Entry demographics and pharmacological treatment of migraine patients referred to a tertiary care pain clinic.

The aim was to examine pharmacological treatment of migraine patients admitted to a tertiary care pain clinic. A retrospective review of 100 consecutive migraine patients admitted to The Wasser Pain Management Centre was conducted. Patients included met the 2nd Edition of the International Classification of Headache Disorders criteria for diagnosis of migraine. Data were collected with regard to nicotine and alcohol consumption, family history of migraine headaches, other pain diagnoses and pharmacological treatment. Twenty-two per cent of these patients were male as opposed to 78% female. The mean age of patients admitted for migraine was 43.4 years. Of the patients admitted, 48% had tried at least one triptan in the past and only 31% were actively using triptan(s). The most commonly used triptan in the past had been sumatriptan, whereas the most common triptan used on admission was rizatriptan. Opiate use was much more prevalent; 72% of admitted patients were using an opiate and 27% used multiple opiates. A significant number of patients had not yet been tried on a triptan despite meeting the diagnostic criteria for migraine and having significant disability. More education of the general medical community may be beneficial in implementing a stratified care approach to migraine management.

Erythropoietin: production by a particulate fraction of rat kidney.

An erythropoietic factor was extracted with hypotonic phosphate buffer from the kidneys of hypoxic rats. Normal rat serum enhanced the activity of this factor, which is associated with the light mitochondrial fraction. The data suggest that the renal factor is not physiologically active unless it interacts with a serum carrier or activator, or that the factor may be an enzyme which produces erythropoietin from some serum substrate.

Renal lysosomes: role in biogenesis of erythropoietin.

The "light" mitochondrial pellet obtained from the kidneys of rats previously treated with Triton WR-1339 and rendered hypoxic was separated into subcellular component fractions by sucrose density gradient centrifugation in a zonal rotor. Selected fractions were pooled, disrupted by osmotic lysis and repeated freeze-thawing, and incubated in the presence and absence of normal rat serum. The incubation mixtures were assayed for erythropoiesis-stimulating activity (erythropoietin). High specific activity was identified only in fractions rich in lysosomes. Biochemical analysis of reference enzymes for the identification of lysosomes and mitochondria, supplemented by electron microscopic examination of the various separated fractions, supports the observed requirement for lysosomal constituents in the formation of erythropoietin by the kidney.

Ethanol modulation of opiate receptors in cultured neural cells.

The mouse neuroblastoma-rat glioma hybrid cell line NG108-15 was used to study the acute and chronic interaction of ethanol with intact neural cells. In the short term, ethanol inhibited opiate receptor binding, but after long-term exposure the cells exhibited an apparent adaptive increase in the number of opiate binding sites; this was reversible when ethanol was withdrawn. High concentrations of ethanol (200 mM) increased opiate binding after 18 to 24 hours, whereas lower concentrations (25 to 50 mM) produced similar changes after 2 weeks. This model system has potential for exploring the cellular and molecular mechanisms underlying ethanol intoxication, tolerance, and withdrawal.

Erythropoiesis in the rat: differential rates of DNA synthesis and cell proliferation.

Direct in vivo estimates of DNA synthesis time in early and late erythroblasts were obtained by using the H(3)- and C(14)-thymidine double-la-beling technique. A double-emulsion autoradiographic procedure was used to resolve the two isotopes. Early erythroblasts were found to proliferate at a rate about five times that of late cells. This results primarily from a shorter mean DNA synthesis time in early cells (2.5 hours) than in late cells (6.5 hours).

Renal erythropoietic factor: role of ions and vasoactive agents in erythropoietin formation.

Evidence is provided for the existence of a renal erythropoietic factor, devoid of vasopressor activity, which upon interaction in vitro with normal serum yields erythropoietin. When undialyzed serum is used, erythropoietin inactivation develops in the incubation mixtures, and this inactivation appears to be dependent on an enzymatic component in preparations of the factor and on ions in serum.

Hepatic regeneration and erythropoietin production in the rat.

The regenerating liver produces erythropoietin in response to hypoxia. The amounts of erythropoietin produced in animals subjected to hepatectomy are significantly higher than those observed in sham-operated animals. Hepatic erythropoietin production appears to be dependent upon the stage of regeneration with the highest levels being produced during the period of greatest proliferation and increase in liver mass.

Nucleoside deaminase: an enzymatic marker for stress erythropoiesis in the mouse.

