Determination of apparent association constants of steroid-cyclodextrin inclusion complexes using a modification of the Hummel-Dreyer method.

A modified Hummel-Dreyer method was used to calculate the apparent association constants of steroid-cyclodextrin inclusion complexes. An external calibration technique was employed, using the y-intercept from a plot of peak area versus concentration to correct for sample solvent effects. Mobile phase temperature and sample diluent organic content were found to be critical factors affecting the accuracy and reproducibility of the results. For four of six sets of data, the modified Hummel-Dreyer method yielded statistically equivalent results to another HPLC method for determining apparent association constants. Limitations of the modified Hummel-Dreyer method are discussed. In particular, the accuracy of the method is poor when measuring small apparent association constants.

Tissue-specific and developmental effects of the easily shocked mutation on ethanolamine kinase activity and phospholipid composition in Drosophila melanogaster.

The easily shocked (eas) gene of Drosophila melanogaster encodes ethanolamine kinase (EK), the first step in phosphatidylethanolamine (PE) synthesis via the CDP-ethanolamine pathway Flies mutant for eas display a complex neurological phenotype. In this paper we look at the contribution of EK to lipid metabolism during Drosophila development with the goal of linking the eas biochemical defect with the organismal phenotype. Using a chromatography-based assay, EK activity was detected in wild-type flies throughout development. Most of the activity in the adult was present in heads, which is primarily tissue of neural origin. Flies mutant for eas showed severely reduced levels of activity at each stage assayed. Using standard extraction methods and thin layer chromatography, phospholipid composition was assayed in wholeflies and in heads. While PE levels were decreased significantly in both tissues, heads also had significantly less phosphatidylserine (PS). Therefore, decreases in both phospholipids may play a role in producing the aberrant phenotype in eas flies.

Rit, a non-lipid-modified Ras-related protein, transforms NIH3T3 cells without activating the ERK, JNK, p38 MAPK or PI3K/Akt pathways.

The biological functions of Rit (Ras-like protein in tissues) and Rin (Ras-like protein in neurons), members of a novel branch of Ras-related GTP-binding proteins that are approximately 50% identical to Ras, have not been characterized. Therefore, we assessed their activity in growth control, transformation and signaling. NIH cells stably expressing a constitutively activated mutant of Rit [Rit(79L)] (analogous to the oncogenic mutant H-Ras(61L)) demonstrated strong growth transformation, proliferating rapidly in low serum and forming colonies in soft agar and tumors in nude mice. Although Rit(79L) alone did not promote morphologically transformed foci, it cooperated with both Raf and Rho A to form Rac/Rho-like foci. Rin [Rin(78L)] cooperated only with Raf. Rit(79L) but not Rin(78L) stimulated transcription from luciferase reporter constructs regulated by SRF, NF-kappaB, Elk-1 and Jun. However, neither activated ERK, JNK or p38, or PI3-K/Akt kinases in immune complex kinase assays. Interestingly, although Rit lacks any known recognition signal for C-terminal lipidation, Rit-transformed cell growth and survival in low serum is dependent on a farnesylated protein, as treatment with farnesyltransferase inhibitors caused apoptosis. Rin cooperated with Raf in focus assays but did not otherwise function in these assays, perhaps due to a lack of appropriate effector pathways in NIH3T3 fibroblasts for this neural-specific Ras family member. In summary, although Rit shares most core effector domain residues with Ras, our results suggest that Rit uses novel effector pathways to regulate proliferation and transformation.

Intervention assessment in an Indian health service pharmacy.

OBJECTIVE: To document and evaluate pharmacists' interventions in a setting that has complete and immediate access to patient information. DESIGN: Descriptive report evaluating self-reported interventions made by pharmacists during the conduct of routine dispensing activities. The data collection period was from February 15 to April 1, 1994. SETTING: Ambulatory care facility offering medical and dental care to high school residents, Native Americans, and Alaska Natives in Northwestern Oregon. MAIN OUTCOME MEASURES: Intervention rate per 100 new prescriptions dispensed. Each intervention was evaluated with regard to the information used to initiate it, when during the dispensing process it was initiated, and the intervention type. Outside evaluators determined the clinical significance of the interventions, including potential adverse health consequences, the likelihood of their occurrence, and the level of medical care that would have been required to treat the problem. RESULTS: Of 2,535 orders screened, 104 interventions (4.1%) were collected; 71% of these occurred during chart screening. Pharmacists most often used the medication order itself (60.6%) to detect prescribing problems, followed by other records in the patient's chart (29.8%). Outside evaluators identified 47.1% of the 104 interventions as clinically significant. The most common adverse health consequence prevented was inadequate control of the patient's condition. Outside evaluators also found that the most common level of corrective care that would have been needed if the intervention had not occurred, was a scheduled physician office visit (59.2%). CONCLUSION: This information suggests that pharmacists who have access to patient information may intervene at higher rates and that more of their interventions may be deemed clinically significant. However, larger, double-blinded, case-controlled studies are needed to definitively draw these conclusions.

