Lactate dehydrogenase (LDH) gene duplication during chordate evolution: the cDNA sequence of the LDH of the tunicate Styela plicata

L-Lactate dehydrogenase (L-LDH, E.C. 1.1.1.27) is encoded by two or three loci in all vertebrates examined, with the exception of lampreys, which have a single LDH locus. Biochemical characterizations of LDH proteins have suggested that a gene duplication early in vertebrate evolution gave rise to Ldh-A and Ldh-B and that an additional locus, Ldh-C arose in a number of lineages more recently. Although some phylogenetic studies of LDH protein sequences have supported this pattern of gene duplication, others have contradicted it. In particular, a number of studies have suggested that Ldh-C represents the earliest divergence among vertebrate LDHs and that it may have diverged from the other loci well before the origin of vertebrates. Such hypotheses make explicit statements about the relationship of vertebrate and invertebrate LDHs, but to date, no closely related invertebrate LDH sequences have been available for comparison. We have attempted to provide further data on the timing of gene duplications leading to multiple vertebrate LDHs by determining the cDNA sequence of the LDH of the tunicate Styela plicata. Phylogenetic analyses of this and other LDH sequences provide strong support for the duplications giving rise to multiple vertebrate LDHs having occurred after vertebrates diverged from tunicates. The timing of these LDH duplications is consistent with data from a number of other gene families suggesting widespread gene duplication near the origin of vertebrates. With respect to the relationships among vertebrate LDHs, our data are not consistent with previous claims that Ldh-C represented the earliest divergence. However, the precise relationships among some of the main lineages of vertebrate LDHs were not resolved in our analyses.      

New energy transfer dyes for DNA sequencing.

We have synthesized a set of four energy transfer dyes and demonstrated their use in automated DNA sequencing. The donor dyes are the 5- or 6-carboxy isomers of 4'-aminomethylfluorescein and the acceptor dyes are a novel set of four 4,7-dichloro-substituted rhodamine dyes which have narrower emission spectra than the standard, unsubstituted rhodamines. A rigid amino acid linker, 4-aminomethylbenzoic acid, was used to separate the dyes. The brightness of each dye in an automated sequencing instrument equipped with a dual line argon ion laser (488 and 514 nm excitation) was 2-2.5 times greater than the standard dye-primers with a 2 times reduction in multicomponent noise. The overall improvement in signal-to-noise was 4- to 5-fold. The utility of the new dye set was demonstrated by sequencing of a BAC DNA with an 80 kb insert. Measurement of the extinction coefficients and the relative quantum yields of the dichlororhodamine components of the energy transfer dyes showed their values were reduced by 20-25% compared with the dichlororhodamine dyes alone.     

Variable Temperature Stress in the Nematode Caenorhabditis elegans (Maupas) and Its Implications for Sensitivity to an Additional Chemical Stressor

A wealth of studies has investigated how chemical sensitivity is affected by temperature, however, almost always under different constant rather than more realistic fluctuating regimes. Here we compared how the nematode Caenorhabditis elegans responds to copper at constant temperatures (8–24°C) and under fluctuation conditions of low (±4°C) and high (±8°C) amplitude (averages of 12, 16, 20°C and 16°C respectively). The DEBkiss model was used to interpret effects on energy budgets. Increasing constant temperature from 12–24°C reduced time to first egg, life-span and population growth rates consistent with temperature driven metabolic rate change. Responses at 8°C did not, however, accord with this pattern (including a deviation from the Temperature Size Rule), identifying a cold stress effect. High amplitude variation and low amplitude variation around a mean temperature of 12°C impacted reproduction and body size compared to nematodes kept at the matching average constant temperatures. Copper exposure affected reproduction, body size and life-span and consequently population growth. Sensitivity to copper (EC₅₀ values), was similar at intermediate temperatures (12, 16, 20°C) and higher at 24°C and especially the innately stressful 8°C condition. Temperature variation did not increase copper sensitivity. Indeed under variable conditions including time at the stressful 8°C condition, sensitivity was reduced. DEBkiss identified increased maintenance costs and increased assimilation as possible mechanisms for cold and higher copper concentration effects. Model analysis of combined variable temperature effects, however, demonstrated no additional joint stressor response. Hence, concerns that exposure to temperature fluctuations may sensitise species to co-stressor effects seem unfounded in this case.     

Optimum spectral windows to minimize quantum noise of ratiometric intracellular fluorescent probes

When fluorescent indicators are used to measure intracellular ligands in single cells, the quality of the data is usually limited by quantum (shot) noise. For indicators which shift excitation or emission wavelengths upon ligand binding, a ratiometric method is usually employed. In choosing the spectral windows for excitation or collection of fluorescence, there is a trade-off between maximum sensitivity to ligand binding, and maximum collection of light. We show that there is a well-defined optimum choice of windows which minimizes the error caused by quantum noise in the estimated ligand concentration. An algorithm for determining these optimum windows is presented. As an example, we consider the measurement of intracellular calcium by indo-1 fluorescence emission ratio in cardiac myocytes. The optimum wavelength bands for collection of fluorescence are considerably wider than those commonly employed. The use of these windows in a pulsed-excitation time-resolved calcium measurement instrument resulted in improved signal to noise ratio of the calcium signal.     

