Structure-function analysis of PrsA reveals roles for the parvulin-like and flanking N- and C-terminal domains in protein folding and secretion in Bacillus subtilis

The PrsA protein of Bacillus subtilis is an essential membrane-bound lipoprotein that is assumed to assist post-translocational folding of exported proteins and stabilize them in the compartment between the cytoplasmic membrane and cell wall. This folding activity is consistent with the homology of a segment of PrsA with parvulin-type peptidyl-prolyl cis/trans isomerases (PPIase). In this study, molecular modeling showed that the parvulin-like region can adopt a parvulin-type fold with structurally conserved active site residues. PrsA exhibits PPIase activity in a manner dependent on the parvulin-like domain. We constructed deletion, peptide insertion, and amino acid substitution mutations and demonstrated that the parvulin-like domain as well as flanking N- and C-terminal domains are essential for in vivo PrsA function in protein secretion and growth. Surprisingly, none of the predicted active site residues of the parvulin-like domain was essential for growth and protein secretion, although several active site mutations reduced or abolished the PPIase activity or the ability of PrsA to catalyze proline-limited protein folding in vitro. Our results indicate that PrsA is a PPIase, but the essential role in vivo seems to depend on some non-PPIase activity of both the parvulin-like and flanking domains.     

Preservation of dried liposomes in the presence of sugar and phosphate

It has been well established that sugars can be used to stabilize liposomes during drying by a mechanism that involves the formation of a glassy state by the sugars as well as by a direct interaction between the sugar and the phospholipid head groups. We have investigated the protective effect of phosphate on solute retention and storage stability of egg phosphatidylcholine (egg PC) liposomes that were dried (air-dried and freeze-dried) in the presence of sugars and phosphate. The protective effect of phosphate was tested using both glucose (low T(g)) and sucrose (high T(g)) by measuring leakage of carboxyfluorescein (CF), which was incorporated inside the vesicles. Liposomes that were dried with glucose or phosphate alone showed complete leakage after rehydration. However, approximately 30% CF-retention was obtained using mixtures of phosphate and glucose. Approximately 75% CF-retention was observed with liposomes that were dried with sucrose. The solute retention further increased to 85% using mixtures of phosphate and sucrose. The pH of the phosphate buffer prior to drying was found to have a strong effect on the solute retention. Fourier transform infrared spectroscopy studies showed that phosphate and sugars form a strong hydrogen bonding network, which dramatically increased the T(g). The HPO(4)(2-) form of phosphate was found to interact stronger with sugars than the H(2)PO(4)(-) form. The increased solute retention of liposomes dried in the sugar phosphate mixtures did not coincide with improved storage stability. At temperatures below 60 degrees C the rate of solute-leakage was found to be strikingly higher in the presence of phosphate, indicating that phosphate impairs storage stability of dried liposomes.     

CC chemokine ligand 18, an atopic dermatitis-associated and dendritic cell-derived chemokine, is regulated by staphylococcal products and allergen exposure

Atopic dermatitis is a chronic inflammatory skin disease with a steadily increasing prevalence. Exposure to allergens or bacterial superantigens triggers T and dendritic cell (DC) recruitment and induces atopic skin inflammation. In this study, we report that among all known chemokines CCL18/DC-CK1/PARC represents the most highly expressed ligand in atopic dermatitis. Moreover, CCL18 expression is associated with an atopic dermatitis phenotype when compared with other chronic inflammatory skin diseases. DCs either dispersed within the dermis or clustering at sites showing perivascular infiltrates are abundant sources of CCL18. In vitro, microbial products including LPS, peptidoglycan, and mannan, as well as the T cell-derived activation signal CD40L, induced CCL18 in monocytes. In contrast to monocytes, monocyte-derived, interstitial-type, and Langerhans-type DCs showed a constitutive and abundant expression of CCL18. In comparison to Langerhans cells, interstitial-type DCs produced higher constitutive levels of CCL18. In vivo, topical exposure to the relevant allergen or the superantigen staphylococcal enterotoxin B, resulted in a significant induction of CCL18 in atopic dermatitis patients. Furthermore, in nonatopic NiSO4-sensitized individuals, only relevant allergen but not irritant exposure resulted in the induction of CCL18. Taken together, findings of the present study demonstrate that CCL18 is associated with an atopy/allergy skin phenotype, and is expressed at the interface between the environment and the host by cells constantly screening foreign Ags. Its regulation by allergen exposure and microbial products suggests an important role for CCL18 in the initiation and amplification of atopic skin inflammation.     

