Glyco-engineered anti-EGFR mAb elicits ADCC by NK cells from colorectal cancer patients irrespective of chemotherapy.

BACKGROUND: The epidermal growth factor receptor (EGFR) is overexpressed in colorectal cancer (CRC), and is correlated with poor prognosis, making it an attractive target for monoclonal antibody (mAb) therapy. A component of the therapeutic efficacy of IgG1 mAbs is their stimulation of antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells bearing the CD16 receptor. As NK cells are functionally impaired in cancer patients and may be further compromised upon chemotherapy, it is crucial to assess whether immunotherapeutic strategies aimed at further enhancing ADCC are viable. METHODS: CRC patients before, during and after chemotherapy were immunophenotyped by flow cytometry for major white blood cell populations. ADCC-independent NK cell functionality was assessed in cytotoxicity assays against K562 cells. ADCC-dependent killing of EGFR(+) A431 cancer cells by NK cells was measured with a degranulation assay where ADCC was induced by GA201, an anti-EGFR mAb glyco-engineered to enhance ADCC. RESULTS: Here, we confirm the observation that NK cells in cancer patients are dysfunctional. However, GA201 was able to induce robust NK cell-dependent cytotoxicity in CRC patient NK cells, effectively overcoming their impairment. CONCLUSIONS: These findings support the evaluation of the therapeutic potential of GA201 in combination with chemotherapy in CRC patients.

The intrinsic factor-vitamin B12 receptor, cubilin, is a high-affinity apolipoprotein A-I receptor facilitating endocytosis of high-density lipoprotein.

Cubilin is the intestinal receptor for the endocytosis of intrinsic factor-vitamin B12. However, several lines of evidence, including a high expression in kidney and yolk sac, indicate it may have additional functions. We isolated apolipoprotein A-I (apoA-I), the main protein of high-density lipoprotein (HDL), using cubilin affinity chromatography. Surface plasmon resonance analysis demonstrated a high-affinity binding of apoA-I and HDL to cubilin, and cubilin-expressing yolk sac cells showed efficient 125I-HDL endocytosis that could be inhibited by IgG antibodies against apoA-I and cubilin. The physiological relevance of the cubilin-apoA-I interaction was further emphasized by urinary apoA-I loss in some known cases of functional cubilin deficiency. Therefore, cubilin is a receptor in epithelial apoA-I/HDL metabolism.

Improved diagnostic criteria for digital flexor tendon sheath pathology using contrast tenography.

BACKGROUND: Pathology of the digital flexor tendon sheath is a significant cause of lameness in the horse. Imaging is important to identify lesions and inform on prognosis prior to tenoscopic surgery. OBJECTIVES: To use a large population to evaluate 1) the sensitivity and specificity of digital flexor tendon sheath (DFTS) contrast radiographs in diagnosing manica flexoria (MF) tears, deep digital flexor tendon (DDFT) tears and constriction of the palmar/plantar annular ligament (PAL) using novel criteria; 2) predisposition to pathology in signalment and limb affected. STUDY DESIGN: Multicentre retrospective cohort study. METHODS: The medical records of 206 horses with lameness localised to the DFTS, contrast radiographs and subsequent tenoscopic surgery were reviewed. Breed and limb predispositions were evaluated for pathology of the DDFT, MF and PAL constriction. Contrast radiographs of the DFTS were reviewed by four masked operators and for each pathology the sensitivity, specificity and interobserver variability were calculated. RESULTS: Contrast tenography was a sensitive test for MF tears (92% confidence interval [CI] 88.4-94.4%; specificity 56%, CI 51.1-61.1%) and specific for diagnosing DDFT tears (73%, CI 68.6-76.8%; sensitivity 54%, CI 47.8-60.2%) but had a lower sensitivity (71%, CI 65.1-75.9% ) and specificity (45%, CI 39.1-52.0%) for PAL constriction. It had good to substantial interobserver agreement for MF and DDFT tears (Krippendorff's alpha 0.68 and 0.46 respectively). Ponies (57%) and cobs (58%) were significantly more likely to be affected with MF tears (other breeds 20-39%, P = 0.003) and Thoroughbreds (50%), warmbloods (45%) and draught breeds (48%) were more likely to have DDFT tears (other breeds 22-34%, P = 0.01). MF tears and PAL constriction were overrepresented in the hindlimbs compared to DDFT tears in forelimbs. MAIN LIMITATIONS: No standardisation of contrast radiographs was possible. The subjectivity of diagnosis of PAL constriction may also have led to bias. Radiographs were read as JPEGS reducing ability to manipulate images. CONCLUSIONS: Contrast radiography of the DFTS is accurate in the pre-operative diagnosis of DFTS pathologies. Different pathologies are overrepresented in certain breeds and limbs.

