Patients With Atopic Dermatitis Sensitized to Pet Dander Mount IgE and T-Cell Responses to Mammalian Cystatins, Including the Human Self-Protein.

BACKGROUND: Immediate and delayed-type hypersensitivity reactions to pet-borne allergens are common in atopic diseases. In atopic dermatitis (AD), controversy surrounds the contribution to the disease of cross-reactivity to self-proteins. Human cystatin A and the cat allergen Fel d 3 belong to the cystatins, an evolutionary conserved protein family. The objective of the present study was to assess crossreactivity between mammalian cystatins and to analyze T-cell responses to cystatin in AD patients sensitized to pet dander. METHODS: cDNA coding for dog cystatin was cloned from dog skin. Sera from 245 patients with IgE-mediated sensitization to cat and dog dander were tested for IgE binding to recombinantly expressed feline, canine, and human cystatin. Of these, 141 were also diagnosed with AD. RESULTS: Cystatin-specific IgE was detected in 36 patients (14.7%), of whom 19 were considerably affected by AD. Within the AD patients, 9 had measurable IgE against all 3 cystatins. Cystatin-sensitized AD patients did not differ from non-cystatin-sensitized patients in terms of disease severity, age, or total IgE levels. T-cell cytokine measurements showed elevated IL-4 levels after stimulation with feline and human cystatin. CONCLUSIONS: The humoral response suggests that in addition to Fel d 3, the homologous protein from dog might play a role in allergy. Furthermore, human cystatin appears to be capable of driving a type 2 immune response in sensitized AD patients and may therefore be considered a so-called autoallergen, as proposed for other evolutionary conserved proteins.

Phosphorylation of tyrosine residues 31 and 118 on paxillin regulates cell migration through an association with CRK in NBT-II cells.

Identification of signaling molecules that regulate cell migration is important for understanding fundamental processes in development and the origin of various pathological conditions. The migration of Nara Bladder Tumor II (NBT-II) cells was used to determine which signaling molecules are specifically involved in the collagen-mediated locomotion. We show here that paxillin is tyrosine phosphorylated after induction of motility on collagen. Overexpression of paxillin mutants in which tyrosine 31 and/or tyrosine 118 were replaced by phenylalanine effectively impaired cell motility. Moreover, stimulation of motility by collagen preferentially enhanced the association of paxillin with the SH2 domain of the adaptor protein CrkII. Mutations in both tyrosine 31 and 118 diminished the phosphotyrosine content of paxillin and prevented the formation of the paxillin-Crk complex, suggesting that this association is necessary for collagen-mediated NBT-II cell migration. Other responses to collagen, such as cell adhesion and spreading, were not affected by these mutations. Overexpression of wild-type paxillin or Crk could bypass the migration-deficient phenotype. Both the SH2 and the SH3 domains of CrkII are shown to play a critical role in this collagen-mediated migration. These results demonstrate the important role of the paxillin-Crk complex in the collagen-induced cell motility.

Functional evidence for an RNA template in telomerase.

The RNA moiety of the ribonucleoprotein enzyme telomerase from the ciliate Euplotes crassus was identified and its gene was sequenced. Functional analysis, in which oligonucleotides complementary to portions of the telomerase RNA were tested for their ability to prime telomerase in vitro, showed that the sequence 5' CAAAACCCCAAA 3' in this RNA is the template for synthesis of telomeric TTTTGGGG repeats by the Euplotes telomerase. The data provide a direct demonstration of a template function for a telomerase RNA and demarcate the outer boundaries of the telomeric template. Telomerase can now be defined as a specialized reverse transcriptase.

Origin and environmental setting of ancient agriculture in the lowlands of Mesoamerica.

