Genetic analysis of chloroplast c-type cytochrome assembly in Chlamydomonas reinhardtii: One chloroplast locus and at least four nuclear loci are required for heme attachment

Chloroplasts contain up to two c-type cytochromes, membrane-anchored cytochrome f and soluble cytochrome c6. To elucidate the post-translational events required for their assembly, acetate-requiring mutants of Chlamydomonas reinhardtii that have combined deficiencies in both plastid-encoded cytochrome f and nucleus-encoded cytochrome c6 have been identified and analyzed. For strains ct34 and ct59, where the phenotype displays uniparental inheritance, the mutations were localized to the chloroplast ccsA gene, which was shown previously to be required for heme attachment to chloroplast apocytochromes. The mutations in another eight strains were localized to the nuclear genome. Complementation tests of these strains plus three previously identified strains of the same phenotype (ac206, F18, and F2D8) indicate that the 11 ccs strains define four nuclear loci, CCS1-CCS4. We conclude that the products of the CCS1-CCS4 loci are not required for translocation or processing of the preproteins but, like CcsA, they are required for the heme attachment step during assembly of both holocytochrome f and holocytochrome c6. The ccsA gene is transcribed in each of the nuclear mutants, but its protein product is absent in ccs1 mutants, and it appears to be degradation susceptible in ccs3 and ccs4 strains. We suggest that Ccsl may be associated with CcsA in a multisubunit "holocytochrome c assembly complex," and we hypothesize that the products of the other CCS loci may correspond to other subunits.     

A neural network-based methodology for machining knowledge acquisition

The paper presents results of a study on collecting machining strategies for machining assistants and process planning. These efforts are being conducted at the NMSU-Integrated Manufacturing Systems Laboratory (IMSL). Goals of the project aim at improving and advancing the solicitation, documentation, and automation of machining knowledge/data acquisition, and integration with CAD/CAM/CAE systems. This paper emphasizes the knowledge acquisition phase of the study utilizing artificial neural networks.     

The silane interphase of composites: Effects of process conditions on γ-aminopropyltriethoxysilane

FT-IR spectroscopy was used to collect spectra of γ-aminopropyltriethoxy silane (γ-APS) coupling agent deposited on KBr plates, modeling the silane interphase of composites, to study the effects of heating the γ-APS film under various environmental conditions. This coupling agent is used in fiber-reinforced epoxy composites. An aminebicarbonate salt forms when the sample is dried in a moist carbon dioxide environment giving rise to a series of bands from 2800 to 2000 centimeter⁻¹ (cm⁻¹) and bands at 1637 cm⁻¹, 1330 cm⁻¹, 696 cm⁻¹, and 663 cm⁻¹. The primary amine group is split into two bands at 1572 cm⁻¹ and 1486 cm⁻¹ in the NH₃⁺ form. Heating removes the aminebicarbonate salt at temperatures above 95°C and condenses the γ-APS polymer to a polysilsesquioxane film. The aminebicarbonate salt partially reforms if the γ-APS film is stored in a moist CO₂ environment. At temperatures above 120°C the primary amine of γ-APS is oxidized in air to imine groups. In the process of evolving the CO₂ the polymer is further condensed and the amine groups can be converted to imines if the temperatures exceeds 120°C. Both processes reduce the reactivity of the coupling agent with the epoxy resin. The structure of the silane interphase must be controlled during the processing of the composite to yield materials with maximum properties.     

Ontogeny of gonadotropin, testosterone, and inhibin secretion in normal boys through puberty based on overnight serial sampling

