Purification and cloning of cytotoxic ribonucleases from Rana catesbeiana (bullfrog).

Ribonucleases with antitumor activity are mainly found in the oocytes and embryos of frogs, but the role of these ribonucleases in frog development is not clear. Moreover, most frog ribonuclease genes have not been cloned and characterized. In the present study, a group of ribonucleases were isolated from Rana catesbeiana (bullfrog). These ribonucleases in mature oocytes, namely RC-RNase, RC-RNase 2, RC-RNase 3, RC-RNase 4, RC-RNase 5 and RC-RNase 6, as well as liver-specific ribonuclease RC-RNase L1, were purified by column chromatographs and detected by zymogram assay and western blotting. Characterization of these purified ribonucleases revealed that they were highly conserved in amino acid sequence and had a pyroglutamate residue at their N-termini, but possessed different specific activities, base specificities and optimal pH values for their activities. These ribonucleases were cytotoxic to cervical carcinoma HeLa cells, but their cytotoxicities were not closely correlated to their enzymatic specific activities. Some other amino acid residues in addition to their catalytic residues were implicated to be involved in the cytotoxicity of the frog ribonucleases to tumor cells. Because the coding regions lack introns, the ribonuclease genes were cloned by PCR using genomic DNA as template. Their DNA sequences and amino acid sequences are homologous to those of mammalian ribonuclease superfamily, approximately 50 and approximately 25%, respectively.

In vitro thermal stress induces apoptosis and reduces development of porcine parthenotes.

The precise physiological causes that result in reduced development of oocytes after heat shock (HS) are not clear. In this study, apoptosis, heat shock protein70 (hsp70), and in vitro development of porcine oocytes were evaluated after HS. Porcine cumulus-oocyte complexes (COCs) were subjected to in vitro maturation for 42 h. The matured oocytes were then heated at 41.5 degrees C for 0 h (control, C0h), 1 h (HS1h), 2 h (HS2h), or 4 h (HS4h). An additional group of oocytes was cultured for 4 h without HS (control, C4h). In Experiment 1, expression of hsp70 was detected by Western-blotting and no difference between controls and HS groups was observed. In Experiment 2, apoptosis of matured oocytes after HS was examined by Annexin V-FITC and TUNEL. No significant TUNEL-positive signals were detected in the heated oocytes compared to the controls, but the intensity of Annexin V-FITC labeling among different groups increased with length of HS and in vitro culture (P<0.05). Oocytes were parthenogenetically activated by an electric pulse plus 6-DMAP (Experiment 3). Mean (+/-S.E.M.) embryonic development in HS2h (cleavage: 42+/-29%; blastocyst: 11+/-10%) and HS4h (cleavage: 36+/-28%; blastocyst: 11+/-8%) were decreased when compared to those in C0h (cleavage: 63+/-12%; blastocyst: 24+/-14%) and C4h (cleavage: 66+/-8%; blastocyst: 21+/-11%). Numbers of blastocysts with TUNEL-positive signals were similar among groups, but the signals increased before the eight-cell stage in HS groups (P<0.05). In conclusion, developmental competence of matured pig oocytes was compromised after heat shock, but it was not closely associated with the expression of oocyte hsp70. However, there may be a link between apoptosis and developmental competence of porcine oocytes.

Size regulation does not cause the composition of mouse chimaeras to become unbalanced.

Mouse chimaeras made by aggregating two 8-cell stage embryos undergo size regulation shortly after implantation. Thus chimaeric pups are approximately normal size at birth despite their origin from two complete embryos. Chimaeras of some strain combinations are genotypically unbalanced such that cells of one strain almost always predominate. For example, the BALB/c inbred strain often makes a low contribution to chimaeras. This genotypic imbalance in the composition could arise by selection against BALB/c cells. Selection may be particularly acute at the time of size regulation. To investigate if the mechanism(s) responsible for size regulation could cause the low contribution of BALB/c cells, we compared the composition of an unbalanced series of chimaeras, produced by aggregating two complete 8-cell stage embryos, with a similar series of chimaeras made by aggregating two half 8-cell stage embryos. In each case the unbalanced strain combination was BALB/c<-->[(C57BL x CBA/Ca)F1 x TGB] and parallel studies were undertaken with a genotypically balanced strain combination. For each chimaera, the composition of the fetus, placenta and extraembryonic membranes were determined at E12.5. When two half embryos were aggregated the BALB/c strain still made a poor contribution to all the tissues of the mid-gestation conceptus. This implies that this strain combination remained unbalanced even when size regulation was absent or minimal. Therefore, size regulation did not play a major role in reducing the contribution of BALB/c cells and producing the phenotypic imbalance in the chimaeras.