Archaeological research in the Gulf Coast of Tabasco reveals the earliest record of maize cultivation in Mexico. The first farmers settled along beach ridges and lagoons of the Grijalva River delta. Pollen from cultivated Zea appears with evidence of forest clearing about 5100 calendar years B.C. (yr B.C.) [6200 (14)C years before the present (yr B.P.)]. Large Zea sp. pollen, typical of domesticated maize (Zea mays), appears about 5000 calendar yr B.C. (6000 yr B.P.). A Manihot sp. pollen grain dated to 4600 calendar yr B.C. (5800 yr B.P.) may be from domesticated manioc. About 2500 calendar yr B.C. (4000 yr B.P.), domesticated sunflower seeds and cotton pollen appear as farming expanded.

Telomere terminal transferase activity from Euplotes crassus adds large numbers of TTTTGGGG repeats onto telomeric primers.

A telomere terminal transferase activity was identified in developing macronuclear extracts from Euplotes crassus. The activity was essentially unregulated in vitro: up to 50 tandem repeats of the Euplotes telomeric repeat sequence TTTTGGGG were added onto synthetic telomeric oligonucleotide primers. Both the structure of the telomere substrate and its 3'-terminal sequence were recognized. The activity was destroyed by low concentrations of RNase A.

Patients With Atopic Dermatitis Sensitized to Pet Dander Mount IgE and T-Cell Responses to Mammalian Cystatins, Including the Human Self-Protein

Background: Immediate and delayed-type hypersensitivity reactions to pet-borne allergens are common in atopic diseases. In atopic dermatitis (AD), controversy surrounds the contribution to the disease of cross-reactivity to self-proteins. Human cystatin A and the cat allergen Fel d 3 belong to the cystatins, an evolutionary conserved protein family. The objective of the present study was to assess crossreactivity between mammalian cystatins and to analyze T-cell responses to cystatin in AD patients sensitized to pet dander. Methods: cDNA coding for dog cystatin was cloned from dog skin. Sera from 245 patients with IgE-mediated sensitization to cat and dog dander were tested for IgE binding to recombinantly expressed feline, canine, and human cystatin. Of these, 141 were also diagnosed with AD. Results: Cystatin-specific IgE was detected in 36 patients (14.7%), of whom 19 were considerably affected by AD. Within the AD patients, 9 had measurable IgE against all 3 cystatins. Cystatin-sensitized AD patients did not differ from non–cystatin-sensitized patients in terms of disease severity, age, or total IgE levels. T-cell cytokine measurements showed elevated IL-4 levels after stimulation with feline and human cystatin. Conclusion: The humoral response suggests that in addition to Fel d 3, the homologous protein from dog might play a role in allergy. Furthermore, human cystatin appears to be capable of driving a type 2 immune response in sensitized AD patients and may therefore be considered a so-called autoallergen, as proposed for other evolutionary conserved proteins. (AU)

Antecedentes: Las reacciones de hipersensibilidad de tipo inmediato y retardado a los alérgenos que están en las mascotas son comunes en las enfermedades atópicas. En este estudio, en pacientes con dermatitis atopica (DA), se analiza la reactividad cruzada con las autoproteínas y su contribución a la enfermedad. Tanto la cistatina A humana como el alérgeno felino Fel d 3 pertenecen a la familia de las cistatinas, una familia de proteínas conservadas evolutivamente. El objetivo del presente estudio fue evaluar la reactividad cruzada entre las cistatinas de mamíferos y analizar la respuestas de las células T a la cistatina en pacientes con DA sensibilizados a la caspa de las mascotas. Métodos: El ADNc que codifica la cistatina de perro se clonó a partir de piel de perro. Se analizaron sueros de 245 pacientes con sensibilización por IgE a la caspa de gato y perro para determinar la unión de IgE a cistatina felina, canina y humana expresada de forma recombinante, respectivamente. De estos 245 pacientes, 141 fueron diagnosticados de DA. Resultados: Se detectó IgE específica frente a cistatina en el 14,7% (36) de los pacientes, de los cuales 19 padecían DA. Dentro de los pacientes con DA, 9 tenían IgE medible contra las tres cistatinas. Los pacientes con DA sensibilizados frente a cistatina no difirieron de los pacientes no sensibilizados con cistatina en términos de gravedad de la enfermedad, edad o niveles totales de IgE. El análisis de citocinas de células T reveló niveles elevados de IL-4 después de la estimulación con cistatina felina y humana. Conclusión: La respuesta humoral sugiere que, además de Fel d 3, la proteína homóloga de perro también podría desempeñar un papel en la alergia. Además, la cistatina humana parece ser capaz de promover una respuesta inmune de tipo 2 en pacientes con DA sensibilizados y, por lo tanto, puede considerarse un autoalérgeno, como se ha propuesto para otras proteínas conservadas evolutivamente. (AU)

