[Effectiveness of instrument disinfectants under increased protein exposure and for standing times up to two weeks].

To examine the possibility of prolongation of the standing times of instrument disinfectants, in vitro tests under high albumin exposure and tests in clinical practice were done. Concerning in vitro tests: The bactericidal effectiveness of 7 commercial preparations for instrument disinfection with different basic substances was determined in quantitative suspension tests and of 8 preparations in practice-oriented tests according to the guidelines for testing and approval of instrument disinfectants by the DGHM (German Society for Hygiene and Microbiology). Tests were carried out under increased albumin exposure and at prolonged standing times up to 12 days. The instrument disinfectants showed microbicidal efficacy up to 12 days at working concentrations slightly increased in relation to the concentrations listed by the DGHM. Concerning tests in clinical practice: In 4 clinical wards (intensive care unit, cystoscopy unit, urological operation unit, gastroenterological endoscopy unit with ERPC) the preparations for instrument disinfection were tested up to 12 days in respect to the protein exposure and the disinfecting activity of the working solution. The working solution was not renewed during the 12 days. The inserted instruments were recorded. Concentration of the disinfectants and the protein concentration were daily determined. Disinfecting activity was examined by practice-oriented microbiological tests. The highest protein exposure was 0.098% after a standing time of 12 days. A loss in concentration of disinfectants could not be found. Nevertheless independent from the protein exposure there was a loss in disinfecting activity of the tap water diluted specimens from the clinical practice. Loss in activity could not be seen in the specimen from laboratory parallel tests, which were diluted by water of standard hardness. The loss in disinfecting activity of the tap water diluted preparation from the gastroenterological endoscopy unit versus a dilution by water of standard hardness could be confirmed in control experiments (practice-oriented tests with S. aureus over a period of 5 days).


[Mercury concentration in blood and urine--before and after the placement of dental amalgam fillings].

70 patients of dentist's surgery were given MOD amalgam fillings (non-gamma-2 amalgam) for molars. They were allocated for comparison to four groups defined by their treatment, i.e. the number of old and new restorations and whether a rubber dam was used. Blood and urine samples were collected at regular intervals before and during a 14-day period after treatment and tested for mercury concentration (Hg). Over the observation period the groups with the highest exposure (1-2 old restorations replaced by new ones) showed a tendency, in contrast to the less exposed groups (1 new filling with or without dam), towards increases (p less than 0.10) in group average Hg values of approx. 0.2 microgram/L (blood) and 0.3 microgram/g creatinine (urine) as acute treatment effects. The highest values recorded before and after the treatment, 3.3 micrograms/L (blood) and 16.5 micrograms/L (urine) are higher than normal but do not indicate any increase in the risk to health especially if they are not persistent.


[RoTrac capillary pore membranes for laboratory filtration. I. Degermination filtration].

RoTrac capillary pore membranes (CPM) are produced by means of the nuclear track technology. Thus a defined and in wide ranges independent adjunction of the different membrane parameters (diameter, density, shape and inclination of pores) is possible. The wanted uniform separation diameter of the membrane can exactly be chosen according to the size of the microorganisms to be rejected. By dead end filtration experiments with E. coli and Serratia marcescens the suitability of RoTrac-CPM in bacteria removal filtration was proven. Blocking was very strong for membranes with pore diameters in size range of the microorganisms (approximately 0.45 micron). Though the filtrate had immense reduced bacteria counts (from 10(7)-10(8) to 10-100 bacteria/ml), it was generally not sterile. For membranes with a pore diameter of 0.2 micron and smaller blocking was essentially lesser. Here filtrate was always sterile. Flux (and thus the filterable volume) corresponds to values of competitive membranes. Compared with those the proven possibility of simple cleaning is an advantage, because rejection and blocking of symmetrical CPM occur directly on the membrane surface. This is promising for use of CPM in cross flow filtration.


An enzymatic procedure for the confirmation of total coliforms and Escherichia coli enumeration from water.

A new procedure for the confirmation of total coliforms and Escherichia coli from presumptive most probable number (MPN) test is described. The procedure utilizes an enzymatic test apparatus composed of a couple of devices, one charged with ONPG-medium for the detection of beta-galactosidase, the other with PNPG-medium for the detection of beta-glucuronidase. The results obtained demonstrate that the procedure is sensitive, specific and accurate. Further, it presents some advantages from the practical point of view: the cost of the devices is relatively low, their use is extremely simple and short time consuming, a single thermostatic apparatus adjusted at 36 degrees C for the incubation of the devices is only required, the results can be obtained after 18 h without any apparatus for the lecture (e.g. UV apparatus), they are not submitted to subjective interpretation.


Dibenzo[a,g]quinolizin-8-ones: synthesis, estrogen receptor affinities, and cytostatic activity.

A number of acetoxy-substituted dibenzo[a,g]quinolizin-8-ones were synthesized by the reaction of 1-oxoisoquinolines with substituted homophthalic acid anhydride. All of the derivatives with acetoxy groups in positions 3 and 10 bind to the estrogen receptor. Relative binding affinities (RBA) ranged from 1.8 to 5.6 (estradiol: RBA = 100) when the substituent at C-6 was a short alkyl group. Introduction of additional oxygen functions in the 2- and/or 11-position decreased binding affinities. Analyses of the enantiomers of 6-methyl (6b) and 6-ethyl (6c) derivatives revealed that the receptor binding is mainly due to one optical isomer (e.g. (-)-6b, 9.9; (+)-6b, 0.6). In hormone-sensitive human MCF-7 breast cancer cells, compounds with one acetoxy group in each aromatic ring strongly inhibited cellular growth. Despite marked differences in receptor affinity, the enantiomers displayed similar activities in this cell culture. In hormone-independent MDA-MB 231 mammary tumor cells, only a weak cytostatic effect was recorded at 10(-5) M. In the immature mouse uterine weight test, minimal estrogenic activity was observed. At higher doses, a significant anti-estrogenic effect became evident. It is assumed that the estrogen antagonism is responsible for the specific cytostatic effect in MCF-7 breast cancer cells.


Synthesis and biological evaluation of a series of flavones designed as inhibitors of protein tyrosine kinases.

A series of flavones has been prepared, which are variously substituted in the 3,3',4',5 and 7 positions with halo-, alkoxy-, nitro-, amino-, hydroxy-, acyloxy- and azido-groups, for evaluation of their cytotoxicity to ANN-1 cells (3T3 murine fibroblasts transformed with the Abelson murine leukaemia virus) which contain a tyrosine kinase. This cytotoxicity was compared to their non-transformed 3T3 counterparts. 3'-Amino-4'-methoxyflavone was the most cytotoxic compound (IC50 = 1.6 microM) and was less inhibitory to the non-transformed parent 3T3 cell line (IC50 = 8 microM). The compound was inactive at 50 microM in assays of the inhibition of the cell-associated Abelson protein tyrosine kinase but inhibited an epidermal growth factor (EGF) protein tyrosine kinase by 42% at 50 microM. Quercetin (3,3',4',5,7-pentahydroxyflavone) was the most potent inhibitor of the Abelson protein tyrosine kinase but showed no selective inhibition of the growth of ANN-1 cells compared to the parent 3T3 cell line. Different structure-activity relationships were observed between the results of the cytotoxicity assays and inhibition of protein tyrosine kinases. Inhibitors of the Abelson protein tyrosine kinase which were competitive with respect to ATP showed different potencies for inhibition of the EGF receptor kinase.


Synthesis, DNA binding interactions and biological activity of bis platinum (II) complexes of N,N,N',N'-tetrakis(2-aminoethyl)diamines.

A series of dimers of the monofunctional platinum species [Pt(dien)Cl]+, linked by a variety of flexible (polymethylene) and more rigid chains, was prepared and evaluated for DNA interactions and cytotoxic activity. The polymethylene-linked dimers were prepared by acylation of N(1),N(3)-bistrityldiethylenetriamine with alpha, omega-dicarboxylic acid chlorides, followed by reduction with diborane. Platination of these ligands was achieved with K2PtI4 prepared in situ, followed by anion exchange. Solutions of the bis(Pt(dien)Cl)2+ complexes were stable, and shown to be pure by 195Pt NMR, but solid products could not be isolated. All of the bis(Pt(dien)Cl)2+ complexes unwound closed circular supercoiled DNA more efficiently than the monomer, and were more efficient than the difunctional platinum complex cisplatin at cross-linking linearized plasmid DNA, as measured on non-denaturing agarose gels. None of the bis(Pt(dien)Cl)2+ complexes were as cytotoxic as cisplatin in both the wild-type and platinum-resistant P388 murine leukaemia cell lines. The more rigid analogues were equitoxic in both sensitive and cisplatin-resistant cells, but none showed in vitro activity against the P388 tumour.


Structure-activity relationships for substituted 9-oxo-9,10-dihydroacridine-4-acetic acids: analogues of the colon tumour active agent xanthenone-4-acetic acid.

A series of 9-oxo-9,10-dihydroacridine-4-acetic acids (acridone-4-acetic acids) were prepared by Jourdan-Ullmann condensation of 2-halobenzoic acids with 2-aminophenylacetic acids, followed by H2SO4-induced cyclodehydration of the resulting 2-[2-(carboxymethyl)phenylamino]benzoic acids. These were evaluated for their ability to induce haemorrhagic necrosis in transplanted colon 38 tumours in mice, using a short-term histology assay. The results broadly paralleled those seen previously for xanthenone-4-acetic acids, with 1-, 2- and 7-substitution being dystherapeutic, and substitution at the 5- and 6-positions by lipophilic groups increasing activity. While some analogues were as active as xanthenone-4-acetic acids in the histology assay and gave significant growth delays against colon 38 tumours in vivo, as a class the 9-oxo-9,10-dihydroacridine-4-acetic acids were generally less potent than the xanthenone-4-acetic acids.


Role of lipophilicity in the in vitro antitumour activity of a series of new mitosene compounds.

The antitumour activity of a series of mitosene compounds in various in vitro tumour models was evaluated in terms of physico-chemical parameters. Lipophilicity, measured as log P, seemed to be important for in vitro antitumour activity in three different cell lines. The in vitro activity of this series of mitosenes in an A204 and a L1210 cell line demonstrated a clear bilinear dependence on log P with optimal activity at log P values of 2.8 and 3.3 respectively. Compounds not able to be activated to bifunctional alkylating species did not fit into this correlation. Although mitosenes have to be activated reductively to alkylating species, no correlation was found between the half-wave reduction potential (E 1/2) and the in vitro activity. This lack of correlation may be caused by the relatively small range of E 1/2-values within this series of mitosene compounds. Our results indicate that penetration of the antitumour mitosenes into the cell and the site of activation is an important process that leads to antitumour activity and that within the range of compounds studied the structural variations are less important for bioreductive activation.


