Technical note: Quantifying and characterizing behavior in dairy calves using the IceTag automatic recording device.

The objectives of the current study were 1) to validate the IceTag (http://www.icerobotics.com) automatic recording device for measuring lying, standing, and moving behavior in dairy calves, and 2) to improve the information yield from this device by applying a filtering procedure allowing for the detection of lying versus upright. The IceTag device provides measures of intensity (I) of lying, standing, and activity measured as percent lying, percent standing, and percent active, but does not directly measure lying, standing, and moving behavior because body movements occurring while lying (e.g., shifts in lying position) and while upright (e.g., grooming) are recorded as activity. Therefore, the following 3-step procedure was applied. First, thresholds for I were determined by choosing the cutoff that maximized the sum of sensitivity (Se) and specificity (Sp). Second, a lying period criterion (LPC) was established empirically, and IceTag data were filtered according to the LPC, providing information on the posture of the animal as lying versus being upright. Third, a new threshold of I was estimated for moving activity conditional on the animal being upright. IceTag recordings from 9 calves were compared with video recordings during a 12-h period and analyzed using 2 x 2 contingency tables. Data from the first 4 calves were used to determine an LPC, whereas the remaining 5 calves served for validation of the procedure. An optimal LPC was found by modeling the deviance between IceTag and video recordings as a function of the LPC and choosing the LPC threshold that minimized the deviance. The IceTag device was found to accurately measure the high-prevalence behaviors (lying and standing; Se+Sp >1.90) and less accurately measure the low-prevalence behavior (moving; Se+Sp = 1.39). Application of the 3-step procedure using an optimal LPC estimate of 24.8 s resulted in an improved description of calf behavior, yielding a valid representation of the number and duration of lying and upright periods (Se+Sp = 2.00) within a precision of 0 to 49 s (95% confidence interval). In group-housed dairy calves, valid measures of the number and duration of lying and upright periods may be obtained from the IceTag device when applying the presented filtering procedure to the data. Measures regarding locomotion, on the other hand, should be used with caution.

Moss systems biology en route: phytohormones in Physcomitrella development.

The moss Physcomitrella patens has become a powerful model system in modern plant biology. Highly standardized cell culture techniques, as well as the necessary tools for computational biology, functional genomics and proteomics have been established. Large EST collections are available and the complete moss genome will be released soon. A simple body plan and the small number of different cell types in Physcomitrella facilitate the study of developmental processes. In the filamentous juvenile moss tissue, developmental decisions rely on the differentiation of single cells. Developmental steps are controlled by distinct phytohormones and integration of environmental signals. Especially the phytohormones auxin, cytokinin, and abscisic acid have distinct effects on early moss development. In this article, we review current knowledge about phytohormone influences on early moss development in an attempt to fully unravel the complex regulatory signal transduction networks underlying the developmental decisions of single plant cells in a holistic systems biology approach.

Molecular tools to study Physcomitrella patens.

The moss Physcomitrella patens has become a suitable model plant system for the analysis of diverse aspects of modern plant biology. The research strategies have been influenced by the implementation of state-of-the-art cell culture and molecular biology techniques. The forthcoming completion of the Physcomitrella genome sequencing project will generate many open questions, the examination of which will rely on a diverse set of molecular tools. Within this article, we intend to introduce the essential cell culture and molecular biology techniques which have been adopted in recent years to make Physcomitrella amenable to a wide range of genetic analyses. Many research groups have made valuable contributions to improve the methodology for the study of Physcomitrella. We would like to apologise to all colleagues whose important contributions could not be cited within this manuscript.

Glyco-engineering of moss lacking plant-specific sugar residues.

The commercial production of complex pharmaceutical proteins from human origin in plants is currently limited through differences in protein N-glycosylation pattern between plants and humans. On the one hand, plant-specific alpha(1,3)-fucose and beta(1,2)-xylose residues were shown to bear strong immunogenic potential. On the other hand, terminal beta(1,4)-galactose, a sugar common on N-glycans of pharmaceutically relevant proteins, e.g., antibodies, is missing in plant N-glycan structures. For safe and flexible production of pharmaceutical proteins, the humanisation of plant protein N-glycosylation is essential. Here, we present an approach that combines avoidance of plant-specific and introduction of human glycan structures. Transgenic strains of the moss Physcomitrella patens were created in which the alpha(1,3)-fucosyltransferase and beta(1,2)-xylosyltransferase genes were knocked out by targeted insertion of the human beta(1,4)-galactosyltransferase coding sequence in both of the plant genes (knockin). The transgenics lacked alpha(1,3)-fucose and beta(1,2)-xylose residues, whereas beta(1,4)-galactose residues appeared on protein N-glycans. Despite these significant biochemical changes, the plants did not differ from wild type with regard to overall morphology under standard cultivation conditions. Furthermore, the glyco-engineered plants secreted a transiently expressed recombinant human protein, the vascular endothelial growth factor, in the same concentration as unmodified moss, indicating that the performed changes in glycosylation did not impair the secretory pathway of the moss. The combined knockout/knockin approach presented here, leads to a new generation of engineered moss and towards the safe and flexible production of correctly processed pharmaceutical proteins with humanised N-glycosylation profiles.