Prevention by tetrandrine of spontaneous development of diabetes mellitus in BB rats.

Treatment of BB rats with the plant alkaloid tetrandrine (20 mg.kg-1.day-1), a novel anti-inflammatory compound, from 35 to 120 days of age reduced the cumulative incidence of spontaneous diabetes from 75.5 to 10.9% (P less than 0.001). Dose-response studies with 0, 5, 10, and 20 mg.kg-1.day-1 of tetrandrine from 35 to 130 days resulted in spontaneous diabetes in 84.2, 63.1, 31.6, and 5.3% of the rats, respectively. When the start of treatment with 20 mg.kg-1.day-1 tetrandrine was delayed until 70 days of age, there was a significant reduction of the incidence of diabetes from 63.1 to 28.6% (P less than 0.01). Histological examination of the pancreases from tetrandrine-treated rats showed a lesser degree of insulitis than controls (P less than 0.01). Drug toxicity was not seen in the rats, as assessed by appearance, behavioral change, organ histology, and blood chemistry. These results provide some hope that tetrandrine may be of value in preventing diabetes and treating newly diagnosed diabetic subjects, either by itself or in combination with a more potent immunosuppressive agent.

Recovery from disuse osteopenia coincident to restoration of muscle strength in mdx mice.

The objective of this study was to compare the tibial structure and the strength of the tibia during muscle weakness and after recovery in mdx mice (which demonstrate X-linked muscular dystrophy and subsequent muscle regeneration) and age-matched control mice. The extent of disuse atrophy produced by muscle weakness and recovery following restoration of normal muscle strength could then be determined. The tibiae adjacent to weakened tibialis anterior muscles of 4-week-old mdx mice had significantly reduced radiographic density (p < 0.05) and cortical thickness (p < 0.001), and increased porosity (p < 0.001) compared to age-matched controls, suggesting development of disuse osteopenia. Significantly less force was required to break mdx tibiae than age-matched control tibiae (p < 0.05). In addition, Sharpey's fiber density was reduced (p < 0.001), suggesting a weakened attachment of the tibialis anterior muscle to bone. At 12 weeks, during the period of muscle regeneration, mdx tibial cortical thickness (p < 0.001) and porosity (p < 0.01) remained significantly lower, but percent calcium and Sharpey's fiber and radiographic densities were significantly greater (p < 0.001) than in age-matched controls, suggesting that bone mineralization and muscle attachment strength had increased to above normal levels in parallel with recovery of strength by the attached muscle. By 18 weeks, mdx tibial cross-sectional area, cortical thickness, and porosity remained significantly less (p < 0.001) than normal. Although Sharpey's fiber density was greater than in age-matched controls (p < 0.001) by 18 weeks, mdx tibial percent calcium (p < 0.005) and Sharpey's fiber density (p < 0.001) were significantly reduced from levels in 12-week-old mdx animals. There was significantly less deformation of the tibia prior to fracture in mdx than control tibiae at 18 weeks of age, suggesting tibial brittleness. Thus, at the site of attachment of mdx muscle to osteopenic bone, the remodelling which accompanies recovery of muscle strength is atypical, and produces an attachment of greater strength than function appears to require. These observations suggest that data are needed regarding bone mass and muscle-bone attachments in humans with disuse osteopenia, DMD, and other neuromuscular diseases.

