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The influence of consensus on the assessment of physical disease in chronic community psychiatric patients.

Physical disease is a frequent concomitant of mental disease. The majority of chronic psychiatric patients have physical complaints. Screening physical disease on a regular basis in an ambulatory setting for chronic psychiatric patients is often impossible if only for lack of medical manpower and facilities. Especially for the nonmedical personnel of a community psychiatric unit (CPU) for chronic patients, approaching and handling physical complaints and diseases poses a formidable problem. This paper addresses the issue of screening physical disease from a completely different viewpoint than that of the often advocated but just as often infeasible routine physical examination and laboratory workup. It is argued that consensus (i.e., agreement and mutual understanding) between patient and therapist on the physical well-being of the patient may increase the accuracy with which a CPU therapist, irrespective of his/her discipline, assesses physical disease. This hypothesis was tested in a sample of 156 chronic ambulatory psychiatric patients. Consensus between patient and CPU therapist was measured by means of a questionnaire. The CPU therapist assessment on physical disease in both the consensus and no consensus group was compared with those of two external medical observers; the patients' general practitioner and a specialist in internal medicine, who followed a protocolized physical screening procedure. The results tend to support the proposed hypothesis. Replication of this study using a larger samples is recommended in order to confirm the value of consensus in the assessment of physical disease in at least some subgroups of chronic ambulatory psychiatric patients and for the implications that consensus could have for daily community psychiatric practice.

Adult↗

Estrogen receptor affinity and location of consensus and imperfect estrogen response elements influence transcription activation of simplified promoters.

We have examined the ability of estrogen receptor (ER) to bind and bend DNA fragments containing the Xenopus laevis vitellogenin A2 estrogen response element (ERE), which contains a palindromic, consensus ERE sequence, the X. laevis vitellogenin B1 ERE2, which contains a 1-bp mismatch in the 5'-end of the half-palindrome, and the human pS2 ERE, which contains a 1-bp mismatch in the 3'-end of the half-palindrome. ER binding induced a 65 degrees bend in DNA fragments containing the consensus ERE, the vitellogenin B1 ERE2, or the pS2 ERE. However, ER affinity for the consensus ERE was 2-fold greater than for either the vitellogenin B1 ERE2 or the pS2 ERE. When Chinese hamster ovary (CHO) cells were transfected with reporter plasmids containing either the consensus ERE, the vitellogenin B1 ERE2, or the pS2 ERE separated from the TATA sequence by 26 helical turns, exposure to 10 nm 17 beta-estradiol increased transcription 12.7-, 2.4-, and 3.8-fold, respectively. Increasing the spacing between the ERE and TATA sequence to three helical turns decreased the ability of the consensus ERE to activate transcription by 55% and increased the ability of the pS2 ERE to activate transcription by 35% but had no significant effect on vitellogenin B1 ERE2 activity. Further increasing the distance between the ERE and TATA sequence to 3.6 helical turns restored the activity of promoters containing the consensus ERE and pS2 ERE but decreased the activity of the promoter containing the relatively weak vitellogenin B1 ERE2. These data support the idea that 1) the affinity of ER for the ERE, 2) the location of an ERE within the promoter, and 3) the magnitude and orientation of DNA bends induced by binding of ER or other proteins are important in transcription activation of estrogen-responsive genes.

Animals↗

Assessing the usefulness of anticardiolipin antibody assays: a cautious approach is suggested by high variation and limited consensus in multilaboratory testing.

An anticardiolipin antibody (ACA) assay has become a laboratory standard for the detection of antiphospholipid antibodies. We evaluated data from a quality assurance program to assess ACA assay usefulness. Cross-laboratory (n = 56) testing of 12 serum samples yielded interlaboratory coefficients of variation (CVs) for lgG ACA and IgM ACA that were higher than 50% in 17 (71%) of 24 cases. The situation for testing consensus was of equal concern. Total consensus occurred in 6 (25%) of 24 cases. General consensus (interlaboratory agreement, 90% or more) was obtained in 10 (42%) of 24 cases. In the majority of test cases, laboratories could not agree on whether a serum sample tested was ACA-positive or ACA-negative. Differing method-related issues also were evident; some methods tended toward higher or lower ACA values. Method-based majority consensus differed from participant majority consensus on many test occasions. Exceedingly high interlaboratory result variation, combined with a general lack of test result grading consensus and method-based variation, indicate that a cautious clinical approach toward laboratory findings is prudent. Laboratory tests should be repeated at least once before making a clinical diagnosis of any anti-phospholipid syndrome-like disorder.