Informing patients: a guide for providing patient health information.

OBJECTIVE: To understand and address patients' need for information surrounding ambulatory-care visits. DESIGN: The authors conducted two patient focus groups regarding patient education. The first covered general information needs of patients and the second explored their reactions to a computer-generated patient handout that was developed in response to the results of the first focus group and implemented in a clinic. RESULTS: Participants sought information about their health--generally after the encounter with their caregiver. They wanted a permanent record of personal health data and relevant educational information. Participants recommended that the information be concise, clear, and illustrated with graphics if appropriate. Receiving health-related information from their providers favorably affected the participants' trust in, relationship with, and confidence in their physicians. When given printouts with graphic trends depicting their responses to therapy, participants reported that they were more motivated to adhere to a treatment plan and were more satisfied with their care. Based on the results of the focus groups, we developed a set of attributes (P.A.T.I.E.N.T.) to guide the development of patient and consumer health information. CONCLUSIONS: Patients participating in our focus groups felt that providing printed summary information to patients at the end of a clinic visit improves their understanding of their care, enhances their relationships with providers, improves their satisfaction with care, and motivates them to adhere to treatment plans. Further empirical studies are necessary to test their perceptions.

The use of electronic medical records: communication patterns in outpatient encounters.

OBJECTIVE: To assess physician-patient communication patterns associated with use of an electronic medical record (EMR) system in an outpatient setting and provide an empirical foundation for larger studies. DESIGN: An exploratory, observational study involving analysis of videotaped physician-patient encounters, questionnaires, and medical-record reviews. SETTING: General internal medicine practice at an academic medical center. PARTICIPANTS: Three physicians who used an EMR system (EMR physicians) and three who used solely a paper record (control physicians). A total of 204 patient visits were included in the analysis (mean, 34 for each physician). MAIN OUTCOME MEASURES: Content analysis of whether physicians accomplished communication tasks during encounters; qualitative analysis of how EMR physicians used the EMR and how control physicians used the paper chart. RESULTS: Compared with the control physicians, EMR physicians adopted a more active role in clarifying information, encouraging questions, and ensuring completeness at the end of a visit. A trend suggested that EMR physicians might be less active than control physicians in three somewhat more patient-centered areas (outlining the patient's agenda, exploring psychosocial/ emotional issues, discussing how health problems affect a patient's life). Physicians in both groups tended to direct their attention to the patient record during the initial portion of the encounter. The relatively fixed position of the computer limited the extent to which EMR physicians could physically orient themselves toward the patient. Although there was no statistically significant difference between the EMR and control physicians in terms of mean time across all visits, a difference did emerge for initial visits: Initial visits with EMR physicians took an average of 37.5 percent longer than those with control physicians. SUMMARY: An EMR system may enhance the ability of physicians to complete information-intensive tasks but can make it more difficult to focus attention on other aspects of patient communication. Further study involving a controlled, pre-/post-intervention design is justified.

Use of computer-based records, completeness of documentation, and appropriateness of documented clinical decisions.

