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PubMed1993

The prevention and management of urinary tract infections among people with spinal cord injuries. National Institute on Disability and Rehabilitation Research consensus statement. January 27-29, 1992.

The Urinary Tract Infection Consensus Conference brought together researchers, clinicians, and consumers to arrive at consensus on the best practices for preventing and treating urinary tract infections in people with spinal cord injuries; the risk factor and diagnostic studies that should be done; indications for antibiotic use; appropriate follow-up management; and needed future research. Urinary tract infection (UTI) was defined as bacteriuria (102 bacteria/ml of urine) with tissue invasion and resultant tissue response with signs and/or symptoms. Asymptomatic bacteriuria represents colonization of the urinary tract without symptoms or signs. Risk factors include: over-distention of bladder, vesicoureteral reflux, high pressure voiding, large post-void residuals, presence of stones in urinary tract, and outlet obstruction. Possible physiologic/structural, behavioral, and demographic risk factors were identified also, Indwelling catheterization, including suprapubic, and urinary diversion and the drainage methods most likely to lead to persistent bacteriuria. Infection risk is reduced with intermittent catheterization, but more severely disabled people who require catheterization by others are at greater risk for UTIs. Clean self-intermittent catheterization does not pose a greater risk of infection than sterile self-intermittent catheterization and is much more economic. However, care must be given to proper cleansing of reusable catheters. Quantitative urine-culture criteria for the diagnosis of bacteriuria include: catheter specimens from individuals on intermittent catheterization > or = 10(2) cfu/ml; clean-void specimens from catheter-free males using condom collection devices > or = 10(4) cfu/ml; and specimens from indwelling catheters of any detectable concentration. Dip stick screening tests may offer promise as an early warning system of UTI since they can be self-administered. Symptomatic UTI should be treated with antibiotics for 7 to 14 days. Longer courses have not been beneficial. In patients with symptomatic UTIs, it is not necessary to wait for the results of cultures before starting treatment. Asymptomatic bacteriuria need not be treated with antibiotics. There is little evidence presently to support the use of antibiotics to prevent infections. Following a recent episode of febrile UTI, possible contributing prior events should be reviewed. The upper tracts should be evaluated (imaging studies) to identify possible abnormalities. A common concern among people with spinal cord injuries is that physicians will alter bladder management programs without regard to lifestyle needs. Social/vocational flexibility may be more important to them than a state-of-the-art bladder management program. Future research should focus on obtaining more representative samples and investigate psycho-social-vocational implications as well as additional clinical-medical factors.

Anti-Bacterial Agents↗
PubMed1993

Drug treatment of depression in the frail elderly: discussion of the NIH Consensus Development Conference on the Diagnosis and Treatment of Depression in Late Life.

The priorities for future research from the NIH Consensus Development Conference on the Diagnosis and Treatment of Depression in Late Life, as republished in the previous article, included recommendations to "conduct clinical trials and observational studies of treatment in the very old, the elderly in institutional setting, and the elderly with medical illnesses," that is, in the frail elderly. The present article reviews recent research in this field and outlines the potential for future developments. The importance of these areas of investigation follows from epidemiological findings suggesting that the prevalence of major depression in community populations, in general, decreases as a function of age but that depressions of all types occur more frequently in the "oldest-old," in patients seen in medical care settings, and in those with chronic disease and disability. The psychopharmacological literature, as summarized for the Consensus Conference in the review by Salzman and the meta-analyses of Klawansky, Greenhouse, and Schneider, indicates that antidepressant medications remain effective in elderly patients with moderate to severe degrees of major depression. Questions remain, however, about the value of drug treatment for those depressions that are most common in late life, including those that occur in extremely old patients and in patients with significant medical illness.

Aged↗
PubMed1996

Physical activity and cardiovascular health. NIH Consensus Development Panel on Physical Activity and Cardiovascular Health.

