Temperature-dependent binding of monoclonal antibodies to C hordein

The consensus octapeptide repeat motif of the barley seed storage protein C hordein, Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln, forms the epitope of two anti-prolamin monoclonal antibodies (Mabs), IFRN 0061 and 0614. The Mabs were found to exhibit unusual temperature-dependent binding characteristics, recognising C hordein and a peptide corresponding to the consensus repeat at 5°C but not at 37°C, as determined by enzyme-linked immunosorbent assay (ELISA). The K_d of IFRN 0614 for the consensus peptide was found to be 1.2×10¹² mol⁻¹ at 12°C, but no constant could be calculated at 37°C due to a lack of binding. Similar ELISA binding characteristics were observed with an anti-C hordein polyclonal antiserum and a Mab raised to the consensus peptide. Circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy showed that the protein and the consensus peptide exist in a temperature-dependent equilibrium of poly-L-proline II type structures and β-turn conformations. Whilst thermodynamic and kinetic effects may reduce antibody binding at higher temperatures, they cannot account for the complete loss of Mab recognition at higher temperatures. It seems likely that the Mabs preferentially recognise the Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln motif when presented in a conformation which may correspond to the poly-L-proline II type conformation which dominates the CD and FTIR spectra at 4-12°C.     

Identification of an epitope in the C terminus of normal prion protein whose expression is modulated by binding events in the N terminus

We have characterized the epitopes of a panel of 12 monoclonal antibodies (Mabs) directed to normal human cellular prion protein (PrP(C)) using ELISA and Western blotting of recombinant PrP or synthetic peptide fragments of PrP. The first group of antibodies, which is represented by Mabs 5B2 and 8B4, reacts with PrP(23-145), indicating that the epitopes for these Mabs are located in the 23 to 145 N-terminal region of human PrP. The second group includes Mabs 1A1, 6H3, 7A9, 8C6, 8H4, 9H7 and 2G8. These antibodies bind to epitopes localized within N-terminally truncated recombinant PrP(90-231). Finally, Mabs 5C3, 2C9 and 7A12 recognize both PrP(23-145) and PrP(90-231), suggesting that the epitopes for this group are located in the region encompassing residues 90 to 145. By Western blotting with PepSpot(TM), only three of Mabs studied (5B2, 8B4 and 2G8) bind to linear epitopes that are present in 13-residue long synthetic peptides corresponding to human PrP fragments. The remaining nine Mabs appear to recognize conformational epitopes. Two N terminus-specific Mabs were found to prevent the binding of the C terminus-specific Mab 6H3. This observation suggests that the unstructured N-terminal region may influence the local conformation within the folded C-terminal domain of prion protein.     

Inhibitory properties and antigenic specificity of monoclonal antibodies to pancreatic colipase

To understand the mechanism by which colipase acts as a protein cofactor for anchoring pancreatic lipase at triacylglycerol/water interface, we have used an immunochemical approach. Ten monoclonal antibodies (Mabs) against porcine pancreatic procolipase were produced. Purified immunoglobulins and Fab fragments were studied for their capacity to inhibit colipase-dependent lipase activity. These studies were carried out by using procolipase, the secretory form of the cofactor, and its trypsin-treated form obtained by removal of the amino terminal pentapeptide by trypsin. Reactivities of Mabs with both forms of the cofactor were also studied by immunoenzymatic methods. Mabs 6.1, 49.20. 75.8, 270.13 and 419.1 were found to inhibit lipolysis by preventing the binding of procolipase or trypsin-treated colipase to the lipid substrate. Mab 72.11 inhibited procolipase binding but had no effect on trypsin-treated colipase. Mab 72.11 reacted with procolipase in ELISA but showed no reactivity with trypsin-treated colipase. Finally, preincubation of Mab 72.11 with porcine procolipase prevented specific cleavage at the Arg5-Gly6 bond by trypsin. It could be concluded, that the five first residues of procolipase are structural elements of the antigenic determinant recognized by Mab 72.11. Results of ELISA additivity tests (cotitrations) further indicated that epitopes for Mabs 6.1, 72.11, 270.13 and 419.1 and for Mabs 49.20 and 75.8 are located in two distinct antigenic regions of the procolipase molecule. It appears then that the lipid binding domain of the pancreatic lipase protein cofactor comprises two regions. The first region corresponds to the amino terminal fragment of the protein. The second region is likely identical with the peptide segment at position 51-59 as previously hypothesized from NMR and spectrophotometric studies. Studies carried out on procolipase chemically modified at tyrosine residues provided evidence that epitopes for Mabs 49.20 and 75.8 are in or close to the region which contains tyrosines at positions 55 and 59, and that the two peptide regions essential for interfacial binding are spatially adjacent in the procolipase and the trypsin-treated form of the cofactor. General conclusions are in accordance with the location of antigenic regions of procolipase determined by predictive methods.     