Efficient FLPe recombinase enables scalable production of helper-dependent adenoviral vectors with negligible helper-virus contamination.

Helper-dependent (HD), high-capacity adenoviruses are one of the most efficient and safe gene therapy vectors, capable of mediating long-term expression. Currently, the most widely used system for HD vector production avoids significant contamination with helper virus by using producer cells stably expressing a nuclear-targeted Cre recombinase and an engineered first-generation helper virus with parallel loxP sites flanking its packaging signal. The system requires a final, density-based separation of HD and residual helper viruses by ultracentrifugation to reduce contaminating helper virus to low levels. This separation step hinders large-scale production of clinical-grade HD virus. By using a very efficient recombinase, in vitro-evolved FLPe (ref. 14), to excise the helper virus packaging signal in the producer cells, we have developed a scalable HD vector production method. FLP has previously been shown to mediate maximum levels of excision close to 100% compared to 80% for Cre (ref. 15). Utilizing a common HD plasmid backbone, the FLPe-based system reproducibly yielded HD virus with the same low levels of helper virus contamination before any density-based separation by ultracentrifugation. This should allow large-scale production of HD vectors using column chromatography-based virus purification.

The apolipoprotein epsilon4 allele determines prognosis and the effect on prognosis of simvastatin in survivors of myocardial infarction : a substudy of the Scandinavian simvastatin survival study.

BACKGROUND: Carriers of the epsilon4 allele of the apolipoprotein E gene are at a higher risk of coronary heart disease than individuals with other genotypes. We examined whether the risk of death or a major coronary event in survivors of myocardial infarction depended on apolipoprotein E genotype and whether the benefits of treatment with simvastatin differed between genotypes. METHODS AND RESULTS: Cox proportional hazards models were used to analyze 5.5 years of follow-up data from 966 Danish and Finnish myocardial infarction survivors enrolled in the Scandinavian Simvastatin Survival Study. A total of 16% of the 166 epsilon4 carriers in the placebo group died compared with 9% of the 312 patients without the allele, which corresponds to a mortality risk ratio of 1.8 (95% confidence interval, 1.1 to 3.1). The risk ratio was unaffected by considerations of sex, age, concurrent angina, diabetes, smoking, and serum lipids in multivariate analyses. Simvastatin treatment reduced the mortality risk to 0.33 (95% confidence interval, 0.16 to 0.69) in epsilon4 carriers and to 0.66 (95% confidence interval, 0. 35 to 1.24) in other patients (P=0.23 for treatment by genotype interaction). Apolipoprotein E genotype did not predict the risk of a major coronary event. Baseline serum levels of lipoprotein(a) also predicted mortality risk and could be combined with epsilon4-carrier status to define 3 groups of patients with different prognoses and benefits from treatment. CONCLUSIONS: Myocardial infarction survivors with the epsilon4 allele have a nearly 2-fold increased risk of dying compared with other patients, and the excess mortality can be abolished by treatment with simvastatin.

Effects of apoE gene polymorphism on Lp(a) concentrations depend on the size of apo(a): a study of 466 white men.

