A functional alternative splicing mutation in human tryptophan hydroxylase-2.

The brain serotonergic system has an essential role in the physiological functions of the central nervous system and dysregulation of serotonin (5-HT) homeostasis has been implicated in many neuropsychiatric disorders. The tryptophan hydroxylase-2 (TPH2) gene is the rate-limiting enzyme in brain 5-HT synthesis, and thus is an ideal candidate gene for understanding the role of dysregulation of brain serotonergic homeostasis. Here, we characterized a common, but functional single-nucleotide polymorphism (SNP rs1386493) in the TPH2 gene, which decreases efficiency of normal RNA splicing, resulting in a truncated TPH2 protein (TPH2-TR) by alternative splicing. TPH2-TR, which lacks TPH2 enzyme activity, dominant-negatively affects full-length TPH2 function, causing reduced 5-HT production. The predicted mRNA for TPH2-TR is present in postmortem brain of rs1386493 carriers. The rs13864923 variant does not appear to be overrepresented in either global or multiplex depression cohorts. However, in combination with other gene variants linked to 5-HT homeostasis, this variant may exhibit important epistatic influences.

Measuring Nonapoptotic Caspase Activity with a Transgenic Reporter in Mice.

The protease caspase-3 is a key mediator of apoptotic programmed cell death. But weak or transient caspase activity can contribute to neuronal differentiation, axonal pathfinding, and synaptic long-term depression. Despite the importance of sublethal, or nonapoptotic, caspase activity in neurodevelopment and neural plasticity, there has been no simple method for mapping and quantifying nonapoptotic caspase activity (NACA) in rodent brains. We therefore generated a transgenic mouse expressing a highly sensitive and specific fluorescent reporter of caspase activity, with peak signal localized to the nucleus. As a proof of concept, we first obtained evidence that NACA influences neurophysiology in an amygdalar circuit. Then focusing on the amygdala, we were able to quantify a sex-specific persistent elevation in caspase activity in females after restraint stress. This simple in vivo caspase activity reporter will facilitate systems-level studies of apoptotic and nonapoptotic phenomena in behavioral and pathologic models.

A cancer rainbow mouse for visualizing the functional genomics of oncogenic clonal expansion.

Field cancerization is a premalignant process marked by clones of oncogenic mutations spreading through the epithelium. The timescales of intestinal field cancerization can be variable and the mechanisms driving the rapid spread of oncogenic clones are unknown. Here we use a Cancer rainbow (Crainbow) modelling system for fluorescently barcoding somatic mutations and directly visualizing the clonal expansion and spread of oncogenes. Crainbow shows that mutations of ß-catenin (Ctnnb1) within the intestinal stem cell results in widespread expansion of oncogenes during perinatal development but not in adults. In contrast, mutations that extrinsically disrupt the stem cell microenvironment can spread in adult intestine without delay. We observe the rapid spread of premalignant clones in Crainbow mice expressing oncogenic Rspondin-3 (RSPO3), which occurs by increasing crypt fission and inhibiting crypt fixation. Crainbow modelling provides insight into how somatic mutations rapidly spread and a plausible mechanism for predetermining the intratumor heterogeneity found in colon cancers.

The antigenic binding site(s) of antibodies to factor XII associated with the antiphospholipid syndrome.

Phospholipid binding proteins, including factor XII (FXII), are known to be targeted by antiphospholipid antibodies (aPA). Factor XII antibodies (FXIIab) have been described in some patients with the antiphospholipid syndrome (APS) and have been shown to lead to reduced levels of FXII. The antigenic binding site(s) and the pathophysiological effects of FXIIab are unknown. In an attempt to elucidate the binding site of these antibodies, immobilized plasma kallikrein was used to cleave FXII into its 52-kDa heavy-chain (HCFXII) and 28-kDa light-chain (LCFXII) components. Plasma samples from 12 female patients with definite APS and FXIIab were investigated for the presence of antibodies to FXII, HCFXII and LCFXII. All but one patient's plasma reacted to FXII, HCFXII and LCFXII in a similar manner. One patient gave markedly reduced positivity to HCFXII and LCFXII, suggesting that the FXIIab in this patient had a higher affinity for the intact FXII molecule. To further investigate the antigenic binding site(s) of FXII, 150 biotinylated peptides of the known FXII sequence were synthesized using a Multipin(TM) peptide synthesis procedure. The IgG and IgM fractions of the 12 patients' plasma were purified by affinity chromatography. The synthesized peptides were captured on streptavidin plates and individual patients' purified FXIIab assayed against the peptides in a modified enzyme-linked immunosorbent assay (ELISA). Two regions were identified as possible antigenic binding site(s) for FXIIab: one in the growth factor domain and the other in the catalytic domain.