Temperature dependent development of Lygus hesperus (Hemiptera: Miridae) nymphs

Lygus hesperus Knight (Hemiptera: Miridae) is a key pest of fruit and vegetable crops, forages, and cotton (Gossypium spp.) in the western United States. Accurate models describing relationships between temperature and L. hesperus development are critical to the study of seasonal L. hesperus population dynamics. Development of L. hesperus nymphs was assessed at nine constant temperatures from 10 to 37.8 degrees C. The relationships between temperature and development for each L. hesperus instar, and for the entire nymphal stage, were best described by six-parameter biophysical models indicating both low- and high-temperature inhibition of development. Development rates asymptotically approached zero with decreasing temperature in the lower thermal range, and decreased with increasing temperatures above 32.2 degrees C. Nymphs did not survive from egg hatch to adulthood at either 10 or 37.8 degrees C, and nymph mortality was > 90% at both 12.8 and 35.0 degrees C. The fifth instar exhibited the longest stadium, whereas the shortest stadia were associated with the second and third instars. Development rates of males and females did not differ, and the ratio of males to females was not different from 1:1 at any temperature. Our temperature-dependent development rate models for L. hesperus nymphs will facilitate control of insect physiological age in controlled laboratory experiments, and should be useful in planning and interpreting field studies on L. hesperus population dynamics.     

Captures of boll weevils (Coleoptera: Curculionidae) in traps associated with different habitats

Programs to eradicate the boll weevil, Anthonomus grandis grandis Boheman, from cotton, Gossypium hirsutum L., in the United States rely heavily on pheromone traps for monitoring weevil populations in both active and posteradication maintenance programs. Modifications to trapping protocols that increase trap effectiveness should contribute to this eradication effort. Between October 1996 and May 1997 and between September 1997 and April 1998, we compared trap effectiveness, indicated by the numbers of captured weevils, in relation to selected habitat types. Each study period was divided into fall, winter, and spring seasons. Traps were closely associated with seven habitat types, including four types with prominent erect vegetation (brush-lined irrigation canal, brush, sugarcane, and resaca or ox-bow lake) and three types with only low-growing or sparse erect vegetation (irrigation drainage canal, unimproved pasture, and fallow fields). Captures of male and female weevils were statistically similar regardless of season or trapping habitat. Although captures differed significantly among habitats, these differences varied among seasons. Trapping habitats with prominent vegetational features generally produced higher weekly captures of weevils than habitats lacking these features. Also, captures in traps associated with prominent vegetation indicated seasonal differences in weevil activity, with highest captures occurring during the fall. Traps associated with habitats lacking prominent vegetation did not statistically demonstrate seasonal differences. Our results indicate that immediate trap surroundings strongly influence the effectiveness of the boll weevil pheromone trap. These results also suggest that effectiveness of current trapping programs may be improved through purposeful association of traps with selected vegetational features.     

Global health policies that support the use of banked donor human milk: a human rights issue

Background

The past ten years have witnessed a rising trend in the prevalence and duration of breastfeeding in Italy, but breastfeeding rates increase in an unequal way; they are higher in the North of Italy than in the South. The purpose of this study was to describe the experiences, expectations and beliefs of a sample of mothers, and to identify differences, if any, between the North and the South of Italy.

Methods

The study was conducted in two regions of Italy, Friuli Venezia Giulia in the Northeast and Basilicata in the South. Two hundred and seventy-nine mothers of infants and children 6 to 23 months of age were interviewed using an 85-item questionnaire including closed and open questions on infant feeding experiences and beliefs, sources of information and support, reasons for intended and actual choices and practices, and some demographic and social variables. Face-to-face interviews were conducted between May 2001 and September 2002. Quantitative and qualitative methods were used for data analysis.

Results

The distribution of the mothers by age, education, employment and parity did not differ from that of the general population of the two regions. The reported rates of initiation and duration of breastfeeding were also similar: 95% started breastfeeding, exclusive breastfeeding was 32% at three and 9% at six months, with 64% and 35% of any breastfeeding, respectively. Some differences were reported in the rates of full breastfeeding, reflecting different ages of introduction of non-nutritive fluids. These, as well as nutritive fluids – including infant formula – and complementary foods, were introduced far too early. Advice on infant feeding was generally provided by health professionals and often was not based on up-to-date recommendations. Mothers were generally aware of the advantages of breastfeeding, but at the same time reported problems that they were not able to solve alone or through social and health system support. Most mothers would welcome the support of a peer counsellor. More mothers in Basilicata than in Friuli Venezia Giulia reported difficulties with breastfeeding related to returning to work and were not familiar with their rights on breastfeeding and maternity leave.

Conclusion

Programmes for the protection, promotion and support of breastfeeding in these and similar regions of Italy should concentrate on better training of health professionals with regards to lactation management, communication, and counselling skills. The addition of trained peer counsellors could reinforce the work done by the health system and, through community involvement, could help change social prejudice in the mid- and long-term. The differences between regions should be taken into account in formulating these programmes to avoid increasing, and possibly to decrease, the current gaps.