Characterization of inclusion complexes of betamethasone-related steroids with cyclodextrins using high-performance liquid chromatography.

HPLC was used to study the inclusion complexes formed between various beta- and gamma-cyclodextrins and a series of corticosteroids related to betamethasone. Apparent association constants were measured in acetonitrile-water for a set of 13 steroids. An increase in the stability of the steroid-cyclodextrin complex is observed at lower concentrations of acetonitrile. The effects of the nature of the halide at the 9-position, the location of a double bond within the C-ring, substitution at the 9- and 11-positions, and modification of the D-ring of the steroid backbone were studied. The 11- and 17-positions were found to be critically involved in the inclusion process. Larger apparent association constants were obtained with gamma-cyclodextrin (gamma-CD) than with beta-cyclodextrin (beta-CD) due to the increased diameter of the gamma-CD cavity. Van't Hoff plots were constructed to examine the thermodynamic properties of the inclusion process. Plots constructed using retention factors were found to be nonlinear when gamma-CD was present in the mobile phase. This is due to an increase in the strength of the inclusion complex as temperature decreases. Plots constructed using apparent association constants were linear, indicating that the mechanism of inclusion does not change over the range of temperatures studied (10 to 80 degrees C). Enthalpy-entropy compensation was observed for 11 of the 13 steroids studied. The usefulness of cyclodextrins to achieve the separation of steroids in HPLC is discussed and a practical application for the analysis of a steroid and three potential impurities is described.

Cardiac tamponade caused by serous pericardial effusion in patients on extracorporeal membrane oxygenation.

PURPOSE: Extracorporeal membrane oxygenation (ECMO) has been successful in the treatment of critically ill children; however, its use has been accompanied by a broad range of complications. The authors describe the presentation, clinical course, treatment, and outcome of 4 patients on ECMO in whom pericardial tamponade developed caused by a serous effusion. METHODS: A retrospective review of patients placed on ECMO at our institution from 1993 to 1997 was performed. The case histories of 4 patients in whom pericardial tamponade developed caused by a serous effusion were reviewed in detail. RESULTS: The first patient presented with hypotension while on venovenous (VV) ECMO. The hypotension improved with fluid resuscitation. The patient was converted from (VV) to venoarterial (VA) ECMO when hypotension recurred. After a third episode of hypotension, a narrow pulse pressure was noted, and echocardiography results confirmed a pericardial effusion. The diagnosis was recognized earlier in the course of the subsequent 3 patients. All 4 patients were treated with aspiration of serous fluid from the pericardium with an over-the-needle plastic catheter that was left in place. More than 1 aspiration was required in all cases. All 4 patients survived. CONCLUSIONS: The authors have identified a group of ECMO patients with pericardial tamponade caused by serous effusion with good response to treatment. A high index of suspicion and early echocardiography is warranted to confirm the diagnosis in a patient with hypotension on ECMO.

Pharmacological inhibition of Ras-transformed epithelial cell growth is linked to down-regulation of epidermal growth factor-related peptides.

