Analysis of chemotaxis when the fraction of responsive cells is small--application to mammalian sperm guidance

The detection of chemotaxis-related changes in the swimming behavior of mammalian spermatozoa in a spatial chemoattractant gradient has hitherto been an intractable problem. The difficulty is that the fraction of responsive cells in the sperm population is very small and that the large majority of the cells, though non-responsive, are motile too. Assessment of the chemotactic effects in a spatial gradient is also very sensitive to the quality of sperm tracking. To overcome these difficulties we propose a new approach, based on the analysis of the distribution of instantaneous directionality angles made by spermatozoa in a spatial gradient versus a no-gradient control. Although the use of this parameter does not allow identification of individual responding cells, it is a reliable measure of directionality, independent of errors in cell tracking caused by cell collisions, track crossings, and track splitting. The analysis identifies bias in the swimming direction of a population relative to the gradient direction. It involves statistical chi(2) tests of the very large sample of measured angles, where the critical chi(2) values are adjusted to the sample size by the bootstrapping procedure. The combination of the newly measured parameter and the special analysis provides a highly sensitive method for the detection of a chemotactic response, even a very small one.     

Optimization of extraction of high-ester pectin from passion fruit peel (Passiflora edulis flavicarpa) with citric acid by using response surface methodology

A central composite design was employed to optimize the extraction of pectin with citric acid. The independent variables were citric acid concentration (0.086–2.91% w/v) and extraction time (17–102 min). The combined effect of these variables on the degree of esterification was investigated. Results have shown that the generated regression models adequately explained the data variation and significantly represented the actual relationship between the independent variables and the responses. Besides that, the citric acid concentration was the most important factor to affect the degree of esterification, as it exerted a significant influence on the dependent variable. Lower citric acid concentration increased the pectin degree of esterification. The surface response showed the relationships between the independent variables, and thus responses were generated. Through this surface, the satisfactory condition of 0.086% w/v citric acid for 60 min was established for extraction of high-ester yellow passion fruit pectin.     

Removal of Prymnesium parvum (Haptophyceae) and its toxins using clay minerals

Laboratory experiments were conducted to examine the ability of several clay minerals from Sweden to remove the fish-killing microalga, Prymnesium parvum Carter, from suspension. In their commercial form (i.e. after incineration at 400 °C), seawater slurries (salinity = 26) of the three minerals tested were generally ineffective at removing P. parvum from culture within a range of 0.01 to 0.50 g/L, and after 2.5 h of flocculation and settling. Dry bentonite (SWE1) displayed the highest removal efficiency (RE) at 17.5%, with 0.50 g/L. Illite (SWE3) averaged only 7.5% RE between 0.10 to 0.50 g/L, while kaolinite (SWE2) kept the cells suspended instead of removing them. Brief mixing of the clay-cell suspension after SWE1 addition improved RE by a factor of 2.5 (i.e. 49% at 0.50 g/L), relative to no mixing. The addition of polyaluminum chloride (PAC, at 5 ppm) to 0.50 g/L SWE1 also improved RE to 50% relative to SWE1 alone, but only minor improvements in RE were seen with SWE2 and SWE2 combined with PAC. In further experiments, P. parvum grown in NP-replete conditions were removed in greater numbers than cells in N- or P-limited cultures, at 0.10–0.25 g/L of SWE1 and 5 ppm PAC. With 0.50 g/L, RE converged at 40% for all three culture conditions. The toxin concentration of NP-replete cultures decreased from 24.2 to 9.2 μg/mL (60% toxin RE) with 0.10–0.50 g/L SWE1 treatment and 5 ppm PAC. A strong correlation was found between cell and toxin RE (r²=0.995). For N-limited cultures, toxin RE ranged between 21 and 87% with the same clay/PAC concentrations, although the correlation between cell and toxin removal was more moderate (r²=0.746) than for NP-replete conditions. Interestingly, the toxin concentration within the clay-cell pellet increased dramatically after treatment, suggesting that clay addition may stimulate toxin production in N-stressed cells. For P-limited cultures, toxin concentration also decreased following clay/PAC treatment (i.e. 36% toxin RE), but toxin removal was poorly correlated to cell removal (r²=0.462). To determine whether incineration affected SWE1’s removal ability, a sample of its wet, unprocessed form was tested. The RE of wet bentonite (SWE4) was slightly better than that of SWE1 (31% versus 17%, respectively, at 0.50 g/L), but when 5 ppm PAC was added, RE increased from 10 to 64% with 0.05 g/L of SWE4, and increased further to 77% with 0.50 g/L. There were no significant differences in RE among NP-replete, N-limited and P-limited cultures using PAC-treated SWE4. Finally, RE varied with P. parvum concentration, reaching a maximum level at the lowest cell concentration (1×10³ cells/mL): 100% RE with 0.10 and 0.50 g/L SWE4 + 5 ppm PAC. RE dropped as cell concentration increased to 1×10⁴ and 5×10⁴ cells/mL, but rose again when concentration increased to 1×10⁵ cells/mL, the concentration used routinely for the removal experiments above. Based on these results, SWE4 with PAC was the most effective mineral sample against P. parvum. Overall, these studies demonstrated that clay flocculation can be effective at removing P. parvum and its toxins only under certain treatment conditions with respect to cell concentration, clay type and concentration, and physiological status.     

