Regulation of tubulin and actin mRNA production in rat brain: expression of a new beta-tubulin mRNA with development.

The expression of alpha-tubulin, beta-tubulin, and actin mRNA during rat brain development has been examined by using specific cDNA clones and in vitro translation techniques. During brain maturation (0 to 80 days postnatal), these mRNA species undergo a significant decrease in abundance. The kinetics of this decrease varies between the cerebrum and the cerebellum. These mRNAs are most abundant in both tissues during week 1 postnatal, each representing 10 to 15% of total mRNA activity. Both alpha- and beta-tubulin mRNA content decreases by 90 to 95% in the cerebrum after day 11 postnatal, and 70 to 80% decreases in the cerebellum after day 16. Actin sequences also decrease but to a lesser extent in both tissues (i.e., 50%). These decreases coincide with the major developmental morphological changes (i.e., neurite extension) occurring during this postnatal period. These studies have also identified the appearance of a new 2.5-kilobase beta-tubulin mRNA species, which is more predominant in the cerebellar cytoplasm. The appearance of this form occurs at a time when the major 1.8-kilobase beta-tubulin mRNA levels are declining. The possibility that the tubulin multigene family is phenotypically expressed and then this expression responds to the morphological state of the nerve cells is discussed.

Domains of beta-tubulin essential for conserved functions in vivo.

The relationship between the primary sequence of tubulins and their properties in cells was studied by gene transfection experiments. Previously, we studied a chimeric beta-tubulin formed from chicken beta-tubulin-2 sequences in the amino-terminal portion and the highly divergent Saccharomyces cerevisiae TUB2 sequences in the carboxy-terminal 25% of the molecule. In the cytoplasm of cultured animal cells, this protein incorporates into all microtubule structures and assembles with the same efficiency as endogenous tubulin. We show that the protein products of chimeric genes with an increasing proportion of yeast sequence, extending 5' of the carboxy-terminal 25%, are abnormal in two ways. First, they assemble with a significantly lower efficiency than the original chimeric protein or the endogenous tubulins. Second, they are less stable in the cytoplasm. The results suggest that the position of the yeast sequences is crucial in determining the properties of the molecule. Results of analyses of 1 deletion mutation and 10 linker insertions in the original chimeric tubulin suggest that those changes made outside the carboxyl terminus completely disrupt assembly activity, while those made in the carboxyl terminus do not.

Differential expression of two neural cell-specific beta-tubulin mRNAs during rat brain development.

We recently demonstrated that beta-tubulin mRNA expression is regulated during rat brain development. This is manifested by a dramatic decrease in both 1.8- and 2.9-kilobase (kb) mRNAs when extensive neurite elongation is occurring. Coincident with these decreases is the increased production of a 2.5-kb mRNA. (J.F. Bond and S.R. Farmer, Mol. Cell. Biol. 3:1333-1342, 1983). In the present study, we have isolated and characterized three different cDNAs corresponding to beta-tubulin mRNAs (R beta T.1, R beta T.2, and R beta T.3). Hybridization of 3' untranslated region subclones of R beta T.1 and R beta T.2 cDNAs to RNA from a variety of rat tissues and cells revealed that these two cDNAs are neural cell specific. R beta T.1 corresponds to an abundant 1.8-kb mRNA expressed only at early stages of rat brain development. R beta T.2 corresponds to the 2.5-kb mRNA expressed at later stages. These data strongly suggest that there is differential expression of the beta-tubulin multigene family during rat brain development.

Recombinant Fel d.I: Expression, purification, IgE binding and reaction with cat-allergic human T cells.

This study describes the properties of the two recombinantly expressed polypeptide chains of Fel d I, the major allergen produced by the domestic cat (Felis domesticus). An inframe linker encoding polyhistidine has been added to the 5' ends of the Fel d I chains 1 and 2 cDNAs to facilitate purification using Ni2+ ion affinity chromatography. This method provides high yields in a single step of rchain 1 and rchain 2 of Fel d I with a > 90% level of purity. Polymerase chain reaction (PCR) methods were used to introduce a thrombin cleavage site (LVPR decreases GS) at the N-terminus of both chains. Thrombin cleavage of rchain 1 and rchain 2 followed by HPLC purification of the cleavage products allowed the isolation of each recombinant chain with only two additional residuals (GS) at the N-terminus of the native sequence. Amino acid sequencing analysis of the N-terminus and mass spectrometry of these polypeptides demonstrated that they are highly pure and full-length. Direct ELISA assays showed that IgE from cat-allergic patients binds to both rchain 1 and rchain 2 of Fel d I, demonstrating that both these chains contribute to the allergenicity of this heterodimeric protein. An examination of the reactivity of T cells derived from cat-allergic patients revealed that both polypeptide chains contribute to the T cell response to this allergen. Consequently, it is concluded that the immunological response to Fel d I is composed of a reaction at both the B and T cell level to each of the two chains that constitute the native allergen.

