Clusters of cortical spreading depolarizations in a patient with intracerebral hemorrhage: a multimodal neuromonitoring study.

BACKGROUND: Spontaneous intracerebral hemorrhage (ICH) is associated with high morbidity and mortality. Cortical spreading depolarizations (CSDs) increase brain matrix metalloproteinase (MMP)-9 activity leading to perihematomal edema expansion in experimental ICH. METHODS: The purpose of this report is to describe cerebral metabolic changes and brain extracellular MMP-9 levels in a patient with CSDs and perihematomal edema expansion after ICH. RESULTS: We present a 66-year-old male patient with ICH who underwent craniotomy for hematoma evacuation. Multimodal neuromonitoring data of the perihematomal region revealed metabolic distress and increased MMP-9 levels in the brain extracellular fluid during perihematomal edema progression. At the same time, subdural electrocorticography showed clusters of CSDs, which disappeared after ketamine anesthesia on day six. Perihematomal edema regression was associated with decreasing cerebral MMP-9 levels. CONCLUSIONS: This novel association between clusters of CSDs, brain metabolic distress, and increased MMP-9 levels expands our knowledge about secondary brain injury after ICH. The role of ketamine after this devastating disorder needs further studies.

Transgenic tilapia and the tilapia genome.

The tilapia fish (Oreochromis niloticus) has an important place in the aquaculture of the developing world. It is also a very useful laboratory animal, and readily lends itself to the transgenic technology. Through the use of reporter genes, a range of potential gene promoters have been tested in tilapia, both through transient and stable expression of the reporter construct. Using the transgenic technology, growth enhanced lines of tilapia have been produced. These fish have no abnormalities and offer a considerable growth advantage for future exploitation. It is however crucial that transgenic fish, to be exploited in aquaculture, be sterile, and various methods of achieving sterility are considered. These include triploidy, gene knock out of crucial hormone encoding genes via homologous recombination, and knock down of the function of the same genes via ribozyme or antisense technologies. Transgenic tilapia also offer the potential for exploitation as biofactories in the production of valuable pharmaceutical products, and this is also discussed.

Copy number related transgene expression and mosaic somatic expression in hemizygous and homozygous transgenic tilapia (Oreochromis niloticus).

Three lines of transgenic tilapia (Oreochromis niloticus) fish were generated with a construct containing a lacZ reporter gene spliced to a 4.7 kb 5' regulatory region of a carp beta actin gene. All these three lines contain different copy numbers of transgenes and the levels of lacZ expression were found to be related to transgene copy number. Mosaic patterns of somatic lacZ expression were observed in these three lines which differed between lines but were consistent within a line. We also observed that expression of the reporter gene in homozygous transgenic fish was approximately two-fold greater than in the hemizygous transgenics. Analysis of expression of the reporter gene on a tissue-to-tissue basis demonstrated that lacZ expression of the reporter gene in stably transformed fish occured with variable intensity in different organs and tissues and was also sometimes variable in different cells of the same tissue in Gland G2 generations of the transgenic lines.

The retinoblastoma susceptibility gene product/Sp1 signalling pathway is modulated by Ca2+/calmodulin kinases II and IV activity.

To investigate the possible link between Ca2+ signalling and cell cycle control we analysed Ca2+/calmodulin kinases (CamK) interaction with the retinoblastoma susceptibility gene product/SP1 pathway. CamK II and IV activate c-fos transcription through a short promoter region (-99 to -53) containing the retinoblastoma control element (RCE) and a cAMP response element (CRE) related sequences. Deletion analysis revealed that the RCE is a major CamK responsive element and is sufficient to confer CamK and Ca2+ regulation to a minimal promoter. Direct interactions between SP1 and RCE were confirmed by gel shift experiments. Using transient transfection experiments, we show that CamK-dependent transcription is regulated by the retinoblastoma (Rb) susceptibility gene product and the p107 Rb related protein. However, the stimulatory effects of CamKs and Rb on c-fos are blocked by overexpression of both proteins. These effects appear to be directly mediated by SP1 as shown by the use of a Gal4/SP1 fusion proteins. In conclusion, CamK II and IV, two major Ca2+-dependent intracellular effectors, may represent a molecular link between this second messenger transduction pathway and effectors that control cell cycle progression through Rb/SP1 signalling pathway.

Activation of gene transcription by tilapia prolactin variants tiPRL188 and tiPRL177.

