Kinetics of photo-active bacteriorhodopsin analog 3,4-didehydroretinal.

Kinetics of the photo-induced processes of the transient states of the 3,4-didehydroretinal (3,4-dhr) modified bacteriorhodopsin (bR) was studied by a flash photolysis method in a water suspension at room temperature. The excitation initiated a photocycle with several transient intermediates similar to the trans photocycle of native bR. The main observation of the study was that although major part (80%) of the population of the M state relaxed via the O intermediate as in natural bR, 20% relaxed directly to the bR ground state in 200 ms.

Color sensitive retina based on bacteriorhodopsin.

Bacteriorhodopsin (BR), a membrane protein of a microorganism Halobacterium salinarium has been studied since the 80's as a potential material for information technology. The information processing applications of BR employ either photochromic or photoelectric properties of the protein. In this study we discuss about design principles and describe our study of the use of bacteriorhodopsin as a sensor material for a color sensitive artificial retina. This retina includes low-level processing of input information. The design of a color sensitive matrix element, the self-organizing color adaptation algorithm and a system model for the retina are presented.

Color recognition with bacteriorhodopsin.

We have studied opto-electric properties of wild type bacteriorhodopsin and its two artificial variants. We have measured opto-electric responses with respect to wavelength for all three proteins and we describe the use of the proteins for color detection. Opto-electric responses of proteins to set of colored lights were measured and it has been shown that bacteriorhodopsin and its variants can be used to recognize color. A simple equation for estimating opto-electric response to arbitrary spectrum is given.

Complementary DNA cloning of the predominant allergen of bovine dander: a new member in the lipocalin family.

BACKGROUND: A number of allergenic proteins in animal danders have been characterized at the molecular level, but little is known of their biologic functions. We have found that the prevalence of IgE antibodies among patients with cattle-associated asthma is highest against a dander protein referred to as BDA20. OBJECTIVE: The study was performed to characterize the molecular structure of BDA20,* the predominant allergen in bovine dander. METHODS: Clones encoding allergens were identified and isolated from a complementary DNA library by immunoblotting and DNA hybridization and sequenced. Recombinant proteins were produced in Escherichia coli. Immunoreactivity of the recombinant proteins and amino acid sequences of peptides obtained from native BDA20 after Lys-C cleavage were used to identify clones coding for BDA20. RESULTS: In this article we report the cDNA and amino acid sequences of BDA20. Homology comparisons showed that BDA20 belongs to the family of lipocalins. CONCLUSIONS: The results link a dander allergen to a group of functionally important proteins. Lipocalins are present in various body fluids and secretions of several animal species in which they function as carriers of small hydrophobic molecules, such as retinoids and pheromones. If allergenicity proves to be a property shared by lipocalins, our results will have considerable implications for allergen research.

Immune reactivity of cow-asthmatic dairy farmers to the major allergen of cow (BDA20) and to other cow-derived proteins. The use of purified BDA20 increases the performance of diagnostic tests in respiratory cow allergy.

BACKGROUND: Cow dust is one of the most important inducers of occupational allergic diseases in Finland. For example, in 1991 it accounted for almost 40% of the new occupational asthma cases. OBJECTIVE: This study compares the performance of the purified major cow allergen (BDA20) and crude bovine epithelial extract (BEA) in diagnostic tests and examines the role of milk allergy-associated bovine proteins (bovine serum albumin, alpha-lactalbumin, beta-lactoglobulin, casein) in respiratory cow allergy. METHODS: The humoral responses of cow-asthmatic and healthy farmers to the various components of BEA were analysed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The levels of specific IgE and IgG antibodies were quantificated with enzyme-linked immunosorbent assays (ELISAs). The cellular responses were analysed with antigen-specific lymphocyte proliferation tests. RESULTS: The specific anti-BDA20 IgE measurement was found to be best in distinguishing between the asthmatic farmers and their healthy colleagues. It proved possible to determine a cut-off value that gave the analysis a specificity and sensitivity of 100%; the distinction between the two groups was highly significant (P < 0.0001). In the lymphocyte proliferation analysis, cow asthma was more closely associated with reactivity to BDA20 than to BEA. In the measurement of anti-BDA20 and anti-BEA IgG antibody levels, considerable overlap between the groups was observed, suggesting that these antibodies are not directly involved in cow allergy. When proteins associated with milk allergy were used as test reagents, no statistically significant differences could be observed between the groups, except for anti-casein IgE antibodies the level of which, however, overlapped considerably between the farmer groups. CONCLUSION: These findings suggest that purified BDA20 is better than BEA for diagnosing cow asthma and that proteins associated with milk allergy are of only marginal significance in this disease.

