Measurement of immunoglobulin synthesis using the ELISPOT assay

We describe a method for the detection of specific antibody-producing cells from either in vitro or in vivo immunization. These techniques are especially useful for detecting antibodies from developing hybridomas. We have successfully used the system to detect isotype-specific antibodies to a variety of bacterial antigens which were produced by heterohybridomas.     

Individual Variation in Advertisement Calls of Territorial Male Green Frogs, Rana clamitans: Implications for Individual Discrimination

Individuals of many territorial species discriminate between familiar territorial neighbors and unfamiliar strangers based on individual differences in acoustic signals. Many anuran amphibians are territorial and use primarily acoustic communication during social interactions, but evidence for acoustically mediated individual discrimination is available only for one species. As a first step in research designed to investigate individual discrimination in a second species of territorial frog, we examined patterns of within-male and among-male variability in 198 advertisement calls of 20 male green frogs, Rana clamitans . The aim was to determine which acoustic properties could be used by males to recognize their territorial neighbors and to estimate limits of reliability afforded by these properties for identifying different neighbors. All of the call properties that we examined exhibited significant inter-individual variation. Discriminant function analyses assigned between 52% and 100% of calls to the correct individual, depending on sample size and the call properties included in the model. This suggests that there is sufficient among-male variability to statistically identify individuals by their advertisement calls. The call properties of fundamental frequency and dominant frequency contributed the most towards discrimination among individuals. Based on their natural history and behavior and the results reported here, we suggest that male green frogs likely discriminate between strangers and adjacently territorial neighbors based on individual variation in advertisement calls.     

Comparative Genomic Sequence Analysis of the FXR Gene Family: FMR1, FXR1, and FXR2

Mutations in the X-linked gene FMR1 cause fragile X syndrome, the leading cause of inherited mental retardation. Two autosomal paralogs of FMR1 have been identified, and are known as FXR1 and FXR2. Here we describe and compare the genomic structures of the mouse and human genes FMR1, FXR1, and FXR2. All three genes are very well conserved from mouse to human, with identical exon sizes for all but two FXR2 exons. In addition, the three genes share a conserved gene structure, suggesting they are derived from a common ancestral gene. As a first step towards exploring this hypothesis, we reexamined the Drosophila melanogaster gene Fmr1, and found it to have several of the same intron/exon junctions as the mammalian FXRs. Finally, we noted several regions of mouse/human homology in the noncoding portions of FMR1 and FXR1. Knowledge of the genomic structure and sequence of the FXR family of genes will facilitate further studies into the function of these proteins.     

Structuring features of lake districts: landscape controls on lake chemical responses to drought