Antibodies, Anticardiolipin↗

Canadian consensus guidelines for the diagnosis and management of acromegaly.

Acromegaly is a chronic condition associated with considerably increased morbidity and mortality if left unchecked. In December 2004, a national meeting was held to discuss the diversity in clinical practice across the country in diagnosing and treating patients with acromegaly, as well as to seek consensus on a number of management principles. The group reviewed recent guidelines and discussed issues of diagnosis, treatment, monitoring and treating comorbidities to seek a Canadian consensus on the management of this rare disorder. Consensus was that diagnosis should include clinical and biochemical findings, but is hinged on establishing GH hypersecretion with IGF-I and OGTT testing. Treatment has traditionally included surgical resection or debulking, along with adjunctive medical therapy (primarily somatostatin analogues), if necessary, to normalize GH levels. The option of primary medical therapy in managing this condition has recently emerged and can be justified for non-surgical candidates or for those in whom surgery is not expected to be curative. Overall, improved screening practices and superior epidemiological data are required, since timely diagnosis and appropriate treatment are crucial for reducing the potentially debilitating effects of this chronic, progressive disease. The current evidence also supports the need for long-term follow-up of disease activity and comorbidities in diagnosed patients. A national meeting was held to discuss the diversity in clinical practice across the country in diagnosing and treating patients with acromegaly, as well as to seek consensus on a number of management principles. After brief reviews of the most recent Canadian guidelines and the 2004 guidelines published by the American Association of Clinical Endocrinologists, the group was asked to specifically examine the issues of diagnosis, treatment, monitoring and treating comorbidities and seek a Canadian consensus on practice. This paper summarizes the working group's findings and the points of consensus that were achieved.

Acromegaly↗

Urothelial neoplasia of the urinary bladder--comparison of interobserver variability for WHO Classification 1972 with WHO/ISUP Consensus Classification 1998.

BACKGROUND: Classification of urothelial bladder tumours is an important factor in the treatment and prognosis of these lesions. Over the years many classifications have been proposed for this purpose. The objective of this study was to classify urothelial neoplasms of the urinary bladder using the latest WHO/ISUP Consensus Classification 1998 and WHO Classification 1972 and compare the two regarding interobserver variability. METHODS: This study included 100 consecutive biopsy specimens of urothelial neoplasms of the urinary bladder diagnosed at the department of Histopathology, Armed Forces Institute of Pathology, Rawalpindi. These were classified according to WHO Classification 1972 and WHO/ISUP Consensus Classification 1998 by 2 groups of pathologists independently. The tumour categories for WHO classification 1972; papilloma, and transitional cell carcinoma (TCC) grades I, II and III were compared with the WHO/ISUP Consensus Classification entities of papilloma, papillary neoplasm of low malignant potential, low grade and high grade papillary carcinomas. Kappa statistics were used to evaluate interobserver variability. Chi square test was used to calculate significance. RESULTS: There was agreement on 80 tumours between the two groups of histopathologists when using WHO classification 1972 while there was agreement on 95 tumours using WHO/ISUP consensus classification. The value of Kappa for WHO Classification was 0.68 (good agreement) whereas for WHO/ISUP Consensus Classification it was 0.91 (excellent agreement). The difference between the two systems was statistically significant (p<0,001). Kappa values were less for benign and borderline lesions using both systems. CONCLUSIONS: WHO/ISUP Consensus Classification 1998 showed less interobserver variability than WHO Classification 1972 in the evaluation of bladder tumours. It was found easier to apply by both groups. There was less agreement on the benign and borderline lesions using both the classifications.

Carcinoma, Papillary↗

The carboxyl domain of zinc fingers of the Evi-1 myeloid transforming gene binds a consensus sequence of GAAGATGAG.

