A sensitive two-site enzyme-linked immunosorbent assay for measurement of the major Alternaria alternata allergen Alt a 1.

BACKGROUND: Alt a 1 is the major allergen in Alternaria alternata, one of the most important fungi associated with allergic diseases. Mold allergenic extracts show considerable heterogeneity, and thus accurate standardization of these extracts is essential to guarantee their quality. OBJECTIVE: To develop an Alt a 1-specific assay and to evaluate the correlation of Alt a 1 content with the IgE-binding activity of A. alternata extracts. METHODS: Recombinant Alt a 1 was produced as nonfusion protein from a polymerase chain reaction-cloned complementary DNA Alt a 1 sequence. Natural Alt a 1 was purified from spent culture medium. Monoclonal and polyclonal antibodies directed to Alt a 1 were produced and used to construct a specific Alt a 1 enzyme-linked immunosorbent assay (ELISA). RESULTS: The ELISA developed was highly reproducible and sensitive, with a detection limit lower than 0.5 ng/mL and a practical working range of 0.5 to 50 ng/mL. The assay was able to detect an Alt a 1-like protein in Stemphylium extracts. Identical parallel dose-response curves were observed when natural Alt a 1 and recombinant Alt a 1 were used as standard. A good correlation was obtained between Alt a 1 content of 13 A. alternata extracts and their IgE-binding activity. Alt a 1 was responsible for 70% of the IgE-binding activity of the whole extract. CONCLUSIONS: This sensitive and specific Alt a 1 assay allows the quantification of this major mold allergen and represents a useful tool for the standardization of A. alternata extracts in mass units. It also provides a reliable indication of the allergenic activity of the whole extract.     

Apolipoprotein(a) phenotypes are reliable biomarkers for familial aggregation of coronary heart disease

Lipoprotein(a) [Lp(a)] is an atherogenic and prothrombotic molecule formed by the covalent binding of the highly polymorphic apolipoprotein(a) [apo(a)] to apoprotein B-100 of LDL. High Lp(a) concentrations are a recognized genetic risk factor for coronary heart disease (CHD) and have been shown to be related with a familial clustering of ischemic cardiac events. Nevertheless, the association between apolipoprotein(a) isoforms and a positive familial history of CHD has received far less attention. In this report, we explored the distribution of apo(a) phenotypes in 127 CHD subjects with a family history of coronary events and in 92 CHD patients without such a history. Twenty-two apo(a) isoforms were detected by a high-resolution immunoblotting method. In univariate analysis, the percentage of subjects with at least one small sized apo(a) isoform was significantly higher in CHD patients with a positive family history than in those without (P<0.01). Multivariate analysis showed that apo(a) isoforms of low molecular weight were the best predictors of familial aggregation of cardiac ischemia. We conclude that apo(a) size polymorphism is strongly associated with a familial history of CHD and is more efficient than Lp(a) plasma concentrations in predicting the familial clustering of coronary disease. When detected by high-resolution techniques, apo(a) phenotypes are objective laboratory markers that can substitute for a knowledge of a positive family history of CHD and should be used, together with Lp(a) levels, to better assess the familial predisposition to coronary events.     

On the probability that a novel variant is a disease-causing mutation

When a novel variant is found in a patient and not in a group of controls, it becomes a candidate for the disease-causing mutation in that patient. At present, no sampling theory exists for assessing the probability that the novel SNP might actually be a neutral variant. We have developed a population genetics-based method for calculating a P-value for a mutation-detection effort. Our method can be applied to a heterozygous patient, a homozygous patient, with or without inbreeding, or to a patient who is a compound heterozygote. Additionally, the method can be used to calculate the probability of finding a neutral variant at frequencies that differ between a group of patients and a group of controls, given some length of sequence examined. This method accounts for the multiple testing that is inherent in identification of variants through sequencing, to be used in subsequent case-control analyses. We show, for example, that for complete resequencing of 10 kb, the probability of finding a neutral variant in a patient and not in 50 controls is about 15%. Thus, discovery of a variant in a patient and not in a group of controls is, on its own, very weak evidence of involvement with disease.     