Activation of the interleukin 2 receptor: a possible role for tyrosine phosphatases.

Engagement of interleukin-2 (IL-2) mediates the heterodimeridation of the common beta chain (beta(c)) and common gamma chain (gamma(c)) of the IL-2 receptor (IL-2R). This is sufficient and necessary for receptor activation and signal transduction. It is generally held that the IL-2R is activated by the trans-activity of the protein tyrosine kinases (PTKs) Jak1 and Jak3 associated with beta(c) and gamma(c) respectively. Transduction of proliferative signals requires Jak3 activity. A Jak3 independent signalling pathway involving p56(lck), generating anti-apoptotic signals, can be observed and requires the PROX domain of gamma(c). p56(lck) can be activated by dephosphorylation of an inhibitory carboxyl terminal phosphorylated tyrosine residue (Y505). We propose that this is mediated by a PROX domain associated protein tyrosine phosphatase (PTP). Activation of p56(lck) alone is insufficient for transduction of proliferative signals and thus works in concert with Jak3 mediated receptor activation. This indicates that both gamma(c) domains are vital for signal transduction.

DNA probes in human disease.

Nucleic acid probes are able to detect the presence of particular sequences in a sample down to the level of a few hundred molecules. They can discriminate between similar sequences to a resolution of better than one part in 10(9). They are capable of detecting inherited defects in tissues where the phenotype is not being expressed, and in cases where the biochemical aberration is not understood. They can characterize acquired diseases in somatic cells (both tumours and infectious agents). Additionally, they can be used to characterize multifactorial (either polygenic or requiring an environmental stimulus to interact with a genetic predisposition) diseases. Nucleic acid 'fingerprints' provide an unequivocal identification of the origin of cells which may be applied in criminal law, civil law, and in the follow up to bone marrow transplants. In spite of this tremendous potential, there is still a large gap between their use in research laboratories and their widespread application in pathology laboratories. There are two basic reasons for this. The first is the number of labour-intensive steps involved in the various 'blotting' techniques which greatly reduces the rate at which assays may be performed. The second is the need to use probes labelled with isotopes which are short-lived and may require stringent safety measures to be employed. Recent work both in this laboratory and elsewhere is designed to circumvent both these problems.

An improved method for generating single-chain antibodies from hybridomas.

Cloning the correct VL kappa gene from hybridomas derived from MOPC-21 can be problematic because such cell lines variably express a transcript which is aberrantly rearranged at the VJ recombination site. Cellular levels of the aberrant transcript can exceed that of productive light chain RNA, so a large proportion of the VL gene-derived products obtained on PCR amplification of hybridoma cDNA may not encode a functional protein. We have developed a method in which antibody variable region genes are recovered from hybridoma cDNA using a unique set of V gene family-specific primers; the V region genes are then spliced by PCR, in the form 5'-VL-LINKER-VH-3' (where the linker encodes [GlyGlyGlyGlySer]3), and cloned into an expression vector under control of T7 RNA polymerase. Plasmid DNA is isolated from colonies, and the insert is expressed in an in vitro rabbit reticulocyte lysate-based coupled transcription/translation system, in a microtiter plate format. Since aberrantly rearranged VL kappa genes contain a translation termination codon at amino acid position 105, only constructs containing the correctly rearranged gene produce a protein of the predicted size. We demonstrate the method by producing the single-chain form of OKT9, a murine IgG1 which binds to the human transferrin receptor, and extend the results to show that the protein generated by the in vitro expression system retains the antigen binding properties of the parent antibody. Our method will be generally useful for screening single-chain antibodies for function prior to large scale production in vivo.

