Inversion of the roles of the nucleophile and acid/base catalysts in the covalent binding of epoxyalkyl xyloside inhibitor to the catalytic glutamates of endo-1,4-beta-xylanase (XYNII): a molecular dynamics study

X-Ray crystal structures have revealed that 2, 3-epoxypropyl-beta-D-xyloside reacts with endo-1,4-beta-xylanase (XYNII) by forming a covalent bond with Glu86. In contrast, 3, 4-epoxybutyl-beta-D-xyloside forms a covalent bond with Glu177. In the normal enzyme reaction Glu86 acts as the catalytic nucleophile and Glu177 as the acid/base catalyst. To rationalize the observed reactivity of the two mechanism-based inhibitors, we carried out eight 300 ps molecular dynamics simulations for different enzyme-inhibitor complexes. Simulations were done for both stereo isomers (R and S) of the inhibitors and for enzyme in which the protonation state of the nucleophile and acid/base catalyst was normal (Glu86 charged, Glu177 neutral) and in which the roles of the catalytic residues were reversed (Glu86 neutral, Glu177 charged). The number of reactive conformations found in each simulation was used to predict the reactivity of epoxy inhibitors. The conformation was considered to be a reactive one when at the same time (i) the proton of the catalytic acid was close (<2.9/3.4/3.9 A) to the oxirane oxygen of the inhibitor, (ii) the nucleophile was close to the terminal carbon of the oxirane group (<3.4/3.9/4.4 A) and (iii) the nucleophile approached the terminal carbon from a reactive angle (<30/45/60 degrees from an ideal attack angle). On the basis of the number of reactive conformations, 2,3-epoxypropyl-beta-D-xyloside was predicted to form a covalent bond with Glu86 and 3, 4-epoxybutyl-beta-D-xyloside with Glu177, both in agreement with the experiment. Thus, the MD simulations and the X-ray structures indicate that in the covalent binding of 3, 4-epoxybutyl-beta-D-xyloside the roles of the catalytic glutamates of XYNII are reversed from that of the normal enzyme reaction.     

A multipurpose vector system for the screening of libraries in bacteria, insect and mammalian cells and expression in vivo

We have constructed a novel tetra-promoter vector (pBVboostFG) system that enables screening of gene/cDNA libraries for functional genomic studies. The vector enables an all-in-one strategy for gene expression in mammalian, bacterial and insect cells and is also suitable for direct use in vivo. Virus preparation is based on an improved mini Tn7 transpositional system allowing easy and fast production of recombinant baculoviruses with high diversity and negligible background. Cloning of the desired DNA fragments or libraries is based on the recombination system of bacteriophage lambda. As an example of the utility of the vector, genes or cDNAs of 18 different proteins were cloned into pBVboostFG and expressed in different hosts. As a proof-of-principle of using the vector for library screening, a chromophoric Thr⁶⁵-Tyr-Gly⁶⁷-stretch of enhanced green fluorescent protein was destroyed and subsequently restored by novel PCR strategy and library screening. The pBVboostFG enables screening of genome-wide libraries, thus making it an efficient new platform technology for functional genomics.     

Gastric Pit Cell Hyperplasia and Glandular Atrophy in Carbonic Anhydrase IX Knockout Mice: Studies on Two Strains C57/BL6 and BALB/C

Carbonic anhydrase (CA) isoenzyme IX is a hypoxia-inducible enzyme, which is expressed in the human and rodent gastrointestinal tract and overexpressed in several different tumors. Functionally, it has probably an effect on proliferation and differentiation of gastrointestinal epithelial cells. It may also participate in gastric morphogenesis, since a recent study has shown gastric pit cell hyperplasia and glandular atrophy in CA IX-knockout mice. However, it is not known whether CA IX produces morphological changes in the gastric mucosa, which can turn into a dysplasia or malignancy in the presence of some carcinogenic factors. High-salt diet is considered such a factor which has been shown to modulate Helicobacter pylori-associated carcinogenesis. We produced two strains of CA IX-knockout mice, C57/BL6 and BALB/c, and the mice ate either standard or high-salt feed for 20 weeks. Stomach samples were collected from 40 Car9^−/− knockout mice and 37 wildtype littermates, and the tissue sections were examined for histology. CA IX-deficiency caused gastric pit cell hyperplasia and glandular atrophy in both BALB/c and C57/BL6 strains. Excess dietary salt had no significant effect on the severity of pit cell hyperplasia. No dysplasia was found in any of the groups. In C57/BL6 mice, CA IX-deficiency was associated with gastric submucosal inflammation. The results indicate that CA IX-deficiency provides a useful model to study the mechanisms of gastric morphogenesis and epithelial integrity. Further studies are needed to see whether CA IX has a role in the regulation of immune response. The findings suggest that although CA IX-deficiency is not a tumor-promoting factor per se, it induces glandular atrophy in the body mucosa, a lesion which is considered to be a preneoplastic alteration in the stomach.     