Polymorphisms in the genes for the low-density lipoprotein (LDL) receptor ligands, apolipoprotein E (apoE), and apolipoprotein B (apoB) are associated with variation in plasma levels of LDL cholesterol. Lp(a) lipoprotein(a) [Lp(a)] is LDL in which apoB is attached to a glycoprotein called apolipoprotein(a) [apo(a)]. Apo(a) has several genetically determined isoforms differing in molecular weight, which are inversely correlated with Lp(a) concentrations in blood. The interaction of apo(a) with triglyceride-rich lipoproteins differs with the size of apo(a), and therefore the effects of apoE gene polymorphism on Lp(a) levels could also depend on apo(a) size. We have investigated the possible effect of genetic variation in the apoE and apoB genes on plasma Lp(a) concentrations in 466 white men with different apo(a) phenotypes. Overall there was no significant association between the common apoE polymorphism and Lp(a), but in the subgroup with apo(a)-S4, concentrations of Lp(a) differed significantly among the apoE genotypes (P = 0.05). Lp(a) was highest in the apoE genotypes epsilon 2 epsilon 3 and epsilon 3 epsilon 3 and lowest in genotype epsilon 3 epsilon 4, and the apoE polymorphism was estimated to account for about 2.4% of the variation in Lp(a). In contrast, in the subgroup with apo(a)-S2 Lp(a) was significantly lower (P = 0.04) in apoE genotype epsilon 2 epsilon 3 than in genotype epsilon 3 epsilon 3. Lp(a) concentrations did not differ among the XbaI (P = 0.65) or SP 24/27 (P = 0.26) polymorphisms of the apoB gene. The expected effects of both apoE and apoB polymorphism on LDL levels were significant in the whole population sample and in subjects with large-sized apo(a) isoforms (P < 0.01), whereas no effect was seen in those with low molecular weight apo(a) isoforms. We conclude that the influence of apoE genotypes on Lp(a) concentrations depends on the size of the apo(a) molecule in Lp(a), possibly because both apo(a)-S4 and apoE4 have high affinity for triglyceride-rich lipoproteins and may be taken up and degraded rapidly by remnant receptors.

Transcriptional targeting to anterior pituitary lactotrophic cells using recombinant adenovirus vectors in vitro and in vivo in normal and estrogen/sulpiride-induced hyperplastic anterior pituitaries.

The use of pituitary cell type-specific promoters is a powerful molecular tool to achieve pituitary cell type-specific transcriptional targeting of transgenes encoded by viral vectors. It has recently been proposed that transcriptional targeting of therapeutic genes could be harnessed as a gene therapy strategy for the treatment of pituitary disease. We describe the successful use of the human PRL promoter (hPrl) encoded within recombinant adenovirus vectors to target transgene expression of Herpes Simplex Virus Type 1-Thymidine Kinase (HSV1-TK) or beta-galactosidase to lactotrophic cells in vitro and in vivo. Functionally, the restriction of expression of HSV1-TK to lactotrophic tumor cells, using the hPrl promoter, resulted in the cell type-specific induction of apoptosis in the lactotrophic GH3 tumor cell line, in the presence of ganciclovir (GCV). In the corticotrophic AtT20 cell line, we detected neither HSV1-TK expression, nor apoptosis in the presence of GCV. The hPrl promoter encoded within a recombinant adenoviral vector also restricted transgene expression to lactotrophic cells in primary anterior pituitary (AP) cultures, and importantly, within the anterior pituitary gland in vivo. When the HSV1-TK driven by hPrl promoter was used in an in vivo model ofestrogen/sulpiride lactotroph induced hyperplasia within the AP in situ, the treatment was not effective in either reducing the weight of the gland, the number of lactotrophic cells within the transduced area in vivo, or the circulating PRL levels. This is in contrast to the human cytomegalovirus promoter (hCMV) driving expression of HSV1-TK in the same experimental paradigm, which was effective in reducing pituitary weight and circulating PRL levels. Our results have important implications in the design of gene therapy strategies for pituitary tumors. We demonstrate that both the choice of the in vivo animal model, i.e. adenoma in the AP gland in situ, and the particular gene therapy strategy chosen, i.e. use of strong ubiquitous promoters vs. weaker but cell type-specific promoters, determine the experimental therapeutic outcome.

'Complete' spinal cord injury does not block perceptual responses to genital self-stimulation in women.

