Behavioural responses related to increasing core body temperature of grazing dairy cows experiencing moderate heat stress.

Exposure to direct solar radiation, high ambient temperature, lack of wind movement, coupled with own metabolic heat production, makes grazing dairy cows vulnerable to heat stress. In pastures, it would be beneficial to monitor heat stress by observable changes in behaviour. We hypothesised that grazing dairy cows exhibit behavioural changes due to increasing heat load in temperate climate. Over two consecutive summers, 38 full-time grazing Holstein dairy cows were investigated in 12 experimental periods of up to 3 consecutive days where the cows were repeatedly exposed to various levels of moderate heat load determined by the comprehensive climate index (CCI). The CCI defines the ambient climate conditions, combining air temperature, relative humidity, solar radiation and wind speed. Vaginal temperature (VT) was automatically measured as an indicator of heat stress. In addition, as a less invasive method, we investigated if reticular temperature (RET) can be indicative of heat stress on pastures. Walking activity, lying-, feeding, and ruminating durations were recorded continuously with sensors. Respiration rate (RR), proximity to and competition at the water trough, social licking, self-licking, inter-individual distance, and fly intensity were directly observed. Data were analysed in the morning (0900-1100 h) and during the hottest time of day when cows were on pasture (1230-1430 h). The VT and RET showed similar patterns in relation to the CCI, suggesting that RET can be suitable for continuous monitoring of heat stress on pastures. In the morning, the cow's VT and RET did not relevantly react to the CCI. During the period 1230-1430 h, the cow's mean VT (mean vaginal temperature (VTMEAN); range: 37.7-40.3 °C) and mean RET (mean reticular temperature; range: 37.0-41.1 °C) were positively related to the mean CCI (mean comprehensive climate index) in this period (mean ± SD: 25.9 ± 5.71 °C). For cows with greater VTMEAN, an increased mean RR and decreased durations of walking, lying, feeding, and ruminating were found. These cows were also more likely to be in proximity to the water trough and to have small inter-individual distances. Changes in these traits seem to reflect behavioural adaptations to heat stress in a temperate climate and could be used to detect the heat stress in individual dairy cows on pastures.

Short-term physiological responses to moderate heat stress in grazing dairy cows in temperate climate.

Even in temperate climate regions, an increase in ambient temperature and exposure to solar radiation can cause heat stress in lactating dairy cows. We hypothesised that grazing dairy cows exhibit short-term physiological changes due to increasing heat load under moderate climate conditions. Over two consecutive summers, 38 lactating Holstein dairy cows were studied in a full-time grazing system. Data were collected in 10 experimental periods of up to three consecutive days with a moderate comprehensive climate index (CCI). The individual animals' vaginal temperature (VT), heart rate, and locomotor activity data were automatically monitored with sensors. Blood samples and proportional whole milk samples were collected at afternoon milking. The concentrations of beta-hydroxybutyrate, glucose, non-esterified fatty acids, urea nitrogen, plasma thyroxine and triiodothyronine were analysed in blood plasma, and fat, protein, lactose, urea nitrogen, cortisol, Na+, K+, and Cl- concentrations were analysed in milk. The daily distribution of VT recordings greater than 39 °C showed a circadian rhythm with a proportion of recordings of 2% and lower during the night and a percentage of 10% or higher in the afternoon. The cows' maximal daily vaginal temperature (VTMAX) between 0830 and 1430 h was positively related to the mean daily CCI in the same time period (CCIMEAN; mean and SD 23.6 ± 5.4 °C). Cows with greater VTMAX had an increased mean heart rate, plasma glucose and milk cortisol concentrations and decreased concentrations of plasma thyroxine and triiodothyronine. The concentration of Na+ in milk was lower, and the concentration of K+ in milk tended to be higher in cows with increased VTMAX. For beta-hydroxybutyrate, non-esterified fatty acids and urea nitrogen concentrations in plasma and fat and lactose concentrations in milk no relationships were found in terms of increasing VT. For milk urea nitrogen and protein concentrations, the proportion of total variance explained by inter-individual or -period variance was high. In conclusion, changes observed in milk and blood likely reflected short-term physiological responses to moderate heat stress. In particular, milk cortisol and Na+ may be useful traits for timely monitoring of heat stress in individual cows because their inter-individual variances were relatively small and samples can be collected non-invasively.