Synthesis and biochemical evaluation of analogues of aminoglutethimide based on phenylpyrrolidine-2,5-dione.

A series of (aminophenyl)pyrrolidine-2,5-diones has been prepared that bear structural similarities to aminoglutethimide (1, 3-(4-aminophenyl)-3-ethylpiperidine-2,6-dione). The inhibitory activity of these compounds was evaluated toward human placental aromatase and bovine adrenal cholesterol side chain cleavage (CSCC) enzyme assay systems. Selective, competitive inhibition of the aromatase enzyme system was demonstrated by 5 (3-(4-aminophenyl)-1-methyl-pyrrolidine-2,5-dione, Ki = 1.75 microM), 6 (3-(4-aminophenyl)-1,3-dimethylpyrrolidine-2,5-dione, Ki = 1.75 microM), 7 (3-(4-aminophenyl)-3-methylpyrrolidine-2,5-dione, Ki = 0.8 microM), and 8 (3-(4-aminophenyl)-3-ethylpyrrolidine-2,5-dione, Ki = 1.0 microM). Compound 15 (3-(4-aminophenyl)pyrrolidine-2,5-dione) proved unexpectedly difficult to prepare following standard methods and was only moderately inhibitory toward aromatase (IC50 = 20 microM). Compound 16 (3-(4-aminophenyl)-3-ethyl-1-methylpyrrolidine-2,5-dione) was weakly inhibitory toward testosterone aromatization and totally inactive toward androstenedione aromatization. These compounds were either weak or ineffective inhibitors of the CSCC enzyme systems, while 1 gave Ki values toward aromatase and CSCC enzymes of 0.68 and 14 microM, respectively. The unsubstituted phenylpyrrolidinediones were inactive in either system, and the 4-nitrophenyl derivatives exhibited weak, nonselective inhibition, indicating the importance of the primary amine moiety for potent inhibition of aromatase activity.

Antibodies to factor XII are distinct from antibodies to prothrombin in patients with the anti-phospholipid syndrome.

Patients with the anti-phospholipid syndrome (APS) have antiphospholipid antibodies (aPA) which are often targeted towards phospholipid binding proteins such as beta2-glycoprotein I and prothrombin. Antibodies to factor XII (FXIIabs) have also been identified in some patients with APS. Factor XII (FXII) is a member of the kringle family of proteins which include plasminogen and prothrombin. Antibodies to prothrombin have been associated with myocardial infarction and have been shown to cross react with plasminogen. Sixteen patients with APS and FXIIabs were investigated for the presence of antibodies to prothrombin, by enzyme linked immunosorbent assay in a calcium (Ca++) independent assay. All sixteen showed different antibody binding patterns than those observed for antibodies to FXII. Eight patients were further investigated using surface plasmon resonance (SPR) for antibody binding to covalently bound FXII and to covalently bound prothrombin in both Ca++ dependent and independent systems. Of three patients demonstrating antibody binding to FXII by SPR, none demonstrated antibody binding to prothrombin in a Ca++ independent system with one demonstrating antibody binding to prothrombin that was Ca++ dependent. Of five patients who did not bind FXII by SPR, one demonstrated antibody binding to prothrombin in a Ca++ independent system while two demonstrated antibody binding to prothrombin in a Ca++ dependent system. Antibodies to FXII in patients with APS appear to be distinct from antibodies to prothrombin.

The physiological disposition of 14 C-methsuximide in the rat.

1. (14)C-Methsuximide (N-methyl-(14)C-2-methyl-2-phenylsuccinimide) was rapidly absorbed from the small intestine of the rat (t(1/2) 17.4 min).2. The drug was rapidly and fairly evenly distributed throughout the body with peak blood and tissue levels occurring 1 h after oral administration. At all times, adrenals, body fat, kidneys and liver had higher levels of (14)C-methsuximide than other tissues and the drug freely traversed the blood-brain barrier. However, radioactivity disappeared rapidly from most tissues after the initial phase of the distribution.3. During 24 h, 26% of the orally administered radioactivity was recovered in urine and 29% appeared in expired air as (14)CO(2). The excretion of (14)CO(2) indicated N-demethylation of (14)C-methsuximide to 2-methyl-2-phenylsuccinimide. 2.7% of an administered dose of methsuximide was excreted unchanged in 24 h urine and 2.7% appeared as 2-methyl-2-phenylsuccinimide. 2-Methyl-2-phenylsuccinimide was also detected as a urinary metabolite of methsuximide in man.4. 2-Methyl-2-phenylsuccinimide possesses anticonvulsant activity and it is suggested that this metabolite contributes to the overall anticonvulsant activity and toxicity of methsuximide.5. Rat urine also contained a radioactive substance with similar chromatographic properties to one of the products of alkaline hydrolysis of methsuximide. This compound may arise from the spontaneous decomposition of the parent drug in vivo.

