Identification of four nuclear transport signal-binding proteins that interact with diverse transport signals.

The transport of proteins into the nucleus requires not only the presence of a nuclear transport signal on the targeted protein but also the signal recognition proteins and the nuclear pore translocation apparatus. Complicating the search for the signal recognition proteins is the fact that the nuclear transport signals identified share little obvious homology. In this study, synthetic peptides homologous to the nuclear transport signals from the simian virus 40 large T antigen, Xenopus oocyte nucleoplasmin, adenovirus E1A, and Saccharomyces cerevisiae MAT alpha 2 proteins were coupled to a UV-photoactivable cross-linker and iodinated for use in an in vitro cross-linking reaction with cellular lysates. Four proteins, p140, p100, p70, and p55, which specifically interacted with the nuclear transport signal peptides were identified. Unique patterns of reactivity were observed with closely related pairs of nuclear transport signal peptides. Competition experiments with labeled and unlabeled peptides demonstrated that heterologous signals were able to bind the same protein and suggested that diverse signals use a common transport pathway. The subcellular distribution of the four nuclear transport signal-binding proteins suggested that nuclear transport involves both cytoplasmic and nuclear receptors. The four proteins were not bound by wheat germ agglutinin and were not associated tightly with the nuclear pore complex.

Effect of basic and nonbasic amino acid substitutions on transport induced by simian virus 40 T-antigen synthetic peptide nuclear transport signals.

A previous study demonstrated the ability of a synthetic peptide homologous to the simian virus 40 T-antigen nuclear transport signal to induce the nuclear transport of carrier proteins and the dependence of peptide-induced transport on a positive charge at the lysine corresponding to amino acid 128 of T antigen. In this investigation synthetic peptides were utilized to examine the effect on transport of amino acid substitutions within the T-antigen nuclear transport signal. Nuclear transport was evaluated by immunofluorescence after microinjection of protein-peptide conjugates into the cytoplasm of mammalian cells. Substitution of other basic amino acids at position 128 revealed a hierarchy for nuclear transport. The rate of nuclear transport was most rapid when a lysine was at position 128 followed in descending order by arginine, D-lysine, ornithine, and p-aminophenylalanine. Peptide-induced nuclear transport was dependent upon a positively charged amino acid at positions 128 and 129, since substitutions of neutral asparagines at these positions abolished transport. However, partial transport was observed with the peptide having an asparagine at position 128 when a high number of peptides were conjugated to the carrier protein.

Dependence of the murine antibody response to an anti-CDR2 VH peptide on immunogen formulation.

A peptide corresponding to the second complementarity determining region of the heavy chain (CDR2 VH) from a murine anti-CD4 monoclonal antibody, designated L202, was synthesized by solid phase methodology in a number of different antigenic forms, for the purpose of comparing the effectiveness of different adjuvant-carrier systems in the induction of a murine antibody response against the immunizing peptide and parent antibody molecule. Two of the synthetic constructs contained the palmitoyl and N-palmitoyl-cysteinyl-S-(2,3-palmitoyloxy)-propanediol (PAM3Cys) moieties, respectively, attached to the peptide amino terminus with the immunogen comprising liposomal formulations of each. A third immunogen consisted of the CDR2 VH peptide admixed with the PAM3Cys non-covalently and incorporated into liposomes (PAM3Cys + CDR2 VH). A fourth composition comprised the CDR2 VH peptide conjugated to KLH via the sulfhydryl of an added N terminal cysteine (KLH-CDR2 VH) and injected with Complete Freund's adjuvant (CFA). A fifth immunogen consisted of the CDR2 VH peptide synthesized on an octameric, branched polylysine core as a multiple antigenic peptide (MAP-CDR2 VH) injected in the presence of Freund's adjuvant. Groups of five mice were injected intramuscularly with each of these immunogens and bled at two week intervals. The highest anti-peptide gamma-immunoglobulin (IgG) responses (against uncoupled peptide by ELISA) after 56 days were obtained with mice receiving the PAM3Cys-CDR2 VH peptide. However, when screened against the CDR2 V(H) peptide present as the MAP derivative by ELISA, IgG raised against the cognate MAP-CDR2 peptide was much more reactive than IgG raised against the liposomal PAM3Cys-CDR2 VH immunogen. In either case, IgG raised against the KLH-CDR2VH conjugate was poorly reactive. These differences in reactivity to the two forms of the CDR2 VH peptide by ELISA did not correspond to major differences in reactivities to the intact L202 Ab by ELISA. Although the IgG against the MAP immunogen was slightly more reactive than the other antisera against the l202 Ab, all titers were less than 1:100. These data illustrate some limitations of using anti-peptide responses as indicators of potential reactivity against the native protein, but suggest that alternate formulations including lipoidal peptides are more effective than corresponding KLH-peptide conjugates in eliciting Ab responses against poorly immunogenic epitopes.