OBJECTIVE: To investigate whether using a computer-based patient record (CPR) affects the completeness of documentation and appropriateness of documented clinical decisions. DESIGN: A blinded expert panel of four experienced internists evaluated 50 progress notes of patients who had chronic diseases and whose physicians used either a CPR or a traditional paper record. MEASUREMENTS: Completeness of problem and medication lists in progress notes, allergies noted in the entire record, consideration of relevant patient factors in the progress note's diagnostic and treatment plans, and appropriateness of documented clinical decisions. RESULTS: The expert reviewers rated the problem lists and medication lists in the CPR progress notes as more complete (1.79/2.00 vs 0.93/2.00, P < 0.001, and 1.75/2.00 vs. 0.91/2.00, P < 0.001, respectively) than those in the paper record. The allergy lists in both records were similar. Providers using a CPR documented consideration of more relevant patient factors when making their decisions (1.53/2.00 vs. 1.07/2.00, P < 0.001), and documented more appropriate clinical decisions (3.63/5.00 vs. 2.50/5.00, P < 0.001), compared with providers who used traditional paper records. CONCLUSIONS: Physicians in our study who used a CPR produced more complete documentation and documented more appropriate clinical decisions, as judged by an expert review panel. Because the physicians who used the CPR in our study volunteered to do so, further study is warranted to test whether the same conclusions would apply to all CPR users and whether the improvement in documentation leads to better clinical outcomes.

Measuring the effects of reminders for outpatient influenza immunizations at the point of clinical opportunity.

OBJECTIVE: To evaluate the influence of computer-based reminders about influenza vaccination on the behavior of individual clinicians at each clinical opportunity. DESIGN: The authors conducted a prospective study of clinicians' influenza vaccination behavior over four years. Approximately one half of the clinicians in an internal medicine clinic used a computer-based patient record system (CPR users) that generated computer-based reminders. The other clinicians used traditional paper records (PR users). MEASUREMENTS: Each nonacute visit by a patient eligible for an influenza vaccination was considered an opportunity for intervention. Patients who had contraindications for vaccination were excluded. Compliance with the guideline was defined as documentation that a clinician ordered the vaccine, counseled the patient about the vaccine, offered the vaccine to a patient who declined it, or verified that the patient had received the vaccine elsewhere. The authors calculated the proportion of opportunities on which each clinician documented action in the CPR and PR user groups. RESULTS: The CPR and PR user groups had different baseline compliance rates (40.1 and 27.9 per cent, respectively; P<0.05). Both rates remained stable during a two-year baseline period (P = 0.34 and P = 0.47, respectively). The compliance rates in the CPR user group increased 78 per cent from baseline (P<0.001), whereas the rates for the PR user group did not change significantly (P = 0.18). CONCLUSIONS: Clinicians who used a CPR with reminders had higher rates of documentation of compliance with influenza-vaccination guidelines than did those who used a paper record. Measurements of individual clinician behavior at the point of each clinical opportunity can provide precise evaluation of interventions that are designed to improve compliance with guidelines.

Clinical decision support systems for the practice of evidence-based medicine.

BACKGROUND: The use of clinical decision support systems to facilitate the practice of evidence-based medicine promises to substantially improve health care quality. OBJECTIVE: To describe, on the basis of the proceedings of the Evidence and Decision Support track at the 2000 AMIA Spring Symposium, the research and policy challenges for capturing research and practice-based evidence in machine-interpretable repositories, and to present recommendations for accelerating the development and adoption of clinical decision support systems for evidence-based medicine. RESULTS: The recommendations fall into five broad areas--capture literature-based and practice-based evidence in machine--interpretable knowledge bases; develop maintainable technical and methodological foundations for computer-based decision support; evaluate the clinical effects and costs of clinical decision support systems and the ways clinical decision support systems affect and are affected by professional and organizational practices; identify and disseminate best practices for work flow-sensitive implementations of clinical decision support systems; and establish public policies that provide incentives for implementing clinical decision support systems to improve health care quality. CONCLUSIONS: Although the promise of clinical decision support system-facilitated evidence-based medicine is strong, substantial work remains to be done to realize the potential benefits.

Surgical resection for metastatic melanoma to the liver: the John Wayne Cancer Institute and Sydney Melanoma Unit experience.