Cryopreservation of Grapevine (Vitis spp.) Embryogenic Cell Suspensions by Encapsulation–Vitrification

Embryogenic cell suspensions of two grapevine rootstocks: 110 Ritcher (V. berlandieri × V. rupestris), 41B (V. vinifera × V. berlandieri) and several table grape and wine cultivars (Vitis vinifera) were successfully cryopreserved by the encapsulation–vitrification method. Embryogenic cell suspensions were precultured for 3 days in liquid MGN medium supplemented with daily increasing sucrose concentrations of 0.25, 0.5, 0.75 M. Precultured cells were encapsulated and directly dehydrated with a highly concentrated vitrification solution prior to immersion in liquid nitrogen for 1 h. After rewarming at 40 °C for 3 min, cryopreserved cells were post-cultured on solid MGN medium supplemented with 2.5 g l^–1 activated charcoal. Surviving cells were transferred to solid MGN medium for regrowth or solid MG medium for embryo development and then to solid WPM for plant regeneration. Optimal viability was 42–76% of cryopreserved cells when cell suspensions were precultured with a final sucrose concentration of 0.75 M and dehydrated with PVS2 at 0 °C for 270 min. Biochemical analysis showed that sucrose preculture caused changes in levels of total soluble protein and sugars in cell suspensions. Although the increase in fresh weight was significantly lower in cryopreserved cells than in control cells, the growth pattern of the cryopreserved cells and control cells was the same after two subcultures, following re-establishment in cell suspensions. Protocol developed in this study suggests a universal and highly efficient cryopreservation system suitable for several genetically diversed Vitis species.     

Retail Survey of Brazilian Milk and Minas Frescal Cheese and a Contaminated Dairy Plant To Establish Prevalence, Relatedness, and Sources of Listeria monocytogenes Isolates

A study was designed to recover Listeria monocytogenes from pasteurized milk and Minas frescal cheese (MFC) sampled at retail establishments (REs) and to identify the contamination source(s) of these products in the corresponding dairy processing plant. Fifty milk samples (9 brands) and 55 MFC samples (10 brands) were tested from REs located in Juiz de Fora, Minas Gerais, Brazil. All milk samples and 45 samples from 9 of 10 MFC brands tested negative for L. monocytogenes; however, “brand F” of MFC obtained from REs 119 and 159 tested positive. Thus, the farm/plant that produced brand F MFC was sampled; all samples from the milking parlor tested negative for L. monocytogenes, whereas several sites within the processing plant and the MFC samples tested positive. All 344 isolates recovered from retail MFC, plant F MFC, and plant F environmental samples were serotype 1/2a and displayed the same AscI or ApaI fingerprints. Since these results established that the storage coolers served as the contamination source of the MFC, plant F was closed so that corrective renovations could be made. Following renovation, samples from sites that previously tested positive for the pathogen were collected from the processing environment and from MFC on multiple visits; all tested negative for L. monocytogenes. In addition, on subsequent visits to REs 159 and 119, all MFC samples tested negative for the pathogen. Studies are ongoing to quantify the prevalence, levels, and types of L. monocytogenes in MFC and associated processing plants to lessen the likelihood of listeriosis in Brazil.     

Long-range oxidative damage in duplex DNA: the effect of bulged G in a G–C tract and tandem G/A mispairs

Two series of duplex DNA oligomers were prepared having an anthraquinone derivative (AQ) covalently linked at a 5′-terminus. Irradiation of the AQ at 350 nm leads to injection of an electron hole (radical cation) into the DNA. The radical cation migrates through the DNA causing reaction primarily at G_n sequences. In one series, GA tandem mispairs are inserted between GG steps to assess the effect of the mispair on the transport of the radical cation, reaction (damage) caused by the radical cation at the mispair, and repair of the resulting damage by formamidopyrimidine DNA glycosylase (Fpg). In the second series, a bulged guanine in a G₃C₂ sequence is interposed between the GG steps. These experiments reveal that neither G/A tandem mispairs nor bulged guanines are significant barriers to long-range charge migration in DNA. The radical cation does not cause reaction at guanines in the G/A tandem mispair. Reaction does occur at the bulged guanine, but it is repaired by Fpg.     

Dinophysis blooms in the deep euphotic zone of the Baltic Sea: do they grow in the dark?

In situ growth rates of the toxin-producing dinoflagellate Dinophysis norvegica collected in the central Baltic Sea were estimated during the summers of 1998 and 1999. Flow cytometric measurements of the DNA cell cycle of D. norvegica yielded specific growth rates (μ) ranging between 0.1 and 0.4 per day, with the highest growth rates in stratified populations situated at 15–20 m depth. Carbon uptake rates, measured using ¹⁴C incubations followed by single-cell isolation, at irradiances corresponding to depths of maximum cell abundance were sufficient to sustain growth rates of 0.1–0.2 per day. The reason for D. norvegica accumulation in the thermocline, commonly at 15–20 m depth, is thus enigmatic. Comparison of depth distributions of cells with nutrient profiles suggests that one reason could be to sequester nutrients. Measurements of single-cell nutrient status of D. norvegica, using nuclear microanalysis, revealed severe deficiency of both nitrogen and phosphorus as compared to the Redfield ratio.It is also possible that suitable prey or substrate for mixotrophic feeding is accumulating in the thermocline. The fraction of cells containing digestive vacuoles ranged from 2 to 22% in the studied populations. Infection by the parasitic dinoflagellate Amoebophrya sp. was observed in D. norvegica in all samples analysed. The frequency of infected cells ranged from 1 to 3% of the population as diel averages, ranging from 0.2 to 6% between individual samples. No temporal trends in infection frequency were detected. Estimated loss rates based on observed infection frequencies were 0.5–2% of the D. norvegica population daily, suggesting that these parasites were not a major loss factor for D. norvegica during the periods of study.     

Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.