A Single-Strand Annealing Protein Clamps DNA to Detect and Secure Homology

Repair of DNA breaks by single-strand annealing (SSA) is a major mechanism for the maintenance of genomic integrity. SSA is promoted by proteins (single-strand-annealing proteins [SSAPs]), such as eukaryotic RAD52 and λ phage Redβ. These proteins use a short single-stranded region to find sequence identity and initiate homologous recombination. However, it is unclear how SSAPs detect homology and catalyze annealing. Using single-molecule experiments, we provide evidence that homology is recognized by Redβ monomers that weakly hold single DNA strands together. Once annealing begins, dimerization of Redβ clamps the double-stranded region and nucleates nucleoprotein filament growth. In this manner, DNA clamping ensures and secures a successful detection for DNA sequence homology. The clamp is characterized by a structural change of Redβ and a remarkable stability against force up to 200 pN. Our findings not only present a detailed explanation for SSAP action but also identify the DNA clamp as a very stable, noncovalent, DNA–protein interaction.     

Activation of human T cells with the physiological regulator of protein kinase C

We investigated whether sn-1,2-dioctanoylglycerol (diC8) activates highly purified human T cells. diC8's signaling activity was also compared with that of 12-O-tetradecanoylphorbol-13-acetate (TPA). diC8 and ionomycin were synergistic in promoting T-cell proliferation. The proliferative response was dependent upon an operational interleukin-2 (IL-2) system and exhibited a high degree of specificity; sn-1,2-diC8 was twice as active as racemic-1,2-diC8, and diC8 and TPA were not synergistic. diC8's signaling activity differed from that of TPA. diC8, unlike TPA, failed to elicit IL-2 receptors or proliferation, independently of ionomycin. diC8 also failed to promote the proliferation of T cells signaled with anti-CD3 or -CD2 monoclonal antibodies. Two different inhibitors of PKC, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine or staurosporine, inhibited T-cell proliferation induced with diC8 and ionomycin, but not with TPA and ionomycin. These observations, in addition to demonstrating the differential activity of diC8 and TPA, document a signaling role for diacylglycerol in the activation of normal T cells.     

Factors influencing the egg production of Ascaris lumbricoides: relationship to weight, length and diameter of worms

Fifty children aged 6 to 13 years and infected with Ascaris lumbricoides were selected for the study. The number of eggs laid daily by a female Ascaris increased with increase in its length, weight and diameter. Female worms became mature and started laying eggs when they reached a length of 118 mm. Adult female worms measuring 3.7 mm or more in diameter were found to be mature. The minimum weight of a worm producing eggs was 1.1 g. On average the number of eggs produced by the female decreased with increase in the worm load.     

In vitro identification and characterisation of iron chelating catechol-containing natural products and derivatives

BioMetals - Iron is an essential component for multiple biological processes. Its regulation within the body is thus tightly controlled. Dysregulation of iron levels within the body can result in...     

Cryo-electron microscopy structures of the N501Y SARS-CoV-2 spike protein in complex with ACE2 and 2 potent neutralizing antibodies

The recently reported “UK variant” (B.1.1.7) of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-Å resolution cryo-electron microscopy (cryo-EM) structure of the complex between the ACE2 receptor and N501Y spike protein ectodomains that shows Y501 inserted into a cavity at the binding interface near Y41 of ACE2. This additional interaction provides a structural explanation for the increased ACE2 affinity of the N501Y mutant, and likely contributes to its increased infectivity. However, this mutation does not result in large structural changes, enabling important neutralization epitopes to be retained in the spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to 2 representative potent neutralizing antibody fragments.     

Evidence for Compromised Insulin Signaling and Neuronal Vulnerability in Experimental Model of Sporadic Alzheimer’s Disease

Evidence from animal studies categorizes sporadic Alzheimer’s disease (sAD) as a metabolic syndrome with accompanying cognitive deficits. Given that glial cells act as “silent partners” to neurons by providing trophic support and defense, the present study investigated the role of glia in sAD pathology. A streptozotocin (STZ)-induced glial-neuronal co-culture model of sAD was used to study the metabolic status of the two cell types. Real time RT-PCR and Western blotting results indicated that amyloid precursor protein (APP) and β-secretase (BACE1) were highly expressed in co-cultured neurons than in monocultures. Increased amyloidogenesis was accompanied by decreased expression of mediators in insulin signaling pathway that included insulin receptor (IR), insulin receptor substrate 2 (IRS2), insulin-like growth factor 2 (IGF2), insulin-like growth factor 1 receptor (IGF1R), total-glycogen synthase kinase 3β (t-GSK3β), and phosphorylated-GSK3β^ser9 (p-GSK3β^ser9), suggesting that neuronal cells are more prone to metabolic variability when cultured in the presence of glial cells. Findings from the sAD model induced by intracerebroventricular (ICV) injection of STZ revealed that increased amyloid beta (Aβ) load in the hippocampus was potentially responsible for the hyperphosphorylation of tau at ser³⁹⁶. Furthermore, impaired cognitive functions and decreased dendritic spine density and axonal thinning in CA1 region of hippocampus were associated with decreased IR and p-GSK3β^ser9/t-GSK3β expression. Taken together, the present study provides evidence that glia mediated response and insulin signaling defects drive pathological changes in sAD and represent potential targets for delaying sAD progression.     

Knowledge-based interaction potentials for proteins.

We discuss the derivation of atomic-level potentials of mean force from the known protein structures and their applicability for structural evaluation applications. In the derivation process, rigorous density estimation methodology is used to estimate the probability density functions (PDFs) for the distributions of interatomic distances in the protein structures. Potentials of mean force are then derived from these density functions using simple Boltzmann's relation. We also test the potentials against pairs of current and superseded protein structures in the Protein Data Bank. Using PDF potentials to evaluate each structure pair, we are able to identify, with high accuracy, which of the two structures is of higher resolution or better quality. This result shows that the PDF potentials are sensitive to details in protein structures as the current and superseded atomic coordinates generally do not differ by more than 1 A in root-mean-square deviation, and that the PDF potentials could potentially be used for X-ray structure refinement and protein structure prediction.     

Apical cell surface expression of rat dipeptidyl peptidase IV in transfected Madin-Darby canine kidney cells.

Dipeptidyl peptidase IV (DPPIV) is a type II membrane glycoprotein that is predominantly localized to the apical plasma membrane in various epithelial cells. In order to understand in more detail the biogenesis and sorting of DPPIV, the cDNA for rat DPPIV was inserted into a mammalian plasmid expression vector so that DPPIV expression was driven by a control region composed of the SV40 early promoter region fused to the enhancer of the Rous sarcoma virus. Madin-Darby canine kidney cells transfected with this construct were found to express the DPPIV protein. In these transfected cells, the majority of DPPIV was present on the apial cell surface. This observation suggests that the information for apical surface localization is inherent in the DPPIV molecule itself and that this sorting information is decipherable in the epithelial cells of a different species. DPPIV is transported efficiently from the endoplasmic reticulum to the Golgi apparatus as assessed by pulse-chase experiments. Furthermore, evidence is presented which suggests that the majority of DPPIV is sorted intracellularly to the apical cell surface. The same protein has, however, been reported to be sorted by an indirect pathway through transcytosis from the basolateral to the apical cell surface in hepatocytes (Bartles, J.R., Feracci, H., M., Stinger, B., and Hubbard, A.L. (1987) J. Cell Biol. 105, 1241-1251). This study suggests that the same protein can take two different pathways in different cell types for its correct apical cell surface localization.