Identification and functional characterization of cation-chloride cotransporters in plants

Chloride (Cl(-)) is an essential nutrient and one of the most abundant inorganic anions in plant tissues. We have cloned an Arabidopsis thaliana cDNA encoding for a member of the cation-Cl(-) cotransporter (CCC) family. Deduced plant CCC proteins are highly conserved, and phylogenetic analyses revealed their relationships to the sub-family of animal K(+):Cl(-) cotransporters. In Xenopus laevis oocytes, the A. thaliana CCC protein (At CCC) catalysed the co-ordinated symport of K(+), Na(+) and Cl(-), and this transport activity was inhibited by the 'loop' diuretic bumetanide, a specific inhibitor of vertebrate Na(+):K(+):Cl(-) cotransporters, indicating that At CCC encodes for a bona fide Na(+):K(+):Cl(-) cotransporter. Analysis of At CCC promoter-beta-glucuronidase transgenic Arabidopsis plants revealed preferential expression in the root and shoot vasculature at the xylem/symplast boundary, root tips, trichomes, leaf hydathodes, leaf stipules and anthers. Plants homozygous for two independent T-DNA insertions in the CCC gene exhibited shorter organs such as inflorescence stems, roots, leaves and siliques. The elongation zone of the inflorescence stem of ccc plants often necrosed during bolt emergence, while seed production was strongly impaired. In addition, ccc plants exhibited defective Cl(-) homeostasis under high salinity, as they accumulated higher and lower Cl(-) amounts in shoots and roots, respectively, than the treated wild type, suggesting At CCC involvement in long-distance Cl(-) transport. Compelling evidence is provided on the occurrence of cation-chloride cotransporters in the plant kingdom and their significant role in major plant developmental processes and Cl(-) homeostasis.     

Temperature regulation in a hypometabolic primate, the slow loris (Nycticebus coucang).

Six slow loris were exposed to air temperatures between 10 degrees C and 40 degrees C. Rectal temperature was stable (mean, 34.8 degrees C) at air temperatures between 17 degrees C and 31 degrees C; at higher air temperatures, the animals became hyperthermic. Oxygen consumption was minimal at air temperatures of 31.4-36.6 degrees C; the mean value (0.250 ml O2 g-1 h-1) was only 36% of the expected level for a eutherian Mammal. The slow loris increased its heat production at lower air temperatures. Thermal polypnea occurred in response to heat, and some of the animals were able to dissipate their entire metabolic heat production at lower air temperatures. Thermal polypnea occurred in response to heat, and some of the animals were able the combined thermal conductance of the tissues and haircoat was 73% of the predicted values. It was concluded that, in spite of its low metabolic rate, the slow loris had effective responses to moderate cold, and that, in addition, it was well adapted to a hot climate.     

Relationship between ultrasonic properties and structural changes in the mesophyll during leaf dehydration

The broad-band ultrasonic spectroscopy technique allows the determination of changes in the relative water content (RWC) of leaves with contrasting structural features. Specifically, the standardized frequency associated with the maximum transmittance (f/f(o)) is strongly related to the RWC. This relationship is characterized by the existence of two phases separated by an inflexion point (associated with the turgor loss point). To obtain a better understanding of the strong relationship found between RWC and f/f(o), this work has studied the structural changes experienced by Quercus muehlenbergii leaves during dehydration in terms of ultrasounds measurements, cell wall elasticity, leaf thickness, leaf density, and leaf structure. The results suggest that the decrease found in f/f(o) before the turgor loss point can be attributed to the occurrence of changes in the estimation of the macroscopic effective elastic constant of the leaf (c(33)), mainly associated with changes in the bulk modulus of elasticity of the cell wall (ε). These changes are overriding or compensating for the thickness decreases recorded during this phase. On the other hand, the high degree of cell shrinkage and stretching found in the mesophyll cells during the second phase seem to explain the changes in the acoustic properties of the leaf beyond the turgor loss point. The formation of large intercellular spaces, which increased the irregularity in the acoustic pathway, may explain the increase of the attenuation coefficient of ultrasounds once the turgor loss point threshold is exceeded. The direct measurement of c(33) from ultrasonic measurements would allow a better knowledge of the overall biomechanical properties of the leaf further than those derived from the P-V analysis.     