OBJECTIVE: To provide physicians and the general public with a responsible assessment of the relationship between physical activity and cardiovascular health. PARTICIPANTS: A nonfederal, nonadvocate, 13-member panel representing the fields of cardiology, psychology, exercise physiology, nutrition, pediatrics, public health, and epidemiology. In addition, 27 experts in cardiology, psychology, epidemiology, exercise physiology, geriatrics, nutrition, pediatrics, public health, and sports medicine presented data to the panel and a conference audience of 600 during a 2-day public session. Questions and statements from conference attendees were considered during the open session. Closed deliberations by the panel occurred during the remainder of the second day and the morning of the third day. EVIDENCE: The literature was searched through MEDLINE and an extensive bibliography of references was provided to the panel and the conference audience. Experts prepared abstracts with relevant citations from the literature. Scientific evidence was given precedence over clinical anecdotal experience. CONSENSUS PROCESS: The panel, answering predefined questions, developed their conclusions based on the scientific evidence presented in open forum and the scientific literature. CONSENSUS STATEMENT: The panel composed a draft statement that was read in its entirety and circulated to the experts and the audience for comment. There-after, the panel resolved conflicting recommendations and released a revised statement at the end of the conference. The panel finalized the revisions within a few weeks after the conference. CONCLUSIONS: All Americans should engage in regular physical activity at a level appropriate to their capacity, needs, and interest. Children and adults alike should set a goal of accumulating at least 30 minutes of moderate-intensity physical activity on most, and preferably all, days of the week. Most Americans have little or no physical activity in their daily lives, and accumulating evidence indicates that physical inactivity is a major risk factor for cardiovascular disease. However, moderate levels of physical activity confer significant health benefits. Even those who currently meet these daily standards may derive additional health and fitness benefits by becoming more physically active or including more vigorous activity. For those with known cardiovascular disease, cardiac rehabilitation programs that combine physical activity with reduction in other risk factors should be more widely used.

Adult↗
PubMed1998

NIH Consensus Conference. Acupuncture.

OBJECTIVE: To provide clinicians, patients, and the general public with a responsible assessment of the use and effectiveness of acupuncture to treat a variety of conditions. PARTICIPANTS: A nonfederal, nonadvocate, 12-member panel representing the fields of acupuncture, pain, psychology, psychiatry, physical medicine and rehabilitation, drug abuse, family practice, internal medicine, health policy, epidemiology, statistics, physiology, biophysics, and the representatives of the public. In addition, 25 experts from these same fields presented data to the panel and a conference audience of 1200. Presentations and discussions were divided into 3 phases over 2 1/2 days: (1) presentations by investigators working in areas relevant to the consensus questions during a 2-day public session; (2) questions and statements from conference attendees during open discussion periods that were part of the public session; and (3) closed deliberations by the panel during the remainder of the second day and morning of the third. The conference was organized and supported by the Office of Alternative Medicine and the Office of Medical Applications of Research, National Institutes of Health, Bethesda, Md. EVIDENCE: The literature, produced from January 1970 to October 1997, was searched through MEDLINE, Allied and Alternative Medicine, EMBASE, and MANTIS, as well as through a hand search of 9 journals that were not indexed by the National Library of Medicine. An extensive bibliography of 2302 references was provided to the panel and the conference audience. Expert speakers prepared abstracts of their own conference presentations with relevant citations from the literature. Scientific evidence was given precedence over clinical anecdotal experience. CONSENSUS PROCESS: The panel, answering predefined questions, developed their conclusions based on the scientific evidence presented in the open forum and scientific literature. The panel composed a draft statement, which was read in its entirety and circulated to the experts and the audience for comment. Thereafter, the panel resolved conflicting recommendations and released a revised statement at the end of the conference. The panel finalized the revisions within a few weeks after the conference. The draft statement was made available on the World Wide Web immediately following its release at the conference and was updated with the panel's final revisions within a few weeks of the conference. The statement is available at http://consensus.nih.gov. CONCLUSIONS: Acupuncture as a therapeutic intervention is widely practiced in the United States. Although there have been many studies of its potential usefulness, many of these studies provide equivocal results because of design, sample size, and other factors. The issue is further complicated by inherent difficulties in the use of appropriate controls, such as placebos and sham acupuncture groups. However, promising results have emerged, for example, showing efficacy of acupuncture in adult postoperative and chemotherapy nausea and vomiting and in postoperative dental pain. There are other situations, such as addiction, stroke rehabilitation, headache, menstrual cramps, tennis elbow, fibromyalgia, myofascial pain, osteoarthritis, low back pain, carpal tunnel syndrome, and asthma, in which acupuncture may be useful as an adjunct treatment or an acceptable alternative or be included in a comprehensive management program. Further research is likely to uncover additional areas where acupuncture interventions will be useful.

