Changes in type I keratin expression associated with HPV16 transformation of human epidermal keratinocytes.

We have used immunoblotting to compare expression of type I keratins in two strains of normal human epidermal keratinocytes (v and u) and their HPV16-transformed derivatives (vp and up). The levels of keratins 14 and 17 were similar in all four cell strains, whereas keratins 18 and 19 were more abundant in vp and up than in the normal parental keratinocytes. Keratin 13 was more abundant in the transformed cells than in the parentals; in addition, expression in v was higher than in u, and expression in vp was higher than in up, suggesting strain-specific variation in expression. Keratin 16 was the only keratin to be more highly expressed in v and u than in vp and up; this is consistent with the reduced capacity of the transformants for stratification and terminal differentiation. Double-label immunofluorescence of vp and up showed that more cells expressed involucrin than keratin 16. We conclude that HPV16 transformation results in marked changes in keratin expression. The increased expression of keratin 18, a keratin that is normally expressed in simple epithelia, fits well with reports of increased keratin 18 expression in invasive squamous cell carcinomas of skin and other keratinocyte-derived tumours.


Patterns of phenotypic expression of human junctional, gingival and reduced enamel epithelia in vivo and in vitro.

Epithelia differ regionally in their patterns of phenotypic expression. The junctional epithelium (JE) that attaches the oral mucosa to the teeth is a unique tissue that shows a pattern of differentiation unlike other oral epithelia and forms basal lamina against the non-vital tooth surface. The mechanisms that establish this unusual phenotype and the developmental origin of this epithelium are both uncertain. The formation of JE by downgrowth of the oral gingival epithelium (OGE) during tooth eruption has been suggested but morphological studies indicate that it may be derived from the reduced enamel epithelium (REE) that covers the crown of the unerupted tooth. These epithelia of potential origin differ in their developmental histories: intrinsic differences between them could thus significantly influence the phenotype of an epithelium formed from them. The patterns of phenotypic expression of specimens of dissected JE, OGE and REE, and of cell cultures of these epithelia grown under standardized conditions, were examined (1) by immunocytochemistry using monoclonal antibodies with specificity for individual cytokeratins, vimentin and ICAM-1, and (2) by two-dimensional SDS-PAGE and immunoblotting. The results indicated that, in vivo, OGE expressed keratin markers typical of differentiating mucosal epithelium; JE and REE, in contrast, lacked expression of most such markers but expressed keratins typical of simple epithelia together with some undefined keratin peptides. All epithelia showed changes in vitro but OGE remained different from JE and REE. OGE lost expression of the differentiation markers K1, K10 and K13; it acquired some expression of K19, but less than JE and REE. Cultures of JE and REE retained some expression of ICAM-1 and K8 and K18, and consistently acquired high levels of vimentin expression. These findings indicate that differences persist in standardized culture conditions and that these are apparently of an intrinsic nature. They support a concept of the origins of JE from REE and suggest that the unusual in vivo phenotype of JE results partly from intrinsic differences acquired during its development.


Irreversible inhibition of transglutaminases by sulfonium methylketones: optimization of specificity and potency with omega-aminoacyl spacers.

Sulfonium methylketones, of structure Cbz-Phe-NH(CH2)nCOCH2S+ (CH3)2, n > 2, are specific and potent inactivators of transglutaminases. The length of the -(CH2)n-spacer moiety, n = 1-5, is a critical determinant for both the specificity and potency of the inactivator. The dipeptidyl analog Cbz-Phe-Gly-(CH2)nS+ (CH3)2, n = 1, is a more powerful inactivator of the thiol proteinase cathepsin B, k/K > 3 x 10(5) M-1 min-1, than of transglutaminases, ki(app)/Ki(app) < 1.5 x 10(4) M-1 min-1. In contrast, the gamma-aminobutyryl analog, n = 3, is a very potent transglutaminase inactivator with ki(app)/Ki(app) = 3.1 x 10(6) M-1 min-1, but does not inactivate cathepsin B. In cell studies, the gamma-aminobutyryl and epsilon-aminohexyl analogs inhibited the transglutaminase-mediated process of ionophore-induced cross-linked envelope formation by human malignant keratinocytes and the order of potency was related to that found for enzyme inhibition. The sulfonium methylketones, in equilibrium with the resonance stabilized ylides, are chemically inert towards glutathione under ambient conditions demonstrating the potential utility of this novel class of transglutaminase inhibitors for the study of enzyme inhibition in cellular environments.


Inactivation of gamma-aminobutyric acid aminotransferase by various amine buffers.

It is hypothesized that buffers capable of forming a Schiff base with the PLP of gamma-aminobutyric acid aminotransferase (GABA-AT) may lead to denaturation and inactivation of the enzyme. On the basis of this hypothesis three new methods for the selective destruction of GABA-AT in GABAse (a commercial bacterial source of a mixture of GABA-AT and succinic semialdehyde dehydrogenase [SSDH]) and from pig brain are described: (1) dialysis against a primary or secondary amine buffer; (2) gel filtration with a primary or secondary amine buffer as eluent; (3) inactivation with gabaculine followed by dialysis or gel filtration with pyrophosphate buffer. The SSDH activity in GABAse, which remains unchanged by all of these methods, may then be used in a coupled assay to measure the activity of GABA-AT from different sources. These results also suggest that the use of primary and secondary amine buffers should be avoided when inhibitors are being tested with GABA-AT.


Studies on aldose reductase inhibitors from fungi. I. Citrinin and related benzopyran derivatives.

The fungal metabolites, citrinin (4,6-dihydro-8-hydroxy-3,4,5-trimethyl-6- oxo-3H-2-benzopyran-7-carboxylic acid) and DHMI (3,4-dihydro-6-methoxy-3,7-dimethyl-1H-2-benzopyran-8-ol), as well as certain synthetic derivatives, have been evaluated for aldose reductase inhibitory activity using a rat lens enzyme preparation. Citrinin and its reduction product, dihydrocitrinin, were found to have significant activity (IC50 approximately 10 microM), whereas the other compounds were 3-10 times less potent. Kinetic studies showed that citrinin was not an irreversible inhibitor of the enzyme, as might be expected of a quinone methide. Spectroscopic (NMR) evidence is presented for the existence of citrinin predominantly in the form of its hemi-acetal in aqueous solutions, suggesting that it is this benzo[c]pyran derivative which interacts with the enzyme, rather than the quinone methide form.