The level of nucleoside deaminase was determined in extracts of mouse tissues obtained during a period of accelerated erythropoiesis induced by hypoxia, hemorrhage, or the injection of phenylhydrazine. Under these conditions a striking (10- to 100-fold) elevation of the enzyme activity occurred in the spleen. Similar results were obtained with the injection of purified erythropoietin. In control animals, only a trace of nucleoside deaminase activity was detected in the blood. During the reticulocyte response which followed erythropoietic stimulation, there was a sharp increase in the blood level of nucleoside deaminase, which rose up to 120 times that of control animals. By differential centrifugation, the enzyme was localized to the reticulocyte-rich fraction. Erythrocyte nucleoside deaminase remained elevated even after the reticulocyte count had fallen to normal in the phenylhydrazine-treated mice or to zero after the cessation of hypoxia. There was a very gradual decline in the enzyme activity in the blood which fell to the barely detectable control levels about 45 days after the initial reticulocyte response, a time period which corresponds to the survival of the mouse red blood cell. The persistence of high levels of nucleoside deaminase for the full life span of a generation of erythrocytes formed during stress, viewed in contrast to the virtual absence of the enzyme from normal erythrocytes of all ages, represents an enzymatic difference between the normal red blood cell and the cell produced under conditions of accelerated erythropoiesis.

Characterization of an Arabidopsis Mutant That Is Nonresponsive to Inducers of Systemic Acquired Resistance.

Systemic acquired resistance (SAR) is a general defense response in plants that is characterized by the expression of pathogenesis-related (PR) genes. SAR can be induced after a hypersensitive response to an avirulent pathogen or by treatment with either salicylic acid (SA) or 2,6-dichloroisonicotinic acid (INA). To dissect the signal transduction pathway of SAR, we isolated an Arabidopsis mutant that lacks the expression of an SA-, INA-, and pathogen-responsive chimeric reporter gene composed of the 5[prime] untranslated region of an Arabidopsis PR gene, [beta]-1,3-glucanase (BGL2), and the coding region of [beta]-glucuronidase (GUS). This mutant, npr1 (nonexpresser of PR genes), carries a single recessive mutation that abolishes the SAR-responsive expression of other PR genes as well. While SA-, INA-, or avirulent pathogen-induced SAR protects wild-type plants from Pseudomonas syringae infection, the mutant cannot be protected by pretreatment with these inducers. The insensitivity of npr1 to SA, INA, and avirulent pathogens in SAR induction indicates that these inducers share a common signal transduction pathway. Moreover, in npr1, the localized expression of PR genes induced by a virulent Pseudomonas pathogen is disrupted, and the lesion formed is less confined. These results suggest a role for PR genes in preventing the proximal spread of pathogens in addition to their suggested role in SAR.

Granulopoiesis in "preleukemic" mice with anemia induced by Rauscher leukemia virus, variant a.

To understand further the hematopoietic dyscrasias induced by a variant (a) of Rauscher leukemia virus (RLV), we used Escherichia coli endotoxin to stress the hematopoietic system of control and RLV/a-infected BALB/c mice. During the preleukemic stages of virus infection, there was slight splenomegaly without peripheral blood erythroblastosis. Granulocyte release and tissue mobilization mechanisms appeared unaffected by the RLV/a infection. Both RLV/a-infected and control mice reacted to endotoxin with peripheral granulocytosis and peritoneal granulocyte mobilization, though the circulating granulocyte levels in RLV/a-treated mice initially were lower than those in controls. Spleen of RLV/a-infected animals were larger than those of controls, but both responded to endotoxin with elevated numbers of granulocytes and erythroblasts. Since numbers of bone marrow erythroblasts in both groups of mice were decreased after endotoxin, stem cell competition and/or shunting of stem cells from marrow to spleen may have been involved. Endotoxin also induced rapid falls in hematocrit levels in both groups. These studies suggested that RLV/a-infected mice can be a model to study 1) erythropoietic dysfunction uncomplicated by defective granulopoietic release and tissue mobilization control mechanisms, and 2) progression of evolving granulocytic leukemia.

Stimulation of erythropoietic differentiation in BALB/c mice infected with Rauscher leukemia virus.

The function of phenylhydrazine (PHZ) hemolysis in ameliorating the anemia induced in mice by a slow-acting strain of Rauscher leukemia virus (RLV-A) was described. After cessation of treatment with PHZ, mid-stage RLV-A-infected, anemic mice responded with massive reticulocytoses and a rebound in hematocrit above control levels. RLV-infected mice, subjected to PHZ-induced hemolysis or phlebotomy, produced high levels of plasma erythropoietin (Ep); this suggested that Ep mediated the PHZ-induced differentiation. In addition, administration of exogenous Ep induced a wave of erythroid maturation in RLV-infected anemic mice, which indicated that virus-infected erythroid precursors could still respond to the hormone governing normal differentiation.

Genetic variation among 82 pharmacogenes: The PGRNseq data from the eMERGE network.

