UV damage endonuclease employs a novel dual-dinucleotide flipping mechanism to recognize different DNA lesions

Repairing damaged DNA is essential for an organism’s survival. UV damage endonuclease (UVDE) is a DNA-repair enzyme that can recognize and incise different types of damaged DNA. We present the structure of Sulfolobus acidocaldarius UVDE on its own and in a pre-catalytic complex with UV-damaged DNA containing a 6-4 photoproduct showing a novel ‘dual dinucleotide flip’ mechanism for recognition of damaged dipyrimidines: the two purines opposite to the damaged pyrimidine bases are flipped into a dipurine-specific pocket, while the damaged bases are also flipped into another cleft.     

Enhancement of therapeutic protein in vivo activities through glycoengineering

Delivery of protein therapeutics often requires frequent injections because of low activity or rapid clearance, thereby placing a burden on patients and caregivers. Using glycoengineering, we have increased and prolonged the activity of proteins, thus allowing reduced frequency of administration. Glycosylation analogs with new N-linked glycosylation consensus sequences introduced into the protein were screened for the presence of additional N-linked carbohydrates and retention of in vitro activity. Suitable consensus sequences were combined in one molecule, resulting in glycosylation analogs of rHuEPO, leptin, and Mpl ligand. All three molecules had substantially increased in vivo activity and prolonged duration of action. Because these proteins were of three different classes (rHuEPO is an N-linked glycoprotein, Mpl ligand an O-linked glycoprotein, and leptin contains no carbohydrate), glycoengineering may be generally applicable as a strategy for increasing the in vivo activity and duration of action of proteins. This strategy has been validated clinically for glycoengineered rHuEPO (darbopoetin alfa).     

Crystal structure of the DNA repair enzyme ultraviolet damage endonuclease

The ultraviolet damage endonuclease (UVDE) performs the initial step in an alternative excision repair pathway of UV-induced DNA damage, nicking immediately adjacent to the 5' phosphate of the damaged nucleotides. Unique for a single-protein DNA repair endonuclease, it can detect different types of damage. Here we show that Thermus thermophilus UVDE shares some essential structural features with Endo IV, an enzyme from the base excision repair pathway that exclusively nicks at abasic sites. A comparison between the structures indicates how DNA is bound by UVDE, how UVDE may recognize damage, and which of its residues are involved in catalysis. Furthermore, the comparison suggests an elegant explanation of UVDE's potential to recognize different types of damage. Incision assays including point mutants of UVDE confirmed the relevance of these conclusions.     

Desmosomal cadherins utilize distinct kinesins for assembly into desmosomes

The desmosomal cadherins, desmogleins (Dsgs) and desmocollins (Dscs), comprise the adhesive core of intercellular junctions known as desmosomes. Although these adhesion molecules are known to be critical for tissue integrity, mechanisms that coordinate their trafficking into intercellular junctions to regulate their proper ratio and distribution are unknown. We demonstrate that Dsg2 and Dsc2 both exhibit microtubule-dependent transport in epithelial cells but use distinct motors to traffic to the plasma membrane. Functional interference with kinesin-1 blocked Dsg2 transport, resulting in the assembly of Dsg2-deficient junctions with minimal impact on distribution of Dsc2 or desmosomal plaque components. In contrast, inhibiting kinesin-2 prevented Dsc2 movement and decreased its plasma membrane accumulation without affecting Dsg2 trafficking. Either kinesin-1 or -2 deficiency weakened intercellular adhesion, despite the maintenance of adherens junctions and other desmosome components at the plasma membrane. Differential regulation of desmosomal cadherin transport could provide a mechanism to tailor adhesion strength during tissue morphogenesis and remodeling.     

<Emphasis Type="Italic">In situ</Emphasis> Quantification of Phytoplankton in Reservoirs Using a Submersible Spectrofluorometer

A submersible in situ spectrofluorometer, which permits the differentiation of four algal groups (green algae, diatoms, cryptophytes and cyanobacteria), was used for phytoplankton monitoring in five reservoirs with varying levels of eutrophication and composition of their phytoplankton communities. Data obtained in situ were compared to standard laboratory methods for phytoplankton quantification; concentration of chlorophyll a and microscopy analysis. A high correlation (r = 0.95, n = 96) between chlorophyll a levels using different methods was found in all types of phytoplankton community. Taxonomic analyses and cell counts were closely related to the ratio of algal classes measured by the in situ spectrofluorometer. The submersible device used in the study measures in a continuous mode, which is advantageous in comparison with discrete sampling. This method appears to be a good tool for water quality management and can be used in the detection of natural horizontal and vertical variability in phytoplankton communities or for the early detection of cyanobacterial blooms. The device used in this study is recommended as a screening tool that enables more effective sampling that can be focused on the localities and depths where changes in phytoplankton composition occur.     