HYPOTHESIS: Metastatic melanoma to the liver is not incurable; complete surgical resection can achieve long-term survival in selected patients. BACKGROUND: Metastases to the liver are diagnosed in 10% to 20% of patients with American Joint Committee on Cancer stage IV melanoma. Surgical resection has not been generally accepted as a therapeutic option, as most patients will have other sites of disease that limit their survival to a median of only 4 to 6 months. However, there is little information on outcomes following resection in those patients with disease limited to the liver. PATIENTS AND METHODS: Review of the prospective melanoma databases at the John Wayne Cancer Institute, Santa Monica, Calif, and the Sydney Melanoma Unit, Sydney, Australia, identified 1750 patients with hepatic metastases, of whom 34 (2%) underwent exploration with intent to resect the metastases. Prognostic factors within the group of patients who underwent resection were examined by univariate and multivariate analysis, and median disease-free survival (DFS) and overall survival (OS) were calculated. RESULTS: Of 34 patients undergoing exploratory celiotomy, 24 (71%) underwent hepatic resection and 10 (29%) underwent exploration but not resection. Eighteen patients (75%) underwent complete surgical resection, while the remaining 6 underwent palliative or debulking procedures with incomplete resection. The operative resections included lobectomy (n=14), segmentectomy (4), nonanatomic resection (5), and extended lobectomy (1). The median number of resected lesions was 1, and median lesion size was 5 cm (range, 0.7-22 cm). The median disease-free interval between initial diagnosis of melanoma and development of hepatic metastases was 58 months (range, 0-264 months). Median DFS and OS estimates in the 24 patients who underwent surgical resection were 12 months (range, 0-147 months) and 28 months (range, 2-147 months), respectively. Five-year DFS and OS in this group were 12% and 29%. Macroscopically, complete resection of disease (P =.001) and histologically negative resection margins (P =.03) significantly improved DFS by univariate analysis. Patients rendered surgically free of disease also tended to have improved OS (P =.06). Median OS was 28 months for patients who underwent surgical resection compared with 4 months for patients who underwent exploration only (P<.001). CONCLUSIONS: Resection of metastatic melanoma to the liver may improve DFS and OS in selected patients, similar to resection of other metastatic sites. Therefore, patients with limited metastatic sites, including the liver, who can be rendered free of disease should be considered for complete surgical resection, as their prognosis is otherwise dismal.

Matrix metalloproteinase inhibitors reduce phorbol ester-induced cutaneous inflammation and hyperplasia.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteases which play key roles in extracellular matrix remodeling, connective tissue damage, inflammation and cell proliferation in a variety of tissues. Since MMP inhibitors have been recently shown to decrease proliferation of vascular smooth-muscle cells, and to prevent neutrophil infiltration in response to alkali burns, we sought to determine whether MMPs play a role in the pathogenesis of inflammatory or hyperproliferative skin disorders. The effects of a specific MMP inhibitor and its analogues on phorbol dibutyrate (PdiBu)-induced inflammation and epidermal hyperplasia in murine skin were assessed. Topical GM 6001, a hydroxamic acid analog with potent inhibitory activity against several MMPs, markedly inhibited PdiBu-induced increases in both ear thickness and ear punch-biopsy weight in a dose-dependent manner 30 h after topical application of PdiBu. Maximal inhibition (75%) was obtained at a dose of 100 micrograms/cm2 (P < 0.01). Moreover, histologic analysis revealed that GM 6001 decreased both the inflammatory cellular infiltrates and epidermal hyperplasia induced by PdiBu. Whereas similar results were found for GM 1489, an analog of GM 6001, acetohydroxamic acid, containing the critical metal ligand group but without the amino acid side chains necessary for binding to the MMPs, did not alter the response to PdiBu inflammation/hyperplasia. These results show that the MMP inhibitors, GM 6001 and GM 1489, are effective in reducing both the inflammatory and hyperproliferative responses that occur following topical phorbol ester application, suggesting a potential role for MMPs in cutaneous inflammatory dermatoses. Moreover, the delivery of this class of inhibitors across intact stratum corneum implies that MMP inhibition could provide an approach to the topical treatment of inflammatory dermatoses.

The HIPAAcratic oath: do no harm to patient data.