Sleep phase advance and lithium to sustain the antidepressant effect of total sleep deprivation in bipolar depression: new findings supporting the internal coincidence model?

Recent European studies suggested that sleep phase advance (SPA) could sustain the effects of total sleep deprivation (TSD) both with or without a combined antidepressant drug treatment. Previous studies by our group showed that an ongoing lithium treatment could enhance and sustain the effect of repeated TSD. In the present study we studied the effect of a single TSD followed by 3 days SPA (beginning with sleep allowed from 17:00 until 24:00, with daily shiftbacks of 2 h) in consecutively admitted bipolar depressed inpatients who were taking a chronic lithium salts treatment (n=16) or who were devoid of psychotropic medications (n=14). Changes in mood during treatment were recorded with self administered visual analogue scales and with Hamilton rating scale for depression. Results showed that SPA could sustain the acute antidepressant effect of TSD, and that lithium enhanced the effect of the chronobiological treatment. According to the internal coincidence model, the better clinical effects observed in lithium-treated patients could be due to the phase delaying effect of lithium on biological rhythms, leading to a better synchronization of biological rhythms with the sleep-wake cycle.

Large-scale identification of virulence genes from Streptococcus pneumoniae.

Streptococcus pneumoniae is the major cause of bacterial pneumonia, and it is also responsible for otitis media and meningitis in children. Apart from the capsule, the virulence factors of this pathogen are not completely understood. Recent technical advances in the field of bacterial pathogenesis (in vivo expression technology and signature-tagged mutagenesis [STM]) have allowed a large-scale identification of virulence genes. We have adapted to S. pneumoniae the STM technique, originally used for the discovery of Salmonella genes involved in pathogenicity. A library of pneumococcal chromosomal fragments (400 to 600 bp) was constructed in a suicide plasmid vector carrying unique DNA sequence tags and a chloramphenicol resistance marker. The recent clinical isolate G54 was transformed with this library. Chloramphenicol-resistant mutants were obtained by homologous recombination, resulting in genes inactivated by insertion of the suicide vector carrying a unique tag. In a mouse pneumonia model, 1.250 candidate clones were screened; 200 of these were not recovered from the lungs were therefore considered virulence-attenuated mutants. The regions flanking the chloramphenicol gene of the attenuated mutants were amplified by inverse PCR and sequenced. The sequence analysis showed that the 200 mutants had insertions in 126 different genes that could be grouped in six classes: (i) known pneumococcal virulence genes; (ii) genes involved in metabolic pathways; (iii) genes encoding proteases; (iv) genes coding for ATP binding cassette transporters; (v) genes encoding proteins involved in DNA recombination/repair; and (vi) DNA sequences that showed similarity to hypothetical genes with unknown function. To evaluate the virulence attenuation for each mutant, all 126 clones were individually analyzed in a mouse septicemia model. Not all mutants selected in the pneumonia model were confirmed in septicemia, thus indicating the existence of virulence factors specific for pneumonia.

Interaction of normal and mutant SRY proteins with DNA.

In mammals, sex determination is caused by the Y-chromosome gene SRY. The DNA-binding domain of human SRY protein is similar to those of the chromatin protein HMG1. Like HMG1, SRY binds to kinked DNA structures, and bends linear DNA sharply upon binding. We analysed the biochemical properties of mutant SRY proteins from five patients with complete gonadal dysgenesis: two bind and bend DNA almost normally, two bind inefficiently but bend DNA normally, and one binds DNA with almost normal affinity but produces a different angle. The mutations with moderate effect on complex formation can be transmitted to progeny, the ones with severe effects on either binding or bending are de novo. The angle induced by SRY depends on the exact DNA sequence, thus discriminating different target sites. We suggest that the exact spatial arrangement of the nucleoprotein complex organized by SRY in chromatin is essential for the expression of genes involved in testis differentiation.