Inappropriate expression of the Evi-1 zinc finger gene is associated with myeloid leukemia and myelodysplastic syndromes in mice and humans and has been hypothesized to contribute to pathology by blocking myeloid differentiation. Evi-1 contains two domains of zinc fingers, an amino-terminal domain of seven fingers and a carboxyl domain of three fingers. The first domain binds a consensus sequence of GA(C/T)AAGATAAGATAA in binding and amplication reactions or GATA repeat containing regions of genomic DNA. The experiments described here, establish a consensus sequence for the carboxyl domain of zinc fingers consisting of GAAGATGAG. Unlike the first domain, the consensus sequence established for the carboxyl domain is identical to that which would be predicted by the current rules relating to C2H2 zinc fingers and DNA recognition. Substitution of sequences in finger 8 with those in finger 9, demonstrate that the individual fingers bind the predicted region of the consensus sequence. In an attempt to engineer binding of constructs containing the carboxyl domain, a variety of mutations were made in the middle finger that would be predicted to change the consensus sequence in specific ways. Remarkably, most of the mutations were deleterious and destroyed specific DNA binding. Although Evi-1 contains potential transcriptional activation domains, it was not able to activate gene transcription from CAT constructs containing the consensus sequence.

Animals↗

The medical and surgical determinants of heart transplantation outcomes: the results of a consensus survey in the United States.

Heart transplantation may well be the most successful transplantation procedure performed today. One-year patient survival rates now exceed 80%. Many factors are thought to account for differences in outcomes among transplantation centers. No attempt has been made to assess consensus among transplantation program directors regarding the major determinants of patient outcome. In the National Cooperative Transplantation Study we evaluated consensus through a detailed survey of all heart transplantation programs active in the United States in 1988. Of the eligible programs, 104 (91%) returned completed surveys. Data on the medical and surgical determinants of outcomes have been analyzed descriptively. Considerable consensus occurred among program directors about the importance of several factors. For example, over 90% of the respondents felt that heart biopsy should be used as the standard rejection monitoring technique and that left and right heart catheterization should be performed annually with coronary arteriography. Over 60% believed that the availability of a left ventricular assist device for temporary use would also enhance patient outcome. Several approaches were considered to have little beneficial effect on outcome. These included cytoimmunologic monitoring and electrocardiography as standard rejection monitoring techniques. Nearly one half of the respondents opposed steroid discontinuation after transplantation. On several other approaches there was a lack of consensus including the use of heterotopic heart transplantation and conversion from cyclosporine because of renal dysfunction. Consensus conferences are now regarded as a means by which technologic innovations can be evaluated and medical practice guidelines can be set. This analysis suggests that consensus is a useful approach toward assessing medical and surgical practices in heart transplantation.

Data Collection↗

Consensus statement on the use of intravenous immunoglobulin therapy in the treatment of autoimmune mucocutaneous blistering diseases.

OBJECTIVES: The purpose of the meeting of the Consensus Development Group was to critically evaluate the current published data on the use of intravenous immunoglobulin (IVIg) therapy in the treatment of autoimmune mucocutaneous blistering diseases (AMBDs) and to discuss the industrial preparation and safety features of this biologic agent. PARTICIPANTS: The participants were physicians who frequently treat patients with these diseases and included dermatologists, oral medicine specialists, ophthalmologists, and immunologists. The members of the group provided input and discussion in their areas of expertise. The participants were invited attendees. EVIDENCE: Data samples included only published information in the English-language literature. The expert opinions and experience of the members of the Consensus Development Group were vital to the discussion. CONSENSUS PROCESS: A consensus was achieved by an open discussion and cumulative agreement on all issues relevant to the use of IVIg therapy in the treatment of AMBDs. Special emphasis was placed on indications for its use, determination of outcome parameters, and development of a protocol for its therapeutic use. We also focused on its safety and on prevention of adverse effects. CONCLUSION: This consensus statement outlines the scope of IVIg treatment; provides guidelines for its use, including indications, prescreening, premedications, dose, frequency, and monitoring; and defines the end point of therapy.

Chemistry, Pharmaceutical↗

Depression and Bipolar Support Alliance consensus statement on the unmet needs in diagnosis and treatment of mood disorders in late life.