BACKGROUND & AIMS: Posttranslational farnesylation is required for Ras activation. Farnesyl transferase inhibitors (FTIs) selectively block protein farnesylation and reduce the growth of many Ras-transformed cells in vitro and in vivo. Activated Ras transforms rat intestinal epithelial (RIE-1) cells by a mechanism distinct from NIH 3T3 fibroblasts in that an epidermal growth factor receptor (EGFR) autocrine loop contributes significantly to the Ras-transformed RIE-1 phenotype. METHODS: The ability of FTIs to block growth of Ras-transformed RIE-1 cells was evaluated, and these results were correlated with decreased EGFR ligand production. RESULTS: FTI L744,832 caused a selective, dose-dependent, reversible blockade in proliferation of H-Ras-transformed RIE-1 cells, whereas control cell lines, K-Ras-transformed cells, and activated raf-transfected RIE cells were unaffected. The growth-inhibitory effects of L744,832 correlated with loss of farnesylated H-Ras protein and a marked reduction in transforming growth factor (TGF)-alpha and amphiregulin expression. Inhibition of proliferation of H-Ras RIE-1 cells by L744,832 was overcome by exogenous TGF-alpha, and enhanced growth inhibition was achieved by EGFR blockade in combination with L744,832. + CONCLUSIONS: These data suggest that one mechanism by which FTIs inhibit growth of H-Ras-transformed epithelial cells is by reducing Ras-induced EGFR ligand production.

Ras, but not Src, transformation of RIE-1 epithelial cells is dependent on activation of the mitogen-activated protein kinase cascade.

Src transformation of NIH3T3 mouse fibroblasts has been shown to be dependent on Ras function. Since we recently showed that the signaling pathways that mediate Ras transformation of RIE-1 rat intestinal epithelial cells are distinct from those that cause Ras transformation of fibroblasts, we utilized three approaches to determine if Src transformation of RIE-1 cells is dependent on Ras. First, although both Ras and Src cause upregulation of an epidermal growth factor (EGF) receptor-dependent autocrine growth loop, only Ras transformation required this activity. Second, whereas both Src and Ras caused upregulation of the p42 and p44 mitogen-activated protein kinases (MAPKs), only Ras transformation was blocked by the inhibition of MAPK activation by treatment with the PD 98059 MEK inhibitor. Third, treatment with the farnesyltransferase inhibitor FTI-277 blocked Ras, but not Src, transformation. Taken together, these observations suggest that Src transformation of RIE-1 cells is not dependent on Ras. Finally, we determined that Ras activation of Raf-independent pathways alone is sufficient to cause growth transformation of RIE-1 cells. Thus, both Ras and Src cause transformation of RIE-1 cells via pathways distinct from those required to cause transformation of NIH3T3 cells.

The seizure locus encodes the Drosophila homolog of the HERG potassium channel.

Mutations in the seizure (sei) locus cause temperature-induced hyperactivity, followed by paralysis. Gene cloning studies have established that the seizure gene product is the Drosophila homolog of HERG, a member of the eag family of K+ channels implicated in one form of hereditary long QT syndrome in humans. A series of five null alleles with premature stop codons are all recessive, but viable. A missense mutation in the sei gene, which changes the charge at a conserved glutamate residue near the outer mouth of the pore, has a semidominant phenotype, suggesting that the mutant seizure protein acts as a poison in a multimeric complex. Transformation rescue of a null allele with a cDNA under the control of an inducible promoter demonstrates that induced expression of seizure potassium channels in adults rescues the paralytic phenotype. This rescue decays with a t1/2 of approximately 1-1.5 d after gene induction is discontinued, providing the first estimate of ion channel stability in an intact, multicellular animal.

A maternal product of the Punch locus of Drosophila melanogaster is required for precellular blastoderm nuclear divisions.

The Punch locus of Drosophila melanogaster encodes the pteridine biosynthesis enzyme guanosine triphosphate cyclohydrolase. One class of Punch mutants is defective for a maternal function that results in embryonic death. We demonstrate here that the embryos exhibit nuclear division defects during the precellular blastoderm stage of development. These defects include abnormal nuclear distribution, mitotic asynchrony, and persisting chromatin bridges. Daughter nuclei that do not complete chromosome separation nevertheless initiate new interphase and mitotic cycles. As a result, interconnected mitotic figures are observed. Mitotic spindles and nuclear envelopes appear essentially normal. A mutant phenocopy was induced in wild-type embryos by treatment with the guanosine triphosphate cyclohydrolase inhibitor, 2,4-diamino-6-hydroxypyrimidine, at a very early cleavage stage. Furthermore, an inhibitor of a terminal step in pteridine biosynthesis produced an identical phenotype. Immunolocalization experiments define expression of Punch protein in nurse cells during oogenesis. The protein is packaged into granules as it is transported into the oocyte cytoplasm. As syncytial blastoderm nuclear divisions proceed, Punch protein levels decrease and disappear by cellularization. Defects in the expression of the protein in Punch maternal effect mutants correlate well with the early phenotypes. These results show that a Punch product is directly involved in early nuclear divisions and suggest a possible role in chromosome separation.