Hydroperoxidic inhibitor of horse liver alcohol dehydrogenase activity, tightly bound to the enzyme-NAD+ complex, characteristically degrades the coenzyme.

The strong inhibition of horse liver alcohol dehydrogenase (HLAD) by p-methylbenzyl hydroperoxide (XyHP) is only transient, XyHP behaves also as a pseudo-substrate of the enzyme and in the presence of NAD+, is degraded by HLAD to (as yet unidentified) non-inhibiting products while the NAD+ is converted to a derivative similar to the "NADX", originally observed in an analogous reaction of HLAD with hydrogen peroxide. The apparent KM for XyHP is approximately 10(4) times smaller than that for H2O2. The catalytic constant kcat for HLAD degradation of XyHP is two orders of magnitude less than that for ethanol dehydrogenation. XyHP inhibits both directions of the alcohol-aldehyde interconversion with equal potency. The first step of the inhibition mechanism is a tight binding of XyHP to the binary HLAD-NAD+ complex.


4-(Aminomethyl)-1-aryl-2-pyrrolidinones, a new class of monoamine oxidase B inactivators.

Both 4-(aminomethyl)-1-phenyl-2-pyrrolidinone (4a) and 4-(aminomethyl)-1- (methoxyphenyl)-2-pyrrolidinone (4b) hydrochlorides were synthesized via a six-step sequence, which represents a general approach to 1,4-disubstituted 2-pyrrolidinones. Both of these compounds inactivated monoamine oxidase B and represent the first in a new class of monomamine oxidase inactivators.


Ferrocenopapain, an organometallic protein formed by site-specific inactivation of papain using chloroacetylferrocene.

Papain is rapidly, specifically and irreversibly inactivated by chloroacetylferrocene in a time-dependent, two-stage process which involves initial, physical complexation between the enzyme and the organometallic reagent followed by chemical reaction. The kinetics of the process show saturation kinetics with respect to inactivator concentration. The modification occurs with a 1:1 stoichiometry and the degree of loss of enzymatic activity is directly reflected in the loss of thiol groups in the active-site that are accessible to Ellman's reagent. The modification is faster at higher pH. The ferrocenium ion of the modified papain can be generated at low pH by anaerobic treatment with ferric nitrate and shows a 180 percent increased lifetime at pH 3.3 compared to the acetylferrocenium ion under identical conditions. The spectral properties of the ferrocenopapain indicate that the ferrocene is in an unusual environment at the protein active-site and the implications of this are discussed.


Time-dependent inhibition of pea cotyledon diamine oxidase by some hydrazides.

The inhibition of diamine oxidase (EC 1.4.3.6) from pea cotyledons (PDAO) by some hydrazides has been studied. It was found that PDAO is inhibited in a time-dependent manner at pH = 7.0 by the hydrazides of acetic, benzoic, nicotinic, isonicotinic, picolinic and 3,4-dihydro-4-oxophtalazine-1-carboxylic acids, by 1-(carboxymethyl)trimethylammonium chloride hydrazide (Girard's reagent T), 1-(carboxymethyl)pyridinium chloride hydrazide (Girard's reagent P) and oxalic acid dihydrazide. The inhibition was partially reversible. Rate constants for enzyme inactivation were in the range 0.29-1.95 min-1. The hydrazides give apparent noncompetitive inhibition at pH = 7.0, but for isonicotinic hydrazide, this changes to competitive inhibition at pH = 8.0. Apparent inhibition constants (K1APP) for the hydrazides with PDAO are in the range 0.005-1.5 mmol l-1.


Observations on the conformational changes in the structure of a cell surface protease, followed by its ability to be recognised by competitive and non-competitive inhibitors.

Lung tumour cells possess a cell surface protease which can be inhibited by a cytoplasmic protein inhibitor extracted from these cells. The dissociation of this enzyme-inhibitor complex on the surface of tumour cells in sections treated with 10(-4) M sodium dodecyl sulphate has been studied. The dissociation of the inhibitor and regain of enzymic activity was followed by the use of a fluorescent probe which binds to the active centre of the cell surface enzyme in a competitive manner.


Additional peptidyl diazomethyl ketones, including biotinyl derivatives, which affinity-label calpain and related cysteinyl proteinases.

Calpain, the calcium-activated cysteinyl proteinase, can be irreversibly inactivated by peptidyl diazomethyl ketones in which the peptide portion contains a penultimate leucine residue. Some new derivatives of this type have been synthesized and examined for their rates of inactivation of chicken gizzard and human platelet calpain. Two derivatives containing a C-terminal biotin residue, Biot-Aca-Leu-TyrCHN2 and Biot-Aca-Leu-Leu-TyrCHN2, have also been prepared in the expectation that their application to the study of the function of calpain and related proteases will prove fruitful.


Exploring the hexokinase glucose binding site through correlation analysis and molecular modeling of glucosamine inhibitors.

A series of N-substituted glucosamines has been designed, synthesized, and tested as inhibitors of yeast hexokinase. All derivatives exhibited competitive inhibition kinetics with respect to glucose. Quantitative structure-activity relationships were derived from the resulting inhibition data. The most significant equation demonstrated the existence of highly specific steric effects for the seven meta-substituted benzoylglucosamines included in the relationship. Molecular modeling of potential complexes between the inhibitors and the hexokinase substrate binding site strongly suggests that the steric effects arise from potential contacts with two amino acid residues lying in the region occupied by the amide substituents.


Cholinesterase studies with (R) (+)- and (S)(-)-5-(1,3,3-trimethylindolinyl)-N-(1-phenylethyl)carbamate.

A limited number of carbamates have been found useful for treatment of cholinergic symptoms with pyridostigmine and physostigmine being the main focus. In recent years 5-(1,3,3-trimethylindolinyl)N,N-dimethylcarbamate (I) has received considerable attention in the Chinese literature for a similar role. We report on the first synthesis of stereoisomers of an analog of (I). The isomers prepared were (R)(+)-5-(1,3,3-trimethylindolinyl)-N-(1-phenylethyl)carbamate (II) and (S)(-)-5-(1,3,3-trimethylindolinyl)-N-(1-phenylethyl)carbamate (III). The pKa value for each isomer was 6.8. Eel acetylcholinesterase inhibition studies were carried out at 25.0 degrees C over the pH range of 6.0 to 9.0. They reflect the first pH profiles using enantiomorphs of a cholinesterase inhibitor. The inhibition potencies for (II) and (III) over the range examined were similar. At pH 7.60 the ki for II = 7.38 x 10(3) M-1 min-1 (SD = 398) and for (III) the ki = 6.67 x 10(3) M-1 min-1 (SD = 355). In accord with the findings of Wilson and Bergmann20 on physostigmine our results indicate that the protonated form of (II) and (III) is the more potent inhibitor.


