Serum protein synthesis in the early chick embryo

Isolated yolk-sacs of chick embryos secreted serum proteins when incubated in buffered chick Ringer's solution. The presence of serum transferrin, two embryo-specific alpha-globulins, and a prealbumin were demonstrated by acrylamide gel analysis. Yolk-sacs from embryos explanted at 11-13 somites (40 hr preincubation) and cultured for 48 hr secreted in addition a protein with the mobility of serum albumin. Incubation of yolk-sacs in the presence of radioactive valine indicated that serum proteins were synthesized as early as the primitive streak stage. By incubating isolated yolk-sacs and embryos from 48-hr explants in the presence of radioactive valine, the synthesis of serum proteins was found to be restricted to the yolk-sac at this stage of development. Culturing explants on various nutrient proteins as well as protein starvation medium altered the relative synthesis of several serum proteins. We have proposed that morphological and biochemical changes in embryos resulting from altered nutrition may be mediated by the proteins of the serum.     

Isolation and some properties of a 34-kDa-membrane protein that may be responsible for ribosome binding in rat liver rough microsomes.

We have isolated, by hydroxyapatite chromatography with a non ionic detergent and a high salt concentration, a non-glycosylated, membrane protein with a relative molecular weight of 34 kDa that had previously been found to be a major constituent of the membrane protein fraction showing ribosome-binding activity derived from rat liver rough microsomes (RM). The isolated 34 kDa protein (p34), when incorporated into a liposome model membrane, exhibited significant binding activity toward ribosomes, its binding properties being similar to those observed with intact RM. Immunochemical analyses using antibodies directed against p34 suggested that it is a membrane-embedded RM surface protein, which is specifically localized in ribosome-attached organelles and widely distributed among mammalian tissues. These results would constitute evidence that p34 is a likely candidate for an RM ribosome-binding protein.     

Localization of ras antigenicity in rat hepatocyte plasma membrane and rough endoplasmic reticulum fractions

We have examined the antigenicity of plasma membrane (PM) and rough microsomal (RM) fractions from rat liver using anti-ras monoclonal antibodies 142-24EO5 and Y13-259 and immunochemistry as well as electron microscope immunocytochemistry. Proteins immunoprecipitated with monoclonal antibody 142-24E05 were separated using single-dimensional gradient-gel electrophoresis. The separated proteins were then blotted onto nitrocellulose sheets and incubated with [alpha-32P]GTP. Radioautograms of blots indicated the presence of specific 21.5- and 22-kDa labeled proteins in the PM fraction. A 23.5-kDa [alpha-32P] GTP-binding protein was detected in immunoprecipitates of both PM and RM fractions. Monoclonal antibody Y13-259 reacted only with the 21.5-kDa [alpha-32P] GTP-binding protein in the plasma membrane fraction. When anti-ras monoclonal antibody 142-24E05 and the immunogold technique were applied to membrane fractions using a preembedding immunocytochemical method, specific labeling was observed in association with both vesicular structures and membrane sheets in the PM fraction but only with electron-dense vesicular structures in the RM fraction. Thus ras antigenicity is associated with hepatocyte plasma membranes and ras-like antigenicity is probably associated with vesicular (secretory/endocytic) elements in both plasma membrane and rough microsomal preparations.     

Immunodetection of the bacteriocin lacticin RM: analysis of the influence of temperature and Tween 80 on its expression and activity

Immunoassays with specific antibodies offer higher sensitivity than do bioassays with indicator strains in the detection and quantification of several bacteriocins. Here we present the purification of lacticin RM and the production of specific polyclonal antibodies to a synthetic peptide resembling an internal fragment of the mature bacteriocin. The specificity and sensitivity of the generated polyclonal antibodies were evaluated in various immunoassays. The detection limits of lacticin RM were found to be 1.9, 0.16, and 0.18 micro g ml(-1) for Western blot, immuno-dot blot, and noncompetitive indirect enzyme-linked immunosorbent assays, respectively. Immunoassay sensitivities were 12.5-fold higher than that of the agar diffusion test (ADT). The production of lacticin RM showed temperature dependency, with 3, 4.2, 12.7, 28.9, 37.8, and 12 micro g ml(-1) at 37, 30, 20, 15, 10, and 4 degrees C, respectively. Temperature-stability analysis demonstrated that lacticin RM is sensitive to mild temperature, but the loss of activity does not seem to result from protein degradation. Tween 80 increased the concentration of lacticin RM eightfold and probably affected the results of the ADT either by enhancing the activity of lacticin RM or by increasing the sensitivity of the indicator strain. The use of antibodies for the specific detection and quantification of lacticin RM can expand our knowledge of its production and stability, with important implications for further investigation and future application.     

Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. II. Transmembrane disposition and characterization of glycoproteins

Rat liver microsomal glycoproteins were purified by affinity chromatography on concanavalin A Sepharose columns from membrane and content fractions, separated from rough microsomes (RM) treated with low concentrations of deoxycholate (DOC). All periodic acid-Schiff (PAS)-positive glycoproteins of RM showed affinity for concanavalin A Sepharose; even after sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis, most of the microsomal glycoproteins bound [125I]concanavalin A added to the gels, as detected by autoradiography. Two distinct sets of glycoproteins are present in the membrane and content fractions derived from RM. SDS acrylamide gel electrophoresis showed that RM membranes contain 15--20 glycoproteins (15--22% of the total microsomal protein) which range in apparent mol wt from 23,000 to 240,000 daltons. A smaller set of glycoproteins (five to seven polypeptides), with apparent mol wt between 60,000 and 200,000 daltons, was present in the microsomal content fraction. The disposition of the membrane glycoproteins with respect to the membrane plane was determined by selective iodination with the lactoperoxidase (LPO) technique. Intact RM were labeled on their outer face with 131I and, after opening of the vesicles with 0.05% DOC, in both faces with 125I. An analysis of iodination ratios for individual proteins separated electrophoretically showed that in most membrane glycoproteins, tyrosine residues are predominantly exposed on the luminal face of the vesicles, which is the same face on which the carbohydrate moieties are exposed. Several membrane glycoproteins are also exposed on the cytoplasmic surface and therefore have a transmembrane disposition. In this study, ribophorins I and II, two integral membrane proteins (mol wt 65,000 and 63,000) characteristic of RM, were found to be transmembrane glycoproteins. It is suggested that the transmembrane disposition of the ribophorins may be related to their possible role in ribosome binding and in the vectorial transfer of nascent polypeptides into the microsomal lumen.     

CD4+ T Cells Support Production of Simian Immunodeficiency Virus Env Antibodies That Enforce CD4-Dependent Entry and Shape Tropism In Vivo

CD4⁺ T cells rather than macrophages are the principal cells infected by human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) in vivo. Macrophage tropism has been linked to the ability to enter cells through CCR5 in conjunction with limiting CD4 levels, which are much lower on macrophages than on T cells. We recently reported that rhesus macaques (RM) experimentally depleted of CD4⁺ T cells before SIV infection exhibit extensive macrophage infection as well as high chronic viral loads and rapid progression to AIDS. Here we show that early-time-point and control Envs were strictly CD4 dependent but that, by day 42 postinfection, plasma virus of CD4⁺ T cell-depleted RM was dominated by Envs that mediate efficient infection using RM CCR5 independently of CD4. Early-time-point and control RM Envs were resistant to neutralization by SIV-positive (SIV⁺) plasma but became sensitive if preincubated with sCD4. In contrast, CD4-independent Envs were highly sensitive to SIV⁺ plasma neutralization. However, plasma from SIV-infected CD4⁺ T cell-depleted animals lacked this CD4-inducible neutralizing activity and failed to neutralize any Envs regardless of sCD4 pre-exposure status. Enhanced sensitivity of CD4-independent Envs from day 42 CD4⁺ T cell-depleted RM was also seen with monoclonal antibodies that target both known CD4-inducible and other Env epitopes. CD4 independence and neutralization sensitivity were both conferred by Env amino acid changes E84K and D470N that arose independently in multiple animals, with the latter introducing a potential N-linked glycosylation site within a predicted CD4-binding pocket of gp120. Thus, the absence of CD4 T cells results in failure to produce antibodies that neutralize CD4-independent Envs and CD4-pretriggered control Envs. In the absence of this constraint and with a relative paucity of CD4⁺ target cells, widespread macrophage infection occurs in vivo accompanied by emergence of variants carrying structural changes that enable entry independently of CD4.     