Physician executives need to prepare their organizations for the next great system-wide challenge--HIPAA, the Health Insurance Portability and Accountability Act of 1996. Organizations will have to plan for and execute a compliance program with the same vigor and system-wide participation as they did for Y2K. This article provides a brief overview of HIPAA, emphasizing the privacy and security components that will be the biggest challenge for physician executives. Physician leaders must become actively involved in the policymaking process to ensure a balanced approach to protecting the confidentiality of health information, while giving providers optimal access to data to make informed decisions on patient care and management. Ignoring HIPAA is simply not an option.

Palmitic acid in chicken granulosa cell death-lipotoxic mechanisms mediate reproductive inefficacy of broiler breeder hens.

In vivo and in vitro approaches were used to elucidate mechanisms of palmitate-induced cytotoxicity of follicle granulosa cells in fuel-overloaded broiler hens. In contrast to their energy-restricted counterparts, broiler breeder hens fed ad libitum for 2 wk had dyslipidemia, atresia within hierarchical ovarian follicles, and a 34% reduction in egg production (P < 0.05). Based on vital staining of freshly isolated granulosa cells with annexin V/propidium iodide, there were increases in apoptosis consistent with suppressed Akt activation (P < 0.05). Supplementing primary granulosa cell cultures with 0.5 mM palmitate for 48 or 96 h increased apoptosis (P < 0.05). Palmitate-induced cell death was accompanied by increased acyl-CoA oxidase, carnitine palmitoyl transferase-1, serine palmitoyl transferase, and sphingomyelinase transcripts and increased concentrations of proinflammatory interleukin-1ß (P < 0.05). Triacsin-C inhibition of fatty acyl-CoA synthesis blunted interleukin-1ß production and rescued granulosa cultures from palmitate-induced cell death. That there was partial to complete prevention of cell death with addition of the free radical scavenger pyrrolidine dithiocarbamate, the sphingomyelinase inhibitor imipramine, or the de novo ceramide synthesis inhibitor fumonisin B1, supported the notion that palmitate-induced granulosa cell cytotoxicity operated through a palmitate-derived metabolite. Palmitoyl-CoA may be channeled into ß-oxidation and/or into bioactive metabolites that increase free radical generation, an inflammatory response, and ceramide production. In conclusion, palmitate-derived metabolites activated apoptotic machinery in avian granulosa cells, which caused ovarian follicular atresia and reduced egg production in fuel-overloaded broiler breeder hens.

Expressed transcripts associated with high rates of egg production in chicken ovarian follicles.

The purpose of this study was to characterize differentially expressed transcripts associated with varying rates of egg production in Taiwan country chickens. Ovarian follicles were isolated from two strains of chicken which showed low (B) or high (L2) rates of egg production, then processed for RNA extraction and cDNA library construction. Three thousand and eight forty clones were randomly selected from the cDNA library and amplified by PCR, then used in microarray analysis. Differentially expressed transcripts (P<0.05, log(2)> or = 1.75) were sequenced, and aligned using GenBank. This analysis revealed 20 non-redundant sequences which corresponded to known transcripts. Eight transcripts were expressed at a higher level in ovarian tissue prepared from chicken strain B, and 12 transcripts were expressed at a higher level in L2 birds. These differential patterns of expression were confirmed by semi-quantitative RT-PCR. We show that transcripts of cyclin B2 (cycB2), ferritin heavy polypeptide 1 (FTH1), Gag-Pol polyprotein, thymosin beta4 (TB4) and elongation factor 1 alpha1 (EEF1A1) were enriched in B strain ovarian follicles. In contrast, thioredoxin (TXN), acetyl-CoA dehydrogenase long chain (ACADL), inhibitor of growth family member 4 (ING4) and annexin II (ANXA2) were expressed in at higher levels in the L2 strain. We suggest that our approach may lead to the isolation of effective molecular markers that can be used in selection programs in Taiwan country chickens.

Major issues in user interface design for health professional workstations: summary and recommendations.