Anti-peptide reagent identifies a primary-structure-dependent, cross-reactive idiotype expressed on heavy and light chains from a murine monoclonal anti-CD4.

A synthetic peptide corresponding to the second complementarity determining region (CDR2) of the immunoglobulin (Ig) variable (V) region heavy (H) chain (CDR2VH) of anti-Leu3a, a murine monoclonal antibody specific for the human CD4 molecule, was used to elicit the production of specific rabbit anti-peptide antibodies. The rabbit anti-peptide antiserum was tested for reactivity against the immunizing peptide, anti-Leu3a, and a panel of mouse monoclonal anti-CD4. Only the immunizing peptide and anti-Leu3a were recognized by ELISA, whereas the H chains of anti-Leu3a and five other monoclonal anti-CD4 preparations were recognized by Western blot analysis. These data suggest that linear structures corresponding to the CDR2VH are not normally exposed on the surface of these monoclonal antibodies and become accessible only upon unfolding of the Ig molecule. In addition, Western blot analysis demonstrated that the anti-CDR2VH peptide antiserum was able to recognize the Ig light (L) chain of anti-Leu3a. This reactivity to both H and L chains from anti-Leu3a was ascribed to a homologous five amino acid sequence region shared by the two chains. The region of homology was associated with the third framework (FR3) of the L chain and was included as a portion of the sequence in the CDR2VH synthetic peptide. This observation was confirmed by the ability of the CDR2VH anti-peptide antiserum to bind the L chains of three mouse myeloma proteins that exhibited the five amino acid sequence region of homology within their respective FR3. Together, these data provide information on the structural basis of idiotypes shared by the H and L chains from the same antibody molecule and indicate that five amino acids might be sufficient to define a minimal continuous idiotypic determinant.

Anti-idiotypic antibodies of a predefined specificity generated against CDR3VH synthetic peptides define a private anti-CD4 idiotype.

A synthetic peptide corresponding to the third complementarity determining region (CDR) of the heavy chain (CDR3VH) of anti-Leu3a, a monoclonal anti-CD4 antibody which inhibits HIV gp120 binding to CD4, was used to elicit specific anti-peptide antibodies in rabbits. The anti-peptide antisera showed anti-idiotypic antibody (anti-Id) activity and recognized both the immunizing peptide and the intact cognate protein by ELISA. In addition, the antisera reacted with isolated heavy chains of anti-Leu3a by Western blot analysis. The lack of reactivity with a panel of monoclonal anti-CD4 antibodies suggested that the anti-peptide antisera recognize a private idiotype (Id) associated with the anti-Leu3a CDR3VH region. Further studies demonstrated the inability of the rabbit antisera to inhibit the binding of anti-Leu3a to the CD4 molecule. In addition, soluble recombinant CD4 was unable to inhibit the binding of the rabbit anti-peptide antisera to anti-Leu3a indicating that the CDR3VH region may not be involved in CD4 recognition. Anti-Id containing sera from mice, rabbits and nonhuman primates immunized with the intact anti-Leu3a molecule did not bind the CDR3VH synthetic peptide, suggesting that the corresponding region of anti-Leu3a may not represent an immunodominant idiotypic determinant in thes e species. These results suggest the potential use of synthetic peptides corresponding to immunoglobulin variable (V) region amino acid sequences in generating anti-Id reagents of a predefined specificity. In addition, V-region synthetic peptides may be useful in mapping the idiotopes recognized by an anti-Id response to the cognate molecule.

Fine specificity of the murine antibody response to HIV-1 gp160 determined by synthetic peptides which define selected epitopes.