The C3/465 glycan hole cluster in BG505 HIV-1 envelope is the major neutralizing target involved in preventing mucosal SHIV infection

Stabilized HIV-1 envelope (Env) trimers elicit tier 2 autologous neutralizing antibody (nAb) responses in immunized animals. We previously demonstrated that BG505 SOSIP.664.T332N gp140 (BG505 SOSIP) immunization of rhesus macaques (RM) provided robust protection against autologous intra-vaginal simian-human immunodeficiency virus (SHIV) challenge that was predicted by high serum nAb titers. Here, we show that nAb in these protected RM targeted a glycan hole proximal to residue 465 in gp120 in all cases. nAb also targeted another glycan hole at residues 241/289 and an epitope in V1 at varying frequencies. Non-neutralizing antibodies directed at N611-shielded epitopes in gp41 were also present but were more prevalent in RM with low nAb titers. Longitudinal analysis demonstrated that nAb broadened in some RM during sequential immunization but remained focused in others, the latter being associated with increases in nAb titer. Thirty-eight monoclonal antibodies (mAbs) isolated from a protected RM with an exceptionally high serum neutralization titer bound to the trimer in ELISA, and four of the mAbs potently neutralized the BG505 Env pseudovirus (PV) and SHIV. The four neutralizing mAbs were clonally related and targeted the 465 glycan hole to varying degrees, mimicking the serum. The data demonstrate that the C3/465 glycan hole cluster was the dominant neutralization target in high titer protected RM, despite other co-circulating neutralizing and non-neutralizing specificities. The isolation of a neutralizing mAb family argues that clonotype expansion occurred during BG505 SOSIP immunization, leading to high titer, protective nAb and setting a desirable benchmark for HIV vaccines.     

反胶束中单宁酶的光学行为和稳定性

为了测定反胶束系统中单宁酶的光学行为和增溶方式,采用紫外分光光度法和荧光扫描技术对AOT水／异辛烷组成的反胶束体系中单宁酶和水相中单宁酶的光学行为进行研究,同时研究了不同反应体系中单宁酶的稳定性,并对单宁酶在反胶束体系中的增溶方式进行探讨。结果表明：反胶束体系与水相中的单宁酶,其光学行为存在很大差别。反胶束体系有利于单宁酶的稳定,脂肪醇作为反应底物,其碳链的增长有利于单宁酶在反胶束中的稳定性。单宁酶是以嵌入反胶束膜或与反胶束内膜接触的方式增溶的。     

THE LOCALIZATION OF SPECTRIN ON THE INNER SURFACE OF HUMAN RED BLOOD CELL MEMBRANES BY FERRITIN-CONJUGATED ANTIBODIES

Spectrin, a major protein constituent of mammalian red blood cell membrane preparations, has been localized on the inner surface of human red blood cell membranes by techniques that utilized specific ferritin-conjugated antibodies and fixation of membranes shortly after hemolysis so as to allow penetration of the ferritin-antibody labels. The labeling of spectrin was shown to be specific by the following criteria. (a) Nonhomologous ferritin-conjugated antibodies did not specifically bind to either membrane surface. (b) Blocking the membrane-bound spectrin with excess unconjugated antispectrin antibodies prevented ferritin-antibody labeling. (c) Removal of spectrin by treating the membrane preparation with a low ionic strength buffer containing ethylenediaminetetraacetate and β-mercaptoethanol prevented labeling by specific ferritin-conjugated antibodies.     

