Multi-temperature effects on Hill reaction activity of barley chloroplasts

1. 1. The relationship between temperature and Hill reaction activity has been investigated in chloroplasts isolated from barley (Hordeum vulgare L. cv. Abyssinian).

2. 2. An Arrhenius plot of the photoreduction of 2,6-dichlorophenolindophenol (DCIP) showed no change in slope over the temperature range 2–38 °C. The apparent Arrhenius activation energy (E_a) for the reaction was 48.1 kJ/mol.

3. 3. In the presence of an uncoupler of photophosphorylation, methylamine, the E_a for DCIP photoreduction went through a series of changes as the temperature was increased. Changes were found at 9, 20, 29 and 36 °C. The E_a was highest below 9 °C at 63.7 kJ/mol. Between 9 and 20 °C the E_a decreased to 40.4 kJ/mol and again to 20.2 kJ/mol between 20 and 29 °C. Between 29 and 36 °C there was no further increase in activity with increasing temperature. The temperature-induced changes at 9, 20 and 29 °C were reversible. At temperatures above 36 °C (2 min) a thermal and largely irreversible inactivation of the Hill reaction occurred.

4. 4. Temperature-induced changes in E_a were also found when ferricyanide was substituted for DCIP or gramicidin D for methylamine. The addition of an uncoupler of photophosphorylation was not required to demonstrate temperature-induced changes in DCIP photoreduction following the exposure of the chloroplasts to a low concentration of cations.

5. 5. The photoreduction of the lipophilic acceptor, oxidized 2, 3, 5, 6-tetramethyl-p-phenylenediamine, also showed changes in E_a in the absence of an uncoupler.

6. 6. The temperature-induced changes in Hill activity at 9 and 29 °C coincided with temperature-induced changes in the fluidity of chloroplast thylakoid membranes as detected by measurements of electron spin resonance spectra. It is suggested that the temperature-induced changes in the properties and activity of chloroplast membranes are part of a control mechanism for regulation of chloroplast development and photosynthesis by temperature.

Suspension array technology: evolution of the flat-array paradigm.

Suspension arrays of microspheres analyzed using flow cytometry offer a new approach to multiplexed assays for large-scale screening applications. By optically encoding micron-sized polymer particles, suspension microarrays can be created to enable highly multiplexed analysis of complex samples. Each element in the array is comprised of a subpopulation of particles with distinct optical properties and each array element bears a different surface receptor. Nucleic acids, proteins, lipids or carbohydrates can serve as receptors to support the analysis of a wide range of biomolecular assemblies, and applications in genomic and proteomic research are being developed. Coupled with recent innovations for rapid serial analysis of samples, molecular analysis with microsphere arrays holds significant potential as a general analysis platform for both research and clinical applications.     

Cryopreservation of first-stage and infective third-stage larvae of Strongyloides stercoralis

Infective third-stage larvae of Strongyloides stercoralis were frozen over liquid nitrogen and remained infective to dogs when thawed. Successful cryopreservation depended on a 30-60-min incubation in a cryoprotectant (10% DMSO and 10% dextran) before freezing and thawing the frozen larvae into RPMI. First-stage larvae could also be frozen by this method. Thawed first-stage larvae remained viable and continued their development to third-stage larvae, which were shown to be infective to dogs.     

Self-assembling Shell Proteins PduA and PduJ have Essential and Redundant Roles in Bacterial Microcompartment Assembly

Protein self-assembly is a common and essential biological phenomenon, and bacterial microcompartments present a promising model system to study this process. Bacterial microcompartments are large, protein-based organelles which natively carry out processes important for carbon fixation in cyanobacteria and the survival of enteric bacteria. These structures are increasingly popular with biological engineers due to their potential utility as nanobioreactors or drug delivery vehicles. However, the limited understanding of the assembly mechanism of these bacterial microcompartments hinders efforts to repurpose them for non-native functions. Here, we comprehensively investigate proteins involved in the assembly of the 1,2-propanediol utilization bacterial microcompartment from Salmonella enterica serovar Typhimurium LT2, one of the most widely studied microcompartment systems. We first demonstrate that two shell proteins, PduA and PduJ, have a high propensity for self-assembly upon overexpression, and we provide a novel method for self-assembly quantification. Using genomic knock-outs and knock-ins, we systematically show that these two proteins play an essential and redundant role in bacterial microcompartment assembly that cannot be compensated by other shell proteins. At least one of the two proteins PduA and PduJ must be present for the bacterial microcompartment shell to assemble. We also demonstrate that assembly-deficient variants of these proteins are unable to rescue microcompartment formation, highlighting the importance of this assembly property. Our work provides insight into the assembly mechanism of these bacterial organelles and will aid downstream engineering efforts.     

Area security unit in a psychiatric hospital.