In vitro release of arachidonic acid and in vivo responses to respirable fractions of cotton dust.

It was considered that the fall in lung function seen after exposure to cotton dust may be attributable in part to the activity of arachidonic acid metabolites, such as leucotrienes as well as to the more established release of histamine by cotton dust. However, we found that cotton and barley dusts elicited poor release of arachidonic acid from an established macrophage like cell line compared with that observed with other organic dusts. In the experimental animal, pulmonary cellular responses to both cotton and barley dust were similar to those evoked by moldy hay and pigeon dropping dusts, although after multiple doses a more severe response was seen to cotton and barley. Since both moldy hay and pigeon droppings elicit a greater arachidonic acid release than cotton or barley, a role for arachidonic acid in inducing the cellular response is less likely than other factors. There are limitations to our conclusions using this system, i.e., the arachidonic acid may be released in a nonmetabolized form, although it is noted that the two dusts with the greatest arachidonic acid release produce their clinical responses in humans largely by hypersensitivity mechanisms.

Urinary excretion and pharmacokinetics of acrolein and its parent drug cyclophosphamide in bone marrow transplant patients.

The urinary excretion and pharmacokinetics of acrolein (ACRO) and its parent drug cyclophosphamide (CP) were investigated in 16 randomly selected bone marrow transplant (BMT) recipients when CP was used for conditioning. Patients suffering from aplastic anemia (n = 3) received a 4-day course of CP at a dose of 50 mg/kg daily infused intravenously (i.v.) over 1 h. Patients with leukemia (n = 13) were given either a combination of busulphan followed by CP at a dose of 50 mg/kg infused i.v. over 1 h for 4 days, or CP at a dose of 60 mg/kg by i.v. infusion over 1 h daily for 2 days followed by total body irradiation. Serial plasma samples and urine were collected after the start of the first CP dose. CP was analyzed by capillary gas chromatography, whereas ACRO was measured in urine by liquid chromatography. The plasma concentration-time data for CP conformed to the two-compartment model and the mean and s.e.m. values of alpha, beta, Vss, total clearance, and renal clearance observed were 1.29 (0.31) h(-1), 0.17 (0.03) h(-1), 0.67 (0.13) l/kg, 0.14 (0.02) l/h x kg, and 0.0188 (0.0052) l/h x kg, respectively. The mean and s.e.m. values of fraction of CP excreted in the form of ACRO during this interval (fmu) and ratio of the 24-h urinary concentration of ACRO/creatinine (Cmu(n)) were 1.96 (0.35%) and 9.11 (2.19) microg of ACRO/mg of creatinine, respectively. Two patients developed hemorrhagic cystitis (HC). Each of these two patients excreted significantly (P < 0.01) more ACRO in the first and second 4-h urine collection periods. However, there was no significant difference in fmu or Cmu(n) of ACRO between either of these two patients and the rest. This suggests that the rate of appearance of ACRO in urine is more crucial for developing HC than the cumulative amount excreted.

Substituted 1-[benzofuran-2-yl)-phenylmethyl]-imidazoles as potent inhibitors of aromatase in vitro and in female rats in vivo.

Substituted 1-[(benzofuran-2-yl)-phenylmethyl]-imidazoles are a new class of potent aromatase inhibitor with in vitro IC50 values < 10 nM for certain members using human placental enzyme. At a dose of 2 mg/kg in PMSG-stimulated rats, selected compounds effectively reduce the oestradiol levels by 82-98%.

Pharmacokinetics of S- and R-enantiomers of aminoglutethimide following oral administration of racemic drug in breast cancer patients.