Monoclonal Antibody NM2 Recognizes the Protein Kinase C Phosphorylation Site in B-50 (GAP-43) and in Neurogranin (BICKS)

Abstract: Mouse monoclonal B-50 antibodies (Mabs) were screened to select a Mab that may interfere with suggested functions of B-50 (GAP-43), such as involvement in neurotransmitter release. Because the Mab NM2 reacted with peptide fragments of rat B-50 containing the unique protein kinase C (PKC) phosphorylation site at serine-41, it was selected and characterized in comparison with another Mab NM6 unreactive with these fragments. NM2, but not NM6, recognized neurogranin (BICKS), another PKC substrate, containing a homologous sequence to rat B-50 (34–52). To narrow down the epitope domain, synthetic B-50 peptides were tested in ELISAs. In contrast to NM6, NM2 immunoreacted with B-50 (39–51) peptide, but not with B-50 (43–51) peptide or a C-terminal B-50 peptide. Preabsorption by B-50 (39–51) peptide of NM2 inhibited the binding of NM2 to rat B-50 in contrast to NM6. NM2 selectively inhibited phosphorylation of B-50 during endogenous phosphorylation of synaptosomal plasma membrane proteins. Preabsorption of NM2 by B-50 (39–51) peptide abolished this inhibition. In conclusion, NM2 recognizes the QASFR peptide in B-50 and neurogranin. Therefore, NM2 may be a useful tool in physiological studies of the role of PKC-mediated phosphorylation and calmodulin binding of B-50 and neurogranin.     

The peptide-substrate-binding domain of collagen prolyl 4-hydroxylases is a tetratricopeptide repeat domain with functional aromatic residues

Collagen prolyl 4-hydroxylases catalyze the formation of 4-hydroxyproline in -X-Pro-Gly-sequences and have an essential role in collagen synthesis. The vertebrate enzymes are alpha2beta2 tetramers in which the catalytic alpha-subunits contain separate peptide-substrate-binding and catalytic domains. We report on the crystal structure of the peptide-substrate-binding domain of the human type I enzyme refined at 2.3 A resolution. It was found to belong to a family of tetratricopeptide repeat domains that are involved in many protein-protein interactions and consist of five alpha-helices forming two tetratricopeptide repeat motifs plus the solvating helix. A prominent feature of its concave surface is a deep groove lined by tyrosines, a putative binding site for proline-rich Tripeptides. Solvent-exposed side chains of three of the tyrosines have a repeat distance similar to that of a poly-L-proline type II helix. The aromatic surface ends at one of the tyrosines, where the groove curves almost 90 degrees away from the linear arrangement of the three tyrosine side chains, possibly inducing a bent conformation in the bound peptide. This finding is consistent with previous suggestions by others that a minimal structural requirement for proline 4-hydroxylation may be a sequence in the poly-L-proline type II conformation followed by a beta-turn in the Pro-Gly segment. Site-directed mutagenesis indicated that none of the tyrosines was critical for tetramer assembly, whereas most of them were critical for the binding of a peptide substrate and inhibitor both to the domain and the alpha2beta2 enzyme tetramer.     

