Genetic characterization and in vivo image analysis of novel zebrafish Danio rerio pigment mutants.

This study reports the isolation and characterization of a new type of transparent zebrafish Danio rerio mutant called pinky (pk), which has been visually isolated from a spontaneous mutation in a D. rerio colony. The pk larvae possess complex mutations affecting pigmentation because of missing pigment cells or a dramatic reduction in the chromatophore number. The pk displays a totally colourless phenotype and adult body transplant with no other obvious external morphological abnormalities, except for a red retina. The molecular analysis results in several candidate genes, hps1, ap3m2 and rabggta, implicated in the Hermansky-Pudlak syndrome (HPS) genes associated with HPS in pk. To demonstrate its applications of deep-tissue imaging, this study examines green fluorescent protein alone or with other fluorescent proteins to investigate their capability for using multilabelling purposes in live adult pk. In this study, pk is particularly valuable for tissue cell labelling and internal organogenesis studies because of its optical clarity in the adult body.

A comparison of critical structure dose and toxicity risks in patients with low grade gliomas treated with IMRT versus proton radiation therapy.

Proton therapy offers dosimetric advantage of decreased dose to non-target tissues. This study explored the potential benefits of proton radiation therapy versus photon based intensity modulated radiation therapy (IMRT) for patients with low grade gliomas (LGG) through dosimetric comparison and biological modeling of potential radiation-induced toxicities. Eleven patients were treated with fractionated proton radiation therapy on a prospective protocol assessing for feasibility and treatment toxicity of proton radiation therapy in patients with LGG. IMRT treatment plans were created for each patient using the same CT planning data set and defined structures. The prescription dose to clinical target volume (CTV) was 54 Gy(RBE). The toxicity risk of IMRT and protons was estimated based upon equivalent uniform dose (EUD) and normal tissue complication probability (NTCP) modeling. The risk of secondary tumors for each modality was estimated. Proton EUD for most immediate normal tissue structures was between 10-20 Gy lower than the EUD delivered by IMRT. However, the difference in NTCP was negligible for both modalities. The mean excess risk of proton radiation-induced second tumor in the brain per 10,000 cases per year is 47 (range 11-83), while the mean risk for IMRT is 106 (range 70-134). The mean ratio of excess risk IMRT/protons is 2.2 (range 1.6-6.5), demonstrating that the risk of secondary tumors is consistently higher for IMRT. Proton therapy effectively reduces the dose to surrounding normal tissues in LGG patients. IMRT has a twofold higher risk of secondary intracranial tumors as compared to proton therapy. In most cases, NTCP is negligible for both modalities. The benefit of proton therapy over IMRT may be more substantial in patients with tumors in proximity to critical structures.

Immunomagnetic reduction assay for nervous necrosis virus extracted from groupers.

Nervous necrosis virus (NNV) is the cause of viral nervous disease, which is a serious constraint on production for grouper aquaculture. Real-time PCR is commonly used to detect and quantify NNV, has the disadvantages of being expensive and technically demanding. In this study, an immunomagnetic reduction (IMR) assay was developed as a rapid and cost-effective alternative to real-time PCR. This method used magnetic nanoparticles conjugated with antibodies specific for viral surface antigens to detect NNV in grouper tissue samples. The association of NNV with the antibody-conjugated magnetic particles resulted in a reduction in magnetic signal, which was strongly correlated with the concentration of NNV, as determined by real-time PCR. Grouper larvae were prepared for testing using a viral extraction buffer which provided a rapid, 15-min method of extracting viral antigens and had an extraction efficiency of higher than 80%. In addition, this study proposes using magnetic nanoparticles as labeling markers and as an assaying reagent for NNV. The magnetic nanoparticles are functionalized with antibodies against the viral surface of NNV and are able to associate specifically with NNV. The reduction of the magnetic signals comes from the association between magnetic particles and NNV, and relates to the concentration of NNV. The results show that the detected concentrations of NNV are highly correlated to those detected by real-time PCR.

Involvement of the terminus of grouper betanodavirus capsid protein in virus-like particle assembly.

Dragon grouper, Epinephelus lanceolatus, nervous necrosis virus (DGNNV) consists of 180 copies of capsid protein that encapsulates a bipartite genome of single-stranded (+)RNAs. Expressing the open reading frame (ORF) of RNA2 in Escherichia coli forms virus-like particles (VLPs) that resembles native virus. Deleting N- and C-termini revealed different impacts on VLP formation. Deletion of 35 or 52 residues at the N-terminus completely ruined the VLP assembly, presumably due to removal of positively charged residues for binding RNAs. When deletions were restricted to 4, 16, or 25 N-terminal residues, the assembly of VLPs remained. The ability of VLP formation diminished when 4 to 11 C-terminal residues were deleted. The termini that can be deleted without seriously destructing the VLPs are 25 and 3 residues at N- and C-termini, respectively.