Rational design of immunotoxins: current progress and future prospects.

Immunotoxins are hybrid protein molecules created by the chemical or genetic fusion of an antibody and a protein toxin. Advances in the molecular engineering of monoclonal antibodies and toxins now permit the generation of a variety of immunotoxin types with properties optimized for the therapy of different malignancies. Novel research promises a new generation of protein therapeutics in which the properties of intravascular transport, selective cell binding, internalization, translocation to the cytosol, subcellular localization and selective catalytic action can be manipulated to order. Immunotoxins that combine selective tumour binding with a tumour-specific mechanism of action, such as enzymatic inactivation of cellular products responsible for maintaining the malignant state, could have a potent and selective anti-tumour action.


Avian scleroderma: evidence for qualitative and quantitative T cell defects.

T cell activation is dependent upon calcium influx and protein kinase C activation, with subsequent lymphocyte proliferation dependent upon IL-2. Abnormalities in T cell proliferation, including abnormal calcium influx and defective protein kinase C activation, have been identified in aged mice and humans and many autoimmune diseases including diabetes, lupus and scleroderma. Since UCD line 200 chickens, which spontaneously develop a scleroderma-like disease, have both thymic defects and a diminished peripheral blood lymphocyte response to IL-2, we have further investigated T cell function in these birds. Interestingly, line 200 T cells respond poorly in vitro to a variety of diversely acting T cell mitogens including concanavalin A, phytohemagglutinin and anti-chicken CD3 monoclonal antibody. Moreover, they do not respond well even to phorbol myristate acetate in conjunction with ionomycin. Addition of exogenous IL-2-containing supernatant concurrently with mitogenic stimulation also had no significant effect. Analysis of intracellular free calcium demonstrated that the lymphocytes from diseased birds had a reduced influx of calcium (or release for intracellular stores) following stimulation. These data clearly reflect a unique defect in T cell activation associated with avian scleroderma. Analysis of chicken CD3, CD4 and CD8 expression revealed a 39% decrease in peripheral blood CD4+ cells in scleroderma birds, although this decrease was not sufficient to explain the 80-90% decrease observed in proliferation assays and calcium influx. Our data support the hypothesis that avian scleroderma is mediated via abnormal function of lymphocyte co-stimulatory molecules or intracellular calcium regulators.


The murine Sm-D autoantigen: multiple genes, genetic polymorphism, evolutionary conservation and lack of intervening sequences in the coding region.

Antibodies to the Sm nuclear antigen are diagnostic of systemic lupus erythematosus (SLE). MRL/Mp-lpr/lpr mice develop a similar illness, and a proportion also develop anti-Sm. To understand better anti-Sm reactivity in this murine model, we have cloned the murine Sm-D autoantigen. One cDNA clone was 517 bp long with an open reading frame of 357 nucleotides, encoding a 13.3 kDa protein of 119 amino acids. At the nucleotide level, the murine Sm-D cDNA was 89.8% homologous with human Sm-D (94% in the coding region), yet there was identity at the protein level, including a Gly-Arg nine-fold repeated C-terminus motif. Southern blot analysis of PstI-digested genomic DNA from seven mouse strains demonstrated a 7.8 kb band in every strain; in addition, a 2.8 kb band was seen in AKR/J, LG/J and MRL/Mp-lpr/lpr. PCR amplification of genomic DNA showed a single Sm-D gene product of 360 bp, which indicated a lack of intervening sequences. The Sm-D protein is thus highly conserved in evolution, probably owing to its essential role in the physiology of the cell.


The requirements for growth of in vivo activated autoimmune B cells are similar to those of in vitro generated lipopolysaccharide B cell blasts and dissimilar to anti-IgM plus IL-4 induced B lymphoblasts.

The requirements for growth of in vivo activated B cells (natural blasts) from autoimmune NZB/W mice and of B cells from the same animals activated in vitro with either LPS or anti-IgM plus IL4 (mimicking 'in vitro' antigen induced TH cell-B cell interaction) were studied comparatively. The proliferation of natural and LPS blasts was inhibited by anti-IgM antibodies and augmented by recombinant IL-5. In contrast, anti-IgM stimulated the growth of anti-IgM plus IL-4 primed B cells but was without effect on the proliferative responses in the presence of IL-5. The growth inhibition induced by anti-IgM signalling on natural and LPS blasts seemed to be due to cross-linking of sIg rather than to binding of anti-IgM antibodies to the Fc receptors since a similar effect was observed with the F(ab)'2 fragment of this molecule. Maximum proliferation was obtained by a combination of IL-4 and IL-5 in natural and LPS blasts, whereas peak responses in anti-IgM plus IL-4 blasts were achieved by a combination of anti-IgM and IL-4. Lymphoblasts recovered after preculturing natural blasts in medium alone (more differentiated in vivo activated B cells) displayed high spontaneous proliferation which was strongly inhibited by anti-IgM. This inhibition was reversed partially by IL-4 and totally by IL-5. To define better the role of BLy+ cells in the spleen of NZB/W mice, purified Ly1+ and Ly1- cells, obtained by separation using magnetic beads, were analysed. The growth of both cell subpopulations was inhibited by anti-IgM and enhanced by IL-5. Cytotoxic elimination of Ly1+ cells from the primed B blast populations did not modify the proliferative pattern of these cells. Our results show that the growth requirements of in vivo activated autoimmune B cells resemble those of LPS blasts and differ from those following stimulation with anti-IgM plus IL-4, suggesting that B cells in systemic autoimmune diseases may have been activated by polyclonal stimulation. Nevertheless, other mechanisms for autoimmune B cell activation cannot be ruled out by the present experimental approach.


Quantitative analyses comparing all major spleen cell phenotypes in BB and normal rats: autoimmune imbalance and double negative T cells associated with resistant, prone and diabetic animals.

The BB rat is a model of spontaneous autoimmune diabetes. To characterize quantitatively all known immune cell subsets involved in disease pathogenesis, FACS analysis of spleen cells was performed in diabetes-prone (DP) and acutely diabetic (D) BB rats and compared with diabetes-resistant (DR) BB and normal Wistar-Furth (WF) strains. We observed increased percentages of splenic NK cells in DP and D animals compared with DR rats using an NK-specific monoclonal antibody. We found increased proportions of splenic macrophages in the T-lymphopenic DP and D rats and low macrophage contents in DR spleens compared with WF spleens. We observed that percentages of the CD4-CD8- T cell receptor alpha/beta+ (double-negative) T cell subset were strikingly increased in the lymphopenic DP and D animals, compared with DR animals. We observed increased percentages of activated splenic CD5+ T cells expressing the IL-2 receptor and MHC class II antigen in DP and D rats compared with DR animals. Our studies suggest that (a) splenic NK cells and macrophages quantitatively appear to be involved in the pathogenesis of diabetes; (b) double-negative T cells escape from the T cell depletion process; (c) a marked increase of activated splenic T cells suggests diabetes is associated with general T cell activation processes; and (d) an altered balance among the different immune cell subsets may in part explain the pathogenesis of diabetes, since marked relative changes are observed when comparing the DR strain to the DP strain in both the prediabetic and diabetic stages.


Rheumatoid arthritis synovial fluid enhances T cell effector functions.

Rheumatoid arthritis is a chronic autoimmune joint disease of unknown etiology. T cells are believed to be important in the pathogenesis of rheumatoid arthritis since they infiltrate the joints and express several activation markers, such as MHC class II and IL-2R. In this study we have elucidated the effect on freshly isolated T cells of rheumatoid arthritis synovial fluid (RA-SF), which contains in vivo produced cytokines and enzymes. The mouse mixed lymphocyte culture (MLC) has been used as a model and specific cytotoxicity was evaluated against 51Cr-labelled sensitive target cells. Studies have shown that RA-SF contains a B cell differentiation activity that can cross-react between the human and murine species. Here we have shown that the addition of RA-SF strongly potentiates cytotoxic activity as well as lymphokine production by allogeneic activated effector T cells. The enhanced cytotoxicity induced by RA-SF was found to be due to a combined effect of increased cytotoxic T lymphocyte (CTL) precursor frequency, measured by limiting dilution analysis, and a more efficient killing on a per cell basis. Kinetic studies show that RA-SF must be added within 48 h after initiation of the MLC, otherwise the effect is lost. The target cell specificity of RA-SF was studied, using enriched CD4+ or CD8+ responder cells in the MLC. It was found that RA-SF could act directly on the CD8+ cells and potentiate their development to cytotoxic effector cells: this activity was not found when CD4+ responder cells were used instead. RA-SF could, on the other hand, greatly enhance IL-2 production by CD4+ responder cells. We suggest that B and T cell activity in RA-SF is important in the propagation of chronic inflammation in the joints of patients with rheumatoid arthritis.


Use of a molecularly cloned human SS.B antigen to detect anti-SS.B antibodies.

The aim of this study was to examine the utility of diagnostic assays based on recombinant SS.B/La (rSS.B). Using this antigen, we have developed an ELISA and an immunoblot and compared these recombinant antigen-based assays with traditional thymus extract-based counterimmunoelectrophoresis (CIEP). Using the recombinant ELISA, the incidence of anti-SS.B in 184 normal blood donors was 2.2% (four sera). These four sera were all low titre, i.e., 3-5 SD above the mean. Of 38 sera positive for anti-SS.B by CIEP, 37 were positive in both recombinant assays (97.4% concordance). Anti-SS.B titre in CIEP correlated strongly with results of the rSS.B-based ELISA, but the ELISA was 3,000-fold more sensitive. In an analysis of 152 autoimmune sera containing anti-DNA, anti-RNP, anti-centromere, anti-SS.A/Ro or anti-cardiolipin, all of which were negative for anti-SS.B/La by CIEP, the recombinant assays detected 17 new anti-SS.B positives. These positive results were found only in sera which had previously been characterised by CIEP as anti-SS.A/Ro positive. Anti-SS.B/La antibodies detected by recombinant SS.B assays were found to be highly predictive of primary Sjögren's syndrome. Our results show that rSS.B can have an important role in the design of sensitive and specific assays for anti-SS.B. The diagnostic significance of anti-SS.B/La as a guide to primary Sjögren's syndrome is not diminished by the increased sensitivity of recombinant SS.B assays.


Evidence that a putative anti-idiotypic monoclonal antibody may actually be recognizing circulating immune complexes.

