RATE OF O2 PRODUCTION DERIVED FROM PULSE‐AMPLITUDE‐MODULATED FLUORESCENCE: TESTING THREE BIOOPTICAL APPROACHES AGAINST MEASURED O2‐PRODUCTION RATE1

Light absorption by phytoplankton is both species specific and affected by photoacclimational status. To estimate oxygenic photosynthesis from pulse‐amplitude‐modulated (PAM) fluorescence, the amount of quanta absorbed by PSII needs to be quantified. We present here three different biooptical approaches to estimate the fraction of light absorbed by PSII: (1) the factor 0.5, which implies that absorbed light is equally distributed among PSI and PSII; (2) the fraction of chl a in PSII, determined as the ratio between the scaled red‐peak fluorescence excitation and the red absorption peak; and (3) the measure of light absorbed by PSII, determined from the scaling of the fluorescence excitation spectra to the absorption spectra by the “no‐overshoot” procedure. Three marine phytoplankton species were used as test organisms: Prorocentrum minimum (Pavill.) J. Schiller (Dinophyceae), Prymnesium parvum cf. patelliferum (J. C. Green, D. J. Hibberd et Pienaar) A. Larsen (Haptophyceae), and Phaeodactylum tricornutum Bohlin (Bacillariophyceae). Photosynthesis versus irradiance (P vs. E) parameters calculated using the three approaches were compared with P versus E parameters obtained from simultaneously measured rates of oxygen production. Generally, approach 1 underestimated, while approach 2 overestimated the gross O₂‐production rate calculated from PAM fluorescence. Approach 3, in principle the best approach to estimate quanta absorbed by PSII, was also superior according to observations. Hence, we recommend approach 3 for estimation of gross O₂‐production rates based on PAM fluorescence measurements.     

DNA fusion-gene vaccination in patients with prostate cancer induces high-frequency CD8+ T-cell responses and increases PSA doubling time

We report on the immunogenicity and clinical effects in a phase I/II dose escalation trial of a DNA fusion vaccine in patients with prostate cancer. The vaccine encodes a domain (DOM) from fragment C of tetanus toxin linked to an HLA-A2-binding epitope from prostate-specific membrane antigen (PSMA), PSMA_27–35. We evaluated the effect of intramuscular vaccination without or with electroporation (EP) on vaccine potency. Thirty-two HLA-A2⁺ patients were vaccinated and monitored for immune and clinical responses for a follow-up period of 72 weeks. At week 24, cross-over to the immunologically more effective delivery modality was permitted; this was shown to be with EP based on early antibody data, and subsequently, 13/15 patients crossed to the +EP arm. Thirty-two HLA-A2^? control patients were assessed for time to next treatment and overall survival. Vaccination was safe and well tolerated. The vaccine induced DOM-specific CD4⁺ and PSMA₂₇-specific CD8⁺ T cells, which were detectable at significant levels above baseline at the end of the study (p = 0.0223 and p = 0.00248, respectively). Of 30 patients, 29 had a measurable CD4⁺ T-cell response and PSMA₂₇-specific CD8⁺ T cells were detected in 16/30 patients, with or without EP. At week 24, before cross-over, both delivery methods led to increased CD4⁺ and CD8⁺ vaccine-specific T cells with a trend to a greater effect with EP. PSA doubling time increased significantly from 11.97 months pre-treatment to 16.82 months over the 72-week follow-up (p = 0.0417), with no clear differential effect of EP. The high frequency of immunological responses to DOM-PSMA₂₇ vaccination and the clinical effects are sufficiently promising to warrant further, randomized testing.     

