Dexamethasone-Induced FKBP51 Expression in CD4+ T-Lymphocytes Is Uniquely Associated With Worse Asthma Control in Obese Children With Asthma.

Introduction: There is evidence that obesity, a risk factor for asthma severity and morbidity, has a unique asthma phenotype which is less atopic and less responsive to inhaled corticosteroids (ICS). Peripheral blood mononuclear cells (PBMC) are important to the immunologic pathways of obese asthma and steroid resistance. However, the cellular source associated with steroid resistance has remained elusive. We compared the lymphocyte landscape among obese children with asthma to matched normal weight children with asthma and assessed relationship to asthma control. Methods: High-dimensional flow cytometry of PBMC at baseline and after dexamethasone stimulation was performed to characterize lymphocyte subpopulations, T-lymphocyte polarization, proliferation (Ki-67+), and expression of the steroid-responsive protein FK506-binding protein 51 (FKBP51). T-lymphocyte populations were compared between obese and normal-weight participants, and an unbiased, unsupervised clustering analysis was performed. Differentially expressed clusters were compared with asthma control, adjusted for ICS and exhaled nitric oxide. Results: In the obese population, there was an increased cluster of CD4+ T-lymphocytes expressing Ki-67 and FKBP51 at baseline and CD4+ T-lymphocytes expressing FKBP51 after dexamethasone stimulation. CD4+ Ki-67 and FKBP51 expression at baseline showed no association with asthma control. Dexamethasone-induced CD4+ FKBP51 expression was associated with worse asthma control in obese participants with asthma. FKBP51 expression in CD8+ T cells and CD19+ B cells did not differ among groups, nor did polarization profiles for Th1, Th2, Th9, or Th17 percentage. Discussion: Dexamethasone-induced CD4+ FKBP51 expression is uniquely associated with worse asthma control in obese children with asthma and may underlie the corticosteroid resistance observed in this population.

A novel xyloglucan-specific endo-beta-1,4-glucanase: biochemical properties and inhibition studies.

A novel xyloglucan-specific endo-beta-1,4-glucanase gene (xeg5A) was isolated, cloned, and expressed in Esherichia coli. The enzyme XEG5A consisted of a C-terminal catalytic domain and N-terminal sequence of approximately 90 amino acid residues with unknown function. The catalytic domain assumed an (alpha/beta)(8)-fold typical of glycoside hydrolase (GH) family 5, with the two catalytic residues Glu240 and Glu362 located on opposite sides of the surface groove of the molecule. The recombinant enzyme showed high specificity towards tamarind xyloglucan and decreasing activity towards xyloglucan oligosaccharide (HDP-XGO), carboxymethyl cellulose, and lichenan. Tamarind xyloglucan was hydrolyzed to three major fragments, XXXG, XXLG/XLXG, and XLLG. The hydrolysis followed the Michaelis-Menten kinetics, yielding K (m) and V (max) of 3.61 +/- 0.23 mg/ml and 0.30 +/- 0.01 mg/ml/min, respectively. However, the hydrolysis of HDP-XGO showed a decrease in the rate at high concentrations suggesting appearance of excess substrate inhibition. The addition of XXXG resulted in linear noncompetitive inhibition on the hydrolysis of tamarind xyloglucan giving a K (i) of 1.46 +/- 0.13 mM. The enzyme was devoid of transglycosylase activities.

Karyotypic evolutions of cancer species in rats during the long latent periods after injection of nitrosourea.

BACKGROUND: A century of research has established that cancers arise from tissues exposed to carcinogens only after long latencies of years to decades and have individual clonal karyotypes. Since speciation from known precursors also depends on long latencies and new species also have individual karyotypes, we and others have recently proposed that carcinogenesis is a form of speciation. According to this theory karyotypic evolutions generate new cancer species from normal cells as follows: Carcinogens induce aneuploidy (Figure 1). By unbalancing thousands of genes aneuploidy automatically destabilizes the karyotype and thus catalyzes random karyotypic variations. Selections of variants with proliferative phenotypes form non-clonal hyperplasias with persistently varying karyotypes. Very rare karyotypic variations form new cancer species with individual clonal karyotypes. Despite destabilization by the resulting congenital aneuploidies, cancer karyotypes are stabilized within narrow margins of variation by clonal selections for cancer-specific autonomy. Because all non-cancerous aneuploidies are unstable, all aneusomies of prospective cancers are joined in single-steps, rather than gradually. Since this mechanism is very inefficient, it predicts long latent periods from carcinogens to cancers and individual clonal cancer karyotypes. RESULTS: Here we have tested the predicted roles of karyotypic evolutions during the time course of carcinogenesis in an established experimental system. In this system injection of nitrosourea induces in female rats non-invasive mammary hyperplasias ("tumors") after two or more months, and invasive carcinomas after six or more months. Accordingly four specific predictions were tested: (1) Invasive cancers are late and carry individual clonal karyotypes and phenotypes, (2) Persistent hyperplasias carry non-clonal karyotypes, (3) Non-clonal hyperplasias generate clonal cancers spontaneously but rarely, (4) Cancer-karyotypes arise with all individual clonal aneusomies in single-steps. All four predictions were experimentally confirmed. CONCLUSIONS: Our results along with the literature reveal a coherent karyotypic mechanism of carcinogenesis: Carcinogens induce aneuploidy. The inherent instability of aneuploidy automatically catalyzes new karyotypic variations. Aneuploid karyotypes with proliferative phenotypes form varying non-clonal hyperplasias. Rare variations form cancer species with individual clonal karyotypes, which are stabilized by clonal selection for autonomy. The low odds of this mechanism explain the long latencies of carcinogenesis, the individuality and karyotypic clonality of cancers.