Murine monoclonal antibodies (mAb) reacting with affinity-purified antihistone antibodies (AHA) from serum of a patient with systemic lupus erythematosus (SLE) were obtained. One of them, 8B3, was initially considered to recognize idiotypic (Id) determinants in AHA since (a) it reacted with AHA but not with control IgG; (b) this reactivity could be inhibited using affinity-purified AHA, but not with control IgG or whole serum; (c) affinity-isolated 8B3+ antibodies showed antihistone activity and not other activities tested so far; (d) antihistone activity due to 8B3+, but not that of 8B3- from the same serum, could be fully inhibited by the presence of 8B3 mAb in the antihistone assay and (e) serum levels of 8B3 reactivity were higher than normal in SLE patients with AHA (56%), in contrast with SLE patients without AHA (6%). From these results it was deduced that 8B3 defined a cross-reactive Id shared by a subset of AHA in SLE patients. However, the present results suggest that (a) 8B3 mAb did not recognize AHA or Ig, but did recognize a 55 kDa histone-binding protein; (b) this 55 kDa protein was present free at low concentration in all human sera, but also associated with IgG in 8B3+ SLE sera and (c) these complexes are responsible for the false positive results in the antihistone assay as shown for DNA/anti-DNA complexes. Thus, mAbs recognizing the non-Ig moiety of circulating immune complexes may resemble anti-Id antibodies with features of the so-called epibodies. These immune complexes may be responsible for false positive results and caution should be exercised in the interpretation of results.


Expression of inactive stage anti-dsDNA idiotypes on anti-ssDNA antibodies in a lupus patient during active stage of lupus cerebritis.

The possibility that idiotypes (Ids) defined on anti-double stranded DNA (dsDNA) antibodies during active and inactive stages of lupus (1/84 Id and 4/90 Id, respectively) were expressed on anti-DNA antibodies during a subsequent active period (9/90) of the disease was investigated in a lupus patient with lupus cerebritis. Using rabbit (R)-anti-Ids specific to 1/84 Id and 4/90 Id in inhibition assays, the 4/90 Id was shown to be expressed on the framework regions of anti-single stranded DNA (ssDNA) but poorly on co-existing anti-dsDNA antibodies of active (9/90) stage. The 1/84 Id was poorly expressed on both types of 9/90 anti-DNA antibodies. While the 9/90 anti-ssDNA significantly bound to immobilized ssDNA and several single-stranded polynucleotides, only ssDNA inhibited the binding of the anti-ssDNA to ssDNA, suggesting its monospecificity toward ssDNA. Western blot analysis following isoelectric focusing showed that a spectrotype pattern of 4/90 Id-positive 9/90 anti-ssDNA IgG was similar to that of the 4/90 anti-dsDNA, suggesting that they are of related clonal origin. The present study suggests the idiotypic heterogeneity of anti-DNA antibodies and the shift of antigen specificity within an idiotypically related anti-DNA population during exacerbation of the disease.


Galactose terminating oligosaccharides of IgG in patients with primary Sjögren's syndrome.

Using a simple but novel ELISA, we have screened 40 serum samples from patients with primary Sjögren's syndrome and 34 normal controls for IgG glycosylation deficiencies, identified by their specific ricin binding. Elevated levels of asialylated IgG were detected in 24 patients. The extent of asialylation was significantly higher in the patients with extraglandular manifestations than in the others. Interestingly, the correlation of asialylated IgG was apparent only with Raynaud's phenomenon and arthritis, and not other extraglandular manifestations. Strong correlations (P less than 0.01) were noted between asialylated IgG and rheumatoid factor or IgA-containing immune complexes.


Intravenous cocaine challenges during desipramine maintenance.

Intravenous challenges with placebo and cocaine doses ranging from 0.125 to 0.5 mg/kg were administered to five subjects using a within subjects design during placebo and active desipramine (DMI) maintenance at a fixed dose of 150 mg daily for at least 10 days. The "high" reported after cocaine infusion was not altered by DMI, but "desire for cocaine" after a single dose was attenuated. Together with the results of clinical trials of DMI for cocaine abuse, these laboratory results suggest that DMI may reduce cocaine craving both during and between cocaine binges. Physiologically, baseline heart rate was higher on DMI, but the incremental heart rate response to cocaine was attenuated.


Selective breeding for increased cholinergic function: biometrical genetic analysis of muscarinic responses.

Biometric genetic analyses of behavioral and physiologic responses known to be related to muscarinic cholinergic receptors (hypothermia, hypoactivity, inhibited avoidance, and reduced responding for water) were studied in genetic crosses and backcrosses of the Flinders sensitive line (FSL) and Flinders resistant line (FRL) of rats. The FSL rats were more sensitive to the direct muscarinic agonists, arecoline and oxotremorine, and to the indirect agonist, physostigmine, than any other group. The next most sensitive group was the F1 x FSL backcross, followed by the F2, F1, F1 x FRL backcross, and the FRL, in that order. These differences between the genetic groups could be accounted for completely by either solely additive or additive plus dominance genetic factors. When dominance genetic factors contributed to the differences among groups (6 out of 15), the F1 responded like the FRL rats. The variance of the responses measured made it impossible to obtain reliable estimates of the number of genes involved in many instances; when such estimates were possible, several genes (greater than or equal to 3) appeared to be involved. We conclude that muscarinic sensitivity in rats is under genetic control, with the greatest contribution coming from additive genetic factors. Because the FSL rat appears to be a genetic animal model of depression, the finding of several genes influencing muscarinic responses may help account for the difficulties investigators have had in locating a single major gene or biological marker for human depressive disorders.


Bromocriptine reverses the elevation in intracranial self-stimulation thresholds observed in a rat model of cocaine withdrawal.

Cocaine use frequently occurs in episodic prolonged binges. Following such a cocaine binge, the user suffers from severe depression mixed with irritability, anxiety, anergia and anhedonia. These symptoms constitute the cocaine withdrawal syndrome. Since cocaine's rewarding effects are mediated by enhanced dopaminergic neurotransmission in the mesocorticolimbic system, it is possible that a long-acting dopamine agonist might block the withdrawal effects associated with the termination of a prolonged bout of cocaine self-administration. An animal model of post-cocaine anhedonia was developed using the elevation in intracranial self-stimulation (ICSS) thresholds following the termination of prolonged periods of cocaine self-administration as a measure of an animal's "anhedonic" state. In the present study, an attempt was made to reverse the postcocaine elevation in ICSS thresholds with acute administration of a dopaminergic agonist, bromocriptine. Rats were allowed to self-administer cocaine for 24 hours continuously. Four hours after the termination of the self-administration session, animals were injected with either vehicle or bromocriptine (1, 2, or 4 mg/kg, IP). Two hours later (6 hours post cocaine), the animals' self-stimulation thresholds were assessed. Confirming previous work, treatment with the vehicle following a cocaine "binge" resulted in elevated ICSS thresholds compared to predrug baseline levels or to control rats' thresholds. Bromocriptine, at doses that had no effect on ICSS thresholds in control rats, reversed the postcocaine anhedonia in a dose-related manner. These results indicate that bromocriptine-like drugs (pharmacological agents that enhance dopaminergic neurotransmission) may be able to ameliorate some of the effects of cocaine withdrawal on mood and motivational state. In addition, the results of the present study indicate that the proposed animal model of cocaine withdrawal could be useful in the discovery and development of new pharmacotherapies for cocaine withdrawal.


Cerebral glucose metabolism in patients with summer seasonal affective disorder.

Positron emission tomography scans of nine patients diagnosed with summer seasonal affective disorder (SSAD) were compared with scans of 45 normal control subjects to investigate differences in brain glucose metabolism. All subjects performed an auditory discrimination task beginning several minutes before injection of F-18-deoxyglucose and continuing for 30 minutes after injection. Regional glucose metabolic rates were extracted from 60 rectangular regions of interest measured in five planes selected as atlas matches from 28 total slices. Statistically significant differences between patients with SSAD and normal control subjects were found in cerebral glucose metabolic rate and also in normalized regional glucose metabolic rates in the orbital frontal cortex and in the left inferior parietal lobule.


Human hallucinogen interactions with drugs affecting serotonergic neurotransmission.

The absence of relevant human research studies of hallucinogenic drugs has not curtailed their unsupervised use. Two cases are presented that suggest decreased sensitivity to the serotonergic hallucinogens psilocybin and LSD induced by drugs with effects on serotonergic neurotransmission, allopurinol and fluoxetine. These reports suggest that hallucinogens' effects in humans are mediated by serotonergic receptors.


Suppressor T cells: some answers but more questions.

Many properties of suppressor T cells and the antigen-binding factors derived from them have evaded molecular genetic definition. Here, Martin Dorf and colleagues discuss recent data in the context of a growing awareness of the molecules and principles involved.


NK cells and T cells: mirror images?

The expression of MHC class I molecules protects cells against lysis by natural killer (NK) cells. It is possible that NK cells are 'educated' to recognize self MHC class I molecules and that the combination of self peptide and MHC class I molecule blocks NK-mediated lysis. Here, Rogier Versteeg compares and contrasts models of education and self-nonself discrimination by T cells and NK cells, and presents a hypothesis for the evolution of T cells from NK cells.


One explanation for F1 antiparent responses.

The phenomenon of hybrid resistance, in which F1 offspring reject parental grafts, remains a perplexing problem. Here, Tina Dalianis and Lars Ahrlund-Richter propose that one component of the F1 antiparent response results from competition between the two sets of parentally derived major histocompatibility complex (MHC) molecules for 'promiscuous' peptides. Lack of tolerance in the F1 animal results from insufficient presentation of these peptides on the MHC molecule that has lower affinity for the peptides.


Immunosuppression by ultraviolet B radiation: initiation by urocanic acid.

Irradiation with UV-B, a component of natural sunlight, initiates systemic immunosuppression of delayed-type hypersensitivity responses. This may be a fundamental regulatory mechanism, controlling the interaction between mammals and potentially deleterious environmental UV radiation. Here, Frances Noonan and Edward De Fabo assess the evidence that suppression is initiated by the photoisomerization of trans-urocanic acid (UCA) in the stratum corneum, discuss the significance of this mechanism for skin cancer outgrowth and propose applications for UCA in transplantation.


Autoimmunity and the ovary.

The ovary was first documented as a target of autoimmunity over three decades ago yet today the aetiology and pathogenesis of autoimmune-mediated premature ovarian failure (POF) are poorly understood. Here, Roy and Helga Moncayo provide a brief overview of human autoimmune-mediated POF and the animal models of POF. They propose a model for the development of the disease, highlighting the role of supraphysiological levels of gonadotropins in inducing selectively antigenic mature ovarian elements.


Human T-cell activation deficiencies.

The increasing understanding of T-cell activation is paralleled by the recognition of a growing range of 'experiments of nature' that cause T-cell activation deficiencies. Analysis of these deficiencies is, in turn, contributing to the understanding of T-cell function in vivo. Here, José Regueiro, Antonio Arnaiz-Villena and colleagues review current knowledge of structural and functional T-cell defects and the implications of these for T-cell biology.


