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With an eye toward the development of novel atypical antipsychotic agents, we have studied the structure-affinity relationships of N,N'-di-o-tolylguanidine (DTG, 3) and its congeners at the haloperidol-sensitive sigma receptor. A number of DTG analogues were synthesized and evaluated in in vitro radioligand displacement experiments with guinea pig brain membrane homogenates, using the highly sigma-specific radioligands [3H]-3 and [3H]-(+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine and the phencyclidine (PCP) receptor specific compounds [3H]-N-[1-(2-thienyl)-cyclohexyl]piperidine and [3H]-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10- imine. The affinity of N,N'-diarylguanidines for the sigma receptor decreases with increasing steric bulk of ortho substituents larger than C2H5. Hydrophobic substituents are generally preferred over similarly positioned hydrophilic ones. Furthermore, electroneutral substituents are preferred over strongly electron donating or withdrawing groups. Significant binding to the sigma receptor is usually retained as long as at least one side of the guanidine bears a preferred group (e.g. 2-CH3C6H5). Replacement of one or both aryl rings with certain saturated carbocycles (e.g. cyclohexyl, norbornyl, or adamantyl) leads to a significant increase in affinity. By combining the best aromatic and best saturated carbocyclic substituents in the same molecule, we arrived at some of the most potent sigma ligands described to date (e.g. N-exo-2-norbornyl-N'-(2-iodophenyl)guanidine, IC50 = 3 nM vs [3H]-3). All of the compounds tested were several orders of magnitude more potent at the sigma receptor than at the PCP receptor, with a few notable exceptions. This series of disubstituted guanidines may be of value in the development of potential antipsychotics and in the further pharmacological and biochemical characterization of the sigma receptor.

TGF alpha is one member of a family of soluble growth factors that are derived from integral-membrane precursors. The mature form of TGF alpha is released from its transmembrane precursor (proTGF alpha) by a protease that, in many tumor cells, is inefficient or limiting. We have previously established that, in the absence of processing, membrane-anchored proTGF alpha is biologically active and can interact with the EGF receptor on adjacent cells, thereby inducing the receptor's intrinsic tyrosine kinase activity. We further showed that this interaction leads to immediate downstream signal transduction as evidenced by Ca2+ mobilization. To extend these observations, and to investigate its transforming potential, we infected normal rat kidney (NRK) cells with retroviral expression vectors that encode mutated forms of proTGF alpha containing amino acid substitutions at the proteolytic cleavage sites. NRK cells harboring these mutant constructs do not secrete mature growth factor, but do express biologically active proTGF alpha on the cell surface as shown by their ability to induce the autophosphorylation of EGF receptor on neighboring A431 cells in co-culture. Expression of the mutant proTGF alpha molecules promoted the anchorage-independent growth of NRK cells in soft agar, and caused them to be tumorigenic when injected into nude mice. These results demonstrate that an interaction between EGF receptor and the integral membrane precursor to TGF alpha can provide a mitogenic stimulus that leads to transformation. They further suggest that the accumulation of proTGF alpha on the surface of some transformed cells has physiological relevance.