Use of an inducible reporter gene system for the analysis of auxin distribution in the moss Physcomitrella patens.

The plant hormone auxin plays a major role in a variety of growth and developmental responses, even in the more ancient plants-for example, cell differentiation in mosses. Nevertheless, almost nothing is known about the distribution of auxin during moss development. To address this question, we characterised auxin distribution in the moss Physcomitrella patens using auxin-inducible reporter gene systems. Stable transgenic Physcomitrella plants were produced expressing the beta-glucuronidase (GUS) gene driven by the auxin-inducible promoters GH3 and DR5, respectively. Both fusions showed remarkable differences with respect to auxin-induced promoter strength and expression kinetics. A detailed characterisation of the GUS expression pattern in different developmental stages revealed that the highest auxin concentrations were in dividing and ontogenetic young cells.

Visualization of a cytoskeleton-like FtsZ network in chloroplasts.

It has been a long-standing dogma in life sciences that only eukaryotic organisms possess a cytoskeleton. Recently, this belief was questioned by the finding that the bacterial cell division protein FtsZ resembles tubulin in sequence and structure and, thus, may be the progenitor of this major eukaryotic cytoskeletal element. Here, we report two nuclear-encoded plant ftsZ genes which are highly conserved in coding sequence and intron structure. Both their encoded proteins are imported into plastids and there, like in bacteria, they act on the division process in a dose-dependent manner. Whereas in bacteria FtsZ only transiently polymerizes to a ring-like structure, in chloroplasts we identified persistent, highly organized filamentous scaffolds that are most likely involved in the maintenance of plastid integrity and in plastid division. As these networks resemble the eukaryotic cytoskeleton in form and function, we suggest the term "plastoskeleton" for this newly described subcellular structure.

Effect of lattice defects on Hele-Shaw flow over an etched lattice.

We examine the patterns formed by injecting nitrogen gas into the center of a horizontal, radial Hele-Shaw cell filled with paraffin oil. We use smooth plates and etched plates with lattices having different amounts of defects (0-10 %). In all cases, a quantitative measure of the pattern ramification shows a regular trend with injection rate and cell gap, such that the dimensionless perimeter scales with the dimensionless time. By adding defects to the lattice, we observe increased branching in the pattern morphologies. However, even in this case, the scaling behavior persists. Only the prefactor of the scaling function shows a dependence on the defect density. For different lattice defect densities, we examine the nature of the different morphology phases.

The early growth response protein (EGR-1) regulates interleukin-2 transcription by synergistic interaction with the nuclear factor of activated T cells.

The early growth response-1 gene (EGR-1) is induced by a wide range of stimuli in diverse cell types; however, EGR-1-regulated genes display a highly restricted pattern of expression. Recently, an overlapping Sp1.EGR-1 binding site has been identified within the interleukin-2 (IL-2) gene promoter directly upstream of the binding site for the nuclear factor of activated T cells (NFAT). We used transfection assays to study how the abundantly and constitutively expressed Sp1 protein and the immediate early EGR-1 zinc finger protein regulate IL-2 gene expression. Here, we identify EGR-1 as an important activator of the IL-2 gene. In Jurkat T cells, EGR-1 but not Sp1 acts as a potent coactivator for IL-2 transcription, and in combination with NFATc, EGR-1 increases transcription of an IL-2 reporter construct 200-fold. Electrophoretic mobility shift assays reveal that recombinant EGR-1 and NFATc bind independently to their target sites within the IL-2 promoter, and the presence of both sites on the same DNA molecule is required for EGR-1.NFATc.DNA complex formation. The transcriptional synergy observed here for EGR-1 and NFATc explains how the abundant nuclear factor EGR-1 contributes to the expression of restrictively expressed genes.

The human zinc finger protein EGR-4 acts as autoregulatory transcriptional repressor.