Enhanced immunoreactivity and preferential heterodimer formation of reassociated Fel d I recombinant chains.

In this study we have addressed the question of whether reassociating the two recombinant protein chains that comprise the major cat dander allergen, Fel d I, would change the overall IgE and allergic patient T cell immunoreactivity compared to the native molecule. To accomplish this, the chains were combined under reducing and denaturing conditions, then allowed to reassociate by dilution and extensive dialysis against a physiological buffer. An initial examination of the reaction products using quantitative capture ELISA demonstrated comparable reactivity to Fel d I. Further analysis, using a pool of cat allergic patient plasma, showed that the products of the reassociation reaction (rFel d I) also possessed an enhanced IgE binding capacity. Depletion ELISA results gave only a 5% difference in reactivity between rFel d I and the native protein versus a 20% difference with the mixture of the two chains. Comparative secondary T cell stimulation assays were subsequently performed using cat allergic patient peripheral blood lymphocytes. Here the results demonstrated no loss of reactivity with the reassociated chains as compared to Fel d I or the two mixed recombinant chains. To biochemically characterize the products of the reassociation reaction we have performed reverse phase HPLC and then analysed the isolated fractions by mass spectrometry. It was clear from these results that like the native Fel d I, the products of the reassociation reaction favored heterodimer formation, with no homodimer being detected. This implies that the reassociated protein chains had preferentially adopted a native-like conformation.

Native and recombinant Fel dI as probes into the relationship of allergen structure to human IgE immunoreactivity.

To delineate the relationship between the structural conformation and the stability of an allergen and its antigenicity, we have chosen the major allergen from cat dander, Fel dI. From protein sequence analysis data we have examined the structure of the naturally occurring Fel dI and we have found it to exist as an anti-parallel heterodimer. We have used ELISA, RAST, Western blot and histamine release techniques to compare the IgE reactivity of a set of cat allergic patient samples to purified, native Fel dI and the E. coli expressed chains 1 and 2. Results from these studies demonstrate a significant level of IgE reactivity to all forms when examined for direct binding. However, both blot and ELISA competition assays show a much higher reactivity to Fel dI in solution compared to the separate recombinant chains and this is supported by the histamine release data. Although native Fel dI chain 2 contains an N-linked carbohydrate moiety, this does not seem to play a role in the reactivity of IgE to chain 2. Denaturation of Fel dI with alkali conditions leads to a dramatic decrease in IgE reactivity, even though measurable changes to the backbone structure of the protein are minimal. One proposed explanation is that both chains possess a core region determined by their primary structures and that the major IgE epitopes are dependent upon them. The relative reactivity amongst these allergen forms varied with the method of analysis, implying that the conformational requirements for IgE antibody binding are best studied by the application of more than one experimental protocol. Results from these qualitative analyses afford insight into the allergenicity of this exceptionally stable cat pelt protein.

Purification and immunochemical characterization of recombinant and native ragweed allergen Amb a II.

The complete sequence of a cDNA encoding Amb a II and its relationship to the Amb a I family of allergens has recently been described [Rogers et al. (1991) J. Immun. 147, 2547-2552; Griffith et al. (1991a), Int. Archs Allergy appl. Immun. 96, 296-304]. In this study, we present results generated with rabbit antipeptide antisera that recognize Amb a II or Amb a I, but not both. The specificity of two anti-Amb a II antipeptide sera, anti-RAE-50.K and anti-RAE-51.K, was verified on Western blots of recombinant Amb a II and Amb aI.1. These two sera, directed against separate regions of the Amb a II molecule, detected three individual 38-kDa Amb a II isoforms on 2D Western blots of aqueous ragweed pollen extract. These Amb a II isoforms have pI in the 5.5-5.85 range and can be easily distinguished from Amb a I isoforms with pI in the 4.5-5.2 range detected by an anti-Amb a I specific peptide antiserum. The Amb a II isoforms have also been individually purified from pollen, positively identified as Amb a II by amino acid sequencing, and visualized as separate bands on IEF gels. An analysis of Amb a II cDNA sequences generated by PCR led to the prediction of three Amb a II isoforms with pI of 5.74, 5.86 and 5.97 that are very similar to the pI deduced from 2D Western blot analysis. Recombinant Amb aI.1 and Amb a II have been expressed in E. coli, purified in their denatured form, and examined by ELISA for their capacity to bind pooled allergic human IgE. Purified native Amb a and Amb a II from pollen were shown to have very similar IgE-binding properties. In contrast, Amb a II had a markedly reduced IgE-binding capacity as compared to Amb a I.1. These data suggest that recombinant Amb a I.1 and Amb a II, isolated in a denatured form, differ significantly in their IgE-binding properties whereas the native molecules isolated from pollen do not.

