Some theoretical considerations regarding the effects of steric hindrance and intrinsic global coupling on the flexibility of Fc-anchored immunoglobulins

Nanosecond fluorescence depolarization studies reported in the accompanying companion paper showed that the long rotational correlation time, phi L, increased somewhat when rabbit IgG anti-dansyl antibodies were anchored in staphylococcal protein A (SpA) soluble complexes. The increases in phi L upon anchoring IgG probably resulted from "global coupling" effects caused by: increased steric hindrance of the antibody segments in the SpA complexes and intrinsic structural constraints already present in the monomeric IgG. Global coupling results from a restriction in the angular range of a flexible segment and is manifest when flexible motions alone cannot depolarize all of the fluorescence, so that the slower global tumbling of the entire particle is also required. Such effects cannot be resolved directly from experimental anisotropy data, however, because only a single long correlation time, phi L, is well defined over the limited time range of most fluorophores. In this paper, estimates of the anisotropy contributions from flexible and global motions of the IgG-SpA complexes are determined by contrasting theoretical and measured decays. For this analysis it was assumed that each of the experimental phi L-values is a weighted composite of the rotational correlation time associated with the less restricted flexible motions of the Fab arms, phi F, and the correlation time associated with global tumbling of the entire particle, phi G. A general two-exponential expression was used to relate phi F and phi G to phi L. This approach was meaningful because phi G-values of the various SpA complexes had been calculated from hydrodynamic measurements. The theoretical decays clearly show that, even if phi G is much longer than phi F, these two rotational motions still cannot be resolved over the experimentally accessible time range. Families of emission anisotropy decay curves for IgG antibodies with different amounts of intrinsic global coupling and for anchored antibodies with different amounts of steric hindrance were simulated by varying the preexponential weighting factors of the flexible and global terms. By comparing the calculated curves with the measured decays, it is evident that the rabbit IgG anti-dansyl antibodies do not have much intrinsic global coupling, but rather they are highly flexible. The curves also indicate that even for the exceptionally compact IgG4-SpA2 17-S complex, which showed the most steric hindrance in electron micrographs, the appropriate phi G weighting factor is only 0.28. Thus, as supposed earlier, the anchored antibodies exhibit considerable segmental flexibility. In closing, the above concepts are used to examine the results of     

Rotational dynamics of immunoglobulin G antibodies anchored in protein A soluble complexes

The rotational dynamics of rabbit IgG anti-dansyl antibodies anchored in staphylococcal protein A (SpA) soluble complexes were studied by both steady-state and nanosecond fluorescence spectroscopy. To aid in the interpretation of the anisotropy data, the results of recently reported hydrodynamic and electron microscopic studies of IgG-SpA complexes were used to calculate global tumbling times of the various complexes and to estimate the steric hindrance of the antibody Fab segments. The anisotropy decays, fitted to the sum of two exponentials, indicated that the Fab arms of antibodies bound to SpA by their Fc regions exhibit considerable flexibility. For the different IgG-SpA mixtures examined, changes in the IgG preexponential anisotropy weighting factors, fS and fL, and the short rotational correlation time, phi S, were relatively small. On the other hand, the long rotational correlation time, phi L, increased systematically when the percentage of larger IgG-SpA complexes in a mixture was increased. The greatest restriction of Fab flexibility was observed for antibodies anchored in the exceptionally compact IgG4-SpA2 complexes. Available electron microscopic data suggest that increases in phi L correlate with increased steric hindrance of the antibody segments. Both native and hinge-disulfide-cleaved IgG experienced similar percentage increases in phi L when bound in SpA complexes. In agreement with our earlier interpretation, the results of this study provide rather striking evidence that phi L mainly represents flexible motions of the Fab segments and not global tumbling: the phi L-values of IgG bound in the various SpA complexes ranged from 101 to 162 nsec, whereas the calculated global tumbling times of the different complexes ranged from about 300 to 3000 nsec.     

