Subcellular localization of the Iitracellular survival-enhancing Eis protein of Mycobacterium tuberculosis.

Mycobacterium tuberculosis is a facultative intracellular pathogen that has evolved the ability to survive and multiply within human macrophages. It is not clear how M. tuberculosis avoids the destructive action of macrophages, but this ability is fundamental in the pathogenicity of tuberculosis. A gene previously identified in M. tuberculosis, designated eis, was found to enhance intracellular survival of Mycobacterium smegmatis in the human macrophage-like cell line U-937 (J. Wei et al., J. Bacteriol. 182:377-384, 2000). When eis was introduced into M. smegmatis on a multicopy vector, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the appearance of a unique 42-kDa protein band corresponding to the predicted molecular weight of the eis gene product. This band was electroeluted from the gel with a purity of >90% and subjected to N-terminal amino acid sequencing, which demonstrated that the 42-kDa band was indeed the protein product of eis. The Eis protein produced by M. tuberculosis H37Ra had an identical N-terminal amino acid sequence. A synthetic polypeptide corresponding to a carboxyl-terminal region of the deduced eis protein sequence was used to generate affinity-purified rabbit polyclonal antibodies that reacted with the 42-kDa protein in Western blot analysis. Hydropathy profile analysis showed the Eis protein to be predominantly hydrophilic with a potential hydrophobic amino terminus. Phase separation of M. tuberculosis H37Ra lysates by the nonionic detergent Triton X-114 revealed the Eis protein in both the aqueous and detergent phases. After fractionation of M. tuberculosis by differential centrifugation, Eis protein appeared mainly in the cytoplasmic fraction but also in the membrane, cell wall, and culture supernatant fractions as well. Forty percent of the sera from pulmonary tuberculosis patients tested for anti-Eis antibody gave positive reactions in Western blot analysis. Although the function of Eis remains unknown, evidence presented here suggests it associates with the cell surface and is released into the culture medium. It is produced during human tuberculosis infection and therefore may be an important M. tuberculosis immunogen.

Identification of a Mycobacterium tuberculosis gene that enhances mycobacterial survival in macrophages.

Intracellular survival plays a central role in the pathogenesis of Mycobacterium tuberculosis. To identify M. tuberculosis genes required for intracellular survival within macrophages, an M. tuberculosis H37Rv plasmid library was constructed by using the shuttle vector pOLYG. This plasmid library was electroporated into Mycobacterium smegmatis 1-2c, and the transformants were used to infect the human macrophage-like cell line U-937. Because M. smegmatis does not readily survive within macrophages, any increased intracellular survival is likely due to cloned M. tuberculosis H37Rv DNA. After six sequential passages of M. smegmatis transformants through U-937 cells, one clone (p69) was enriched more than 70% as determined by both restriction enzyme and PCR analyses. p69 demonstrated significantly enhanced survival compared to that of the vector control, ranging from 2.4- to 5.3-fold at both 24 and 48 h after infection. DNA sequence analysis revealed three open reading frames (ORFs) in the insert of p69. ORF2 (1.2 kb) was the only one which contained a putative promoter region and a ribosome-binding site. Deletion analysis of the p69 insert DNA showed that disruption of ORF2 resulted in complete loss of the enhanced intracellular survival phenotype. This gene was named the enhanced intracellular survival (eis) gene. By using an internal region of eis as a probe for Southern analysis, eis was found in the genomic DNA of various M. tuberculosis strains and of Mycobacterium bovis BCG but not in that of M. smegmatis or 10 other nonpathogenic mycobacterial species. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that all M. smegmatis eis-containing constructs expressed a unique protein of 42 kDa, the predicted size of Eis. The expression of this 42-kDa protein directly correlated to the enhanced survival of M. smegmatis p69 in U-937 cells. These results suggest a possible role for eis and its protein product in the intracellular survival of M. tuberculosis.

The new JCAHO pain standards: implications for pain management nurses.