Potential therapeutic recombinant proteins comprised of peptides containing recombined T cell epitopes.

The complete primary structure of Fel d I2 has been determined and shown to be comprised of two separate polypeptide chains (designated chain 1 and 2). Overlapping peptides covering the entire sequence of both chains of Fel d I have been used to map the major areas of human T cell reactivity. The present study describes three non-contiguous T cell reactive regions of < 30 aa in length that were assembled in all six possible configurations using PCR and recombinant DNA methods. These six recombinant proteins comprised of defined non-contiguous T cell epitope regions artificially combined into single polypeptide chains have been expressed in E. coli, highly purified, and examined for their ability to bind to human cat-allergic IgE and for human T cell reactivity. Several of these recombined T cell epitope-containing polypeptides exhibit markedly reduced IgE binding as compared to the native Fel d I. Importantly, the human T cell reactivity to individual T cell epitope-containing regions is maintained even though each was placed in an unnatural position as compared to the native molecule. In addition, T cell responses to potential junctional epitopes were not detected. It was also demonstrated in mice that s.c. injection of T cell epitope-containing polypeptides inhibits the T cell response to the individual peptides upon subsequent challenge in vitro. Thus, these recombined T cell epitope-containing polypeptides, which harbor multiple T cell reactive regions but have significantly reduced reactivity with allergic human IgE, constitute a novel potential approach for desensitization to important allergens.

Comparison of information processing technologies.

OBJECTIVE: To examine the type of information obtainable from scientific papers, using three different methods for the extraction, organization, and preparation of literature reviews. DESIGN: A set of three review papers was identified, and the ideas represented by the authors of those papers were extracted. The 161 articles referenced in those three reviews were then analyzed using 1) a formalized data extraction approach, which uses a protocol-driven manual process to extract the variables, values, and statistical significance of the stated relationships; and 2) a computerized approach known as "Idea Analysis," which uses the abstracts of the original articles and processes them through a computer software program that reads the abstracts and organizes the ideas presented by the authors. The results were then compared. The literature focused on the human papillomavirus and its relationship to cervical cancer. RESULTS: Idea Analysis was able to identify 68.9 percent of the ideas considered by the authors of the three review papers to be of importance in describing the association between human papillomavirus and cervical cancer. The formalized data extraction identified 27 percent of the authors' ideas. The combination of the two approaches identified 74.3 percent of the ideas considered important in the relationship between human papillomavirus and cervical cancer, as reported by the authors of the three review articles. CONCLUSION: This research demonstrated that both a technically derived and a computer derived collection, categorization, and summarization of original articles and abstracts could provide a reliable, valid, and reproducible source of ideas duplicating, to a major degree, the ideas presented by subject specialists in review articles. As such, these tools may be useful to experts preparing literature reviews by eliminating many of the clerical-mechanical features associated with present-day scientific text processing.

Gas chromatographic determination of cadaverine, putrescine, and histamine in foods.

A GLC procedure for the quantitative determination of the diamines putrescine and cadaverine has been developed, using their perfluoropropionyl derivatives. The amines were extracted from foods with methanol; an interval standard, hexanediamine, was added and a dry residue of their hydrochloride salts was prepared. The salts were derivatized with perfluoropropionic anhydride by heating for 30 min at 50 degrees C. The reaction mixture was separated on an alumina column to remove excess reagent, and the derivatives were eluted with a solution of 30% ethyl acetate in toluene. GLC separations were performed on a 3% OV-225 column held at 180 degrees C. The retention times were 4.3, 5.7, and 7.0 min for the derivatives for putrescine, cadaverine, and the internal standard, respectively. Less than 1 microgram diamine/g tissue could be quantitated, using either an electron capture detector or a nitrogen-specific detector. The procedure was applied to cheese and a variety of fishery products. An increase in the diamines correlated with the presence of decomposition in some of the products. A collaborative study of the method is planned.

Fluorometric determination of histamine in tuna: development of method.