OBJECTIVES: To review progress made during the past decade in late-life mood disorders and to identify areas of unmet need in health care delivery and research. PARTICIPANTS: The Consensus Development Panel consisted of experts in late-life mood disorders, geriatrics, primary care, mental health and aging policy research, and advocacy. EVIDENCE: (1) Literature reviews addressing risk factors, prevention, diagnosis, treatment, and delivery of services and (2) opinions and experiences of primary care and mental health care providers, policy analysts, and advocates. CONSENSUS PROCESS: The Consensus Development Panel listened to presentations and participated in discussions. Workgroups considered the evidence and prepared preliminary statements. Workgroup leaders presented drafts for discussion by the Consensus Development Panel. The final document was reviewed and edited to incorporate input from the entire Consensus Development Panel. CONCLUSIONS: Despite the availability of safe and efficacious treatments, mood disorders remain a significant health care issue for the elderly and are associated with disability, functional decline, diminished quality of life, mortality from comorbid medical conditions or suicide, demands on caregivers, and increased service utilization. Discriminatory coverage and reimbursement policies for mental health care are a challenge for the elderly, especially those with modest incomes, and for clinicians. Minorities are particularly underserved. Access to mental health care services for most elderly individuals is inadequate, and coordination of services is lacking. There is an immediate need for collaboration among patients, families, researchers, clinicians, governmental agencies, and third-party payers to improve diagnosis, treatment, and delivery of services for elderly persons with mood disorders.

Age Factors↗

Development of performance indicators for the primary care management of pediatric epilepsy: expert consensus recommendations based on the available evidence.

PURPOSE: To use available evidence and expert consensus to develop performance indicators for the evaluation and management of pediatric epilepsy. METHODS: We used a three-step process to develop the performance indicators. First, research findings were compiled into evidence tables focusing on different clinical issues. Second, an advisory panel of clinicians, educational and public health experts, and families of children with epilepsy reviewed the evidence. The advisory group used the evidence to draft a preliminary set of performance indicators for pediatric epilepsy management. Third, 13 internationally recognized experts in pediatric neurology or epilepsy rated the value of these indicators on a 5-point scale [1 (essential) to 5 (not necessary)] in a two-round Delphi process. Positive consensus was reached if >or=80% of experts gave an indicator a "1" rating and negative consensus if >80% gave an indicator a "5" rating. Indicators that achieved positive consensus during either round of the Delphi process constituted the final set of indicators. RESULTS: Of the 68 draft performance indicators, the expert panel members achieved positive consensus on 30 performance indicators: eight indicators related to diagnostic strategies and seizure classification, nine related to antiepileptic drug use, six related to cognitive and behavioral issues, six related to quality of life, and three related to specialty referrals. CONCLUSIONS: We identified 30 potential indicators for evaluating the care provided to pediatric patients with epilepsy. The next step is to examine the relation of these performance indicators to clinical outcomes and health care utilization among pediatric patients with epilepsy.

Adult↗

The Expert Consensus Guideline Series. Treatment of behavioral emergencies.

OBJECTIVES: Behavioral emergencies are a common and serious problem for consumers, their communities, and the healthcare settings on which they rely to contain, assess, and ultimately help the individual in a behavioral crisis. Partly because of the inherent dangers of this situation, there is little research to guide provider responses to this challenge. Key constructs such as agitation have not been adequately operationalized so that the criteria defining a behavioral emergency are vague. The significant progress that has been made for some disease states with better treatments and higher consumer acceptance has not penetrated this area of practice. A significant number of deaths of patients in restraint has focused government and regulators on these issues, but a consensus about key elements in the management of behavioral emergencies has not yet been articulated by the provider community. The authors assembled a panel of 50 experts to define the following elements: the threshold for emergency interventions, the scope of assessment for varying levels of urgency and cooperation, guiding principles in selecting interventions, and appropriate physical and medication strategies at different levels of diagnostic confidence and for a variety of etiologies and complicating conditions. METHOD: In order to identify issues in this area on which there is consensus, a written survey with 808 decision points was developed. The survey was mailed to a panel of 52 experts, 50 of whom completed it. A modified version of the RAND Corporation 9-point scale for rating appropriateness of medical decisions was used to score options. Consensus on each option was defined as a non-random distribution of scores by chi-square "goodness-of-fit" test. We assigned a categorical rank (first line/preferred choice, second line/alternate choice, third line/usually inappropriate) to each option based on the 95% confidence interval around the mean rating. Guideline tables were constructed describing the preferred strategies in key clinical situations. RESULTS: The expert panel reached consensus on 83% of the options. The relative appropriateness of emergency interventions was ascertained for a continuum of behaviors. When asked about the frequency with which emergency interventions (parenteral medication, restraints, seclusion) were required in their services, 47% of the experts reported that such interventions were necessary for 1%-5% of patients seen in their services and 32% for 6%-20%. In general, the consensus of this panel lends support to many elements of recent Health Care Financing Administration regulations, including the timing of clinician assessment and reassessment and the intensity of nursing care. However, the panel did not endorse the concept of "chemical restraint," instead favoring the idea that medications are treatments for target behaviors in behavioral emergencies even when the causes of these behaviors are not well understood. Control of aggressive behavior emerged as the highest priority during the emergency; however, preserving the physician-patient relationship was rated a close second and became the top priority in the long term. Oral medications, particularly concentrates, were clearly preferred if it is possible to use them. Benzodiazepines alone were top rated in 6 of 12 situations. High-potency conventional antipsychotics used alone never received higher ratings than benzodiazepines used alone. A combination of a benzodiazepine and an antipsychotic was preferred for patients with suspected schizophrenia, mania, or psychotic depression. There was equal support for high-potency conventional or atypical antipsychotics (particularly liquids) in oral combinations with benzodiazepines. Droperidol emerged in fourth place in some situations requiring an injection. CONCLUSIONS: To evaluate many of the treatment options in this survey, the experts had to extrapolate beyond controlled data in comparing modalities with each other or in combination. Within the limits of expert opinion and with the expectation that future research data will take precedence, these guidelines provide some direction for addressing common clinical dilemmas in the management of psychiatric emergencies and can be used to inform clinicians in acute care settings regarding the relative merits of various strategies.