Biochemical characterisation of a pancreatic elastase inhibitor from the leech Hirudinaria manillensis.

The jawed leech, Hirudinaria manillensis is closely related to Hirudo medicinalis, both belonging to the same family Arhynchobdellida. From Hirudo, two potent peptide inhibitors, hirudin (a thrombin inhibitor) and eglin (an elastase/chymotrypsin inhibitor) have been characterised in detail. During our studies to isolate thrombin inhibitor from the leech Hirudinaria a potent inhibitor, analogous to eglin, was also detected. Results indicate that this inhibitor, which we have named 'GELIN', is significantly different from eglin. Gelin was isolated and purified to homogeneity by ion exchange chromatography and reverse phase HPLC. The isoelectric point of Gelin was estimated to be 4.55, in contrast to 6.45 for eglin. The molecular weight of Gelin was similar to eglin, as estimated by SDS-PAGE. Amino-terminal sequence analysis of the first 29 residues show no sequence homology with eglin or any other serine protease inhibitors. Circular dichroism studies showed that the secondary structure of Gelin has no helix, 58% beta sheets and 42% random structures compared to 19% helix, 56% beta sheets and 25% random structures in eglin. Like eglin, Gelin inhibits elastase, cathepsin G and chymotrypsin but has little or no activity towards plasmin, thrombin, pepsin and trypsin. These data suggest that the elastase inhibitors from these two species of leech are fundamentally different in structure, indicative of independent evolutionary origin.


Observations on the inhibition of serum and cell surface enzymes by eicosapentaenoic acid.

The relationship between serum and tumour cell surface proteolytic enzymes and the development of muscle breakdown in cancer cachexia has been studied in a murine model of the condition (MAC16). The surface of the MAC16 tumour cells carried a proteolytic enzyme referred to as guanidinobenzoatase (GB). Serum from mice also contained an enzyme (referred to as MSE) which cleaved the trypsin inhibitor 4-methylumbelliferyl-p-guanidinobenzoate as a true substrate, but there was no relationship with weight loss or the presence or absence of tumour and the level of this serum enzyme. Polyunsaturated fatty acids (PUFAs) were shown to be inhibitors of MSE at microM concentrations and one PUFA, eicosapentaenoic acid (EPA) was found to be a non-competitive inhibitor of both MSE and GB. The effect of EPA was specific since other proteolytic enzymes, trypsin, esterase and tissue plasminogen activator were unaffected by concentrations inhibiting GB and MSE. MSE and GB are two different enzymes which possess some common properties. However, GB is likely to be significant for tumour development since MSE is also found in normal mouse serum.


Some 1-, and 3-substituted 3-(4'-aminophenyl)pyrrolidine-2,5-diones as selective inhibitors of aromatase.

1-Alkyl-3-(4'aminophenyl)pyrrolidine-2,5-diones (7,R = C3H7-C7H15) are potent inhibitors of aromatase in vitro, the 1-hexyl (Ki = 62nM) being about 100-fold more potent than aminoglutethimide (AG), and more selective in their ratio of aromatase:CSCC inhibitory potency. The 1-pentyl, 1-hexyl and 1-heptyl derivatives are more stable to liver microsomal metabolism in vitro than AG possibly due to inhibition of the liver cytochrome P450s. 1,3-Dialkyl-3(4'-aminophenyl)pyrrolidine-2,5-diones (9) have been synthesised by a novel method. Although the higher homologues (di-pentyl and di-hexyl) are more potent in vitro as inhibitors of aromatase than AG, they are less active than their 1-alkyl counterparts with the same alkyl substituent.


Blood serum angiotensin-converting enzyme, alfa2-macroglobulin and triglycerides in patients with diabetes.

Blood serum activity of ACE, alfa2-macroglobulin as well as triglycerides concentration was determined in 90 patients with diabetes and in 40 healthy persons. Taking into account criteria established by WHO the patients with diabetes were divided into two groups. The first group consisted of 32 patients with diabetes of type I (juvenile one); the second group consisted of 58 patients with diabetes of type II (adult one). It was found that blood serum level of all tested parameters is statistically increased (p < or = 0.001) in comparison with control group. In patients with diabetes of type I the level of alfa2-macroglobulin and ACE was higher than in those with diabetes of type II. The serum contents of triglycerides in both groups of patients was comparable. No correlation between parameters determined in patients with diabetes was found.


Evidence for commissurally projecting parvalbumin-immunoreactive basket cells in the dentate gyrus of the rat.

The fluorescent retrograde tracer, fluorogold, was used to identify commissurally projecting neurons in the hippocampus and dentate gyrus. After injection of fluorogold into the hippocampus, the contralateral hippocampus was evaluated for fluorogold-immunoreactive or fluorescent neurons. In addition to observing labeled hilar neurons and CA3 pyramidal cells that previously have been reported to send commissurally projecting axons to the contralateral hippocampus, the authors unexpectedly found a population of fluorogold-labeled cells in the granule cell layer with the morphology and location of GABA-immunoreactive basket cells. Immunocytochemical staining revealed that all fluorogold-labeled cells of the granule cell layer were immunoreactive for parvalbumin. However, not all parvalbumin cells, shown previously to be a subset of GABA neurons, were fluorogold-labeled. The association between fluorogold transport and parvalbumin immunoreactivity was unique for these cells of the granule cell layer. In the adjacent hilus, relatively few of the many fluorogold-labeled cells were parvalbumin- or GABA-immunoreactive. These results (1) identify a population of presumed inhibitory neurons that apparently form commissural projections; (2) document that all of these cells contain the calcium-binding protein parvalbumin; and (3) indicate that the vast majority of commissurally projecting hilar neurons are neither parvalbumin- nor GABA-immunoreactive.


