Proteomic and computational analysis of secreted proteins with type I signal peptides from the Antarctic archaeon Methanococcoides burtonii

LC-MS/MS was used to identify secreted proteins in the Antarctic archaeon Methanococcoides burtonii. Seven proteins possessing a classical class 1 signal peptide were identified in the supernatant from cultures grown at 4 and 23 degrees C. The proteins included a putative S-layer cell surface protein, cell surface protein involved with cell adhesion, and trypsin-like serine protease. Protease activity was detected in the secreted fraction, and the signal peptide cleavage site of the protease was confirmed using Edman sequencing. The expression profile of putative cell surface proteins suggests a requirement for cell interactions during growth at low temperature. Sequences of the secreted proteins were used to compile a dataset containing a further 32 predicted secreted proteins from the Methanosarcinaceae. Many of these proteins were also S-layer cell surface proteins with a variety of predicted roles, particularly in cell-cell interaction. Computational analysis of signal peptides revealed a preference for lysine in the n-region, leucine in the h-region, and a eucaryal-type cleavage site, highlighting the mosaic nature of signal peptides in Archaea. This is the first study to experimentally characterize secreted proteins from a cold-adapted archaeon and provides new insight and a functional dataset for studying secretion in Archaea.     

The influence of water level on macrophyte growth and trophic interactions in eutrophic Mediterranean shallow lakes: a mesocosm experiment with and without fish

1. Water‐level fluctuations are typical of lakes located in the semi‐arid Mediterranean region, which is characterised by warm rainy winters and hot dry summers. Ongoing climate change may exacerbate fluctuations and lead to more severe episodes of drought, so information on the effects of water level on the functioning of lake ecosystems in such regions is crucial. 2. In eutrophic Lake Eymir, Turkey, we conducted a 4‐month (summer) field experiment using cylindrical 0.8‐m‐ (low‐water‐level) and 1.6‐m‐deep (high‐water‐level) mesocosms (kept open to the sediment and atmosphere). Fish (tench, Tinca tinca, and bleak, Alburnus escherichii) were added to half of the mesocosms, while the rest were kept fishless. Ten shoots of Potamogeton pectinatus were transplanted to each mesocosm. 3. Sampling for physicochemical variables, chlorophyll a (chl‐a), zooplankton and per cent plant volume inhabited (PVI%) by macrophytes was conducted weekly during the first 5 weeks, and subsequently biweekly. Macrophytes were harvested on the last sampling date. During the course of the experiment, the water level decreased by 0.41 ± 0.06 m. 4. Throughout the experiment, fish affected zooplankton abundance (?), nutrient concentrations (+), chl‐a (+) and water clarity (?) most strongly in the low‐water‐level mesocosms and the zooplankton community shifted towards dominance of small‐sized forms. The fishless mesocosms had a higher zooplankton/phytoplankton ratio, suggesting higher grazing. 5. Greatest macrophyte growth was observed in the low‐water‐level fishless mesocosms. However, despite high nutrient concentrations and low water clarity, macrophytes were also abundant in the fish mesocosms and particularly increased following a water‐level decrease from midsummer onwards. Macrophyte growth was poor in the high‐water‐level mesocosms, even in the fishless ones with high water clarity. This was ascribed to extensive periphyton development reducing light availability for the macrophytes. 6. Our results indicate that a reduction in water level during summer may help maintain the growth of macrophytes in Mediterranean eutrophic shallow lakes, despite a strong negative effect of fish predation on water clarity. It is therefore probable that an expected negative effect of global climate change on water clarity because of eutrophication and enhanced top‐down control of fish may be, at least partly, counteracted by reduced water level, provided that physical disturbance is not severe.     

Nifedipine loaded chitosan microspheres prepared by emulsification phase-separation

A high yield of nifedipine-chitosan microspheres could be obtained using an emulsification phase-separation method. A high level of entrapment of nifedipine in the microspheres was achieved. The microspheres exhibited excellent swelling properties. Differential scanning calorimetry, X-ray diffractometry, and scanning electron microscopy confirmed that at 1.84% loading, nifedipine was dispersed molecularly. The microspheres exhibited faster release at low loadings compared to high loadings. Fitting the data to the coupled Fickian/case II equation, showed that at low loadings polymer relaxation coefficients (k₂) were high. As the polymer content increased in the microspheres, the value of n (diffusional exponent characteristic of the release mechanism) approached one, which is indicative of zero order.     