Diurnal and Seasonal Variations in Chlorophyll a Fluorescence in Two Mediterranean-Grassland Species Under Field Conditions

Seasonal and daily variations in chlorophyll (Chl) fluorescence were studied in two representative species of Mediterranean grasslands, Tuberaria guttata (an annual) and Chamaemelum nobile (a perennial), in order to assess physiological responses to climatically induced stresses during the growing season. The photochemical efficiency of photosystem (PS) 2 in dark-adapted leaves was measured by the Chl fluorescence ratio Fv/Fp. This ratio decreased progressively from December to July, as the effects of increasing solar radiation and summer drought became more severe. The seasonal decline was observed particularly as a depression of morning and midday values, when photoinhibition was more evident. In both species, the extent of this diurnal depression increased with midday irradiance throughout winter and spring. After sunset, there was complete recovery to optimum values. Towards the end of the life cycle, increased irradiance did not affect the midday decline further but Fv/Fp measurements in the morning and evening never regained their optimum values, indicating the accumulation of photodamage in the reaction centres of PS2. The half-rise time of Fp (T1/2), used to estimate the size of the plastoquinone pool, showed little daily variation in C. nobile throughout the most important part of its seasonal cycle. However, towards the end of its life cycle (June and July) T1/2 values ranged from ca. 200 ms before sunrise to near zero at midday on the same day. The annual species, T. guttata, showed similar disregulation in energy transmission rate both at the seedling stage and at end of its life cycle. Thus seedlings and reproductive plants in particular are sensitive to environmental conditions (extremes of temperature and drought) and cannot maintain consistent electron flow throughout the day.     

Comparison of a recombinant-antigen enzyme immunoassay with Treponema pallidum hemagglutination test for serological confirmation of syphilis

A recombinant-antigen enzyme immunoassay (EIA), BioSCREEN anti-Treponema pallidum, was compared favorably with the T. pallidum hemagglutination test, in the detection of specific antibodies in different groups of sera from patients with primary (n = 38), secondary (n = 10), early latent (n = 28) and congenital syphilis (n = 2), patients with leptospirosis ( n= 8), infectious mononucleosis (n = 7), hepatitis (n = 9), diabetes mellitus (n = 11), rheumatoid arthritis (n = 13), leprosy (n = 11), tuberculosis (n = 9), HIV/Aids ( n= 12), systemic lupus erythematosus (n = 4), rheumatic fever (n = 3), old-persons (n = 9), pregnant women (n = 29) and blood donors (n = 164). The coincidence between them was 95.1%. The sensitivity and specificity of the EIA were 93.3% and 95.5%, respectively. Fifteen serum specimens belonging to old-persons, pregnant women, blood donors, and patients with human leptospirosis, hepatitis, diabetes mellitus, tuberculosis and rheumatic fever gave false-positive results by Venereal Disease Research Laboratory and/or Rapid Plasma Reagin. The EIA can be used as alternative method for the serological confirmation of syphilis.     

RNA quality in frozen breast cancer samples and the influence on gene expression analysis – a comparison of three evaluation methods using microcapillary electrophoresis traces

Background  

Assessing RNA quality is essential for gene expression analysis, as the inclusion of degraded samples may influence the interpretation of expression levels in relation to biological and/or clinical parameters. RNA quality can be analyzed by agarose gel electrophoresis, UV spectrophotometer, or microcapillary electrophoresis traces, and can furthermore be evaluated using different methods. No generally accepted recommendations exist for which technique or evaluation method is the best choice. The aim of the present study was to use microcapillary electrophoresis traces from the Bioanalyzer to compare three methods for evaluating RNA quality in 24 fresh frozen invasive breast cancer tissues: 1) Manual method = subjective evaluation of the electropherogram, 2) Ratio Method = the ratio between the 28S and 18S peaks, and 3) RNA integrity number (RIN) method = objective evaluation of the electropherogram. The results were also related to gene expression profiling analyses using 27K oligonucleotide microarrays, unsupervised hierarchical clustering analysis and ontological mapping.     

