Versatility and specialization in labrid fishes: ecomorphological implications

Summary The term specialized has been used to describe species that possess unique functional attributes and/or a narrow, stereotyped range of attributes, but there are few comparative functional analyses of specialists and generalists. If species with functional morphological specializations are capable of functioning over a broad range, the link between morphology and ecology may be relaxed under certain environmental conditions. In this study, high-speed films of jaw movements during prey capture were compared statistically for three coexisting coral reef fish species in the family Labridae, one trophic specialist and two trophic generalists. The trophic specialist possessed a unique functional feature related to the movement of the hyoid in the floor of the mouth, while the trophic generalists were not observed to possess any functional specializations. All three species showed functional versatility in that they were able to adjust their prey capture mechanism in response to the evasive potential of the prey. The functional versatility of trophic specialists has implications for ecomorphological studies, since species characterized as possessing unique functional or morphological features may demonstrate marked flexibility in ecological variables such as diet or foraging behavior, decreasing the likelihood of identifying correlations between morphology and ecology.     

Nicastrin, presenilin, APH-1, and PEN-2 form active gamma-secretase complexes in mitochondria

Mitochondria are central in the regulation of cell death. Apart from providing the cell with ATP, mitochondria also harbor several death factors that are released upon apoptotic stimuli. Alterations in mitochondrial functions, increased oxidative stress, and neurons dying by apoptosis have been detected in Alzheimer's disease patients. These findings suggest that mitochondria may trigger the abnormal onset of neuronal cell death in Alzheimer's disease. We previously reported that presenilin 1 (PS1), which is often mutated in familial forms of Alzheimer's disease, is located in mitochondria and hypothesized that presenilin mutations may sensitize cells to apoptotic stimuli at the mitochondrial level. Presenilin forms an active gamma-secretase complex together with Nicastrin (NCT), APH-1, and PEN-2, which among other substrates cleaves the beta-amyloid precursor protein (beta-APP) generating the amyloid beta-peptide and the beta-APP intracellular domain. Here we have identified dual targeting sequences (for endoplasmic reticulum and mitochondria) in NCT and showed expression of NCT in mitochondria by immunoelectron microscopy. We also showed that NCT together with APH-1, PEN-2, and PS1 form a high molecular weight complex located in mitochondria. gamma-secretase activity in isolated mitochondria was demonstrated using C83 (alpha-secretase-cleaved C-terminal 83-residue beta-APP fragment from BD8 cells lacking presenilin and thus gamma-secretase activity) or recombinant C100-Flag (C-terminal 100-residue beta-APP fragment) as substrates. Both systems generated an APP intracellular domain, and the activity was inhibited by the gamma-secretase inhibitors l-685,458 or Compound E. This novel localization of NCT, PS1, APH-1, and PEN-2 expands the role and importance of gamma-secretase activity to mitochondria.     

p53 and p21 regulate error-prone DNA repair to yield a lower mutation load

Regulation of mutation rates is critical for maintaining genome stability and controlling cancer risk. A special challenge to this regulation is the presence of multiple mutagenic DNA polymerases in mammals. These polymerases function in translesion DNA synthesis (TLS), an error-prone DNA repair process that involves DNA synthesis across DNA lesions. We found that in mammalian cells TLS is controlled by the tumor suppressor p53, and by the cell cycle inhibitor p21 via its PCNA-interacting domain, to maintain a low mutagenic load at the price of reduced repair efficiency. This regulation may be mediated by binding of p21 to PCNA and via DNA damage-induced ubiquitination of PCNA, which is stimulated by p53 and p21. Loss of this regulation by inactivation of p53 or p21 causes an out of control lesion-bypass activity, which increases the mutational load and might therefore play a role in pathogenic processes caused by genetic instability.     

Susceptibility of dopamine D5 receptor targeted mice to cysteamine

BACKGROUND: Recently we demonstrated that gastric mucosa of rats can synthesize, store and release dopamine. Out of five different subtypes, mRNA of D5 (=D1b) dopamine receptor is very abundant in the gastric epithelium. D1 receptor selective dopamine agonists have been shown to protect against experimental gastro-duodenal lesions. AIMS: To test the hypothesis that protective effects of dopamine involve D5 receptors, mucosal lesions were induced in D5 receptor deficient (KO) and wild-type (WT) mice using cysteamine. Morphology and gastric acid secretion of D5 KO mice were also studied. METHODS: Single doses of 600 mg/kg, 300 mg/kg cysteamine or vehicle were administered subcutaneously to fasted animals. After 24 h, number and severity of gastro-duodenal lesions were analyzed. Basal and histamine-induced maximal gastric acid output were measured by a stomach-sac wash-through method. RESULTS: All the KOs in the 600 mg/kg cysteamine group died within 4 h showing symptoms of toxicity while three out of four WTs survived (P<0.05). Mortality after 300 mg/kg cysteamine was significantly higher in KOs versus the WTs: 6/14 versus 2/11, P<0.05. Gastric lesion-index was also significantly higher in KOs (median, middle quartile): four (3-9) versus 0 (0-0), P<0.05. Duodenal lesions did not develop from this single dose of cysteamine in either genotype. Basal and histamine-induced maximal gastric acid output were comparable in the two genotypes. CONCLUSIONS: This study demonstrates that loss of D5 receptor causes mucosal vulnerability and increased toxicity of cysteamine in genetically manipulated mice. Thus, D5 receptor subtype is indeed likely to be involved in protective effects of dopamine in the stomach.     