Lack of good user interfaces has been a major impediment to the acceptance and routine use of health-care professional workstations. Health-care providers, and the environment in which they practice, place strenuous demands on the interface. User interfaces must be designed with greater consideration of the requirements, cognitive capabilities, and limitations of the end-user. The challenge of gaining better acceptance and achieving widespread use of clinical information systems will be accentuated as the variety and complexity of multi-media presentation increases. Better understanding of issues related to cognitive processes involved in human-computer interactions is needed in order to design interfaces that are more intuitive and more acceptable to health-care professionals. Critical areas which deserve immediate attention include: improvement of pen-based technology, development of knowledge-based techniques that support contextual presentation, and development of new strategies and metrics to evaluate user interfaces. Only with deliberate attention to the user interface, can we improve the ways in which information technology contributes to the efficiency and effectiveness of health-care providers.

The effects of embryo stage and cell number on the composition of mouse aggregation chimaeras.

Studies with intact preimplantation mouse embryos and some types of chimaeric aggregates have shown that the most advanced cells are preferentially allocated to the inner cell mass (ICM) rather than the trophectoderm. Thus, differences between 4-cell and 8-cell stage embryos could contribute to the tendency for tetraploid cells to colonise the trophectoderm more readily than the ICM in 4-cell tetraploid<-->8 cell diploid chimaeras. The aim of the present study was to test whether 4-cell stage embryos in 4-cell diploid<-->8-cell diploid aggregates contributed equally to all lineages present in the E12.5 conceptus. These chimaeras were compared with those produced from standard aggregates of two whole 8-cell embryos and aggregates of half an 8-cell embryo with a whole 8-cell embryo. As expected, the overall contribution of 4-cell embryos was lower than that of 8-cell embryos and similar to that of half 8-cell stage embryos. In the 4-cell<-->8-cell chimaeras the 4-cell stage embryos did not contribute more to the trophectoderm than the ICM derivatives. Thus, differences between 4-cell and 8-cell embryos cannot explain the restricted tissue distribution of tetraploid cells previously reported for 4-cell tetraploid<-->8-cell diploid chimaeras. It is suggested that cells from the more advanced embryo are more likely to contribute to the ICM but, for technical reasons, are prevented from doing so in simple aggregates of equal numbers of whole 4-cell and whole 8-cell stage embryos.

Knowledge-based support for a physician's workstation.

We describe knowledge-based support for a Physician's Workstation prototype. Our knowledge base uses a qualitative simulation model of patient physiology. We present the motivation behind our design, discuss the components of the knowledge base, and show how the knowledge base supports a physician's workstation in the patient management process. We describe a graphical knowledge base editor used by the domain expert for knowledge acquisition, and a graphical knowledge base presenter which monitors the qualitative simulation during patient event processing.

ActiveGuidelines: integrating Web-based guidelines with computer-based patient records.

Use of the World Wide Web provides an efficient means to disseminate guidelines, but integrating them into the workflow at the point of care remains elusive. We developed a method, ActiveGuidelines (AGL), of integrating web-based guidelines with computer-based patient record (CPR) systems. An ActiveGuideline is an HTML document containing special tags that are interpreted by a CPR as actions (e.g., medication order, test order, referral, patient instructions). In our usage scenario, the CPR automatically displays ActiveGuidelines relevant to the current patient context. After reviewing the guideline, the user selects recommended orders directly from the ActiveGuideline. The selected orders are automatically transmitted to the CPR and executed as regular orders. An ActiveGuideline editor facilitates easy conversion of existing HTML-formatted guidelines into ActiveGuidelines. We believe that integrating patient-specific ActiveGuidelines within a CPR system will improve utilization of clinical guidelines in routine patient care.

Traditional medical records as a source of clinical data in the outpatient setting.

We conducted an observational study at a university hospital clinic to determine the success with which physicians find patient information using traditional hospital records as the source of data. We recorded 168 consecutive patient cases presented to attending physicians by internal medicine residents, and analyzed the transcripts to identify questions indicating that the physicians could not find patient information in the medical record. In 81 percent of the cases, physicians could not find all the patient information that they desired during a patient's visit. We performed thematic analysis to generate a set of 15 prototypical questions asked by physicians regarding patient information. The multiauthored medical record system we studied did not provide effective access to patient information for physicians making clinical decisions in an outpatient setting. Improved methods for addressing prototypical questions arising in routine practice are needed.