Monoclonal antibodies to the 27-34K insulin-like growth factor binding protein

Monoclonal antibodies were prepared against the 27-34K insulin-like growth factor (IGF)-binding protein purified from human placenta/decidua and designated placental protein 12 (PP12). Four different antibodies were characterized. Each recognized the major band at 32K on immunoblots of the purified PP12 preparation and amniotic fluid. In liquid phase RIA, IGF-I did not affect the binding of [125I] PP12 to one antibody (Mab 6303), it slightly increased the binding to two antibodies (Mab 6301 and 6304), and it slightly decreased the binding to one antibody (Mab 6302). All antibodies immunoprecipitated the cross-linked PP12-[125I] IGF-I complex, but Mab 6302 considerably less effectively than the others. Preincubation of PP12 with Mab 6302 completely inhibited the binding of [125I] IGF-I to PP12, whereas preincubation with Mab 6303 had no effect, and Mab 6301 as well as Mab 6304 increased it. These results suggest that Mab 6302 binds to an epitope at or near to the IGF-binding site, whereas the other antibodies react at other sites of the PP12 molecule. Conformational changes in PP12 probably account for the IGF-I-induced increase in the binding of Mabs 6301 and 6304 to [125I] PP12, and vice versa, for Mabs 6301- and 6304-induced increase in the binding of [125I] IGF-I to PP12.     

Monoclonal antibodies to lymphocytes of rainbow trout (Oncorhynchus mykiss)

Monoclonal antibodies (Mabs) to lymphocytes of rainbow trout have been developed by immunisation with synthetic peptides, prepared from selected parts of the alpha- and beta-gene sequences of the T-cell receptor (TCR). Mab 1C2 (TCR beta immunisation) identified lymphocytes in blood (11%), spleen (18%) and in thymus (9%) in flow cytometry analysis (FCM). Immune complexes of lymphocytes coupled to Mab 1C2 was used for further immunisations resulting in numerous supernatants reactive with lymphocytes in FCM, of which Mabs 7A5 and 8H4 were selected for further characterisation. Mab 7A5 identified 31% of lymphocytes in blood and 9% in the spleen. Mab 8H4 labelled 61% and 85% of lymphocytes in the same organs. Mab 8H4 reacted with the majority of the lymphocytes in the thymus (98%). Mabs 1C2, 7A5 and 8H4 recognised surface markers on both Ig(-) and Ig(+) lymphocytes in peripheral blood and in spleen in double staining experiments. An increased proportion of Ig(-) lymphocytes were identified when Ig(+) lymphocytes were eliminated by immunomagnetic separation. No cross-reactivity of Mabs 1C2, 7A5 or 8H4 to anti-thrombocyte Mabs was detected. Mab 1C2 captured molecules of about 40 and also of 55-60kDa, in an immunoprecipitation assay. Mab 7A5 recognised an antigen of approximately 75-80kDa and Mab 8H4 identified proteins of about 70, 100 and 150kDa. Immunohistochemical staining by Mab 8H4 of fixed thymus, revealed a strong labelling of lymphoid cells in the outer zones of thymus. The 8H4 positive lymphoid cells surrounds circular structures, which were not labelled by Mab 8H4. These distinctly appearing structures have a similar shape as nurse cells described in mammals.     

Pine wood nematode (PWN)-secretory antigen 571 as a biomarker for the PWN and its monoclonal antibodies for detecting PWN and its infected pine tree

The biochemical characteristics of pine wood nematode (PWN)-secretory antigen 571 (PWN-SA571) identified from the PWN secretome and PWN-infected pine tree extracts (PWNIPT) were investigated. PWN-SA571 has homologs in various nematodes in diverse families and a signal peptide at the amino-terminal end. Since it was not possible to express the full-length PWN-SA571 protein, two peptides showing high antigenicity were synthesized and used as antigens for generating monoclonal antibodies (Mabs). Mab-secreting fusion hybridoma cell lines were selected based on immunoreactivity to free peptide and BSA-conjugated peptide (BSA pep) antigens by enzyme-linked immunosorbent assay for establishing Mab-secreting hybridoma lines. Ascites containing PWN-SA571-specific Mabs showed stronger immunoreactivity to PWNIPT than to healthy pine tree extracts. In addition, PWN-SA571-specific Mabs could recognize less than 20 ng of BSA pep in lateral flow assays. Furthermore, purified biotin-conjugated PWN-SA571-specific Mab IgG or IgM were able to detect BSA pep (<15 ng) and PWN extracts (<600 ng). Taken together, these results demonstrate that PWN-SA571-specific Mabs could detect PWN or PWNIPT extracts by ELISA, LFA, or dot blot analysis.     