Genetic variation can affect drug response in multiple ways, although it remains unclear how rare genetic variants affect drug response. The electronic Medical Records and Genomics (eMERGE) Network, collaborating with the Pharmacogenomics Research Network, began eMERGE-PGx, a targeted sequencing study to assess genetic variation in 82 pharmacogenes critical for implementation of "precision medicine." The February 2015 eMERGE-PGx data release includes sequence-derived data from ∼5,000 clinical subjects. We present the variant frequency spectrum categorized by variant type, ancestry, and predicted function. We found 95.12% of genes have variants with a scaled Combined Annotation-Dependent Depletion score above 20, and 96.19% of all samples had one or more Clinical Pharmacogenetics Implementation Consortium Level A actionable variants. These data highlight the distribution and scope of genetic variation in relevant pharmacogenes, identifying challenges associated with implementing clinical sequencing for drug treatment at a broader level, underscoring the importance for multifaceted research in the execution of precision medicine.

Membrane-bound protein kinase activity in acetylcholine receptor-enriched membranes.

Membrane protein phosphorylation may be a general regulatory mechanism mediating the response of cells to exogenous metabolic and physical signals. We have determined that the membrane-bound acetylcholine receptor is the major substrate phosphorylated in situ by a nearby membrane protein kinase. Moreover, these same membranes also contain phosphoprotein phosphatase activity which dephosphorylates the membrane-bound receptor. These findings suggest that reversible phosphorylation of the actylcholine receptor may be critical for receptor function at the synapse. Therefore, it is necessary to define the properties of the enzymes which mediate this phosphorylation-dephosphorylation mechanism. In this report we describe the properties of the first component of this system, the membrane-bound protein kinase in receptor-enriched membranes from the electric organ of Torpedo californica. Only ATP is effective as a phosphate donor for this cyclic AMP-independent membrane kinase; GTP does not support phosphorylation of the receptor. Both casein and histone can also be phosphorylated by the membrane protein kinase, but casein is a better substrate. Although phosphorylation of the receptor appears to be regulated by cholinergic ligands and K+, casein phosphorylation is not specifically affected by these agents. Moreover, while phosphorylation of the acetylcholine receptor is maximal in receptor=enriched membranes, casein phosphorylation is similar in all membrane fractions prepared from the electric organ. Taken together, these findings suggest that the membrane protein kinase activity in receptor-enriched membranes is similar to most other membrane kinases. Therefore, the unique characteristics of membrane-bound acetylcholine receptor phosphorylation appear to be determined by the receptor and its availability as a substrate for the membrane kinase.

Phenylhydrazine-induced leukocytosis in the rat.

Rats were injected with phenylhydrazine (PHZ) for periods of up to 6 months, during which time a marked leukocytosis was induced. The highest leukocyte counts occurred within 4-5 days following injection. An initial injection of 4 mg/100 gm body weight evoked a mean total leukocyte count of 129 X 10(3) cells/microliter. Successive weekly injections of 2 mg/100 gm resulted in a mean total leukocyte count of 70 X 10(3) cells/microliter compared to a mean total leukocyte count of 12.5 X 10(3) cells/microliter in saline-injected rats. Lymphocytes and monocytes accounted for approximately 75% of the total cell counts in both the PHZ-treated and control rats. The presence of increased numbers of mononuclear cells was confirmed by Percoll gradient separation and by phase-contrast microscopy. Although a leukocytosis was evident when using the automated Coulter electronic cell counter, it was not discernible when blood samples were counted manually in a hemocytometer by light microscopy. Histological examination of the thymus, lymph nodes, and spleen of the PHZ-treated rats indicated that lymphocytes and monocytes were mobilized from these sites. Lymphocyte depletion was evident, and germinal centers were found in all these lymphoid organs, indicating that PHZ induced a lymphopoietic response. A possible autoimmune etiology for PHZ-induced red blood cell destruction is discussed.

Mechanisms of extrarenal erythropoietin (Ep) production.

Evidence is presented for production, by the subtotally hepatectomized (hepx) rat, of a factor which induces morphological and physiological hyperactivity of the Kupffer (K) cells and increased formation of Ep by the regenerating liver. This factor, originally termed hepatopoietin (Hp), and more recently the hepatic erythropoietic factor (HEF), is detectable in higher concentration in the hepatic venous blood than in blood draining other organs, thus supporting its hepatic origin. Production of the HEF is best related to hyperactivity of the K cells and not to the parenchymal (P) cells. The HEF can be demonstrated by administering serum from hepx donors to normal rats which respond with increased production of Ep when nephrectomized (nephrx) and rendered hypoxic. Removal of the kidneys from hepx donors further augments the Ep response to this serum in recipients. Subtotal hepx also evokes the production of a renal inhibitory factor (RIF) which reduces the ability of the liver to function as an extrarenal source of Ep. This inhibitor is found in renal venous blood and not in blood draining other organs. It is suggested that the RIF reduces the hepatic Ep response to hypoxia by diminishing the production and/or activity of the HEF. The RIF possesses no anti-Ep activity and its appearance and actions are not influenced by accumulation of metabolic wastes (as in the nephrx or ureterally-ligated rat). Erythroblastic nests have been observed in regenerating livers at 24-48 hr after subtotal hepx. It would seem that removal of a considerable part of the liver, which stimulates hepatic regeneration, confers upon this organ an increased ability to produce Ep and to function as a hematopoietic inductive microenvironment (HIM) for erythropoiesis.