Evolutionary conservation in the DNA-binding and -bending properties of HMG-boxes from SRY proteins of primates.

Mammalian sex determination is caused by the Y-chromosome gene SRY, which encodes a protein containing a DNA-binding domain (HMG-box) of about 70 amino acids (aa). The HMG-box is very conserved in a wide variety of mammals; conversely, the flanking non-box regions show a high degree of aa sequence divergence, even between closely related species. The HMG-box of human SRY binds sequence-specifically to linear DNA and produces a sharp bend; it also interacts with high affinity to kinked DNA structures irrespective of their sequences. Point mutations associated with sex reversal in XY human females fall within the HMG-box and either affect the affinity for DNA or modify the geometry of the DNA-protein complex. Here, we show that the DNA-binding and -bending properties of the HMG-boxes of SRY from human and seven different primates are extremely similar to each other. Together with other data, this suggests that the inability of mouse and human SRY to substitute for each other is due to differences in the conserved HMG-box, rather than the non-conserved flanking sequences.

Sex-reversing mutations affect the architecture of SRY-DNA complexes.

The testis determining factor, SRY, is a DNA binding protein that causes a large distortion of its DNA target sites. We have analysed the biochemical properties of the DNA binding domains (HMG-boxes) of mutant SRY proteins from five patients with complete gonadal dysgenesis. The mutant proteins fall into three categories: two bind and bend DNA almost normally, two bind inefficiently but bend DNA normally and one binds DNA with almost normal affinity but produces a different angle. The mutations with moderate effect on complex formation can be transmitted to male progeny, the ones with severe effects on either binding or bending are de novo. The angle induced by SRY depends on the exact DNA sequence and thus adds another level of discrimination in target site recognition. These data suggest that the exact spatial arrangement of the nucleoprotein complex organized by SRY is essential for sex determination.

Protein HU binds specifically to kinked DNA.

We have purified the main four-way junction DNA-binding protein of Escherichia coli, and have found it to be the well-known HU protein. HU protein recognizes with high-affinity one of the angles present in the junction, a molecule with the shape of an X. Other DNA structures characterized by sharp bends or kinks, like bulged duplex DNAs containing unpaired bases, are also bound. HU protein appears to inhibit cruciform extrusion from supercoiled inverted repeat (palindromic) DNA, either by constraining supercoiling or by trapping a metastable interconversion intermediate. All these properties are analogous to the properties of the mammalian chromatin protein HMG1. We suggest that HU is a prokaryotic HMG1-like protein rather than a histone-like protein.

SRY, like HMG1, recognizes sharp angles in DNA.

HMG boxes are DNA binding domains present in chromatin proteins, general transcription factors for nucleolar and mitochondrial RNA polymerases, and gene- and tissue-specific transcriptional regulators. The HMG boxes of HMG1, an abundant component of chromatin, interact specifically with four-way junctions, DNA structures that are cross-shaped and contain angles of approximately 60 and 120 degrees between their arms. We show here also that the HMG box of SRY, the protein that determines the expression of male-specific genes in humans, recognizes four-way junction DNAs irrespective of their sequence. In addition, when SRY binds to linear duplex DNA containing its specific target AACAAAG, it produces a sharp bend. Therefore, the interaction between HMG boxes and DNA appears to be predominantly structure-specific. The production of the recognition of a kink in DNA can serve several distinct functions, such as the repair of DNA lesions, the folding of DNA segments with bound transcriptional factors into productive complexes or the wrapping of DNA in chromatin.