Acupuncture Therapy↗
PubMed1998

Effective medical treatment of opiate addiction. National Consensus Development Panel on Effective Medical Treatment of Opiate Addiction.

OBJECTIVE: To provide clinicians, patients, and the general public with a responsible assessment of the effective approaches to treat opiate dependence. PARTICIPANTS: A nonfederal, nonadvocate, 12-member panel representing the fields of psychology, psychiatry, behavioral medicine, family medicine, drug abuse, epidemiology, and the public. In addition, 25 experts from these same fields presented data to the panel and a conference audience of 600. Presentations and discussions were divided into 3 phases over 2 1/2 days: (1) presentations by investigators working in the areas relevant to the consensus questions during a 2-day public session; (2) questions and statements from conference attendees during open discussion periods that are part of the public session; and (3) closed deliberations by the panel during the remainder of the second day and morning of a third day. The conference was organized and supported by the Office of Medical Applications of Research, National Institutes of Health. EVIDENCE: The literature was searched through MEDLINE and other National Library of Medicine and online databases from January 1994 through September 1997 and an extensive bibliography of 941 references was provided to the panel and the conference audience. Experts prepared abstracts for their presentations as speakers at the conference with relevant citations from the literature. Scientific evidence was given precedence over clinical anecdotal experience. CONSENSUS PROCESS: The panel, answering predefined questions, developed its conclusions based on the scientific evidence presented in open forum and the scientific literature. The panel composed a draft statement that was read in its entirety and circulated to the experts and the audience for comment. Thereafter, the panel resolved conflicting recommendations and released a revised statement at the end of the conference. The panel finalized the revisions within a few weeks after the conference. The draft statement was made available on the World Wide Web immediately following its release at the conference and was updated with the panel's final revisions. CONCLUSIONS: Opiate dependence is a brain-related medical disorder that can be effectively treated with significant benefits for the patient and society, and society must make a commitment to offer effective treatment for opiate dependence to all who need it. All persons dependent on opiates should have access to methadone hydrochloride maintenance therapy under legal supervision, and the US Office of National Drug Control Policy and the US Department of Justice should take the necessary steps to implement this recommendation. There is a need for improved training for physicians and other health care professionals. Training to determine diagnosis and treatment of opiate dependence should also be improved in medical schools. The unnecessary regulations of methadone maintenance therapy and other long-acting opiate agonist treatment programs should be reduced, and coverage for these programs should be a required benefit in public and private insurance programs.

Cost of Illness↗
PubMedDate not supplied

Guidelines for nonemergency use of parenteral phenytoin products: proceedings of an expert panel consensus process. Panel on Nonemergency Use of Parenteral Phenytoin Products.

This document summarizes the proceedings of an expert panel consensus process addressing the nonemergency use of parenteral phenytoin products for management of seizures in pediatric and adult patients. The algorithm and consensus statements developed by the expert panel emphasize strategies for lowering the probability of adverse events associated with the use of parenteral phenytoin products. Specific patient characteristics are defined to guide administration and monitoring of parenteral phenytoin therapy. The algorithm provides a decision pathway for the selection of the product and the route of administration of phenytoin sodium or fosphenytoin sodium after it has been determined that a parenteral phenytoin product is appropriate. Key factors covered in the algorithm include a list of patient characteristics and considerations necessary to prevent parenteral phenytoin adverse effects during selection of administration route and recommendations for monitoring of parenteral phenytoin therapy once it has been initiated. Situations requiring rapid attainment of high phenytoin concentrations, such as in the management of acute seizures, are not addressed in these guidelines.

Adolescent↗
PubMed2000

Prevention of venous thromboembolism: adherence to the 1995 American College of Chest Physicians consensus guidelines for surgical patients.