The precursor for transforming growth factor-alpha (TGF-alpha) is a membrane glycoprotein that can establish contact with epidermal growth factor/TGF-alpha receptors on adjacent cells or can be cleaved to release TGF-alpha that diffuses into the medium. Cleavage of pro-TGF-alpha occurs at Ala/Leu-Ala/Leu-Ala-Val-Val sites located at each end of the mature TGF-alpha sequence. To characterize the cleavage process of pro-TGF-alpha and the role of glycosylation in this process, we have introduced a pro-TGF-alpha expression vector in wild type Chinese hamster ovary (CHO) cells and in the mutant CHO cell clone ldlD that has a reversible defect in protein glycosylation. Analysis of metabolically labeled and cell surface-labeled products immunoprecipitated with antibodies directed against the extracellular TGF-alpha sequence and the cytoplasmic pro-TGF-alpha C-terminal domain shows that cleavage of pro-TGF-alpha in wild type CHO cells occurs in two steps. Both processing steps occur after pro-TGF-alpha reaches the cell surface. In the first step, pro-TGF-alpha rapidly (t1/2 = 30 min) loses the amino-terminal segment that precedes the TGF-alpha sequence. In the second step, pro-TGF-alpha is cleaved at the carboxyl terminus of the TGF-alpha sequence releasing this factor into the medium. This second step is slow (t1/2 = 2 h). The action of pancreatic elastase added to CHO-TGF-alpha cells mimics the first step but not the second one. Synthesis, cell surface exposure, rate of cleavage, and generation of bioactive TGF-alpha in ldlD-TGF-alpha cells are not markedly affected by the lack of N-acetylgalactosamine-dependent protein O-glycosylation or galactose-dependent glycan chain modification. The results indicate that, despite their similarity in amino acid sequence, the two cleavage sites that flank TGF-alpha may be processed with different kinetics which can lead to retention of pro-TGF-alpha on the cell surface.

Somatic cell hybrids have been instrumental in the recognition of specific chromosomes containing genes capable of suppressing the malignant phenotype. As a first step towards the identification of possible suppressor genes in prostate cells, we created hybrids by fusing normal prostate cells with malignant HeLa cells. Similar to hybrids made with other combinations of normal and malignant cells, the normal phenotype was dominant and the malignant phenotype was suppressed. The phenotype of the nontumorigenic hybrids after injection into nude mice resembled that of normal keratinocyte X HeLa hybrids, and tiny, nonprogressive keratinized nodules were produced. One hybrid clone was tumorigenic, possible due to the loss of a normal suppressor gene, and displayed glandular as well as squamous elements. Further characterization of these hybrids should permit isolation of specific suppressor genes, as well as promote recognition of elements that regulate the glandular phenotype.

Assessment of perinatal brain oedema is complicated by normal changes in brain water that accompany the marked physiological, biochemical and morphological alterations occurring during this phase of development. Multiexponential analysis of transverse decay curves (TDCs), derived from 128 echo CPMG images, of white matter (WM) made oedematous by either exposure of animals to triethyltin (TET) or cryogenic cortical lesions revealed a second, slower decay component not apparent in controls. More significantly, an obvious difference was noted between the TET and cryogenic lesion fast decay components which might serve as a basis to differentiate non-invasively cytotoxic and vasogenic oedemas.

The extent of contrast manipulation and the assessment of characteristic artifacts in imaging studies of brain and knee as performed with novel variants of the Rapid Acquisition Relaxation Enhanced (RARE) sequence are reported. Methods of ordering the phase encoding within one or two echo trains are proposed for manipulating T2 contrast. Options for minimizing artifacts associated with the various schemes are discussed. The extent of T1 contrast manipulation in RARE sequences is explored by varying repetition rates in a signal averaging scheme and by applying inversion pulses prior to data acquisition. The results demonstrate that RARE sequences can be utilized for obtaining good quality images with a range of tissue contrast options similar to those associated with slower spin-echo methods. They also suggest that RARE applications need not be confined to highlighting long T2 fluid spaces, an application already well documented.

A fast imaging technique, missing pulse steady-state free precession (MP-SSFP), is described. MP-SSFP is one of a class of steady-state free precession techniques in which every nth RF pulse is missing. MP-SSFP has been implemented for the case where every third RF pulse is omitted: [-theta 1-r-theta 2-r-echo-r-]. A RF-refocused echo forms at the time of the missing pulse. This echo is less sensitive to field inhomogeneities than the gradient-recalled echoes used in most fast imaging methods. An analytical expression is obtained for the signal strength as a function of the flip angles theta 1 and theta 2, the interpulse interval tau, and the amount of interpulse dephase. The expression shows that theta 1 and theta 2 provide two degrees of freedom to optimize the signal-to-noise ratio and improve tissue contrast. Two different cases, theta 1 = theta 2 and theta 1 = -theta 2, are described to demonstrate the difference in contrast. The first case gives a strong signal from spins with short T1 and short T2 while the second case has contrast very similar to a conventional SSFP technique. The theoretical expression predicts that the signal consists of multiple components which may be observed experimentally by adjusting the gradient pulses.