1. Within a lake district of relatively homogeneous geomorphology, the responses of lakes to climate are influenced by the complexity of the hydrogeologic setting, position in the landscape, and lake‐specific biological and physical features. We examined lake chemical responses to drought in surface water‐ and groundwater‐dominated districts to address two general questions. (1) Are spatial patterns in chemical dynamics among lakes uniform and synchronous within a lake district, suggesting broad geomorphic controls; variable in a spatially explicit pattern, with synchrony related to landscape position, suggesting hydrologic flowpath controls; or spatially unstructured and asynchronous, suggesting overriding control by lake‐specific factors? (2) Are lake responses to drought a simple function of precipitation quantity or are they dictated by more complex interactions among climate, unique lake features, and hydrologic setting? 2. Annual open‐water means for epilimnetic concentrations of chloride, calcium, sulfate, ANC, DOC, total nitrogen, silica, total phosphorus, and chlorophyll a measured between 1982 and 1995 were assembled for lakes in the Red Lake and ELA districts of north‐western Ontario, the Muskoka – Dorset district in south‐central Ontario, and the Northern Highland district of Wisconsin. Within each district, we compared responses of lakes classified by landscape position into highland or lowland, depending on relative location within the local to regional hydrologic flow system. Synchrony, defined as a measure of the similarity in inter‐annual dynamics among lakes within a district, was quantified as the Pearson product‐moment correlation (r) between two lakes with observations paired by year. To determine if solute concentrations were directly related to interannual variations in precipitation quantity, we used regression analysis to fit district‐wide slopes describing the relationship between each chemical variable and annual (June to May) and October to May (Oct–May) precipitation. 3. Among lakes in each of the three Ontario districts, the pattern of chemical response to interannual shifts in precipitation was spatially uniform. In these surface water‐ dominated districts, solute concentrations were generally a simple function of precipitation. Conservative solutes, like calcium and chloride, tended to be more synchronous and were negatively related to precipitation. Solutes such as silica, total phosphorus, and chlorophyll a, which are influenced by in‐lake processes, were less synchronous and relationships with precipitation tended to be positive or absent. 4. In the groundwater‐dominated Northern Highland lakes of Wisconsin, we observed spatial structure in drought response, with lowland lakes more synchronous than highland lakes. However, there was no evidence for a direct relationship between any solute and precipitation. Instead, increases in the concentration of the conservative ion calcium during drought were not followed by a symmetrical return to pre‐drought conditions when precipitation returned to normal or above‐average values. 5. For calcium, time lags in recovery from drought appeared related to hydrologic features in a complex way. In the highland Crystal Lake, calcium concentrations tracked lake stage inversely, with a return to pre‐drought concentrations and lake stage five years after the drought. This pattern suggests strong evaporative controls. In contrast, after five years of normal precipitation, calcium in the lowland Sparkling Lake had not returned to pre‐drought conditions despite a rebound in lake stage. This result suggests that calcium concentrations in lowland lakes were controlled more by regional groundwater flowpaths, which track climatic signals more slowly. 6. Temporal dynamics driven by climate were most similar among lakes in districts that have a relatively simple hydrology, such as ELA. Where hydrologic setting was more complex, as in the groundwater‐dominated Northern Highland of Wisconsin, the expression of climate signals in lakes showed lags and spatial patterns related to landscape position. In general, we expect that landscape and lake‐specific factors become increasingly important in lake districts with more heterogeneous hydrogeology, topography or land use. These strong chemical responses to climate need to be considered when interpreting the responses of lakes to other regional disturbances.     

ESTIMATION OF AGE-SPECIFIC PROBABILITIES OF FIRST RETURN AND ANNUAL SURVIVAL RATES FOR THE MALE GRAY SEAL (HALICHOERUS GRYPUS) ON SABLE ISLAND FROM CAPTURE-RECAPTURE DATA

We used a longitudinal capture-recapture study to estimate the age-specific probabilities of first return to the breeding colony and annual survival rates for male gray seals ( Halichoerus grypus ), based on resightings of seals branded as young on Sable Island. We estimated that the average age of first returns for seals born in 1969–1970 to be 9.1 (SE 0.4) yr; for seals born in 1973–1974 it is estimated to be 9.8 (SE 0.2) yr. The estimated annual survival rate of these males was estimated to be 0.976 (SE 0.003).     

SECONDARY SEX CHARACTERISTICS IN SPINACIA OLERACEA L.: QUANTITATIVE EVIDENCE FOR THE EXISTENCE OF AT LEAST THREE SEXUAL MORPHS

Spinacia oleracea L. was grown hydroponically across an experimental salinity gradient with treatments of 0.0, 1.5, 3.0, 4.5, and 6.0 grams of NaCl/liter of nutrient medium. Plant height, root biomass, vegetative biomass, fruit morphology, average fruit weight, and fruit mass: volume ratio were examined in each treatment to determine if differences other than primary sex characteristics exist between male, female, and monoecious plants. Average fruit weight was the only parameter examined that was not useful as a secondary sex characteristic. Orthogonal contrasts examining plant height, vegetative biomass, and root biomass of plants were distinct from one another across the salinity gradient. Similarly, female and monoecious plants differed significantly from one another in terms of root biomass and the average mass: volume ratio of the fruit each sexual morph produced, as well as the proportion of smooth to wrinkled fruit produced. The data indicate that male, female, and monoecious plants are three distinct sexual morphs.     

Semi-automated imaging system to quantitate Her-2/neu membrane receptor immunoreactivity in human breast cancer.

