Optimization of heterocyclic 4-hydroxystyryl derivatives for histological localization of endogenous and immunobound peroxidase activity

Assisted by the development of light excitation and measuring techniques and the commercial availability of highly sensitive equipment, luminescent labels are sensitive detection tools for life sciences research. By contrast to a wide variety of well established chromogenic techniques, fluorescent labels for detecting peroxidase (PO) have been confined to only a few substrates. We describe here novel fluorescent substrates of PO derived from heterocyclic 4-hydroxy styrenes as useful tools for detecting endogenous and exogenous targets in fixed cells and tissues. Excellent localization, high staining sensitivity, outstanding photostability, and exceptionally low background staining were achieved by optimizing the substrate through chemical synthesis. Structure/staining behavior relationships are discussed. By contrast to tyramine-fluorochrome conjugates employed in the catalyzed reporter deposition (CARD) technique, reporting and anchoring functions are no longer separated. Consequently, enzymatic cross-linking of the substrate yields an altered fluorochrome with different properties. Spectral properties and anchoring capability are interdependent and influenced by environmental effects and pH. We screened overall staining capability of 4-hydroxy styryl derivatives using an iterative semi-empirical approach, and ascertained optimal substitution patterns for high PO staining specificity and high fluorescence response. Reliable staining performance was achieved with alkyl chains of short or medium length at the positively charged nitrogen, whereas introducing polar groups often impaired the staining specificity of PO. Catalytic cross-linking of heterocyclic 4-hydroxy-styryl derivatives is a promising approach for permanent fluorescent staining of PO in fixed cells and tissues, and complements the CARD technique. Histochemical and immunohistochemical applications are presented using conventional and confocal fluorescence microscopes with different excitation sources. Spectral properties of selected stains are discussed. Novel stains also are of potential interest as “reactive-tracers” for living cells under multi-photon laser excitation conditions, because they exhibit pronounced nonlinear optical properties.     

Environmental effects on progesterone profile measures of dairy cow fertility

Environmental effects on fertility measures early in lactation, such as the interval from calving to first luteal activity (CLA), proportion of samples with luteal activity during the first 60 days after calving (PLA) and interval to first ovulatory oestrus (OOE) were studied. In addition, traditional measurements of fertility, such as pregnancy to first insemination, number of inseminations per service period and interval from first to last insemination were studied as well as associations between the early and late measurements. Data were collected from an experimental herd during 15 years and included 1106 post-partum periods from 191 Swedish Holsteins and 325 Swedish Red and White dairy cows. Individual milk progesterone samples were taken twice a week until cyclicity and thereafter less frequently. First parity cows had 14.8 and 18.1 days longer CLA (LS-means difference) than second parity cows and older cows, respectively. Moreover, CLA was 10.5 days longer for cows that calved during the winter season compared with the summer season and 7.5 days longer for cows in tie-stalls than cows in loose-housing system. Cows treated for mastitis and lameness had 8.4 and 18.0 days longer CLA, respectively, compared with healthy cows. OOE was affected in the same way as CLA by the different environmental factors. PLA was a good indicator of CLA, and there was a high correlation (-0.69) between these two measurements. Treatment for lameness had a significant influence on all late fertility measurements, whereas housing was significant only for pregnancy to first insemination. All fertility traits were unfavourably associated with increased milk production. Regression of late fertility measurements on early fertility measurements had only a minor association with conception at first AI and interval from first to last AI for cows with conventional calving intervals, i.e. a 22 days later, CLA increased the interval from first to last insemination by 3.4 days. Early measurements had repeatabilities of 0.14-0.16, indicating a higher influence by the cow itself compared with late measurements, which had repeatabilities of 0.09-0.10. Our study shows that early fertility measurements have a possibility to be used in breeding for better fertility. To improve the early fertility of the cow, there are a number of important factors that have to be taken into account.     

Molecular cytogenetic characterization of ring chromosome 4 in a female having a chromosomally normal child

We present the clinical and molecular findings of mosaic ring chromosome 4. The patient was referred to us for infertility and short stature. Results of three repeated cytogenetic analyses from lymphocytes showed a similar mosaic karyotype with multiple cell-lines [46,XX,r(4)/45,XX,-4/46,XX,dic r(4)/47,XX,r(4),+r(4)/46,XX]. FISH showed deletion of the 4p subtelomeric region and the 4q telomeric region from the ring chromosome 4. The breakpoints were mapped using molecular analysis. Parental karyotypes were normal. During the course of this study, the patient became pregnant without assisted reproductive technology. The result of amniocentesis performed at 16 weeks gestation showed a normal karyotype. Delivery was uncomplicated. This is the first report, to our knowledge, of the presence of ring chromosome 4 having various mosaic conditions in a female having a chromosomally normal fetus.     

Design and application of compliant mechanisms for surgical tools

This paper introduces the benefits of exploiting elasticity in the engineering design of surgical tools, in general, and of minimally invasive procedures, in particular. Compliant mechanisms are jointless mechanisms that rely on elastic deformation to transmit forces and motion. The lack of traditional joints in these single-piece flexible structures offers many benefits, including the absence of wear debris, pinch points, crevices, and lubrication. Such systems are particularly amenable to embedded sensing for haptic feedback and embedded actuation with active-material actuators. The paper provides an overview of design synthesis methods developed at the Compliant Systems Design Laboratory and focuses specifically on surgical applications. Compliant systems have potential to integrate. well within the constraints of laparoscopic procedures and telerobotic surgery. A load-path representation is used within a genetic algorithm to solve two gripper example problems. In addition, the paper illustrates the design and construction of an organ (kidney) manipulator for use in minimally invasive procedures.     