The newly approved Joint Commission on Accreditation of Healthcare Organizations (JCAHO) pain management standards present an important opportunity for widespread and sustainable improvement in pain assessment and management. Unrelieved pain is a major, yet avoidable, public health problem. Despite 20 years of work by educators, clinicians, and professional organizations and the publication of clinical practice guidelines, there have been, at best, modest improvements in pain management practices. Multiple barriers found in the health care system, and among health care professionals, patients, and families, continue to impede progress. In August 1997 a collaborative project was initiated to integrate pain assessment and management into the standards, intent statements, and examples of implementation of JCAHO--a rare opportunity to improve pain management in health care facilities throughout the country. After review by multiple JCAHO committees and advisory groups and critique by an expert panel, the JCAHO Board of Commissioners approved the revisions in May 1999. The revisions are published in the 2000-2001 standards manuals and will be effective January 1, 2001, for all patient care organizations accredited by JCAHO--ambulatory care, behavioral health, health care networks, home care, hospitals, long-term care, and long-term care pharmacies. An evaluation of the impact of the revisions is currently being completed, and education of the JCAHO surveyors and health care professionals is underway. Nurses, especially those with expertise in pain management, are valuable resources as health care organizations change their pain assessment and management processes to meet the new standards.

Trends in medical use and abuse of opioid analgesics.

CONTEXT: Pain often is inadequately treated due in part to reluctance about using opioid analgesics and fear that they will be abused. Although international and national expert groups have determined that opioid analgesics are essential for the relief of pain, little information has been available about the health consequences of the abuse of these drugs. OBJECTIVE: To evaluate the proportion of drug abuse related to opioid analgesics and the trends in medical use and abuse of 5 opioid analgesics used to treat severe pain: fentanyl, hydromorphone, meperidine, morphine, and oxycodone. DESIGN AND SETTING: Retrospective survey of medical records from 1990 to 1996 stored in the databases of the Drug Abuse Warning Network (source of abuse data) and the Automation of Reports and Consolidated Orders System (source of medical use data). PATIENTS: Nationally representative sample of hospital emergency department admissions resulting from drug abuse. MAIN OUTCOME MEASURES: Medical use in grams and grams per 100,000 population and mentions of drug abuse by number and percentage of the population. RESULTS: From 1990 to 1996, there were increases in medical use of morphine (59%; 2.2 to 3.5 million g), fentanyl (1168%; 3263 to 41,371 g), oxycodone (23%; 1.6 to 2.0 million g), and hydromorphone (19%; 118,455 to 141,325 g), and a decrease in the medical use of meperidine (35%; 5.2 to 3.4 million g). During the same period, the total number of drug abuse mentions per year due to opioid analgesics increased from 32,430 to 34,563 (6.6%), although the proportion of mentions for opioid abuse relative to total drug abuse mentions decreased from 5.1% to 3.8%. Reports of abuse decreased for meperidine (39%; 1335 to 806), oxycodone (29%; 4526 to 3190), fentanyl (59%; 59 to 24), and hydromorphone (15%; 718 to 609), and increased for morphine (3%; 838 to 865). CONCLUSIONS: The trend of increasing medical use of opioid analgesics to treat pain does not appear to contribute to increases in the health consequences of opioid analgesic abuse.

Separate contributions of UhpA and CAP to activation of transcription of the uhpT promoter of Escherichia coli.

Activation of promoters by multiple transcription factors might occur through favorable contacts of the activators with themselves or RNA polymerase, or by changes in DNA geometry that enhance formation of the transcription complex. Transcription of the Escherichia coli uhpT gene, encoding the organophosphate transporter, requires the response regulator UhpA and is stimulated by the global regulator protein CAP. CAP binds to the uhpT promoter at a single site, centered at -103.5 bp relative to the start of transcription, and UhpA binds to multiple sites between positions -80 and -32. Overexpression of UhpA did not reduce the degree of CAP stimulation of uhpT-lacZ expression, showing that CAP action is more complex than enhancement of the binding of UhpA. Footprinting experiments demonstrated that UhpA and CAP modestly stimulated each other's binding to the uhpT promoter, but did not affect the positioning of the binding sites. An in vitro transcription system was used to examine the contribution of each transcription factor at the uhpT promoter. Action of UhpA and CAP proteins was not affected by template supercoiling. Kinetic analyses of productive and abortive initiation showed that CAP acted both to stabilize by fivefold the open promoter complexes formed in the presence of UhpA and to enhance by twofold the rate of their formation. These results indicate that open complex formation requires UhpA and that CAP stabilizes the open complex.