Tracing of axonal connections by rhodamine-dextran-amine in the rat hippocampal-entorhinal cortex slice preparation.

In order to demonstrate axonal connections preserved in rat temporal cortex slices the authors used rhodamine-dextran-amine as a tracer. The slices contained the neocortical areas Te2 and Te3, the medial and lateral entorhinal cortices (MEC and LEC), the subicular regions, and the dentate gyrus and hippocampus proper. Rhodamine-dextran-amine crystals were placed by microinjection into a given area. Following this local lesioning the dye was permitted to diffuse and migrate intraaxonally in antero- and retrograde directions for about 8 hours. The slices were then formaldehyde-fixed and analyzed by fluorescence microscopy. Most of the known connections within and between the entorhinal cortex and the hippocampus and dentate gyrus were preserved in the slice preparation, provided that the slices were cut with a near horizontal orientation corresponding to plates 99-108 in Paxinos and Watson (1986). Only the lateral perforant path between the LEC and the hippocampus could not be followed to its full extent. The authors conclude that most aspects of the intrinsic synaptic organization of the temporal lobe can be reliably studied in hippocampal-entorhinal cortex slice preparations.


Lesion-induced synapse reorganization in the hippocampus of cats: sprouting of entorhinal, commissural/associational, and mossy fiber projections after unilateral entorhinal cortex lesions, with comments on the normal organization of these pathways.

This study evaluates whether three forms of sprouting occur in the hippocampus of the cat following unilateral entorhinal cortex (EC) lesions: (1) sprouting of projections from the EC contralateral to the lesion; (2) sprouting of the commissural/associational system; and (3) sprouting of mossy fibers. Tract tracing techniques were used to define the normal organization of the entorhinal cortical projection system, the commissural/associational (C/A) systems, and the mossy fiber projections in normal cats. The same techniques were then used to evaluate whether there were changes in these projections in animals with long-standing unilateral EC lesions. The projections from the entorhinal cortex were evaluated autoradiographically following injections of 3H proline into the entorhinal area. The projections of the C/A system were traced using the Fink-Heimer technique after lesions of the hippocampal commissures, and by using autoradiographic techniques after injections of 3H proline into the hippocampus. The distribution of mossy fibers was evaluated using the Timm's stain. The results reveal that unilateral lesions of the EC in cats lead to the same sorts of sprouting that have been described in rats. There is: (1) an increase in the density of the crossed projection from the surviving EC to the contralateral dentate gyrus that had been deprived of its normal EC inputs; (2) an expansion of the terminal field of the C/A projection system into portions of the molecular layer of the dentate gyrus normally occupied by EC projections; and (3) an increase in supragranular mossy fibers in some animals. The mossy fiber sprouting was especially prominent when the lesions encroached upon the hippocampus. The studies also reveal additional details about the normal organization of hippocampal pathways in cats. The most important points are: (1) there is a crossed projection from the entorhinal cortex to the contralateral dentate gyrus; and (2) there is a complex laminar organization of the commissural and associational terminal fields in the molecular layer of the dentate gyrus that appears to be related to the point of origin of the projections along the septotemporal axis of the hippocampus. This heretofore unrecognized aspect of the laminar organization of C/A terminations has important implications for the temporal competition hypothesis, which has been advanced to account for the development of these afferent systems.


Mossy cell axonal projections to the dentate gyrus molecular layer in the rat hippocampal slice.

The ipsilateral associational pathway connects different septotemporal levels of the dentate gyrus. Neurons of the dentate hilus project hundreds of micrometers from the cells of origin to the inner molecular layer. The authors hypothesized that mossy cells, the major cell type of the hilus, also project locally to the inner molecular layer. Within a 400 microns slice, mossy cells were (1) recorded intracellularly while the inner molecular layer was stimulated to test for antidromic responses, and (2) labeled with biocytin and examined with light and electron microscopy for axonal projections into the molecular layer. The authors found that mossy cells can be antidromically activated by inner molecular layer stimulation and that axonal projections to the molecular layer can be visualized within a 400 microns hippocampal slice. In 13 of 19 intracellularly labeled and electrophysiologically characterized mossy cells, collaterals could be traced into the molecular layer. These results suggest that mossy cells contribute to the ipsilateral associational pathway and also participate in local recurrent circuitry to influence granule cell activity.


Region-specific age effects on AMPA sensitivity: electrophysiological evidence for loss of synaptic contacts in hippocampal field CA1.

The effects of aging on the responsiveness of hippocampal neurons to iontophoretic application of L-glutamate and AMPA were studied in vitro. There were no effects of age on neuronal responses to L-glutamate; however, CA1 pyramidal cells of old rats, but not granule cells in the fascia dentata, showed both a smaller reduction in extracellularly-recorded synaptic responses following application of AMPA (presumably mediated by depolarization), and smaller extracellular "DC" fields (measured by subtracting the DC potentials at the dendrite and soma following AMPA application in the dendrites). To examine the cellular bases of this age-related alteration in AMPA sensitivity, two additional electrophysiological approaches were used: (1) measurement of the amplitude ratios of extracellular EPSP and fiber potential components of the Schaffer collateral-CA1 response; (2) measurement of intracellularly recorded unitary EPSPs and quantal analysis of their fluctuations. The interpretations that would be placed on four hypothetical possible outcomes of such experiments are outlined and assessed in relation to the experimental data. The pattern of results obtained in the present experiments supports the following conclusions: In old rats, individual Schaffer collateral synapses do not appear to have altered AMPA receptor properties, as neither the mean size of the unitary synaptic response nor the apparent quantal size differs between age groups; however, the data do support the conclusion that there are fewer synapses per Schaffer collateral branch in old versus young CA1 pyramidal cells.


Bv: a distinct category of B sub-group among Chinese blood donors in Hong Kong.

During the period 1984-1989, a total of 46 examples of Bv phenotype were encountered out of a total of 567,210 donors, giving an incidence of 1 in 12,330 among the Chinese in Hong Kong. The Bv determinant corresponds to the portion of the B antigen that is present on rabbit red cells, and gives a negative reaction with polyclonal anti-B reagents absorbed with rabbit red cells that still react with B3. Some potent monoclonal anti-B reagents confirm the absence of a B epitope from Bv red cells even by adsorption and elution techniques. The failure of some monoclonal anti-B reagents to detect Bv demonstrates the need to select or blend monoclonal anti-B reagents for use in typing Oriental bloods. Cell-conversion techniques failed to convert O cells to B cells using Bv serum with the appropriate substrate, whereas sera from most of the other B variants were capable of doing so. The Bv phenotype, therefore, represents a distinct category of B subgroups that is easily distinguishable from B3 and other B variants.


