the concentration was increased. A higher . concentration in the medium was needed to increase the ATP at 5 ?C than at 25?C. This would suggest that deficiency occurs more readily at low than at high temperatures. More ¹⁴C wasincorporated into photosynthetic metabolites within the chloroplastsat 5 ?C than at 25 ?C, indicating decreased assimilate exportwhen the temperature was low. Dihydroxyacetone phosphate waspreferentially exported when the concentration enabled a high rate of photosynthesis at 25 ?C. However, underconditions of deficiency, either due to low concentration in the medium or due to low temperature, 3-phosphoglycerate was preferred for export. The results suggest that the relatively high photosyntheticrates at low temperature are due to increased concentrationsof photosynthetic metabolites. The assimilate export at lowtemperature seems to be decreased due to decreased concentrationsof dihydroxyacetone phosphate in the stroma. Preferential exportof 3-phosphoglycerate at low temperature or at low concentration in the medium may be a consequence of high stromalconcentrations of this metabolite. On the other hand, it couldalso be due to decreased stromal pH. Key words: Chloroplast, Photosynthesis, Phosphate, Temperature, Translocation, ATP, Wheat     

The heat shock response of an antarctic alga is evident at 5°C

A subtidal seaweed collected in antarctic waters, Plocamium cartilagineum (L. Dix.), displayed induction of mRNAs encoding the 70 kDa heat shock protein (HSP70) and the ubiquitin polyprotein (UBI) when incubated at 5°C. Maximal induction of HSP70 mRNA was observed when the alga was incubated at 10°C for 1 h. Incubations at higher temperatures or for longer periods reduced the amount of HSP70 mRNA detected. Incubations at 20°C or greater resulted in cell death. These data indicate that dispite the unusually low temperature of induction, this macrophyte exhibits a heat shock response similar to that of other organisms at temperatures 5 to 10°C above usual growth conditions.     

Inhibition of hepatitis C virus NS5A by fluoro-olefin based γ-turn mimetics

The HCV non-structural protein NS5A has been established as a viable target for the development of direct acting antiviral therapy. From computational modeling studies strong intra-molecular hydrogen bonds were found to be a common structural moiety within known NS5A inhibitors that have low pico-molar replicon potency. Efforts to reproduce these γ-turn-like substructures provided a novel NS5A inhibitor based on a fluoro-olefin isostere. This fluoro-olefin containing inhibitor exhibited picomolar activity (EC(50)=79 pM) against HCV genotype 1b replicon without measurable cytotoxicity. This level of activity is comparable to the natural peptide-based inhibitors currently under clinic evaluation, and demonstrates that a peptidomimetic approach can serve as a useful strategy to produce potent and structurally unique inhibitors of HCV NS5A.     

Effects of polyamines on proliferation and IgM productivity of human–human hybridoma,HB4C5 cells

Immunoglobulin production stimulating activity of polyamines was investigated. Spermidine, thermine and triethylenetetraamine (TETA) stimulated IgM production of human–human hybridoma, HB4C5 cells under serum-free condition. IgM production of HB4C5 cells was accelerated 5.9-, 5.3-, and 3.7-fold by spermidine at 4.5 mM, thermine at 2 mM and TETA at 2.5 mM, respectively. However, putrescine did not enhance IgM production. Spermidine enhanced IgM productivity of the hybridoma cells in spite of its growth suppression activity. TETA also inhibited cell proliferation and the effect on the acceleration of IgM productivity disappeared during 5 days because of its cytotoxicity. On the other hand, thermine facilitated IgM productivity of the hybridoma cells without growth suppression. The laser confocal microscopic analysis revealed that IgM content inside HB4C5 cells was increased by thermine. This result suggests that thermine facilitates IgM synthesis in hybridoma cells.     

Mechanism of interaction of the cyanine dye DiS−C3-(5) with renal brush-border vesicles

Summary The equilibrium binding mechanism and kinetics of binding of diS–C₃-(5) (3,3-dipropylthiodicarbocyanine iodide) to rabbit renal brush-border membrane vesicles (BBMV) were examined using steady-state and time-resolved fluorescence, and fluorescence stopped-flow methods. In aqueous solution, diS–C₃-(5) exists as a monomer at concentrations <5 m with fluorescence emission peak at 670 nm (excitation 622 nm), anisotropyr=0.102, and lifetime =1.2 nsec (23°C). Upon addition of increasing BBMV (voltage clamped to 0 mV using K⁺/valinomycin), the 670 nm emission peak decreases, corresponding to formation of a nonfluorescent membrane dimer, and subsequently a new emission peak at 695 nm increases, corresponding to membrane monomer. Dynamic depolarization studies show that aqueous diS–C₃-(5) rotation is unhindered with a rotational rateR=0.57 nsec^–1 while membrane monomer is hindered with steady-state anisotropyr=0.190, lifetime =2.1 nsec,R=0.58 nsec^–1 and limiting anisotropyr =0.11. Based on equilibrium fluorescence titrations, the membrane monomer-dimer (M-D) dissociation constant,K _d=[M]²/[D][BBMV], is 0.0013, where BBMV is expressed as membrane phospholipid concentration. Three distinct kinetic processes are identified by stopped-flow experiments in which BBMV are mixed with diS–C₃-(5). There is rapid binding of diS–C₃-(5) to the membrane to form bound monomer with a 6-msec exponential time constant. The membrane monomer at the membrane outer surface then aggregates to form bound dimer at the outer surface with a concentration independent time constant of 30 msec. The overall dimerization reaction probably consists of a rate-limiting reorientation process (30 msec) followed by a rapid dimerization which occurs on a nanosecond time scale. Finally, there is a 0.8 to 1 sec translocation of membrane dimer between symmetric sites at the inner and outer membrane surfaces. The translocation reaction is the step which is probably sensitive to changes in transmembrane electrical potential.