A left-handed 3(1) helical conformation in the Alzheimer Abeta(12-28) peptide

We show for the first time that the secondary structure of the Alzheimer beta-peptide is in a temperature-dependent equilibrium between an extended left-handed 3(1) helix and a flexible random coil conformation. Circular dichroism spectra, recorded at 0.03 mM peptide concentration, show that the equilibrium is shifted towards increasing left-handed 3(1) helix structure towards lower temperatures. High resolution nuclear magnetic resonance (NMR) spectroscopy has been used to study the Alzheimer peptide fragment Abeta(12-28) in aqueous solution at 0 degrees C and higher temperatures. NMR translation diffusion measurements show that the observed peptide is in monomeric form. The chemical shift dispersion of the amide protons increases towards lower temperatures, in agreement with the increased population of a well-ordered secondary structure. The solvent exchange rates of the amide protons at 0 degrees C and pH 4.5 vary within at least two orders of magnitude. The lowest exchange rates (0.03-0.04 min(-1)) imply that the corresponding amide protons may be involved in hydrogen bonding with neighboring side chains.     

Inhibition of the catalytic properties of Staphylococcus aureus nuclease by monoclonal antibodies

Monoclonal antibodies (Mab) specific for Staphylococcus aureus nuclease (nuclease) were examined for their capacity to inhibit the enzyme-mediated cleavage of DNA. Within a panel of 22 anti-nuclease Mab produced by hybridoma cell lines derived from SJL/J, A/J or BALB/c mice, only five were capable of modifying nuclease activity. Of the five, only one protected DNA from enzymatic degradation whereas the others reduced the rate of the enzymatic reaction. When mixed together, partially inactivating Mabs were frequently more efficient inhibitors than when used individually. It was shown by competitive binding assay that nuclease could be bound simultaneously to more than one Mab. Mixtures of five inactivating Mabs were able to completely block the nuclease activity. Although the actual mechanism for Mab nuclease inactivation is not known, the present data are consistent with simple steric hindrance for the formation of the DNA-nuclease complex by bulky Mab molecules bound to epitopes close to, but distinct from, nuclease catalytic sites. A mathematical model for Mab binding and inactivation of nuclease, taking into account multiple binding events for one or two Mabs interacting with nuclease, was used to derive affinities and maximum reductions of the enzymatic rate (details on the derivation of the equations and on the hypotheses of the model are given in an appendix). This analysis showed that the observed cooperative effects were dependent on the formation of multi-molecular complexes in which nuclease is bound simultaneously to two (or more) different Mabs. It also shows that the formation of cyclic complexes, if allowed, might result in very high apparent affinities. Since in screening of hybridoma fusions, the probability of finding such pairs of monoclonal antibodies would be low, this phenomenon may explain the fact that no Mab, or mixture of Mabs, matched the polyclonal antisera in capacity to block nuclease enzymatic activity.Abbreviations Nuclease Staphylococcus aureus, Foggi Strain, nuclease - Ig immunoglobulin, Mab(s)monoclonal antibody(ies) - ELISA enzyme linked immunosorbent assay - RIA radioimmunoassay     

Expression of human apolipoprotein A-I epitopes in high density lipoproteins and in serum

