Expression of hypoxia-inducible genes is suppressed in altered gravity due to impaired nuclear HIF1α accumulation.

Extravehicular activities, the backbone of manned space exploration programs, set astronauts into mild hypoxia. Unfortunately, microgravity aggravates threatening symptoms of hypoxia such as vision impairment and brain edema. Hypoxia-inducible factors (HIFs) sense cellular hypoxia and, subsequently, change the cells' expression profile instantaneously by rapidly translocating-most likely cytoskeleton-dependently-into the nucleus and subsequently forming transcription complexes with other proteins. We tested the hypothesis that this fundamental process could be altered by sudden changes in gravitational forces in parabolic flights using a newly developed pocket-size cell culture lab that deoxygenizes cells within 15 min. Sudden gravity changes (SGCs 1g-1.8g-0g-1.8g-1g) during hypoxic exposure suppressed expression of the HIF1α-dependent genes investigated as compared with hypoxia at constant 1g. Normoxic cells subjected to SGCs showed reduced nuclear but not cytoplasmatic HIF1α signal and appeared to have disturbed cytoskeleton architecture. Inhibition of the actin-dependent intracellular transport using a combination of myosin V and VI inhibitors during hypoxia mimicked the suppression of the HIF1α-dependent genes observed during hypoxic exposure during SGCs. Thus, SGCs seem to disrupt the cellular response to hypoxia by impairing the actin-dependent translocation of HIF1α into the nucleus.

Methemoglobinemia, Increased Deformability and Reduced Membrane Stability of Red Blood Cells in a Cat with a CYB5R3 Splice Defect.

Methemoglobinemia is an acquired or inherited condition resulting from oxidative stress or dysfunction of the NADH-cytochrome b5 reductase or associated pathways. This study describes the clinical, pathophysiological, and molecular genetic features of a cat with hereditary methemoglobinemia. Whole genome sequencing and mRNA transcript analyses were performed in affected and control cats. Co-oximetry, ektacytometry, Ellman's assay for reduced glutathione concentrations, and CYB5R activity were assessed. A young adult European domestic shorthair cat decompensated at induction of anesthesia and was found to have persistent methemoglobinemia of 39 ± 8% (reference range < 3%) of total hemoglobin which could be reversed upon intravenous methylene blue injection. The erythrocytic CYB5R activity was 20 ± 6% of normal. Genetic analyses revealed a single homozygous base exchange at the beginning of intron 3 of the CYB5R3 gene, c.226+5G>A. Subsequent mRNA studies confirmed a splice defect and demonstrated expression of two mutant CYB5R3 transcripts. Erythrocytic glutathione levels were twice that of controls. Mild microcytosis, echinocytes, and multiple Ca2+-filled vesicles were found in the affected cat. Erythrocytes were unstable at high osmolarities although highly deformable as follows from the changes in elongation index and maximal-tolerated osmolarity. Clinicopathological presentation of this cat was similar to other cats with CYB5R3 deficiency. We found that methemoglobinemia is associated with an increase in red blood cell fragility and deformability, glutathione overload, and morphological alterations typical for stress erythropoiesis.

Identification of shedders of elephant endotheliotropic herpesviruses among Asian elephants (Elephas maximus) in Switzerland.

Elephants, particularly Asian (Elephas maximus), are threatened by lethal elephant hemorrhagic disease (EHD) due to elephant endotheliotropic herpesviruses (EEHV). At least five of seven known EEHV types have been associated to EHD, with types 1, 4, and 5 predominantly affecting Asian elephants. In Switzerland, at least three Asian elephants have been lost due to EHD but nothing is known about the present EEHV1 circulation. Moreover, the prevalence of other EEHV types has never been assessed. Intermittent shedding of EEHV can be monitored through collecting trunk secretions and analyzing them by PCR methods that discriminate the different EEHV types. To identify EEHV shedders, seven of eight Asian elephants in a Swiss zoo were trained to provide trunk wash samples. These were collected at intervals over a period of four months and tested by PCR for presence of EEHV1 through 6. Moreover, the quality of each sample was assessed by testing for the elephant TNF-alpha gene. Overall, 57% of the samples were valid with five of seven participating elephants identified as EEHV shedders. Two of those shed virus only once, whereas the other three, all closely related among each other, shed virus on multiple occasions. One of the frequent shedders had been in very close contact to all of the three EHD victims. Therefore, we speculate that this particular animal may represent the virus source in all three cases. However, when subtyping was conducted, the presently circulating virus was identified as EEHV1B, while the virus subtype causing EHD had been 1A in all three cases. In addition to four animals excreting EEHV1, a recently introduced animal was observed to shed EEHV3/4. We suggest that the policy of trunk washing to identify and characterize EEHV-shedders is to be endorsed in zoos with ongoing or planned elephant breeding programs.