Local secretory response by the mouse uterine epithelium to the presence of a blastocyst or a blastocyst-like bead

Summary The ultrastructure of the uterine secretion appearing locally at the site of a blastocyst or a blastocyst-like bead was compared to the ultrastructure of the secretion present in a sterile horn of similarly treated mice. The secretion consisted of a homogeneous substance with small vesicles. The secretion was sparse and only slightly electron dense in a sterile horn, while it was more abundant and denser in a horn with a luminal object. Around a blastocyst, the secretion appeared more dense than it did around a bead, while the small vesicles of the secretion were more frequently occurring around a bead than around a blastocyst. It is concluded that a luminal object like a blastocyst is capable of eliciting a local secretory activity in the uterine epithelium, and it is suggested that this secretion is rich in hydrolytic enzymes.     

Glycosylation and palmitoylation of Wnt-3a are coupled to produce an active form of Wnt-3a

Wnt-3a is a representative ligand that activates the beta-catenin-dependent pathway in Wnt signaling and is modified with glycans and palmitate. In this study, we analyzed the relationship between glycosylation and lipidation of Wnt-3a. Secretion of a Wnt-3a mutant that lacks glycosylation (Wnt-3a NQ) was impaired. Wnt-3a C77A, which lacks palmitoylation at Cys77, was secreted with similar efficiency to wild-type Wnt-3a (Wnt-3a WT), but did not induce the internalization of low-density lipoprotein receptor-related protein 6 (LRP6). Furthermore, removal of palmitate from Wnt-3a suppressed the ability to bind to its receptors Frizzled8 and LRP6. Wnt-3a C77A was glycosylated to an extent similar to Wnt-3a WT, while Wnt-3a NQ was not modified with palmitate. Expression of porcupine, which is a putative acyltransferase, enhanced palmitoylation of Wnt-3a WT greatly, but that of Wnt-3a NQ slightly. While Wnt-3a WT was present in both the endoplasmic reticulum (ER) and Golgi, Wnt-3a NQ was located to the ER only. Furthermore, Wnt-3a was not palmitoylated but was glycosylated in the cells treated with Brefeldin A, which inhibits transport of vesicles from the ER to the Golgi. These results indicate that glycosylation of Wnt-3a precedes palmitoylation and that both modifications are necessary for secretion of an active Wnt-3a.     

miR-34a 通过Snail 诱导肺癌EMT 及促进其转移的分子机制

目的:探讨miR-34a 在肺癌组织中的表达情况以及miR-34a 在肺癌细胞侵袭和迁移过程中的作用及其机制。方法:qPCR 检测肺癌和正常肺组织中miR-34a 的表达情况;使用miR-34a-mimic 和miR-34a-inhibitor 过表达和沉默miR-34a,qPCR 检测沉默和过表达效果;Western blot 检测沉默和过表达miR-34a 后Snail 蛋白的表达情况;荧光素酶报告基因检测miR-34a 与Snail 的相互作用;Transwell 侵袭实验检测miR-34a 的表达对肺癌细胞侵袭能力的影响;划痕实验检测miR-34a 的表达对肺癌细胞迁移能力的影响;Western blot 检测E-Cadherin、Vimentin 和Twist 蛋白的表达情况。结果:与正常肺组织相比,肺癌组织中miR-34a 表达明显降低;且晚期、低分化和有淋巴结转移的肺癌组织miR-34a 表达明显较早期、高分化和无淋巴结转移的肺癌组织低;miR-34a-mimic miR-34a-inhibitor 可以有效抑制和过表达miR-34a 的表达;miR-34a 能与Snail 的3忆UTR 特异性结合;miR-34a 可以调控肺癌H1650 细胞的侵袭迁移能力;过表达miR-34a 上调E-Cadherin,同时下调Vimentin 和Twist 蛋白的表达,沉默miR-34a 则相反。结论:miR-34a 在肺癌中表达明显降低,且跟肺癌分期分级以及淋巴结转移与否密切相关,同时miR-34a 可以通过上皮间质转化调节肺癌细胞侵袭和迁移能力。     

Genetic polymorphism of rabbit VHa region: a new allotype, a108

We report a new rabbit IgVH allotype, designated a108, which was detected following studies of the progeny of a wild rabbit (Oryctolagus cuniculus) from Zembra Island (Tunisia). This allotype seems to be specific of this island, since it has not been detected in other wild rabbit populations (in Spain, Portugal or France). Its determinants are located on the Fd fragment of the immunoglobulin (Ig) heavy chain and it behaves like the product of an allele at the alpha locus. Allotype a108 was strongly related serologically to a1 and a107, and to a lesser extent to a101, a109 and a3 allotypes. We also detected determinants shared among the a1, a107, a108, a101 and a109 allotypes. These determinants were expressed by a large fraction of Ig molecules from rabbits of allotype a1, a107 and a108 and by a very small fraction of Ig from rabbits of allotype a101 and a109.     