Enhancing Stability of Exenatide-Containing Pressurized Metered-Dose Inhaler Via Reverse Microemulsion System

The dispersibility and stability issues of peptide drugs during preparation and storage hinder the widespread adoption of pressurized metered-dose inhaler (pMDI). This study aimed to develop a reverse microemulsion (RM) of exenatide (EXE) pMDI through a liquid-based bottom-up method, thus to overcome the stability issue of peptide drugs encountered in traditional top-down methods, such as milling down and high-pressure homogenization. In this study, Pluronic® L64 (L64) was chosen as a surfactant to prepare the EXE-RM pMDI formulations with the assistance of ethanol. The results showed RM possessed a particle size of 123.80?±?2.91 nm with 0.121?±?0.024 PdI and a satisfied fine-particle fraction of 41.30?±?3.73% measured by a next-generation impactor. In addition, the dispersion stability of RM pMDI was maintained after storage at 4 °C for 50 days. The secondary structure of EXE was maintained during the preparation process. Moreover, the results indicated that L64 was compatible with cells and could improve the penetration of EXE through cell monolayers. Through the liquid-based bottom-up method, EXE-RM pMDI was successfully prepared and exhibited favorable stability and aerodynamic performance. This study offers a preparation strategy to enhance the stability of peptides in pMDIs.     

Immunodetection of the Bacteriocin Lacticin RM: Analysis of the Influence of Temperature and Tween 80 on Its Expression and Activity

Immunoassays with specific antibodies offer higher sensitivity than do bioassays with indicator strains in the detection and quantification of several bacteriocins. Here we present the purification of lacticin RM and the production of specific polyclonal antibodies to a synthetic peptide resembling an internal fragment of the mature bacteriocin. The specificity and sensitivity of the generated polyclonal antibodies were evaluated in various immunoassays. The detection limits of lacticin RM were found to be 1.9, 0.16, and 0.18 μg ml⁻¹ for Western blot, immuno-dot blot, and noncompetitive indirect enzyme-linked immunosorbent assays, respectively. Immunoassay sensitivities were 12.5-fold higher than that of the agar diffusion test (ADT). The production of lacticin RM showed temperature dependency, with 3, 4.2, 12.7, 28.9, 37.8, and 12 μg ml⁻¹ at 37, 30, 20, 15, 10, and 4°C, respectively. Temperature-stability analysis demonstrated that lacticin RM is sensitive to mild temperature, but the loss of activity does not seem to result from protein degradation. Tween 80 increased the concentration of lacticin RM eightfold and probably affected the results of the ADT either by enhancing the activity of lacticin RM or by increasing the sensitivity of the indicator strain. The use of antibodies for the specific detection and quantification of lacticin RM can expand our knowledge of its production and stability, with important implications for further investigation and future application.     

Exchange kinetics and composition of endocytic membranes in terms of plasma membrane constituents: a morphometric study in macrophages

Intracellular membrane traffic, during endocytosis in mouse bone marrow-derived macrophages, was studied quantitatively by morphometric and kinetic analysis. Three functionally different markers were used: Horseradish peroxidase (HRP) served as a fluid-phase (FP) marker (1000 micrograms HRP/ml in the presence of mannan) or as a receptor-mediated (RM) membrane marker (25 micrograms HRP/ml) and, third, plasma membrane (PM) glycoconjugates, enzymatically labeled with [3H]galactose at the cell surface, served as a covalent membrane marker. The cell surface was labeled with [3H]galactose, followed by either FP or by RM uptake of HRP. The kinetics of the intracellular appearance of the markers were measured as the membrane area stained by HRP-reaction product and as the number of autoradiographic grains associated with these membranes. The following compartments were distinguished: PM, coated vesicles (VI), pinosomes or endosomes (VII), secondary lysosomes (VIII), and HRP-negative vesicles (EV). Tubular structures of VII became labeled with HRP only during RM uptake. The markers flowed first into VI and VII, and after 5 min into VIII. EV became labeled with the covalent membrane marker starting from 5 min. The ratio of autoradiographic grain number to HRP-stained membrane area remained constant with time although substantially different for the various compartments, viz. 100% (VI), 50% (VII and EV) and 30% (VIII) as compared to the PM (100%). This indicated that endosomes were only partially derived from internalized PM and that secondary lysosomes contained a substantial pool of PM constituents. The observed kinetics suggested that once every 30 to 40 min the entire PM was internalized, the bulk of which was recycled after 4 min from a prelysosomal compartment(s) leaving only 12 to 20% for recycling via membranes of secondary lysosomes after a residence time of 24 to 33 min.     

Inositol 1,4,5-trisphosphate receptors. Localization in epithelial tissue.