The genome of Chelonid herpesvirus 5 harbors atypical genes.

The Chelonid fibropapilloma-associated herpesvirus (CFPHV; ChHV5) is believed to be the causative agent of fibropapillomatosis (FP), a neoplastic disease of marine turtles. While clinical signs and pathology of FP are well known, research on ChHV5 has been impeded because no cell culture system for its propagation exists. We have cloned a BAC containing ChHV5 in pTARBAC2.1 and determined its nucleotide sequence. Accordingly, ChHV5 has a type D genome and its predominant gene order is typical for the varicellovirus genus within the alphaherpesvirinae. However, at least four genes that are atypical for an alphaherpesvirus genome were also detected, i.e. two members of the C-type lectin-like domain superfamily (F-lec1, F-lec2), an orthologue to the mouse cytomegalovirus M04 (F-M04) and a viral sialyltransferase (F-sial). Four lines of evidence suggest that these atypical genes are truly part of the ChHV5 genome: (1) the pTARBAC insertion interrupted the UL52 ORF, leaving parts of the gene to either side of the insertion and suggesting that an intact molecule had been cloned. (2) Using FP-associated UL52 (F-UL52) as an anchor and the BAC-derived sequences as a means to generate primers, overlapping PCR was performed with tumor-derived DNA as template, which confirmed the presence of the same stretch of "atypical" DNA in independent FP cases. (3) Pyrosequencing of DNA from independent tumors did not reveal previously undetected viral sequences, suggesting that no apparent loss of viral sequence had happened due to the cloning strategy. (4) The simultaneous presence of previously known ChHV5 sequences and F-sial as well as F-M04 sequences was also confirmed in geographically distinct Australian cases of FP. Finally, transcripts of F-sial and F-M04 but not transcripts of lytic viral genes were detected in tumors from Hawaiian FP-cases. Therefore, we suggest that F-sial and F-M04 may play a role in FP pathogenesis.

The subcellular localization of TRPP2 modulates its function.

TRPP2, also known as polycystin-2, is a calcium permeable nonselective cation channel that is mutated in autosomal dominant polycystic kidney disease but has also been implicated in the regulation of cardiac development, renal tubular differentiation, and left-to-right (L-R) axis determination. For obtaining further insight into how TRPP2 exerts tissue-specific functions, this study took advantage of PACS-dependent trafficking of TRPP2 in zebrafish larvae. PACS proteins recognize an acidic cluster within the carboxy-terminal domain of TRPP2 that undergoes phosphorylation and mediate retrieval of TRPP2 to the Golgi and endoplasmic reticulum (ER). The interaction of human TRPP2 with PACS proteins can be inhibited by a Ser812Ala mutation (TRPP2(S812A)), thereby allowing TRPP2 to reach other subcellular compartments, and enhanced by a Ser812Asp mutation (TRPP2(S812D)), thereby trapping TRPP2 in the ER. It was found that the TRPP2(S812A) mutant rescued cyst formation of TRPP2-deficient zebrafish larvae to the same degree as wild-type TRPP2, whereas the TRPP2(S812D) mutant was significantly more effective in normalizing the distorted body axis of TRPP2-deficient fish. Surprisingly, the TRPP2(S812D) mutant rescued the abnormalities of L-R asymmetry more effectively than either wild-type or TRPP2(S812A), suggesting that the ER localization of TRPP2 plays an important role in the development of normal L-R asymmetry. Taken together, these findings support the hypothesis that TRPP2 assumes distinct subcellular localizations to exert tissue-specific functions.