cDNA cloning and protein analysis of a bovine dermal allergen with homology to psoriasin.

Immunoscreening of a cDNA library from bovine skin led to isolation of clones coding for an allergen named BDA11. Sequence analysis of the clones revealed that they can encode a protein of 11.6 kDa with a predicted pI of 5.19. Allergenicity of BDA11 was verified by the IgE reactivity in cattle-allergic patients' sera with the recombinant protein produced in Escherichia coli. A biochemically purified native allergen of 11 kDa from bovine dander was identified as BDA11 by peptide sequencing. Homology comparisons showed that BDA11 had a 63.4% amino acid identity with human psoriasin. Psoriasin is a calcium-binding protein expressed in keratinocytes, and it is strongly up-regulated in psoriatic skin. BDA11 also had segments homologous with calcium-binding proteins from three other species.

Homology of a bovine allergen and the oligomycin sensitivity-conferring protein of the mitochondrial adenosine triphosphate synthase complex.

We have characterized bovine allergens by constructing and analyzing a complementary DNA library from bovine skin. Clones producing proteins that reacted with IgE antibodies from persons with allergy were purified and sequenced. One set of the allergen-coding clones showed an almost complete homology with the bovine oligomycin sensitivity-conferring protein of the mitochondrial adenosine triphosphate synthase complex. The IgE antibodies adsorbed with the recombinant allergen reacted with an 11 kd protein in the cow dander extract. Binding of the IgE from patients allergic to the recombinant allergen expressed in Escherichia coli confirmed the allergen-coding ability of the complementary DNA sequence. The prevalence of the IgE-positive sera among patients with cow allergy and control subjects suggests that the recombinant allergen represents one of the minor allergens in cow dander. This is the first time a mammalian allergen has been identified as a protein with a known function.

trans activation by the full-length E2 proteins of human papillomavirus type 16 and bovine papillomavirus type 1 in vitro and in vivo: cooperation with activation domains of cellular transcription factors.

Papillomaviral E2 genes encode proteins that regulate viral transcription. While the full-length bovine papillomavirus type 1 (BPV-1) E2 peptide is a strong trans activator, the homologous full-length E2 product of human papillomavirus type 16 (HPV-16) appeared to vary in function in previous studies. Here we show that when expressed from comparable constructs, the full-length E2 products of HPV-16 and BPV-1 trans activate a simple E2- and Sp1-dependent promoter up to approximately 100-fold in human keratinocytes and other epithelial cells as well as human and animal fibroblasts. Vaccinia virus-expressed, purified full-length HPV-16 and BPV-1 E2 proteins bound a consensus E2 site with high specific affinities (Kd = approximately 10(-9) M) and stimulated in vitro transcription up to six- to eightfold. In vivo and in vitro trans activation by either E2 protein required cooperation with another activator, such as Sp1, or other factors that interact with papillomavirus promoters, such as AP-1, Oct-1, nuclear factor 1/CTF, transcriptional enhancer factor 1, or USF. The glutamine-rich domain B of Sp1 or the mutually unrelated activation domains of other transcription factors were necessary and sufficient for cooperation with either E2 factor. We conclude that like BPV-1 E2, the HPV-16 E2 protein has the potential to function as a strong activator of viral gene expression in cooperation with cellular transcription factors.

Affinity purification of the major bovine allergen by a novel monoclonal antibody.