A metamodel of theories of psychotherapy: a guide to their analysis,comparison, integration and use

Every theory of psychotherapy is composed of a myth and an associated ritual. A myth is a conceptual scheme for explaining clinical problems. A ritual is based upon a myth and is a model of the clinical change process. This article goes far beyond the observation that theories of psychotherapy consist of a myth and a ritual to propose that they share a common underlying structure. The central purpose of this article is to delineate this structure. This structure constitutes a metamodel of theories of psychotherapy. The article shows how the metamodel is a fresh conceptual tool (a) for understanding, analysing, comparing and contrasting, and integrating the basic concepts and principles of theories of psychotherapy and (b) for building case formulations and treatment plans from a theory of psychotherapy. Copyright © 2003 John Wiley & Sons, Ltd.     

Metabolism of Lp(a): assembly and excretion

Lp(a) is one of the most atherogenic lipoproteins, and we know much more about the pathophysiology of Lp(a) than about its physiological function and metabolism. From our previous investigations and the new results reported here, we propose the following model of Lp(a) metabolism: apo(a) is biosynthesized in liver cells and the size of the isoform determines its rate of synthesis and excretion. Specific kringle-4 domains in apo(a), mainly T-6 and T-7, bind in a first step to circulating LDL, followed by the stabilization of the newly formed Lp(a) complex by a disulfide bridge. Circulating Lp(a) interacts specifically with kidney cells, or possibly other tissues, causing cleavage of 2/3–3/4 of the N-terminal part of apo(a) by a collagenase-type protease. Part of the apo(a) fragments is found in the urine, but there are indications that they in fact represent the biologically active form of apo(a). The core portion of Lp(a) in turn is cleared by the LDL-receptor or another specific binding system of the liver. Strategies for reducing plasma Lp(a) levels with medication should aim at interfering with the assembly of Lp(a) on one hand and the stimulation of apo(a) fragmentation on the other hand.     

Fine-needle aspiration cytology of a lipoblastoma: a case report

A lipoblastoma is a rare benign tumor of immature white fat, and more than 90% of lipoblastomas occur before the age of 3 years. The diagnosis of a lipoblastoma is mostly dependent on a histopathological examination of a surgically excised specimen. However, an accurate preoperative diagnosis is essential for the planning of surgery, particularly for a lesion of the head and neck area. We experienced a case of a cervical lipoblastoma of a 23-month-old boy. A preoperative fine-needle aspiration biopsy showed the sample as moderately cellular and showed fragments of mature and immature adipose tissues containing a large number of capillary vessels. There were numerous lipoblast-looking cells with a multivacuolated cytoplasm, and the nuclei were small, compressed by vacuoles, and centrally located. According to the cytological findings, the lesion was diagnosed as a benign adipose tumor suggestive of a lipoblastoma. Subsequent surgical excision confirmed the diagnosis of the fine-needle aspiration biopsy. The cytologic features of lipoblastoma are not well known because of the rarity of the lesion. However, the fine-needle aspiration cytological features of a lipoblastoma are sufficiently characteristic to make a specific preoperative diagnosis.     