Stability of atenolol in an extemporaneously compounded oral liquid.

The stability of atenolol in an oral liquid stored for 40 days under various conditions was studied. A liquid preparation of atenolol 2 mg/mL was prepared by triturating 50-mg atenolol tablets with a commercially available oral diluent. Twelve 30-mL portions in vials were prepared. Three vials were refrigerated (5 degrees C) and shaken immediately before analysis, three were stored at room temperature (25 degrees C) and shaken, three were refrigerated and not shaken, and three were stored at room temperature and not shaken. One sample from each vial was assayed in duplicate on days 0, 15, 30, and 40 by stability-indicating high-performance liquid chromatography. The concentration of atenolol remained above 90% of the original concentration in each vial under each of the four sets of conditions. Microbial growth occurred in a few cultured samples, and pH changed minimally. Atenolol 2 mg/mL in an oral liquid was stable for up to 40 days when stored at 5 or 25 degrees C and shaken or at 5 or 25 degrees C and not shaken.

Direct sensing platinum ultramicrobiosensors for glucose.

Miniaturized glucose biosensors were constructed from disk-shaped ultramicroelectrodes (UMEs) (diameter = 10 microns and 25 microns), cylindrical UMEs (diameter = 25 microns), and platinum black modified UMEs. Glucose oxidase (E.C.1.1.3.4) was immobilized by glutaraldehyde cross-linking. All ultramicrobiosensors (UMBs) had response times (100% response) of less than 1 min, and had linear response over the human clinical range for glucose (3-7 mM). Response to glucose was linear over a greater concentration range at cylindrical UMBs, compared to similarly prepared disk-shaped UMBs, typically from 3-20 mM glucose. Thin, electrochemically polymerized films were used to prevent signals due to interfering species. A variety of different films, and electropolymerization conditions were examined. Poly(1,3-diaminobenzene) (1,3-DAB) was found to be the most effective at preventing signals due to interferents. Poly(1,3-DAB) was used to protect the biosensor from fouling. A 25 microns biosensor, with poly(1,3-DAB), was used for the direct measurement of glucose in complex samples.

Organization and expression of the Drosophila FMRFamide-related prohormone gene.

The Drosophila FMRFamide gene is comprised of two exons separated by a 2.8-kb intervening sequence. The first exon consists of the 5' untranslated region and is spliced to the initiator methionine codon. The second exon encodes the prohormone and the 3' untranslated region of the mRNA. The promoter region contains a TATA box 30 nucleotides upstream from a consensus transcription start site. Immunohistochemical studies using antibodies generated against synthetic peptides and in situ hybridization histochemistry define a set of about 40 neurons in the brain and ventral ganglia that express the prohormone gene.

Molecular and developmental genetics of the Punch locus, a pterin biosynthesis gene in Drosophila melanogaster.

Punch (Pu), the gene encoding the pterin biosynthetic enzyme GTP cyclohydrolase in Drosophila, is a complex locus. Mutations fall into several complementation classes that correspond to classes of mutants with distinct morphological and protein phenotypes. Two of these classes are developmentally specific, with mutants in each having defects in discrete subsets of the known functions of the locus. Defined functions of the locus include a role in embryonic nuclear divisions using initially a maternal Pu product, the synthesis of pterin cofactors that are required for catecholamine biosynthesis beginning in late embryogenesis, and the production of pterin-screening pigments in the developing adult eye. Mutant phenotypes include an interruption in synchronous nuclear divisions in precellular blastoderm embryos, a segment pattern phenotype in late embryos, failure to pigment and cross-link embryonic cuticular structures and failure to synthesize red eye pigments. Molecular analysis reveals that the locus is large, a minimum of 29 kb as defined by Southern mapping of Pu mutants. This region is transcriptionally extremely active, encoding at least 16 developmentally regulated transcripts. One transcript has been shown to be responsible for the production of the adult eye GTP cyclohydrolase on the basis of developmental profile, location with respect to the mapping of eye-specific Pu mutants, absence in eye-specific mutants, and hybrid-selection in vitro translation experiments. Several other transcripts are candidates for Pu vital functions, as suggested by their pattern of expression and their derivation from regions to which lethal Pu mutations map.