Serological and clinical aspects of granulocyte antibodies leading to alloimmune neonatal neutropenia.

Eighteen cases of alloimmune neonatal neutropenia (ANN) were analysed for their clinical and serological properties. Pregnancy was normal in all cases, but a 50% incidence of abortion is recorded. With the exception of two premature babies, all newborns were delivered at term. Omphalitis and mild infections of the skin were predominantly present. None of the new-borns died by overwhelming sepsis. The average duration of neutropenia was 11 weeks (range 3-28 weeks). Intravenous IgG therapy was followed by transient remission in 2 of 4 affected newborns. Antibody differentiation revealed in five sera NA1-, in four sera NA2- and in two sera NB1-specific antibodies. In two sera only HLA antibodies were detectable. Complement activating antibodies were determined in 72% of the sera. Screening for granulocyte-specific antibodies in 1016 postpartum sera of unselected women revealed a total of 11 sera (1.1%) reacting selectively with granulocytes, but only four (0.4%) were directed against a known granulocyte-specific antigen. None of the new-born of mothers alloimmunized to granulocyte antigens developed neutropenia, which suggests an incidence of ANN below 0.1%.


Differences in the characteristics of human monoclonal and polyclonal anti-D as revealed by immunochemical investigations: human monoclonal antibodies share specificities with natural antibodies.

To determine the basis of the tissue cross-reactions shown by some human monoclonal anti-Rh D antibodies, we have investigated the tissue reactivities of 48 further human monoclonal antibodies (mAb) against D and other Rh antigens, and compared them with those of normal and anti-D sera and immunoglobulin preparations, and affinity-purified polyclonal anti-D antibodies. Although we were unable to detect any tissue reactivities associated with the D-binding fraction of polyclonal antisera or prophylactic immunoglobulin, the non-erythroid cell types identified by the tissue-reactive human anti-Rh mAb of both IgM and IgG class were those recognized by antibodies present in both normal and anti-D sera. These results indicate: (a) that the tissue specificities of human anti-Rh mAb are similar to those of natural antibodies, and (b) that there are immunochemical differences between polyclonal and monoclonal anti-D antibodies, at least of IgG class, which may be relevant to the use of the latter in the prevention of haemolytic disease of the new-born by immune prophylaxis.


The effect of glycosylation trimming enzyme inhibitors on monoclonal antibody recognition of alpha-sialoglycoprotein epitopes.

The human erythrocyte membrane contains four sialoglycoproteins, denoted alpha, beta, gamma and delta (also known as glycophorins A, C, D and B respectively), of which alpha-sialoglycoprotein (alpha-SGP) is the most predominant species. The extracellular portion of alpha-SGP is heavily glycosylated with approximately 15 O-linked carbohydrate side-chains and a single N-linked group. We have used inhibitors of carbohydrate trimming enzymes to investigate the contribution of this single N-glycan moiety towards the recognition of a range of antibody binding sites on alpha-SGP. Two erythromyeloid cell lines, K562 and HEL, were cultured in the presence of these inhibitors and altered binding of antibodies to epitopes adjacent to the N-glycan was observed. Digoxigenin-coupled lectins were used to stain cytocentrifuge preparations and Western blots of cell lysates in order to confirm that modification of N-linked carbohydrate side-chains had been achieved. We suggest that the N-glycan side chain of alpha-SGP has a role in conferring conformational stability upon epitopes which lie in its vicinity.


Serum concentration of alpha-2-macroglobulin, alpha-1-antitrypsin and alpha-1-antichymotrypsin [correction of alpha-2-antichymotrypsin] in patients with Hodgkin's disease.

In twenty four patients diagnosed with Hodgkin's disease we have determined several antiproteases. Our findings have shown the decrease of concentration of alpha-2-macroglobulin and increase of alpha-1-proteinase inhibitor and alpha-1-antichymotrypsin. The observed changes were statistically significant p < 0.001. We did not find any correlation between the concentration of inhibitors and the clinical stage of carcinoma and histopathological data. The determination of the level of antiproteases in patients with cancer disease may be assumed to reflect partly at least the capability of anticancer mechanism in the host.


[Granulosa cell tumor with liver metastasis treated with partial hepatectomy and chemotherapy. Report of a case].

A patient with granulosa cell tumor recurrence 14 years after primary resection, was treated with partial hepatectomy and adjuvant chemotherapy (vinblastine, cisplatin and bleomycin). She achieved complete remission and is free of disease 18 months after treatment.


Antibody binding to blood group antigens in relation to temperature: scanning electron microscopy of immunogold-labelled erythrocytes.

Binding patterns of mouse monoclonal antibodies (mAb) to P1, Pk, N, I, H, Y or A antigens were visualized in the backscatter electron imaging mode of a scanning electron microscope by indirect immunogold labelling. Experiments were performed at room temperature (RT) and at 4 degrees C. In experiments with anti-P1 and anti-Pk, clusters of immunolabelling particles dominated the immunolabelling pattern much more at RT than at 4 degrees C. By contrast, no clustering was seen with anti-N, even at RT. Clustering was also observed at RT with anti-I, anti-H and anti-Y, and on some Ax and A3 cells with anti-A, but was much reduced at 4 degrees C. Immunolabelling was stronger at 4 degrees C than at RT with all mAb except anti-N and anti-A. The results indicate that glycolipid blood group antigens are more mobile in the membrane of intact erythrocytes at RT than at 4 degrees C, and that the cells bind more antibodies to such antigens at 4 degrees C than at RT. We suggest that antigen immobilization in the cold will reduce cross-linking of antigens and hence increase the number of antibody molecules needed for epitope saturation, leading to increased binding of antibody in the cold. This may be the main reason for cold-enhanced agglutination with human blood group antibodies.


[Specific odor component produced by Mycobacterium lepraemurium on Ogawa yolk medium].

