Role of Pax genes in eye evolution: a cnidarian PaxB gene uniting Pax2 and Pax6 functions

PaxB from Tripedalia cystophora, a cubomedusan jellyfish possessing complex eyes (ocelli), was characterized. PaxB, the only Pax gene found in this cnidarian, is expressed in the larva, retina, lens, and statocyst. PaxB contains a Pax2/5/8-type paired domain and octapeptide, but a Pax6 prd-type homeodomain. Pax2/5/8-like properties of PaxB include a DNA binding specificity of the paired domain, activation and inhibitory domains, and the ability to rescue spa(pol), a Drosophila Pax2 eye mutant. Like Pax6, PaxB activates jellyfish crystallin and Drosophila rhodopsin rh6 promoters and induces small ectopic eyes in Drosophila. Pax6 has been considered a "master" control gene for eye development. Our data suggest that the ancestor of jellyfish PaxB, a PaxB-like protein, was the primordial Pax protein in eye evolution and that Pax6-like genes evolved in triploblasts after separation from Cnidaria, raising the possibility that cnidarian and sophisticated triploblastic eyes arose independently.     

Catalysis,specificity, and ACP docking site of Streptomyces coelicolor malonyl-CoA:ACP transacylase

Malonyl-CoA:ACP transacylase (MAT), the fabD gene product of Streptomyces coelicolor A3(2), participates in both fatty acid and polyketide synthesis pathways, transferring malonyl groups that are used as extender units in chain growth from malonyl-CoA to pathway-specific acyl carrier proteins (ACPs). Here, the 2.0 A structure reveals an invariant arginine bound to an acetate that mimics the malonyl carboxylate and helps define the extender unit binding site. Catalysis may only occur when the oxyanion hole is formed through substrate binding, preventing hydrolysis of the acyl-enzyme intermediate. Macromolecular docking simulations with actinorhodin ACP suggest that the majority of the ACP docking surface is formed by a helical flap. These results should help to engineer polyketide synthases (PKSs) that produce novel polyketides.     

Cardiovascular response to punching tempo

Eighteen trained volunteers (12 men and 6 women: age = 22.0 +/- 2.8 years, height = 170.79 +/- 7.67 cm, weight = 71.54 +/- 12.63 kg) participated in 2-minute, randomized fitness boxing trials, wearing 0.34-kg punching gloves, at various tempos (60, 72, 84, 96, 108, and 120 b.min(-1)). During each trial, oxygen uptake (VO(2)), heart rate (HR), and ventilation (VE) were measured continuously. A rating of perceived exertion (RPE) was attained at the conclusion of each trial. Subjects were able to attain VO(2) values ranging from 26.83 to 29.75 ml.kg(-1).min(-1), which correspond to 67.7-72.5% of VO(2)max. The HR responses yielded results ranging from 167.4 to 182.2 b.min(-1), or 85 to 93% of HRmax. No significant difference (p > 0.05) was seen with VO(2) between trials, although a significant difference (p < 0.05) was observed with HR, VE, and RPE. It appears that boxing speed is associated with increased VE, HR response, and perceived effort but not with VO(2). Energy expenditure values ranged from 9.8 to 11.2 kcal.min(-1) for the boxing trials. These results suggest that fitness boxing programs compare favorably with other exercise modalities in cardiovascular response and caloric expenditure.     

Phospholipid hydroperoxide glutathione peroxidase induces a delay in G1 of the cell cycle

Phospholipid hydroperoxide glutathione peroxidase (PhGPx) is an antioxidant enzyme that reduces cellular phospholipid hydroperoxides (PLOOHs) to alcohols. Cellular peroxide tone has been implicated in cell growth and differentiation. By reducing the PLOOH level in the cell membrane, PhGPx regulates the peroxide tone and thereby might be involved in cell growth. We hypothesized that overexpression of PhGPx in human breast cancer cells would decrease their growth rate. We stably transfected MCF-7 cells (Wt) with L-PhGPx and measured cell doubling time, plating efficiency, and cell cycle phase transit times. P-4 cells (8-fold increase in PhGPx activity) showed a 2-fold increase in doubling time; doubling time increased directly with PhGPx activity (r = 0.95). The higher the PhGPx activity, the lower the plating efficiency (r = -0.86). The profile of other antioxidant enzymes was unchanged. Overexpression of PhGPx lowered the steady-state level of PLOOH (by > 60%). Results from bromodeoxyuridine pulse-chase experiments and flow cytometry indicate that PhGPx induced a delay in MCF-7 proliferation that was primarily due to a slower progression from G1 to S. These results support the hypothesis that PhGPx plays a regulatory role in the progression of MCF-7 cells from G1 to S possibly by regulating the steady-state levels of PLOOH. These data suggest that PhGPx can lower the peroxide tone, which might change the cellular redox environment resulting in a delay in G1 transit. Thus, PhGPx could be an important factor in cell growth.     