BACKGROUND: The American College of Chest Physicians addressed the dilemma of identifying optimal therapy for venous thromboembolism (VTE) prophylaxis and published their Fourth Consensus Conference on Antithrombotic Therapy in 1995, with recommendations for prophylactic therapy. Despite these recommendations, appropriate VTE prophylactic therapy is underused. OBJECTIVES: To examine routine practices in the prevention of VTE in high-risk surgical patients and to determine the extent of adoption of grade A prophylactic therapies as recommended by the American College of Chest Physicians. METHODS: Retrospective medical record review in 10 teaching or community-based hospitals located in the United States. Medical charts of 1907 patients were randomly selected for review from the population of patients who underwent high-risk major abdominal surgery, total hip replacement, hip fracture repair, or total knee replacement between January 1, 1996, and February 28, 1997. RESULTS: Of 1907 patients, VTE prophylaxis was used in 89.3%; use was 93.7% in each of the 3 orthopedic surgery groups and 75.2% in the high-risk major abdominal surgery group. The percentage of patients receiving grade A therapy was highest in the hip replacement group (84.3%) vs. the other groups (knee replacement, 75.9%; hip fracture repair, 45.2%; abdominal surgery, 50.3%). CONCLUSIONS: The use of grade A prophylaxis was related to the type of surgery, with the highest use seen in total hip replacement and the lowest in hip fracture repair. One in 4 patients who underwent high-risk major abdominal surgeries failed to receive any form of VTE prophylaxis. Publication of consensus statements alone may be insufficient to ensure the incorporation of important new clinical information into routine practice.

Adult↗
PubMed2000

Pulmonary artery catheterization and clinical outcomes: National Heart, Lung, and Blood Institute and Food and Drug Administration Workshop Report. Consensus Statement.

OBJECTIVE: The efficacy and safety of the pulmonary artery catheter are under scrutiny because of its association with increased morbidity and mortality in observational studies. In response, the National Heart, Lung, and Blood Institute (NHLBI) and the US Food and Drug Administration (FDA) conducted the Pulmonary Artery Catheterization and Clinical Outcomes workshop in Alexandria, Va, on August 25 and 26, 1997, to develop recommendations regarding actions to improve pulmonary artery catheter utility and safety. PARTICIPANTS: The NHLBI and FDA planning task force selected a workshop chairperson, subcommittee chairs, and participants. Approximately 85 participants were selected for their collective expertise in critical care, pulmonary medicine, cardiovascular medicine and surgery, pediatrics, nursing, biostatistics, and medical economics. The meeting was open to industry representatives and other government and lay observers. This workshop was funded by the NHLBI and the FDA's Division of Devices. EVIDENCE: Published reports relating to the efficacy and safety of the pulmonary artery catheter, especially consensus documents developed by professional societies. CONSENSUS PROCESS: The planning task force disseminated materials, held teleconferences, and developed draft position papers prior to the workshop. These were modified during the workshop and thereafter in the course of several teleconferences, and presented to the entire group for final modifications and approval. CONCLUSIONS: A need exists for collaborative education of physicians and nurses in performing, obtaining, and interpreting information from the use of pulmonary artery catheters. This effort should be led by professional societies, in collaboration with federal agencies, with the purpose of developing and disseminating standardized educational programs. Areas given high priority for clinical trials were pulmonary artery catheter use in persistent/refractory congestive heart failure, acute respiratory distress syndrome, severe sepsis and septic shock, and low-risk coronary artery bypass graft surgery. JAMA. 2000;283:2568-2572

Catheterization, Swan-Ganz↗
PubMed1997

Evaluation of mental retardation: recommendations of a Consensus Conference: American College of Medical Genetics.

A Consensus Conference utilizing available literature and expert opinion sponsored by the American College of Medical Genetics in October 1995 evaluated the rational approach to the individual with mental retardation. Although no uniform protocol replaces individual clinician judgement, the consensus recommendations were as follows: 1. The individual with mental retardation, the family, and medical care providers benefit from a focused clinical and laboratory evaluation aimed at establishing causation and in providing counseling, prognosis, recurrence risks, and guidelines for management. 2. Essential elements of the evaluation include a three-generation pedigree: pre-, peri-, and post-natal history, complete physical examination focused on the presence of minor anomalies, neurologic examination, and assessment of the behavioral phenotype. 3. Selective laboratory testing should, in most patients, include a banded karyotype. Fragile X testing should be strongly considered in both males and females with unexplained mental retardation, especially in the presence of a positive family history, a consistent physical and behavioral phenotype and absence of major structural abnormalities. Metabolic testing should be initialed in the presence of suggestive clinical and physical findings. Neuroimaging should be considered in patients without a known diagnosis especially in the presence of neurologic symptoms, cranial contour abnormalities, microcephaly, or macrocephaly. In most situations MRI is the testing modality of choice. 4. Sequential evaluation of the patient, occasionally over several years, is often necessary for diagnosis, allowing for delineation of the physical and behavioral phenotype, a logical approach to ancillary testing and appropriate prognostic and reproductive counseling.

