Characterization of circadian oscillations in alanine dehydrogenase activity in non-dividing populations of Euglena gracilis (z)

Possible factors that could generate the circadian oscillations in alanine dehydrogenase (EC 1.4.1.1.) activity observed in cultures of non-dividing Euglena gracilis (Z) have been examined in an effort to learn more about the basic timekeeping mechanism of biological clocks. No differences in K_m, pH optimum or electrophoretic mobility could be demonstrated between enzyme extracted from the minimum part of the 24-h oscillation in activity and that extracted from the maximum part. Also, no evidence for the presence of activators or inhibitors was found in mixing experiments. The effect of cycloheximide on the rhythm was examined; it was shown that the oscillation ceases in the presence of the inhibitor, but that if the inhibitor is removed after 12 h, the rhythm resumes with no apparent change in phase. Analyses of gel scans of enzyme preparations partially purified by (NH₄)₂SO₄ fractionation and polyacrylamide gel electrophoresis indicated that there was more alanine dehydrogenase protein present at the maximum part of the cycle than there was at the minimum part. In view of these and other data, an operational model of a circadian biological clock is discussed.     

Spontaneous Aggregation and Cytotoxicity of the β-Amyloid Aβ1–40: A Kinetic Model

The time dependency of the spontaneous aggregation of the fibrillogenic β-Amyloid peptide, Aβ^1–40, was measured by turbidity, circular dichroism, HPLC, and fluorescence polarization. The results by all methods were comparable and they were most consistent with a kinetic model where the peptide first slowly forms an activated monomeric derivative (AM), which is the only species able to initiate, by tetramerization, the formation of linear aggregates. The anti-Aβ antibody 6E10, raised against residues 1–17, at concentrations of 200–300 nM delayed significantly the aggregation of 50 μM amyloid peptide. The anti–Aβ antibody 4G8, raised against residues 17–24, was much less active in that respect, while the antibody A162, raised against the C-terminal residues 39–43 of the full-length Aβ was totally inactive at those concentrations. Concomitant with the aggregation experiments, we also measured the time dependency of the Aβ^1–40–induced toxicity toward SH-EP1 cells and hippocampal neurons, evaluated by SYTOX Green fluorescence, lactate dehydrogenase release, and activation of caspases. The extent of cell damage measured by all methods reached a maximum at the same time and this maximum coincided with that of the concentration of AM. According to the kinetic scheme, the latter is the only transient peptide species whose concentration passes through a maximum. Thus, it appears that the toxic species of Aβ^1–40 is most likely the same transient activated monomer that is responsible for the nucleation of fibril formation. These conclusions should provide a structural basis for understanding the toxicity of Aβ^1–40 in vitro and possibly in vivo.     

A radioimmunoassay for epinephrine and norepinephrine in tissues and plasma

An I¹²⁵ radioimmunoassay (RIA) has been developed for the measurement of plasma and tissue epinephrine (E) and norepinephrine (NE). The assay utilizes an antibody which specifically binds metanephrine. E and NE are detected by conversion to metanephrine with the enzymes catechol-0-methyltransferase and phenylethanol-amine-N-methyltransferase. The assay is very specific and will allow the measurement of E and NE in less than 500 μl of normal human plasma. E and NE concentrations were determined by both the RIA and a radioenzymatic assay in canine, human and rat biologic samples. The correlation coefficients between the two assays were .962 for E and .956 for NE. The RIA is sensitive, specific, precise and significantly less costly and time consuming than present radioenzymatic methods.     

Studies on ulcerative dermal necrosis of salmonids

Fourteen fresh run salmon Satmo salar L. with early extant lesions of ulcerative dermal necrosis (UDN) were kept in separate tanks and treated with zinc free malachite green. Ten of the fish were held at 10° C and 4 at 2° C. The treatment precluded infection with Saprolegnia fungus and allowed natural resolution of the lesions. There was a marked difference in rate of healing between warm and cold water conditions.
Histological examination of healing lesions at different stages showed that there was a primary invasion of cuboidal epithelium over the collagen scar followed by a phase of disorganized proliferation which eventually organized itself into normal epithelium. Melanocytes were very obvious in the dermis of healing lesions.     