The human EGR-4 (AT133) gene represents one member of a family of four related zinc finger proteins, that are simultaneously and coordinately induced in resting cells upon growth stimulation. In order to characterise the function of the EGR-4 zinc finger protein, we have expressed the protein in the eukaryotic baculovirus system. The recombinant EGR-4 protein has a molecular mass of 78 kDa, as demonstrated by SDS-PAGE and Western blotting. DNA binding studies revealed that the EGR-4 protein binds to the EGR consensus motif GCGTGGGCG, but not to the G-rich regulatory ZIP-element of the human IL-2 gene, that is a binding site for EGR-1. EGR-4 functions as transcriptional repressor. Overexpression of EGR-4 mediates repression of a minimal c-fos promoter through a threefold EGR consensus site. Furthermore the EGR-4 protein displays autoregulatory activities. This protein downregulates expression of its own gene promoter in a dose dependent manner. A G-rich region in the EGR-4 promoter, located at position -106 to -82, could be identified as binding site for the recombinant EGR-4 protein. A comparison of the two related zinc finger proteins EGR-4 and EGR-1 revealed for each protein distinct and specific DNA binding- and transcriptional regulatory activities.

Coordinate expression and distinct DNA-binding characteristics of the four EGR-zinc finger proteins in Jurkat T lymphocytes.

The Early Growth Response Genes (EGR-1 to AT133/EGR-4) encode a family of proteins that are composed of three homologous consecutive zinc fingers of the Cys2-His2 type and different flanking sequence. Upon growth stimulation of resting cells the four EGR-genes are simultaneously transcribed. We have analyzed the expression of the four EGR-proteins in Jurkat T cells and show by Western blot analysis that the four EGR-proteins are coordinately induced upon treatment with a combination of PHA and PMA. As the individual proteins are reported to bind to identical target sequences, we have analyzed the DNA-binding of the native proteins. Using nuclear extract in which we have demonstrated expression of all four EGR-proteins, only EGR-1, but no other member of this protein family is found to bind to the EGR-consensus site (GCG GGG GCG). In addition, DNA-binding of both native EGR-1 and of recombinant EGR-1 and AT133/EGR-4 proteins expressed in insect cells was analyzed. This comparison revealed distinct binding properties of recombinant EGR-1 and AT133/EGR-4 to oligonucleotides that include the EGR-consensus sites. The distinct binding affinities suggest that in vivo EGR-proteins bind to different target sequences and that each EGR-protein regulates distinct target genes. This is underlined by demonstrating that EGR-1 but not AT133/EGR-4 binds to a related G-rich promoter element with the sequence GGG GTG GGG. This G-rich sequence serves as an overlapping binding site for the two zinc finger proteins EGR-1 and Sp1. As similar overlapping binding sites for EGR-1 and Sp1 have been identified in several human and mouse gene promoters, we raise the question whether the Sp1 binding sites described in a large number of eukaryotic gene promoters also represent binding sites for EGR-1.

A regulatory element in the human interleukin 2 gene promoter is a binding site for the zinc finger proteins Sp1 and EGR-1.

Activation of the interleukin 2 (IL-2) gene after antigen recognition is a critical event for T cell proliferation and effector function. Prior studies have identified several transcription factors that contribute to the activity of the IL-2 promoter in stimulated T lymphocytes. Here we describe a novel regulatory element within the IL-2 promoter located immediately upstream of the nuclear factor of activated T cell (NFAT) domain. This region (termed the zinc finger protein binding region (ZIP)) serves as binding site for two differently regulated zinc finger proteins: the constitutively expressed transcription factor Sp1 and the inducible early growth response protein EGR-1. In unstimulated cells which do not secrete IL-2, only Sp1 binds to this region, while in stimulated IL-2 secreting cells the inducible EGR-1 protein recognizes this element. In Jurkat T cells, the ZIP site serves as an activator for IL-2 gene expression, and a combination of ZIP and NFAT binding sites is required for maximal IL-2 promoter activity. These results suggest a critical role of the ZIP site for IL-2 promoter activity.

Reverse pressure gradients across the teat canal related to machine milking.

Miniature pressure transducers were inserted through the udder cistern wall of 10 cows and placed into the teat cistern and, in addition, beneath the teat end. Data were sampled every millisecond and collected during 59 sequences of manual teat handling pre- and postmilking, 575 attachments, 384 sequences of 30-s milking, and 623 sequences of detachment. Attachment and detachment were mainly done during overmilking in short sequences lasting 8 to 20 s. Reverse pressure gradients across the teat canal occurred during manual teat handling (54%), attachment of the milking unit (29%), milking (1%), and detachment (26%). Overall risk included empty teats. Risk factors at pre- and postmilking teat handling were the compression of teats and the following movement back to normal shape. When the diameter of the mouthpiece orifice of the liner was larger than the teat diameter, the frequency of reverse pressure gradients at attachment was halved compared with attachment of more narrow liners. The method of attaching the milking unit on empty teats without the risk of creating reverse pressure gradients was not identified. Reverse pressure gradients in empty teats may be avoided during detachment of liners if the mouthpiece orifice diameter is greater than the teat diameter. Detachment with the liner in open position reduced the risk of reverse pressure gradients compared with that from the closed position.