Renal and extrarenal production of erythropoietin following exchange transfusion with plasma or a perfluorocarbon-polyol.

The extrarenal production of erythropoietin (Ep) was measured in young adolescent rats following exchange-transfusion with normal rat plasma or the whole blood replacement fluid, Fluosol-43. In the plasma-transfused anephric groups, the peak extrarenal Ep response to the severe degree of anemic hypoxia represented 23% of that evoked in renal-intact groups. In contrast, the peak extrarenal production of Ep in Fluosol-transfused renoprival groups was almost equal to those serum Ep levels produced in renal-intact groups. Histological studies revealed significant fluorocarbon uptake by hepatic macrophages that coincided with the time of peak production of Ep. Thus, while intense levels of hypoxia are required to stimulate the extrarenal production of Ep, these data suggest that as Fluosol-43 is taken up by the hepatic macrophages, Ep production by these cells is stimulated.

Erythropoietin secretion by isolated rat Kupffer cells.

Hepatic parenchymal and Kupffer cells were isolated from regenerating rat liver and maintained in primary culture in an attempt to identify the cell type responsible for extrarenal erythropoietin (Ep) production. Conditioned media from Kupffer cell cultures were shown to contain Ep. Kupffer cells were cultured in the presence and absence of serum, but only cultures with serum contained bioactive Ep. The addition of latex beads to Kupffer cell cultures increased the amount of Ep secreted. Parenchymal-cell-conditioned media were negative for Ep activity. Isolated liver macrophages might be useful in vitro for the study of Ep elaboration.

Measurement of erythropoietin in chloroleukemic rats.

Erythropoietin (Ep) levels were measured in Shay chloroleukemic rats at various stages of anemia. Serum Ep was shown to increase logarithmically as the anemia became more severe. This increase in Ep levels was similar to that observed in normal rats subjected to acute blood loss. Significant levels of Ep were also demonstrated in ascitic fluid extracted from the peritoneal cavity of leukemic rats. These results indicate that the anemia of this disease is not due to a diminished production of Ep.

Erythropoietin in renal and hepatic lymph of conscious ewes.

Spontaneously flowing fistulae were established in the efferent lymphatics of popliteal, prescapular and prefemoral nodes and lumbar trunk or in the afferent lymphatics draining the kidney and liver of sheep. Lymph was collected from these sites over various time intervals and assayed for erythropoietin (Ep) content. The objective of the study was to establish the anatomic site(s) of Ep production. Normal lymph did not contain detectable titers of Ep, nor did renal lymph or blood plasma from a sheep systematically treated with cobaltous chloride. Renal lymph did contain measurable levels of Ep following renal artery constriction, unilateral hydronephrosis or phenylhydrazine-induced hemolytic anemia. Phenylhydrazine treatment also produced elevated Ep levels in lymph from the liver but not in lymph efferent from either popliteal or prescapular nodes. These results indicate that Ep is generated primarily in the kidney and that the liver may be an extrarenal source of the hormone. The surgical techniques used in this study offer distinct advantages in examining the composition and physiology of lymph in sheep.

Increased erythropoiesis and 2'5'-A polymerase activity in the marrow and spleen of phenylhydrazine-injected rats.

The rate of CFUE production in adult rats varies considerably according to whether the animals are rendered anemic by short or long term treatment with phenylhydrazine. The bone marrow responds during 2 months of phenylhydrazine injections with a progressive increase in CFUE, the spleen shows a rapid and larger rise after 3 daily injections followed by a decline to near zero, control numbers, after prolonged administration of the drug, and the liver never develops erythropoietic activity. Endogenous colonies, that is, colonies that grow in plasma clots without added erythropoietin, are most numerous in bone marrow after phenylhydrazine treatment, are always few in the spleen and are never present in cultures of liver cells. In both marrow and spleen, there is a direct correlation between the rate of erythropoiesis and 2'5'-A polymerase activity. These findings suggest that production of the enzyme may be an early event in erythroid cell differentiation.