BACKGROUND: A priori hypothesis: vaginal and/or cervical self-stimulation will not produce perceptual responses in women with "complete" spinal cord injury (SCI) at or above the highest level of entry of the hypogastric nerves (T10-12) but will produce perceptual responses if SCI is below T-10. DESIGN: Women with complete SCI were assigned to a group with "upper" (T-10 and/or above) (n = 6) or "lower" (below T-10) (n = 10) SCI; uninjured women (n = 5) constituted a control group. Perceptual response to vaginal and/or cervical self-stimulation was quantified as magnitude of analgesia to calibrated finger compressive force. SETTING: Rutgers, The State University of New Jersey, Human Physiology Laboratory, College of Nursing, Newark. PARTICIPANTS: Consecutive samples of first 16 of 34 women with SCI who responded to nationwide advertisements, met inclusion criteria, and volunteered; control group was the first 5 respondents. INTERVENTION: Vaginal or cervical (cervix uteri) self-stimulation applied for 12 minutes, interspersed with non-stimulation periods, while measuring analgesia. MAIN OUTCOME MEASURE: Quantify analgesia magnitude to vaginal or cervical self-stimulation. RESULTS: Significant analgesia was produced in the uninjured group and the group with lower SCI, supporting the hypothesis. Unexpectedly, significant analgesia was also produced in the group with upper SCI. Women in the group with upper SCI also experienced menstrual discomfort, awareness of vaginal and/or cervical stimulation per se, and orgasms. CONCLUSIONS: (1) Genitospinal visceral afferent pathways function in the women in the group with upper SCI, although unrecognized by the American Spinal Injury Association criteria, and/or (2) there exists a functional genital afferent pathway that bypasses the spinal cord and projects directly to the brain, which we propose to be via the vagus nerves.

Isoform-specific binding of human apolipoprotein E to the non-amyloid beta component of Alzheimer's disease amyloid.

The non-A beta component (NAC) of Alzheimer's disease amyloid is a newly discovered 35 amino acid peptide found to be closely linked to the beta-amyloid fibrils in senile plaques. Apolipoprotein E (apoE) is another prominent constituent of senile plaques. In vitro studies have shown that apoE binds beta-amyloid (A beta) with high avidity, but it is unknown to what extent apoE interacts with NAC. We examined the interactions between apoE and NAC and found that apoE bound synthetic NAC, forming a complex that resisted reducing agents and separation on SDS-PAGE. The complex could be formed using apoE from either purified human very low density lipoprotein (VLDL) particles, unfractionated human cerebrospinal fluid (CSF), or recombinant protein. The binding was established within 15 min upon mixing, and the interaction between NAC and apoE was dose-dependent and specific as revealed by competition experiments. The NAC-apoE complex was affected by non-physiological pH, but not by reducing agents such as DTT or beta-mercaptoethanol. ApoE exists in different isoforms of which the apoE3 genotype is the most frequent. Notably, the apoE4 genotype has been linked to late-onset Alzheimer's disease. This study presents evidence that apoE3 as well as apoE4 bind NAC, but the binding to apoE4 is about twice as strong as to apoE3. The isoform-specific binding of NAC to apoE may thus play an important role in amyloidogenesis and in the sequestering of apoE in senile plaques during the progress of Alzheimer's disease.

Fts insertional mutant of Salmonella typhimurium.

A temperature-sensitive filamentation (fts) Salmonella typhimurium mutant was isolated after transposon mutagenesis with mini-Tn 10dTc. The mutant was unable to form colonies after 20 h incubation at 37 degrees C on LB agar. Colonies appeared, however, after longer incubation at the restrictive temperature. Filamentation affected only part of the bacterial population. Rapid mapping using Mu dP22 hybrid phages revealed that the mutation, ftsD220, lies within minutes 68.5 and 73.6 on the genetic map. Further analysis revealed that the ftsD220 mapped at min 73 and that it is linked to cysG (6%) and to aroB (39%). Complementation tests suggested that the ftsD220 mutation is not homologous to a Escherichia coli ftsH mutation.

Expression and purification of an Epstein-Barr virus encoded 23-kDa protein and characterization of its immunological properties.