Enzyme‐immunoassay for the measurement of luteinizing hormone in the serum of African elephants (Loxodonta africana)

Circulating patterns of progesterone and luteinizing hormone (LH) in the elephant have been well characterized, and routine monitoring of these hormones is now viewed as a valuable tool for making informed decisions about the reproductive management of elephants in captivity. Currently, LH monitoring in elephants is done with radio‐immunoassays (RIAs); unfortunately, the use of radioactive materials in RIAs limits their application to institutions with laboratory facilities equipped for the storage and disposal of radioactive waste. Enzyme‐immunoassays (EIAs) offer an inexpensive and more zoo‐friendly alternative to RIA. This work reports on an EIA capable of quantifying circulating LH in African elephants. The EIA employs a biotin label and microtiter plates coated with goat anti‐mouse gamma globulin. LH surges in African elephants (n=3) increased fivefold over baseline concentrations (1.00±0.1 ng/ml vs. 0.2±0.1 ng/ml) and occurred 19.3±0.2 days apart. Ovulatory LH surges were associated with an increase in serum progestogens from 4.8±0.4 ng/ml to 11.7±0.4 ng/ml. The ability to quantify reproductive hormones in elephants via EIA is an important step in the process of making endocrine monitoring more accessible to zoos housing these species. Zoo Biol 21:403–408, 2002. © 2002 Wiley‐Liss, Inc.     

Agrobacterium-mediated transformation of the temperate grass Brachypodium distachyon (genotype Bd21) for T-DNA insertional mutagenesis

Brachypodium distachyon is a promising model system for the structural and functional genomics of temperate grasses because of its physical, genetic and genome attributes. The sequencing of the inbred line Bd21 ( http://www.brachypodium.org ) started in 2007. However, a transformation method remains to be developed for the community standard line Bd21. In this article, a facile, efficient and rapid transformation system for Bd21 is described using Agrobacterium -mediated transformation of compact embryogenic calli (CEC) derived from immature embryos. Key features of this system include: (i) the use of the green fluorescent protein (GFP) associated with hygromycin selection for rapid identification of transgenic calli and plants; (ii) the desiccation of CEC after inoculation with Agrobacterium ; (iii) the utilization of Bd21 plants regenerated from tissue culture as a source of immature embryos; (iv) the control of the duration of the selection process; and (v) the supplementation of culture media with CuSO₄ prior to and during the regeneration of transgenic plants. Approximately 17% of CEC produced transgenic plants, enabling the generation of hundreds of T-DNA insertion lines per experiment. GFP expression was observed in primary transformed Bd21 plants (T₀) and their progeny (T₁). The Mendelian inheritance of the transgenes was confirmed. An adaptor-anchor strategy was developed for efficient retrieval of flanking sequence tags (FSTs) of T-DNA inserts, and the resulting sequences are available in public databases. The production of T-DNA insertion lines and the retrieval of associated FSTs reported here for the reference inbred line Bd21 will facilitate large-scale functional genomics research in this model system.     

Molecular Diversity of Rumen Methanogens from Sheep in Western Australia

The molecular diversity of rumen methanogens in sheep in Australia was investigated by using individual 16S rRNA gene libraries prepared from the rumen contents obtained from six merino sheep grazing pasture (326 clones), six sheep fed an oaten hay-based diet (275 clones), and five sheep fed a lucerne hay-based diet (132 clones). A total of 733 clones were examined, and the analysis revealed 65 phylotypes whose sequences (1,260 bp) were similar to those of cultivated methanogens belonging to the order Methanobacteriales. Pasture-grazed sheep had more methanogen diversity than sheep fed either the oaten hay or lucerne hay diet. Methanobrevibacter strains SM9, M6, and NT7 accounted for over 90% of the total number of clones identified. M6 was more prevalent in grazing sheep, and SM9, despite being found in 16 of the 17 sheep, was more prevalent in sheep fed the lucerne-based diet. Five new species were identified. Two of these species exhibited very little sequence similarity to any cultivated methanogens and were found eight times in two of the six sheep that were grazing pasture. These unique sequences appear to represent a novel group of rumen archaea that are atypical for the rumen environment.     

Cofactor‐induced reversible folding of Flavodoxin‐4 from Lactobacillus acidophilus

Flavodoxins in combination with the flavin mononucleotide (FMN) cofactor play important roles for electron transport in prokaryotes. Here, novel insights into the FMN‐binding mechanism to flavodoxins‐4 were obtained from the NMR structures of the apo‐protein from Lactobacillus acidophilus (YP_193882.1) and comparison of its complex with FMN. Extensive reversible conformational changes were observed upon FMN binding and release. The NMR structure of the FMN complex is in agreement with the crystal structure (PDB ID: 3EDO ) and exhibits the characteristic flavodoxin fold, with a central five‐stranded parallel β–sheet and five α‐helices forming an α/β‐sandwich architecture. The structure differs from other flavoproteins in that helix α2 is oriented perpendicular to the β‐sheet and covers the FMN‐binding site. This helix reversibly unfolds upon removal of the FMN ligand, which represents a unique structural rearrangement among flavodoxins.     

Improved protein identification through the use of unstained gels

In this work, a method for improved protein identification of low-abundance proteins using unstained gels, in combination with robotics and matrix-assisted laser desorption/ionization tandem mass spectrometry, has been developed and evaluated. Omitting the silver-staining process resulted in increased protein identification scores, an increase in the number of peptides observed in the MALDI mass spectrum, and improved quality of the tandem mass spectrometry data.