In this report, we assess the humoral immune response in inbred strains of mice immunized with baculovirus-derived recombinant HIV-1 gp160 (rgp160). Six inbred strains of mice were each immunized with two different concns (5 and 50 micrograms) of rgp160, and the antibody response to rgp160 and synthetic peptides which define distinct gp160 epitopes was examined. Within a given inbred strain of mice, no significant difference in antibody titers to gp160 was observed in those groups receiving either 5 or 50 micrograms of rgp160 per injection. Following three immunizations with rgp160, differences in anti-gp160 titers were observed among the various inbred strains; however, these differences became less apparent after additional injections with rgp160. In addition, each mouse strain exhibited a unique reactivity pattern to seven gp160 epitopes defined by synthetic peptides. Multiple injections with rpg160 were required to induce responses to certain gp160 epitopes. The observed differences in the fine specificity of the humoral immune response to distinct gp160 epitopes among the six inbred strains suggest a genetic basis for regulating the antibody response to these epitopes. This apparent regulation can be overcome by multiple injections with rgp160.

Conformational studies of the N-terminal lipid-associating domain of human apolipoprotein C-I by CD and 1H NMR spectroscopy.

A peptide comprising the N-terminal 38 residues of human apolipoprotein C-I (apoC-I(1-38)) was synthesized using solid-phase methods and its solution conformation studied by CD and 1H NMR spectroscopy. The CD data indicate that apoC-I(1-38) has a similar helical content (55%) in the presence of saturating amounts of SDS or egg yolk lysophosphatidylcholine. A structural ensemble of SDS-bound apoC-I(1-38) was calculated from 464 NOE-based distance restraints using distance geometry methods. ApoC-I(1-38) adopts a helical structure between residues V4 and K30 and an extended C-terminus from Q31 when associated with SDS. The region K12-G15 undergoes slow conformational exchange as indicated by above-average amide resonance linewidths, large temperature coefficients, and fast exchange (< 2 h) of backbone amide protons with deuterium. The mobility of K12-G15 is reflected in the poorly defined dihedral angles of K12 and E13 in the calculated ensemble of structures. The average structure of apoC-I(1-38) is curved toward its hydrophobic face with bends of 125 degrees, centered at K12/E13, and 150 degrees, centered at K21. This curvature appears to be driven by the interaction of two hydrophobic clusters, one formed by residues L8, L11, F14, and L18, and the other by L25, I26, and I29, with the amphiphile SDS. Based on our present structural definition of apoC-I(1-38) and the previously obtained structure of the fragment apoC-I(35-53), we propose the secondary structure of intact apolipoprotein C-I.

Structural studies of a baboon (Papio sp.) plasma protein inhibitor of cholesteryl ester transferase.

A 38-residue protein associated with cholesteryl ester transfer inhibition has been identified in baboons (Papio sp.). The cholesteryl ester transfer inhibitor protein (CETIP) corresponds to the N-terminus of baboon apoC-I. Relative to CETIP, baboon apoC-I is a weak inhibitor of baboon cholesteryl ester transferase (CET). To study the structural features responsible for CET inhibition, CETIP was synthesized by solid-phase methods. Using sodium dodecyl sulfate (SDS) to model the lipoprotein environment, the solution structure of CETIP was probed by optical and 1H NMR spectroscopy. Circular dichroism data show that the protein lacks a well-defined structure in water but, upon the addition of SDS, becomes helical (56%). A small blue shift of 8 nm was observed in the intrinsic tryptophan fluorescence of CETIP in the presence of saturating amounts of SDS, suggesting that tryptophan-23 is not buried deeply in the lipid environment. The helical nature of CETIP in the presence of SDS was confirmed by upfield 1Halpha secondary shifts and an average solution structure determined by distance geometry/simulated annealing calculations using 476 NOE-based distance restraints. The backbone (N-Calpha-C=O) root-mean-square deviation of an ensemble of 17 out of 25 calculated structures superimposed on the average structure was 1.06+0.30 A using residues V4-P35 and 0.51+/-0.17 A using residues A7-S32. Although the side-chain orientations fit the basic description of a class A amphipathic helix, both intramolecular salt bridge formation and "snorkeling" of basic side chains toward the polar face play minor, if any, roles in stabilizing the lipid-bound amphipathic structure. Conformational features of the calculated structures for CETIP are discussed relative to models of CETIP inhibition of cholesteryl ester transferase.