Characterization of virus-like particles assembled in a recombinant baculovirus system expressing the capsid protein of a fish nodavirus.

Betanodaviruses are causative agents of neurological disorders in several species of fish. We cloned and sequenced the RNA2 segment of two grouper viruses isolated from Epinephelus malabaricus (malabaricus grouper nervous necrosis virus, MGNNV) and Epinephelus lanceolatus (dragon grouper nervous necrosis virus, DGNNV). The sequences of the two RNAs were 99% identical and comparison with previously sequenced RNA2 segments of fish nodaviruses striped jack nervous necrosis virus, Atlantic halibut virus, sea bass encephalitis virus, and greasy grouper nervous necrosis virus (GGNNV) revealed that MGNNV and DGNNV were most closely related to GGNNV. No correlation of sequence with geographical habitat was detected. The MGNNV coat protein, the gene product of RNA2, was expressed in Sf21 cells with a recombinant baculovirus system and virus-like particles (VLPs) spontaneously formed. Two types of VLPs were observed: a slower sedimenting particle was RNase-sensitive and stain-permeable, while the faster sedimenting particle survived RNase treatment and was not stain-permeable. An image reconstruction of the latter, obtained with electron cryomicroscopy data, revealed a morphology consistent with T = 3 quasi-symmetry but with features significantly different from insect nodavirus structures at the same resolution. This assembly system allows the first biophysical comparisons of fish and insect nodavirus structure, assembly, and stability.

Expression of polyphosphate kinase inhibits the glucose uptake in Escherichia coli.

This paper examines the effects of phosphate pool and expression of polyphosphate kinase on glucose uptake by expressing the polyphosphate kinase under the control of lac promoter. The E. coli transformant of pL1, containing an IPTG controllable element for polyphosphate kinase expression, showed that the total intracellular phosphate significantly increased. However, the rate of glucose uptake by the resting plasmid-bearing cells with IPTG induction significantly decreased. These findings suggest that the polyphosphate can not directly function as an energy source in E. coli or at least not as a good energy supplier.

Reversible inhibition of tomato fruit senescence by antisense RNA.

Ethylene controls fruit ripening. Expression of antisense RNA to the rate-limiting enzyme in the biosynthetic pathway of ethylene, 1-aminocyclopropane-1-carboxylate synthase, inhibits fruit ripening in tomato plants. Administration of exogenous ethylene or propylene reverses the inhibitory effect. This result demonstrates that ethylene is the trigger and not the by-product of ripening and raises the prospect that the life-span of plant tissues can be extended, thereby preventing spoilage.

Critical Care Scoring System--new concept based on hemodynamic data.

Based on the rationale that hemodynamic, oxygen transport, and perfusion derangements describe physiologically important abnormalities in critically ill patients, we devised a Critical Care Scoring System (CCSS). The database includes a retrospective analysis of 318 patients managed with pulmonary artery catheters during 1986 to 1988. For comparison, Acute Physiology and Chronic Health Evaluation (APACHE II) score was computed for these patients. CCSS includes a total of 17 variables with a weighted score of 71. The mortality rates for increases by 5 points were observed and were statistically significant (p less than .0001). The same was not true for APACHE II. Although neither of the scores could predict ICU days, both reflected the number of life-threatening complications. Serial CCSS that was computed at 24 +/- 4 h (CCSS-C) provided documentation for improvement in patient condition secondary to therapeutic interventions. Statistically improved mortality prediction was observed with CCSS-C. Since 70 (45.7%) of 153 patients died with an APACHE II score less than 14, it appears that this score is deficient in a specific group of patients, i.e., those requiring invasive monitoring. For patients with circulatory or respiratory failure, CCSS may offer a better tool to predict mortality and evaluate therapy.

[Effects of dietary K on blood pressure, prostaglandin, and kallikrein in renovascular hypertensive rats].

The effects of dietary K (food and tap water both containing 1% KCl) on blood pressure and renal prostaglandin-kallikrein-kinin system were investigated in Wistar rats made hypertensive by constriction of left renal artery. Dietary K attenuated the development of hypertension and increased urine volume accompanied by increased excretion of K, but by uninfluenced excretion of Na. Dietary K also increased the urinary excretion of kallikrein, PGE2 and aldosterone in Goldblatt hypertensive rats. There was no significant difference in the values of plasma Na between the two groups with and without dietary K. These results suggest that dietary K may attenuate the development of hypertension, increase urine volume via the mechanism of enhancing production of renal PGE2 and kallikrein in hypertensive rats.