Since 1974 a psychiatric hospital security unit, designed to serve the whole catchment area, has cared for mentally ill (mostly psychotic) patients with disturbed behaviour that cannot be managed in open wards. There are a few long-term dangerous patients but most stay only briefly. The admission of women to the unit was not followed by the expected reduction in violence. The unit has facilities for occupational therapy, physical recreation, work, and study, which are particularly important for those who are too dangerous to leave it. The unit''s calming influence depends as much on the supportive effect of the high staff ratio as on the use of tranquillisers. This type of unit is not suitable for patients with personality disturbances who "act out" or for mentally abnormal offenders; but it functions well as a crisis centre for the disturbed mentally ill, and there is an increasing demand for its services.     

Complementary strand relocation may play vital roles in RecA-based homology recognition

RecA-family proteins mediate homologous recombination and recombinational DNA repair through homology search and strand exchange. Initially, the protein forms a filament with the incoming single-stranded DNA (ssDNA) bound in site I. The RecA–ssDNA filament then binds double-stranded DNA (dsDNA) in site II. Non-homologous dsDNA rapidly unbinds, whereas homologous dsDNA undergoes strand exchange yielding heteroduplex dsDNA in site I and the leftover outgoing strand in site II. We show that applying force to the ends of the complementary strand significantly retards strand exchange, whereas applying the same force to the outgoing strand does not. We also show that crystallographically determined binding site locations require an intermediate structure in addition to the initial and final structures. Furthermore, we demonstrate that the characteristic dsDNA extension rates due to strand exchange and free RecA binding are the same, suggesting that relocation of the complementary strand from its position in the intermediate structure to its position in the final structure limits both rates. Finally, we propose that homology recognition is governed by transitions to and from the intermediate structure, where the transitions depend on differential extension in the dsDNA. This differential extension drives strand exchange forward for homologs and increases the free energy penalty for strand exchange of non-homologs.     

Porcine E. coli: Virulence-Associated Genes,Resistance Genes and Adhesion and Probiotic Activity Tested by a New Screening Method

We established an automated screening method to characterize adhesion of Escherichia coli to intestinal porcine epithelial cells (IPEC-J2) and their probiotic activity against infection by enteropathogenic E. coli (EPEC). 104 intestinal E. coli isolates from domestic pigs were tested by PCR for the occurrence of virulence-associated genes, genes coding for resistances to antimicrobial agents and metals, and for phylogenetic origin by PCR. Adhesion rates and probiotic activity were examined for correlation with the presence of these genes. Finally, data were compared with those from 93 E. coli isolates from wild boars.Isolates from domestic pigs carried a broad variety of all tested genes and showed great diversity in gene patterns. Adhesions varied with a maximum of 18.3 or 24.2 mean bacteria adherence per epithelial cell after 2 or 6 hours respectively. Most isolates from domestic pigs and wild boars showed low adherence, with no correlation between adhesion/probiotic activity and E. coli genes or gene clusters. The gene sfa/foc, encoding for a subunit of F1C fimbriae did show a positive correlative association with adherence and probiotic activity; however E. coli isolates from wild boars with the sfa/foc gene showed less adhesion and probiotic activity than E. coli with the sfa/foc gene isolated from domestic pigs after 6 hour incubation.In conclusion, screening porcine E. coli for virulence associated genes genes, adhesion to intestinal epithelial cells, and probiotic activity revealed a single important adhesion factor, several probiotic candidates, and showed important differences between E. coli of domestic pigs and wild boars.     

Distribution of amiloride-sensitive sodium channels in the oral cavity of the hamster

The distribution of amiloride-sensitive sodium channels (ASSCs) in taste buds isolated from the oral cavity of hamsters was assessed by patch clamp recording. In contrast to the case for rats, taste cells from the fungiform, foliate and vallate papillae and from the soft palate all contain functional ASSCs. The differential distribution of ASSCs between the hamster and the rat may be important for understanding the physiology underlying the differing behavioral responses of these species to sodium salts.      

Experimental exposure of rainbow trout Oncorhynchus mykiss (Walbaum) to the infective stages of the sea louse Lepeophtheirus salmonis (Krøyer) influences the physiological response to an acute stressor

The influence of infection with the juvenile stages of the sea louse, Lepeophtheirus salmonis (Kr?yer) on the response of rainbow trout Oncorhynchus mykiss (Walbaum) to a net confinement protocol was investigated. The experiment consisted of two groups of seawater-adapted rainbow trout, one which was exposed to a total of 4000 nauplii/copepodid stages of L. salmonis 30, 25 and 14 days prior to confinement. Confinement elicited a greater stress response in the lice-exposed fish, than in the controls, as seen by higher plasma cortisol and glucose levels. A reduced spleen somatic index in exposed fish following 6 h confinement coincided with increased erythrocyte and lymphocyte numbers in the blood. Circulating lymphocyte numbers were significantly reduced in both groups 24 h post-confinement, when a lower alternative complement activity was recorded in control fish. Prior to confinement, lice-exposed fish had an elevated serum lysozyme activity and reduced oxygen radical production by blood leukocytes. Following confinement, lysozyme activity was gradually reduced in lice-exposed trout. During confinement, oxygen radical production decreased in control fish and increased in infested fish. Overall, transient exposure to juvenile lice altered the response to a second stressor, which has implications for management procedures of L. salmonis exposed fish.