Serodiagnosis of Epstein-Barr virus (EBV) infection is currently based on the detection of antibodies to distinct EBV antigens by immunofluorescence and enzyme-linked immunosorbent assay-based tests, or in part on the detection of heterophile antibodies by the Paul-Bunnell-Davidson heterophile assay. In the past few years, the specificity and the sensitivity of serodiagnostic assay systems has been improved considerably by the use of purified recombinant EBV antigens. Screening of EBV-positive sera for antigenic reactivities by immunoprecipitation with extracts of EBV-positive cells revealed a 23-kDa protein (p23) that was recognized by antibodies from all EBV carriers tested. Open reading frame BLRF2 was identified as the coding region for this protein. After cloning and high-level expression of the BLRF2 open reading frame as DHFR fusion protein in Escherichia coli, the recombinant protein was purified to near homogeneity with the help of continuous elution electrophoresis. Sera from both EBV-positive and -negative donors were screened by immunoblot analysis and enzyme-linked immunosorbent assay for IgM and IgG antibodies against the EBV-encoded protein p23. Since anti-p23 antibodies were not detectable in 30 of 30 EBV-negative sera, and 294 of 302 EBV-positive sera had either IgM and/or IgG antibody responses to this protein, recombinant p23 seems to be a useful diagnostic marker for EBV-infection.

Increase of serotonin in plasma during onset of halothane-induced malignant hyperthermia in pigs.

The levels of serotonin (5-HT) were determined in platelet-free and platelet-rich plasma before and during the onset of halothane-induced malignant hyperthermia (MH) in genetically MH-susceptible pigs. During MH onset, the free (i.e. physiologically active) levels of 5-HT in plasma rose concomitantly with the increases in muscle tone, body temperature, venous pCO2 and plasma lactate. Since pharmacological stimulation of 5-HT2 receptors has recently been shown to trigger MH in susceptible pigs, the finding of increased 5-HT plasma levels during onset of halothane-induced MH may indicate that 5-HT is involved in the mechanisms by which volatile anesthetics trigger this myopathic syndrome.

Lack of prophylactic or therapeutic efficacy of 5-HT2A receptor antagonists in halothane-induced porcine malignant hyperthermia.

During halothane-induced malignant hyperthermia (MH), plasma levels of serotonin (5-hydroxytryptamine, 5-HT) increase in pigs. Administration of 5-HT agonists which stimulate the 5-HT2A subreceptor triggers MH in susceptible pigs. A possible link between MH induced by 5-HT2A receptor agonists and halothane could be an increase of second messengers such as phosphoinositides (inositol polyphosphates), which have recently been implicated in the abnormal regulation of skeletal muscle calcium release in MH. If so, antagonists of 5-HT2A receptors which are linked to phosphoinositide turnover should be capable of preventing, retarding or attenuating halothane-induced MH. This possibility was investigated in the present study in MH susceptible pigs, using dantrolene for comparison, Development of MH triggered by a halothane challenge (inhalation of 3% halothane for 15 min) was completely prevented by dantrolene, 3.5 mg/i.v., whereas the 5-HT2A receptor antagonists ritanserin (0.5-10 mg/kg i.v.) or ketanserin (0.5-10 mg/kg i.v.) exerted no prophylactic effect. In pigs in which dantrolene, ritanserin or ketanserin where given in combination with hyperventilation after development of MH, dantrolene exerted therapeutic efficacy, whereas neither ritanserin nor ketanserin were effective treatments. The data indicate that 5-HT is not critically involved in the mechanisms of halothane-induced MH, at least under the conditions of the present experimental study.

Generation of analytic plasma lipoprotein profiles using two prepacked superose 6B columns.

A simple, low-priced chromatographic system to generate plasma lipoprotein profiles from total human plasma was tested with plasma from normolipidemic subjects and patients with heterozygous familial hypercholesterolemia, hyper-alpha-lipoproteinemia, lipoprotein lipase deficiency, familial dysbetalipoproteinemia and familial lecithin-cholesterol-acyl-transferase deficiency. The system appears to be a good alternative to more expensive high-pressure liquid chromatography systems, notably in lipoprotein laboratories already provided with equipment for column chromatography. If a microplate photometer and a computer is available in the laboratory, the measurement of various lipids in 70-80 eluant fractions from the columns can be simplified.

Differential ontogenesis of three DOI-induced behaviors in mice.