A role model program to promote institutional changes for management of acute and cancer pain.

This report describes an 18-month project to make acute and cancer pain management an institutional priority in Southeastern Wisconsin health-care facilities. Facility-based teams, each of which included a nurse in a leadership position, were recruited to participate in a project based on the Cancer Pain Role Model Program. The project was conducted in three stages: (a) a 1-day conference focusing on basic pain management issues and clinical standards, (b) a preceptorship at the Medical College of Wisconsin, and (c) a follow-up conference focusing on institutional change. Participants completed an Action Plan, outlining activities aimed at changing practice in their facility. Participants from 17 of the 32 participating facilities partially or completely met their Action Plan goals. Lack of ongoing facility commitment, staff turnover and facility closures were cited as reasons for failure to meet goals. Nurses in key positions, provided with strong institutional commitment and given suitable educational training and nurturing, are ideally suited to help facilitate changes in institutional pain practices.

Protein phosphorylation affects binding of the Escherichia coli transcription activator UhpA to the uhpT promoter.

Expression of the Escherichia coli sugar phosphate transporter UhpT is induced by extracellular glucose 6-phosphate through a transmembrane signaling process dependent on the sensor kinase UhpB and the UhpT homolog, UhpC. These proteins are thought to regulate the phosphorylation of the transcription activator, UhpA. To examine the effect of protein phosphorylation on the binding of UhpA to target sequences in the uhpT promoter region, the UhpA protein was overexpressed and purified. Purified UhpA was phosphorylated by acetyl phosphate in a reaction that was dependent on Mg2+ and on the presence of aspartate 54, the site of phosphorylation in homologous response regulators. Gel electrophoretic mobility shift and DNase I and hydroxyl radical protection assays showed that UhpA bound specifically to the region of the uhpT promoter extending from -80 to -50 bp, relative to the transcription start site. At higher concentrations of UhpA, binding was extended to the -32 region. Binding to the -64 element exhibited positive cooperativity and was stimulated severalfold by phosphorylation of UhpA, whereas extension to the downstream region was more strongly affected by phosphorylation. The consensus sequences for the high affinity UhpA-binding sites in the -64 element and for the downstream, low affinity sites are proposed. The pattern of in vitro binding by UhpA agreed with the in vivo observations that phosphorylation-independent assembly of the transcription initiation complex can occur at elevated concentrations of UhpA.

Effective pain management in terminal care.

Pharmacologic therapy is the mainstay of pain management in the terminally ill cancer patient. Upwards of 90% of the pain of cancer can be controlled by relatively simple means (i.e. oral, rectal, or transdermal analgesics). More invasive procedures may be needed in a small percentage of patients. Effective management of pain in the elderly requires recognition of age-related changes in drug pharmacokinetics and an awareness of drug side effects that may be particularly problematic in older patients. Careful attention to the basic principles of drug use in the elderly will enhance effective pain management and quality of life.

Teaching cancer pain management: durability of educational effects of a role model program.

BACKGROUND: Inadequate management of cancer related pain has resulted primarily from attitudinal barriers and a lack of knowledge about clinical assessment, the administration of analgesics, and therapeutic interventions. METHODS: Fifty health-care providers (13 physicians, 21 nurses, and 16 pharmacists), working as a team, participated in a Role Model Program that presented principles of cancer pain management. A questionnaire that evaluated the fund of knowledge and attitudes regarding cancer pain management was administered prior to the 1-day workshop, at the end of the workshop, and at 4 and 12 months follow-up. The workshop consisted of lectures and discussion groups; in the discussion groups, concepts were clarified, cases presented, and barriers to optimal cancer pain management identified. RESULTS: Significant improvements in attitudes (P < 0.01), knowledge (P < 0.01), and total scores (P < 0.002) were observed when the preworkshop responses were compared with those obtained immediately after instruction. Scores on the questionnaire were the same or slightly better at both 4 and 12 months in follow-up, demonstrating no loss in acquired knowledge or attitude. Comparison of the postworkshop scores with those at 12 months follow-up were significantly better in attitude (P < 0.03), and in total score (P < 0.01); improvements in knowledge also approached significance (p < 0.06). These represented continuing improvements because significant differences in the attitude scores (P < 0.05) and the total score (P < 0.05) were observed when the 4-month and 12-month follow-up responses were evaluated. The effectiveness of the program in the transference of knowledge was also measured; in the first year of the program, more than 4500 health-care professionals were subsequently informed about cancer pain management from the Role Model Participants. CONCLUSIONS: Significant improvements were observed immediately in both attitude and knowledge of cancer pain management principles after the 1-day Role Model Workshop. These improvements continued, as determined at 4 and 12 months follow-up. The Role Model Participants were highly motivated to share the learned principles of cancer pain management with other health-care professionals. These results are consistent with other Role Model Programs that both instruct and involve the participants. The Role Model Program is an efficient and effective means of educating health-care professionals in the concepts of cancer pain management.