The origin and function of tumor-associated macrophages.

Tumor-associated macrophages (TAM) have a complex relationship with the neoplastic cells of the tumor. On the one hand, the two cell types produce reciprocal growth factors and may be considered to have a symbiotic relationship. On the other hand, TAM can be activated to inhibit tumor growth and destroy neoplastic cells. Here, Alberto Mantovani and colleagues describe this delicate balance and the prospects for its therapeutic manipulation.


Lipopolysaccharide antagonists.

Bacterial lipopolysaccharide (LPS) is a potent and pleiotropic stimulus of immune cells. LPS has important clinical relevance because it has a direct role in the pathogenesis of Gram-negative bacterial infection. The lipid A moiety of LPS is responsible for the toxic effects of LPS. The identification of structural analogs and precursors of lipid A, which are apparently competitive antagonists of the biological actions of LPS, is strong evidence that the actions of LPS are mediated by a specific LPS receptor or family of receptors. Identification and analysis of these LPS receptors with LPS antagonists should help to define the pathways of cellular activation by LPS and lead to the development of novel anti-LPS strategies in the therapy of bacterial diseases.


[Phadiatop in the diagnosis of skin allergy to pneumoallergens].

Usually, Phadiotop is presented as an in vitro multitest to pneumoallergens that are indicated in respiratory allergy. Now, in skin allergy atopic dermatitis and reaginic urticaria-Quincke's oedema, the responsibility of pneumoallergens, particularly mites, is clear. 47 Subjects were studied. Clinical history, skin test nd specific IgE gave confirmation of allergy to pneumoallergens. This was proved by the subsequent clinical development, with, in most cases, spectacular improvement after desensitization.8 section. We have compared Phadiotop with these different clinical criteria, skin tests and Cap Rast IgE. The results favour Phadiotop and confirm its value and indication in skin allergy to pneumoallergens.


[Multitest Fx5 in food allergy].

A comparative study was made of three in vivo and in vitro diagnostic methods for food allergy: Fx5 multitest (Pharmacia); Measurement of specific IgE CAP (Pharmacia); Skin tests (Prick Tests). 20 patients, from 3 to 71 years (mean 24.4 years), were selected by clinical suggestion (asthma, rhinitis, atopic dermatitis, urticaria and/or Quincke's oedema). The Fx5 test used six food allergens: wheat, egg, cow's milk, soya, peanut and fish. The Cap Rast for each substance was evaluated, as was Fx5, by a radio-immunological method. The Prick Tests made with the six allergens used were considered to be positive when the diameter of the weal was greater than that produced by a reference test with histamine. The results were considered as a comparison between Fx5 and Cap Rast to each of the foods, between Fx5 and prick Test with five foods and finally between CAP RAST and Prick Test. Correlation Fx5--Cap rast was better and more useful in the diagnosis of food allergy than skin tests.


[Skin tests in allergic vasculitis].

For etiologic checking of cutaneous vasculitis, skin testing may be of valuable help: epicutaneous patch-tests with delayed lecture (48 h.) performed with contact haptens (rubber allergens, azo dyes, resins ...) or drugs in pigmented progressive purpuric skin eruptions; prick-tests submitted to delayed (6-8 h.) clinic and histologic lecture, performed with miscellaneous drug or food-derived, microbial or environmental antigens in necrotizing vasculitis. In these conditions skin testing should be approached with great caution, because of the occurrence of non relevant positive reactions and the risk of precipitation of cutaneous or disseminated lesions of vasculitis.


Immunologic mechanisms in secretory otitis media--recent concepts. Part I.

Increasing evidence has been accumulated in the last few years associating immunological mechanisms in the pathogenesis of secretory otitis media. If immunological processes were found to be involved in both of the previously accepted experimental pathogenic models (Eustachian tube obstruction: microbiologically induced) of the disease, recently published data has provided evidence for a third, not Eustachian tube--or microbiological-dependent, exclusively immunological, experimental model. This suggests that immunological mechanisms are probably always involved in the pathogenesis of a disease that is known to be multifactorial in origin, and thus eventually make Immunology the single most important factor in secretory otitis media. In the present article these recent concepts are extensively reviewed by the authors.


[Reevaluation of environmental factors in allergy to mites: bedding not a factor?].

Prospective multicentric study was conducted among parents of children referred in three allergy units. Unlike current opinions: carpets are less frequent in bedrooms of mite's allergic children. foam bedding don't play any role, local humidity non significantly increases the mite's allergy risk. the sunlight absence is significantly correlated with this hypersensitivity. These data, mainly inconsistent with general conviction, could be explained by socio economic diversity of patients.


[Clinical effectiveness and tolerance of loratadine versus cetirizine in the treatment of seasonal allergic rhinitis].

A double blind multicentre study of seasonal rhinitis (108 patients) has compared Loratadine and Cetirizine. The results of clinical scores are significantly good for both products. Only tolerance is different and in favour of Loratadine, since sleepiness was found in 9.5% of patients treated with Cetirizine, and 3.6% with Loratadine.


Stability of fungal alpha-amylase in sodium dodecylsulfate.

Unfolding of a fungal alpha-amylase in aqueous sodium dodecylsulfate (SDS) solution was examined by SDS-polyacrylamide gel electrophoresis (PAGE). When the alpha-amylase was incubated with 1% SDS at room temperature and subjected to SDS-PAGE, it showed a much higher mobility than expected from the molecular weight. Circular dichroic and gel filtration analyses indicated that the protein is apparently in the native conformation upon incubation with 1% SDS. When the protein was heated in the presence of 1% SDS at 90 degrees C for 10 min, it had a lower mobility in SDS-PAGE and showed characteristics of an unfolded protein by circular dichroism and gel filtration. The melting temperatures of the protein were determined in the absence and presence of SDS by incubating it for 10 min at various temperatures. The melting temperatures were 70, 55, and 49 degrees C in the presence of 0, 1, and 2% SDS, respectively. The observed small shift of the melting temperatures by SDS suggests that the destabilizing action of SDS on the alpha-amylase is weak. However, the unfolding in SDS is not reversible process, since prolonged incubation of the protein with 1% SDS at 50 degrees C gradually increased the amount of unfolded protein. This indicates that the SDS-induced unfolding of the alpha-amylase is a slow process.


Conformation of concanavalin A and its fragments in aqueous solution and organic solvent-water mixtures.

The conformations of concanavalin A (con A), an all-beta protein, and its three CNBr-cleaved fragments were studied by CD. Con A in buffer showed a 197 nm maximum and a 223 nm minimum, which were red-shifted by 6-7 nm from those of regular all-beta proteins and beta-sheet of (Lys)n. Fragment 1 (residue 1-42) resembled an unordered form with a CD maximum at 200 nm, but fragments 2 (residues 43-129) and 3 (residues 130-237) showed a regular CD spectrum with two extrema at 192-193 nm (+) and 214-216 nm (-). Equimolar mixture of the three fragments showed some degree of interaction, but did not reconstitute the conformation of native con A, probably because of the loss of bound Ca2+ and Mn2+ ions in the fragments. In ethanol-, methanol-, and dioxane-water mixed solvents, con A and its fragments remained as beta-sheet. In contrast, addition of trifluoroethanol and sodium dodecyl sulfate induced alpha-helix at the expense of beta-sheet for con A and its fragments in aqueous solution. In 80% trifluoroethanol, the induced helicities exceeded their sequence-predicted helix-potentials, but in 10 mM sodium dodecyl sulfate the helicities agreed well with corresponding predictions.


Structural analysis of seminal and serum human transferrin by second derivative spectrometry and fluorescence measurements.

Denaturation of human seminal transferrin (HSmT) compared with human serum transferrin (HSrT) was followed to check structural differences between these two proteins. Second derivative UV spectroscopy indicated that treatment with 6 M guanidine hydrochloride (Gnd.HCl) induced greater structural changes in HSrT than in HSmT and, in particular; (i) the exposure value of tyrosinyl residues was almost 2.5-fold higher in native HSmT than in native HSrT; and (ii) a much more pronounced movement of tryptophanyl residues toward a higher polar environment could be noticed in HSrT after incubation with denaturing agent. Fluorescence measurements showed that: (i) a shift of the maximum emission wavelength of HSmT occurred (maximum emission was centered at 333 nm instead of 323 nm as for HSrT; excitation = 280 nm); (ii) the intrinsic tryptophan fluorescence intensity of HSmT increased after 36 hr in the range of 1.5-4.0 M of denaturant, whereas an opposite behavior was found for HSrT in the range 0.0-2.0 M; and (iii) the wavelength maximum of fluorescence emission changed in a biphasic manner for HSrT and, conversely, under the same experimental conditions, HSmT gave a linear and parallel increase of fluorescence emission after 1 and 36 hr. We can conclude that this different behavior of HSmT with respect to HSrT might be due mainly to the fact that both the number and the exposure of tyrosinyl and tryptophanyl residues are different. Lately, these effects are discussed in relationship with the fact that HSmT contains less than half disulphide bridges than HSrT.


Dependence of reaction rate of 5,5'-dithiobis-(2-nitrobenzoic acid) to free sulfhydryl groups of bovine serum albumin and ovalbumin on the protein conformations.

The effect of protein conformations on the reaction rate of Ellman's reagent, 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) with sulfhydryl (SH) groups of proteins was examined. The stopped-flow method was applied to follow the reaction of DTNB with SH group of two proteins, bovine serum albumin (BSA) and ovalbumin (OVA), at various concentrations of guanidine hydrochloride and urea. The rates for both the proteins were faster in guanidine than in urea. The rate sharply depended on the protein conformations, which were monitored by changes of helix contents on the basis of the circular dichroism measurements. The reaction rate of DTNB with SH groups of BSA was maximal around 2 M guanidine and 5 M urea. On the other hand, the reaction rate of DTNB with OVA was maximal at 3.5 M guanidine, while it gradually increased with an increase in the urea concentration. The amount of reactive SH group participating in the reaction with DTNB was also estimated by the absorbance change at 412 nm. The magnitudes of absorbance change for the reaction with free SH groups of OVA at low concentrations of the denaturants were appreciably smaller than those for BSA with one free SH group. Most of the four SH groups of OVA might react with DTNB above 5 M guanidine, although only a part of them did even at 9 M urea.


An evaluation of different enzymatic cleavage methods for recombinant fusion proteins, applied on des(1-3)insulin-like growth factor I.