The 50 amino acid form of TGF-alpha is cleaved from a conserved integral membrane glycoprotein by a protease that, in many tumor cells, appears to be limiting. To test whether the membrane-bound precursor has biological activity in the absence of processing, we introduced amino acid substitutions at the proteolytic cleavage sites. BHK cells transfected with expression vectors containing these altered sequences do not secrete detectable levels of mature TGF-alpha into the medium, but express high levels of proTGF-alpha at the cell surface. Coincubation of these BHK cells with A431 cells demonstrates that membrane-bound proTGF-alpha may bind to EGF receptors on the surface of contiguous cells, induce receptor autophosphorylation, and thereby produce a rapid rise in A431 intracellular calcium levels. Thus, proTGF-alpha can be biologically active in the absence of processing, a fact that may have implications for the integral membrane precursors of related growth factors.

Recent advances in culture techniques have enabled routine establishment and propagation of epithelial cells derived from normal and malignant tissues of the human prostate. Comparative studies of the responses of normal and cancer-derived cell populations to various growth and differentiation factors in vitro were undertaken to examine the possibility that cancer cells might respond differentially. Clonal growth assays in serum-free medium demonstrated that optimal proliferation of normal as well as cancer cell strains was generally dependent on the presence of cholera toxin, epidermal growth factor, pituitary extract, hydrocortisone, insulin, and high levels of calcium in the culture medium, and on the use of collagen-coated dishes. Only one cancer strain responded aberrantly to epidermal growth factor and hydrocortisone. Putative differentiation factors (transforming growth factor-beta and vitamin A) inhibited the growth of all normal and cancer strains. The origin of a cancer-derived cell strain that responded similarly to normal strains was verified by positive labeling with a prostate cancer-specific antibody, validating the conclusion from these studies that normal and cancer prostatic epithelial cells are not distinguishable on the basis of responses to the tested factors.

Psychological problems in 170 adolescence that were seen in the Singapore Child Psychiatry Clinic were compared with 2 other reported series from the United Kingdom and the United States of America. The course and prognosis appear similar to what has been observed in the West. The emotional or neurotic conditions have the best outlook while the psychotics have an improvement rate of not more than 45%.

The aim of this study was to examine the relationship between behavioural deviance and parental involvement in childrearing and disciplinary practices in a sample of 401 toddlers. Mothers of each subject were interviewed to assess for the degree of parental involvement and the disciplinary methods used with the child. A Behavioural Screening Questionnaire (BSQ) was used to assess for behavioural deviance in the toddlers. Results indicated that father involvement in childrearing was significantly lower than mother involvement (p = 0.0001). Although consistent with cultural expectations, both low paternal involvement and punitive types of discipline were significantly related to behavioural deviance.

Nuclear magnetic resonance (NMR) imaging was employed to study 10 patients with Alzheimer's disease (AD) and seven healthy elderly control subjects. Coronal sections were used to make volumetric measurements of the hippocampus, ventricles, subarachnoid space, and brain parenchyma. The hippocampal volume (normalized relative to the size of the lenticular nucleus) was reduced by 40% in the AD group compared to the controls, with no overlap between the two groups. Overall measures of brain atrophy and ventricular and sulcal enlargement also showed significantly different group means, although with overlap between the two groups. Hippocampal atrophy did not correlate with either overall brain atrophy or dementia severity, although the degree of brain atrophy was correlated with dementia severity. These results show that NMR is capable of providing in vivo quantification of diminished hippocampal size in AD which is not correlated with overall brain atrophy and which may differentiate AD from normal aging.