Proteins synthesized by inducer T cells: evidence for a mitogenic peptide shared by inducer molecules that stimulate different cell types

Inducer T lymphocytes synthesize and secrete peptides that stimulate growth and differentiation of many cell types, including lymphocytes and monocytes that kill foreign organisms, B lymphocytes, mast cells and hematopoietic precursor cells. To define these inducer molecules more precisely, we have generated clones of these T cells as a source of homogeneous material for biochemical analysis. These clones synthesize peptides that stimulate T and B cells to divide and that also induce the latter cells to secrete immunoglobulin. Inducer cells synthesize a 14 kilodalton growth polypeptide that stimulates T and B lymphocytes, as well as other cell types, to divide. This 14 kilodalton peptide is normally associated with different, larger peptides that appear to focus its mitogenic activity to one or another target cell.     

The ftsA gene product: a possible connection between DNA replication and septation in Escherichia coli

The study of Escherichia coli strain D-2, which harbours the ftsA2(ts) allele, has shown that temperature-induced filaments of this strain can divide, at 30 degrees C, in the absence of DNA replication and translation. Strain D-2 is thermosensitive during a period coincident with that in which the termination protein should be synthesized and exert its action. The ftsA gene product, which participates in the structure of the septum, needs for its synthesis a short period of DNA replication. The FtsA protein could be involved in a mechanism that coordinates chromosome replication and cell division by a pathway different from and independent of the SOS-induced response.     

Drought enhances maize chilling tolerance. II. Photosynthetic traits and protective mechanisms against oxidative stress

In the present research we studied the photosynthetic traits and protective mechanisms against oxidative stress in two maize ( Zea mays L.) genotypes differing in chilling sensitivity (Z7, tolerant and Penjalinan, sensitive) subjected to 5°C for 5 days, with or without pretreatment by drought. The drought pretreatment decreased the symptoms of chilling injury in Penjalinan plants estimated as necrotic leaf area and maximum quantum yield of photosystem II. Furthermore, drought pretreatment diminished the level of lipid peroxidation caused by chilling in Penjalinan plants. After one day of recovery from chilling the Z7 and drought-pretreated Penjalinan plants showed higher net photosynthesis rates than the non-drought-pretreated Penjalinan plants, thereby decreasing the probability of generating reactive oxygen species. The greater net photosynthesis was correlated with the greater NADP-malate dehydrogenase activity. No differences in either the de-epoxidation state of the xanthophyll cycle or the antioxidant enzyme activities were found among the chilled groups of plants. However, a drastic decrease in ascorbate content was observed in chilled Penjalinan plants without drought pretreatment. As we found an increase of H₂O₂ content after drought pretreatment, we suggest its involvement as a signal in the drought-enhanced chilling tolerance of maize.     

Lack of correlation between surface expression and currents in epileptogenic AB-calmodulin binding domain Kv7.2 potassium channel mutants

Heteromers of Kv7.2/Kv7.3 subunits constitute the main substrate of the neuronal M-current that limits neuronal hyper-excitability and firing frequency. Calmodulin (CaM) binding is essential for surface expression of Kv7 channels, and disruption of this interaction leads to diseases ranging from mild epilepsy to early onset encephalopathy. In this study, we addressed the impact of a charge neutralizing mutation located at the periphery of helix B (K526N). We found that, CaM binding and surface expression was impaired, although current amplitude was not altered. Currents were reduced at a faster rate after activation of a voltage-dependent phosphatase, suggesting that phosphatidylinositol-4,5-bisphosphate (PIP₂) binding was weaker. In contrast, a charge neutralizing mutation located at the periphery of helix A (R333Q) did not affect CaM binding, but impaired trafficking and led to a reduction in current amplitude. Taken together, these results suggest that disruption of CaM-dependent or CaM-independent trafficking of Kv7.2/Kv7.3 channels can lead to pathology regardless of the consequences on the macroscopic ionic flow through the channel.