Aggression↗

The Expert Consensus Guideline Series. Treatment of depression in women.

OBJECTIVES: Women constitute two-thirds of patients suffering from common depressive disorders. The treatment of depression in women is therefore a substantial public health concern. High-quality, empirical data on depressive disorders specific to women are limited. As a result, there are no comprehensive evidence-based practice guidelines on the best treatment approaches for these illnesses. We conducted a consensus survey of expert opinion on the treatment of 4 depressive conditions specific to women: premenstrual dysphoric disorder (PMDD), depression in pregnancy, postpartum depression in a mother choosing to breast-feed, and depression related to perimenopause/menopause. METHOD: After reviewing the literature and convening a work group of leading experts, we prepared a written survey covering a total of 858 treatment options in 117 specific clinical situations. Depression severity (mild to severe) was specified for most clinical situations. Treatment options included a broad range of pharmacological, psychosocial, and alternative medicine approaches. Most options were scored using a modified version of the RAND 9-point scale for rating appropriateness of medical decisions. We identified 40 national experts, 36 (90%) of whom completed the survey. Consensus on each option was defined as a non-random distribution of scores by chi-square "goodness-of-fit" test. We assigned a categorical rank (first line/preferred choice, second line/alternate choice, third line/usually inappropriate) to each option based on the 95% confidence interval around the mean rating. Guideline tables indicating preferred treatment strategies were then developed for key clinical situations. RESULTS: The expert panel reached consensus on 76% of the options, with greater consensus in situations involving severe symptoms. For women with severe symptoms in each of the 4 central disorder areas we asked about, the first-line recommendation was for antidepressant medication combined with other modalities (generally psychotherapy), paralleling existing guidelines for severe depression in general populations. For milder symptoms in each situation, the panel was less uniform in recommending antidepressants. For the initial treatment of milder symptoms, the panel either gave equal endorsement to other treatment modalities (e.g., nutritional or psychobehavioral approaches in PMDD; hormone replacement in perimenopause) or preferred psychotherapy over medication (in conception, pregnancy, or lactation). In all milder cases, however, antidepressants were recommended as at least second-line options. Among antidepressants, selective serotonin reuptake inhibitors (SSRIs) as a class were recommended as first-line treatment in all situations. The specific SSRIs that were preferred depended on the particular clinical situation. Tricyclic antidepressants were highly rated alternatives to SSRIs in pregnancy and lactation. CONCLUSIONS: The experts reached a high level of consensus on the appropriateness of including both antidepressant medication, specifically SSRIs, and nonpharmacological modalities in treatment plans for severe depression in 4 key clinical situations unique to women. To evaluate many of the treatment options in this survey, the experts had to extrapolate beyond controlled data in comparing modalities with each other or in combination. Within the limits of expert opinion and with the expectation that future research data will take precedence, these guidelines provide some direction for addressing common clinical dilemmas in women. They can be used to inform clinicians and educate patients regarding the relative merits of a variety of interventions.