A monoclonal antibody was developed to the 20 kd major allergen of cow by immunizing mice with crude dander extract. The monoclonal antibody did not exhibit cross-reactivity to cat, dog, and horse dander extracts when studied by ELISA inhibition. The antibody was used in affinity chromatography for the purification of the 20 kd allergen from cow dander extract. Purity of the allergen was estimated to be 88%, and allergenic reactivity was verified by IgE immunoblotting and skin prick tests. After further purification with size-exclusion chromatography, the allergen was almost 100% pure. The isoelectric point of the double-purified allergen was determined to be 4.1. The amino acid composition was characterized by the predominance of acidic amino acids.

Transcriptional enhancer factor (TEF)-1 and its cell-specific co-activator activate human papillomavirus-16 E6 and E7 oncogene transcription in keratinocytes and cervical carcinoma cells.

The human papillomavirus (HPV)-16 oncogenes, E6 and E7, are transcribed preferentially in keratinocytes and cervical carcinoma cells due to a 5' enhancer. An abundant peptide binding to a 37 nt enhancer element was purified from human keratinocytes by sequence-specific DNA chromatography. This protein was identified as transcriptional enhancer factor (TEF)-1 by complex mobility, binding to wild-type and mutant SV40 and HPV-16 enhansons and antigenic reactivity with two anti-TEF-1 antibodies. TEF-1 is cell-specific, but its transactivation also depends on a limiting, cell-specific TEF-1 'co-activator'. We show that both TEF-1 and the TEF-1 co-activator are active in human keratinocytes and essential for HPV-16 transcription. TEF-1 binding in vivo was necessary for HPV-16 P97 promoter activity. Excess TEF-1 and chimeric GAL4-TEF-1 specifically inhibited the P97 promoter by 'squelching', indicating that HPV-16 transcription also requires a limiting TEF-1 co-activator. TEF-1 and the TEF-1 co-activator functions mirrored HPV-16 transcription by their presence in keratinocytes and cervical carcinoma cells and their absence from lymphoid B-cells, but also functioned in liver cells where the HPV-16 promoter is inactive. TEF-1 and its associated co-activator are thus part of a complex mechanism which determines the restricted cell range of the HPV-16 E6 and E7 oncogene promoter.

Transcriptional activation of the human papillomavirus-16 P97 promoter by an 88-nucleotide enhancer containing distinct cell-dependent and AP-1-responsive modules.

The P97 promoter upstream of the oncogenic early genes of human papillomavirus (HPV)-16 is active in keratinocytes and in cervical carcinoma cells due to a 5' keratinocyte-dependent cis enhancer. In this study, we have mapped the main enhancer activity to an 88-nucleotide (nt) fragment composed of multiple cis elements. A 63-nt promoter-proximal enhancer core was sufficient for P97 activation in a human keratinocytic cell line, HaCaT, and in cervical carcinoma cells. Although the enhancer functioned poorly in hepatoma cells or in fibroblasts, nuclear extracts from different cells protected similar cis elements from DNase I digestion. Two protected half-palindromic NF-I/CTF sites within the 63-nt core were necessary for its function; one represents a "cytokeratin element" (CK), a previously described 8-nt sequence shared with cytokeratin gene promoters. Both sites formed complexes of the same apparent size and relative binding affinity with NF-I/CTF-like factor(s) present in all cells tested. Although cell-dependent P97 activation could be determined by similar, yet distinct NF-I/CTF-like proteins, adjacent cis elements in the enhancer core were also required for function, and may thus interact with additional transcription factors. A 25-nt distal module with two AP-1 sites increased enhancer activity and cooperated with cis elements of the proximal core. Each AP-1 site as well as a third AP-1 site near the promoter bound c-Jun and Jun/Fos in vitro, and was activated by c-Jun and c-Fos in transfections. In addition to cell type-dependent activation, HPV-16 P97 transcription may therefore respond to growth factors and oncogene products via the AP-1 pathway.

Sandwich hybridization in solution: a rapid method to screen HPV 16 DNA in cervical scrapes.