Diagnosis, Differential↗
PubMed1999

Proximal promoter of the rat brain creatine kinase gene lacks a consensus CRE element but is essential for the cAMP-mediated increased transcription in glioblastoma cells.

Our previous studies have shown that transcription of brain creatine kinase (CKB) mRNA in U87-MG glioblastoma cells is stimulated by a forskolin-mediated increase in cyclic AMP (cAMP) via a pathway involving protein kinase A (PKA) and the activation of Galphas proteins. In this report, we have employed transient transfection to investigate the rat CKB gene elements essential for the cAMP-mediated induction of rat CKB transcription in human U87 cells and have mapped the transcription start site of the induced CKB transcripts. We found that the level of induced transcription from the transfected genomic rat CKB gene was the same whether transcription was driven by 2.9 kb of CKB promoter plus 5' flanking sequence or the 0.2 kb CKB promoter, suggesting that the proximal CKB promoter was essential. Also, the level of induced transcription of the chloramphenicol acetyl transferase (CAT) reporter gene driven by the 2.9 kb CKB promoter was the same as with the 0.2 kb CKB promoter. Analyses of a series of 5' deletions of the 0.2 kb proximal CKB promoter showed that the sequences between -80 bp and +1 bp were essential for the cAMP-mediated induction of CKB transcription, despite the absence of a consensus cAMP response element (CRE) sequence in that region. In agreement, gel mobility shift assays showed that nuclear extracts from U87 cells contained a protein(s) which bound specifically to a [32P]CKB DNA probe containing the -60 bp to +1 bp sequence. Mapping the 5' end of the CKB transcripts showed that the initiation of the cAMP-induced transcription occurred almost exclusively from the downstream transcription start site, apparently under the initiation direction of the nonconsensus (-28) TTAA element and not the consensus (-60) TATAAATA element. The results are discussed with regard to nuclear protein factors which may be involved, and the possible cAMP-mediated increase in CKB transcription during myelinogenesis, since the differentiation of oligodendrocytes has previously been shown to be accelerated by increased intracellular cAMP.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Glycosaminoglycan-protein interactions: definition of consensus sites in glycosaminoglycan binding proteins.

Although interactions of proteins with glycosaminoglycans (GAGs), such as heparin and heparan sulphate, are of great biological importance, structural requirements for protein-GAG binding have not been well-characterised. Ionic interactions are important in promoting protein-GAG binding. Polyelectrolyte theory suggests that much of the free energy of binding comes from entropically favourable release of cations from GAG chains. Despite their identical charges, arginine residues bind more tightly to GAGs than lysine residues. The spacing of these residues may determine protein-GAG affinity and specificity. Consensus sequences such as XBBBXXBX, XBBXBX and a critical 20 A spacing of basic residues are found in some protein sites that bind GAG. A new consensus sequence TXXBXXTBXXXTBB is described, where turns bring basic interacting amino acid residues into proximity. Clearly, protein-GAG interactions play a prominent role in cell-cell interaction and cell growth. Pathogens including virus particles might target GAG-binding sites in envelope proteins leading to infection.

Amino Acid Sequence↗
PubMed2000

Glucose-6-phosphate transporter and receptor functions of the glucose 6-phosphatase system analyzed from a consensus defined by multiple alignments.

The cDNA encoding the protein (P46) that is mutated in glycogen storage disease type-1b (GSD-1b) has been previously cloned by homology with bacterial sequences of the uhp (upper hexose phosphate) system. Hydropathic profiles, transmembrane-prediction analysis, and a multiple alignment of 14 sequences related to P46 (with percentage of identity around 30%) allowed to identify two large domains in the proteins linked by a large variable loop. Highly conserved transmembrane (TM) segments, TM1 and TM4 in the first domain and TM5 in the second one, were identified almost in all the integral proteins related to P46. The multiple alignment allowed definition of a consensus involving the 14 sequences related to P46. The detailed comparison of the consensus with the UhpT (the bacterial G6P transporter) and with UhpC (the bacterial G6P receptor) sequences reveals that the P46 protein could carry both G6P receptor and transporter functions.