Tuna extracts are treated with an anion exchange resin to remove interfering materials, histamine is derivatized with o-phthaladehyde, and the fluorescence of the resulting compound is measured fluorometrically. Replicate analyses of acceptable and decomposed tuna packed in oil or water agreed within 1 mg at a level of 10 mg/100 g and within 12 mg at a level of 100 mg/100 g. Recoveries of histamine added to fish were greater than 90 and greater than 83% at levels of 10 and 100 mg/100 g, respectively. The new method is more rapid and specific and is simpler than previous methods because no liquid-liquid extractions or chromatographic separations of histamine are required. The sensitivity of the method allows quantitation of less than 10 mg histamine/100 g sample. The accuracy and precision of the fluorometric method are comparable to those of the official AOAC colorimetric method.

A chicken-yeast chimeric beta-tubulin protein is incorporated into mouse microtubules in vivo.

The role of divergent primary sequences in restricting tubulin function was tested in vivo by a gene transfection experiment. A chicken-yeast chimeric beta-tubulin DNA was introduced into 3T3 cells using the transfection vector pSV2. The 5' end of this gene, from chicken, is similar but not identical with that of mouse beta-tubulins; the 3' end, from yeast, contains a carboxyl terminus that is very different from other known beta-tubulin sequences. The chimeric protein is incorporated efficiently into each of the microtubule structures and each of the microtubules in the host cells. The presence of the protein has no apparent effect on either growth rate or cell morphology. The results suggest that the divergent sequences in this chimeric tubulin molecule place no restrictions on its activities in mouse cells.

An increased frequency of IgE-producing B cell precursors contributes to the elevated levels of plasma IgE in atopic subjects.

BACKGROUND: The production of specific IgE, which underlies the allergic response, may be a normal correlate of the immune response to a certain class of antigen (allergens), or could represent a unique response driven by regulatory signals that are absent in non-allergic individuals. If atopic subjects do possess a regulatory environment favoring IgE production, they may display not only allergen-specific IgE, but also higher levels of total IgE and higher frequencies of IgE-producing B lymphocytes. OBJECTIVE: To address the contribution of antibody-producing cell number to the circulating IgE titre in atopic vs non-atopic subjects. METHODS: Frequency determination by limiting dilution of EBV transformants and Poisson distribution analysis. Titres of total and allergen-specific IgM, IgG, and IgE by specific ELISA. RESULTS: In contrast to findings reported by others, the atopic subjects had a significantly higher frequency of IgE-producing B cells than non-atopics (0.79% of total Ig-producing cells, as compared with 0.17% for the control group; P < 0.01), suggesting that one factor contributing to the high plasma IgE titres in atopic subjects is the high frequency of B lymphocytes with the potential to produce IgE. Although only the atopic subjects produced allergen-specific IgE, the frequency of specific IgE-producing B cells was undetectable in both groups. CONCLUSION: Atopic subjects have higher frequencies of IgE-producing B cell precursors than non-atopics. A correlation exists between IgE-producing B cell frequency and levels of circulating IgE.

Comparison of the levels of the major allergens Der p I and Der p II in standardized extracts of the house dust mite, Dermatophagoides pteronyssinus.

Allergy to the house dust mite Dermatophagoides pteronyssinus is mediated by IgE to the major allergens Der p I and Der p II in the majority of mite-allergic patients. In recent years, standardized preparations of D. pteronyssinus, commercially available from several sources, have become widely used for the diagnosis and immunotherapy of mite allergy. As standardization implies uniformity of allergen composition and potency, we directly compared the absolute and relative quantities of Der p I and Der p II in six different commercial standardized extracts of D. pteronyssinus. Our findings reveal variability in levels of both Der p I and Der p II, producing ratios of Der p I/Der p II ranging from 1.1/1 to 6/1. Although the content of minor allergens in the extracts was not evaluated here, their contribution to the overall reactivity of mite-allergic patients to the commercial extracts was judged to be minimal. This was demonstrated by showing that plasma depleted of reactivity to both Der p I and Der p II had virtually no residual IgE directed against extract components. The variation in the proportion of Der p I and Der p II among different D. pteronyssinus extracts is likely to influence their biological effectiveness. Patients with reactivity against only Der p I or Der p II, who were found to comprise approximately one-third of the mite-allergic population, may not respond optimally to extracts containing relatively low levels of the allergen to which they are sensitive.

Definition of the human T-cell epitopes of Fel d 1, the major allergen of the domestic cat.