Polyacrylamide disc gel electrophoresis was performed on unconcentrated cerebrospinal fluid and serum from 132 neurological patients and from 33 patients without neurological disease. The presence of unique, discrete immunoglobulin bands in the gamma-globulin (cathodal) region of the cerebrospinal fluid gel was assessed as a diagnostic test for multiple sclerosis. The procedure exhibited a specificity for multiple sclerosis of 94% and a sensitivity of 72%, and is easier to perform than most of the other published procedures used to demonstrate oligoclonal immunoglobulins in cerebrospinal fluid. It is recommended for use in the clinical chemistry laboratory as a valuable adjunct to the diagnosis of multiple sclerosis.

Teratogenicity of the anticonvulsant drug phenytoin is thought to involve its bioactivation by cytochromes P-450 to a reactive arene oxide intermediate. We hypothesized that phenytoin also may be bioactivated to a teratogenic free radical intermediate by another enzymatic system, prostaglandin synthetase. To evaluate the teratogenic contribution of this latter pathway, an irreversible inhibitor of prostaglandin synthetase, acetylsalicylic acid (ASA), 10 mg/kg intraperitoneally (ip), was administered to pregnant CD-1 mice at 9:00 AM on Gestational Days 12 and 13, 2 hr before phenytoin, 65 mg/kg ip. Other groups were pretreated 2 hr prior to phenytoin administration with either the antioxidant caffeic acid or the free radical spin trapping agent alpha-phenyl-N-t-butylnitrone (PBN). Caffeic acid and PBN were given ip in doses that respectively were up to 1.0 to 0.05 molar equivalents to the dose of phenytoin. Dams were killed on Day 19 and the fetuses were assessed for teratologic anomalies. A similar study evaluated the effect of ASA on the in vivo covalent binding of radiolabeled phenytoin administered on Day 12, in which case dams were killed 24 hr later on Day 13. ASA pretreatment produced a 50% reduction in the incidence of fetal cleft palates induced by phenytoin (p less than 0.05), without significantly altering the incidence of resorptions or mean fetal body weight. Pretreatment with either caffeic acid or PBN resulted in dose-related decreases in the incidence of fetal cleft palates produced by phenytoin, with maximal respective reductions of 71 and 82% at the highest doses of caffeic acid and PBN (p less than 0.05). Caffeic acid and PBN also significantly reduced the incidence of fetal resorptions produced by phenytoin, but not the fetal weight loss. In viable embryos, ASA pretreatment reduced the covalent binding of phenytoin to embryonic protein by 43% (p less than 0.05). Binding of phenytoin to embryonic resorptions was equally high with and without ASA pretreatment, and within each treatment group was 3- to 10-fold higher than that in the respective placentas and associated viable embryos (p less than 0.05). These results suggest that prostaglandin synthetase may contribute to the enzymatic bioactivation of phenytoin to a teratogenic free radical intermediate.

This spectrophotometric method for the direct determination of potassium in serum or plasma is based on the selective complexing of potassium by a specific macrocyclic polyether, with the subsequent formation of an ion-pair with a colored anion. The colored anion is extracted into an organic solvent, clarified by centrifugation, and then measured at 415 nm. The absorbance of the chromogen varies linearly with [K+] to at least 15 mmol/L. Results of this colorimetric method (y) correlate well with the results obtained by a flame-photometric method (y = 1.04x - 0.22, r = 0.97, n = 81), with CVs ranging from 2 to 4%. We observed no interferences from lipemia, added bilirubin, or various electrolytes. We also evaluated the use of this reagent in a new automated blood analyzer developed by Abbott, a two-dimensional centrifugal system (Clin Chem 31:1457-1463, 1985). Potassium determined with this system (y) correlated well with results by flame photometry: y = 1.02x + 0.02 (r = 0.94, n = 168). With this system one can use whole-blood specimens in measuring potassium.

Unlike adult depression, childhood depression is still a controversial concept in psychiatry. This paper explores and discusses the various issues involved in the controversy. Empirical evidence from epidemiological surveys and clinical studies is examined systematically. A brief survey of the literature over the last fifty years is given. The latest clinical study by Pearce (1974) is highlighted as it demonstrates how the condition can be systematically investigated. Despite conflicting findings, the available evidence, on balance, suggests that childhood depression does exist; it is rare in middle childhood, rarer still in the pre-school period of life but more common in adolescence. Although research findings have begun to give support to the diagnosis of childhood depression, its validity is far from being proven. Obviously, further research is needed.