Immortality of cancers: a consequence of inherent karyotypic variations and selections for autonomy.

Immortality is a common characteristic of cancers, but its origin and purpose are still unclear. Here we advance a karyotypic theory of immortality based on the theory that carcinogenesis is a form of speciation. Accordingly, cancers are generated from normal cells by random karyotypic rearrangements and selection for cancer-specific reproductive autonomy. Since such rearrangements unbalance long-established mitosis genes, cancer karyotypes vary spontaneously but are stabilized perpetually by clonal selections for autonomy. To test this theory we have analyzed neoplastic clones, presumably immortalized by transfection with overexpressed telomerase or with SV40 tumor virus, for the predicted clonal yet flexible karyotypes. The following results were obtained: (1) All immortal tumorigenic lines from cells transfected with overexpressed telomerase had clonal and flexible karyotypes; (2) Searching for the origin of such karyotypes, we found spontaneously increasing, random aneuploidy in human fibroblasts early after transfection with overexpressed telomerase; (3) Late after transfection, new immortal tumorigenic clones with new clonal and flexible karyotypes were found; (4) Testing immortality of one clone during 848 unselected generations showed the chromosome number was stable, but the copy numbers of 36% of chromosomes drifted ± 1; (5) Independent immortal tumorigenic clones with individual, flexible karyotypes arose after individual latencies; (6) Immortal tumorigenic clones with new flexible karyotypes also arose late from cells of a telomerase-deficient mouse rendered aneuploid by SV40 virus. Because immortality and tumorigenicity: (1) correlated exactly with individual clonal but flexible karyotypes; (2) originated simultaneously with such karyotypes; and (3) arose in the absence of telomerase, we conclude that clonal and flexible karyotypes generate the immortality of cancers.

Comparative characterization of a bifunctional endo-1,4- ß-mannanase/1,3-1,4- ß-glucanase and its individual domains.

A fusion gene isolated from a microbial metagenome encodes a N-terminal endo-1,4- ß-mannanase and a C-terminal 1,3-1,4- ß -glucanase,. The full-length gene and the individual N- and C-domains were separately cloned and expressed in E coli. The purified whole enzyme hydrolyzed glucomannan, galactomannan, and ß-glucan with Km and kcat values 2.2, 2.6, 3.6 mg/ml, and 302, 130, 337 min -1 , respectively. The hydrolysis of ß-glucan by the C domain enzyme decreased significantly with added glucomannan to the reaction, suggesting inhibition effect. Analogous result was not observed with the N domain enzyme when ß-glucan was added to the reaction. The whole enzyme did not show improvement of efficiency compared to the individual or additive total hydrolysis of the two domain enzymes using single or mixed substrates.

Individual karyotypes at the origins of cervical carcinomas.