Modulation of proximal signaling in normal and transformed B cells by transmembrane adapter Cbp/PAG

The transmembrane protein Cbp/PAG (Csk binding protein/phospho-protein associated with glycosphingolipid-enriched microdomains) has a negative regulatory role in T cell activation as an adapter for C-terminal Src kinase, Csk. In T cells, membrane docking of Csk is promoted by binding to FynT-phosphorylated Cbp/PAG (pTyr317) to allow targeting of substrates residing in lipid rafts. Here, we investigate a potential parallel position for Cbp/PAG and the Src kinase Lyn in early B cell receptor signaling. Using normal and transformed B cells, we have compared signal profiles of BCR-triggered responses created by phospho-specific flow cytometry. In human normal B cells, our data show that reduced Cbp/PAG levels leads to enhanced and prolonged activation of proximal signaling mediators, while over-expression of the adapter in normal, EBV-transformed cells results in reduced calcium flux. Taken together, our findings support a negative regulatory function for Cbp/PAG in proximal BCR signaling in these cells.     

Sex ratio variation in harvested moose (<Emphasis Type="Italic">Alces alces</Emphasis>) calves: does it reflect population calf sex ratio or selective hunting?

During the last 30 years, the proportion of males in the calf harvest of moose (Alces alces) in Norway has decreased, indicating a decline in proportions of males recruited to the autumn populations. At the same time, the percentages of exclusive calf hunting permits and of calves shot have increased. The change in calf sex ratio may thus simply be the result of hunter preferences for slightly larger (6.2% higher body mass) male calves combined with fewer opportunities for selective hunting due to increasing hunting quotas of calves. We examined this hypothesis by analyzing the variation in sex, number of siblings, carcass mass, date, and location of kill of 16,330 moose calves harvested during 1970–2004. In the presence of hunting selection for larger calves, we predicted larger proportions of male calves to be harvested in populations with large sexual size dimorphism among calves. Similarly, we expected more males to be harvested from twin than single litters because hunters then can more easily compare twins and select the larger calf, which is more often a male. Increasing proportions of single female calves were also expected to occur in the daily harvest as the accumulated number of harvested calves increased and the proportion of calves left in the population decreased. We found no positive relationship between the proportion of male calves and the level of sexual size dimorphism, no clear difference in sex ratio between harvested single and twin calves, and no increase in the proportion of single female calves as the accumulated number of calves in the harvest increased. This suggests that the spatiotemporal variation in the harvest calf sex ratio in Norway most likely reflects differences in population calf sex ratios prior to the hunting season and not varying degrees of hunting selectivity.     

Structural perturbations in DNA caused by bis-intercalation of ditercalinium visualised by atomic force microscopy

Atomic force microscopy (AFM) has been used to examine perturbations in the tertiary structure of DNA induced by the binding of ditercalinium, a DNA bis-intercalator with strong anti-tumour properties. We report AFM images of plasmid DNA of both circular and linearised forms showing a difference in the formation of supercoils and plectonemic coils caused at least in part by alterations in the superhelical stress upon bis-intercalation. A further investigation of the effects of drug binding performed with 292 bp mixed-sequence DNA fragments, and using increment in contour length as a reliable measure of intercalation, revealed saturation occurring at a point where sufficient drug was present to interact with every other available binding site. Moment analysis based on the distribution of angles between segments along single DNA molecules showed that at this level of bis-intercalation, the apparent persistence length of the molecules was 91.7 ± 5.7 nm, approximately twice as long as that of naked DNA. We conclude that images of single molecules generated using AFM provide a valuable supplement to solution-based techniques for evaluation of physical properties of biological macromolecules.     

The influence of DNA stiffness upon nucleosome formation

The rotational and translational positioning of nucleosomes on DNA is dependent to a significant extent on the physicochemical properties of the double helix. We have investigated the influence of the axial flexibility of the molecule on the affinity for the histone octamer by substituting selected DNA sequences with either inosine for guanosine or diaminopurine for adenine. These substitutions, respectively, remove or add a purine 2-amino group exposed in the minor groove and, respectively, decrease and increase the apparent persistence length. We observe that for all sequences tested inosine substitution, with one exception, increases the affinity for histone binding. Conversely diaminopurine substitution decreases the affinity. In the sole example where replacement of guanosine with inosine decreases the persistence length as well as the affinity for histones, the substitution concomitantly removes an intrinsic curvature of the DNA molecule. We show that, to a first approximation, the binding energy of DNA to histones at 1M NaCl is directly proportional to the persistence length. The data also indicate that a high local flexibility of DNA can favour strong rotational positioning.