Antidepressive Agents↗

The NIH Consensus Development Conference on the Diagnosis and Treatment of Depression in Late Life: an overview.

The National Institutes of Health (NIH) Consensus Development Conference on the Diagnosis and Treatment of Depression in Late Life took place on November 4-6, 1991. The full statement of the Consensus Panel, chaired by Arnold J. Friedhoff, M.D., was published in a Consensus Statement and in the Journal of the American Medical Association. The purpose of this article is to provide a brief overview of the planning for the Consensus Development Conference, with particular emphasis on the questions posed by the National Institute of Mental Health (NIMH) Planning Committee to members of the panel. This is followed by a brief synopsis of the panel's opinions on issues raised by the NIMH Planning Committee, as well as their opinions on the most promising issues for future research in late-life depression. The main purposes of the NIH Consensus Development Conference were first, to translate research findings into guidelines for clinical practice, so far as this is possible, and second, to suggest promising directions for the Nation's research agenda in late-life depression.

Aged↗

Report of the Sinus Consensus Conference of 1996.

Retrospective data from sinus floor augmentation bone grafts were collected from 38 surgeons for 1007 sinus grafts that involved the placement of 2997 implants over a 10-year period, with the majority of the implants followed for 3 years or more postrestoration. There were 229 implant failures reported. Various root-form implants and grafting modalities were used. A consensus conference was organized to evaluate the data and reach a consensus on optimal treatment protocols. The complete database demonstrated a 90.0% success rate for implants placed in sinus grafts with at least 3 years of function. Differences in grafting materials, implant surfaces, and timing protocols were statistically analyzed. However, the database was so multivariate and multifactorial that it was difficult to draw definitive conclusions; these must await controlled prospective studies. The consensus conference therefore developed and voted on multiple consensus statements derived by committee review for bone graft materials, type of implants, timing for implant placement, failure analysis, radiographic analysis, indications/contraindications, prosthetics, and nomenclature. Several consensus statements were obtained, the most significant being that the sinus graft should now be considered a highly predictable and effective therapeutic modality.

Bone Substitutes↗

Angelman syndrome 2005: updated consensus for diagnostic criteria.

In 1995, a consensus statement was published for the purpose of summarizing the salient clinical features of Angelman syndrome (AS) to assist the clinician in making a timely and accurate diagnosis. Considering the scientific advances made in the last 10 years, it is necessary now to review the validity of the original consensus criteria. As in the original consensus project, the methodology used for this review was to convene a group of scientists and clinicians, with experience in AS, to develop a concise consensus statement, supported by scientific publications where appropriate. It is hoped that this revised consensus document will facilitate further clinical study of individuals with proven AS, and assist in the evaluation of those who appear to have clinical features of AS but have normal laboratory diagnostic testing.

Angelman Syndrome↗

Thermodynamics of the interactions of lac repressor with variants of the symmetric lac operator: effects of converting a consensus site to a non-specific site.

What are the thermodynamic consequences of the stepwise conversion of a highly specific (consensus) protein-DNA interface to one that is nonspecific? How do the magnitudes of key favorable contributions to complex stability (burial of hydrophobic surfaces and reduction of DNA phosphate charge density) change as the DNA sequence of the specific site is detuned? To address these questions we investigated the binding of lac repressor (LacI) to a series of 40 bp fragments carrying symmetric (consensus) and variant operator sequences over a range of temperatures and salt concentrations. Variant DNA sites contained symmetrical single and double base-pair substitutions at positions 4 and/or 5 [sequence: see text] in each 10 bp half site of the symmetric lac operator (Osym). Non-specific interactions were examined using a 40 bp non-operator DNA fragment. Disruption of the consensus interface by a single symmetrical substitution reduces the observed equilibrium association constant (K(obs)) for Osym by three to four orders of magnitude; double symmetrical substitutions approach the six orders in magnitude difference between specific and non-specific binding to a 40 bp fragment. At these adjacent positions in the consensus site, the free energy effects of multiple substitutions are non-additive: the first reduces /deltaG(obs)o/ by 3 to 5 kcal mol(-1), approximately halfway to the non-specific level, whereas the second is less deleterious, reducing /deltaG(obs)o/ by less than 3 kcal mol(-1). Variant-specific dependences of K(obs) on temperature and salt concentration characterize these LacI-operator interactions. In general, binding constants and standard free energies of binding both exhibit characteristic extrema near 290 K. As a consequence, both the enthalpic and entropic contributions to stability of Osym and variant complexes change from positive (i.e. entropy driven) at lower temperatures to negative (i.e. enthalpy driven) at higher temperatures, indicating that the heat capacity change upon binding, deltaC(obs)o, is large and negative. In general, /deltaC(obs)o/ decreases as the specificity and stability of the variant complex decreases. Stabilities of complexes of LacI with Osym and all variant operators are strongly [salt]-dependent. Binding constants for the variant complexes exhibit a power-dependence on [salt] that is larger in magnitude (i.e. more negative) than for Osym, but no obvious trend relates changes in contributions from the polyelectrolyte effect and the observed reductions in stability (delta deltaG(obs)o). These variant-specific thermodynamic signatures provide novel insights into the consequences of converting a consensus interface to a less specific one; such insights are not obtained from comparisons at the level of delta deltaG(obs)o. We propose that this variant-specific behavior arises from a strong effect of operator sequence on the extent of induced conformational changes in the protein (and possibly also in the DNA site) which accompany binding.