Amino Acid Motifs↗
PubMed2005

Definition of organ involvement and treatment response in immunoglobulin light chain amyloidosis (AL): a consensus opinion from the 10th International Symposium on Amyloid and Amyloidosis, Tours, France, 18-22 April 2004.

We undertook this study to develop uniformly accepted criteria for the definition of organ involvement and response for patients on treatment protocols for immunoglobulin light-chain amyloidosis (AL). A consensus panel was convened comprising 13 specialists actively involved in the treatment of patients with amyloidosis. Institutional criteria were submitted from each, and a consensus was developed defining each organ involved and the criteria for response. Specific criteria have been developed with agreed on definitions of organ and hematologic response as a result of discussions at the 10th International Symposium on Amyloid and Amyloidosis held in Tours, France, April 2004. These criteria now form the working definition of involvement and response for the purposes of future data collection and reporting. We report criteria that centers can now use to define organ involvement and uniform response criteria for reporting outcomes in patients with light-chain AL.

Amyloid↗
PubMed1997

Banff schema for grading liver allograft rejection: an international consensus document.

A panel of recognized experts in liver transplantation pathology, hepatology, and surgery was convened for the purpose of developing a consensus document for the grading of acute liver allograft rejection that is scientifically correct, simple, and reproducible and clinically useful. Over a period of 6 months pertinent issues were discussed via electronic communication media and a consensus conference was held in Banff, Canada in the summer of 1995. Based on previously published data and the combined experience of the group, the panel agreed on a common nomenclature and a set of histopathological criteria for the grading of acute liver allograft rejection, and a preferred method of reporting. Adoption of this internationally accepted, common grading system by scientific journals will minimize the problems associated with the use of multiple different local systems. Modifications of this working document to incorporate chronic rejection are expected in the future.

Acute Disease↗
PubMed2005

Towards consensus in the long-term management of relapse prevention in schizophrenia.

APPROACH TO DEVELOPING GUIDANCE: When developing guidance for the long-term management of schizophrenia, one approach is to adopt a proactive strategy that sets out clear treatment goals and strategies. This should involve a broad view being taken, embracing overall mental and physical well-being rather than simply the absence of illness. Although relapse prevention is an important goal of any long-term management strategy, there are other aspects that need to be considered, such as reintegration into society, regaining independence and quality of life. CURRENT TREATMENT: To help achieve these goals, a range of interventions can be incorporated into long-term management strategies for schizophrenia, including pharmacological interventions, psychosocial therapies and alliance-building initiatives. The current UK National Institute for Clinical Excellence guidelines already recommend that continuous therapy should be practised using an atypical (second-generation) antipsychotic drug, whenever possible, in preference to older typical drugs. The launch of the first long-acting atypical antipsychotic is an interesting new advance that may benefit many patients with schizophrenia. Psychosocial interventions, particularly family-based therapies, as well as cognitive behavioural and compliance therapies, when used alongside antipsychotics, have been shown to reduce relapse rates dramatically and to assist in social reintegration. In addition, forging collaborative alliances with patients and their carers can help to demystify schizophrenia and empower patients to take responsibility for their illness. CONSENSUS STATEMENT: This article outlines a consensus reached by a panel of leading UK healthcare professionals working with schizophrenia brought together to discuss long-term management strategies.

Antipsychotic Agents↗
PubMed1993

Protein product of the somatic-type transcript of the Hoxa-4 (Hox-1.4) gene binds to homeobox consensus binding sites in its promoter and intron.