When Mycobacterium lepraemurium is grown on the 1% Ogawa yolk medium, it produces a specific odor. This odor was not observed in other easily cultivable acid-fast bacilli. Therefore, identification of the components responsible for the specific odor produced by M. lepraemurium was attempted. The odor components were extracted for overnight with sterilized and distilled water from the Ogawa yolk medium on which M. lepraemurium had been cultivated for two months. The odor components in the extract was adsorbed on refined charcoal. After washing with distilled water for three times, the charcoal was dried. Then the odor components were eluted from the charcoal with ethanol and the eluate was condensed under nitrogen gas flow at 40 degrees C. The condensate was analyzed by Gas-Chromatography-Mass-Spectrum (GC-MS). Phenylethanol and phenylacetic acid were identified as major odor components. A mixture of authentic phenylacetic acid, its methyl and ethyl esters, smelled similar to the odor of cultivated medium of M. lepraemurium. Thus, phenylacetic acid was identified as the key odor component produced by M. lepraemurium. When initial isolation culture of M. lepraemurium from murine leproma was cultivated on the Ogawa yolk medium by adding phenylacetic acid, growth inhibition was brought by the compound.


[Modification of Harada's method for rapid staining of mycobacteria].

Harada employed periodic acid-carbol pararosaniline and periodic acid-methenamine silver stain for demonstrating chromophobic bacilli which do not get stained with conventional carbol fuchsin or counter stain. This staining method takes considerable time for complete oxidation with periodic acid. We have succeeded in reducing the oxidation time by using hydrogen peroxide treatment prior to periodic acid and with the use of acidified sodium hydrogen sulfite treatment before carbol pararosaniline stain. We also found that in methenamine silver stain, combined use of semicarbazide and microwave treatment can shorten the whole staining time up to four hours without losing ito sensitivity.


Some biological properties of the human amniotic membrane interferon.

Human amniotic interferon was investigated to define the species specificity of its antiviral action and to compare its anti-cellular and NK cell stimulating activities with those of other human interferons. The antiviral effect was titrated in bovine (RV-IAL) and monkey (VERO) cells. Amniotic interferon exhibited, in bovine cells, 5% of the activity seen in monkey cells, while alpha interferon displayed 200%. No effect was detected with either beta or gamma interferon in bovine cells. Daudi cells were exposed to different concentrations of various interferons and the cell numbers were determined. The anticellular effect of the amniotic interferon reached its peak on the third day of incubation. Results suggested a higher activity for alpha and gamma interferons and a lower activity for beta when compared to amniotic interferon. Using total mononuclear cells as effector cells and K 562 as target cells in a 51Cr release assay, it was demonstrated that low concentrations of amniotic interferon consistently stimulated NK cell activity in cells derived from several donors, the results indicating a higher level of activity with this interferon than with alpha and beta interferons.


Kinetics of antigen specific and non-specific polyclonal B-cell responses during lethal Plasmodium yoelii malaria.

In order to study the kinetics and composition of the polyclonal B-cell activation associated to malaria infection, antigen-specific and non-specific B-cell responses were evaluated in the spleens of mice infected with Plasmodium yoelii 17XL or injected with lysed erythrocytes or plasma from P. yoelii infected mice or with P. falciparum culture supernatants. Spleen/body weight ratio, numbers of nucleated spleen cells and Immunoglobulin-containing and Immunoglobulin-secreting cells increased progressively during the course of infection, in parallel to the parasitaemia. A different pattern of kinetics was observed when anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell plaque forming cells response were studied: maximum values were observed at early stages of infection, whereas the number of total Immunoglobulin-containing and Immunoglobulin-secreting cells were not yet altered. Conversely, at the end of infection, when these latter values reached their maximum, the anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell specific responses were normal or even infranormal. In mice injected with Plasmodium-derived material, a higher increase in antigen-specific PFC was observed, as compared to the increase of Immunoglobulin-containing and Immunoglobulin-secreting cell numbers. This suggested a "preferential" (antigen-plus mitogen-induced) stimulation of antigen-specific cells rather than a generalized non-specific (mitogen-induced) triggering of B-lymphocytes. On the basis of these and previous results, it is suggested that the polyclonal B-cell activation that takes place during the course of infection appears as a result of successive waves of antigen-specific B-cell activation.


Antigenic differences among Leishmania amazonensis isolates and their relationship with distinct clinical forms of the disease.

Immunoblot analysis was used to investigate antigenic differences among clinical isolates of Leishmania amazonensis and their role in the etiology of the disease. Western blots of promastigote homogenates were analyzed with either monoclonal antibodies (MAbs) specific for the L. mexicana complex (M-4, M-6, M-9, and M-11) or polyclonal sera from L. amazonensis infected patients with the various forms of clinical disease. In the case of the MAbs, no significant variation was observed among the strains of L. amazonensis, isolated from cases of cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), diffuse cutaneous leishmaniasis (DCL), visceral leishmaniasis (VL) or post kala-azar dermal leishmaniasis (PKDL), in either the relative mobility (Mr) or the quantitative amount (intensity) of the antigenic determinants. In the case of the sera of the infected patients, the patterns of antigenic reactivity of these strains revealed that, despite showing the presence of shared antigens, differences were observed between some of the antigenic components of the various isolates of L. amazonensis that were recognized by a single serum. Differences were also demonstrated between the antigenic determinants of a single isolate of L. amazonensis that were recognized by the different patients' sera. No apparent association was consistently found, however, between the Mr components identified in these isolates and the clinical form of the disease or the geographical area of isolation. In addition, the spectrum of antigens recognized by the sera from patients with the same clinical form were not identical; although in some instances, similar Mr antigens were shared.(ABSTRACT TRUNCATED AT 250 WORDS)


Pivotal role of hepatocellular regeneration in the ultimate hepatotoxicity of CCl4 in chlordecone-, mirex-, or phenobarbital-pretreated rats.