Bacterial Proteins↗

Coronavirus transcription mediated by sequences flanking the transcription consensus sequence.

In our studies of murine coronavirus transcription, we continue to use defective interfering (DI) RNAs of mouse hepatitis virus (MHV) in which we insert a transcription consensus sequence in order to mimic subgenomic RNA synthesis from the nondefective genome. Using our subgenomic DI system, we have studied the effects of sequences flanking the MHV transcription consensus sequence on subgenomic RNA transcription. We obtained the following results. (i) Insertion of a 12-nucleotide-long sequence including the UCUAAAC transcription consensus sequence at different locations of the DI RNA resulted in different efficiencies of subgenomic DI RNA synthesis. (ii) Differences in the amount of subgenomic DI RNA were defined by the sequences that flanked the 12-nucleotide-long sequence and were not affected by the location of the 12-nucleotide-long sequence on the DI RNA. (iii) Naturally occurring flanking sequences of intergenic sequences at gene 6-7, but not at genes 1-2 and 2-3, contained a transcription suppressive element(s). (iv) Each of three naturally occurring flanking sequences of an MHV genomic cryptic transcription consensus sequence from MHV gene 1 also contained a transcription suppressive element(s). These data showed that sequences flanking the transcription consensus sequence affected MHV transcription.

Animals↗

Features of the 3'-consensus sequence of rotavirus mRNAs critical to minus strand synthesis.

The last seven nucleotides of the 3'-end of rotavirus mRNAs, 5'-UGUGACC-3', are highly conserved and form a cis-acting signal that can promote the synthesis of (-) strand RNA to produce the viral dsRNA genome in vitro. Previous studies have shown that the sequence, location, and strandedness (single- versus double-stranded) of the 3'-consensus sequence of the mRNA affect the efficiency of (-) strand synthesis. In this study, we have used exhaustive mutagenesis of the SA11 gene 8 mRNA and an in vitro replication system to define the importance of each of the residues in the consensus sequence in (-) strand synthesis. The analysis showed that the CC of the consensus sequence was the most critical for (-) strand synthesis. Furthermore, the data revealed that other, but not all, residues of the consensus sequence contributed to efficient (-) strand synthesis in vitro. Mutant gene 8 RNAs supported an intermediate level of (-) strand synthesis when the 15 nt sequence upstream of the CC was replaced with long tracts of poly(A) or poly(U), but not with poly(G). Predictions of the secondary structure of the mutant RNAs suggested that the poly(G)-RNA could not replicate because its 3'-terminus was largely basepaired, instead of extending as a single-stranded tail as is the case for the 3'-termini of the poly(A)- and poly(U)-RNAs and wild-type gene 8 RNA. Subsequent experiments performed with complementary oligonucleotides indicated that efficient RNA replication occurs in vitro only when the last four residues of the 3'-consensus sequence, and most importantly the two terminal C's, existed in a single-stranded form. A single-stranded CC may be crucial for formation of an initiation complex for (-) strand synthesis consisting of viral RdRP, mRNA, and the dinucleotide pGpG.

Animals↗