血清学标志阴性的非甲～戊型肝炎的病原学研究

目的对血清学标志阴性的非甲～戊型肝炎进行病原学研究。方法用ＨＢＶＰＣＲ、ＨＣＶＲＴ－ＰＣＲ和ＨＥＶＲＴ－ＰＣＲ分别检测血清学标志阴性的非甲～戊型肝炎患者血清，并对其部分阳性产物进行克隆测序。结果８７例非甲～戊型肝炎血清ＨＢＶＤＮＡ均为阴性，９例（１０．３％）为ＨＣＶＲＮＡ阳性，部分经测序证实为ＨＣＶ１ｂ亚型；余７８例为ＨＢＶＤＮＡ和ＨＣＶＲＮＡ均阴性。该７８例中，１４例因无血清未作ＨＥＶＲＮＡ检测，余６４例中４９例（７６．６％）为ＨＥＶＲＮＡ阴性，１５例（２３．４％）为ＨＥＶＲＮＡ阳性。经序列分析显示，其中９例为典型的中国ＨＥＶ株基因序列，６例变异较大，与典型的中国株基因序列的同源性仅为８０％左右。４９例ＨＢＶＤＮＡ、ＨＣＶＲＮＡ和ＨＥＶＲＮＡ均阴性的血清中１６例（３２．６％）ＨＧＶＲＮＡ阳性。由此可见，该８７例中至少有９例为ＨＣＶ感染，１５例为ＨＥＶ感染，１６例为ＨＧＶ感染。结论对血清学标志阴性的非甲～戊型肝炎的病人应该用ＰＣＲ法进行病原学分型，以明确其诊断     

Evolution of the NS genes of the influenza a viruses. II. Characteristics of the amino acid changes in the NS1 proteins of the influenza a viruses

We compared the amino acid sequences of the NS1 proteins of human, equine, and avian influenza viruses. The ratios of the amino acid substitutions per nucleotide substitutions in the NS1 proteins were about 27–45%, suggesting the existence of constraints on the amino acid changes of the NS1 protein in evolution. As a measure of constraints exerted on the regions of a protein, a changeability index is proposed. There was a highly conserved region between amino acid residues 30 and 50. The C-terminal region of amino acid residue 165 was a continuously changeable region. We have either introduced several nucleotide substitutions to the NS cDNA of the A/Udorn/72 virus in vitro or constructed the recombinant NS cDNAs between the A/Udorn/72 and A/chick/Japan/24 viruses, and then expressed them in animal cells. We have found that the amino acid substitutions introduced to the low-conserved region of the NS1 protein affected the stability and nuclear localization of the NS1 protein. One of the chimeric proteins between the A/Udorn/72 and A/chick/Japan/24 viruses did not move to the nucleus of the cell and remained in the cytoplasm.     

Expression and evaluation of Chikungunya virus E1 and E2 envelope proteins for serodiagnosis of Chikungunya virus infection

Purpose

Chikungunya virus (CHIKV) causes endemic or epidemic outbreaks of CHIKV fever, which is a mosquitoe-transmitted viral disease in Africa, India, South-East Asia, and recently Southern Europe. Currently, serological diagnostic tests such as hemagglutination inhibition test (HI test), in-house IgM capture enzyme-linked immunosorbent assays (ELISA), and indirect immunofluorescence test were used for diagnosis of chikungunya fever, which are based on whole virus antigens.

Materials and Methods

CHIKV E1, and E2 envelope proteins for the CHIKV-specific serodiagnostic reagents for chikungunya fever were expressed in baculovirus expression system. The seroreactivity of recombinant CHIKV E1 and E2 envelope proteins were evaluated using sera panels of patients from Laboratoire Marcel Merieux by indirect IgM capture ELISA.

Results

The recombinant CHIKV E1 and E2 envelope protein showed sensitivity of 77.5% and 90%, respectively. The specificities of both CHIKV E1 and E2 envelope proteins were 100%.

Conclusion

The recombinant CHIKV E1 and E2 envelope proteins could be a useful diagnostic reagent for CHIKV infection.     

NEP1-40促进高苯丙氨酸损伤大鼠神经元Nogo A表达

目的 了解神经元NgR受体拮抗剂NEP1-40对高苯丙氨酸(Phe)作用下大鼠大脑皮质层神经元Nogo A蛋白表达的影响.方法 原代培养胎龄16～18 d的SD大鼠皮质神经元,体外模拟高Phe(0.9 mmol/L)环境,并用不同浓度NEP1-40干预;用免疫荧光方法检测神经元轴突生长和生长锥塌陷情况,实时荧光定量PCR和Western blot检测Nogo A mRNA和蛋白表达.结果 Nogo A可以在神经元胞体和突起表达;高Phe作用12、24和48 h神经元NogoA mRNA与α微管蛋白(α-Tub)的相对表达量明显减低(P<0.05);高Phe作用后神经元Nogo A蛋白明显降低(P<0.05);NEP1-40对正常神经元无促进作用,但对高phe作用下的神经元呈浓度依赖性地促进其Nogo A的表达.结论 NEP1-40可以促进高Phe损伤时神经元Nogo A的表达增高,从而促进神经元轴突的生长.     

