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.

Immunohistochemical localization of choline acetyltransferase in the chicken mesencephalon.

Choline acetyltransferase, a specific marker for cholinergic neurons, has been immunohistochemically localized in the mesencephalon and in the caudal diencephalon of the chicken. A complete series of transverse sections through the mesencephalon is presented. In the diencephalon, cholinergic fibers were found in the stria medullaris, the fasciculus retroflexus, and the ventral portion of the supraoptic decussation. The nucleus triangularis and the nucleus geniculatus lateralis, pars ventralis also contained cholinergic fibers. Small cholinergic cell bodies were found in the medial habenula. In the pretectum, cholinergic fibers innervated the nucleus lentiformis mesencephali and the tectal gray. The nucleus spiriformis lateralis also contained cholinergic fibers, while most of the cell bodies in the nucleus spiriformis medialis were cholinergic. In the mesencephalon, labelled fibers were found in the nucleus intercollicularis and in all layers of the optic tectum except the stratum opticum. The highest density of tectal cholinergic fibers was in the stratum griseum et fibrosum superficiale (SGFS), layer f. Radial cells located in SGFS, layer i were also cholinergic. In the isthmic nuclei, cholinergic fibers were found in the pars magnocellularis, while the pars parvicellularis and the nucleus semilunaris contained labelled cells. The oculomotor, Edinger-Westphal, trochlear, and trigeminal motor nuclei all had cholinergic cell bodies. Cholinergic axons were present in the oculomotor and trochlear nerves. In the tegmentum, cell bodies were labelled in the nucleus mesencephalicus profundus, pars ventralis, while the nucleus interpeduncularis had dense cholinergic innervation. Our localization of cholinergic cell bodies and fibers has been compared with earlier autoradiographic and anatomical studies to help define cholinergic systems in the avian brain. For example, the results indicate that the chicken may have a cholinergic habenulointerpeduncular system similar to that reported in the rat. Establishing the cholinergic systems within the avian midbrain is important for designing future neurophysiological and pharmacological studies of cholinergic transmission in this region.

Localization of choline acetyltransferase-like immunoreactivity in the embryonic chick retina.

Putative sites of acetylcholine synthesis in the retina of the embryonic and posthatched chick were localized immunohistochemically with antisera to choline acetyltransferase; the resultant choline acetyltransferase-like immunoreactivity (ChAT-IR) was compared to demonstrated sites of acetyltransferase (AChE) activity, and changes were followed in localization during development. The results confirmed the early and rapid course of development of the chick's retinal cholinergic system described in previous biochemical and morphological studies. Immunoreactivity was first detected at embryonic day 6.5 in cells close to the retina's vitreal surface. By 8 days it was present in cells in two juxtaposed rows; by the ninth day the two rows were separated and immunoreactivity was evident in two subliminae of the inner plexiform layer. On the tenth day distribution was like that in the posthatched chicken, in type I cholinergic cells in the inner nuclear layer and in type II cells in the ganglion cell layer (Millar et al.: Neurosci. Lett. 61:311-316, '85), and similar to that of most vertebrates. Three days before hatching, a third population of weakly immunoreactive cells (type III cells) appeared within the inner nuclear layer. The onset of localizable ChAT-IR occurred in amacrine cells and in their processes, before the period of synaptogenesis. Acetylcholinesterase activity was localized at an earlier age than ChAT-IR, and at all ages was present in more cells. The results obtained support the view that "displaced" cholinergic amacrine cells begin to differentiate at the same time and in the same retinal region as type I cholinergic cells. Separation of the two groups is a consequence of the ramification of processes of amacrine and ganglion cells rather than a result of the secondary migration of cells between layers.

CSF beta-endorphin and the severity of pain.

Beta-endorphin-like immunoreactivity (i beta-EP) was measured in the CSF at myelography of 24 patients suspected of vertebral disk disease. Patients made several ratings of mood and pain for the 24 hours preceding myelography. Composite scores for pain, negative mood, and positive mood were derived by factor analysis. Pain Factor scores were negatively correlated with i beta-EP (r = -0.59, p less than 0.001), indicating a possible role for i beta-EP in the perception of the severity of pain. No significant correlation was shown between Positive or Negative Mood Factor scores and CSF i beta-EP. A physiologic indicator of pain severity is discussed.

Cholinergic neurons of the chicken ciliary ganglion contain somatostatin.