This study was undertaken to examine the pharmacokinetics of both enantiomers of AG--that is, (R-AG) and (S-AG) and respective acetyl metabolites, R-AcAG and S-AcAG--in breast cancer patients. Six patients received a single dose (500 mg) of the racemic drug, and serial plasma samples and urine were collected over a 48-hour period. R-AG, S-AG, R-AcAG, and S-AcAg were measured simultaneously by high-performance liquid chromatography using two serial chiral separation columns with ultraviolet detection. The plasma concentrations of R-AG were about 1.5 times higher than those of S-AG, and the data for both enantiomers exhibited the characteristics of the one-compartment open model. There were no significant differences between R- and S-AG in ka, tmax, V/F, and t1/2. The formation of R- and S-AcAG was rapid, and no correlation was found between the t1/2 values of the AG enantiomers with that of their acetylated metabolites. Overall, 41% of the dose was excreted in urine as AG (15% R-AG and 26% S-AG) and 5.1% as AcAG (2.9% R-AcAG and 2.2% S-AcAG). Renal clearance of S-AG was significantly greater (i.e., 2.3-fold) than that of R-AG and appears to be most likely the cause for the other pharmacokinetic differences observed. Both enantiomers had low renal extraction ratios, suggesting extensive tubular reabsorption of the compounds. However, based on the data obtained, it was concluded that the main factor contributing to the therapeutic effectiveness of racemic AG is the large potency difference between the R- and S- forms (R > S). The pharmacokinetic differences between R-AG and S-AG appear to contribute only marginally to the activity of this drug as an aromatase inhibitor.

Development of tolerance to the CNS effects of aminoglutethimide in mice.

Initially, there is a high incidence of CNS-depressant side-effects when the aromatase inhibitor, aminoglutethimide, is used in the treatment of patients with advanced breast cancer. Tolerance to these effects develops with continued dosing. This study examines the development of tolerance to various indices of CNS depression with the drug in mice. Single doses of aminoglutethimide induced a dose-dependent depression of spontaneous locomotor activity, rotarod performance, righting reflex and body temperature and a dose-related antileptazol activity. On repeated dosing with the drug, tolerance to these various activities occurred. The tolerance was found to be dose-dependent in the rotarod and righting reflex tests and time-dependent in the locomotor and body temperature tests. Although the results do not allow a determination of whether this clearly demonstrated phenomenon in the mouse is primarily functional or dispositional, the slow onset (14 days) for complete tolerance may be indicative of a functional mechanism.

A granulocyte test for detection of cattle heterozygous for mannosidosis.

alpha-Mannosidase and beta-N-acetylglucosaminidase activities were found to differ between lymphocytes and granulocytes isolated from bovine blood. Activities of both enzymes in granulocyte preparations were found to be related to the eosinophil content of the preparations. A procedure is described that allows definition of the mannosidosis genotype of adult cattle by giving consideration to the influence of eosinophil content of granulocyte preparations upon the activity of alpha-mannosidase relative to that of beta-N-acetylglucosaminidase.

Sensitivity differences to 5-HT and carbachol in subsections of the isolated rat stomach fundus strip: an improved preparation.

The rat stomach fundus strip has often been used as a sensitive assay of pharmacological activity at 5-HT receptors. In the present study, we describe an improved preparation based on the finding that the majority of the contractile response to 5-HT and carbachol was present in one particular longitudinal quartile of the tissue. The fundi were dissected into four anatomically consistent sections and labelled accordingly (left ventral = LV; right ventral = RV; left dorsal = LD; right dorsal = RD). A distinct pattern of responsiveness (contraction) to 5-HT and carbachol was observed. For carbachol, the maximum contraction ratios of the sections (LV:LD:RV:RD) were 7.2(+/-0.8):5.8(+/-1.4):4.2(+/-0.46):3.0 (+/-0.22), respectively. For 5-HT, the differences were more pronounced with maximum contraction ratios of 5.6 (+/-0.94): 3.6(+/-0.58):1.4(+/-0.30):1.6(+/-0.22), respectively. When the sections were dissected along the submucosa, the mucosal section responded to carbachol and 5-HT, while the muscularis externa did not respond to these agonists. Selection of the LV section allowed the use of smaller bath volumes, and subsequent bisection facilitated the design of more effective paired experiments.