The expression and immunoreactivity of apolipoprotein (apo) A-I epitopes in high density lipoproteins (HDL) and serum has been investigated using two series of monoclonal antibodies (Mabs) which have been described elsewhere. Series 1 Mabs, identified as 3D4, 6B8, and 5G6, were obtained by immunization and screening with apoA-I, and series 2 Mabs, identified as 2F1, 4H1, 3G10, 4F7, and 5F6, were obtained by immunization and screening with HDL. These Mabs were characterized with respect to their binding to HDL particles in solution. In series 2 Mabs, 2F1, 3G10, and 4F7, which react with apoA-I CNBr-fragments 1 and 2, could precipitate 100% of 125I-labeled HDL, while 4H1 and 5F6, which react with CNBr fragments 1 and 3, precipitated 90 and 60% of 125I-labeled HDL, respectively. Therefore, three distinct epitopes mapped to CNBr fragments 1 and 2 have been identified which are expressed on all HDL particles, indicating that several antigenic do mains exist on apoA-I which have the same conformation on all apoA-I-containing lipoproteins. The Mabs reacting at these sites have significantly higher affinity constants for 125I-labeled HDL than those that failed to precipitate 100% of HDL. This suggests that the high affinity Mabs react with apoA-I epitopes that are both expressed on all lipoproteins and located in thermo-dynamically stable regions of the molecules. All Mabs from series 1 precipitated 35% or less of 125I-labeled HDL prepared from freshly collected serum, but the proportion of HDL particles expressing the epitopes for these Mabs doubled or more upon serum storage at 4 degrees C. The time course of the alteration of apoA-I antigen in vitro was measured in three normolipemic donors. Upon storage of serum at 4 degrees C, the immunoreactivity of series 2 Mabs (4H1, 3G10) remained unchanged. However, the immunoreactivity of series 1 Mab 3D4 increased linearly at 38%/day for 4 weeks and by 12 weeks had plateaued at about 280-fold compared to day 1. The immunoreactivity of other series 1 Mabs also increased significantly with time in vitro. This process was partially inhibited in the presence of EDTA and by addition of antioxidants, however, the exact molecular nature of this in vitro alteration of apoA-I antigen was not identified.     

Stabilization of the GDP-bound conformation of Gialpha by a peptide derived from the G-protein regulatory motif of AGS3

The G-protein regulatory (GPR) motif in AGS3 was recently identified as a region for protein binding to heterotrimeric G-protein alpha subunits. To define the properties of this approximately 20-amino acid motif, we designed a GPR consensus peptide and determined its influence on the activation state of G-protein and receptor coupling to G-protein. The GPR peptide sequence (28 amino acids) encompassed the consensus sequence defined by the four GPR motifs conserved in the family of AGS3 proteins. The GPR consensus peptide effectively prevented the binding of AGS3 to Gialpha1,2 in protein interaction assays, inhibited guanosine 5'-O-(3-thiotriphosphate) binding to Gialpha, and stabilized the GDP-bound conformation of Gialpha. The GPR peptide had little effect on nucleotide binding to Goalpha and brain G-protein indicating selective regulation of Gialpha. Thus, the GPR peptide functions as a guanine nucleotide dissociation inhibitor for Gialpha. The GPR consensus peptide also blocked receptor coupling to Gialphabetagamma indicating that although the AGS3-GPR peptide stabilized the GDP-bound conformation of Gialpha, this conformation of Gialpha(GDP) was not recognized by a G-protein coupled receptor. The AGS3-GPR motif presents an opportunity for selective control of Gialpha- and Gbetagamma-regulated effector systems, and the GPR motif allows for alternative modes of signal input to G-protein signaling systems.     

Two monoclonal antibodies to actin: one muscle selective and one generally reactive

Two IgG1, kappa monoclonal antibodies (Mab) against actin have been obtained from a fusion in which chicken gizzard actin was used as the immunogen. One Mab, designated B4, shows a preferential reactivity toward enteric smooth muscle actin but also cross-reacts with skeletal, cardiac, and aorta actins on the basis of immunoblots, ELISA assays, and indirect immunofluorescence. However, this antibody does not react with either cytoplasmic actin in any of these assay systems. A second Mab, designated C4, reacts with all six known vertebrate isoactins as well as Dictyostelium discoideum and Physarum polycephalum actins. Thus B4 Mab appears to react with an epitope that is at least partially shared among the muscle actins but not found in cytoplasmic actins, while C4 Mab binds to an antigenic determinant that has been highly conserved among the actins. The binding sites of both Mabs on skeletal actin overlap that of pancreatic DNase I. Both antibodies bind a SV8 proteolytic product comprising the amino-terminal two-thirds of the actin molecule, and their epitopes appear to overlap since C4 can compete for the binding of B4 to skeletal actin. Neither antibody is able to prevent actin polymerization.     