Different enzymatic methods for cleavage of recombinant fusion proteins were compared. To find an efficient cleavage method, five different fusion proteins were produced. The fusion proteins differed only in the linker region between the fusion partner and the desired product, human des(1-3)insulin-like growth factor I. A cleavage study was performed with enterokinase, plasmin, thrombin, urokinase, and recombinant H64A subtilisin. Significant cleavage was obtained using thrombin, H64A subtilisin, and enterokinase. Thrombin cleavage was studied on a larger scale and des(1-3)IGF-I was recovered at a final yield of 3 mg/L growth medium. Thrombin and enterokinase were also studied as immobilized proteases and they cleaved the fusion proteins with retained activity. To further improve thrombin cleavage, a continuous reactor was constructed, consisting of a closed system with a thrombin column and an ion exchange column in series. Here, the fusion protein circulated while free des(1-3)IGF-I was bound to the ion exchange column after release from the fusion protein. In the reactor, thrombin was as efficient as the free enzyme but gave a diminished rate of product degradation.


In vivo and in vitro methylation of lysine residues of Euglena gracilis histone H1.

We have earlier identified and purified two protein-lysine N-methyltransferases (Protein methylase III) from Euglena gracilis [J. Biol. Chem., 260, 7114 (1985)]. The enzymes were highly specific toward histone H1 (lysine-rich), and the enzymatic products were identified as epsilon-N-mono-, di- and trimethyllysines. These earlier studies, however, were carried out with rat liver histone H1 as the in vitro substrate. Presently, histone H1 has been purified from Euglena gracilis through Bio-Rex 70 and Bio-Gel P-100 column chromatography. The Euglena histone H1 showed a single band on SDS-polyacrylamide gel electrophoresis and behaved like other histone H1 of higher animals, whereas it had a much higher Rf value than the other histones H1 in acid/urea gel electrophoresis. When the Euglena histone H1 was [methyl-3H]-labeled in vitro by a homologous enzyme (one of the two Euglena protein methylase III) and analyzed on two-dimensional gel electrophoresis, three distinctive subtypes of histone H1 were shown to be radiolabeled, whereas five subtypes of rat liver histone H1 were found to be labeled. Finally, by the combined use of a strong cation exchange and reversed-phase Resolve C18 columns on HPLC, we demonstrated that Euglena histone H1 contains approximately 9 mol% of epsilon-N-methyllysines (1.40, 1.66, and 5.62 mol% for epsilon-N-mono-, di- and trimethyllysines, respectively). This is the first demonstration of the natural occurrence of epsilon-N-methyllysines in histone H1.


Purification and sequence determination of canine relaxin.

Relaxin immunological activity has been observed in the plasma of pregnant bitches, and preliminary studies in our laboratory indicated that the highest relaxin concentrations were found in placentas. Therefore, canine placentas were collected at term and also from spay and relaxin was purified by methods developed for equine relaxin. Tissue was prepared by homogenization and purification on a C18 column. The preparation was further purified by stepwise elution ion-exchange chromatography, gel filtration, and gradient elution ion-exchange chromatography. One predominant peak in relaxin immunoactivity was collected. Canine relaxin was found to be larger than either porcine or equine relaxin as determined by SDS-PAGE. It migrated faster under reducing conditions, indicating a subunit structure. Purified canine relaxin was used for tracer and standard in a canine radioimmunoassay (RIA) using an antiporcine relaxin antibody. Concentrations of relaxin immunoactivity using the canine assay were up to 300-fold higher in placental preparations than those measured in the porcine relaxin assay. Sequence analysis of canine relaxin revealed a structure similar to other relaxins in the presence and placement of cystine residues.


Pepsin fragmentation of botulinum type E neurotoxin: isolation and characterization of 112, 48, 46, and 16 kD fragments.

Controlled digestion of approximately 150 kD single chain botulinum type E neurotoxin with pepsin at pH 6.0 produced 112, 48, 46, and 16 kD fragments. These were chromatographically purified; their locations in the approximately 1300 amino acid residue long neurotoxin were determined by identifying the amino terminal 10 residues of 112 and 48 kD fragments, 50 residues of 46 kD fragment, and 59 residues of 16 kD fragment. The 48 and 112 kD fragments contain the N-terminal segment of the neurotoxin (i.e., residue no. 1 to approximately 425 and 1 to approximately 990, respectively), the 46 kD fragment corresponds to approximately 407 residues of the C-terminal region, and the 16 kD fragment contains the approximately 140 residues from a segment nearer to the C-terminus. The 48 kD fragment is similar to the approximately 50 kD N-terminal light chain of the approximately 150 kD dichain neurotoxin, which is generated by tryptic cleavage of the approximately 150 kD single chain neurotoxin, and is separated from the approximately 100 kD C-terminal heavy chain by dithiothreitol (DTT) reduction of an intrachain disulfide bond in the presence of 2 M urea (Sathyamoorthy and DasGupta, J. Biol. Chem. 260, 10461, 1985). The pepsin-generated 48 kD fragment, unlike the light chain, was isolated without exposure to DTT and urea. The single chain 112 kD fragment following trypsin digestion yielded 48 and 60 kD fragments that were separable after DTT reduction of the intrachain disulfide which links them. The N-terminal residues of the smaller fragment were identical to that of the single chain 150 kD neurotoxin; the single chain 112 kD fragment is therefore the neurotoxin minus the approximately 50 kD C-terminal half of the heavy chain. The biological activities of the 48 and 112 kD fragments can be demonstrated in permeabilized PC12 cells (Lomneth et al., J. Neurochem. 57, 1413, 1991); they inhibit norepinephrine release.


Biophysical analysis of phaseolin denaturation induced by urea, guanidinium chloride, pH, and temperature.

The structural stability of phaseolin was determined by using absorbance, circular dichroism (CD), fluorescence emission, and fluorescence polarization anisotropy to monitor denaturation induced by urea, guanidinium chloride (GdmCl), pH changes, increasing temperature, or a combination thereof. Initial results indicated that phaseolin remained folded to a similar extent in the presence or absence of 6.0 M urea or GdmCl at room temperature. In 6.0 M GdmCl, phaseolin denatures at approximately 65 degrees C when probed with absorbance, CD, and fluorescence polarization anisotropy. The transition occurs at lower temperatures by decreasing pH. Kinetic measurements of denaturation using CD indicated that the denaturation is slow below 55 degrees C and is associated with an activation energy of 52 kcal/mol in 6.0 M GdmCl. In addition, kinetic measurement using fluorescence emission indicated that the single tryptophan residue was sensitive to at least two steps of the denaturation process. The fluorescence emission appeared to reflect some other structural perturbation than protein denaturation, as fluorescence inflection occurred approximately 5 degrees C prior to the changes observed in absorbance, CD, and fluorescence polarization anisotropy.


Microenvironment of tryptophan residues in beta-lactoglobulin derivative polypeptide-sodium dodecyl sulfate complexes.

The changes of microenvironment of tryptophan residues in beta-lactoglobulin A and its cyanogen bromide (CNBr) fragments with the binding of sodium dodecyl sulfate (SDS) were studied with measurements of the rates of N-bromosuccinimide (NBS) modification reactions by stopped-flow photometry. Two tryptophan residues of carboxyamidomethylated (RCM) beta-lactoglobulin A in the states of their complexes with SDS were clearly distinguishable by their differences in NBS modification rates. We confirmed by experiments with CNBr fragments containing trytophan residue. The modification rates of Trp 19 in RCM beta-lactoglobulin A-SDS complexes were about 10-fold smaller than those expected for tryptophan residues exposed entirely to the aqueous solvent. The Trp 61 was hardly changed. The change of rate constants for Trp 19 was virtually consistent with those observed when N-acetyl-L-trytophan ethylester was dissolved in SDS micelles. For various species of polypeptide-SDS complexes, all tryptophan residues were reactive to NBS and also, for some of them, the differences in NBS modification rates were observed between tryptophan residues on a common polypeptide chain. These results suggest micellar and heterogeneous bindings of SDS to polypeptides.


Similarity between average distance maps of structurally homologous proteins.

A similarity between average distance maps (Kikuchi et al., 1988a)--that is, predicted contact maps of two tertiary structurally homologous proteins--is examined. Comparisons of shapes of average distance maps (we refer to this as ADM) are made by superpositions of ADMs for two homologous proteins. Also, we compare shapes of actual contact maps for the pair of proteins. We search a optimal superposition mode of each pair of maps showing that two proteins are most similar. It is concluded that two ADMs are also similar when actual tertiary structures between two proteins show similarity. A criterion for similarity of maps is also proposed. The possibility of application of this method to detect weak homology between protein structures is discussed.


Sequence and structural relationships in the cytokine family.

The sequences of nine different cytokines, growth hormone, and prolactin have been aligned and their secondary structure predicted. The alignment reveals that each exon has a characteristic sequence pattern shared by all cytokines. The most striking sequence similarity is observed in exon 4, where the residue pair Phe-Leu is conserved in many cytokines. In addition, there are discreet homologous regions between two specific growth factors, including a high degree of homology between granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3). The secondary structure analysis predicts that exon 3 of all cytokines has an antiparallel helix-turn-helix motif, which is likely to form the central helical segments of a four alpha-helical bundle-type structure. Based on the secondary structure and the disulfide-bonding pattern, the topological connectivity for a number of cytokines has been predicted.


Rat liver low M(r) phosphotyrosine protein phosphatase isoenzymes: purification and amino acid sequences.

Two low M(r) phosphotyrosine protein phosphatases have been isolated from rat liver. The enzymes were previously known as low M(r) acid phosphatases, but several recent studies have demonstrated that this family of enzymes possesses specific phosphotyrosine protein phosphatase activity. We determined the complete amino acid sequences of the two isoenzymes and named them AcP1 and AcP2. Both consist of 157 amino acid residues, are acetylated at the NH2-terminus, and have His as the COOH-terminus. The molecular weights calculated from the sequences are 18,062 for AcP1 and 17,848 for AcP2. They are homologous except in the 40-73 zone, where about 50% of residues are different. This fact suggests that the two isoenzymes are produced by an alternative splicing mechanism. There is no homology between these two isoenzymes and the receptor-like phosphotyrosine protein phosphatases LAR, CD45, human placenta PTPase 1B, and rat brain PTPase-1. AcP1 and AcP2 are also distinct from rat liver PTPase-1 and PTPase-2, since these last enzymes have higher molecular weights. AcP1 differs from AcP2 with respect to (1) substrate affinity and (2) its sensitivity to activators and inhibitors, thus suggesting a their different physiological function.


A customized non-axial external beam technique for treatment of prostate carcinomas.