Comparison of in vitro and in vivo haemotoxic effects of aminoglutethimide and glutethimide.

The aromatase inhibitor, aminoglutethimide, causes a blood dyscrasia in about 1% of women taking the drug for the treatment of advanced breast cancer. The parent compound, glutethimide, is known not to possess this toxic action. The effect of both drugs on bone (femur) marrow cells of mice and rats was examined by means of standard methods for the colony forming assay for granulocyte-macrophage (GM-CFC) and erythroid cells (CFU-E). There were no significant effects on the cell colonies up to drug concentrations of at least 10 mug/ml. However, above this level concentration-dependent effects were observed. For mouse GM-CFC, 50 and 30% of the cell colonies died when incubated with glutethimide (33 mug/ml) and aminoglutethimide (100 mug/ml), respectively. Similar results for the two drugs were obtained with the mouse CFU-E and with both cell types from the rat. The results indicate that the inhibitory effects were most likely to be non-specific. For the in vivo study, glutethimide and aminoglutethimide were given orally (50 mg/kg, daily). After 3 wk of this regimen in mice, there was a significant fall (about 50%) in both white blood cell and platelet counts. This is similar to the blood picture changes induced by aminoglutethimide in women. No haematological effects were observed in rats receiving this drug and glutethimide was without effect in either species. Thus, the toxic effects of aminoglutethimide appear to be more selective in vivo than in vitro. It is proposed that N-hydroxylation of aminoglutethimide, which proceeds in mouse and man but not rat, is an important step in the toxic mechanism. Overall the results indicate that the in vitro assessment used in this study was not a suitable predictor of the in vivo haemotoxic effects of aminoglutethimide.

Are there compartment syndromes in some patients with idiopathic back pain?

Palpable rigidity of the epaxial (paraspinal) muscles, lordotic flattening, and spinal flexion accompanying back pain generally are ascribed to epaxial muscle spasm. However, palpable rigidity without muscle spasm occurs in compartment syndromes and epaxial muscle contractions extend the spine, increasing lordosis. Epaxial compartment syndromes are proposed as a possible cause of palpable rigidity, lordotic flattening, and spinal flexion accompanying idiopathic back pain. This article demonstrates the following: existence of an epaxial compartment by latex and dye injections; simulation of epaxial compartment syndromes in unembalmed cadavers by saline injections; and a "Bourdon tube effect" producing spinal flexion with lordotic flattening during epaxial compartment syndrome simulation in embalmed cadavers. In addition, resting and exercising epaxial compartment pressures were measured in 18 normal volunteers with a slit catheter.

Dose-response and time-response biochemical and histological study of potassium dichromate-induced nephrotoxicity in the rat.

This study provides quantitative toxicological data on potassium dichromate-induced renal damage and considers the possible difficulties arising from the non-invasive in vivo assessment of renal damage, with particular attention to enzymuria. Renal damage induced in male Wistar rats by single sc injections of potassium dichromate was assessed 52 to 72 hr after doses ranging from 3 to 20 mg potassium dichromate/kg body weight and throughout a 9-day period following a dose of 20 mg potassium dichromate/kg. The earliest and most sensitive non-invasive functional change in the dose-response and time-response studies was an elevation in the rate of urinary excretion of protein. Evidence of tissue damage was observed with elevations in the urinary excretion rates of the brush border enzymes, gamma-glutamyltransferase, alkaline phosphatase and leucine aminopeptidase, the cytosolic enzymes, aspartate aminotransferase and lactate dehydrogenase and the lysosomal enzyme, N-acetyl-beta-D-glucosaminidase. Such changes occurred as early as the abnormal urinary protein excretion, but returned to control or sub-control values sooner. Urinary brush border enzyme excretion returned to control values within 48 hr following potassium dichromate injection, despite histological and histochemical evidence of extensive renal damage and renal dysfunction. Elevations in plasma aspartate aminotransferase and lactate dehydrogenase levels were observed, but histochemical and isoenzyme studies would be needed to determine the source of these increases. The simplest and most persistent indicators of renal damage were the urinary excretion of protein and N-acetyl-beta-D-glucosaminidase.