Photosensitizing potential of ofloxacin.

BACKGROUND: The relative phototoxic risk of ofloxacin, one of the newer fluoroquinolones, was compared with that of an active control of known but low phototoxic risk, naproxen. METHODS: A randomized, controlled, open-label trial was used with a standardized phototoxic assay completed at baseline, midway through, and at the termination of the 12-day trial. The trial was held at a dermatology research laboratory located at a large tertiary referral and teaching hospital. Thirty healthy volunteers who met the inclusion criteria and met none of the exclusion criteria were enrolled. Twenty-seven patients completed the trial. Three subjects failed to complete the trail. One subject developed an exaggerated response to the initial photoexposure and was dropped from the study. The other two subjects failed to return for follow-up visits. RESULTS: Both ofloxacin and the active control agent, naproxen, significantly increased the subjects' response to the tested solar and ultraviolet irradiation. There was, however, no significant difference between the responses observed for ofloxacin versus naproxen at any time. CONCLUSIONS: Ofloxacin possesses a definite but low potential to cause phototoxic reactions in humans. These study data, in concert with surveillance data, suggest a hierarchy of phototoxic risk among the fluoroquinolones: fleroxacin >> lomefloxacin, pefloxacin >> ciprofloxacin > enoxacin, norfloxacin, ofloxacin. The impact that phototoxicity risk will have on selecting the optimum member of a large drug family appears to be substantial in outpatient and ambulatory settings and minimal in inpatient settings.

Design and implementation of a cost-accounting system in hospital pharmacy.

The design and implementation of a cost-accounting system in a hospital pharmacy department is described. Pharmacy resource use (labor, drugs, supplies, and overhead), or pharmacy's intermediate products, was clearly defined in terms of dosage forms (10 groupings representing variable labor and supplies) and drug products (more than 100 categories that incorporate cost and volume of use for 3000 line items). Costs were defined as variable or nonvariable (fixed), based on whether they were related to a specific medication order. Labor was divided into variable and fixed components. Time standards were developed using time and motion studies. Variable labor hours were determined as follows: specified hours (the volume of each dosage form multiplied by the standard time for each dosage form); nonspecified hours (time not directly associated with production); hours worked (specified plus nonspecified hours); and hours paid (hours worked plus sick leave and vacation). A standard cost for each drug product was based on the weighted average of volume and cost of the individual line items. The total drug budget was constructed by multiplying the standard cost for each drug product times the projected volume for each drug product. The pharmacy budget was developed by calculating the number and mix of pharmacy products used in association with the projected number and type of cases for the fiscal year. The monthly pharmacy budget reports were assembled with data from the payroll, billing, and cost-accounting systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Digoxin prophylaxis following coronary artery bypass surgery.

The effect of the postoperative administration of digoxin to patients undergoing coronary artery bypass surgery on the incidence of supraventricular arrhythmias was studied. Patients were randomly assigned to a control group (n = 51) or digoxin group (n = 47) on a prospective basis. Patient characteristics were similar in both groups, and no patients were receiving digoxin therapy preoperatively or other antiarrhythmic medications. All patients had normal systolic ejection fractions, renal function, and hepatic function. Eight patients (16%) in the control group developed postoperative arrhythmias while seven patients (15%) in the digoxin group developed supraventricular arrhythmias. This difference was not significant. Two patients in the digoxin group developed digoxin-induced arrhythmias, and two other patients experienced digoxin-related nausea and vomiting, which were resolved with discontinuation of the drug. The postoperative administration of digoxin to patients undergoing coronary artery bypass surgery had no effect on the incidence of supraventricular arrhythmias. The prophylactic use of digoxin therapy in this patient population is not recommended unless there is a history of arrhythmias responsive to digoxin therapy.

The pharmacy computer system at the New England Medical Center.

The pharmacy computer system developed and implemented at the New England Medical Center (NEMC) is described. The system was developed to improve the department's fiscal performance and operations. It operates on NEMC's large mainframe system and has the following components-order entry, unit dose/patient profile, i.v. admixture, and financial/management. The system was designed to interface with the admissions and census system and use the pharmacy department's drug data file. It accepts medication orders from pharmacy satellites and then processes the orders according to the requirements for the i.v. admixture or unit dose procedures. Financial data are collected as a routine part of all medication order processing. The system allows for the entry of multiple medication orders on one cathode ray tube (CRT) screen, thus facilitating order entry. The use of programmed system defaults is described. Order entry, dose selection, and patient profiles are displayed on a CRT. An active order list is printed once daily at midnight to serve as a back-up for the system. The computer system has had a positive impact on the department's operational, clinical, and management functions. Future enhancements of the NEMC system are discussed.