A simplified method for determination of peptide-protein molar ratios using amino acid analysis.

In this report, we have described methods to improve the efficiency of coupling synthetic peptides to keyhole limpet hemocyanin (KLH) and for the analysis of the composition of the resulting peptide-protein conjugates. KLH was first dissolved in buffers containing 3 M guanidine hydrochloride to maintain solubility and derivatized with either of two water soluble, heterobifunctional crosslinkers, m-maleimido-benzoyl-N-hydroxy-sulfosuccinimide ester (SMBS), or sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SSMCC) (300:1 molar excess over KLH). Synthetic peptides containing an amino terminal cysteine were then crosslinked to the modified KLH via sulfhydryl reaction with the crosslinker maleimide groups. Following dialysis to remove free peptide, the amino acid composition of the conjugate was determined. The molar ratio of peptide to protein within the conjugate was obtained by comparing the conjugate composition with that of both the KLH and peptide analyzed separately, and by a multiple regression, least squares analysis of the data. This method is generally applicable to the analysis of the molar ratios of protein-protein conjugates of unknown sequence or composition, and requires only the prior determination of the experimental amino acid composition of each component of the conjugate separately.

Methods for solid phase peptide synthesis which employ a minimum of instrumentation.

Both automated and manual methods of solid phase peptide synthesis employ three basic steps: (a) Attachment of the first amino acid to a resin, (b) peptide synthesis via successive carbodiimide couplings and (c) cleavage and deblocking of the peptide. Instead of an automated peptide synthesizer, one can manually synthesize peptides with a sintered glass funnel as the only required piece of equipment. Following solid phase synthesis, one can cleave and deblock peptides without the use of anhydrous hydrofluoric acid (HF); hence, the need for specialized equipment required for handling HF can also be eliminated. In the procedure described in this report, cleavage and deblocking is carried out with trifluoromethane sulfonic acid (TFMSA) in glass vessels without the need for high pressure Teflon fittings. Since completion of the coupling reaction can be monitored during each cycle when manual methods are employed, one can avoid repetitive couplings and, thereby economize on reagents. Since TFMSA cleavage and deblocking can be carried out in open glass vessels, one can cleave and deblock large numbers of peptides at the same time. With the methods described, one can satisfactorily prepare large quantities of peptides at minimal cost.

Synthetic peptides corresponding to sequences in HIV envelope gp41 and gp120 enhance in vitro production of interleukin-1 and tumor necrosis factor but depress production of interferon-alpha, interferon-gamma and interleukin-2.

Patients with human immunodeficiency virus (HIV) infections have aberrant production of a number of lymphokines and monokines. Envelope glycoproteins are believed to be important in HIV pathogenesis and may influence the production of these cytokines. Therefore, synthetic peptides corresponding to amino acid sequences 735-752 and 846-860 of glycoprotein gp41 and to amino acid sequence 304-328 of gp120 were investigated for their abilities to affect the production of the following cytokines by normal peripheral blood mononuclear cells in the presence of appropriate inducers: interferon (IFN)-alpha, IFN-gamma, interleukin (IL)-1, IL-2, and tumor necrosis factor (TNF). In contrast to cells and inducers alone (or in the presence of a control peptide), gp41 or gp120 synthetic peptides were able to depress the production of IFN-alpha, IFN-gamma and IL-2. In contrast, these peptides produced an elevation of the production of IL-1 and TNF. The effect of the gp41 peptides was more marked than that of gp120 peptides in most cases. These studies indicate that these HIV envelope glycoproteins may be directly responsible for aberrant lymphokine and monokine production in patients infected with this virus and therefore may be at least partially responsible for the pathogenesis of AIDS.

Isolation and characterization of simian immunodeficiency viruses from two subspecies of African green monkeys.