Our previous studies have shown that intraperitoneal administration of DOI [(+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane] simultaneously produces the head-twitch and ear-scratch responses (HTR and ESR, respectively) in mice via activation of 5-HT2A receptors. In the present study, we have investigated the ontogeny of these DOI-induced behaviors in both male and female mice across a wide age range (i.e., postnatal days 7, 14, 18, 22, 28, 35, 42, 63, 120, and 180). We also measured the effects of DOI on the locomotor activity of these mice. In addition to the vehicle, 2 doses of DOI (1 and 2.5 mg/kg) were used in age-matched different male and female groups. The age of onset for significant production of HTR and ESR by both doses of DOI were between postpartum days 14-18 and 18-22, respectively. Maximal HTR frequency to both doses of DOI (66 and 94 HTRs) occurred on postpartum day 28. Thereafter, the HTR frequency tended to decrease with increasing age, but the attenuation did not attain significance. On the other hand, maximal ESR score (37 and 60 ESRs) generally developed between postpartum days 22-35 for the cited doses of DOI. After 35 days of age, the ESR frequency dramatically decreased and, by postnatal day 180, no significant response was obtained to either dose of DOI. Age-matched vehicle-treated male and female control groups exhibited few (1-8) HTRs and ESRs across the age range tested. DOI dose-dependently enhanced locomotor activity in both male and female mice relative to their age- and sex-matched vehicle-treated controls for the first 28 days of life. Thereafter, no significant effect was observed. None of the cited behaviors exhibited gender differences across the age range tested. The present results suggest that DOI-induced changes in HTR, ESR, and locomotor activity develop and mature differentially, but in a similar manner, in male and female mice. Furthermore, unlike DOI-induced HTR, the ability of DOI to produce ear-scratches and to enhance locomotor activity in mice disappears with old age.

Temporal differential adaptation of head-twitch and ear-scratch responses following administration of challenge doses of DOI.

Previously, we reported that administration of the 5-HT2A/C receptor agonist, DOI [(+/-)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane], can simultaneously produce the head-twitch response (HTR) and the ear-scratch response (ESR) in mice. Our recent studies have indicated that the HTR is a 5-HT2A receptor-mediated phenomenon, whereas the ESR is probably a 5-HT2C receptor-mediated event. The HTR and ESR exhibit subsensitivity to a challenge dose of DOI (2.5 mg/kg) administered 24 h after its acute or termination of its chronic (2.5 mg/kg, once daily for 13 days) administration. When the dose interval for the challenge dose of DOI was increased to 48 h, both the acute- and chronically treated mice exhibited a simultaneous supersensitive HTR response and a subsensitive ESR effect. The purpose of the present study was to investigate the dose-response effects of lower challenge doses of DOI 48 h following their respective first injections as well as determining the effects of repeated DOI injections at 2-h intervals for 8 h. Thus, in the present study, initial administration of DOI produced a dose- and time-dependent increase in the mean frequencies of both HTR and ESR. Significant HTRs were observed after administration of the lowest tested dose of DOI (0.25 mg/kg), whereas a robust frequency of ESR was only evident at 1 mg/kg or greater doses of DOI. A 48-h challenge administration of lower doses of DOI (0.25 and 0.5 mg/kg) did not significantly affect their respective first injection HTR scores.(ABSTRACT TRUNCATED AT 250 WORDS)

The head-twitch response in the least shrew (Cryptotis parva) is a 5-HT2- and not a 5-HT1C-mediated phenomenon.

Our initial studies suggested that the 5-HT2/1C agonist (+/-)-1-(2,5-dimethoxy-4-iodophenyl-2-aminopropane [(+/-)-DOI] produces both the head-twitch response (HTR) and the ear-scratch response (ESR) in mice via stimulation of 5-HT2 receptors. However, challenge studies revealed that these behaviors are produced via two different receptors (possibly 5-HT2 and 5-HT1C). Due to a lack of selective agents one cannot designate a particular response for the activation of a specific receptor. The purpose of the present study was to investigate such behaviors in the least shrew, which is more sensitive to (+/-)-DOI than rodents. IP injection of (+/-)-DOI in shrews produced a dose-dependent (bell-shaped) and time-dependent increase in the HTR frequency. The (+/-)-DOI-induced HTR was equipotently and completely attenuated by the 5-HT2/1C antagonists ketanserin and spiperone. The 5-HT1C antagonist with 5-HT2 agonist action, lisuride, also produced the HTR in a bell-shaped dose- and time-dependent fashion. Central injections of both (+/-)-DOI (0.2 microgram) and lisuride (0.5 microgram) also induced the behavior. Both peripheral and central administration of lisuride failed to produce the ESR. (+/-)-DOI significantly induced the ESR only at the highest dose tested (2.5 mg/kg, IP). Centrally administered (+/-)-DOI (0.2 microgram) produced more ESRs relative to vehicle controls; however, the difference did not attain significance. At low doses (0.31 and 0.63 mg/kg), (+/-)-DOI had no effect on locomotor activity, but it significantly attenuated the behavior at larger doses. Both low and high doses of lisuride increased the motor activity. Spiperone dose-dependently suppressed locomotion, whereas ketanserin had no effect. The present results suggest that the HTR is a 5-HT2 receptor-mediated event and changes in locomotor activity do not affect the induced HTR.