Using a polyclonal antiserum raised against the inositol 1,4,5-trisphosphate receptor (IP3R) purified from rat cerebellum, we examined the subcellular distribution of IP3R in canine pancreatic homogenates. IP3R was present primarily in a smooth microsomal fraction (low density), a (high density) rough microsomal (RM) fraction previously shown to consist of highly purified rough endoplasmic reticulum (RER) vesicles, and, to a much lesser extent, in an intermediate density microsomal fraction which did not contain markers for RER or plasma membrane. When the RM fraction was subjected to isopycnic centrifugation on sucrose gradients, IP3R equilibrated at high sucrose densities. When ribosomes were extracted from the RM fraction by treatment with puromycin/high salt, IP3R equilibrated at considerably lighter sucrose densities. This shift in density indicated that IP3R which was present in the RM fraction is associated with the RER. Because of a significant amount of IP3R fractionating into the smooth microsomal fraction (which contains plasma membrane, among other "smooth" membranes) and a considerable amount of IP3R present in the nuclear pellet which is also enriched in plasma membrane, we examined the possibility that IP3R may be present in plasma membrane. Further subfractionation of a crude plasma membrane pellet from rat liver revealed that IP3R coenriched with a plasma membrane marker and strongly suggested an association of IP3R with plasma membrane. The issue of why the same receptor is found in multiple biochemically and morphologically distinct membrane fractions is discussed in terms of the possibility of RER subcompartmentalization and IP3R subtypes. The fractionation pattern of IP3R in pancreas is significantly different from that previously reported for calcium (Ca2+)-binding proteins and an intracellular Ca-ATPase (Nigam, S. K. and Towers, T. (1990) J. Cell Biol. 111, 197-200), raising questions as to links between these latter proteins and IP3 sensitive Ca2+ pools. Nevertheless, although the fractionation patterns are different, all of these proteins are clearly associated with the RER.     

Effects of maternal nutritional status on fetal and placental growth and on fetal urea synthesis in sheep

Fetal and placental growth, and fetal and maternal urea synthesis in late gestation, were studied in 2-year-old Corriedale ewes on a maintenance ration (M) except when subjected to moderate dietary restriction from day 50 to day 100 (RM), day 100 to day 135 (MR) or day 50 to day 135 (RR). In comparison with fetuses of ewes maintained throughout the experiment (MM), RR fetuses were smaller and RM fetuses were larger whereas MR fetuses were unaffected; all restrictions were associated with increased placental size. Fetal urea synthesis at day 133 in the well-nourished ewes (MM) was 21.5 mg N h-1 kg-1 increasing to, respectively, 25.7, 27.3 and 38.8 mg N h-1 kg-1 in groups MR, RM and RR; these values were 1.6, 3.9, 2.2 and 3.8 times the maternal rates of synthesis. On the basis of the observed urea synthesis rates, amino acid oxidation could have accounted for up to, respectively, 32, 38, 40 and 57% of fetal oxygen consumption in groups MM, MR, RM and RR. Amino acids, in addition to their role in tissue accretion, may be key energy substrates for the fetus.     

Neuroacanthocytosis associated with a defect of the 4.1R membrane protein

Background

Neuroacanthocytosis (NA) denotes a heterogeneous group of diseases that are characterized by nervous system abnormalities in association with acanthocytosis in the patients' blood. The 4.1R protein of the erythrocyte membrane is critical for the membrane-associated cytoskeleton structure and in central neurons it regulates the stabilization of AMPA receptors on the neuronal surface at the postsynaptic density. We report clinical, biochemical, and genetic features in four patients from four unrelated families with NA in order to explain the cause of morphological abnormalities and the relationship with neurodegenerative processes.

Case presentation

All patients were characterised by atypical NA with a novel alteration of the erythrocyte membrane: a 4.1R protein deficiency. The 4.1R protein content was significantly lower in patients (3.40 ± 0.42) than in controls (4.41 ± 0.40, P < 0.0001), reflecting weakened interactions of the cytoskeleton with the membrane. In patients IV:1 (RM23), IV:3 (RM15), and IV:6 (RM16) the 4.1 deficiency seemed to affect the horizontal interactions of spectrin and an impairment of the dimer self-association into tetramers was detected. In patient IV:1 (RM16) the 4.1 deficiency seemed to affect the skeletal attachment to membrane and the protein band 3 was partially reduced.

