Parameters affecting the use of the lac repressor system in eukaryotic cells and transgenic animals

Elements of the lactose operon were used to study parameters affecting gene expression in cultured cells and transgenic animals. A Lac repressor protein containing a nuclear transport signal was shown to inhibit expression of a reporter gene by interacting with lac operator sequences. In cultured cells, operator sequence, operator placement and induction parameters were all shown to be important for obtaining tight repression of a reporter gene followed by high level expression upon induction. Induction levels were also dependent on the reporter gene, with the luciferase gene yielding higher induction levels than the chloramphenicol acetyltransferase gene. In transgenic animals, the lacI mRNA was not detected in the C57BL/6 mouse strain until the animal was exposed to a demethylating agent. After 5-azacytidine treatment, expression of lacI mRNA was detected in the brain, heart, kidney, lung and ovary. In the FVB transgenic mouse strain, expression of lacI mRNA was detected without 5-azacytidine treatment in the kidney, liver, lung, and testes. Preliminary experiments with double transgenic animals containing both lacI and operator/luciferase transgenes showed a decrease in luciferase expression compared to the luciferase-only animals in both tissue extracts and transgenic fetal primary cultures, although IPTG induction was not achieved in these animals or primary cultures. The applicability and challenges of the system for regulation of gene expression are discussed.     

Biochemical identification of transmembrane segments of the Ca(2+)-ATPase of sarcoplasmic reticulum

The transmembrane segments of sarcoplasmic reticulum Ca(2+)-ATPase were determined by trypsinization of cytoplasmic side-out intact sarcoplasmic reticulum vesicles. The membrane portion of tryptic digest comprising the transmembrane fragments, joined by the intravesicular segments, was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after labeling with fluorescein 5-maleimide in the presence of sodium dodecyl sulfate. In this way, seven fluorescent bands of tryptic fragments below 11 kDa were observed which were derived from 4 pairs of membrane spanning segments and one hydrophobic sequence at the C-terminal end. Two peptides of 10.8 and 10.6 kDa had the identical N-terminal sequence beginning at Glu826, representing the transmembrane segments M7 and M8 and their connecting loop. A band at 8.1 kDa contained one peptide beginning at Tyr36 (M1/loop/M2). A 7.7-kDa peptide starting at Leu253 (M3/loop/M4) and a 7.3-kDa peptide beginning at Ala752 (M5/loop/M6) were also observed. A band at 6.7 kDa contained two peptides, one beginning at Ser48 (M1/loop/M2) and another beginning at Tyr763 (M5/loop/M6). In addition, a 4-kDa peptide beginning at Met925 was observed. The size of this peptide did not allow for a complete pair of transmembrane segments, but this peptide could have been derived from trypsinolysis between the last pair of membrane spanning segments. These data therefore provide biochemical evidence for at least 8 transmembrane segments and perhaps two more at the C-terminal end of the enzyme.     

Ubiquitin expression in inclusion body myositis. An immunohistochemical study

Ubiquitin has been shown by immunohistochemical studies to be a component of many of the filamentous inclusion bodies that are known in neuropathology. In the current study, we examined the expression of ubiquitin in 14 cases of typical inclusion body myositis, in skeletal muscle specimens from four cases of typical amyotrophic lateral sclerosis, and in muscle specimens from three normal controls. In the cases of inclusion body myositis, rimmed vacuoles were ubiquitin immunoreactive in all cases. Intrasarcoplasmic inclusions were positive in the nine cases that had them. In four cases, there were positive intranuclear inclusions, and in seven, there was homogeneous staining of nuclei. Atrophic fibers and necrotic fibers were positive in 11 and nine cases, respectively. In the cases of amyotrophic lateral sclerosis, atrophic fibers were positive in three cases, and focal nuclear staining was seen in two. In one of the three control cases, a few atrophic fibers had faint sarcoplasmic positivity; no other staining was seen. We conclude that ubiquitin is a component of the inclusions that characterize inclusion body myositis. However, ubiquitin expression in skeletal muscle disease is not pathognomonic of inclusion body myositis.     