A solution hybridization method is introduced as a rapid diagnostic method for demonstration of papillomavirus DNA in cervical scrapes. 32P-Labelled detector probe and the biotinylated capture probes were hybridized with DNA of the specimen after pretreatment by boiling in alkaline SDS. After 4 h of hybridization the hybrids were collected onto avidin coated beads and measured. The sensitivity of the method was 1-5 x 10(5) HPV 16 DNA molecules. Cervical carcinoma cell lines CaSki and SiHa were informative as to the sensitivity of the solution hybridization and the in situ hybridization methods. CaSki cells containing about 700 HPV 16 DNA copies per cell were positive by both methods. SiHa cells with one HPV 16 DNA copy per cell were positive by the sandwich assay but remained negative in the in situ test. A series of 126 cervical scrapes collected from consecutive patients participating in a follow-up study for cervical HPV infection were tested for HPV 16 DNA by both methods. The detection rate of the sandwich test was 19/126 (15%) and that of the in situ method 21/126 (17%) yielding 26 diagnoses altogether. Twelve of these were obtained by one method only. The results obtained by studying the cervical cell lines and repeated specimens taken from constantly HPV 16 positive patients suggested that the two methods can measure different types of infections and thus complement each other in the diagnosis of cervical HPV infections.

Prospective follow-up of cervical HPV infections: life table analysis of histopathological, cytological and colposcopic data.

A total of 532 women with established cervical HPV infection have been prospectively followed (without treatment) since 1981 for a mean of 45 (SD 21) months. The patients were examined by colposcopy, PAP smears and/or punch biopsy every 6 months. The life-table method was applied to analyze the clinical course (i.e. regression and progression) of the HPV lesions, stratified by their colposcopic pattern, PAP smear findings and grade of CIN. During the follow-up, 107 (41.8%) of 256 patients with HPV-NCIN lesion in the first punch biopsy, experienced spontaneous regression. The corresponding proportions for HPV-CIN I, HPV-CIN II and HPV-CIN III lesions were 31.1%, 34.2%, and 20.7%, respectively. In the overall comparison between these four groups, the heterogeneity in the probability of regression was statistically significant (p = 0.0005). Clinical progression was also associated significantly with the histological grade of the lesions in the first biopsy. Progression rate was only 5.8% for HPV-NCIN lesions, as compared to 12.3% for HPV-CIN I, 20% for HPV-CIN II, and 55.2% for HPV-CIN III. The probability of progression varied significantly between the four groups (p less than 0.00001). Cumulative proportion of regression was 46% for patients with PAP smear class I, 84% with class II, and 82% for those with class III, cells, i.e. PAP smear was not of value in predicting the regression. However, PAP smears predicted clinical progression (p = 0.006 overall).(ABSTRACT TRUNCATED AT 250 WORDS)

Factors associated with progression of cervical human papillomavirus (HPV) infections into carcinoma in situ during a long-term prospective follow-up.

In the course of a prospective study of 508 women with papillomavirus (HPV) lesions of the uterine cervix, 66 lesions that progressed into carcinoma in situ (CIS) were identified and treated by conization during a mean follow-up period of 35 months. The lesions were investigated with light microscopy and with in-situ DNA hybridization using 35S-labelled probes for HPV 6, 11, 16, 18, 31 and 33. After radical cone treatment, 11 of the 66 women (16.7%) have presented with a recurrent HPV infection. The recurrence rate increased with the duration of the follow-up period from less than 10% at the mean follow-up of 25 months to 16.7% at the most recent follow-up at 35 months. Most of these 66 HPV lesions (89%) presented with concomitant CIN in the first punch biopsy, but it is noteworthy that the other 11% presented without concomitant CIN. HPV DNA of at least one of the six types examined was found in 73% of the first biopsies and it is noteworthy that the so-called 'low-risk' types, HPV 6 and 11, were found as frequently as the 'high-risk' types, HPV 16 and 18 (18% and 17%, respectively). This would suggest a similarity in the biological behaviour of these two HPV groups. Although the concept of the 'high-risk' and 'low-risk' HPV types may remain at least partially valid, it is imperative to realize that infection by HPV 6 and 11 by no means excludes the possibility for clinical progression into CIS and eventually to an invasive carcinoma.