Proteolysis in heterocyst-forming cyanobacteria: characterization of a further enzyme with trypsin-like specificity, and of a prolyl endopeptidase from Anabaena variabilis.

Soluble extracts of the cyanobacterium Anabaena variabilis ATCC 29413 and an engineered mutant that lacks an intracellular protease cleaving after Lys and Arg (Maldener, Lockau, Cai, and Wolk, Mol. Gen, Genet. 225, 113-120 (1991)) were separated by ion exchange chromatography, and protease profiles determined using azocasein, N alpha-benzoyl-D,L-arginine-4-nitroanilide and N-carbobenzoxy-glycyl-L-proline-4-nitroanilide as substrates. A second enzyme cleaving at the carboxyl side of lysine and arginine, and a prolyl endopeptidase were detected, enriched and characterized. Both proteolytic enzymes appear to be located in the periplasm.

Patients' perceptions of outcomes of a Canadian hospitalization.

Notes that with health care reform moving at tremendous speed throughout Canada, a great deal of interest in outcomes research has been generated. States that the research team consisted of 17 professional practice leaders from eight disciplines. Proposes, through the research, to identify from the perspective of former patients what results they hoped to achieve prior to discharge from hospital and what facilitated and hindered them in achieving these results. Reports that a representative sample was selected for the study. Forty-one former patients each participated in up to two focus groups, with a total of 16 focus groups conducted. Hierarchical analysis revealed themes that fell within the framework of structure, process and outcomes. The findings will assist in ensuring that more appropriate and effective care is offered to patients by a variety of disciplines.

Atypical reactions to halothane in a subgroup of homozygous malignant hyperthermia(MH)-susceptible pigs: indication of a heterogenous genetic basis for the porcine syndrome.

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle. In genetically susceptible pigs, MH can be induced by volatile, halogenated anaesthetics such as halothane. Within a series of pharmacological investigations, a fulminant MH could be induced in 59 of 66 homozygous halothane-susceptible pigs by a challenge with 3% halothane for 15 minutes. The typical MH was characterized by sudden appearance of tachycardia, muscle rigidity with typical extension of the hindlimbs, increase of body temperature, acidosis-caused by rapid increase of CO2 and lactate production-, hyperkalaemia and increased activity of creatine kinase (CK) and aspartate transaminase (AST). In seven homozygous MH-susceptible pigs, this typical MH could not be induced by halothane. These animals responded with sudden appearance of bradyarrhythmia and decrease of arterial pressure. In these MH-atypical pigs (MHA) neither the typical extension of hindlimbs nor a hyperthermia occurred. Compared to a group of 6 MH-susceptible pigs with typical reactions to halothane (MHS), the biochemical alterations were significantly retarded in MHA-pigs. These atypical reactions to halothane could be the effect of decreased cardiac output. Concerning the atypical reactions, we observed a familiar predisposition in MH-susceptible pigs. Although atypical reactions were not found in a group of homozygous halothane-nonsusceptible pigs (MHN), a possible explanation for atypical reactions could be a MH-independent halothane-susceptibility of the myocardium+ in MHA-pigs. On the other side the data may indicate that a primary defect in both the skeletal muscle and also the myocardium is involved in MH. The different reactions to halothane in MH-susceptible pigs could point to a genetic heterogeneity.