A nonaxial four-field technique for primary and boost treatment of prostate carcinoma has been developed in an effort to minimize dose to rectum and bladder. Using a treatment planning CT scan, prostate, rectal and bladder volumes are outlined and corresponding three-dimensional structures are created. An oblique CT image, which bisects the prostate volume and excludes rectum and bladder, is reconstructed. Four beams, consisting of direct laterals and two anterior, inferior obliques, are designed, which lie in the plane of the generated image. Utilizing a beam's-eye-view display and multilevel dose calculations, blocks are designed for each port, which encompass the target volume within a minimum isodose surface. These customized angles result in oblique ports with rectal and bladder sparing that can be comparable to that of direct lateral ports. Dose-volume histogram analysis is used to evaluate the merits of this nonaxial method with other approaches.


High dose rate treatment of a maxillary sarcomatoid carcinoma: a case report.

A 37-year old Native American woman presented with a rare sarcomatoid carcinoma of the left maxilla. She underwent extensive resection, but developed an orbital cavity recurrence. This was treated with external beam radiation therapy. The boost posed dosimetric difficulties due to the anatomic peculiarities of the treatment area. Extensive treatment planning for a high dose rate Iridium 192 source helped overcome these problems. Control of the tumor was achieved in the site of recurrence.


Immobilization and treatment of patients receiving radiation therapy for extremity soft-tissue sarcoma.

The treatment of extremity soft-tissue sarcoma has evolved considerably over the years. Previously, the preferred treatment was radical resection or amputation. Recently, radiation therapy combined with conservative resection has been shown to provide adequate tumor control while preserving a functional limb. Often, a large volume of tissue is irradiated in a manner that requires the patient to be in a reproducible position. Minimizing patient movement is imperative to ensure that the prescribed dose is delivered to the designated target volume. At our institution, Alpha Cradles have been routinely used for eight years to aid in positioning extremity sarcoma patients. The positioning of the patient for tumors at different locations in the extremity and the Alpha Cradle fabrication is described. The positioning device is comfortable for the patient and the accuracy is verified by comparison of simulation films with weekly port films.


Weight consideration in the use of cerrobend beam blocks.

The technique of using customized field blocking to protect sensitive normal tissue during megavoltage radiation treatment is common practice in modern radiation therapy. The introduction of CT-based treatment planning has revolutionized customized field shaping. We carried out a prospective evaluation of 54 cerrobend blocks during a one-month time period. The goals of this study were to analyze the specific block patterns and correlate these with field size, block weight, and field setup. Factors contributing to excessively large and heavy cerrobend blocks defined as > or = 20 lbs. were identified. Twenty-two percent of blocks were found to be excessively large and one-third of these were a consequence of planning decisions. A review of these situations suggests that alternative methods would have avoided the excessive weight. Concerns have been raised regarding the safety of large and heavy cerrobend blocks. These blocks were therefore analyzed in terms of tray sag and tray break-point. Our data suggest that within this clinical range of block weight, neither tray sag nor tray break-point are of significant concern.


Determination of basal dose rates in Paris system of interstitial implants.

The isodose distributions are related to the source configurations in the Paris system of interstitial implants. In this system, the basal dose rates are determined at those points identified relative to the implant. Once these points have been located, the basal dose rates are merely the summation of the dose rate contributions from all the sources. By applying the symmetry properties of the implant, the number of basal dose rate computations and the number of dose rate summations are reduced. The reduction in both the number of computations and the number of summations has facilitated the determination of the average basal dose rate. After the average basal dose rate has been determined, the reference dose rate that is used clinically is calculated. Examples of determining the basal dose rates for a few interstitial implants are presented.


Verification of treatment plans by mathematical formulas for single catheter HDR brachytherapy.

In high dose-rate brachytherapy, 500 to 1000 cGy will be given in a matter of minutes. Therefore, careful treatment planning and accurate verification of the plan is critical. To expedite the plan verification procedure, simple mathematical formulas were derived in our previous paper for single catheter treatments on a Nucletron microSelectron system. In this paper, the usefulness and limitations of these formulas for the plan verification procedure are further discussed.


Immobilization and stabilization of testicular clamshell shields in seminoma patients.

It is standard practice when treating the pelvic and para-aortic lymph nodes of seminoma patients, to use a method to reduce the dose to the testicles due to scattered radiation. A common method is the use of lead testicular shields (clamshells). We have developed a method of immobilizing the patient while at the same time providing a stable and reproducible position for the testicular shield.


Electron beam versus photon beam radiation therapy for the treatment of orbital lymphoid tumors.

Shielding the lens of the eye while treating orbital lymphoid tumors and achieving a relatively homogeneous dose can be a dosimetric challenge. There are many clinical cases of tumors close to one or both eyes, where it is desirable to spare the lens as much as possible. We will show isodose curves comparing direct anterior fields using various electron beam energies to a single 6 mv photon beam to a multifield combination. The presence of an eye shield in a photon beam may impair the dose distribution at the back of the eye to an undesirable degree. The strong in-depth dose drop-off in electrons and the ease with which the beam can be intercepted suggests that an electron beam with an eye shield may offer advantages. We designed a suspended movable lens shield .8 cm wide with a height of 4.5 cm made of cerrobend surrounded by a brass cylinder mounted to a thin plexiglass plate to insert into a Varian electron 6 x 6 cone. A 1.5 cm gap between block and eye surface was used. Verification of dose distribution under the lens shield was obtained by film dosimetry.


Stepwise transformation of primary thyroid epithelial cells by a mutant Ha-ras oncogene: an in vitro model of tumor progression.

Activating mutations of the ras oncogene family occur at high frequency in all stages of thyroid tumorigenesis, both human and experimental. To test the causal nature of this association, and to investigate the biological role of ras mutation, we introduced a mutant c-Ha-ras gene into normal rat thyroid follicular cells using an ecotropic retroviral vector. The major immediate effect was to greatly extend the proliferative lifespan of these cells in culture from less than 3 to more than 15 doublings, without any observable loss of growth-factor dependence or differentiated functions. This in vitro phenotype strongly supports an initiating role for ras mutation in the genesis of benign thyroid tumors (adenomas) in vivo. Spontaneous transformation was observed at low frequency on continuous culture of mutant ras-expressing cells, giving rise to fully immortalized, growth factor-independent, highly tumorigenic lines. Transformation was associated with (i) loss of responsiveness to the growth inhibitor TGF-beta 1, and (ii) greatly increased nuclear levels of p53 protein, which unexpectedly was not due to point mutation in the conserved regions of the p53-coding sequence. We postulate that these two phenomena are causally related to each other and to the transformed phenotype.


Alternative splicing of neurofibromatosis type 1 gene transcript in malignant brain tumors: PCR analysis of frozen-section mRNA.

The neurofibromatosis type 1 (NF1) gene encodes a 360-residue region showing significant homology to the catalytic domains of both mammalian GTPase-activating protein (GAP) and yeast IRA protein. The product of the GAP-related domain of the NF1 gene (NF1-GRD) has been shown to stimulate ras GTPase and consequently to inactivate ras protein. We previously reported that the NF1-GRD has two types of transcripts, type I and type II, which are generated by an alternative splicing mechanism, and that the differential splicing of the NF1-GRD may be related to differentiation of neuroectodermal cells. Here we examined the differential expression of type I and type II transcripts of NF1-GRD in clinical samples of supratentorial malignant brain tumors by the RNA-polymerase chain reaction (PCR) method using frozen tissue sections. Our observations revealed that normal cerebrum predominantly expressed the type II NF1-GRD transcript, whereas primitive neuroectodermal tumors predominantly expressed the type I transcript. Additionally, although the type I/type II ratio in astrocytomas varied widely among tissue samples, all glioblastomas showed higher type I/type II ratios than adjacent brain samples. The RNA-PCR analysis using frozen tissue sections is a useful and sensitive method for detecting genetic markers in clinical tissue samples.


Effect of simultaneous variation of weight, density, temperature and O2 concentration on rainbow trout (Oncorhynchus mykiss) body composition.

The simultaneous effect of weight, initial density (kg/m3, temperature and O2 concentration on rainbow trout body composition (fat, protein, moisture and ash) has been studied. In 3 successive experimental phases fish were kept in different lots of varying initial weight (178-372 g), initial density (7.2-38.8 kg/m3) and temperature (15-20 degrees C). Simple correlations were statistically significant for weight vs fat (r = 0.56; P less than 0.001) and moisture (r = -0.57; P less than 0.001); temperature vs fat (r = 0.73; P less than 0.001) moisture (r = -0.73; P less than 0.001) and ash (r = -0.26; P less than 0.02); and O2 concentration vs fat (r = 0.22; P less than 0.05). Multivariant equations for the different compounds were obtained. Only fat and moisture percentages showed significant differences (rm = 0.75; P less than 0.00005); an inverse relation existing between them (r = -0.94; P less than 0.001). Temperature is the factor which has the strongest influence on fat and moisture when it varies simultaneously with weight, initial density and O2 concentration, which is shown by its equation coefficients (P less than 0.00005).


Distribution of alpha CDCP-immunoreactive neurons in the central nervous system of the snail Helix aspersa.

alpha CDCP is a neuropeptide produced by the caudodorsal cells of Lymnaea stagnalis and encoded by the genes of the egg-laying hormone (ELH). The use of a polyclonal antiserum raised against alpha CDCP resulted in the detection of about 800 immunoreactive neurons in the parietal ganglia and a small population (60 cells) in the cerebral ganglia of Helix aspersa. As the genes of ELH are well conserved among the gastropod species, these data designate the parietal ganglia as a putative source for the egg-laying hormone in Helix aspersa.


Sleeping distance in relation to sexual state in the Arctic blue fox.

The paper provides comparative data concerning sleeping distance in relation to sexual state and hierarchical dominance in the Arctic blue fox. Increasing levels of vulval swelling and electrical resistance of vaginal tract were inversely related to sleeping distance between the highest-ranking male and each female on heat. A mating pair typically slept no further than 50 cm from each other. One month after copulation, the distance was already more than 200-250 cm. It can be concluded that oestrus-associated changes in sexual hormone levels have an influence on observed changes in sleeping distance.


Distribution of radioactivity of 14C-amino acids added to the medium in cells and metabolites in cultures of rumen fungi.

A mixture of L-(U-14C) amino acids was added to cultures of 11 strains of rumen anaerobic fungi belonging to Neocallimastix frontalis, Neocallimastix joyonii, Sphaeromonas communis and Piromonas communis. Fungi were grown in a complex medium with glucose for 4 days. The radioactivity was found in cellular protein (27.7-65.3% of the total radioactivity recovered), lactate (16.9-41.8%), volatile fatty acids (7.4-25.7%) and ethanol (4.6-10.5%). A small amount of radioactivity was recovered in lipids (0.2-1.8%) and CO2 (0.3-1.0%). The results suggest that the assimilation of amino acids by growing fungal cells was quantitatively comparable with their dissimilation to metabolites.