Screening of premalignant cervical lesions for HPV 16 DNA by sandwich and in situ hybridization techniques.

A series of 97 cervical smears and 69 directed punch biopsies derived from 84 consecutive women prospectively followed-up for cervical HPV (human papillomavirus) infections were studied using the sandwich hybridization and in situ hybridization techniques with HPV 16 DNA probes. The aim was to test the sensitivity and applicability of these two techniques in routine diagnosis of cervical HPV infections from smears. As a measure of specimen adequacy, the number of cells recovered in the cervical scrape was determined along with HPV 16 DNA in the sandwich hybridization test using human pro-alpha 2(I)-collagen gene probe. CIN (cervical intraepithelial neoplasia) was suggested in 56% of the patients by the Pap smear, and disclosed in 65% of the biopsies. HPV 16 DNA was present in 57% of cervical scrapes consistent with CIN, i.e., were of Pap smear classes III or IV. Forty percent of the scrapes not suggestive of CIN, i.e., Pap smear classes I or II, also contained HPV 16 DNA. The detection rate for HPV 16 DNA of the sandwich hybridization method was 89% of that of the in situ method in adequate scrapes, but only 43% in cell-poor specimens. The number of HPV 16 DNA-positive scrapes as compared with the total number of diagnoses obtained by studying also the biopsies was 31/36 (69 patients). The results indicate that the cervical scrape as a noninvasive specimen is applicable for screening of cervical HPV infections, and it can be studied with acceptable sensitivity by the rapid sandwich hybridization technique. However, if a punch biopsy is indicated it should be studied using the in situ hybridization technique that allows more sensitive detection of HPV DNA than any other hybridization method and enables the analysis of several HPV types in the same sample instead of only one HPV type in the scrapes.

Phenotypic transformation of primary mouse fibroblasts by BPV 1 DNA.

Cultures of primary fibroblasts of C57BL/6J mice were used as targets for transformation by bovine papillomavirus type 1 (BPV 1) DNA. Although no foci were observed, several lines of transformed cells were established by subculturing. These immortalized cell lines had in vitro growth characteristics in high and low serum media and saturation densities typical of transformed cells. Karyotype analyses revealed extensive aneuploidic changes. In two of the three cell lines analyzed, viral DNA was present in monomeric episomal form, in the third cell line all viral sequences were found in the high molecular weight region of a Southern blot. Despite the transformed phenotype, only one of the cell lines was tumorigenic in nude mice at a low level.

Nucleic acid sandwich hybridization in detection of HPV 16 DNA: technique and its clinical application.

Nucleic acid sandwich hybridization technique was used for detection of HPV 16 specific DNA in cervical scrapes. Alternating HPV 16-PstI fragments were cloned into plasmid pBR322 and phage M13mp10. pBR322-clones were used as 32P-labelled probe reagents and the M13mp10 clones as catching reagents in the assay. The detection limit of the test proved to be 1-5 X 10(5) HPV 16 molecules per test. A weak cross reaction was seen with HPV 31 DNA but not with the other types tested, e.g. HPV 6, 11, 18 and 33. Cervical scrapings obtained from 163 consecutive patients included in a prospective follow up study were analyzed for HPV 16 DNA with the sandwich hybridization method, dot-blot hybridization being used as a reference method. Sandwich hybridization assay detected 25 positive cases out of 163 specimens (15.3%). Of these 6 and 3 additional specimens were positive in dot-blot hybridization assay. HPV 16 DNA was related to higher PAP grades, and HPV 16 appeared more frequently in HPV CIN than HPV NCIN lesions. None of the infections caused by HPV 16 regressed, and 24% progressed during the follow up.

Tumour-associated antigens in primary mouse fibroblasts induced by transformation with bovine papillomavirus type 1.