BACKGROUND: A heterodimeric acidic glycoprotein (Fel d 1) has been defined as the major allergen of the domestic cat. Because T-cell help is required for the initiation and maintenance of allergic responses, it is of importance to determine the T-cell-reactive regions of the Fel d 1 molecule. METHODS: Overlapping peptides corresponding to the two chains of Fel d 1 were tested in proliferation assays on polyclonal T-cell lines and for the ability to bind Fel d 1-specific IgE in ELISA and histamine release assays. RESULTS: Assay of T-cell lines derived from 53 subjects allergic to cats demonstrated that the majority of T-cell reactivity is found in chain 1 of Fel d 1. Two peptides (Fel-1 and Fel-2) containing major epitopes, alone or as a mixture, efficiently activated T cells and exhibited minimal detectable reactivity with IgE by ELISA or histamine release assay. CONCLUSIONS: Two Fel d 1 peptides containing major T-cell epitopes have been identified, have been shown to bind minimal Fel d 1-specific IgE, and are now being tested for the ability to decrease T-cell responses in patients with cat allergy as a new form of immunotherapy.

Cloning of Amb a I (antigen E), the major allergen family of short ragweed pollen.

To determine the structure of Amb a I (previously called antigen E), the major allergen from short ragweed, cDNA from pollen was cloned into lambda gt11 and lambda gt10. One of the three distinct clones isolated from the lambda gt11 library by screening with anti-denatured Amb a I antibodies was used to screen both libraries for other Amb a I sequences. Multiple clones were isolated and sequenced and proved to be highly homologous but nonidentical. The clones could be divided into three groups based on sequence similarity, and in accordance with the International Union of Immunological Societies-approved nomenclature (Marsh, D. G., Goodfriend, L., King, T. P., Lowenstein, H., and Platts-Mills, T. A. E. (1986) Bull. WHO 64, 767-770) they have been designated Amb a I.1, Amb a I.2, and Amb a I.3. Clones within a group have greater than 99% identity, and similarity among groups is 85-90% at the nucleotide level. The amino acid sequence of four peptides (isolated from antigen E obtained from the Research Resources Branch of the National Institutes of Health) containing 132 amino acids was identical to one of the clones (Amb a I.1). The presence of multiple naturally occurring isoelectric forms of Amb a I was demonstrated by two-dimensional gel electrophoresis and Western blotting. Southern blot analysis demonstrates the presence of multiple Amb a I-related sequences in the ragweed genome. Amb a I is therefore not a single molecule but rather a family of closely related proteins.

Amino acid sequence of Fel dI, the major allergen of the domestic cat: protein sequence analysis and cDNA cloning.

The complete primary structure of Fel dI (International Union of Immunological Societies nomenclature), the major allergen produced by the domestic cat, Felis domesticus, was determined by protein sequence analysis and cDNA cloning. Protein sequencing of Fel dI from an immunoaffinity-purified extract of house dust revealed that the allergen is composed of two polypeptide chains. Degenerate oligonucleotides derived from the protein sequence were used in polymerase chain reaction amplification of cat salivary gland cDNA to demonstrate that the two chains are encoded by different genes. Chain 1 of Fel dI shares amino acid homology with rabbit uteroglobin, while chain 2 is a glycoprotein with N-linked oligosaccharides.

Multiple Amb a I allergens demonstrate specific reactivity with IgE and T cells from ragweed-allergic patients.

The relationship between the structure and abundance of an inhaled protein and its potential for causing an allergic response is unknown. This study analyzes Amb a I, a family of related proteins formerly known as Ag E, that comprise the major allergens of short ragweed (Ambrosia artemisiifolia). T cells isolated from ragweed allergic patients were shown to proliferate in response to purified Amb a I.1 protein from pollen in in vitro secondary cultures, demonstrating the presence of T cell stimulatory epitopes in Amb a I.1. Three recombinant forms of Amb a I (Amb a I.1, Amb a I.2, and Amb a I.3) obtained as cDNA derived from pollen mRNA were expressed in bacteria. All three recombinant forms were shown to be specifically recognized by pooled ragweed-allergic human IgE on immunoblots, confirming these gene products are important allergens. An examination of immunoblots probed with sera derived from allergic patients revealed a variation in IgE binding specificity. A minority of patients' IgE exclusively reacted with recombinant Amb a I.1, whereas most patients' IgE reacted with Amb a I.1 as well as Amb a I.2 and Amb a I.3 proteins. A detailed examination of the reactivity of T cells derived from 12 allergic patients to these recombinant Amb a I forms revealed that these allergens are all capable of stimulating T cell proliferation in in vitro assays. It is concluded that the allergic response to ragweed pollen in most allergic patients is composed of a reaction to multiple related Amb a I proteins at both the B and T cell levels.