The positive-acting sulfur regulatory protein CYS3 of Neurospora crassa: nuclear localization,autogenous control,and regions required for transcriptional activation

The positive-acting global sulfur regulatory protein, CYS3, of Neurospora crassa turns on the expression of a family of unlinked structural genes that encode enzymes of sulfur catabolism. CYS3 contains a leucine zipper and an adjacent basic region (b-zip), which together constitute a bipartite sequence-specific DNA-binding domain. Specific anti-CYS3 antibodies detected a protein of the expected size in nuclear extracts of wild-type Neurospora under conditions in which the sulfur circuit is activated. The CYS3 protein was not observed in cys-3 mutants. Nuclear extracts of wild type, but not cys-3 mutants, also showed specific DNA-binding activity identical to that obtained with a CYS3 protein expressed in Escherichia coli. A truncated CYS3 protein that contains primarily the b-zip domain binds to DNA with high specificity and affinity in vitro, yet fails to activate gene expression in vivo, and instead inhibits the function of the wild-type CYS3 protein. Amino-terminal, carboxy-terminal, and internal deletions as well as alanine scanning mutagenesis were employed to identify regions of the CYS3 protein that are required for its trans-activation function. Regions of CYS3 carboxy terminal to the b-zip motif are not completely essential for function although loss of an alanine-rich region results in decreased activity. All deletions amino terminal to the b-zip motif led to a complete loss of CYS3 function. Alanine scanning mutagenesis demonstrated that an unusual proline-rich domain of CYS3 appears to be very important for function and is presumed to constitute an activation domain. It is concluded that CYS3 displays nuclear localization and positive autogenous control in Neurospora and functions as a trans-acting DNA-binding protein.     

Proteasome activity is required for T lymphocyte aggregation after mitogen activation

The proteasome is a multicatalytic complex of proteases involved in T lymphocyte proliferation and activation through multiple mechanisms. In this study, we investigated its role in lymphocyte aggregation. We found that blocking proteasome activity by a proteasome-specific inhibitor lactacystin (LAC) prevented clustering of T lymphocytes after stimulation with various mitogens. Expression of adhesion molecules ICAM-1 and LFA-1 at cell surfaces of activated T cells was decreased after treatment with LAC. Mechanisms by which the proteasome intervenes in the expression of these adhesion molecules were different. LAC inhibited ICAM-1 expression at the mRNA level, whereas LFA-1 inhibition was probably at a post-translational level. Downregulation of these molecules after proteasome inhibition likely contributes to the observed repression of T cell aggregation. Our results show that the proteasome plays an important role in cell-cell interaction during T cell activation.     

Emotion-Motion Interactions in Conversion Disorder: An fMRI Study

Objectives

To evaluate the neural correlates of implicit processing of negative emotions in motor conversion disorder (CD) patients.

Methods

An event related fMRI task was completed by 12 motor CD patients and 14 matched healthy controls using standardised stimuli of faces with fearful and sad emotional expressions in comparison to faces with neutral expressions. Temporal changes in the sensitivity to stimuli were also modelled and tested in the two groups.

Results

We found increased amygdala activation to negative emotions in CD compared to healthy controls in region of interest analyses, which persisted over time consistent with previous findings using emotional paradigms. Furthermore during whole brain analyses we found significantly increased activation in CD patients in areas involved in the ‘freeze response’ to fear (periaqueductal grey matter), and areas involved in self-awareness and motor control (cingulate gyrus and supplementary motor area).

Conclusions

In contrast to healthy controls, CD patients exhibited increased response amplitude to fearful stimuli over time, suggesting abnormal emotional regulation (failure of habituation / sensitization). Patients with CD also activated midbrain and frontal structures that could reflect an abnormal behavioral-motor response to negative including threatening stimuli. This suggests a mechanism linking emotions to motor dysfunction in CD.     

Effect of chronic elevated carbon dioxide on the expression of acid-base transporters in the neonatal and adult mouse

Several pulmonary and neurological conditions, both in the newborn and adult, result in hypercapnia. This leads to disturbances in normal pH homeostasis. Most mammalian cells maintain tight control of intracellular pH (pH(i)) using a group of transmembrane proteins that specialize in acid-base transport. These acid-base transporters are important in adjusting pH(i) during acidosis arising from hypoventilation. We hypothesized that exposure to chronic hypercapnia induces changes in the expression of acid-base transporters. Neonatal and adult CD-1 mice were exposed to either 8% or 12% CO(2) for 2 wk. We used Western blot analysis of membrane protein fractions from heart, kidney, and various brain regions to study the response of specific acid-base transporters to CO(2). Chronic CO(2) increased the expression of the sodium hydrogen exchanger 1 (NHE1) and electroneutral sodium bicarbonate cotransporter (NBCn1) in the cerebral cortex, heart, and kidney of neonatal but not adult mice. CO(2) increased the expression of electrogenic NBC (NBCe1) in the neonatal but not the adult mouse heart and kidney. Hypercapnia decreased the expression of anion exchanger 3 (AE3) in both the neonatal and adult brain but increased AE3 expression in the neonatal heart. We conclude that: 1) chronic hypercapnia increases the expression of the acid extruders NHE1, NBCe1 and NBCn1 and decreases the expression of the acid loader AE3, possibly improving the capacity of the cell to maintain pH(i) in the face of acidosis; and 2) the heterogeneous response of tissues to hypercapnia depends on the level of CO(2) and development.     