The murine Hoxa-4 gene encodes a protein with a homeodomain closely related to those produced by the Antennapedia-like class of Drosophila genes. Drosophila homeodomain proteins can function as transcription factors, binding to several specific DNA sequences. One sequence that is frequently encountered contains a core ATTA motif within a larger consensus sequence, such as CAATTAA. The in vitro synthesized protein product of Hoxa-4 was shown to bind to a subset of restriction fragments of the Hoxa-4 gene itself as determined by gel retardation experiments. Direct examination of the sequences of the fragments bound by Hoxa-4 protein revealed the presence of four regions containing the core ATTA motif. Two regions contained sequences of the CAATTAA class and were located approximately 1 kb upstream from the putative somatic Hoxa-4 promoter and within the intron. Two additional binding sites containing the consensus target sequence involved in autoregulation of Drosophila Deformed gene were identified: one immediately downstream of the putative embryonic transcription start site and one within the intron, respectively. Specific binding of the in vitro produced Hoxa-4 protein to oligonucleotides corresponding to these sequences was observed in gel retardation assays. The same results were obtained with Hoxa-4 protein produced in a Baculovirus expression system. Experiments using oligonucleotides containing base substitutions in positions 1, 3, 4, and 5 in the sequence CAATTAA showed severely reduced binding. The use of truncated mutant Hoxa-4 proteins in gel retardation assays and in transient co-transfection experiments revealed that the intact homeodomain was required for the binding. These results also suggested that the Hoxa-4 gene has the potential to auto-regulate its expression by interacting with the homeodomain binding sites present in the promoter as well as in the intron.

Amino Acid Sequence↗
PubMed2004

Searching for nitrile hydratase using the Consensus-Degenerate Hybrid Oligonucleotide Primers strategy.

Consensus-Degenerate Oligonucleotide Hybrid Primers (CODEHOP) were designed by using the WWW-implemented strategy, based on multiple alignments of nitrile hydratase (NHase) alpha subunit, available from EMBL database. These primers were successfully tested with known NHase-producing bacterial strains such as Agrobacterium tumefaciens DSM 9674, Rhodococcus erythropolis DSM 9675, R. erythropolis 9685 and R. erythropolis 11397 and also allowed amplification from organisms not previously referenced (Variovorax sp. DSM 11402 and Agrobacterium sp. DSM 11401). Hybrid primer utilisation was evaluated by analysing the incorporated sequences in cloned PCR fragments. Several primers from the oligonucleotide pool seemed to participate in the amplification of the correct fragment. Common garden soil was used as a source for both acetonitrile culture enrichment screening and direct DNA extraction. A R. erythropolis strain (CCMI 1005) was isolated by culture enrichment and allowed the PCR amplification of a DNA sequence (AJ548493) identical to the NHase coding sequences commonly described for that species, while the direct use of soil DNA as template led to the CODEHOP detection of two putative NHase coding sequences (AJ548498 and AJ548499), which were significantly different from any other known sequence. The phylogenetic relationship between all sequences obtained and the published NHase coding sequences was assessed by neighbour-joining analysis. The results demonstrate the use of consensus-degenerate primers in NHase detection from organisms known to express it and in the screening for new NHase family members.

Agrobacterium tumefaciens↗
PubMed2005

Proteomics-based consensus prediction of protein retention in a bacterial membrane.

The availability of complete bacterial genome sequences allows proteome-wide predictions of exported proteins that are potentially retained in the cytoplasmic membranes of the corresponding organisms. In practice, however, major problems are encountered with the computer-assisted distinction between (Sec-type) signal peptides that direct exported proteins into the growth medium and lipoprotein signal peptides or amino-terminal membrane anchors that cause protein retention in the membrane. In the present studies, which were aimed at improving methods to predict protein retention in the bacterial cytoplasmic membrane, we have compared sets of membrane-attached and extracellular proteins of Bacillus subtilis that were recently identified through proteomics approaches. The results showed that three classes of membrane-attached proteins can be distinguished. Two classes include 43 lipoproteins and 48 proteins with an amino-terminal transmembrane segment, respectively. Remarkably, a third class includes 31 proteins that remain membrane-retained despite the presence of typical Sec-type signal peptides with consensus signal peptidase recognition sites. This unprecedented finding indicates that unknown mechanisms are involved in membrane retention of this class of proteins. A further novelty is a consensus sequence indicative for release of certain lipoproteins from the membrane by proteolytic shaving. Finally, using non-overlapping sets of secreted and membrane-retained proteins, the accuracy of different signal peptide prediction algorithms was assessed. Accuracy for the prediction of protein retention in the membrane was increased to 82% using a majority-vote approach. Our findings provide important leads for future identification of surface proteins from pathogenic bacteria, which are attractive candidate infection markers and potential targets for drugs or vaccines.

Amino Acid Sequence↗