The three 5S rRNA genes from the human malaria parasite Plasmodium falciparum are linked.

5S rRNA and rDNA from Plasmodium falciparum have been characterized. The 5S rRNA transcripts isolated from erythrocytic stage parasites are composed of three distinct subclasses, 117-119 nucleotides in length, which are identical in sequence with the exception of one or two additional uridine residues at the 3' terminus. Southern blot analysis of genomic DNA identified three 5S rRNA gene classes which are clustered within 1.5 kb of DNA. Cloning and sequence analyses of the 5S rDNA revealed identical coding regions surrounded by divergent extremely A+T rich flanking sequences (greater than 90%). Typical PolIII termination signals (6-8) T residues abut each coding region. Copy number analysis indicates that P. falciparum contains only three 5S rRNA genes, the lowest number reported for any organism.

Heterogeneity and expression of the Plasmodium falciparum 5.8S ribosomal RNA genes.

The number and expression of some of the large ribosomal RNA (rRNA) gene classes present in the human malaria parasite Plasmodium falciparum was determined. Southern blot analyses, using the 5.8S rRNA coding region as a marker, indicate that the P. falciparum genome contains at least 5 distinct subclasses of large rRNA genes. Dideoxy sequencing of the 5.8S rRNA domain and Northern blot analyses demonstrate that only one subclass is transcribed during the parasite's asexual erythrocytic life cycle.

Characterization and complete nucleotide sequence of a 5.8S ribosomal RNA gene from Plasmodium falciparum.

The 5.8S and 5S rRNA components from the FCR-3/The Gambia strain of Plasmodium falciparum have been identified and the complete nucleotide sequence of a 5.8S ribosomal RNA gene determined. Unlike the 5S rRNA species, the 5.8S is a single homogeneous population of molecules of 157 nucleotides. Comparison of its nucleotide sequence with previously reported 5.8S rRNA sequences indicates that it is homologous to these molecules, but distantly related to them. The sequence of the 5.8S rRNA coding region from the pfrib-2 recombinant of the HG13 Gambian isolate of P. falciparum is identical.

Synthesis and an X-ray structure of soluble phenylacetylene macrocycles with two opposing bipyridine donor sites

The synthesis of the shape-persistent macrocycles 10a and 10b with two bipyridine units in opposing sides by Hagihara/Sonogashira cross-coupling chemistry of suitably functionalized building blocks is reported. X-ray analysis of single crystals of 10b shows a layered structure with channels filled with solvent molecules and parts of the flexible chains. with which the cycle is decorated for solubility reasons.

Syntheses and 1H-, 13C- and 15N-NMR spectra of ethynyl isocyanide, H-C triple bond C-N triple bond C, D-C triple bond C-N triple bond C and prop-1-ynyl isocyanide, H3C-C triple bond C-N triple bond C, D3C-C triple bond C-N triple bond C: high resolution infrared specturm of prop-1-ynyl isocyanide

Ethynyl isocyanide, H-C triple bond C-N triple bond C (1a), deuteroethynyl isocyanide, D-C triple bond C-N triple bond C (1b), prop-1-ynyl isocyanide, H3C-C triple bond C-N triple bond C (1c), and trideuteroprop-1-ynyl isocyanide, D3C-C triple bond C-N triple bond C (1d) are synthesized by flash vacuum pyrolysis of suitable organometallic precursor molecules (CO)5Cr(CN-CCl triple bond CClH) (5a), (CO)5Cr(CN-CCI=CClD) (5b), (CO)5Cr(CN-CCl=CCl-CH3) (5c) and (CO)5Cr(CN-CCI=CCl-CD3) (5d), respectively. Compounds 5a-d are formed in two steps by radical alkylation of tetraethyl-ammonium pentacarbonyl(cyano)chromate, NEt4[Cr(CO)5(CN)] (2) by 1,1,2,2,-tetrachloroethane (3a), 1,1,2,2-tetrachloro-1,2-dideuteroethane (3b), 1,1,2,2,-tetrachloropropane (3c), and 1,1,2,2-tetrachloro- 1,3,3,3-tetradeutero-propane (3d) yielding [(CO)5Cr(CN-CCl2-CCl2-H)] (4a), [(CO)5Cr(CN-CCl2-CCl2D)] (4b), [(CO)5Cr(CN-CCl2-CCl2-CH3)] (4c), and [(CO)5Cr(CN-CCl2-CCl2-CD3)] (4d). Dehalogenation of 4a-d using zinc in diethylether/acetic acid gives 5a-d, respectively. A multinuclear NMR study revealed the 1H-, 13C- and 15N-NMR data of 1a and 1c. Molecular spectroscopic data of 1c were determined by high resolution infrared spectroscopy. The by-products of the pyrolysis are the E and Z isomers of the halogenated ethenyl isocyanides H(Cl)C=CCl-NC (6a) and H3C(Cl)C=CCl-NC (6c) which have been characterized by IR, MS and NMR spectroscopy.