Our earlier histomorphometric and biochemical studies suggested that the progressive phase of the interactive toxicity of chlordecone (CD) + CCl4 involves suppression of hepatocellular regeneration. The objective of the present work was to correlate hepatocellular regeneration with CCl4 (100 microliters/kg)-induced hepatotoxicity in rats maintained for 15 days on a normal (N) diet, relative to the regenerative response in rats maintained on a diet containing either 10 ppm CD, 225 ppm phenobarbital (PB), or 10 ppm mirex (M). Hepatocellular regeneration was assessed by measuring DNA and 3H-thymidine (3H-T) incorporation, followed by autoradiographic analysis of liver sections. Hepatotoxicity was assessed by measuring plasma transaminases (aspartate and alanine) followed by histopathological observations of liver sections for necrotic, swollen, and lipid-laden cells. Lethality studies were also carried out to assess the consequence of hepatotoxicity on animal survival. Dietary 10 ppm CD potentiated the hepatotoxicity of CCl4 to a greater extent than PB or M, as evidenced by elevations in plasma enzymes. Although the serum enzymes were significantly elevated in PB rats in contrast to the slight elevations in N and M rats, they returned to normal levels by 96 hr. However, serum enzyme elevations in CD rats were progressive with time until death of the animals. Actual liver injury by CCl4 was greater in PB- than in CD-pretreated rats, as evidenced by histopathological observations. A 100% mortality occurred in CD-pretreated rats at 60 hr after CCl4 administration, whereas no mortality occurred in either N-, M-, or PB-pretreated rats, indicating recovery from liver injury. Hepatocellular nuclear DNA levels were significantly decreased starting at 6 hr after CCl4 administration to CD-pretreated rats, but not in M- or PB-pretreated rats. 3H-T incorporation into nuclear DNA as well as percentage of labeled cells showed a biphasic increase in N rats: 1 at 1-2 hr, and the other at 36-48 hr after CCl4 administration. However, only 1 peak of 3H-T incorporation at 36-48 hr was observed in the CD + CCl4 combination, which was also significantly lower when compared to that observed after the M or PB + CCl4 combination treatments. These findings suggest that there is recovery in N-, PB-, or M-pretreated rats from CCl4-induced injury by virtue of the stimulated hepatocellular regeneration and tissue repair.(ABSTRACT TRUNCATED AT 400 WORDS)


[Experimental approaches to treatment of prostatic adenoma with the luliberin analog surfagon].

Therapeutic efficacy of surfagon, an analog of LH-RH which was injected subcutaneously at a daily dose of 100 micrograms/kg for 30 days, was investigated in ACI male rats with induced prostatic adenoma. This drug was shown to be effective proceeding from a morphological study and measurement of the prostate. Measurement of blood testosterone levels by radioimmunoassay showed a decrease in this level in rats, treated with surfagon, i. e. a therapeutic effect of the drug in prostatic adenoma was accompanied by a decrease in the level of androgens.


Effects of tracer, volume, osmolarity and infection on fluid kinetics during CAPD.

A review is given on various aspects of using the disappearance rate of intraperitoneally administered macromolecules for the determination of fluid kinetics in CAPD patients. The rationale and mathematics for the calculation of transcapillary ultrafiltration and of indirect measurement of lymphatic absorption are described. A comparison is made between autologous haemoglobin, human albumin and dextran 70. Dextran 70 appeared most useful; one brand of human albumin increased solute transport. Lymphatic absorption was higher after the installation of a 3-litre dialysate volume than after a 2-litre one, and also higher during peritonitis than after recovery from infection. A gradual increase in intraperitoneal volume, as obtained with glucose 3.86% dialysate, had no apparent effect on the disappearance rate of dextran 70. It is concluded that intraperitoneally administered dextran 70 is a clinically useful marker for the description of fluid kinetics in CAPD patients under various conditions.


Use of the disappearance rate for the estimation of lymphatic absorption during CAPD.

Several studies are discussed which investigated the usefulness of the disappearance rate of macromolecules from the peritoneal cavity for estimating convective fluid loss from the peritoneal cavity into the peritoneal lymphatic system. It is shown that dextrans are removed from the peritoneal cavity by a size-independent process at a mean rate of 1.37 +/- 0.15 ml/min, whereas the clearance from blood to dialysate of dextrans is size-dependent. The fluid removal rate estimated by the difference in bidirectional transport of inulin (1.79 +/- 0.38 ml/min; p < 0.0005) was of the same order of magnitude as has been found using the removal rate of macromolecules from the peritoneal cavity. Also, the role of local accumulation of macromolecules was studied during continuous administration of dextrans. No differences were found in the dextran disappearance rate before and after saturation of the peritoneal interstitium with dextran (1.1 +/- 0.6 vs. 1.0 +/- 0.4 ml/min). During a study using a hypoosmotic solution we calculated a net transcapillary backfiltration of fluid, whereas the dextran removal rate was in the same order of magnitude as found using commercially available dialysate. In our opinion, the disappearance rate of macromolecules is an estimate of convective fluid loss from the peritoneal cavity into the peritoneal lymphatic system.


Differentiating the language disorder in dementia from dysphasia--the potential of a screening test.

A considerable amount of information is now available about the way in which senile dementia of the Alzheimer type (SDAT) affects language performance. Speech and language therapists may have the requisite knowledge to work in the field, but they have few assessment materials to facilitate improved differential diagnosis between language breakdown in dementia and dysphasia. This study highlights the differential diagnostic potential of a short screening test with a weighted scoring system. The best discriminatory effect was in the areas of action picture description, verbal description and confrontation naming. Neither age nor sex appears to have a significant effect on results, and re-test results contribute little to the test's discriminatory power in terms of differential diagnosis between the two groups. The possibilities for future test usage and development are discussed.


The influence of endotracheal bleomycin administration on phospholipid composition of rat lung surfactant.

The influence of intratracheal bleomycin instillation (10 mg x kg-1) on lung surfactant phospholipids in rats after 7 and 14 days from drug administration was evaluated. It was stated that bleomycin causes a great fall in phosphatidylcholine, disaturated phosphatidylcholine and phosphatidylglycerol content, both in extra- and intracellular pool of surfactant. The data thus presented evidenced that intratracheal bleomycin administration caused a profound changes in the lipid composition of rat lung surfactant.


The influence of endotracheally administered bleomycin on hydroxyproline content and collagenase and elastase activity in rat lungs.

The effect of endotracheal administration of bleomycin (10 mg x kg-1) on hydroxyproline content, and on collagenase and elastase activities in rat lungs was investigated. A significant increase in lung tissue hydroxyproline content on 7th day after bleomycin administration was observed. Similar rise in this amino acid level in bronchoalveolar lavage was observed, however, on 14th day after the drug administration. Collagenase activity was lowered by 17% and 36%, respectively. No changes in elastase activity were found. These data suggest that bleomycin cause the lung fibrosis by restraining the collagenase activity.