Mutations in the E2 and NS5A regions in patients infected with hepatitis C virus genotype 1a and their correlation with response to treatment

Heterogeneity of subgenomic regions of hepatitis C virus (HCV) may be associated with response to interferon (IFN) therapy. The amino acid sequences of the PKR/eIF‐2α phosphorylation homology domain (pePHD), IFN sensitivity determining region (ISDR), PKR binding domain (PKRBD), and variable region 3 (V3) were studied in 19 patients before and after 4 weeks of treatment. All patients were infected with HCV genotype 1a and were treated with pegylated‐IFN and ribavirin. Thirteen patients achieved sustained viral response (responders) and six failed to clear viral RNA (nonresponders). The amino acid sequences in the pePHD and ISDR were identical in responders and nonresponders. However, amino acid substitution at position 2252 of PKRBD was significantly different between responders and nonresponders (P = 0.044). A larger number of mutations were observed in the V3 region of responders (P < 0.001). In this region, the amino acid in position 2364 differed between responders and nonresponders (responders: aspartic acid and serine, nonresponders: asparagine, P = 0.018). The amino acid sequences in the regions which were studied did not change after 4 weeks of treatment. It is concluded that the presence of specific amino acids in position 2252 of PKRBD and position 2364 of V3 might be associated with clinical response to IFN. J. Med. Virol. 83:1332–1337, 2011. © 2011 Wiley‐Liss, Inc.     

丙型肝炎病毒基因组部分E2／NS1蛋白基因在大肠杆菌中的表达

在大肠杆菌中表达了丙型肝炎病毒基因组的部分Ｅ２／ＮＳ１蛋白（氨基酸３６４－５１２）。表达蛋白经ＳＤＳ－ＰＡＧＥ分析，主要表达带的分子量在～４３０００左右。表达蛋白用于检测已用Ｃ３３ｃ及Ｑ２２检测过的血清，发现Ｅ２ＮＳ１引抗体阳性率占Ｃ３３ｃ及Ｑ２２抗体阳性血清的２０％，尚没有发现单独Ｅ２／ＮＳ１抗体阳性的血清。     

Novel mutations of the PRKAR1A gene in patients with acrodysostosis

Acrodysostosis is characterized by a peripheral dysostosis that is accompanied by short stature, midface hypoplasia, and developmental delay. Recently, it was shown that heterozygous point mutations in the PRKAR1A gene cause acrodysostosis with hormone resistance. By mutational analysis of the PRKAR1A gene we detected four different mutations (p.Arg368Stop, p.Ala213Thr, p.Tyr373Cys, and p.Arg335Cys) in four of seven affected patients with acrodysostosis. The combination of clinical results, endocrinological parameters and in silico mutation analysis gives evidence to suppose a pathogenic effect of each mutation. This assumption is supported by the de novo origin of these mutations. Apart from typical radiological abnormalities of the hand bones, elevated thyroid stimulating hormone and parathyroid hormone values as well as short stature are the most common findings. Less frequent features are characteristic facial dysmorphisms, sensorineural hearing loss and mild intellectual disability. These results lead to the conclusion that mutations of PKRAR1A are the major molecular cause for acrodysostosis with endocrinological abnormalities. In addition, in our cohort of 44 patients affected with brachydactyly type E (BDE) we detected only one sequence variant of PRKAR1A (p.Asp227Asn) with an unclear effect on protein function. Thus, we conclude that PRKAR1A mutations may play no major role in the pathogenesis of BDE.     

原核表达戊型肝炎病毒基因重组结构蛋白免疫保护恒河猴的初步实验研究

目的 观察基因重组戊型肝炎病毒（HEV）结构蛋白对恒河猴戊型肝炎野病毒攻击的保护作用。方法 用HEV重组结构蛋白免疫恒河猴，猴血清中抗-HEV升高后，与空白对照组一同用HEV野病毒攻击，采血观察野病毒攻击前后ALT和HEV抗体等的动态变化。结果 野病毒攻击后第3周，空白对照组5只猴ALT均出现明显异常，而免疫接种组5只猴ALT均正常，未观察到明显的肝脏炎症表现，结论 HEV重组结构蛋白免疫恒河猴之后，可以有效地保护HEV野病毒的攻击，该HEV重组结构蛋白可作为戊型肝炎基因工程疫苗的候选蛋白。     

Expression of RAB27B is up-regulated in senescent human cells

Immortal SVts8 cells that express thermolabile SV40 T antigen exhibit a senescence-like phenomenon upon inactivation of the T antigen. By using a cDNA subtractive hybridization technique, RAB27B, a member of the RAB GTPase family, was found to be up-regulated in senescent SVts8 cells. The up-regulation of RAB27B depends on the p53 gene. Enhanced expression was also observed in replicative senescence in normal human fibroblasts.     

Ultrastructural localization of viral antigens using the protein A-gold technique

HSV and DNV viral antigens have been localized by electron microscopy using the protein A-gold technique. The labelling of HSV antigens was detected over the (naked and enveloped) viral particles as well as on the cytoplasm and the nucleoplasm. In contrast, DNV antigens were revealed only over clusters of viral particles in the nucleus. The high sensitivity of the technique and the good ultrastructural preservation allowed a very fine identification of the labelled structures. Thus, the protein A-gold technique can be applied generally for the ultrastructural detection and identification of viral antigens and might be useful for diagnostic purposes.     