Real time in vitro studies of doxorubicin release from PHEMA nanoparticles

Background  

Many anticancer agents have poor water solubility and therefore the development of novel delivery systems for such molecules has received significant attention. Nanocarriers show great potential in delivering therapeutic agents into the targeted organs or cells and have recently emerged as a promising approach to cancer treatments. The aim of this study was to prepare and use poly-2-hydroxyethyl methacrylate (PHEMA) nanoparticles for the controlled release of the anticancer drug doxorubicin.     

Ribozymes that cleave an RNA sequence from human immunodeficiency virus: the effect of flanking sequence on rate

Ribozymes designed to cleave sequences specific to viral RNA may be better antiviral agents than simple antisense oligonucleotides. High catalytic activity with the lowest possible chain length is desired for this purpose. We have synthesized several hammerhead ribozymes that cleave sequences from HIV-1 RNA. On reducing from 20 to 12 the base pairs formed with the substrate, the rate of cleavage at 37 degrees C increased 10-fold. Deletions from the stem/loop structure in the ribozyme also increased the initial rate of reaction.     

Effect of Anionic Salt and Highly Fermentable Carbohydrate Supplementations on Urine pH and on Experimentally Induced Hypocalcaemia in Cows

The objective of this experiment was to determine the effect of dietary grain on calcium homeostasis. Six rumen-fistulated dairy cows with 3 or more previous lactations and no history of parturient paresis were randomly assigned to a sequence of diets in a crossover study with 4 periods of 10 days each. Dietary treatments were: A control ration consisting of wrap grass silage alone (1), the control ration supplemented with ammonium chloride and ammonium sulphate salt solution (2), control ration following a period with supplementation (3) and control ration supplemented with increasing amounts of barley from 4 to 10 kg/cow per day, expected to produce subclinical rumen acidosis (4). Daily intake of the diets was adjusted to 14 kg DM/cow per day. On day 11, the calcium-regulating mechanisms in cows were challenged until recumbency by a standardized intravenous EDTA infusion and cows were left to recover spontaneously. Anion supplementation and the feeding of highly fermentable carbohydrate lowered urine pH below 7.0 due to subclinical acidosis. During spontaneous recovery from EDTA induced hypocalcaemia, the cows more quickly regained a whole blood free calcium concentration of 1.00 mmol/L if they had most recently been supplemented with either anionic salts or with increasing amounts of barley, as compared to the basic ration. It is concluded that so-called slug-feeding or 'steaming up' with highly fermentable carbohydrates before parturition in milk fever susceptible cows enhanced calcium homeostasis similar to the effect seen in cows on anionic diets.     

PACAP is expressed in sympathoexcitatory bulbospinal C1 neurons of the brain stem and increases sympathetic nerve activity in vivo

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an excitatory neuropeptide present in the rat brain stem. The extent of its localization within catecholaminergic groups and bulbospinal sympathoexcitatory neurons is not established. Using immunohistochemistry and in situ hybridization, we determined the extent of any colocalization with catecholaminergic and/or bulbospinal projections from the brain stem was determined. PACAP mRNA was found in tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the C1-C3 cell groups. In the rostral ventrolateral medulla (RVLM), PACAP mRNA was found in 84% of the TH-ir neurons and 82% of bulbospinal TH-ir neurons. The functional significance of these PACAP mRNA positive bulbospinal neurons was tested by intrathecal administration of PACAP-38 in anaesthetized rats. Splanchnic sympathetic nerve activity doubled (110%) and heart rate rose significantly (19%), although blood pressure was unaffected. In addition, as previously reported, PACAP was found in the A1 cell group but not in the A5 cell group or in the locus coeruleus. The RVLM is the primary site responsible for the tonic and reflex control of blood pressure through the activity of bulbospinal presympathetic neurons, the majority of which contain TH. The results indicate 1) that pontomedullary neurons containing both TH and PACAP that project to the intermediolateral cell column originate from C1-C3 and not A5, and 2) intrathecal PACAP-38 causes a prolonged, sympathoexcitatory effect.     

Further evidence for stroma lamellae as a source of Photosystem 1 fractions from spinach chloroplasts

The mechanism of digitonin action on spinach chloroplasts was investigated by thin sectioning. Evidence is presented which shows that digitonin continues to modify membranes for many minutes after the addition of the fixative glutaraldehyde. However, the action of digitonin can be stopped by simultaneous fixation and dilution of the detergent. Such experiments indicate that the initial action of digitonin is to release stroma lamellae which in turn yield a Photosystem 1 fraction. This interpretation is further supported by a significant correlation between the chlorophyll a/chlorophyll b ratio and the ratio of stroma to grana lamellae in spinach chloroplasts.