Ethnicity and lipoprotein(a) polymorphism in Native Mexican populations

BACKGROUND: Lp(a) is a lipoparticle of unknown function mainly present in primates and humans. It consists of a low-density lipoprotein and apo(a), a polymorphic glycoprotein. Apo(a) shares sequence homology and fibrin binding with plasminogen, inhibiting its fibrinolytic properties. Lp(a) is considered a link between atherosclerosis and thrombosis. Marked inter-ethnic differences in Lp(a) concentration related to the genetic polymorphism of apo(a) have been reported in several populations. AIM: The study examined the structural and functional features of Lp(a) in three Native Mexican populations (Mayos, Mazahuas and Mayas) and in Mestizo subjects. METHODS: We determined the plasma concentration of Lp(a) by immunonephelometry, apo(a) isoforms by Western blot, Lp(a) fibrin binding by immuno-enzymatic assay and short tandem repeat (STR) polymorphic marker genetic analysis by capillary electrophoresis. RESULTS: Mestizos presented the less skewed distribution and the highest median Lp(a) concentration (13.25 mg dL(-1)) relative to Mazahuas (8.2 mg dL(-1)), Mayas (8.25 mg dL(-1)) and Mayos (6.5 mg dL(-1)). Phenotype distribution was different in Mayas and Mazahuas as compared with the Mestizo group. The higher Lp(a) fibrin-binding capacity was found in the Maya population. There was an inverse relationship between the size of apo(a) polymorphs and both Lp(a) levels and Lp(a) fibrin binding. CONCLUSION: There is evidence of significative differences in Lp(a) plasma concentration and phenotype distribution in the Native Mexican and the Mestizo group.     

Preferrential rearrangement in normal rabbits of the 3' VHa allotype gene that is deleted in Alicia mutants; somatic hypermutation/conversion may play a major role in generating the heterogeneity of rabbit heavy chain variable region sequences

The rabbit is unique in having well-defined allotypes in the variable region of the heavy chain. Products of the VHa locus, (with alleles a1, a2, and a3), account for the majority of the serum immunoglobulins. A small percentage of the serum immunoglobulins are a-negative. In 1986, Kelus and Weiss described a mutation that depressed the expression of the Ig VH a2 genes in an a1/a2 rabbit. From this animal the Alicia rabbit strain was developed and the mutation was termed ali. We previously showed, using Southern analysis and the transverse alternating field electrophoresis technique, that the difference between the ali rabbit and normal is a relatively small deletion including some of the most 3' VH genes. The most JH proximal 3' VH1 genes in DNA from normal rabbits of a1, a2 and a3 haplotypes encode a1, a2 and a3 molecules respectively, and it has been suggested that these genes are responsible for allelic inheritance of VHa allotypes. The present study suggests that the 3' end of the VH locus probably plays a key role in regulation of VH gene expression in rabbits because VH gene(s) in this region are the target(s) of preferential VDJ rearrangements. This raises the possibility that mechanisms such as somatic gene conversion and hypermutation are at work to generate the antibody repertoire in this species. Our data support the view that the 3' VH1 gene may be the preferred target for rearrangement in normal rabbits, and for the normal chromosome in heterozygous ali animals. However, homozygous ali rabbits with a deletion that removed the a2-encoding VH1 on both chromosomes do survive, rearrange other VH genes and produce normal levels of immunoglobulins as well as a significant percentage of B cells which bear the a2 allotype. This challenges the view that one VH gene, VH1, is solely responsible for the inheritance pattern of VHa allotypes.     

Immunologic analysis of monoclonal and immunoglobulin E antibody epitopes on natural and recombinant Amb a 1

Background Amb a 1 is the major allergen from ragweed pollen and more than 90% of ragweed‐allergic patients react with this protein. Although Amb a 1 was cloned and sequenced in 1991, little is known of the specificity of anti‐Amb a 1 antibodies or of the immunologic properties of the recombinant allergen. Objective To compare binding of monoclonal antibodies (mAb) and IgE antibodies to purified natural Amb a 1 (nAmb a 1) and recombinant Amb a 1 (rAmb a 1). Methods Binding of a panel of anti‐Amb a 1 mAb and IgE antibodies to nAmb a 1 or rAmb a 1 was compared by immunoblotting. Chimeric ELISA was used to measure specific IgE to these allergens using 89 ragweed‐allergic sera from Austria, Italy, Canada and the United States. Results The 8 mAb bound to a 38 kDa Amb a 1 band in ragweed pollen extract and a subset of 5 mAb also bound to the 26 kDa chain of nAmb a 1. A two‐site ELISA was developed using a mAb pair, which was ～10‐fold more sensitive to rAmb a 1. There was a significant correlation between IgE antibody binding to nAmb a 1 and rAmb a 1 (n=89, r=0.79, P<0.001). A subset of ～40% of patients showed greater reactivity to nAmb a 1 than to rAmb a 1. Conclusions The data suggest that there is less reactivity of human IgE to rAmb a 1 compared with nAmb a 1. The development of more sensitive, quantitative ELISA for Amb a 1 will require the production of new mAb especially directed against nAmb a 1.     