Bovine papillomavirus type 1 (BPV 1) DNA was used to transform primary fibroblasts of C57BL/6J mice. Transformation frequency in these cells was much lower than in C127 cells and not associated with the appearance of morphologically distinct foci. However, continuous lines of transformed C57BL/6J cells were developed by serial subculturing of transfected cells. These transformed cell lines showed phenotypic properties associated with transformation including abnormal karyotypes. They contained variable amounts of viral DNA, but the copy number was in the same range as in six C127 transformants tested for comparison. In two cell lines monomeric viral DNA in an episomal form was detected. Slowly migrating viral sequences in these and in the third line were probably episomal concatamers, but the possibility of integration could not be excluded. There was some variation in immunogenicity, but all cell lines induced a cell-mediated immune response in syngeneic mice detected by the chromium release assay. In addition to BPV 1-transformed cell lines, the effector cells also reacted against an unidentified antigen shared by 2 cell lines transformed by SV40 and UV irradiation, respectively.

Tumorigenicity of primary mouse fibroblasts transformed by bovine papillomavirus type 1.

Cell lines transformed by bovine papillomavirus type 1 DNA were established from transfected primary fibroblast cultures of C57BL/6 mice. Southern blot analysis revealed that most of the viral DNA in the cells was in episomal oligomeric form. These cell lines were tested for tumorigenicity in nude mice in comparison to properties associated with malignant transformation including growth in low-serum medium, focus formation in agar, and resistance to natural killer cells. Two of the 5 cell lines were highly tumorigenic, producing spindle cell sarcomas in all animals. Metastatic spreading to local lymph nodes was also observed. The same cell lines also showed anchorage-independent growth in agar, but no correlation was observed between natural killer cell resistance and tumorigenicity. Viral transcripts were detected in two nontumorigenic and in two tumorigenic cell lines.

Cervical smears in assessment of the natural history of human papillomavirus infections in prospectively followed women.

The value of cervical (Papanicolaou) smears in monitoring the natural history of cervical human papillomavirus (HPV) infections was assessed in a series of 513 women prospectively followed since 1981. On each clinic visit, the patients were subjected to colposcopy accompanied by cervical smears and/or punch biopsies. The latter were analyzed by light microscopy for concomitant cervical intraepithelial neoplasia (CIN) and by transmission electron microscopy (TEM) for HPV particles as well as for HPV structural proteins. The stromal immunocompetent cell (ICC) infiltrates were phenotypically characterized using monoclonal antibodies for T-cell subsets, NK and K cells and Langerhans cells. HPV DNA typing was accomplished by Southern blot, spot and in situ hybridization using probes for HPV 6, 11, 16, 18 and 31. Lesions showing only changes consistent with HPV infection (HPV-NCIN) were associated with less severe atypia in cervical smears than were lesions with coexistent CIN (HPV-CIN). Normal smears were observed, however, in 24.7% of the cases with HPV-NCIN lesions, in 11.5% of cases with HPV-CIN I lesions but only exceptionally in cases with HPV-CIN II and III lesions (2.2% and 3.3%). The percentages of the different ICC phenotypes did not correlate with the atypia in cervical smears, but there was a shift towards the lower values of the T-helper/T-suppressor (OKT4+/OKT8+) cell ratio in parallel with increasing atypia. The possibility of latent HPV infection was suggested by the detection of viral particles, HPV antigens and HPV DNA in lesions shedding normal cells in the smears. The high-risk HPV types 16 and 18 were associated with the highest frequency of severely atypical cells; in the majority of cases, the low-risk types HPV 6 and 11 presented with less severe atypia. The first cervical smear seems to be of value as a predictor of the natural history of HPV lesions, as indicated by the fact that regression was inversely and progression directly related to initial cellular atypia. The present results confirm the intimate association between HPV infections and CIN. Although the biologic potential of the HPV infections seems to be dependent on multiple factors, routine cervical smears, because of their potential value in monitoring the natural history of this infection, should constitute an important means in the prospective follow-up of these patients.