Update on the cancer pain role model education program.

Application of traditional educational methods has done little to improve cancer pain management in the United States. This report details the results of the first year of the expanded Wisconsin Cancer Pain Initiative Role Model Program, a novel approach to cancer pain education. One hundred and ninety-six physicians and nurse educators together with their clinical partners attended one of three role model conferences in 1992-1993 and developed Action Plans detailing their proposed educational goals. Results indicate that participants demonstrated significant improvement in cancer pain knowledge as a result of the 1-day conference. Within 12 months of the conference, 64% of Role Model teams completely or partially met their Action Plan goals. In total, 227 educational or clinical practice projects were completed. The Cancer Pain Role Model Program represents an excellent educational program for disseminating cancer pain information and instituting positive changes in clinical practice.

Transcription activation at the Escherichia coli uhpT promoter by the catabolite gene activator protein.

Transport and utilization of sugar phosphates in Escherichia coli depend on the transport protein encoded by the uhpT gene. Transmembrane induction of uhpT expression by external glucose 6-phosphate is positively regulated by the promoter-specific activator protein UhpA and the global regulator catabolite gene activator protein (CAP). Activation by UhpA requires a promoter element centered at -64 bp, relative to the start of transcription, and activation by CAP requires a DNA site centered at position -103.5. This DNA site binds the cyclic AMP-CAP complex in vitro, and its deletion from the promoter reduces transcription activity to 7 to 9% of the wild-type level. Ten uhpT promoter derivatives with altered spacing between the DNA site for CAP and the remainder of the promoter were constructed. Their transcription activities indicated that the action of CAP at this promoter is dependent on proper helical phasing of promoter elements, with CAP binding on the same face of the helix as RNA polymerase does. Five CAP mutants defective in transcription activation at class I and class II CAP-dependent promoters but not defective in DNA binding or DNA bending (positive control mutants) were tested for the ability to activate transcription. These CAPpc mutants exhibited little or no defect in transcription activation at uhpT, indicating that CAP action at uhpTp involves a different mechanism than that which is used for its action at other classes of CAP-dependent promoters.

Proliferation of mast cells in the smooth muscle of denervated rat jejunum.

Mast cell hyperplasia and changes in phenotypic characteristics subsequent to myenteric and extrinsic denervation of a segment of rat jejunum were studied. The myenteric plexus and extrinsic nerves were ablated by serosal application of the cationic surfactant benzyldimethyltetradecylammonium chloride. There was a four-fold increase in the number of mast cells in the smooth muscle layers 15 days after denervation. This increase was sustained for at least 90 days after treatment. No increase in mast cell number was observed in the villus-crypt axis of the jejunum. Berberine sulfate fluorescent detection of heparin-containing mast cells demonstrated that a change in mast cell phenotype occurred between 20 and 90 days after the denervation procedure. The fact that myeloperoxidase activity was the same in denervated and control tissue within 5 days of denervation demonstrates the lack of a chronic inflammatory reaction. Our results suggest that mast cells might play a role in the gut wall re-modeling processes.

State cancer pain initiatives.

Interest and participation in state cancer pain initiatives have grown rapidly in the past 5 yr. Of signal importance to these state efforts is the fact that several national groups have made relief of cancer pain a priority. State cancer pain initiatives will play a key role in disseminating basic pain-management information, in changing practice and ultimately in evaluating the effectiveness of cancer pain control efforts. They are dedicated to making relief of cancer pain a reality. That is a challenge that will occupy them for many years to come.