BACKGROUND: In 1952 Papanicolaou et al. first diagnosed and graded cervical carcinomas based on individual "abnormal DNA contents" and cellular phenotypes. Surprisingly current papilloma virus and mutation theories of carcinomas do not mention these individualities. The viral theory holds that randomly integrated, defective genomes of papilloma viruses, which are often untranscribed, cause cervical carcinomas with unknown cofactors 20-50 years after infection. Virus-free carcinomas are attributed to mutations of a few tumor-suppressor genes, especially the p53 gene. But the paradox of how a few mutations or latent defective viral DNAs would generate carcinomas with endless individual DNA contents, degrees of malignancies and cellular phenotypes is unsolved. Since speciation predicts individuality, we test here the theory that cancers are autonomous species with individual clonal karyotypes and phenotypes. This theory postulates that carcinogens induce aneuploidy. By unbalancing mitosis genes aneuploidy catalyzes chain reactions of karyotypic evolutions. Most such evolutions end with non-viable karyotypes but a few become new cancer karyotypes. Despite congenitally unbalanced mitosis genes cancer karyotypes are stabilized by clonal selections for cancer-specific autonomy. RESULTS: To test the prediction of the speciation theory that individual carcinomas have individual clonal karyotypes and phenotypes, we have analyzed here the phenotypes and karyotypes of nine cervical carcinomas. Seven of these contained papilloma virus sequences and two did not. We determined phenotypic individuality and clonality based on the morphology and sociology of carcinoma cells in vitro. Karyotypic individuality and clonality were determined by comparing all chromosomes of 20 karyotypes of carcinomas in three-dimensional arrays. Such arrays list chromosome numbers on the x-axis, chromosome copy numbers on the y-axis and the number of karyotypes arrayed on the z-axis. We found (1) individual clonal karyotypes and phenotypes in all nine carcinomas, but no virus-specific markers, (2) 1-to-1 variations between carcinoma-specific karyotypes and phenotypes, e.g. drug-resistance and cell morphology, (3) proportionality between the copy numbers of chromosomes and the copy numbers of hundreds of over- and under-expressed mRNAs, (4) evidence that tobacco-carcinogens induce cervical carcinomas via aneuploidy, consistent with the speciation theory. CONCLUSIONS: Since the individual clonal karyotypes of nine carcinomas correlated and co-varied 1-to-1 with complex individual transcriptomes and phenotypes, we have classical genetic and functional transcriptomic evidence to conclude that these karyotypes encode carcinomas - much like the clonal karyotypes that encode conventional species. These individual karyotypes explain the individual "DNA contents", the endless grades of malignancies and the complex individual transcriptomes and phenotypes of carcinomas.

Origin of metastases: subspecies of cancers generated by intrinsic karyotypic variations.

Conventional mutation theories do not explain (1) why the karyotypes of metastases are related to those of parental cancers but not to those of metastases of other cancers and (2) why cancers metastasize at rates that often far exceed those of conventional mutations. To answer these questions, we advance here the theory that metastases are autonomous subspecies of cancers, rather than mutations. Since cancers are species with intrinsically flexible karyotypes, they can generate new subspecies by spontaneous karyotypic rearrangements. This phylogenetic theory predicts that metastases are karyotypically related to parental cancers but not to others. Testing these predictions on metastases from two pancreatic cancers, we found: (1) Metastases had individual karyotypes and phenotypes. The karyotypes of metastases were related to, but different from, those of parental cancers in 11 out of 37 and 26 out of 49 parental chromosomal units. Chromosomal units are defined as intact chromosomes with cancer-specific copy numbers and marker chromosomes that are > 50% clonal. (2) Metastases from the two different cancers did not share chromosomal units. Testing the view that multi-chromosomal rearrangements occur simultaneously in cancers, as opposed to sequentially, we found spontaneous non-clonal rearrangements with as many new chromosomal units as in authentic metastases. We conclude that metastases are individual autonomous species differing from each other and parental cancers in species-specific karyotypes and phenotypes. They are generated from parental cancers by multiple simultaneous karyotypic rearrangements, much like new species. The species-specific individualities of metastases explain why so many searches for commonalities have been unsuccessful.

Functional Cloning and Expression of the Schizophyllum commune Glucuronoyl Esterase Gene and Characterization of the Recombinant Enzyme.

The gene encoding Schizophyllum commune glucuronoyl esterase was identified in the scaffold 17 of the genome, containing two introns of 50 bp and 48 bp, with a transcript sequence of 1179 bp. The gene was synthesized and cloned into Pichia pastoris expression vector pGAPZα to achieve constitutive expression and secretion of the recombinant enzyme in soluble active form. The purified protein was 53 kD with glycosylation and had an acidic pI of 3.7. Activity analysis on several uronic acids and their derivatives suggests that the enzyme recognized only esters of 4-O-methyl-D-glucuronic acid derivatives, even with a 4-nitrophenyl aglycon but did not hydrolyze the ester of D-galacturonic acid. The kinetic values were K(m) 0.25 mM, V(max) 16.3 µM·min(-1), and k(cat) 9.27 s(-1) with 4-nitrophenyl 2-O-(methyl 4-O-methyl-α-D-glucopyranosyluronate)-ß-D-xylopyranoside as the substrate.

Is carcinogenesis a form of speciation?