Using a Lethality Index to Assess Susceptibility of Tribolium confusum and Oryzaephilus surinamensis to Insecticides

We evaluated knockdown caused by four insecticides: alpha-cypermethrin, chlorfenapyr, pirimiphos-methyl and fipronil against adults of Tribolium confusum Jacquelin Duval, the confused flour beetle and Oryzaephilus surinamensis (L.), the sawtoothed grain beetle. Bioassays were conducted on concrete and metal surfaces. Adults of the tested species were exposed on both surfaces treated with the above insecticides at two doses (low and high). Knockdown assessment was done after 15, 30 and 60 min of adult exposure in the treated surfaces. Also, after 1, 3, 5, 7 and 14 d of exposure, a lethality index was calculated with an equation resulting to values from 0 to 100, where 100 indicated complete mortality and 0 complete survival. We also developed a lethality index by ranking each adult on each surface from 0 to 4, 0: adults moved normally, 1: adults were knocked down, but were able to walk for short intervals, 2: adults were knocked down and unable to walk, but with visible movement of antennae etc., 3: adults were knocked down, with very minimal movement of the tarsi and the antennae and 4: adults were dead (no movement). Knockdown of adults immediately after exposure (15–60 min) was higher for pirimiphos-methyl followed by alpha-cypermethrin, for both dose rates tested and species, but only on the metal surface. The lethality index was nearly 100 for all insecticides after 5d of exposure for O. surinamensis, while for T. confusum the adult lethality index was considerably lower for alpha-cypermethrin, suggesting that that recovery from knockdown occurred. Chlorfenapyr was the only insecticide that was more effective on concrete than on metal, while the reverse was noted for the other three insecticides. These results show that knockdown has different levels, which can be used as indicators of insect mortality or recovery.     

Effects of larval competition on survival and growth in Mediterranean fruit flies

1. Larval success was compared when one, two, or three egg clutches were laid in kumquat fruits (≈ 10 ml in volume) either successively on the same day or at the rate of one clutch per day. 2. Increased clutch density was associated with a significant decrease in larval survival rate and non‐significant decreases in larval growth rate and pupal mass. 3. Larval and pupal parameters showed significantly larger variance when clutches were laid on successive days than on the same day, suggesting a competitive advantage for older larvae over younger larvae. 4. The results suggest that, in small fruit, reduced fitness due to larval competition may act against possible fitness benefits due to social facilitation among adult females, hence reducing the likelihood of non‐linear population dynamics caused by processes such as the Allee effect.     

Horizontal transfer of a mitochondrial plasmid

Direct evidence for horizontal transfer of a mitochodnrial plasmid from the discomyceteAscobolus immersus to the pyrenomycetePodospora anserina is presented. Southern blot hybridisation analysis, polymerase chain reaction (PCR) amplification, and DNA sequencing demonstrate transmission of a linear plasmid upon hyphal contact. DNA extraction from isolated organelles indicates a mitochondrial localisation for the plasmid inP. anserina. This is the first report of horizontal gene transfer among unrelated fungi. These results have important evolutionary implications for plasmid propagation in fungi.     

Burn Injury Reduces Neutrophil Directional Migration Speed in Microfluidic Devices

Thermal injury triggers a fulminant inflammatory cascade that heralds shock, end-organ failure, and ultimately sepsis and death. Emerging evidence points to a critical role for the innate immune system, and several studies had documented concurrent impairment in neutrophil chemotaxis with these post-burn inflammatory changes. While a few studies suggest that a link between neutrophil motility and patient mortality might exist, so far, cumbersome assays have prohibited exploration of the prognostic and diagnostic significance of chemotaxis after burn injury. To address this need, we developed a microfluidic device that is simple to operate and allows for precise and robust measurements of chemotaxis speed and persistence characteristics at single-cell resolution. Using this assay, we established a reference set of migration speed values for neutrophils from healthy subjects. Comparisons with samples from burn patients revealed impaired directional migration speed starting as early as 24 hours after burn injury, reaching a minimum at 72–120 hours, correlated to the size of the burn injury and potentially serving as an early indicator for concurrent infections. Further characterization of neutrophil chemotaxis using this new assay may have important diagnostic implications not only for burn patients but also for patients afflicted by other diseases that compromise neutrophil functions.