Characterization of new Punch mutations: identification of two additional mutant classes.

The Punch locus of Drosophila melanogaster which encodes the pteridine biosynthetic enzyme, GTP cyclohydrolase, is genetically complex. Lethal alleles of the locus resolve into an array of interallelic complementation groups, and at least one class of mutations is developmentally specific, affecting GTP cyclohydrolase activity only in the heads of adults. All previously isolated Punch alleles were identified on the basis of a mutant eye color phenotype. By screening mutagenized chromosomes over Punch region deficiencies, we have now isolated new alleles on the basis of lethal and visible phenotypes. Most of these alleles fall into previously identified genetic classes, but two new classes of mutations were also found. One class contains two alleles that behave as dominant lethal mutations in some genetic backgrounds. The other class represents a second developmentally specific set of alleles that affect the function of the Punch locus only during embryogenesis.

An analysis of the embryonic defects in Punch mutants of Drosophila melanogaster.

Punch (Pu), a complex genetic locus, encodes GTP cyclohydrolase, the first enzyme in the pteridine biosynthetic pathway. In the larval and adult stages of the Drosophila life cycle, the function of the locus can be monitored by enzyme assays. Although enzyme activity cannot be detected prior to larval stages, the locus must also have earlier functions since most homozygous Pu mutants die during embryogenesis. In order to assess the role of the locus during this stage of development, morphological examinations of embryos from different classes of Pu mutants were performed. An exact correspondence has been found between genetic and morphological classes of Pu mutations. The locus is required during two periods of embryogenesis. These requirements are genetically separable as shown by mutants with defects specific to each period. An early function utilizes both maternal and zygotic components. Mutants defective for these components have abnormal segment patterns. Late in embryogenesis, a Pu product is necessary for the proper pigmentation of larval cuticle and proper orientation and differentiation of other larval structures, particularly in the head region. A cold-sensitive period corresponds to this later function as determined by temperature-shift experiments. Some of the phenotypes observed correspond to known physiological roles of pteridines; others are unexpected and unexplained.

Tissue-specific and complex complementation patterns in the Punch locus of Drosophila melanogaster.

Mutations in the Punch locus result in loss of GTP cyclohydrolase activity, but all mutations do not affect the enzyme in the same way. There are at least three classes of Punch mutations. One class results in a dominant eye color, recessive lethal phenotype. A second class of mutations also causes a recessive lethal phenotype, but heterozygous mutants have normal eye color. They show loss of GTP cyclohydrolase function in all tissues where activity can be measured. Alleles comprising a third class are recessive eye color mutations that are homozygous viable. Individuals with this third type of mutation show loss of enzyme activity in the eye, but show normal or near-normal activity elsewhere. In order to examine the organization and function of this locus further, we have performed interallelic complementation tests on 25 Punch mutations, monitoring viability and enzyme activity in prepupae and adults. Most allele combinations are lethal. Those that complement do so in ways that are tissue-or stage-specific and unpredictable. Tests of mutants with tissue-specific phenotypes and of individuals mutant for complementing Punch lethal alleles lead us to conclude that Punch is a complex locus, both with respect to its organization and to its products.

Controlled trial of continuous positive airway pressure given by face mask for hyaline membrane disease.

A controlled trial of elective intervention with continuous positive airway pressure (CPAP) was performed on 24 infants with hyaline membrane disease whose arterial oxygen tension (Pao2) fell below 8kPa (60 mmHg) while they were breathing a fractional inspired oxygen concentration (F1O2) greater than 0.60. A face mask was used to apply the CPAP. The progress of the 12 infants who were treated on entry to the trial was compared with that of 12 infants who were treated later. All 12 infants in the early-intervention group and 8 infants in the late-intervention group survived. When CPAP was started, Pao2 increased and the early-treated infants breathed high concentrations of oxygen for a shorter period than the late-treated infants. The 4 infants in the early-intervention group who required mechanical ventilation needed lower mean airway pressures to achieve satisfactory gas exchange than the 7 ventilated infants in the late-intervention group. We conclude that a Pao2 less than 8 kPa while breathing an F1o2 greater than 0.60 is an adequate indication for giving CPAP in hyaline membrane disease, and that early intervention with CPAP allows infants who go on to require mechanical ventilation to be ventilated at lower pressures.