Conclusion

A decreased expression pattern of the 4.1R protein was observed in the erythrocytes from patients with atypical NA, which might reflect the expression pattern in the central nervous system, especially basal ganglia, and might lead to dysfunction of AMPA-mediated glutamate transmission.     

Premotor-Motor Interhemispheric Inhibition Is Released during Movement Initiation in Older but Not Young Adults

Neural interactions between contralateral motor regions are thought to be instrumental in the successful preparation, and execution, of volitional movements. Here we investigated whether healthy ageing is associated with a change in functional connectivity, as indicated by the ability to modulate interhemispheric interactions during movement preparation in a manner that assists rapid movement responses. Thirteen young (mean age 22.2 years) and thirteen older (68.5 years) adults rapidly abducted their left index finger as soon as possible in response to a visual imperative signal, presented 500 ms after a visual warning signal.Interactions between left dorsal premotor cortex (LPMd) and right primary motor cortex (RM1) and between left primary motor cortex (LM1) and RM1 were investigated at six time points between the warning signal and the volitional response using paired-pulse transcranial magnetic stimulation. Relative to the inhibitory interactions measured at rest, both young and older adults released LM1-RM1 inhibition beginning 250 ms after the warning signal, with no significant differences between groups. LPMd-RM1 interactions became facilitatory (from the onset of the imperative signal onwards) in the older, but not the young, group. Regression analyses revealed that for the older adults, modulation of LPMd-RM1 interactions early in the preparation period was associated with faster responses, suggesting that specifically timed modulation of these pathways may be a compensatory mechanism to offset, at least in part, slowing of motor responses. The results suggest a greater reliance on premotor regions during the preparation of simple motor actions with advancing age.     

Endometrial Stromal Cells of Women with Recurrent Miscarriage Fail to Discriminate between High- and Low-Quality Human Embryos

Background

The aetiology of recurrent miscarriage (RM) remains largely unexplained. Women with RM have a shorter time to pregnancy interval than normally fertile women, which may be due to more frequent implantation of non-viable embryos. We hypothesized that human endometrial stromal cells (H-EnSCs) of women with RM discriminate less effectively between high-and low-quality human embryos and migrate more readily towards trophoblast spheroids than H-EnSCs of normally fertile women.

Methodology/Principal Findings

Monolayers of decidualized H-EnSCs were generated from endometrial biopsies of 6 women with RM and 6 fertile controls. Cell-free migration zones were created and the effect of the presence of a high-quality (day 5 blastocyst, n = 13), a low-quality (day 5 blastocyst with three pronuclei or underdeveloped embryo, n = 12) or AC-1M88 trophoblast cell line spheroid on H-ESC migratory activity was analyzed after 18 hours. In the absence of a spheroid or embryo, migration of H-EnSCs from fertile or RM women was similar. In the presence of a low-quality embryo in the zone, the migration of H-EnSCs of control women was inhibited compared to the basal migration in the absence of an embryo (P<0.05) and compared to the migration in the presence of high-quality embryo (p<0.01). Interestingly, the migratory response H-EnSCs of women with RM did not differ between high- and low-quality embryos. Furthermore, in the presence of a spheroid their migration was enhanced compared to the H-EnSCs of controls (p<0.001).

Conclusions

H-EnSCs of fertile women discriminate between high- and low-quality embryos whereas H-EnSCs of women with RM fail to do so. H-EnSCs of RM women have a higher migratory response to trophoblast spheroids. Future studies will focus on the mechanisms by which low-quality embryos inhibit the migration of H-EnSCs and how this is deregulated in women with RM.     