Direct reprogramming of mouse and human fibroblasts into multipotent neural stem cells with a single factor

The generation of induced pluripotent stem cells (iPSCs) and induced neuronal cells (iNCs) from somatic cells provides new avenues for basic research and potential transplantation therapies for?neurological diseases. However, clinical applications must consider the risk of tumor formation by iPSCs and the inability of iNCs to self-renew in culture. Here we report the generation of induced neural stem cells (iNSCs) from mouse and human fibroblasts by direct reprogramming with a single factor, Sox2. iNSCs express NSC markers and resemble wild-type NSCs in their morphology, self-renewal, ability to form neurospheres, and gene expression profiles. Cloned iNSCs differentiate into several types of mature neurons, as well as astrocytes and oligodendrocytes, indicating multipotency. Implanted iNSCs can survive and integrate in mouse brains and, unlike iPSC-derived NSCs, do not generate tumors. Thus, self-renewable and multipotent iNSCs without tumorigenic potential can be generated directly from fibroblasts by reprogramming.     

Crystal structure of Bacillus stearothermophilus UvrA provides insight into ATP-modulated dimerization, UvrB interaction, and DNA binding

The nucleotide excision repair pathway corrects many structurally unrelated DNA lesions. Damage recognition in bacteria is performed by UvrA, a member of the ABC ATPase superfamily whose functional form is a dimer with four nucleotide-binding domains (NBDs), two per protomer. In the 3.2 A structure of UvrA from Bacillus stearothermophilus, we observe that the nucleotide-binding sites are formed in an intramolecular fashion and are not at the dimer interface as is typically found in other ABC ATPases. UvrA also harbors two unique domains; we show that one of these is required for interaction with UvrB, its partner in lesion recognition. In addition, UvrA contains three zinc modules, the number and ligand sphere of which differ from previously published models. Structural analysis, biochemical experiments, surface electrostatics, and sequence conservation form the basis for models of ATP-modulated dimerization, UvrA-UvrB interaction, and DNA binding during the search for lesions.     

Rapid target-specific remodeling of fast-spiking inhibitory circuits after loss of dopamine

In Parkinson's disease (PD), dopamine depletion alters neuronal activity in the direct and indirect pathways and leads to increased synchrony in the basal ganglia network. However, the origins of these?changes remain elusive. Because GABAergic interneurons regulate activity of projection neurons and?promote neuronal synchrony, we recorded from pairs of striatal fast-spiking (FS) interneurons and direct- or indirect-pathway MSNs after dopamine depletion with 6-OHDA. Synaptic properties of?FS-MSN connections remained similar, yet within 3?days of dopamine depletion, individual FS cells doubled their connectivity to indirect-pathway MSNs, whereas connections to direct-pathway MSNs remained unchanged. A model of the striatal microcircuit revealed that such increases in FS innervation were effective at enhancing synchrony within targeted cell populations. These data suggest that after dopamine depletion, rapid target-specific microcircuit organization in the striatum may lead to increased synchrony of indirect-pathway MSNs that contributes to pathological network oscillations and motor symptoms of PD.     

Effect of thirty days of creatine supplementation with phosphate salts on anaerobic working capacity and body weight in men

The purpose of this study was to determine the effect of 30 days of single-dose creatine supplementation with phosphate salts (CPS) on body weight (BW) and anaerobic working capacity (AWC) in men. Using a double-blind design, 32 men randomly received 1 serving of either CPS (5 g Cr + 4 g phosphate) (n = 17) or 20 g of dextrose as placebo (PL) (n = 15) for 30 days. AWC determined from the Critical Power Test and BW were measured at baseline, 10 days, 20 days, 30 days, and 10 days post-supplementation. Results (2 x 5 ANOVA) showed no significant differences between groups for AWC at any time point; however, BW was significantly increased at 10 days in the CPS group (1.0 kg) vs. PL (0.0 kg), and remained elevated for the duration of the study. These findings suggest that a single 5 g x d(-1) dose of CPS for 30 days increases BW but is not effective for increasing AWC in men.