Somatostatin immunoreactivity was studied in the avian ciliary ganglion by immunocytochemistry and radioimmunoassay. Immunoreactivity was localized to small diameter cell bodies of neurons from embryos, newly-hatched and adult preparations. Immunostaining of ganglia with a mixture of antisera to substance P and monoclonal antibody to somatostatin indicated that a number of somatostatin-immunoreactive neurons were surrounded by substance P-immunoreactive boutons, which characteristically terminate on choroidal neurons. Staining with a mixture of antisera to choline acetyltransferase and antibody to somatostatin showed that the somatostatin-immunoreactive neurons were less intensely-stained for choline acetyltransferase than were the neurons lacking somatostatin immunoreactivity. Bundles of nerve fibers showing somatostatin and choline acetyltransferase immunoreactivity were found in the choroid layers of the eye. Radioimmunoassay indicated the presence of somatostatin immunoreactivity in both chick and quail ganglia; the somatostatin immunoreactivity eluted from high pressure liquid chromatography in the same positions as authentic somatostatin 14 and 28. These results show that somatostatin is contained in cholinergic choroidal neurons in the chick and quail ciliary ganglion.

Conditioned morphine tolerance in the rat: absence of a compensatory response and cross-tolerance with stress.

In four separate experiments with rats as subjects, strong evidence was obtained that tolerance development to morphine analgesia occurs most rapidly when morphine delivery is paired with salient contextual cues. However, contextual cues previously paired with morphine did not elicit conditioned drug-compensatory responses when presented to nondrugged animals. These results were obtained by different analgesia assessments, with different drug-administration--analgesia-test latencies, and in environments differing with respect to stress level. Stress level did influence nociceptive response, as it was found that the combination of bright illumination, white noise, and a strong odor resulted in antinociception in the absence of drug. Moreover, rats that had a history of receiving morphine in this stressful context were tolerant to this stress-induced antinociception, but only when morphine was present in their systems. In the final two studies, this antinociception, which was cross-tolerant with morphine, was characterized with respect to naloxone reversibility and brain levels of met- and leu-enkephalin as determined by radioimmunoassay.

Development of enkephalin in the rectum and ganglion of Remak of the chick.

Radioimmunoassay (RIA) and high performance liquid chromatography were used in combination with immunocytochemistry to study the development of met-enkephalin (Met-enk) in the rectum and the ganglion of Remak of the chicken. Met-enk was detected by RIA in the rectum at 5 days of incubation (d.i.). The concentration increased from 5-9 d.i. and did not change significantly thereafter. In contrast, the concentration of Met-enk in Remak's ganglion increased throughout the developmental period of study. Met-enk immunoreactivity first appeared in cell bodies in Remak's ganglion at 6 d.i. and in a small number of processes in the wall of the rectum. By 9 d.i., Remak's ganglion contained many immunoreactive cell bodies, some of which extended processes into the wall of the rectum in the region of the myenteric plexus. Varicosities were first seen in the rectum at 13 d.i. and increased in number and staining intensity with developmental age. The fact that immunoreactive cell bodies persist in Remak's ganglion throughout the course of development and send processes into the rectum suggests that a major portion of enkephalinergic innervation of the rectum is extrinsic. On the other hand, the presence of Met-enk immunoreactivity in both nerve cell bodies and processes in rectal explants stripped of Remak's ganglion suggests that this peptide is also contained in intrinsic neurons in the chick rectum.

Development of enkephalinergic neurons in the gut of the chick.

The content and distribution of Met-enkephalin immunoreactivity in the developing chick gut was studied by radioimmunoassay and immunocytochemistry. Met-enkephalin was detected by radioimmunoassay in the duodenum of the 5-day chick embryo. The concentration in this region increased 4-fold by 13 days of incubation and declined thereafter to the levels found in the 4-week chicken. The concentration of enkephalin in the midgut increased about 2-fold between 9 and 13 days of incubation and remained constant until hatching. In the 7-day duodenum, metenkephalin immunoreactivity was found in a network of darkly stained nodes (accumulations of ganglion cells) faintly stained internodal nerve bundles; this network of immunoreactivity was localized to the myenteric plexus. By 9 days of incubation, the network was more extensive and the intensity of staining was increased. At 13 days of incubation, varicosities were found in the region of the ganglion cells and in internodal nerve bundles. At this time, immunoreactivity was clearly visualized in the submucosal plexus. In the newly hatched chicken, met-enkephalin was found in nerves within the circular smooth muscle, as well as the myenteric and submucosal plexuses. The early appearance of met-enkephalin in the developing chick enteric nervous system suggests this peptide may act as a primary neurotransmitter in the organization and control of gut motility.

Dynorphin (1-13): analgesia, hypothermia, cross-tolerance with morphine and beta-endorphin.

