Structure and regulation of the CDK5-p25(nck5a) complex

CDK5 plays an indispensable role in the central nervous system, and its deregulation is involved in neurodegeneration. We report the crystal structure of a complex between CDK5 and p25, a fragment of the p35 activator. Despite its partial structural similarity with the cyclins, p25 displays an unprecedented mechanism for the regulation of a cyclin-dependent kinase. p25 tethers the unphosphorylated T loop of CDK5 in the active conformation. Residue Ser159, equivalent to Thr160 on CDK2, contributes to the specificity of the CDK5-p35 interaction. Its substitution with threonine prevents p35 binding, while the presence of alanine affects neither binding nor kinase activity. Finally, we provide evidence that the CDK5-p25 complex employs a distinct mechanism from the phospho-CDK2-cyclin A complex to establish substrate specificity.     

A microcalorimetric study of the effect of La3+ on mitochondria isolated from Star-Cross 288 chicken liver tissue cells

The thermogenic curves of the metabolism of mitochondria isolated from the liver of chicken Star-Cross 288 and the effect of La³⁺ on it were studied by using an LKB-2277 BioActivity Monitor, ampoule method, at 37°C. After isolation from the chicken liver tissue, mitochondria still have metabolic activity and can live for a long time by using the stored nutrients. From the thermogenic curves, we obtained the thermokinetic equations under different condition. The kinetic study shows that La³⁺ has changed the metabolism completely.     

Concentrations and flux of rare earth elements in a semifield plot as influenced by their agricultural application

The concentrations of rare earth elements (REs) in atmospheric particles, soil, soil water, surface runoff, and different parts of corn in an experimental plot in the suburb of Beijing, China were measured and the flux of REs was estimated. The concentration of REs in air particles with diameter less than 10 μm is 36 ng/m³ and, by ratio analysis, the origin of REs in the atmosphere was likely local soil. The concentration of soluble REs is 0.69 μg/L in rainwater, 5–7 μg/L in surface runoff, as well as 1–4 μg/L in soil water and the application of RE mixture has no observed influences on their concentrations. The concentration of soluble REs in surface runoff and soil water was dominated by aqueous-solid-phase partitioning. By estimation of the flux, most of the REs applied will remain in the soil phase and continued application of REs will cause an accumulation of REs in agricultural soil.     

Impact of cell growth morphology on retroviral transduction: effect of contact inhibition

Retrovirus-mediated gene transfer is one of the most commonly used methods to deliver, integrate, and express the gene of interest because the retrovirus can insert the desired gene into the chromosome of the target cells with high stability. However, to deliver the gene successfully, the retrovirus requires active division to integrate reversely transcribed DNA into the chromosome of target cells. In this study, we focused on the effect of cell-cell contact inhibition on the efficiency of retroviral transduction with two anchorage-dependent cell lines: NIH 3T3 and 293 cells. These two cell lines have very different cell morphologies and growth patterns on surfaces. Human embryonic kidney epithelial 293 cells tend to stick together after dividing, while NIH 3T3 cells migrate to occupy available surface and spread. Experimental data indicate that the abatement of the transduction rate of 293 cells was initiated in the early stage of the culture, whereas effect of contact inhibition of NIH 3T3 cells on the transduction rate became dominating at the end of the culture period. Experimental results were also quantitatively illustrated by plotting normalized multiplicity of infection (MOI) versus normalized cell density. According to the outcomes, cell inoculation density plays an important role in optimizing the retroviral transduction rate. The optimal time of retroviral transduction should be confined to the accelerating growth phase for 293 cells and at the exponential growth phase for NIH 3T3 cells. The implication drawn from this study is that contact inhibition effect on retroviral transduction should be taken into account for large-scale gene transfer systems such as the microcarrier bioreactor.     

Effect of electroacupuncture on pressor reflex during gastric distension

The effect of electroacupuncture (EA) on the reflex cardiovascular response induced by mechanical distension of the stomach was studied in ventilated male Sprague-Dawley rats anesthetized by ketamine and alpha-chloralose. Repeated balloon inflation of the stomach to produce 20 mmHg tension on the gastric wall induced a consistent rise in mean arterial pressure, while heart rate (372 +/- 22 beats/min) was unchanged. This response was reversed by transection of the splanchnic nerves. Bilateral application of EA (1-2 mA, 2 Hz) at Neiguan-Jianshi acupoints (pericardial meridian, Pe 5-6) over the median nerve for 30 min significantly decreased the pressor response from 33 +/- 6 to 18 +/- 4 mmHg (n = 7, P < 0.05). This effect began after 10 min of EA and continued for 40 min after termination of EA. EA at Zusanli-Shangquxu acupoints (stomach meridian, St 36-37) over the deep peroneal nerve similarly inhibited the pressor response. The effect lasted for 10 min after EA was stopped (n = 6, P < 0.05), while EA at Guangming-Xuanzhong acupoints (gallbladder meridian, GB 37-39) over the superficial peroneal nerve did not inhibit the pressor response. Naloxone injected intravenously (n = 6) immediately after termination of EA or administered by microinjection into the rostral ventrolateral medulla (rVLM) 25 min after initiation of EA (n = 6) reversed the inhibition by EA, suggesting an opiate mechanism, including the rVLM, was involved.     