Cercopithecus aethiops (African Green monkey), a nonhuman primate species distributed throughout subsaharan Africa, has been shown to have high seroprevalence rates of antibodies to simian immunodeficiency virus (SIV), and therefore, has been proposed as a natural reservoir for immunodeficiency viruses. Our laboratories have isolated SIV-like viruses from two East African subspecies of C. aethiops designated grivet and vervet monkeys. Analysis of the structural proteins based on the molecular weights and immunologic cross-reactivity to the prototypic SIV(MAC), HIV-1, and HIV-2 isolates suggests that these viruses are distinctly different. Heterogeneity was observed in the molecular weights of the gag, pol, and env gene products between SIV isolates from vervets [SIV(AGM(VER))] and grivets [SIV(AGM(GRI))]. Phenotypically, SIV(AGM(VER)) isolates were distinguishable from SIV(AGM(GRI)) isolates by the apparent size difference of the major core antigen p24. All SIV(AGM(GRI)) and SIV(AGM(VER)) isolates were found to encode a transmembrane protein of approximately 40 kD (gp40) in contrast to gp32 of SIV(MAC). Furthermore, the transmembrane protein was shown to be encoded by the entire env open reading frame, unlike gp32 of SIC(MAC) or gp36 of SIV(AGM(TYO-1)). These data indicate that viruses from C. aethiops share common features with SIV(MAC) and HIV-1, but represent diverse SIV-like viruses which may vary according to subspecies and geographic location.

Strong association of simian immunodeficiency virus (SIVagm) envelope glycoprotein heterodimers: possible role in receptor-mediated activation.

Soluble forms of a human cell-surface molecule expressed on T lymphocytes (CD4) neutralize diverse strains of both human (HIV) and simian (SIV) immunodeficiency viruses through the induction of envelope shedding and direct competition with cellular CD4 for virus binding. However, we have previously shown that sCD4 enhances infection of simian immunodeficiency viruses from African green monkeys (SIVagm) and have theorized that this enhancement is due to the induction of conformational changes leading to viral fusion (receptor-mediated activation). In this report, we compared the relative association of the envelope glycoproteins of SIVagm with HIV type 1 (HIV-1) in order to determine if a more stable association of SIVagm envelope glycoproteins might account for the differential effects of sCD4 on the infectious process. Monospecific antisera to each of the SIVagm glycoproteins were generated and used to detect stable heterodimers by radioimmunoprecipitation. Standard solubilization buffers containing both ionic and nonionic detergents or saturating concentrations of sCD4 failed to disrupt SIVagm gp120 interactions with the transmembrane protein, gp36, whereas HIV-1 heterodimers were easily dissociated. Higher concentrations of SDS (1%) were necessary to disrupt the SIVagm envelope complexes demonstrating the existence of strong noncovalent interactions between these membrane glycoproteins. In addition, morphometric analysis by electron microscopy revealed that the linear density of SIVagm spikes was stable and resisted shedding when virus was incubated with sCD4 whereas a significant decrease in linear spike density was noted for HIV-1. Based on our original hypothesis, the strong association of SIVagm glycoprotein spikes during soluble receptor binding may allow for highly stable conformational intermediates important for viral fusion, while neutralization of HIV-1 by sCD4 results from less stable envelope associations.

D-dihexanoylphosphatidylcholine is not a pure competitive inhibitor of phospholipase A2 hydrolysis of L-dihexanoylphosphatidylcholine.

The inhibition of Crotalus adamanteus phospholipase A2 hydrolysis of L-dihexanoylphosphatidylcholine by D-dihexanoylphosphatidylcholine was investigated with inhibitor and substrate in the monomeric concentration range. The results showed that the D-enantiomer acts as a partial (not pure) competitive inhibitor of the enzyme. These results suggest that an ESI complex exists, in which hydrolysis of the substrate still occurs. Thus, binding of the D-enantiomer to the enzyme decreases the affinity for the substrate by a factor, alpha, while Vmax is unaffected. The value of alpha was determined to be 4.70 +/- 0.14. These findings complicate the use of D-phosphatidylcholines in mixed micelles with the L-enantiomer as a possible method to investigate the mechanism of interfacial activation of this enzyme.

Dihexanoylphosphatidylethanolamine: effect of head group charge on rates of alkaline and phospholipase A2 catalyzed hydrolyses.

