Ownership of Automobile as Prima Facie Evidence of Responsibility for Negligence of Person Operating It

Generally speaking, absent statutory change, an owner of an automobile is responsible for injuries resulting from its negligent operation by another, only if it is shown that, at the time of the injury, the relationship of principal and agent or master and servant existed between the owner and the operator, and that the operator was then acting in the scope of his employment. Ordinary human experience and knowledge show clearly that in the great majority of cases automobiles are operated by their owners or by some servant or agent on the owner\u27s business. It is equally apparent that in the cases where this is false, the knowledge of the facts and the ability to show them are peculiarly in the cognizance of the owner. Understanding this, the great majority of the courts of the United States recognixe that it is desirable and fair to aid the plaintiff in establishing his case by giving him the assistance of some sort of a presumption of the agency of the driver and that he was in the scope of his employment-either from ownership of the automobile alone, or from ownership plus some other related fact.Generally speaking, absent statutory change, an owner of an automobile is responsible for injuries resulting from its negligent operation by another, only if it is shown that, at the time of the injury, the relationship of principal and agent or master and servant existed between the owner and the operator, and that the operator was then acting in the scope of his employment. Ordinary human experience and knowledge show clearly that in the great majority of cases automobiles are operated by their owners or by some servant or agent on the owner\u27s business. It is equally apparent that in the cases where this is false, the knowledge of the facts and the ability to show them are peculiarly in the cognizance of the owner. Understanding this, the great majority of the courts of the United States recognize that it is desirable and fair to aid the plaintiff in establishing his case by giving him the assistance of some sort of a presumption of the agency of the driver and that he was in the scope of his employment-either from ownership of the automobile alone, or from ownership plus some other related fact

Determination of the Structural Allosteric Inhibitory Mechanism of Dihydrodipicolinate Synthase

Dihydrodipicolinate Synthase (EC 4.3.3.7; DHDPS), the product of the dapA gene, is an enzyme that catalyzes the condensation of pyruvate and S-aspartate-β-semialdehyde (ASA) into dihydrodipicolinate via an unstable heterocyclic intermediate, (4S)-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. DHDPS catalyzes the first committed step in the biosynthesis of ʟ-lysine and meso-diaminopimelate; each of which is a necessary cross-linking component between peptidoglycan heteropolysacharide chains of bacterial cell walls. Therefore, strong inhibition of DHDPS would result in disruption of meso-diaminopimelate and ʟ-lysine biosynthesis in bacteria leading to decreased bacterial growth and cell lysis. Much attention has been given to targeting the active site for inhibition; however DHDPS is subject to natural feedback inhibition by ʟ-lysine at an allosteric site. In DHDPS from Campylobacter jejuni ʟ-lysine is known to act as a partial uncompetitive inhibitor with respect to pyruvate and a partial mixed inhibitor with respect to ASA. Little is known about how the protein structure facilitates the natural inhibition mechanism and mode of allosteric signal transduction. This work presents ten high resolution crystal structures of Cj-DHDPS and the mutant Y110F-DHDPS with various substrates and inhibitors, including the first reported structure of DHDPS with ASA bound to the active site. As a body of work these structures reveal residues and conformational changes which contribute to the inhibition of the enzyme. Understanding these structure function relationships will be valuable for the design of future antibiotic lead compounds. When an inhibitor binds to the allosteric site there is meaningful shrinkage in the solvent accessible volume between 33% and 49% proportional to the strength of inhibition. Meanwhile at the active site the solvent accessible volume increases between 5% and 35% proportional to the strength of inhibition. Furthermore, inhibitor binding at the allosteric site consistently alters the distance between hydroxyls of the catalytic triad (Y137-T47-Y111') which is likely to affect local pKa's. Changes in active site volume and modification of the catalytic triad would inhibit the enzyme during the binding and condensation of ASA. The residues H56, E88, R60 form a network of hydrogen bonds to close the allosteric site around the inhibitor and act as a lid. Comparison of ʟ-lysine and bislysine bound to wt-DHDPS and Y110F-DHDPS indicates that enhanced inhibition of bislysine is most likely due to increased binding strength rather than altering the mechanism of inhibition. When ASA binds to the active site the network of hydrogen bonds among H56, E88 and R60 is disrupted and the solvent accessible volume of the allosteric site expands by 46%. This observation provides some explanation for the reduced affinity of ʟ-lysine in high ASA concentrations. ʟ-Lysine, but not other inhibitors, is found to induce dynamic domain movements in the wt-DHDPS. These domain movements do not appear to be essential to the inhibition of the enzyme but may play a role in cooperativity between monomers or governing protein dynamics. The moving domain connects the allosteric site to the dimer-dimer interface. Several residues at the weak dimer interface have been identified as potentially involved in dimer-dimer communication including: I172, D173, V176, I194, Y196, S200, N201, K234, D238, Y241, N242 and K245. These residues are not among any previously identified as important for formation of the quaternary structure

The nature of cell-cycle checkpoints: facts and fallacies

The concept of checkpoint controls revolutionized our understanding of the cell cycle. Here we revisit the defining features of checkpoints and argue that failure to properly appreciate the concept is leading to misinterpretation of experimental results. We illustrate, using the mitotic checkpoint, problems that can arise from a failure to respect strict definitions and precise terminology

Microtubule disassembly delays the G2–M transition in vertebrates

AbstractWhen cell cultures in growth are treated with drugs that cause microtubules to disassemble, the mitotic index (MI) progressively increases as the cells accumulate in a C-mitosis. For many cell types, however, including rat kangaroo kidney PtK1 cells, the MI does not increase during the first several hours of treatment [1–3] (Figure 1). This ‘lag’ implies either that cells are entering mitosis but rapidly escaping the block, or that they are delayed from entering division. To differentiate between these possibilities, we fixed PtK1 cultures 0, 90 and 270 minutes after treatment with nocodazole, colcemid, lumi-colcemid, taxol or cytochalasin D. After 90 minutes, we found that the numbers of prophase cells in cultures treated with nocodazole or colcemid were reduced by ∼80% relative to cultures treated with lumi-colcemid, cytochalasin D or taxol. Thus, destroying microtubules delays late G2 cells from entering prophase and, as the MI does not increase during this time, existing prophase cells do not enter prometaphase. When mid-prophase cells were treated with nocodazole, the majority (70%) decondensed their chromosomes and returned to G2 before re-entering and completing prophase 3–10 hours later. Thus, a pathway exists in vertebrates that delays the G2–M transition when microtubules are disassembled during the terminal stages of G2. As this pathway induces mid-prophase cells to transiently decondense their chromosomes, it is likely that it downregulates the cyclin A–cyclin-dependent kinase 2 (CDK2) complex, which is required in vertebrates for the early stages of prophase [4]