Neuroprotective role of delta-opioid receptors in cortical neurons

We recently demonstrated that delta-opioid receptor (DOR) activation protects cortical neurons against glutamate-induced injury. Because glutamate is a mediator of hypoxic injury in neurons, we hypothesized that DOR is involved in neuroprotection during O2 deprivation and that its activation/inhibition may alter neuronal susceptibility to hypoxic stress. In this work, we tested the effect of opioid receptor activation and inhibition on cultured cortical neurons in hypoxia (1% O2). Cell injury was assessed by lactate dehydrogenase release, morphology-based quantification, and live/dead staining. Our results show that 1) immature neurons (days 4 and 6) were not significantly injured by hypoxia until 72 h of exposure, whereas day 8 neurons were injured after only 24-h hypoxia; 2) DOR inhibition (naltrindole) caused neuronal injury in both day 4 and day 8 normoxic cultures and further augmented hypoxic injury in these neurons; 3) DOR activation ([D-Ala2,D-Leu5]enkephalin) reduced neuronal injury in day 8 cultures after 24 h of normoxic or hypoxic exposure and attenuated naltrindole-induced injury with prolonged exposure; and 4) mu- or kappa-opioid receptor inhibition (beta-funaltrexamine or nor-binaltorphimine) had little effect on neurons in either normoxic or hypoxic conditions. Collectively, these data suggest that DOR plays a crucial role in neuroprotection in normoxic and hypoxic environments.     

Unisex pheromone detectors and pheromone-binding proteins in scarab beetles

Olfaction was studied in two species of scarab beetle, Anomala octiescostata and Anomala cuprea (Coleoptera: Scarabaeidae: Rutelinae), which are temporarily isolated and use the same sex pheromone compounds, (R)-buibuilactone and (R)-japonilure. Single sensillum recordings in A. octiescostata revealed highly sensitive olfactory receptor neurons (ORNs) (threshold <1 pg) that were tuned to the detection of the green leaf volatile compound (Z)-3-hexenyl acetate. As opposed to similar ORNs in another scarab species, Phyllopertha diversa, in A. octiescostata a diazo analogue elicited much lower neuronal responses than the natural ligand. Detectors for other floral and leaf compounds were also characterized. Extremely stereoselective ORNs tuned to sex pheromone were identified in male and female antennae. Biochemical investigations showed that, in A. octiescostata and A. cuprea, the pheromone-binding proteins (PBPs) isolated from male antennae were identical to PBPs obtained from female antennae. AoctPBP and AcupPBP had seven different amino acid residues. Binding of AoctPBP to (R)-japonilure is shown. PdivOBP1, which is also known to bind to (R)-japonilure, differed from AcupPBP in only two amino acid residues, one at the N-terminus and the other near the C-terminus. The structural features of the Bombyx mori PBP are compared with the sequences of eight known scarab odorant-binding proteins.     

A decade of differential display

It has been 10 years since the invention of differential display (DD), a conceptually simple methodology that allows the detection and identification of differentially expressed genes. In the past decade, the number of publications describing successful applications of DD has outnumbered those using any other competing methodologies, including subtractive hybridization, representational difference analysis, serial analysis of gene expression, and DNA microarrays. This review will provide a glimpse of the current progress made in DD technological development, refinement, and automation. Excellent examples of DD applications in studying a variety of biological problems, in such diverse biological systems as bacteria, yeast, flies, plants, and higher mammals, are presented to provide a roadmap for those who would like to pursue a fruitful gene "fishing" expedition. Some of the fundamental differences between DD and DNA microarrays are also discussed.     

A neutral beta-D-glucan from dates of the date palm,Phoenix dactylifera L

Polysaccharides extracted from Libyan dates with hot water and 0.05 M NaOH were fractionated and purified by ion-exchange and gel-filtration chromatography. According to the methylation and hydrolysis analyses, the results indicate the D-glucan to be linear and to contain both (1-->3)- and (1-->4)-linkages. The anomeric NMR measurements confirm that the sugar residues are beta-glycosidically linked. This is the first report on the isolation of a neutral beta-D-glucan from dates.