8,9-Didehydrofluoranthenes as building blocks for the synthesis of extended polycyclic aromatic hydrocarbons (PAHs).

[reaction: see text] The synthesis of phenyl-substituted 8,9-dibromofluoranthene and p-dodecylphenyl-substituted 8,9-fluoranthene anthranilic acid is presented. Their synthetic potential as 8,9-didehydrofluoranthene precursors is demonstrated in combination with a new biscyclopentadienone by the synthesis of novel phenyl-substituted PAHs with up to 14 annulated rings. The crystal structure of 7,16-diphenylfluorantheno[8,9-k]fluoranthene is given.

Effects of drug-induced pulmonary phospholipidosis on lung mechanics in rats.

Chronic administration of amphiphilic drugs to rats induces pulmonary phospholipidosis (P), a disease characterized by accumulation of phospholipids and large foamy macrophages in alveolar spaces. We investigated whether P induced by chlorphentermine (CPH) causes changes in lung volumes and mechanics in this species. Groups of rats were fed CPH (50 mg.kg-1.day-1) for 1, 2, 3, 5, 9, and 14 wk. After each treatment period, lung volumes and mechanics were studied in the anesthetized, paralyzed, supine rat. Partial pressure-volume (PV) curves were developed at 3 and 6 ml above functional residual capacity (FRC; PV3, PV6), followed by maximal [up to total lung capacity (TLC)] PV curves. FRC was determined by saline displacement. Lungs were then fixed for histopathological examination. A subgroup of animals was allowed a recovery period of 6 wk, after the 9 wk of CPH administration. Pair-fed rats served as controls (CTR) at each time point. Lung weight increased in CPH-treated (CPH-T) rats from 1.5 +/- 0.2 (SD) g at week 1 to 5.8 +/- 1.4 g at week 14, reflecting the development of P. TLC, FRC, transpulmonary pressure at FRC, the shape of maximal PV curves, and static expiratory lung compliance computed from maximal PV data points did not change in CPH-T rats. However, partial PV curves of CPH-T lungs (particularly PV3) were shifted downward and to the right of those of CTR at 2, 3, 5, and 9 wk, indicating increased recoil pressure in phospholipidotic lungs at these time points.(ABSTRACT TRUNCATED AT 250 WORDS)

Low-temperature crystallization and structure determination of N-(trifluoromethyl)formamide, N-(2,2,2-trifluoroethyl)formamide and 2,2,2-trifluoroethyl isocyanide.