Growth hormone response to TRH in male broiler chickens selected for body weight gain or food conversion and reared at either a moderate or a high ambient temperature.

The aim of the present experiment was to study the growth hormone (GH) response upon thyrotropin releasing hormone (TRH) challenge (2 micrograms/kg body weight) in broiler chickens selected for body weight gain (GL line: fat line) or for feed efficiency (FC line: lean line) reared at either a moderate (33-23 degrees C) or high (33 degrees C) ambient temperature. A higher plasma GH level at 5 min after TRH administration was observed in the high temperature conditioned chickens of both lines. Also at high ambient temperature, an enhanced GH decrease between 15 min and 30 min post-injection and a higher acute elimination rate was calculated compared to moderate ambient temperature. A significantly higher GH secretory response was observed in the leaner FC line chickens, which was probably related to the more pronounced pulsatory GH secretion rate in these chickens. There was no difference in GH acute elimination rate between both lines in both environments. No interactions between line and rearing temperature for these parameters of GH dynamics were observed.


Effect of ovarian cystic or haemorrhagic follicles on embryo recovery and survival after transfer in hCG-ovulated rabbits.

The relationship between the presence of cystic and/or haemorrhagic follicles in both donor and recipient does and survival at birth of frozen-thawed embryos (778 embryos transferred) from 3 selected rabbit strains (NZ: New Zealand white; SY and SB: synthetic breeds) were studied. Donor does (SY:108; NZ:99; SB:96) were mated and treated with 25 IU of hCG. Only morphologically normal oviductal morulae (64-66 h) were frozen. Frozen-thawed embryos from each of the 90 donor does were transferred into the oviducts of synchronized recipient does of the same strain 48 h after injection of 25 IU of hCG (SY:31; NZ:28; SB:31). The frequency of follicular anomalies (36 and 43%) was high in both donor and recipient does, respectively, and it was not affected by strain or parity. The follicular anomalies had a negative effect on the percentage of embryos recovered in the oviduct (70 vs 77%) but not on the percentage of recovered embryos catalogued as morphologically normal (97%). The absence of follicular anomalies in recipient does had a significantly favourable effect on the pregnancy rate (63 vs 18%; P less than 0.05) and consequently on embryo survival rate at birth (26 vs 7%; P less than 0.01).


Bovine cumulus expansion and corona-oocyte disconnection during culture in vitro.

Cumulus-oocyte complexes were aspirated from small antral follicles (3-5 mm in diameter) and divided into 2 groups: complexes in which a dark rim of corona cells were visible around the zona pellucida (group 1); and those in which the corona displayed the same density as the rest of the cumulus cell mass (group 2). Cumulus complexes of both groups were evaluated by acetoorcein staining, scanning electron microscopy (SEM) and 3H-uridine uptake during culture in vitro. Germinal vesicle breakdown was initiated at 7 h of culture in group 1 while more than half of the oocytes in group 2 already displayed germinal vesicle breakdown at the time of aspiration. However, whereas more than 80% of the oocytes in group 1 had reached metaphase II at 24 h of culture, almost half of the oocytes in group 2 were arrested in metaphase I after this time interval. As revealed by SEM the complexes of group 2 showed signs of expansion such as elongation of cumulus cells and presence of extracellular matrix already at the time of aspiration. In group 1 these features were noticed at 7-9 h of culture. A high level of metabolic coupling between corona cells and oocyte was maintained up to 9 h of culture in group 1 followed by a decrease to a constant low level at 13 h. In group 2 a high degree of coupling was maintained up to 7 h followed by a gradual decrease to a constant low level at 13 h. It is concluded that cumulus-oocyte complexes with a dark rim of corona cells as judged by stereomicroscopy mature at a higher rate and maintain unexpanded characteristics and efficient corona-oocyte coupling longer than complexes with even density of the cumulus mass. Consequently, the presence of a dark rim of corona cells may be used as a criterion for selection of oocytes for in vitro embryo production.


Effect of dietary lysine on muscle protein turnover in growing chickens.

Day-old male chickens were fed ad libitum isoenergetic diets containing 20% crude protein but differing in their lysine content (from 6.5 up to 11.3 g/kg). At 3 weeks of age, protein fractional synthesis rates in the pectoralis major muscle were determined using a large dose injection of 120 mumol per kg body weight of L-[4-3H] phenylalanine. Protein gain in the pectoralis major was measured between 19 and 23 days of age. Protein breakdown was obtained by calculating the difference between protein synthesis and deposition. Weight gain varied curvilinearly with dietary lysine intake and was maximum for 11.3 g lysine/kg of diet. In birds fed an adequate lysine intake (10.1-11.3 g/kg) protein fractional synthesis and breakdown rates were 23.6-25.9 and 17.8-19.8%/d respectively. Increasing lysine supplementation in the diet resulted in an impairment of protein fractional breakdown rates. By contrast, protein fractional synthesis rates remained unchanged owing mainly to an improvement in the synthesis efficiency (kRNA), until birds were fed an adequate lysine intake. These data suggest that the growth rate reduction of chickens fed lysine deficient diets was due to alterations in both rates of protein synthesis and breakdown in skeletal muscle. A maximum protein deposition is achieved when kRNA was optimal, ie for a dietary lysine content of about 9 g/kg, a value close to the requirement.


Kinetics of particulate and solute marker passage in sheep supplemented with cationomycin and lasalocid antibiotics. Comparisons among methods for calculating mean retention time.

Passage of particulate and solute markers in the digestive tract was studied in sheep fed a roughage-pelleted diet supplemented or not with ionophore antibiotics. Methods of marker administration (continuous infusion vs pulse-dose) and mathematical treatment of data were investigated. Antibiotic supplementation did not affect markers' mean retention time (MRT) significantly, regardless of sampling site, technique of marker administration or MRT calculation methods used. The pulse dose technique led to 20-30% higher estimate of particle markers MRT in the entire gut and stomachs of sheep than did the continuous infusion method. Similar results were obtained between these methods for solute markers MRT at both sampling sites. Total MRT in the digestive tract could be partitioned among compartments using a deterministic model when applied to pulse-dose kinetics. Such a partitioning appeared unsatisfactory with continuous-infusion data. Estimations of particle or solute markers MRT in the rumen from duodenal and faecal marker kinetics were significantly different and rarely correlated.


[Early diagnosis of pregnancy in the cow as assessed by milk ejection induced by luteal oxytocin].

The diagnosis of pregnancy described in this article is based on the observation of milk ejection which, in the case of corpus luteum maintenance, results from the release of luteal oxytocin induced by intravenous administration of a non luteolytic dose of PGF2 alpha. The tests were performed in 410 lactating cows, 18-22 days after insemination (mean 19.95 days, SD 0.75), 3 h prior to the evening milking. A cannula was placed in the left fore-teat; when the cisternal milk flow ceased, a small dose of a prostaglandin F2 alpha analogue (256 micrograms Dinolytic) was injected. If a corpus luteum was present, the alveolar milk flow (1,276 +/- 570 ml) started 86 +/- 35 s later and pregnancy could be presumed. If the corpus luteum was no longer functional, the milk flow did not start again and the cow was considered not pregnant. The accuracy of positive results, ie 72.3%, significantly exceeded that obtained by radioimmunoassay of plasma progesterone used as a reference method, ie 68.6% (P less than 0.05). By contrast, the accuracy of negative results was lower, ie 93.5 vs 100%, (P less than 0.05): of the 195 pregnant cows which proceeded to term, 10 did not respond to the milk ejection test. In spite of these reserves, the test has the advantage of being inexpensive, rapid (about 5 min) and easy to interpret, ie either the milk flows or it does not. In addition, it may be used early, because from day 20 after Al the reliability of prediction of non pregnant cows (67.0%) was significantly higher when compared to the use of progesterone radioimmunoassay (58.6%) (P less than 0.05). With such a test, non pregnant cows can be reinseminated immediately and not only after a 3-week period. This may contribute to reducing the length of the infertile period.


Evidence of a contralateral motor influence on reciprocal inhibition in man.

The role of contralateral movement on both H reflex and reciprocal inhibition was studied. In normal men H reflex was induced by median nerve stimulation. Reciprocal inhibition was achieved through stimulation of the antagonist radial nerve. On this basis the effects of contralateral arm movement were analyzed. Furthermore the putative influence of exteroceptive origin was also verified by means of digit stimulation. Results showed that contralateral arm movement did not affect H reflex amplitude; on the contrary, it was able to enhance reciprocal inhibition induced by extensors on flexors. Study of cutaneous afferents demonstrated that contralateral digit stimulation failed to elicit modifications on both H reflex and reciprocal inhibition. On the other hand, ipsilateral digit stimulation lowered H reflex amplitude and increased the degree of reciprocal inhibition. Experimental findings underline the possibility that an informational array reaches the contralateral IA interneuron: therefore a mutual (bilateral) interaction among IA interneurones may accordingly be hypothesised.


Human brain phenolsulfotransferase. Regional distribution in Parkinson's disease.

Brain phenolsulfotransferase (PST) in 105.000 x g supernatant fractions prepared from post mortem human brain catalyzes the sulfate conjugation of dopamine (DA). Using 50 microM DA, the PST activity was linear up to one hour. The KM value for DA was 3.1 microM. Higher concentrations of DA from 25 b microM up caused inhibition of PST activity. Assessment of regional distribution in normal brain using 20 microM DA concentration revealed the highest PST activities in temporal and frontal cortex. About ten times lower activities were measured in parietal and occipital lobe, amygdala, hypothalamus, and hippocampus, whereas the nucleus accumbens, nucleus basalis of Meynert, caudate nucleus, and substantia nigra showed the lowest activities (about 1% of those in frontal and parietal cortex). In the brains of subjects with Parkinson's disease (PD) treated with levodopa, a significant reduction of PST activities was observed in hypothalamus, frontal and temporal cortex, amygdaloid nucleus, occipital and parietal cortex (between 20 and 38.8% of controls). Depletion of PST activity was less severe in hippocampus (46% of controls), nucleus accumbens, putamen, and substantia nigra (67 and 72% of controls, respectively). No changes were observed in the nucleus basalis of Meynert, while PST activity was increased in the caudate nucleus (174 to 203% of controls). The presented data indicate that on PD brain the PST activity is reduced in areas of the cerebral isocortex and limbic system, while in the basal ganglia it is either mildly reduced (putamen) or increased (caudate nucleus). Selective changes of PST activity in PD brain may indicate an important function of this enzyme in the metabolism and/or storage of DA under pathological conditions.


Efficacy of memantine, an NMDA receptor antagonist, in the treatment of Parkinson's disease.