Flow cytometry and start codon targeted (SCoT) genetic fidelity assessment of regenerated plantlets in Tylophora indica (Burm.f.) Merrill

Tylophora indica (Burm.f.) Merrill. is a pharmacologically important plant, popular for alkaloidal and non-alkaloidal richness. Large scale propagation of T. indica is difficult in the wild as the seeds are small and the frequency of germination is very poor. In the present study, the genome size estimation of in vitro regenerated (indirect, direct and somatic embryo mediated) T. indica was made by flow cytometric method. Clonal fidelity of the regenerants was assessed using a start codon targeted (SCoT) molecular marker. Initially, the explants were inoculated on Murashige and Skoog basal medium supplemented with various concentrations of plant growth regulators like 2,4-dichlorophenoxy acetic acid (2,4-D), Kinetin, 6-benzyl amino purine (BAP) and 1-naphthalene acetic acid either singly or in combinations. The highest callus induction frequency (87.75%) was obtained in 6.7 µM 2,4-D added MS medium which metamorphosed into progressive stages (globular, heart, torpedo, and cotyledonary) of embryos. Mature and healthy somatic embryos efficiently germinated into plantlets on 8.8 µM BAP?+?1.4 µM GA₃ enriched MS medium. Histological and scanning electron microscopic study confirmed the above developing stages. The regenerated shoots were rooted best in 2.45 µM Indole-3-butyric acid supplemented solid MS medium. The plants were hardened and acclimatized with 90% survivability. The flow cytometric 2C DNA content of indirect, direct and somatic embryo derived plants was 1.896 pg, 1.940 pg and 1.926 pg respectively, very similar to the mother plant (1.928 pg). SCoT marker generated a high percentage of monomorphic bands (94%) revealing similarity with the mother plant, thus ensuring genetic fidelity. To the best of our knowledge, this is perhaps the first ever report of 2C DNA content estimation and SCoT marker based genetic homogeneity study in T. indica.

     

A note on the effects of co-mingling piglet litters on pre-weaning growth, injuries and responses to behavioural tests

The purpose of this study was to determine how co-mingling litters affected piglets’ pre-weaning growth, ear injuries, suckling behaviour and responses to behavioural tests used to measure coping abilities. Thirty sows and their respective litters were housed in standard farrowing crates until day 13 after birth. On day13, the partition between two neighbouring pens was removed for 20 litters allowing piglets to interact (forming 10 co-mingled litters). The remaining 10 control litters were kept in standard farrowing crates throughout the experiment. Three focal piglets from each litter were used for data collection. Focal piglets were weighed and ear injuries recorded on days 2, 4, 9, 12, 15 and 18 after birth. There were no differences in piglets’ weight gain before or after co-mingling. Ear injuries were more abundant in co-mingled litters on day 15 (P < 0.05) but these differences disappeared by day 18. Suckling behaviour was recorded on days 5, 8, 10, 14, 16 and 18 after birth. There were no differences in teat fidelity, suckling frequency and mother fidelity between treatments. Three behavioural tests, social challenge, isolation, and backtest, were performed before and after co-mingling. There were no treatment effects on piglets’ response to the isolation test and backtest. Co-mingled piglets showed longer latency for the first aggressive interaction (P < 0.05), spent more time in proximity to one another (P < 0.05) and performed less single bites (P < 0.05) than control piglets during the social challenge. In addition, the duration and frequency of aggressive interactions (P < 0.05) were lower in co-mingled piglets than control piglets. Co-mingling did not affect the frequency of single head thrusts or oral–nasal contact, but did tend to increase the frequency of escape attempts (P < 0.10). Our results suggest that co-mingling litters during lactation affects piglets’ social behaviour, by primarily decreasing aggressive interactions during social challenges.     

Tau Kinetics in Neurons and the Human Central Nervous System

1. Download high-res image (181KB)
2. Download full-size image

     

Effect of carbon dioxide on neonatal mouse lung: a genomic approach.

Despite the deleterious effects associated with elevated carbon dioxide (CO(2)) or hypercapnia, it has been hypothesized that CO(2) can protect the lung from injury. However, the effects of chronic hypercapnia on the neonatal lung are unknown. Hence, we investigated the effect of chronic hypercapnia on neonatal mouse lung to identify genes that could potentially contribute to hypercapnia-mediated lung protection. Newborn mouse litters were exposed to 8% CO(2), 12% CO(2), or room air for 2 wk. Lungs were excised and analyzed for morphometric alterations. The alveolar walls of CO(2)-exposed mice appeared thinner than those of controls. Analyses of gene expression differences by microarrays revealed that genes from a variety of functional categories were differentially expressed following hypercapnia treatment, including those encoding growth factors, chemokines, cytokines, and endopeptidases. In particular and of major interest, the expression level of genes encoding surfactant proteins A and D, as well as chloride channel calcium-activated 3, were significantly increased, but the expression of WNT1-inducible signaling pathway protein 2 was significantly decreased. The significant changes in gene expression occurred mostly at 8% CO(2), but only a few at 12% CO(2). Our results lead us to conclude that 1) there are a number of gene families that may contribute to hypercapnia-mediated lung protection; 2) the upregulation of surfactant proteins A and D may play a role as anti-inflammatory or antioxidant agents; and 3) the effects of CO(2) seem to depend on the level to which the lung is exposed.