Laminin α1-Chain Shows a Restricted Distribution in Epithelial Basement Membranes of Fetal and Adult Human Tissues

Two novel monoclonal antibodies were raised and used to study the expression of laminin (Ln) α1-chain in developing and adult human tissues. In both fetal and adult kidney, a distinct immunoreactivity was seen in basement membranes (BM) of most proximal tubules but not in the distal tubular or glomerular BM or in the basal laminae of blood vessels. Immunoprecipitation of metabolically labeled cultured human renal proximal tubular cells showed an abundant production and deposition of Ln α1-chain to the extracellular matrix, suggestive of an epithelial origin of kidney Ln-1. Quantitative cell adhesion experiments with JAR choriocarcinoma cells showed that purified human Ln-1 is a good substrate for cell adhesion that it is differently recognized by integrin receptors when compared to mouse Ln-1. In fetal and adult testes immunoreactivity was solely confined to BM of the seminiferous epithelium. In the airways BM-confined reaction was only seen in fetal budding bronchial tubules (16–19 weeks) at the pseudoglandular stage of development. In the skin a distinct immunoreactivity was confined to BM of developing hair buds but not in epithelial BMs of adult epidermis or of epidermal appendages. In other adult tissues, immunoreactivity was found in BMs of thyroid, salivary, and mammary glands as well as in BMs of endometrium and endocervix, but not of ectocervix or vagina. No immunoreactivity was found in BMs of most of the digestive tract, including the liver and pancreas, except for BMs of esophageal submucosal glands and duodenal Brunner's glands. In fetal specimens, BMs of the bottoms of the intestinal and gastric glands were positive. Basal laminae of blood vessels were generally negative for Ln α1 chain with the exception of specimens of both fetal and adult central nervous system in which immunoreactivity for Ln α1 chain was prominently confined to capillary walls. The results suggest that outside the central nervous system, Ln α1 chain shows a restricted and developmentally regulated expression in BMs of distinct epithelial tissues.     

Stability of the Associations between Early Life Risk Indicators and Adolescent Overweight over the Evolving Obesity Epidemic

Background

Pre- and perinatal factors and preschool body size may help identify children developing overweight, but these factors might have changed during the development of the obesity epidemic.

Objective

We aimed to assess the associations between early life risk indicators and overweight at the age of 9 and 15 years at different stages of the obesity epidemic.

Methods

We used two population-based Northern Finland Birth Cohorts including 4111 children born in 1966 (NFBC1966) and 5414 children born in 1985–1986 (NFBC1986). In both cohorts, we used the same a priori defined prenatal factors, maternal body mass index (BMI), birth weight, infant weight (age 5 months and 1 year), and preschool BMI (age 2–5 years). We used internal references in early childhood to define percentiles of body size (<50, 50–75, 75–90 and >90) and generalized linear models to study the association with overweight, according to the International Obesity Taskforce (IOTF) definitions, at the ages of 9 and 15 years.

Results

The prevalence of overweight at the age of 15 was 9% for children born in 1966 and 16% for children born in 1986. However, medians of infant weight and preschool BMI changed little between the cohorts, and we found similar associations between maternal BMI, infant weight, preschool BMI, and later overweight in the two cohorts. At 5 years, children above the 90^th percentile had approximately a 12 times higher risk of being overweight at the age of 15 years compared to children below the 50^th percentile in both cohorts.

Conclusions

The associations between early body size and adolescent overweight showed remarkable stability, despite the increase in prevalence of overweight over the 20 years between the cohorts. Using consequently defined internal percentiles may be a valuable tool in clinical practice.     

Genetic structure of an endangered raptor at individual and population levels

The Finnish population of White-tailed Eagle (Haliaeetus albicilla) has gone through two major demographic bottlenecks during the last two centuries. Strong conservation measures have allowed the population to recover, but despite the rapid population growth during recent years the species is still classified as endangered. We studied the genetic population structure at both individual and population levels in an attempt to recognize the processes shaping it. We used 9 microsatellite loci and 473 base pair fragment of the mitochondrial DNA control region on samples collected between the years 2003 and 2007 (N = 489). We found a clear isolation by distance pattern at fine scale (i.e. individual level) which is most likely a result of species’ philopatric behaviour. Although we did not find signs of the recent bottlenecks, we did find evidence of an ancient bottleneck that has occurred most likely over 21,000 years ago, long before the genetic divergence of the two present Finnish subpopulations (one along the Baltic Sea coast line and another in Lapland and easternmost Finland). We conclude that the present population structure is mainly a consequence of the species philopatric behaviour over a long time period instead of recent population bottlenecks. Based on our results, the Finnish population seems to have ongoing immigration from neighbouring populations. Hence, even though the population has recovered mainly through local growth, our results suggest that gene flow from genetically differentiated populations have had an impact as well.     

Antrodia hyalina, a new polypore from Russia, and A. leucaena, new to Europe

A new polypore species, Antrodia hyalina, is described from Russia. It is morphologically similar to Antrodia pulvinascens, but differs in having annual, thinner and softer basidiocarps, solid skeletal hyphae, and cylindrical spores. Antrodia leucaena, originally described from China, is reported as new from Finland and Russia on Populus tremula. Antrodia wangii is regarded as a synonym of A. bondartsevae.     

Biogenic nanoporous silicon carrier improves the efficacy of buparvaquone against resistant visceral leishmaniasis

Visceral leishmaniasis is a vector-borne protozoan infection that is fatal if untreated. There is no vaccination against the disease, and the current chemotherapeutic agents are ineffective due to increased resistance and severe side effects. Buparvaquone is a potential drug against the leishmaniases, but it is highly hydrophobic resulting in poor bioavailability and low therapeutic efficacy. Herein, we loaded the drug into silicon nanoparticles produced from barley husk, which is an agricultural residue and widely available. The buparvaquone-loaded nanoparticles were several times more selective to kill the intracellular parasites being non-toxic to macrophages compared to the pure buparvaquone and other conventionally used anti-leishmanial agents. Furthermore, the in vivo results revealed that the intraperitoneally injected buparvaquone-loaded nanoparticles suppressed the parasite burden close to 100%. By contrast, pure buparvaquone suppressed the burden only by 50% with corresponding doses. As the conclusion, the biogenic silicon nanoparticles are promising carriers to significantly improve the therapeutic efficacy and selectivity of buparvaquone against resistant visceral leishmaniasis opening a new avenue for low-cost treatment against this neglected tropical disease threatening especially the poor people in developing nations.