A mixed-phase immunoassay based on simultaneous binding of fluorescently tagged and PNA-conjugated peptide epitopes on antibodies: quantification on PNA-coated microparticles

A mixed-phase immunoassay based on simultaneous binding of an antibody to its fluorescently tagged peptide epitope and a PNA conjugate of the same peptide has been developed. As a fluorescent marker, a europium(III) chelate allowing time-resolved measurement from a single particle has been employed. The ternary complex formed in solution is immobilized by Watson-Crick base-pairing to a microparticle bearing a PNA sequence complementary to that present in the complex. The concentration of the antibody in the sample may then be determined by a single particle measurement. Accordingly, different antibodies may in principle be addressed by sequence-specific hybridization to different categorized microparticles.     

Amplification of KIT, PDGFRA, VEGFR2, and EGFR in gliomas

Receptor tyrosine kinase aberrations are implicated in the genesis of gliomas. We investigated expression and amplification of KIT, PDGFRA, VEGFR2, and EGFR in 87 gliomas consisting of astrocytomas, anaplastic astrocytomas, oligodendrogliomas, or oligoastrocytomas in tumor samples collected at the time of the diagnosis and in samples of the same tumors at tumor recurrence. Gene amplifications were investigated using either chromogenic in situ hybridization or fluorescence in situ hybridization, and protein expression using immunohistochemistry. In samples collected at glioma diagnosis, KIT and PDGFRA amplifications were more frequent in anaplastic astrocytomas than in astrocytomas, oligodendrogliomas, and oligoastrocytomas [28% versus 5% (P = 0.012) and 33% versus 2% (P = 0.0008), respectively]. VEGFR2 amplifications occurred in 6% to 17% of the gliomas at diagnosis, and EGFR amplifications in 0% to 12%. Amplified KIT was more frequently present in recurrent gliomas than in newly diagnosed gliomas (P = 0.0066). KIT amplification was associated with KIT protein expression and with presence of PDGFRA and EGFR amplifications both at the time of the first glioma diagnosis and at tumor recurrence, and with VEGFR2 amplification at tumor recurrence. Three (4%) primary gliomas and 10 (14%) recurrent gliomas that were evaluable for coamplification of KIT, PDGFRA, and VEGFR2 showed amplification of at least two of these genes; the amplicon contained amplified KIT in all 13 cases. In conclusion, besides glioblastoma, amplified KIT, PDGFRA, and VEGFR may also occur in lower-grade gliomas and in their recurrent tumors. It is currently not known whether specific tyrosine kinase inhibitors are effective in the treatment of such gliomas.     

Winter-time ecology in the Bothnian Bay, Baltic Sea: nutrients and algae in fast ice

Landfast ice algal communities were studied in the strongly riverine-influenced northernmost part of the Baltic Sea, the Bothnian Bay, during the winter-spring transition of 2004. The under-ice river plume, detected by its low salinity and elevated nutrient concentrations, was observed only at the station closest to the river mouth. The bottommost ice layer at this station was formed from the plume water (brine volume 0.71%). This was reflected by the low flagellate-dominated (93%) algal biomass in the bottom layer, which was one-fifth of the diatom-dominated (74%) surface-layer biomass of 88 μg C l⁻¹. Our results indicate that habitable space plays a controlling role for ice algae in the Bothnian Bay fast ice. Similarly to the water column in the Bothnian Bay, average dissolved inorganic N:P-ratios in the ice were high, varying between 12 and 265. The integrated chlorophyll a (0.1–2.2 mg m⁻²) and algal biomass in the ice (1–31 mg C m⁻²) correlated significantly (Spearman ρ = 0.79), with the highest values being measured close to the river mouth in March and during the melt season in April. Flagellates <20 μm generally dominated in both the ice and water columns in February–March. In April the main ice-algal biomass was composed of Melosira arctica and unidentified pennate diatoms, while in the water column Achnanthes taeniata, Scrippsiella hangoei and flagellates dominated. The photosynthetic efficiency (0.003–0.013 (μg C [μg chl a ⁻¹] h⁻¹)(μE m⁻²s⁻¹)⁻¹) and maximum capacity (0.18–1.11 μg C [μg chl a ⁻¹] h⁻¹) could not always be linked to the algal composition, but in the case of a clear diatom dominance, pennate species showed to be more dark-adapted than centric diatoms.     

The solution structure of the invasive tip complex from Afa/Dr fibrils

Afa/Dr family of adhesins are produced by pathogenic Escherichia coli strains that are especially prevalent in chronic diarrhoeal and recurrent urinary tract infections. Most notably, they are found in up to 50% of cystitis cases in children and 30% of pyelonephritis in pregnant women. Afa/Dr adhesins are capped surface fibrils that mediate recognition of the host and subsequent bacterial internalization. Using the newly solved three-dimensional structure of the minimal invasive complex (AfaDE) combined with biochemical and cellular assays, we reveal the architecture of the fibrillar cap and identify a novel mode of synergistic integrin recognition.     