Crystals of N-(trifluoromethyl)formamide, C(2)H(2)F(3)NO, (I), N-(2,2,2-trifluoroethyl)formamide, C(3)H(4)F(3)NO, (II), and 2,2,2-trifluoroethyl isocyanide, C(3)H(2)F(3)N, (III), were grown in situ on an X-ray diffractometer and analysed by single-crystal X-ray diffraction methods at low temperatures. Crystal data: (I) orthorhombic, P2(1)2(1)2(1), a = 4.547 (2) Å, b = 5.947 (3) Å, c = 14.731 (9) Å, V = 398.3 (4) Å(3), Z = 4, M(r) = 113.05, T = 143 K, D(x) = 1.885 Mg m(-3); (II) monoclinic, P2(1)/n, a = 4.807 (1) Å, b = 16.707 (3) Å, c = 6.708 (1) Å, beta = 109.90 (1) degrees, V = 506.6 (2) Å(3), Z = 4, M(r) = 127.07, T = 141 K, D(x) = 1.666 Mg m(-3); (III) orthorhombic, P2(1)2(1)2(1), a = 5.668 (2) Å, b = 9.266 (3) Å, c = 8.626 (2) Å, V = 453.0 (2) Å(3), Z = 4, M(r) = 109.06, T = 163 K, D(x) = 1.599 Mg m(-3). The results showed that in the crystal both formamides (I) and (II) are exclusively present in the form of the Z isomer, although measurements of solutions of (I) have shown that the E isomer prevails [Lentz et al. (1987). Angew. Chem. 99, 951-953]. In addition ab initio calculations for (I) predicted the E isomer to be the more stable one. In compound (III) the isocyanide group is staggered with respect to the trifluoroethyl group. In the crystal packing of (I) and (II) intermolecular N-H.O hydrogen bonds generate infinite chains. In (I), these chains are linked to form sheets by C-H.O contacts. In the crystal structure of (III) each isocyanide dipole is surrounded by four electronegative F atoms with intermolecular C.F contacts between 3.4 and 3.5 Å.

Antimicrobial properties of Honduran medicinal plants.

Ninety-two plants used in the traditional pharmacopoeia of the Pech and neighboring Mestizo peoples of central Honduras are reported. The results of in vitro antimicrobial screens showed that 19 of the extracts from medicinal plants revealed signs of antifungal activity while 22 demonstrated a measurable inhibitory effect on one or more bacterial cultures. Bioassay-guided fractionation of extracts from Mikania micrantha, Neurolaena lobata and Piper aduncum produced weak to moderately active isolates. The broad spectrum of activity of the extracts helps to explain the widespread use of these plants for wound healing and other applications.

Surface characteristics of glass-ionomer cements when treated with cavity varnish.

Two glass-ionomer cements were evaluated. Sixty molds were prepared, 20 with a lining cement, 20 with a base cement, and 20 with a control cement. The samples were tested for hardness; the glass-ionomer materials proved to be significantly harder than the control.

Comparison of marginal adaptation between direct and indirect composites.

The evaluation of composite resin as a posterior restorative material has been slow and attended by several problems. Gap formation has been considered a serious problem by many researchers. The purpose of this in vitro study is to compare the marginal adaptation of posterior composite resins placed by a direct method with those placed by an indirect method utilizing flexible dyes. Conclusions drawn from the results of this study are that both techniques produced marginal gaps. Also there were no differences in the width of marginal gaps in the composite resin restorations placed by our direct or indirect methods, and no marginal gap differences were observed as a result of anatomical location.

The suitability of a rolled BHK21 monolayer system for the production of vaccines against the SAT types of foot-and-mouth disease virus. I. Adaptation of virus isolates to the system, immunogen yields achieved and assessment of subtype cross reactivity.

In an examination of 34 southern African SAT-type foot-and-mouth disease viruses, all but 1 attained satisfactory levels of infectivity within 6 passages in rolled BHK21 monolayer cell cultures. However, there were marked differences between adapted viruses with respect to the mass of immunogen (146S material) produced. Several isolates which consistently produced levels greater than or equal to 2 micrograms/ml were identified. In cross neutralization tests using post-vaccinal sera, SAT-1 and SAT-2 isolates showed considerable diversity and none of the viruses tested would be expected to produce a broad-spectrum response if incorporated into a vaccine. On the other hand, when 2 of the SAT-2 isolates were incorporated into the same vaccine a distinctly broader response resulted.