Primary sequence mapping of human apolipoprotein B-100 epitopes. Comparisons of trypsin accessibility and immunoreactivity and implication for apoB conformation

Differential trypsin-accessibility and monoclonal antibodies (Mabs) to human apolipoprotein (apo) B-100 are both important tools for probing apoB structure and conformation on low-density lipoproteins (LDL). In this study, we have mapped greater than 80% of the C-terminal region (720 residues) of LDL apoB-100 using trypsin digestion. Our results extend our previous data [Yang et al. (1986) Nature (Lond.) 323, 738-742] confirming that the C-terminal region of about 420 residues of apoB-100 is largely inaccessible to trypsin, whereas the part just preceding this region has interspersed trypsin-accessible and inaccessible peptides. We have determined the amino acid sequence of specific apoB-100 peptides containing epitopes recognized by four separate Mabs: two epitopes have been mapped to within 20 residues, one has been mapped to 36 residues, and the last to 80 residues. We used polyclonal antisera to identify 16 overlapping clones of varying lengths of apoB-100 cDNAs extending from the C-terminus of apoB-100 cloned in the expression vector, lambda gt11. These clones were then tested against individual Mabs. By nucleotide sequence analysis of overlapping clones that show differential reactivities to different Mabs, we have mapped the individual epitopes of each Mab to within about 50-150 amino acid residues predicted from the DNA sequences. Confirmation and further fine mapping were accomplished by competition for LDL binding using partially purified fusion proteins and chemically synthesized oligopeptides. Two epitopes (Mabs 7 and 22) were mapped to the C-terminal 20 amino acids of apoB-100, one (Mab 16) to residues 4154-4189, and another (Mab 20) to residues 3926-4005. Mab 16 precipitates more than 80% of LDL particles. Mab 20 precipitates only denatured apoB but not native LDL apoB [Milne et al. (1987) Mol. Immunol. 24, 435]. Mabs 7 and 22 are unique in that they precipitate LDL apoB modified by storage much better than freshly isolated LDL-apoB. Although epitope expression and trypsin-accessibility represent two useful probes for the study of protein conformation, there was no obvious correlation between these two parameters when applied to LDL apoB for the antibodies we have examined.     

The temperature-dependent change in methylation of the Antirrhinum transposon Tam3 is controlled by the activity of its transposase

The Antirrhinum majus transposon Tam3 undergoes low temperature-dependent transposition (LTDT). Growth at 15 degrees C permits transposition, whereas growth at 25 degrees C strongly suppresses it. The degree of Tam3 DNA methylation is altered somatically and positively correlated with growth temperature, an exceptional epigenetic system in plants. Using a Tam3-inactive line, we show that methylation change depends on Tam3 activity. Random binding site selection analysis and electrophoretic mobility shift assays revealed that the Tam3 transposase (TPase) binds to the major repeat in the subterminal regions of Tam3, the site showing the biggest temperature-dependent change in methylation state. Methylcytosines in the motif impair the binding ability of the TPase. Proteins in a nuclear extract from plants grown at 15 degrees C but not 25 degrees C bind to this motif in Tam3. The decrease in Tam3 DNA methylation at low temperature also requires cell division. Thus, TPase binding to Tam3 occurs only during growth at low temperature and immediately after DNA replication, resulting in a Tam3-specific decrease in methylation of transposon DNA. Consequently, the Tam3 methylation level in LTDT is regulated by Tam3 activity, which is dependent on the ability of its TPase to bind DNA and affected by growth temperature. Thus, the methylation/demethylation of Tam3 is the consequence, not the cause, of LTDT.     

Conformational changes in diphtheria toxoids. Analysis with monoclonal antibodies

Monoclonal antibodies (Mab) were raised against CRM197, a non-toxic mutant of diphtheria toxin (DT). The ability of four Mabs to bind DT and the six functional mutants CRM197, CRM176, CRM228, CRM1001, CRM45 and CRM30 was assessed by immunoblotting and by a radioimmunoassay in which the protein antigen in solution competes with labeled CRM197 for the Mab binding site. The results show that the peptides recognized by Mab11.3, Mab53 and Mab23 are accessible in the mutant molecules in solution but not when they are part of the native DT structure, which could therefore be described for this purpose as 'closed' in contrast with an 'open' conformation of CRM197, CRM176 and CRM228. In particular, the behaviour of Mab53 indicates that the single amino acid substitutions in the A fragments of CRM197 and CRM176 also affect the conformation of their B fragments.     