Intracerebroventricular administration of 20, 40 and 60 nmol of dynorphin (1-13) produced analgesia, as assessed by flinch/jump response to footshock, and hypothermia in the rat. Rats developed tolerance to both the analgesic and thermic effects of the 20 nmol dose of dynorphin. Dynorphin and beta-endorphin showed cross-tolerance with respect to their analgesic but not their thermic effects. Dynorphin and morphine also produced cross-tolerant analgesic effects. Naloxone (10 mg/kg, IP) completely blocked the barrel rolling produced by 20 nmol dynorphin but did not alter its analgesic or thermic effects.

Endogenous analgesia in the pregnant rat: an artifact of weight-dependent measures?

It has been reported that pregnancy produces an opioid-mediated, endogenous analgesia in the rat. In an attempt to confirm this finding, we used 5 different analgesic measures to compare the responsiveness of pregnant and non-pregnant female rats to painful stimuli. Pregnant and non-pregnant rats differed only when assessed by measures that were highly correlated with body weight. Furthermore, the reduced pain responsiveness of pregnant rats was not prevented by administration of the opioid antagonists, naloxone or naltrexone. We can find no evidence for an endogenous analgesia of pregnancy; instead, our results suggest that findings of a diminished response to painful stimuli in pregnant rats may be an artifact related to the greater body mass/weight of the pregnant animals.

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.

Attitudes about cancer pain: a survey of Wisconsin's first-year medical students.

A brief questionnaire was administered to 317 first-year students at Wisconsin's two medical schools to assess their attitudes about cancer pain prior to their entry into medical school. Although the students had a realistic perspective of the prevalence and severity of cancer pain, they displayed a number of negative attitudes that if unchanged would contribute to future inadequate pain treatment. Students (a) greatly exaggerated the incidence of psychological dependence (addiction) in patients treated with opioid analgesics, (b) inappropriately timed maximal analgesic therapy to the duration of life expectancy, (c) felt that a majority of patients with cancer currently receive adequate or excessive analgesic therapy, and (d) believed that increasing pain was invariably related to the development of drug tolerance rather than to progression of the disease. Curricular efforts need to be directed at bringing about changes in students' negative attitudes about cancer pain and its 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.

Physician cancer pain education: a report from the Wisconsin Cancer Pain Initiative.

The Wisconsin Cancer Pain Initiative was established in 1986 to address the various public and professional barriers to cancer pain management. This report discusses the initiative's model for physician education, which includes increasing factual knowledge, legitimizing cancer pain as an important treatment priority, and developing clinical role models. Our progress in implementing this education model will be discussed.

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.

The Cancer Pain Role Model Program of the Wisconsin Cancer Pain Initiative.

The Cancer Pain Role Model Program was established in 1990 by the Wisconsin Cancer Pain Initiative to train physicians to be role models for appropriate pain management. Community and academic physicians along with their "clinical partners," a nurse or pharmacist, were recruited to attend a 1-day education conference that included lectures, small group workshops, and development of an action plan. A total of 44 physicians and 55 clinical partners have attended two conferences (1990 and 1991). Participants listed 199 activities on their action plans; postconference surveys have documented initiation of 212 projects aimed at educating others, improving clinical care, resource development, educating self, or research. The Cancer Pain Role Model Program appears to be a promising means of transferring cancer pain information, legitimizing cancer pain as an important treatment issue, and institutionalizing cancer pain management.

Multiple immunoreactive forms of met- and leu-enkephalin in fetal and neonatal rat brain and in rat gut.

We have used radioimmunoassays with carboxyl-specific antisera to study the development of met- and leu-enkephalin in rat brain and gut from 13-days of fetal age through adulthood. Fractionation of HCl extracts of fetal and neonatal brain tissues by HPLC revealed the presence of immunoreactive forms other than met- and leu-enkephalin. For example, HPLC separation of extracts of 16-day fetal brain yielded two peaks of leu-enkephalin-like immunoreactivity. One emerged at the position of leu-enkephalin, the other eluted with about one-third the retention time. There were four peaks of met-enkephalin-like immunoreactivity, one with the retention time characteristic of met-enkephalin, the others with shorter retention times. In contrast, all of the met-enkephalin-like immunoreactivity in adult brain eluted with the retention time characteristic of authentic met-enkephalin and all of the leu-enkephalin-like immunoreactivity eluted in the position of authentic leu-enkephalin. Multiple immunoreactive forms of met- and leu-enkephalin were found in extracts of both fetal and adult gut tissues.

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.