Dihexanoylphosphatidylethanolamine (DiC6-PE) was prepared by phospholipase D catalyzed transphosphatidylation of dihexanoylphosphatidylcholine (DiC6-PC). Below the critical micellar concentration the pKa of the amino group is 9.4 +/- 0.05. The critical micellar concentration of the zwitterionic species is 5.3 +/- 0.2 mM, while that of the anionic species is 11.0 +/- 0.05 mM. Based on the pH dependence of the rate of hydroxide ion catalyzed hydrolysis, the second order rate constant for hydrolysis of the zwitterionic species is 0.70 +/- 0.021 s-1 M-1, while that for the anionic species is 0.040 +/- 0.011 s-1 M-1. The pH-dependence of phospholipase A2 catalyzed hydrolysis at substrate concentrations below the critical micellar concentration shows that the zwitterionic species is the preferred substrate, and the anionic species is either a competitive inhibitor of the hydrolysis of the zwitterionic species or poor substrate. DiC6-PE is hydrolyzed by C. adamanteus at about 1% the rate of DiC6-PC.

[Sex differences of visual attention in cryptogenic partial epilepsy]. / Diferenças sexuais da atenção visual na epilepsia parcial criptogênica.

The performance in visual verbal and non-verbal attentional tests was studied in 14 male and 18 female control subjects and 33 patients with cryptogenic partial epilepsy. The epileptic group was formed of 17 men and 16 women with no evidence of brain damage in CT scan examinations. Interictal epileptiform activity was observed only unilaterally, either in the right (8 male, 8 female) or left (9 male, 8 female) temporal lobes. The performance of epileptic men in visual tests was similar to that of normal men. On the other hand, the performance of epileptic women was worse than that of normal women. These results seem in accordance with the literature that suggests that visual attention in women depends on the functional integrity of both cerebral hemispheres. In men, verbal visual attention is suggested to occur in the left cerebral hemisphere while non-verbal processes, predominantly in the right cerebral hemisphere. Further studies are necessary for the understanding of these sexual differences in cerebral hemispheric asymmetry.

[Quantitative and topographic EEG brain mapping: a study of normal adult population]. / Eletrencefalograma quantitativo e topográfico. (Mapeamento cerebral). Estudo do padrão normal para uma população adulta.

We studied the electric brain activity during wakefulness in 20 Brazilian people through digital EEG and spectral analysis in order to propose a standardization for Brazilian adult population. All this group is healthy with laboratory examinations and mini-mental state (scores higher than 27) evaluation normal. After Fourier fast transformation (FFT) calculation, we found a histogram display with monomodal distribution, with higher values in alpha band. Analyzing the average of these results, different standards from the analogical traditional EEG were found, as the distribution of alpha band and delta activity behavior. The beta 2 and beta 3 behavior showed a diffuse distribution, that is not the usual. By the other hand, other findings are congruent to the analogical EEG as the alpha posterior predominance and the bigger presence of theta activity at the central regions.

[Alpha band coherence analysis of EEG in healthy adult's and Alzheimer's type dementia patients]. / Estudo da coerência do eletrencefalograma para a banda de frequência alfa em indivíduos adultos normais e com provável demência do tipo Alzheimer.

We studied the occipital inter-hemispheric coherence (IHCoh) of EEG (electrodes O1-O2) for alpha band (alpha1 - 8,0 to 10,0 Hz and alpha2 - 10,1 to 12,5 Hz) in healthy adults and Alzheimer's type dementia (ATD) subjects, to observe if there is any significant difference between these two groups that could help in the early diagnosis of ATD. We found a decrease of occipital IHCoh in ATD group for both alpha sub-bands. We believe that Coh analysis of EEG is a powerful auxiliary method in ATD diagnosis.

Atypical pattern related to 14 Hz positive spikes.

We studied two children with a history of headache and a normal physical and neurological examination whose EEG showed an electroencephalographic pattern recently published, the N-shape potential associated with the 14 Hz positive spikes. This graphoelement was observed only during the asleep state.

[Aicardi's syndrome. Report of 2 cases]. / Síndrome de Aicardi. Relato de dois casos.

Aicardi's syndrome is a clinical entity characterized by agenesis of the corpus callosum, severe mental subnormality, seizures (most frequently spasms in flexion), characteristic electroencephalographic changes (burst-suppression pattern), typical chorioretinal lacunae, present only in females. Other associated findings are rib and vertebral dysplasias; frequently found are cortical heterotopias. This syndrome is of hereditary origin, now considered a probable X-linked dominant trait with male lethality. The authors describe two cases of this syndrome with the full clinical picture.