Systematics of the Irish pollan (Coregonus pollan Thompson): an electrophoretic comparison with other Holarctic Coregoninae

Evidence is presented on the genetic relationships of 35 coregonine populations from Alaska, Britain, Ireland, Finland and Sweden. This evidence is derived from an electrophoretic analysis of 27 enzyme loci and additional 'general protein loci'. The Irish pollan C. pollan and the Alaskan C. autumnalis gave identical electrophoretic patterns for all proteins suggesting that they are conspecific and separated only since the last glaciation. C. peled, C. albula and the ' C. lavaretus' complex gave unique patterns for a number of proteins. The F_n C. oxyrhynchus X C albula hybrids, from a lake where no other coregonines occur gave a unique pattern for the enzyme phosphoglucose isomerase but all other enzymes were electrophoretically identical to the C. oxyrhynchus parent and the mean gill raker number was closer to this species than in the F₁ hybrid.     

Kinetics of UV(254) inactivation of selected viral pathogens in a static system

Aims: The objective of this study was to estimate UV₂₅₄ inactivation constants for four viral pathogens: influenza virus type A, porcine respiratory and reproductive syndrome virus (PRRSV), bovine viral diarrhoea virus (BVDV) and reovirus. Methods and Results: Viruses in culture medium were exposed to one of nine doses of UV₂₅₄ and then titrated for infectious virus. Analysis showed that viral inactivation by UV₂₅₄ was more accurately described by a two‐stage inactivation model vs a standard one‐stage inactivation model. Conclusions: The results provided evidence for the existence of two heterogeneous viral subpopulations among the viruses tested, one highly susceptible to UV₂₅₄ inactivation and the other more resistant. Importantly, inactivation constants based on the one‐stage inactivation model would have underestimated the UV₂₅₄ dose required for the inactivation of these viruses under the conditions of the experiment. Significance and Impact of the Study: To improve the accuracy of estimates, it is recommended that research involving the inactivation of micro‐organisms evaluates inactivation kinetics using both one‐stage and two‐stage models. These results will be of interest to persons responsible for microbial agents under laboratory or field conditions.     

A Conjugative Transfer System for the Rumen Bacterium, Butyrivibrio fibrisolvens, Based on Tn916-Mediated Transfer of the Staphylococcus aureus Plasmid pUB110

A limitation of genetic studies of the rumen bacterium, Butyrivibrio fibrisolvens, has been the availability of suitable vectors and transfer systems. Using the conjugative tetracycline resistant transposon, Tn916, the Staphylococcus aureus plasmid, pUB110, and the pUB110-based shuttle vector, pUBLRS, a conjugative transfer system was developed for B. fibrisolvens. B. fibrisolvens donor strains H17c2 and H17c12, containing Tn916 and pUB110 or pUBLRS, respectively, were used in mating experiments with selected B. fibrisolvens strains. Kanamycin resistant transconjugants, containing pUB110, of strains 193, 194, and 195 were detected at a combined average frequency of 7.78 × 10^-7 per donor and 1.11 × 10^-5 per recipient. Transconjugants of strains 193 and 194, containing pUBLRS, were detected at an average frequency of 1.22 × 10^-6 per donor and 4.70 × 10^-8 per recipient. Southern hybridization analysis confirmed the presence of pUB110 and pUBLRS in transconjugants. Results indicated that Tn916 was necessary for mobilization of pUB110 as transconjugants were not detected when the transposon was absent from the donor strains. The ability to mobilize pUB110 and pUBLRS between B. fibrisolvens strains provides a conjugative transfer system that circumvents problems encountered with electroporation.     

Selection and characterization of feather-degrading bacteria from canola meal compost

Canola meal that contains a high level of protein (40% crude protein) was used as compost material for the isolation of feather-degrading bacteria. After 7 and 14 days, bacteria were isolated from compost amended and unamended with soil. Eighty bacterial isolates from canola meal compost were then grown on milk-agar and isolates that produced proteolytic enzymes were identified by the formation of clear haloes around the colonies. A feather medium was chosen for a secondary selection of feather-degrading isolates. Of the eight isolates that hydrolyzed milk protein, five isolates hydrolyzed feathers. Their keratinolytic activities were subsequently confirmed by an assay using azo-keratin as substrate. Seven of the eight bacteria that hydrolyzed milk protein were Bacillus spp, and all five isolates that hydrolyzed feathers were strains of Bacillus licheniformis. Protease inhibition studies indicated that serine proteases are the predominant proteolytic enzymes produced by these feather-degrading isolates. Received 02 April 1999/ Accepted in revised form 17 June 1999     

Evaluation of a three point pressure method for the determination of arterial transmission characteristics

The mathematical expressions required to analyse wave transmission characteristics (frequency dependent phase velocity, attenuation, and reflection) in arteries, under in vivo conditions, are summarized. In addition, a three point pressure method, which theoretically permits experimental determination of the propagation constant (phase velocity and attenuation), was tested under in vitro conditions. It is found to be a potentially powerful tool for in vivo studies if used with the appropriate constraints.