Observations on thermoregulatory ontogeny of mink (Mustela vison)

(1) We measured cooling rate for neonatal mink during a 10min coldroom (3.9 degrees C) exposure and subsequent warming rate during a 20min incubator (37.2 degrees C) exposure, the behaviour of the kits and the changes in their pelage between 1 and 46d of age, in an attempt to monitor the ontogeny of their thermoregulatory capacity. (2) Body weight of the 1d old kits averaged only 12.8+/-2.3g (n=4), but they gained weight rapidly reaching 226.1+/-28.3g (males, n=4) and 207.6+/-16.1g (females, n=4) at 30-31d of age, and 562.3+/-43.2g (males, n=3) and 435.7+/-35.5g (females, n=4) at 45-46d of age. (3) Body cooling rate (C(rate) ( degrees C/min); n=80) was affected by the age (between 1 and 31d), BW, initial rectal temperature (T(r0)), and sex of the kits, in addition to their body posture (P(cold), 1=extended, 2=curled-up) during coldroom exposure. C(rate) ( degrees C/min)=-0.34-0.02age-0.002BW+0.05T(r0)-0.06sex-0.20P(cold) (R(2)=0.75). (4) Body warming rate (W(rate) ( degrees C/min); n=80) was influenced by the age(2) and rectal temperature of the kit after the coldroom exposure (T(r10)). W(rate)( degrees C/min)=1.24+0.0002age(2)-0.04T(r10) (R(2)=0.76). (5) Kit fur fibre length increased from 5.45+/-0.63mm (males, n=2) and 6.20+/-0.20mm (females, n=3) at 22-23d of age to 9.43+/-1.44mm (males, n=4) and 8.70+/-1.89mm (females, n=4) at 30-31d of age, and to 12.93+/-0.47mm (males, n=3) and 11.38+/-0.41mm (females, n=4) at 45-46d of age, the growth averaging about 0.26mm per day. (6) Under normal circumstances newborn mink kits are hypothermic.Their thermoregulation develops only gradually and is dependent on increase in body mass, insulation and behavioural thermoregulation. Their strategy of survival is based on the ability to withstand hypothermia and on the nutrition and warmth provided by the dam.     

No effects of short-term GSM mobile phone radiation on cerebral blood flow measured using positron emission tomography

The present study investigated the effects of 902.4 MHz Global System for Mobile Communications (GSM) mobile phone radiation on cerebral blood flow using positron emission tomography (PET) with the ¹⁵O‐water tracer. Fifteen young, healthy, right‐handed male subjects were exposed to phone radiation from three different locations (left ear, right ear, forehead) and to sham exposure to test for possible exposure effects on brain regions close to the exposure source. Whole‐brain [¹⁵O]H₂O–PET images were acquired 12 times, 3 for each condition, in a counterbalanced order. Subjects were exposed for 5 min in each scan while performing a simple visual vigilance task. Temperature was also measured in the head region (forehead, eyes, cheeks, ear canals) during exposure. The exposure induced a slight temperature rise in the ear canals but did not affect brain hemodynamics and task performance. The results provided no evidence for acute effects of short‐term mobile phone radiation on cerebral blood flow. Bioelectromagnetics 33:247–256, 2012. © 2011 Wiley Periodicals, Inc.     

Net atmospheric impacts of forest bioenergy production and utilization in Finnish boreal conditions

The net CO₂ exchange of forests was investigated to study net atmospheric impact of forest bioenergy production (BP) and utilization in Finnish boreal conditions. Net CO₂ exchange was simulated with a life cycle assessment tool over a 90‐year period and over the whole Finland based on National Forest Inventory data. The difference in the net exchanges between the traditional timber production (TP) and BP regime was considered the net atmospheric impact of forest bioenergy utilization. According to the results, forests became net sources of CO₂ after about 20 years of simulation, and the net exchange was higher in the BP regime than in the TP regime until the middle of the simulation period. From 2040 onwards, the net exchange started to decrease in both regimes and became higher in the TP regime, excluding the last decade of the simulation. The shift of forests to becoming a CO₂ source reflected the decrease in CO₂ sequestration due to the increasing share of recently harvested and seedling stands that are acting as sources of CO₂, and an increase of emissions from degradation of wood products. When expressed in terms of radiative forcing, the net atmospheric impact was on average 19% less for bioenergy compared with that for coal energy over the whole simulation period. The results show the importance of time dependence when considering dynamic forest ecosystems in BP and climate change mitigation. Furthermore, the results emphasize the dualistic role and possibilities of forest management in controlling the build and release of carbon into and from the stocks and in controlling the rate of the build speed, i.e. growth. This information is needed in identifying the capability and possibilities of ecosystems to produce biomass for energy, alongside other products and ecosystem services (e.g. pulp wood and timber), and simultaneously to mitigate climate change.