To investigate hormonal changes occurring in male puberty, we measured LH, FSH, testosterone, and alpha-inhibin immunoactivity in serum samples drawn every 10 min for 8 h (2100-0500 h) from each of 50 normal prepubertal and pubertal boys, aged 8.4-18.8 yr. We measured gonadotropins with ultrasensitive immunofluorometric assays, and testosterone and alpha-inhibin with RIAs. Unlike previous studies, which indexed pubertal development with Tanner stages, we used testicular volume, a more finely graduated indicator of development, to reveal patterns that were obscured when subjects were grouped by Tanner stage. The overnight mean concentration of each hormone increased with testis volume, but the rate of increase on a logarithmic scale slowed as testes grew. Log LH rose precipitously in the late prepubertal and early pubertal periods and plateaued during mid- and late puberty. Based on fitted regression curves, LH increased about 20-fold (from 0.11 IU/L) between testis volumes of 1 and 10 mL, but only an additional 1.5-fold by 30 mL. The developmental trajectory of log testosterone was like that of log LH, but rose less steeply early in puberty. From 0.14 micrograms/L at a testis volume of 1 mL, testosterone increased about 8.5-fold by 10 mL and an additional 3-fold by 30 mL. In contrast, logarithms of overnight mean FSH and alpha-inhibin concentrations rose at a more nearly constant rate throughout puberty. From 0.62 IU/L at a testis volume of 1 mL, the FSH concentration doubled by 10 mL and increased an additional 1.7-fold by 30 mL. From 270 ng/L at a testis volume of 1 mL, inhibin increased 1.5-fold by 10 mL and an additional 1.3-fold by 30 mL. Overnight pulse amplitudes exhibited developmental trajectories similar to those of the corresponding overnight mean concentrations. The number of LH and testosterone pulses during the sampling period averaged 2.2 and 2.1, respectively, at Tanner stage 1 and increased to 4.5 and 3.2, respectively, at Tanner stage 5. The number of FSH and inhibin pulses remained constant throughout puberty, averaging 3.3 and 3.5, respectively. Pairwise correlations among hormone concentrations were strong, reflecting common increasing trends through puberty; however, after accounting for developmental trends, FSH, LH, and testosterone concentrations remained correlated, whereas inhibin was uncorrelated with each of the other three hormones. Measuring gonadotropins with ultrasensitive assays and analyzing the results on a logarithmic scale as a function of testis volume made clear the dramatic hormonal changes that begin before the clinical changes of puberty.     

A framework for extending computer aided process planning to include business activities and computer aided design and manufacturing (CAD/CAM) data retrieval

Computer aided process planning (CAPP) systems have had limited success in integrating business functions and product manufacturing due to the inaccessibility and incompatibility of information residing in proprietary software. While large companies have developed or purchased complex order management and engineering applications, smaller manufacturers continue to use semi-automated and manual methods for managing information throughout the lifecycle of each new product and component. There is a need for reconfigurable and reprogrammable systems that combine advances in computer aided design (CAD/Computer Aided Manufacturing (CAM) technology and intelligent machining with product data management for documentation and cost control. The goal of this research is to demonstrate an architecture in which customer service, CAPP and a costing methodology known as activity based costing (ABC) are incorporated into a single system, thereby allowing companies to monitor and study how expenditures are incurred and which resources are being used by each job. The material presented in this paper is the result of a two year university and industry sponsored research project in which professors and students at the Costa Rica Institute of Technology developed a software application for FEMA Industrial S.A., a local machining and fabrication shop with sixty five employees and both conventional and CNC capabilities. The final results represent not only a significant contribution to local industry and to the students’ education but, also to the continuing growth of CAPP. Implementing better decision making tools and standardizing transactions in digital format would reduce the workload on critical personnel and archive valuable knowledge for analyzing company methods and expertise.     

Macro-Calibration in Sensor/Actuator Networks

We describe an ad-hoc localization system for sensor networks and explain why traditional calibration methods are inadequate for this system. Building upon previous work, we frame calibration as a parameter estimation problem; we parameterize each device and choose the values of those parameters that optimize the overall system performance. This method reduces our average error from 74.6% without calibration to 10.1%. We propose ways to expand this technique to a method of autocalibration for localization as well as to other sensor network applications.     

Serine 257 phosphorylation regulates association of polyomavirus middle T antigen with 14-3-3 proteins

Polyomavirus middle T antigen (MT) is phosphorylated on serine residues. Partial proteolytic mapping and Edman degradation identified serine 257 as a major site of phosphorylation. This was confirmed by site-directed mutagenesis. Isoelectric focusing of immunoprecipitated MT from transfected 293T cells showed that phosphorylation on wild-type MT occurred at near molar stoichiometry at S257. MT was previously shown to be associated with 14-3-3 proteins, which have been connected to cell cycle regulation and signaling. The association of 14-3-3 proteins with MT depended on the serine 257 phosphorylation site. This has been demonstrated by comparing wild-type and S257A mutant MTs expressed with transfected 293T cells or with Sf9 cells infected with recombinant baculoviruses. The 257 site is not critical for transformation of fibroblasts in vitro, since S257A and S257C mutant MTs retained the ability to form foci or colonies in agar. The tumor profile of a virus expressing S257C MT showed a striking deficiency in the induction of salivary gland tumors. The basis for this defect is uncertain. However, differences in activity for the wild type and mutant MT lacking the 14-3-3 binding site have been observed in transient reporter assays.