Temperature-, concentration- and cholesterol-dependent translocation of L- and D-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells

Delineating the mechanisms by which cell-penetrating peptides, such as HIV-Tat peptide, oligoarginines and penetratin, gain access to cells has recently received intense scrutiny. Heightened interest in these entities stems from their ability to enhance cellular delivery of associated macromolecules, such as genes and proteins, suggesting that they may have widespread applications as drug-delivery vectors. Proposed uptake mechanisms include energy-independent plasma membrane translocation and energy-dependent vesicular uptake and internalization through endocytic pathways. In the present study, we investigated the effects of temperature, peptide concentration and plasma membrane cholesterol levels on the uptake of a model cell-penetrating peptide, L-octa-arginine (L-R8) and its D-enantiomer (D-R8) in CD34+ leukaemia cells. We found that, at 4-12 degrees C, L-R8 uniformly labels the cytoplasm and nucleus, but in cells incubated with D-R8 there is additional labelling of the nucleolus which is still prominent at 30 degrees C incubations. At temperatures between 12 and 30 degrees C, the peptides are also localized to endocytic vesicles which consequently appear as the only labelled structures in cells incubated at 37 degrees C. Small increases in the extracellular peptide concentration in 37 degrees C incubations result in a dramatic increase in the fraction of the peptide that is localized to the cytosol and promoted the binding of D-R8 to the nucleolus. Enhanced labelling of the cytosol, nucleus and nucleolus was also achieved by extraction of plasma membrane cholesterol with methyl-beta-cyclodextrin. The data argue for two, temperature-dependent, uptake mechanism for these peptides and for the existence of a threshold concentration for endocytic uptake that when exceeded promotes direct translocation across the plasma membrane.     

Formation of a unique crystal morphology for the poly(ethylene glycol)-poly(epsilon-caprolactone) diblock copolymer

The crystallization behaviors of the poly(ethylene glycol)-poly(epsilon-caprolactone) diblock copolymer with the PEG weight fraction of 0.50 (PEG(50)-PCL(50)) was studied by DSC, WAXD, SAXS, and FTIR. A superposed melting point at 58.5 degrees C and a superposed crystallization temperature at 35.4 degrees C were obtained from the DSC profiles running at 10 degrees C/min, whereas the temperature-dependent FTIR measurements during cooling from the melt at 0.2 degrees C/min showed that the PCL crystals formed starting at 48 degrees C while the PEG crystals started at 45 degrees C. The PEG and PCL blocks of the copolymer crystallized separately and formed alternating lamella regions according to the WAXD and SAXS results. The crystal growth of the diblock copolymer was observed by polarized optical microscope (POM). An interesting morphology of the concentric spherulites developed through a unique crystallization behavior. The concentric spherulites were analyzed by in situ microbeam FTIR, and it was determined that the morphologies of the inner and outer portions were mainly determined by the PCL and PEG spherulites, respectively. However, the compositions of the inner and outer portions were equal in the analysis by microbeam FTIR.     

Antigenic structure of influenza A virus subtype H5 hemagglutinin: mechanism of acquiring stability to monoclonal antibodies in escape-mutants

The analysis of escape mutants of the avian influenza virus of H5 subtype (strain A/Mallard/Pennsylvania/10218/84) revealed the location and structure of two antigenic sites in the hemagglutinin (HA) molecule. Several escape mutants exhibited unusual features in the reactions with monoclonal antibodies (Mabs), being completely resistant in the infectivity neutralization test to the Mabs used for their selection, and retaining the ability to bind the Mabs as revealed by enzyme-linked immunosorbent assay. An enhancement of the binding by an amino acid change in a different antigenic site was demonstrated, as well as a complete abolishment of the binding by a mutation selected by passage in the presence of an excess of the non-neutralizing Mab of high binding ability. The observed effects did not result from the changes in the affinity of the mutant HA toward sialic receptors. The data suggest that one amino acid change in HA may prevent the virus neutralization by different mechanisms for different Mabs: either the binding of the Mab to HA is prevented, or the bound Mab is unable to block the receptor-binding pocket of HA. Different mechanisms of the acquisition of resistance to Mabs in the course of the selection of escape mutants are discussed.