The active site of human C4a anaphylatoxin

The human C4 activation peptide C4a has recently been shown to be biologically active and to share common tissue receptors with human C3a anaphylatoxin. Human C3a and C4a each induce contraction and cause cross-desensitization of isolated guinea-pig ileal strips. The essential active site of C3a is comprised in the model peptide containing the five COOH-terminal residues, Leu-Gly-Leu-Ala-Arg. The anaphylatoxic activities of the corresponding C4a pentapeptide, Ala-Gly-Leu-Gln-Arg, and several other synthetic peptides related to the COOH-terminal sequence of human C4a were examined. The C4a pentapeptide induced contraction of guinea-pig ileum at 1 X 10(-3) M and produced a wheal and flare reaction in human or guinea-pig skin when 2-5 mumols were injected intradermally. The corresponding C3a pentapeptide is 500-fold more active, since it induces contraction of guinea-pig ileum at 3-4 X 10(-6) M and only 4-10 nmole induce a visible skin reaction. Although the C4a pentapeptide is relatively inactive compared to the C3a pentapeptide, two analogs of these peptides, Leu-Gly-Leu-Gln-Arg and Ala-Gly-Leu-Ala-Arg, each exhibited significantly greater activity than Ala-Gly-Leu-Gln-Arg and each analog desensitized ileal smooth muscle towards contraction by either C3a or C4a. Thus it is a combination of two amino acid substitutions, the Ala for Leu-73 and Gln for Ala-76, in the COOH-terminal pentapeptide of C3a that accounts for the markedly reduced activity of C4a. The contribution of the COOH-terminal portion of C4a on its activity was further documented by examining the C4a octapeptide, Lys-Gly-Gln-Ala-Gly-Leu-Gln-Arg and a trialanyl analog, Ala-Ala-Ala-Ala-Gly-Leu-Gln-Arg. The C4a octapeptide, C4a (70-77), exhibited 5-fold greater biologic activity than the C4a pentapeptide, while the trialanyl analog was 40-fold more active. Anaphylatoxic activities of the C4a-(73-77) pentapeptide, C4a-(70-77) octapeptide, and the trialanyl octapeptide analog and their ability to specifically block the action of C3a and C4a on smooth muscle tissue support the conclusion that, as in C3a, the essential active site of C4a resides at its COOH terminus. Since C4a functions as an anaphylatoxin and significant quantities of this mediator may be generated in individuals with hereditary angioneurotic edema (HANE), the hypotheses that the kinin-like activity promoting edema in HANE patients is derived solely from component C2 and/or kininogens should be reappraised. The activities previously assigned to C4a and now confirmed by synthetic C4a analog peptides suggest that the kinin-like activity generated in HANE plasma may be derived in part from C4a.     

MiR-34a inhibits proliferation and migration of breast cancer through down-regulation of Bcl-2 and SIRT1

MicroRNA-34a(miR-34a), a pivotal member of the p53 network, was found to be down-regulated in multiple types of tumors and further reported as a tumor suppressor microRNA. However, the profile and biological effects of miR-34a in breast cancer are still unclear. In this study, we aimed to determine the effect of miR-34a on the growth of breast cancer and to investigate whether its effect is achieved by targeting Bcl-2 and SIRT1. We examined miR-34a levels in breast cancer cell lines and breast cancer specimens by qRT-PCR. Proliferation assay, apoptosis assay, and morphological monitoring were performed to assess the tumor suppression effect of miR-34a in breast cancer cell lines. Western blotting was used to identify the targets of miR-34a. We also investigated the anti-tumor effects of the treatment combining miR-34a with 5-FU in breast cancer cells. We found that miR-34a expression was down-regulated in 5 breast cancer cell lines compared with the immortalized normal mammary epithelial cell line 184A1, and was also down-regulated by almost 50 % in breast cancer samples compared with their corresponding adjacent non-malignant breast tissues. Ectopic restoration of miR-34a in breast cancer cells suppressed cells proliferation, invasion, and induced apoptosis. Bcl-2 and SIRT1 as the targets of miR-34a were found to be in reverse correlation with ectopic expression of miR-34a. Furthermore, the treatment combining miR-34a with 5-FU significantly showed more efficient anti-tumor effects than single treatment of miR-34a or 5-FU. Since miR-34a functions as tumor suppressor microRNA in breast cancer, modulating miR-34a level in breast cancer was suggested to be a new and useful approach of breast cancer therapy.     