Since cancers have individual clonal karyotypes, are immortal and evolve from normal cells treated by carcinogens only after exceedingly long latencies of many months to decades-we deduce that carcinogenesis may be a form of speciation. This theory proposes that carcinogens initiate carcinogenesis by causing aneuploidy, i.e., losses or gains of chromosomes. Aneuploidy destabilizes the karyotype, because it unbalances thousands of collaborating genes including those that synthesize, segregate and repair chromosomes. Driven by this inherent instability aneuploid cells evolve ever-more random karyotypes automatically. Most of these perish, but a very small minority acquires reproductive autonomy-the primary characteristic of cancer cells and species. Selection for autonomy stabilizes new cancer species against the inherent instability of aneuploidy within specific margins of variation. The speciation theory explains five common characteristics of cancers: (1) species-specific autonomy; (2) karyotypic and phenotypic individuality; (3) flexibility by karyotypic variations within stable margins of autonomy; (4) immortality by replacing defective karyotypes from constitutive pools of competent variants or subspecies generated by this flexibility; and (5) long neoplastic latencies by the low probability that random karyotypic alterations generate new autonomous species. Moreover, the theory explains phylogenetic relations between cancers of the same tissue, because carcinogenesis is restricted by tissue-specific transcriptomes. The theory also solves paradoxes of other cancer theories. For example, "aneuploidy" of cancers is now said to be a "paradox" or "cancer's fatal flaw," because aneuploidy impairs normal growth and development. But if the "aneuploidies" of cancers are in effect the karyotypes of new species, this paradox is solved.

AIDS since 1984: no evidence for a new, viral epidemic--not even in Africa.

Since the discoveries of a putative AIDS virus in 1984 and of millions of asymptomatic carriers in subsequent years, no general AIDS epidemic has occurred by 2011. In 2008, however, it has been proposed that between 2000 and 2005 the new AIDS virus, now called HIV, had killed 1.8 million South Africans at a steady rate of 300,000 per year and that anti-HIV drugs could have saved 330,000 of those. Here we investigate these claims in view of the paradoxes that HIV would cause a general epidemic in Africa but not in other continents, and a steady rather than a classical bell-shaped epidemic like all other new pathogenic viruses. Surprisingly, we found that South Africa attributed only about 10,000 deaths per year to HIV between 2000 and 2005 and that the South African population had increased by 3 million between 2000 and 2005 at a steady rate of 500,000 per year. This gain was part of a monotonic growth trajectory spanning from 29 million in 1980 to 49 million in 2008. During the same time Uganda increased from 12 to 31 million, and Sub-Saharan Africa as a whole doubled from 400 to 800 million, despite high prevalence HIV. We deduce from this demographic evidence that HIV is not a new killer virus. Based on a review of the known toxicities of antiretroviral drugs we like to draw the attention of scientists, who work in basic and clinical medical fields, including embryologists, to the need of rethinking the risk-and-benefit balance of antiretroviral drugs for pregnant women, newborn babies and all others who carry antibodies against HIV.

Transgenic oncogenes induce oncogene-independent cancers with individual karyotypes and phenotypes.

Cancers are clones of autonomous cells defined by individual karyotypes, much like species. Despite such karyotypic evidence for causality, three to six synergistic mutations, termed oncogenes, are generally thought to cause cancer. To test single oncogenes, they are artificially activated with heterologous promoters and spliced into the germ line of mice to initiate cancers with collaborating spontaneous oncogenes. Because such cancers are studied as models for the treatment of natural cancers with related oncogenes, the following must be answered: 1) which oncogenes collaborate with the transgenes in cancers; 2) how do single transgenic oncogenes induce diverse cancers and hyperplasias; 3) what maintains cancers that lose initiating transgenes; 4) why are cancers aneuploid, over- and underexpressing thousands of normal genes? Here we try to answer these questions with the theory that carcinogenesis is a form of speciation. We postulate that transgenic oncogenes initiate carcinogenesis by inducing aneuploidy. Aneuploidy destabilizes the karyotype by unbalancing teams of mitosis genes. This instability thus catalyzes the evolution of new cancer species with individual karyotypes. Depending on their degree of aneuploidy, these cancers then evolve new subspecies. To test this theory, we have analyzed the karyotypes and phenotypes of mammary carcinomas of mice with transgenic SV40 tumor virus- and hepatitis B virus-derived oncogenes. We found that (1) a given transgene induced diverse carcinomas with individual karyotypes and phenotypes; (2) these karyotypes coevolved with newly acquired phenotypes such as drug resistance; (3) 8 of 12 carcinomas were transgene negative. Having found one-to-one correlations between individual karyotypes and phenotypes and consistent coevolutions of karyotypes and phenotypes, we conclude that carcinogenesis is a form of speciation and that individual karyotypes maintain cancers as they maintain species. Because activated oncogenes destabilize karyotypes and are dispensable in cancers, we conclude that they function indirectly, like carcinogens. Such oncogenes would thus not be valid models for the treatment of cancers.

