Purification and characterization of a surface-associated carotenoid-binding complex from the photosynthetic prokaryote,Prochlorothrix hollandica

We have identified a water-soluble surface-associated complex from Prochlorothrix hollandica, composed of two polypeptides of 56 and 58 kilodaltons (kDa), zeaxanthin, and lipopolysaccharide. The complex was purified by preparative isoelectric focusing (pI=3.0). The outer membrane lipopolysaccharide co-purified with the complex. Immunocytochemisty employing a polyclonal antibody to the apoproteins exclusively labeled the cell surface. Both zeaxanthin and the protein accumulated under high light intensities, thus we propose that the complex may play a role in photoprotection.     

Determining the optimal system-specific cut-off frequencies for filtering in-vitro upper extremity impact force and acceleration data by residual analysis

The fundamental nature of impact testing requires a cautious approach to signal processing, to minimize noise while preserving important signal information. However, few recommendations exist regarding the most suitable filter frequency cut-offs to achieve these goals. Therefore, the purpose of this investigation is twofold: to illustrate how residual analysis can be utilized to quantify optimal system-specific filter cut-off frequencies for force, moment, and acceleration data resulting from in-vitro upper extremity impacts, and to show how optimal cut-off frequencies can vary based on impact condition intensity. Eight human cadaver radii specimens were impacted with a pneumatic impact testing device at impact energies that increased from 20J, in 10J increments, until fracture occurred. The optimal filter cut-off frequency for pre-fracture and fracture trials was determined with a residual analysis performed on all force and acceleration waveforms. Force and acceleration data were filtered with a dual pass, 4th order Butterworth filter at each of 14 different cut-off values ranging from 60Hz to 1500Hz. Mean (SD) pre-fracture and fracture optimal cut-off frequencies for the force variables were 605.8 (82.7)Hz and 513.9 (79.5)Hz, respectively. Differences in the optimal cut-off frequency were also found between signals (e.g. Fx (medial-lateral), Fy (superior-inferior), Fz (anterior-posterior)) within the same test. These optimal cut-off frequencies do not universally agree with the recommendations of filtering all upper extremity impact data using a cut-off frequency of 600Hz. This highlights the importance of quantifying the filter frequency cut-offs specific to the instrumentation and experimental set-up. Improper digital filtering may lead to erroneous results and a lack of standardized approaches makes it difficult to compare findings of in-vitro dynamic testing between laboratories.     

Novel yeast bioassay for high-throughput screening of matrix metalloproteinase inhibitors

Diverse malfunctions in the expression and regulation of matrix metalloproteinases (MMPs) are often the cause of severe human diseases, bringing the identification of specific MMP inhibitors into major focus, particularly in anticancer treatment. Here, we describe a novel bioassay based on recombinant yeast cells (Pichia pastoris) that express, deliver, and incorporate biologically active human MMP-2 and MMP-9 at the yeast cell surface. Using Sed1p for cell wall targeting and covalent anchorage, a highly efficient bioassay was established that allows high-throughput screening and subsequent validation of novel MMP inhibitors as potential anticancer drugs. In addition, we developed a straightforward synthesis of a new aspartate-derived MMP inhibitor active in the nM range and bearing an amino functionality that should allow the introduction of a wide range of side chains to modify the properties of these compounds.     

Biological characterization of A-ring steroids

Hydroxylated 2,19-methylene-bridged androstenediones were designed as potential mimics of enzyme oxidized intermediates of androstenedione. These compounds exhibited competitive inhibition with low micromolar affinities for aromatase. These inhibitory constants (K_i values) were 10 times greater than the 2,19-methylene-bridged androstenedione constant (K_i = 35–70 nM). However, expansion of the 2,19-carbon bridge to ethylene increased aromatase affinity by 10-fold (K_i = 2 nM). Substitution pf a methylene group with oxygen and sulfur in this expanded bridge resulted in K_i values of 7 and 20 nM, respectively. When the substituent was an NH group, the apparent inhibitory kinetics changed from competitive to uncompetitive. All of these analogs exhibited time-dependent inhibition of aromatase activity following preincubation of the inhibitor with human placental microsomes prior to measuring residual enzyme activity. Part of this inhibition was NADPH cofactor-dependent for the 2,19-methyleneoxy- but not for the 2,19-ethylene-bridged androstenedione. The time-dependent inhibition for these four analogs was very rapid since they exhibited τ₅₀ values, the t_1/2 for enzyme inhibition at infinite inhibitor concentration, of 1 to 3 min. These A-ring-bridged androstenedione analogs represent a novel series of potent steroidal aromatase inhibitors. The restrained A-ring bridge containing CH₂, O, S, or NH could effectively coordinate with the heme of the P450 aromatase to allow the tight-binding affinities reflected by their nanomolar K_i values.     

Mating Type Linked Mutations Which Disrupt the Uniparental Transmission of Chloroplast Genes in Chlamydomonas

In Chlamydomonas reinhardtii, chloroplast genomes are normally transmitted by the mating type plus (mt+) parent and mitochondrial genomes by the mating type minus (mt-) parent. In this paper we describe three new nuclear mutations, designated mat-3-1 to -3, which are tightly linked to the mt+ allele and permit high transmission of chloroplast genomes from the mt- parent, but have no effect on transmission of mitochondrial genomes. We also show that mat-1, reported by others to be a nuclear mutation linked to mt- which promotes transmission of chloroplast genomes by the mt- parent, is probably a vegetative diploid since it contains both mt+ and mt- alleles. Vegetative diploids behave as if they are mt- with respect to mating, but possess a level of chloroplast gene transmission intermediate between that of haploid mt- and mt+ stocks.     

Kinematics,kinetics and muscle activation patterns of the upper extremity during simulated forward falls

The purpose of this study was to explore the effects of fall type and fall height on the kinematics, kinetics, and muscle activation of the upper extremity during simulated forward falls using a novel fall simulation method.Twenty participants were released in a prone position from a Propelled Upper Limb Fall ARrest Impact System. Impacts occurred to the hands from two fall heights (0.05 m and 0.10 m) and three fall types (straight-arm, bent-arm, self-selected). Muscle activation from six muscles (biceps brachii, brachioradialis, triceps brachii, anconeus, flexor carpi radialis and extensor carpi radialis) was collected and upper extremity joint kinematics were calculated.Peak Fx (medio-lateral), as well as Fx and Fz (inferior–superior) load rate increased between the 0.05 m and 0.10 m heights. With respect to fall type, the straight-arm falls resulted in significantly greater Fy (anterior–posterior) impulse and Fy and Fz load rates. The change in elbow flexion angle was greater during the self-selected and bent-arm falls compared to the straight-arm falls; a pattern also seen in the wrist flexion/extension angles. All muscles experienced peak % MVIC prior to the time of the peak force.The results of this study suggest that, to some extent, individuals are capable of selecting an upper extremity posture that allows them to minimize the effects of an impact and it has confirmed the presence of a preparatory muscle activation response.