Sense receptors with electron-dense supporting structures, and centrioles in the nerve fibres of Gieysztoria and Rhinolasius (Platyhelminthes, Rhabdocoela)

The ultrastructure of two types of sense receptors each is described for Gieysztoria and Rhinolasius. In Gieysztoria, one receptor possesses a number of long microvilli, a cilium with a cross-striated rootlet, a septate desmosome, and an electron-dense band apical to the desmosome. The band forms a complex reticulate extension into the base of the receptor. The second receptor is without microvilli, has a cilium with a long cross-striated rootlet, and a septate desmosome with a short basal electron-dense extension or extensions which do not form a complete ring. In Rhinolasius, one receptor possesses a short bulbous cilium without a rootlet, with a septate desmosome of the pleated sheet (comb) type and a weakly developed electron-dense band beneath it. The second receptor has a long cilium with cross-striated rootlet, and a septate desmosome with a weakly developed electron-dense band apical to it. Centrioles were found in nerve fibres of both species.     

Allergy screening with Phadiatop® and CAP Phadiatop® in combination with a questionnaire in adults with asthma and rhinitis

The efficiency of the new screening tests for atopy, Phadiatop and CAP Phadiatop, was studied by comparing their results with a clinical diagnosis of atopy in 100 consecutive adults with asthma and/or rhinitis. Further, the diagnostic efficiency of a combination of Phadiatop and a few standardized questions was studied. The Phadiatop was found to have a specificity of 0.98, and a sensitivity of 0.92 and the CAP Phadiatop a specificity of 0.94 and a sensitivity of 0.96. When the Phadiatop was combined with a few questions, a sensitivity of 1.00 was achieved. It is concluded that Phadiatop and CAP Phadiatop have a higher diagnostic precision than other hitherto used methods for screening of atopic allergy. The place of Phadiatop in a diagnostic flow chart is suggested.     

Basaloid squamous carcinoma with a spindle cell component and osseous metaplasia presenting as a polyp of the hypopharynx

We report a case of basaloid squamous carcinoma with a spindle cell component of the hypopharynx, in a 61-years-old-man. An excisional biopsy of a pedonculated and polypoid tumour was performed by endoscopy. The histologic examination revealed a biphasic tumour with both a basaloid carcinomatous and a spindle cell component. Focally, osseous metaplasia was seen. The spindle cell component demonstrated immunoreactivity with the p63 epithelial marker. The patient was treated with chemotherapy followed by radiotherapy. The patient has been free of disease for one year. It's the ninth case reported in the literature of a biphasic carcinoma with both a basaloid squamous and a spindle cell component and the first case with osseous metaplasia.     

Producing the vowel/a/ prior to speaking inhibits stuttering in adults in the English language

This study investigated the effects of producing and listening to the vowel /a/ on the frequency of overt stuttering moments in eight people who stuttered. Stuttering frequency counts were made for the speech produced in the control condition, and after each of these four experimental conditions: (a) producing a vowel /a/ for 4 s; (b) producing a vowel /a/ for 4 s and waiting for 4 s; (c) listening to a recording of the vowel /a/ for 4 s; and (d) listening to a recording of the vowel /a/ for 4 s and waiting for 4 s. A significant reduction in the stuttering frequency was only observed following production of the vowel /a/ without a 4 s delay (P=0.02), suggesting that the vowel production prior to speech, serves as a temporary fluency enhancer. Its similarity to the occurrence of overt stuttering moments (e.g. discrete part-word repetitions and prolongation's) and its relationship to the fundamental nature of the pathology are discussed.