Cloning and characterization of an exo-xylogucanase from rumenal microbial metagenome.

A novel exo-glucanase gene (xeg5B) was isolated from a rumenal microbial metagenome, cloned, and expressed in E. coli. The 1548 bp gene coded for a protein of 516 amino acids, which assumed an (a/b)(8) fold typical of glycoside hydrolase (GH) family 5. The protein molecule consisted of a loop segment blocking one end of the active site, which potentially provided the enzyme with exo-acting property. The recombinant enzyme showed exclusive specificity towards only xyloglucan and oligoxyloglucan substrates with no detectable activity on unsubstituted linear glucans, CMC, laminarin, and lichenan. The major end products of exhaustive hydrolysis were XX (tetrasaccharide) and XG (trisaccharide). The hydrolysis of tamarind xyloglucan followed the Michaelis-Menten kinetics, yielding K(m) and V(max) of 2.12+/-0.13 mg/ml and 0.17+/-0.01 mg/ml/min (37 degrees C, pH 6.0), respectively.

Functional cloning and expression of a novel Endo-alpha-1,5-L-arabinanase from a metagenomic library.

A novel endo-alpha-L-arabinanase gene (arn2) was isolated, and expressed in E. coli in active form. The recombinant enzyme (ARN2) had optimum activity at pH 6.0 and 45-50( degrees )C with stability between pH 5.0-8.0 and at temperatures up to 40( degrees )C. The recombinant ARN2 catalyzed internal cleavage of alpha-1,5 glycosidic bonds of CM-arabinan, debranched arabinan, linear arabinan, and sugar beet (native) arabinan at rates of decreasing order, and was inactive on wheat arabinoxylan and p-nitrophenyl-alpha-L-arabinofuranoside. Kinetic analysis showed that branching in the arabinan did not significantly affect the apparent K(m) values, and the difference in the reaction rates was likely due to the chemical step after substrate binding. The enzyme hydrolyzed arabino-oligosaccharides of DP> or =6 to smaller oligomers and mostly arabinotriose. Natural and modified arabinans were cleaved to oligomers of various chain lengths, which were progressively hydrolyzed to yield arabinotriose. The pattern of degradation revealed an endo-acting mechanism with arabinotriose as the end product.

Cancer-causing karyotypes: chromosomal equilibria between destabilizing aneuploidy and stabilizing selection for oncogenic function.

The chromosomes of cancer cells are unstable, because of aneuploidy. Despite chromosomal instability, however, cancer karyotypes are individual and quasi-stable, as is evident especially from clonal chromosome copy numbers and marker chromosomes. This paradox would be resolved if the karyotypes in cancers represent chromosomal equilibria between destabilizing aneuploidy and stabilizing selection for oncogenic function. To test this hypothesis, we analyzed the initial and long-term karyotypes of seven clones of newly transformed human epithelial, mammary, and muscle cells. Approximately 1 in 100,000 such cells generates transformed clones at 2-3 months after introduction of retrovirus-activated cellular genes or the tumor virus SV40. These frequencies are too low for direct transformation, so we postulated that virus-activated genes initiate transformation indirectly, via specific karyotypes. Using multicolor fluorescence in situ hybridization with chromosome-specific DNA probes, we found individual clonal karyotypes that were stable for at least 34 cell generations-within limits, as follows. Depending on the karyotype, average clonal chromosome numbers were stable within +/- 3%, and chromosome-specific copy numbers were stable in 70-100% cells. At any one time, however, relative to clonal means, per-cell chromosome numbers varied +/-18% and chromosome-specific copy numbers varied +/-1 in 0-30% of cells; unstable nonclonal markers were found within karyotype-specific quotas of <1% to 20% of the total chromosome number. For two clones, karyotypic ploidies also varied. With these rates of variation, the karyotypes of transformed clones would randomize in a few generations unless selection occurs. We conclude that individual aneuploid karyotypes initiate and maintain cancers, much like new species. These cancer-causing karyotypes are in flexible equilibrium between destabilizing aneuploidy and stabilizing selection for transforming function. Karyotypes as a whole, rather than specific mutations, explain the individuality, fluidity, and phenotypic complexity of cancers.