Predictive equation for acquisition of hepatitis B in hospital workers in a general hospital

A cross-sectional study was performed to obtain risk factors for hepatitis B disease, HBsAg carriers and immunised personnel, among 2470 workers in a general hospital in Madrid, Spain. The data obtained were analyzed with multiple logistic regression to obtain beta coefficients for variables. The results of the analysis show that being a nurse or being regularly exposed to blood are the most important risk factors for hepatitis B acquisition. The length of time working at the same job activity was also a risk factor. The resulting beta coefficients allow the construction for a hepatitis non-immunised, HBsAg carrier and immunised HBV status, which can select subjects for a hepatitis B vaccination program.     

Processing of Yttria-Alumina Coated Silicon Nitride Slips by Slip Casting

Si₃N₄powders coated with 6 wt% Y₂O₃and 4 wt% Al₂O₃were prepared by coprecipitation. The resulting powders were dispersed in water at different pH values and with addition of various amounts of ammonium polyacrylate (NH₄PA) to produce 32 vol% slips. The influence of the amount of NH₄PA solution added and pH on the rheological properties of 32 vol% coated Si₃N₄slips were studied. In addition, the sintered density of cast samples was determined and related to the degree of slip dispersion. The adsorption of the NH₄PA on the coated particle surface was rather high and the surface became saturated near 0.86 mg/m²at pH 9.2. High NH₄PA concentrations (1.7–3 wt%) were necessary to obtain well dispersed 32 vol% coated Si₃N₄slips at pH 9.2. The best stabilization was obtained with the addition of 2.3 wt% NH₄PA; in this condition, the viscosity reached a minimum value of 35 mPa.s at 100 s^–1. The slip viscosity increased with increasing pH from 9.2 to 10.2. Slips with low viscosities gave a more dense packing of cast samples and consequently higher sintered density values.     

Generation of Technology-Independent Retargetable Analog Blocks

This paper introduces a complete methodology for retargeting of analog cells to different sets of specifications. This methodology is technology-independent, thus allowing the migration, from one technology to another, of the circuit under retargeting. Careful integration of the device sizing and layout generation tasks via the incorporation of layout constraints during the sizing process allows to generate fully functional designs in a few minutes. The methodology is illustrated via the retargeting of a fully-differential Miller-compensated two-stage operational amplifier for a new set of specifications and two different technological processes.An erratum to this article can be found at     

A Symbolic Pole/Zero Extraction Methodology Based on Analysis of Circuit Time-Constants

This paper introduces a methodology for symbolic pole/zero extraction based on the formulation of the time-constant matrix of the circuits. This methodology incorporates approximation techniques specifically devoted to achieve an optimum trade-off between accuracy and complexity of the symbolic root expressions. The capability to efficiently handle even large circuits will be demonstrated through several practical circuits.     

Expectations about counseling in Yucatán, Mexico: Toward a "Mexican psychology."

Conducted a study of client expectations of psychological services in Yucatán, Mexico, under the sponsorship of a grant from the Organization of American States. The Expectations about Counseling–Brief form (EAC-B) questionnaire was translated and administered to 488 subjects across age, sex, and experience with psychological services. Subsequent analyses supported the reliability and robustness of the Spanish EAC-B, with factor structure similar to the EAC-B in English. Some relation was found among age, sex, prior counseling experience, and expectations about counseling. Implications of these data for future research and comparison with U.S. subjects are discussed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Synthesis of sliding-mode controllers for nonlinear systems via extended linearization

In this article, a general synthesis method is proposed for the design of discontinuous feedback strategies leading to asymptotically stabilizing sliding regimes. The method is applicable to the class of nonlinear dynamical systems possessing constant equilibrium points. A family of nonlinear stabilizing sliding manifolds, parametrized by generic desired equilibrium point, is specified on the basis of the extended linearization approach. Some examples including simulations are presented for illustrative purposes. Editor: S. Zak