In the tilapia species Oreochromis niloticus, the pituitary releases two forms of prolactins (tiPRL188 and tiPRL177). The binding parameters and the activation of tiPRL-induced JAK2/Stat5 signalling pathway were analysed using a mammalian cell line transiently transfected with the tiPRL receptor (tiPRLR). Our data indicate that the tiPRLR is able to mediate transcriptional activation of the PRL responsive element. At nanomolar concentrations, tiPRL188 activates gene transcription whereas at micromolar concentrations it inhibits luciferase transcription from the lactogenic responsive element. This is consistent with a model of receptor dimerisation. In contrast, the activation by tiPRL177 was only reached at high (microM) concentrations. The transcriptional activities induced by tiPRL177 and tiPRL188 are discussed in the context of the physiology of these hormones.

Identification and modulation of a growth hormone-binding protein in rainbow trout (Oncorhynchus mykiss) plasma during seawater adaptation.

A soluble protein that specifically bound 125I-human growth hormone (hGH) was identified in rainbow trout plasma, using HPLC-gel filtration. The binding affinity of the protein for hGH was 1.2 x 10(9)M-1. 125I-rainbow trout GH (tGH) was also able to bind to the protein albeit with a lower affinity (6.6 x 10(7)M-1) than hGH. Crosslinking experiments using 125I-hGH revealed two specific bands of 150 and 130 kDa. The complex 125I-hGH-BP could be precipitated by a monoclonal anti-GH receptor antibody, suggesting a close relationship between the plasma GH-BP and the GH receptor. A fourfold increase in the hGH binding to the GH-BP was shown 48 h after transfer of the fishes from freshwater to seawater. The increase in binding was related to a high binding capacity without significant changes in binding affinity. These results suggest a potential role of this related GH-BP as an index of GH effects during seawater adaptation in salmonids.

A point mutation in the mitochondrial cytochrome b gene obviates the requirement for the nuclear encoded core protein 2 subunit in the cytochrome bc1 complex in Saccharomyces cerevisiae.

A yeast mutant (cor2-45) in which approximately half of the C terminus of core protein 2 of the cytochrome bc1 complex is lacking due to a frameshift mutation that introduces a stop at codon 197 in the COR2 gene fails to assemble the cytochrome bc1 complex and does not grow on non-fermentable carbon sources that require respiration. The loss of respiration is more severe with this frameshift mutation than with the complete deletion of the COR2 gene, suggesting deleterious effects of the truncated core 2 protein. A search for extragenic suppressors of the nuclear cor2-45 mutation resulted (in addition to the expected nuclear suppressors) in the isolation of a suppressor mutation in the mitochondrial DNA that replaces serine 223 by proline in cytochrome b. Assembly of the cytochrome bc1 complex and the respiratory deficient phenotype of the cor2-45 mutant are restored by the proline for serine replacement in cytochrome b. Surprisingly, this amino acid replacement in cytochrome b corrects not only the phenotype resulting from the cor2-45 frameshift mutation, but it also obviates the need for core protein 2 in the cytochrome bc1 complex since it alleviates the respiratory deficiency resulting from the complete deletion of the COR2 gene. This is the first report of a homoplasmic missense point mutation of the mitochondrial DNA acting as a functional suppressor of a mutation located in a nuclear gene and the first demonstration that the supernumerary core protein 2 subunit is not essential for the electron transfer and energy transducing functions of the mitochondrial cytochrome bc1 complex.

Expression cloning of a cDNA encoding a fish prolactin receptor.

By using an expression cloning strategy, we isolated a single positive clone encoding a tilapia prolactin (PRL) receptor. Tilapia PRL188 was used to screen a freshwater tilapia kidney expression library transfected in COS cells. The tilapia PRL receptor is a mature protein of 606 amino acids. The extracellular domain is devoid of the tandem repeat units present in birds and has two pairs of cysteine residues, a Trp-Ser-Xaa-Trp-Ser motif, and two potential N-glycosylation sites. The cytoplasmic domain contains 372 amino acids, including box 1, a sequence previously shown to be important for signal transduction in mammalian species. Thus, the general structure is similar to the long form of mammalian PRL receptors; however, amino acid comparisons reveal a rather low identity (approximately 37%). Northern blot analysis shows the existence of a single transcript in osmoregulatory tissues and reproductive organs. This localization is in agreement with known functions of PRL in teleosts.