A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis

It is not known how influenza A viruses, important human pathogens, counter PKR activation, a crucial host antiviral response. Here we elucidate this mechanism. We show that the direct binding of PKR to the NS1 protein in vitro that results in inhibition of PKR activation requires the NS1 123-127 amino acid sequence. To establish whether such direct binding of PKR to the NS1 protein is responsible for inhibiting PKR activation in infected cells, we generated recombinant influenza A/Udorn/72 viruses expressing NS1 proteins in which amino acids 123/124 or 126/127 are changed to alanines. In cells infected with these mutant viruses, PKR is activated, eIF-2alpha is phosphorylated and viral protein synthesis is inhibited, indicating that direct binding of PKR to the 123-127 sequence of the NS1 protein is necessary and sufficient to block PKR activation in influenza A virus-infected cells. Unexpectedly, the 123/124 mutant virus is not attenuated because reduced viral protein synthesis is offset by enhanced viral RNA synthesis at very early times of infection. These early viral RNAs include those synthesized predominantly at later times during wild-type virus infection, demonstrating that wild-type temporal regulation of viral RNA synthesis is absent in 123/124 virus-infected cells. Enhanced early viral RNA synthesis after 123/124 virus infection also occurs in mouse PKR-/- cells, demonstrating that PKR activation and deregulation of the time course of viral RNA synthesis are not coupled. These results indicate that the 123/124 site of the NS1A protein most likely functionally interacts with the viral polymerase to mediate temporal regulation of viral RNA synthesis. This interaction would occur in the nucleus, whereas PKR would bind to NS1A proteins in the cytoplasm prior to their import into the nucleus.     

Evaluation of immunoadsorbent electron microscopic techniques for detection of Sindbis virus

Two immunosorbent electron microscopic techniques (ISEM), the protein A coated grid technique (PA-CGT) and the antibody coated grid technique (AB-CGT) were applied and evaluated for the detection of Sindbis virus from infected tissue culture fluids. At optimal conditions, the efficiency of trapping the virions was only about 1.5 higher with the PA-CGT as compared to the AB-CGT, but the PA-CGT was less dependent on the antiserum dilution used in the test. Both methods were suitable for quantitation experiments, since the number of virions trapped was proportional to the virus concentration. The influence of virus incubation time and temperatures, staining solutions, buffers and washing procedures on the trapping efficiency and specificity was further studied with the PA-CGT. Maximal trapping on coated grids was obtained after 3 h incubation of the virus. At room temperature, less debris was found on the grids, as compared to 37 degrees C, and the numbers of virions counted were only slightly lower. The optimal staining solution was alcohol uranyl acetate. The specificity of the PA-CGT was dependent on washing steps with phosphate buffered saline containing bovine serum albumin. With the standard procedure, at room temperature around 3 X 10(7) virions/ml (1 X 10(6) PFU/ml) were specifically detected in about 1.5 h.     

Survival of hepatitis A and E viruses in soil samples

Survival of hepatitis A virus (HAV) and hepatitis E virus (HEV) in soil samples spiked with respective viruses was analysed using real-time PCR. Virus-spiked soil samples were incubated at environmental temperature (ET) and 37°C and processed weekly. Both HAV and HEV were less stable at fluctuating ET than at 37°C. Of the 403 soil samples collected in the vicinity of Mutha river, India, 19.1% and 4.9% were found to be contaminated with HAV and HEV, respectively.     

Emerging hepatitis E virus compared with hepatitis A virus: A new sanitary challenge

Hepatitis A (HAV) and E (HEV) viruses are able to cause liver disease in humans. Among the five classical hepatotropic viruses, they are mainly transmitted via the fecal‐oral route. Historically, many similarities have thus been described between them according to their incidence and their pathogenicity, especially in countries with poor sanitary conditions. However, recent advances have provided new insights, and the gap is widening between them. Indeed, while HAV infection incidence tends to decrease in developed countries along with public health improvement, HEV is currently considered as an underdiagnosed emerging pathogen. HEV autochthonous infections are increasingly observed and are mainly associated with zoonotic transmissions. Extra hepatic signs resulting in neurological or renal impairments have also been reported for HEV, as well as a chronic carrier state in immunocompromised patients, arguing in favor of differential pathogenesis between those two viruses. Recent molecular tools have allowed studies of viral genome variability and investigation of links between viral plasticity and clinical evolution. The identification of key functional mutations in viral genomes may improve the knowledge of their clinical impact and is analyzed in depth in the present review.