The ability of human epithelial cells derived from adult prostatic tissues to undergo clonal growth in culture was examined. In a previously described serum-free culture system, such cells exhibited a density-dependent growth requirement. It was found that raising the level of one of the constituents of the culture medium, bovine pituitary extract, to 100 micrograms/ml permitted excellent clonal growth when as few as 100 cells were inoculated/60-mm2 dish. Raising the levels of supplements other than pituitary extract (cholera toxin, epidermal growth factor, hydrocortisone, or insulin) did not produce this result. The average colony-forming efficiency of cells derived from primary or early passage cultures was approximately 25%. When single cell suspensions were prepared from tissue isolates and directly analyzed for clonal growth, colony-forming efficiencies were approximately 5%, perhaps indicating the proportion of stem cells with proliferative potential in the original isolates. The colony-forming efficiency of a cell population derived from cancer tissue was not significantly different from those of populations derived from normal tissues.

The schizogonic development of Leucocytozoon dubreuili in the kidney proximal tubule cells of the American robin, Turdus migratorius, was studied by electron microscopy. Renal schizogony is initiated by the entry of certain hepatic merozoites into cells of the proximal tubules. Development of the schizont consists of coordinated sequence of events including extensive mitotic nuclear division, multiplication of mitochondria, increase in endoplasmic reticulum and ribosomes, differentiation of membranes, microtubules, micronemes and rhoptries, and cytoplasmic segmentation (cytomere formation). Merozoites form by budding around numerous centers in the schizont and, when mature, are bounded by a single plasma membrane subtended by microtubules. Each merozoite contains a large nucleus, a mitochondrion, and well developed apical complex consisting of 3 polar rings, paired rhoptries, and numerous micronemes. An atypical nuclear division observed in some maturing schizonts was characterized by the multiple fission of a nucleus within a persistent outer nuclear membrane and the absence of mitotic spindle apparatus. Alterations in infected renal cells consisted of disorganization and loss of cytoplasmic organelles and the accumulation of lipofuscin-like inclusions.

This study examines slice selection and diffusion as sources of systematic error in T2 estimates obtained from imaging experiments. The selective refocusing pulses in a multislice, two-echo, spin-warp experiment cause slice profiles of sequential echoes to change in shape. This results in bias in T2 images calculated from the two-echo data set. A method to correct the bias is presented. The effect of diffusion on T2 images obtained with typical two-echo imaging sequences was found to be negligible.

We have developed a new clinical chemistry analyzer, the VISION System, which uses centrifugal force to separate whole blood, measure reagent and plasma volumes, and complete all steps required for a spectrophotometric analysis. The system is based on a multichambered test pack containing liquid reagents, which can be centrifuged in two planes oriented at right angles to each other. The analyzer regulates the temperature, timing and optical measurements for up to 10 different test packs in the same run. We have demonstrated good precision and accuracy on 6 clinical chemistry analytes, 2 enzymes, potassium and theophylline using this system.

The Home Observation for Measurement of the Environment (HOME) Inventory is introduced in this study to examine the home environments of children (three to four and a half years) in Singapore. The Inventory comprises eight subscales and a total of 54 items with a higher score indicating an environment which provides more possibilities for development and growth. Various statistical tests were carried out to determine the validity of the Inventory for use in the local context. These showed the Inventory to be internally consistent and to possess construct validity in particular the HOME Inventory had been validated in correlation tests with demographic variables such as social class index (F = 8.01, p less than 0.0001), people to room ratio (r = -0.37, p less than 0.0001) and a measure of IQ (r = 0.0003). Norms for local toddlers were also provided. It was concluded that the Inventory would play a useful role in detecting children who are physically and intellectually less advanced due to a poorer home environment. This finding could possibly be used in an intervention programme in ameliorating these conditions.