BACKGROUND: Immunocytochemical methods for quantitating Her-2/neu immunoreactivity rest on subjective semi-quantitative interpretations with resulting interobserver, intraobserver, and fatigue variability. METHODS: To standardize and quantitate measurements of Her-2/neu immunoreactivity, we created epithelial-recognition and specific membrane-recognition algorithms, which could image breast cancer cells against a background of stroma, compartmentalize the cancer cell into nucleus, cytoplasm and membrane, and quantitate the degree of Her-2/neu membrane immunoreactivity based on both gray scale intensity and RGB colorimetric determinations. Image acquisition utilized either scanner or microscope with attached camera with a resolution of 20 pixels/10 microm. Areas of 150 whole slides were screened and the regions of interest manually selected for image processing. Three hundred TMA cores were directly processed. Images were acquired by jpg conversion of svs virtual slides or direct jpg photomicrograph capture. Images were then assessed for quality and processed. RESULTS: The digital algorithms successfully created a semi-automated imaging system whose algorithm-based ordinal values for Her-2/neu both strongly correlated with the subjective measurements (intraclass correlation: 0.84; 95% confidence interval: 0.79-0.89) yet exhibited no run variability. In addition, the algorithms generated immunocytochemical measurements of Her-2/neu on an expanded continuous scale, which more reliably distinguished true Her-2/neu positivity from true Her-2/neu negativity (determined by FISH) than subjective or algorithmic ordinal scale measurements. Furthermore, the continuous scale measurements could better resolve different levels of Her-2/neu overexpression than either subjective or algorithmic ordinal interpretation. Other semi-automated analysis systems have been used to measure Her-2/neu and other cellular immunoreactivities, but these either have required proprietary hardware or have been based on luminosity differences alone. In contrast, our algorithms are independent of proprietary hardware and are based on not just luminosity but also many other imaging properties including epithelial recognition and membrane morphology. CONCLUSION: These features provide a more accurate, versatile, and robust imaging analysis platform.     

Corrosion behaviour, metal release and biocompatibility of implant materials coated by TiO2-sol gel chemistry]

Alloys based on titanium or cobalt have been used as implant materials for decades with good success. Because of their natural oxide layer these alloys reveal good corrosion behaviour. In contact with physiological solution metal release takes place, which can cause inflammation. Coatings can improve the corrosion behaviour. In this study Ti6Al4V and Co28Cr6Mo alloys, which are frequently used as implant materials, were tested. Polished discs of these alloys and polished discs, which were coated with TiO2-layers by sol-gel chemistry, were compared regarding their corrosion behaviour and metal ion releasing. The releasing of Al, V, Ti, Co, Cr and Mo was quantified by ICP-MS analysis. The TiO2-coating reduced the release of all ions except of the Al-ion. Both alloys showed a deviating kinetic of ion releasing. In addition, cell response (cell vitality, cell proliferation, endothelial marker CD31 and actin allocation) of osteoblasts and endothelial cells were investigated.     

The use of correlation integrals in the study of localized muscle fatigue of elbow flexors during maximal efforts.

Innovative applications of non-linear time series analysis have recently been used to investigate physiological phenomena. In this study, we investigated the feasibility of using the correlation integral to monitor the localized muscle fatigue process in the biceps brachii during sustained maximal efforts. The subjects performed isometric maximum contractions until failure in elbow flexion (90 degrees from neutral). The median and the 70th percentile frequency of the Surface electromyography (SEMG) power spectrum, the integrated SEMG, and the Correlation Integral (CI) were evaluated during the trials. The linear correlation between these variables and the elbow torque production was used to quantify the ability of a parameter to follow the fatiguing process. The CI had the highest linear correlation with torque (0.77 (0.12SD)), while the spectral indices correlations with torque were much lower. The decreasing trend of the torque production was followed by the spectral indices for only the beginning part of the contraction, while the CI increased sharply after the torque production fell to about 0.60 of the MVC. This suggests that the CI is sensitive to different changes of the SEMG signal during fatigue than the spectral variables.     

The positive and negative conservation impacts of sheep grazing and other disturbances on the vascular plant species and vegetation of lowland subhumid Tasmania

Summary An important conservation question for grazed areas of lowland subhumid Tasmania is ‘what effects do different, practical disturbance regimes have on native vegetation?’ An experiment designed to determine the single and interactive effects of fire and sheep grazing was established at four sites with distinct vegetation types. There were significant interactive effects of fire and sheep grazing on vegetation attributes at all sites. An analysis of published and new data indicated that there were several vascular plant species that appeared dependent on sheep grazing for their persistence in the present landscape, while there were others that were intolerant of this disturbance but required other types of disturbance, such as mowing. However, most native species appeared to survive in a wide variety of disturbance regimes short of ploughing and fertilization. The implications of these results are that a variety of disturbance regimes is necessary to maintain biological diversity in this environment, and that the naturalness of the regime is not necessarily relevant to its use for conservation.