The effect of feeding frequency on consumption of food,absorption efficiency,and gonad production in the sea urchin Lytechinus variegatus

The frequency of feeding in the field is variable in sea urchins, ranging from nearly continuous to diel or intermittent. It is essential to know the effect of feeding interval on physiological and metabolic processes to understand the basis for production. Lytechinus variegatus (50 mm horizontal diameter) were collected in January 1999 and held in closed-circuit aquaria at 25 degrees C and 35 per thousand salinity. After 9 days without food, individuals were fed one of three treatments: food available ad libitum, food available for 1 day every 2 days or food available for 1 day every 4 days for 28 days. The rate of food consumption per day of all individuals was high the first week of feeding. It then decreased to a lower, constant rate in those fed ad libitum but remained high in those fed one day every 2 or 4 days. The total amount eaten was directly related to frequency of feeding. The apparent dry matter digestibility (absorption efficiency) did not vary with frequency of feeding. As the total amount of energy absorbed was directly related to the frequency of feeding, the increase in the rate of food consumption does not compensate for a decrease in frequency of feeding. Gonad production efficiency was directly related to frequency of feeding. Gonad gross production (assimilation) efficiencies were 8.4, 3.9 and 3.4% for individuals fed every day, or fed one day every 2 and 4 days, respectively. The corresponding gonad net assimilation efficiencies were 12.5, 5.5, and 4.8%. A decrease in frequency of food availability results in use of a greater proportion of the food consumed for maintenance and less for gonad production.     

XRCC3 and Rad51 modulate replication fork progression on damaged vertebrate chromosomes

The mechanisms by which the progression of eukaryotic replication forks is controlled after DNA damage are unclear. We have found that fork progression is slowed by cisplatin or UV treatment in intact vertebrate cells and in replication assays in vitro. Fork slowing is reduced or absent in irs1SF CHO cells and XRCC3(-/-) chicken DT40 cells, indicating that fork slowing is an active process that requires the homologous recombination protein XRCC3. The addition of purified human Rad51C-XRCC3 complex restores fork slowing in permeabilized XRCC3(-/-) cells. Moreover, the requirement for XRCC3 for fork slowing can be circumvented by addition of human Rad51. These data demonstrate that the recombination proteins XRCC3 and Rad51 cooperatively modulate the progression of replication forks on damaged vertebrate chromosomes.     

Induced expression and association of the Mona/Gads adapter and Gab3 scaffolding protein during monocyte/macrophage differentiation

Mona/Gads is a Grb2-related, Src homology 3 (SH3) and SH2 domain-containing adapter protein whose expression is restricted to cells of hematopoietic lineage (i.e., monocytes and T lymphocytes). During monocyte/macrophage differentiation, Mona is induced and interacts with the macrophage colony-stimulating factor receptor, M-CSFR (also called Fms), suggesting that Mona could be involved in developmental signaling downstream of the M-CSFR by recruiting additional signaling proteins to the activated receptor. Our present results identify Mona as a specific partner protein for the DOS/Gab family member Gab3 in monocytic/macrophage development. Mona does not interact with Gab2; however, Gab3 also forms a complex with the Mona-related adapter Grb2. Glutathione S-transferase pull-down experiments demonstrate that the Mona and Gab3 interaction utilizes the carboxy-terminal SH3 domain of Mona and the atypical proline-rich domain of Gab3. Mona is known to interact with the phosphorylated Y697 site of the M-CSFR. The M-CSFR mutation Y697F exhibited qualitative and quantitative abnormalities in receptor and Gab3 tyrosine phosphorylation, and Mona induction was greatly reduced. The Y807F M-CSFR mutation is defective in differentiation signaling, but not growth signaling, and also fails to induce Mona protein expression. During M-CSF-stimulated macrophage differentiation of mouse bone marrow cells, Mona and Gab3 expression is coinduced, these proteins interact, and Mona engages in multimolecular complexes. These data suggest that association of Mona and Gab3 plays a specific role in mediating the M-CSFR differentiation signal.     

Altered function in atrium of transgenic mice overexpressing triadin 1

Triadin 1 is a protein in the cardiac junctional sarcoplasmic reticulum (SR) that interacts with the ryanodine receptor, junctin, and calsequestrin, proteins that are important for Ca(2+) release. To better understand the role of triadin 1 in SR-Ca(2+) release, we studied the time-dependent expression of SR proteins and contractility in atria of 3-, 6-, and 18-wk-old transgenic mice overexpressing canine cardiac triadin 1 under control of the alpha-myosin heavy chain (MHC) promoter. Three-week-old transgenic atria exhibited mild hypertrophy. Finally, atrial weight was increased by 110% in 18-wk-old transgenic mice. Triadin 1 overexpression was accompanied by time-dependent changes in the protein expression of the ryanodine receptor, junctin, and cardiac/slow-twitch muscle SR Ca(2+)-ATPase isoform. Force of contraction was already decreased in 3-wk-old transgenic atria. The application of caffeine led to a positive inotropic effect in transgenic atria of 3-wk-old mice. Rest pauses resulted in an increased potentiation of force of contraction after restimulation in 3- and 6-wk-old mice and a reduced potentiation of force of contraction in 18-wk-old transgenic mice. Hence, triadin 1 overexpression triggered time-dependent alterations in SR protein expression, Ca(2+) homeostasis, and contractility, indicating for the first time an inhibitory function of triadin 1 on SR-Ca(2+) release in vivo.