Memantine is a 1-amino-adamantane derivative which has been proposed to be useful in the treatment of Parkinson's disease. Its beneficial effect has been related to its novel properties as an NMDA receptor blocker which can neutralize the effect of glutamate at striatal and subthalamic levels. In the present study, conducted in an open-fashion, 14 parkinsonian patients with motor fluctuations taking L-dopa, were given a supplement of memantine 30 mg/day. After one month, 10 patients completed the treatment (4 discontinued it due to abdominal pain, psychomotor agitation, confusion and dizziness). In 5 patients, the main parkinsonian features improved significantly (1 point or more on the Webster scale). In 6 patients, "off" episodes improved (from daily mean of 273 minutes, to 172 minutes). In summary, memantine addition to parkinsonian features, could form a basis for novel therapeutic strategies directed to neutralize the effects of glutamate at striatal and subthalamic levels.


Peripheral blood cell activities of monoamine oxidase B and superoxide dismutase in Parkinson's disease.

Monoamine oxidase type B (MAO-B) and superoxide dismutase (SOD) are two enzyme systems that are potentially relevant to an oxidative stress model of Parkinson's disease (PD) causation. Activities of MAO-B in platelets (nmol/10(8) cells/hr) and total SOD in lymphocytes (U/mg protein) were assayed among 28 cases of idiopathic PD and 22 controls. As anticipated, MAO-B was lowest in PD cases on selegiline (L-deprenyl) therapy (mean 1.10). There was a slight deficit of MAO-B among male cases not taking selegiline compared to controls (3.78 vs. 4.15), but the opposite trend was observed for females (6.18 vs. 4.16). SOD was slightly higher in cases (7.40), than controls (6.81). Excess SOD among PD cases was seen irrespective of gender, age, or selegiline treatment, although none of the differences was statistically significant. Future research on SOD should take advantage of the availability of assays specific for the cytosolic and mitochondrial forms of the enzyme.


Long-term observation of chronic subcutaneous administration of lisuride in the treatment of motor fluctuations in Parkinson's disease.

Twenty-nine patients with advanced Parkinson's disease were treated with subcutaneous lisuride infusion in addition to a basic therapy consisting of levodopa + PDI in all, and deprenyl in some patients. At the time of the report, 13 patients are still receiving lisuride infusion after 5-36 months, while 16 have dropped out after 0.5-30 months: one because of psychosis, three because of insufficient efficacy, three due to death unrelated to treatment, three because of difficulties in handling the pump as outpatients, and six for other reasons. "Off"-periods and Parkinsonian disability in "off" and in "on" were reduced significantly. These improvements remained constant throughout the observation period. Once the optimal dose regimen is established, only minor adjustments of the doses of lisuride and levodopa are required in the individual case.


Investigation of the mechanism of the effect of tacrine (tetrahydroaminoacridine) on the metabolism of acetylcholine and choline in brain cortical prisms.

The mechanism by which tacrine increases the content and synthesis of acetylcholine (ACh) in cerebrocortical prisms exposed to an irreversible inhibitor of cholinesterases and incubated under resting conditions (Dolezal and Tucek, 1991) is not known. As found in the present experiments, this effect of tacrine is only apparent if its application had been preceded by a period of preincubation, but the preincubation is ineffective if it occurs in the presence of hemicholinium-3. Apparently, choline or a choline-containing compound accumulates in the slices during the preincubation and is then utilized for the enhanced synthesis of ACh in the presence of tacrine. Tacrine did not induce a decrease in the amount of radiolabel that had been incorporated from choline into acid-insoluble compounds, which suggests that the choline which is used for the synthesis of additional ACh does not originate from choline lipids. However, tacrine was found to diminish the efflux of choline from prisms which had been preincubated with an increased concentration of choline in the medium, and from prisms incubated in the presence of hemicholinium-3. It also diminished the efflux of radioactive choline that had accumulated in the prisms during preincubation with a very low concentration of tacrine, when the prisms were subsequently incubated with 4-aminopyridine. It is proposed that the potency of tacrine to increase the content and synthesis of ACh in cerebrocortical prisms whose cholinesterases had been inhibited is due to its ability to diminish the efflux of endogenous choline from the nerve terminals.


Topographic mapping of long latency "cognitive" event-related potentials (P 300): a double-blind, placebo-controlled study with amantadine in mild dementia.

Amantadine is generally used in the prophylaxis of infection with influenza A, in the treatment of Parkinson's disease and in the treatment of neuroleptic side effects. In this study acute effects of amantadine infusions on event-related potentials (ERP) were studied in 20 mildly demented patients diagnosed according to DSM-III-R criteria. Each patient was treated, in randomized order, with 0.2 g amantadine-sulfate in 500 ml NaCl and 500 ml NaCl placebo, i.v. over one hour with an interval of two weeks in-between. ERPs were investigated in an auditory odd-ball paradigm before as well as 5 hours after the infusion. In addition to 17 EEG records, vertical and horizontal EOGs were recorded. After EOG-minimization and visual artifact rejection the peak latencies of the spatial average were determined by an automatic procedure. There was no effect of amantadine on ERP latencies. N1 of the non-target showed a trend towards amplitude augmentation, P2 amplitude was reduced. As compared to placebo, P300 amplitude of targets was significantly augmented by 3.1 microV (30% of pre-treatment value), confirming the hypothesis that amantadine may influence the P 300 amplitude in the sense of an improved availability of cognitive processing resources.


Brain and cerebrospinal fluid cholinesterases in Alzheimer's disease, Parkinson's disease and aging. A critical review of clinical and experimental studies.

Acetylcholinesterase (AChE), an enzyme responsible for the break-down of acetylcholine, is found both in cholinergic and non-cholinergic neurons in the central nervous system. In addition to its role in the catabolism of acetylcholine, AChE have other functions in brain, e.g. in the processing of peptides and proteins, and in the modulation of dopaminergic neurons in the brain stem. Several clinical and experimental studies have investigated AChE in brain and cerebrospinal fluid (CSF) in aging and dementia. The results suggest that brain AChE and its molecular forms show interesting changes in dementia and aging. However, CSF-AChE activity is not a very reliable or sensitive marker of the integrity and function of cholinergic neurons in the basal forebrain complex. Additional work is needed to clarify the role of AChE abnormality in the formation of pathology changes in patients with Alzheimer's disease.


A critical review of the toxicology of glutaraldehyde.

Glutaraldehyde, a low molecular weight aldehyde, has been investigated for toxicity in humans and animals. Examination of this dialdehyde was indicated from previous studies with other aldehydes in which carcinogenicity of formaldehyde and toxicity of acetaldehyde and malonaldehyde have been disclosed. Information gaps concerning the actions of glutaraldehyde have been identified in this review and recommendations are suggested for additional short- and long-term studies. In particular, information regarding irritation of the respiratory tract, potential neurotoxicity, and developmental effects would assist in a complete hazard evaluation of glutaraldehyde. Further study related to disposition, metabolism, and reactions of glutaraldehyde may elucidate the mechanism of action.


Toxicological principles of metal carcinogenesis with special emphasis on cadmium.

Metals are an important and emerging class of carcinogens. At least three metals, specifically nickel, chromium, and arsenic, are confirmed human carcinogens, and several more are suspected to have carcinogenic potential in man. Considering that the list of known human carcinogens of any type is very small, it becomes clear that metals make up a substantial portion of the list. Furthermore, many metals are very potent carcinogens in laboratory animals. Despite this, relatively little attention has been given to the topic of metal carcinogenesis. The reasons for this relative lack of attention are not clear but perhaps are fostered by a perception that, because metals are the simplest of molecules, their mechanism of action must also be simple. This could not be farther from the truth and, although no clear mechanisms have emerged in the area of metal carcinogenesis, it has become apparent that they are anything but simple. Metal carcinogens possess several unique characteristics including a remarkable target site specificity. Detection of the mechanism, or mechanisms, of metal carcinogenesis has, however, proven elusive, in part because of a wide diversity of metallic carcinogenic agents and the intricate nature of metal interactions in biologic systems. The following review explores this broad topic, with special emphasis on toxicological principles including dose-response relationships and potential mechanisms, using cadmium as an example.


Cardiotoxicity of vasodilators and positive inotropic/vasodilating drugs in dogs: an overview.

Standard toxicological studies in dogs using high doses of vasodilators and positive inotropic/vasodilating agents give rise to a species-specific cardiotoxicity. The reason may be the extreme sensitivity of the dog to the pharmacological effects of these drugs; exaggerated pharmacodynamic effects and prolonged disturbance of homeostasis mechanisms often are responsible for the observed organ lesions. An assessment of the toxicological relevance and the risk for patients taking the drugs at therapeutic doses cannot be made without taking into account their pathomechanisms and the pathophysiological basis of the exceptional reaction patterns occurring in dogs. A large series of vasodilating and positive inotropic agents are presented, their pharmacological properties are described, and toxicological effects in dogs are compared. In view of the poor correlation between the distinct cardiac lesions induced in dogs and a lack of comparable toxicity in humans, it appears desirable to reassess the adequacy of the standard toxicological approaches for these substances.


Cardiac troponin T in the diagnosis of myocardial injury.

In the last several decades serum levels of cardiac enzymes and isoenzymes have become the final arbiters by which myocardial damage is diagnosed or excluded. Because conventionally used enzymes are neither perfectly sensitive nor specific, there is need for a new sensitive and cardiospecific marker of myocardial damage. Cardiac troponin T (TnT) is a contractile protein unique to cardiac muscle and can be differentiated by immunologic methods from its skeletal-muscle isoform. An enzyme immunoassay specific for cardiac TnT is now available in a commercial kit for routine use. The biggest advantage of this assay is its cardiospecificity. TnT measurements, however, are also highly sensitive in diagnosis of myocardial injury and accurately discern even small amounts of myocardial necrosis. TnT measurements are, therefore, particularly useful in patients with borderline CK-MB and in clinical settings in which traditional enzymes fail to diagnose myocardial damage efficiently because of lack of specificity--for example, perioperative myocardial infarction or blunt heart trauma. TnT release kinetics reveal characteristics of both soluble, cytoplasmic, and structurally bound molecules. It starts to increase a few hours after the onset of myocardial damage and remains increased for several days. TnT allows late diagnosis of myocardial infarction. The diagnostic efficiency remains at 98% until 6 d after the onset of infarct-related symptoms. TnT is also useful in monitoring the effectiveness of thrombolytic therapy in myocardial infarction patients. The ratio of peak TnT concentration on day 1 to TnT concentration at day 4 discriminates between patients with successful (greater than 1) and failed (less than or equal to 1) reperfusion. TnT measurements are very sensitive and specific for the early and late diagnosis of myocardial damage and could, therefore, provide a new criterion in laboratory diagnosis of the occurrence of myocardial damage.