Pharmacokinetics, toxicity, and functional studies of the selective Kv1.3 channel blocker 5-(4-phenoxybutoxy)psoralen in rhesus macaques

The small molecule 5-(4-phenoxybutoxy)psoralen (PAP-1) is a selective blocker of the voltage-gated potassium channel Kv1.3 that is highly expressed in cell membranes of activated effector memory T cells (TEMs). The blockade of Kv1.3 results in membrane depolarization and inhibition of TEM proliferation and function. In this study, the in vitro effects of PAP-1 on T cells and the in vivo toxicity and pharmacokinetics (PK) were examined in rhesus macaques (RM) with the ultimate aim of utilizing PAP-1 to define the role of TEMs in RM infected with simian immunodeficiency virus (SIV). Electrophysiologic studies on T cells in RM revealed a Kv1.3 expression pattern similar to that in human T cells. Thus, PAP-1 effectively suppressed TEM proliferation in RM. When administered intravenously, PAP-1 showed a half-life of 6.4 hrs; the volume of distribution suggested extensive distribution into extravascular compartments. When orally administered, PAP-1 was efficiently absorbed. Plasma concentrations in RM undergoing a 30-day, chronic dosing study indicated that PAP-1 levels suppressive to TEMs in vitro can be achieved and maintained in vivo at a non-toxic dose. PAP-1 selectively inhibited the TEM function in vivo, as indicated by a modest reactivation of cytomegalovirus (CMV) replication. Immunization of these chronically treated RM with the live influenza A/PR8 (flu) virus suggested that the development of an in vivo, flu-specific, central memory response was unaffected by PAP-1. These RM remained disease-free during the entire course of the PAP-1 study. Collectively, these data provide a rational basis for future studies with PAP-1 in SIV-infected RM.     

Antibodies to the strain-specific and cross-reactive determinants of the haemagglutinin of influenza H3N2 virus. 3. Selection of antigenic variations in vitro and in vivo

Antigenic variants of influenza A/Hong Kong/1/68 (H3N2) were obtained in vitro by letting virus multiply in the allantosis-on-shell system in the presence of anti-haemagglutinin antibodies, prepared from immune goat serum to purified haemagglutinin antigen, and in vivo by giving mice antibody intraperitoneally one day before challenge with a sublethal dose of live virus. In both systems it was shown that the most narrowly reacting strain-specific antibodies selected antigenic variants at an apparently higher rate than a more cross-reactive preparation of antibodies.     

Transfection of HeLa-cells with pEGFP plasmid by impedance power-assisted electroporation

Bioimpedance spectrometry was applied to study cell viability and pEGFP plasmid-transfection efficiency in electroporation (EP) of 20,000 HeLa cells with 0.3 microg DNA in 90 microl low conductivity 0.32 M sucrose medium of pH 7.5. Monopolar rectangular pulses, of field strength 75 V/mm, and pulse length 0.1 ms were applied in 1-16 repetitions with a 10-sec pause interval between pulses. Surviving cells were stained by crystal violet and counted using a confocal microscope. Transfected cells were fixed with 10% formaldehyde and counted as green spots in a fluorescence microscope. In the present investigation we used the method of bioimpedance spectrometry to analyze the effect of EP on survival and transfection ratio of cells in suspension. DC and low-frequency AC currents preferably pass through the medium due to the high impedance of the cell membrane. At frequencies above 10 kHz the impedance of the cell membrane starts to decrease and the impedance value of the cell suspension approach a lower limit value Rinfinity at infinite frequency. Recording of electrical impedance spectra of cells in culture was performed over a frequency range of 10 Hz to 125 kHz, allowing separation of the contribution from extracellular space and that of the cell membranes. A parallel resistance capacitance model of the cell suspension was used to evaluate the response of applying EP pulses. The values of the collective membrane resistance RM decay exponentially (r2=0.995) with the number of applied pulses. The ratio of the extrapolated value of the intact membrane resistance before pulsing, RM,0, and the value RM,N after each pulse makes an index of the effect of electroporation on the cells. The ratio RM,N/RM,0 as well as the relative change of the dissipation factor, tandelta, on the "Loss Change Index" (LCI) fits well a dose-response model (r2=0.98) with the number of applied pulses. The changes in the model parameters membrane resistance DeltaRM=[1-RM,N/RM,o] and loss factor [1-tandelta0/tandeltaN] correlate well with the transfection ratio and fraction of dead cells. Those parameters were used for power-assisted electroporation in monitoring, controlling, and optimizing the EP procedure.