Studies on tachykinin (neurokinin) receptor coupled with inositol phospholipid hydrolysis and salivation in rat salivary glands.

3H-substance P binding to the membranes of rat salivary glands was studied. The Kd and Bmax values were found to be 0.34 nM and 141 fmole/mg protein respectively using established natural occurring tachykinins to displace the binding. The rank order of potency was identified as substance P > neurokinin A > neurokinin B. These natural occurring tachykinins stimulate inositol phospholipid hydrolysis in slices of rat salivary glands, and the rank order of potency was also substance P > neurokinin A > neurokinin B. When salivation was induced by natural occurring tachykinins and acetylcholine (via i.v. route) in anesthetized rats with doses eliciting equivalent salivating responses, atropine blocks acetylcholine- as well as neurokinin B-, but not substance P- or neurokinin A-induced salivation. Based on the results mentioned above, we make a tentative conclusion that multiple receptor subtypes of neurokinins may exist in rat salivary glands. The neurokinins B receptor subtype is likely located presynaptically in the cholinergic nerve endings, whereas substance P and/or neurokinin A receptor subtypes may exist in the glandular tissue. The salivation induced by these neurokinins is likely through the activation of receptors, which are coupled to phosphatidylinositol turnover pathway.


Characterization of some biochemical properties of bradykinin-induced cAMP and cGMP formation in guinea pig ileum.

One of the receptor-mediated events, cyclic nucleotide, i.e. cAMP and cGMP formation induced by bradykinin in guinea pig ileum was investigated in this report. Bradykinin, similar to acetylcholine, produced a rapid rise in the levels of cAMP and cGMP in the ileum. The absolute amount of these cyclic nucleotides induced by the same dosages (10(-8) to 10(-6) M) of bradykinin was greater in cAMP than in cGMP. This increase in cyclic nucleotides was dependent upon the presence of calcium in the medium. Addition of EGTA (0.1 mM) in a calcium free medium resulted in a significant reduction of the levels. The elevation of cAMP and cGMP levels induced by bradykinin in the ileum could not be blocked by either atropine (an anticholinergic agent) or propranolol (a beta-adrenergic blocker). Both of these blockers did not alter the basal levels of two cyclic nucleotides. However bradykinin-induced cAMP formation could be completely blocked by either indomethacin (a prostaglandin synthesis inhibitor) or dexamethasone (a phospholipase A2 inhibitor), This, however, was not true in the case of bradykinin induced cGMP formation. Additionally, both blockers did not create a significant change in the basal levels of these cyclic nucleotides. Bradykinin induced cAMP formation in the ileum was indicated by observed results to likely occur through an indirect process, i.e. the formation and release of prostaglandin in the cell, whereas bradykinin-induced cGMP formation did not. The elevation of these cyclic nucleotides in the cells was observed to be related to the movement of calcium ion across the cell membrane.


The motor actions of natural neurokinins and their specific agonists on the gastrointestinal tract of the rat.

Neurokinins (NK) are a group of peptides that share a common C-terminal and play an important role in control of the motor functions of the mammalian gut. Neurokinin receptors such as NK1, NK2 are certainly present in any segment of gut, whereas the NK3 receptors are probably present in the ileum of the Guinea pig and duodenum of the rat. We measured gastrointestinal responses with natural NK and their very specific agonists to determine the probable receptors in the stomach and small intestine of Sprague-Dawley rats. After overnight fasting, the rats were intubated with a catheter to feed saline liquid meal that contained 10% charcoal. Simultaneously, various doses of NK ranged selected from 10(-10) and 10(-7) mol kg-1 included substance P (SP), neurokinin A (NKA), neurokinin B (NKB), septide, [Nle10]-NKA4-10, senktide, and vehicle were intraperitoneally injected. At 15 min after being fed test meal, the rats were sacrificed. Then we removed entire gut including the stomach to measure the total length of small intestine and transit length of the charcoal, from which the transit ratio was calculated. In comparison with ratios of rats treated with vehicle, the inhibited transit ratios for charcoal were seen among SP at 10(-10) mol kg-1, NKA at 10(-10) and 10(-7) mol kg-1, [Nle10]-NKA4-10 at 10(-7) mol kg-1, and senktide at all doses except 10(-8) mol kg-1. Enhanced transit ratios were seen for septide at the doses 10(-8) and 10(-7) mol kg-1. Likewise the mean total intestinal lengths of rats if they received various treatments of peptides except that rats treated with NKB had somewhat diminished length than those of vehicle-treated rats. Some natural NK and their very specific receptor agonists mainly inhibited rat gastrointestinal charcoal transits. We suggest the probable presence of NK1, NK2 and NK3 receptors in the small intestine and stomach including pylorus of the rat.


RNA metabolism during the sexual differentiation of rat hypothalamus.

Modifications in the basal molecular biology parameters (total concentrations of RNA, DNA, proteins, rRNA, tRNa, free and polysomal bound poly-A+ mRNA) have been determined daily in the growing hypothalamus of male and female rats from day 1 to day 8 after birth. Changes observed in the parameters studied in this work occurred mainly in the first 48 h after birth. In males tRNA and free mRNA (f-mRNA) contents decreased from day 1 to day 2 and then their concentrations remained more or less constant. Total mRNA significantly decreased from day 1 to day 3 and showed a further significant decrease from day 6 to day 8. Polysomal bound-mRNA (b-mRNA) decreased from day 1 to day 3, then increased to day 6, and finally decreased once more from day 6 to day 8. The b-mRNA/f-mRNA, mRNA/rRNA and mRNA/total RNA ratios showed a bimodal behavior with a first peak on day 2, and a second, smaller peak, on days 6-7. The changes observed in the females on the first 2-3 days of life were the inverse of those observed in the males, most of the parameters studied showed a sharp increase from day 1 to day 2 or to day 3 and then a drastic decrease. The only exception to this behavior was the b-mRNA/f-mRNA ratio which showed a small decrease from day 1 to day 2, followed by a continuous increase from day 2 to day 8. b-mRNA concentrations, after the sharp decrease from day 2 to day 3 of life, increased from day 3 to day 7.(ABSTRACT TRUNCATED AT 250 WORDS)


Veratridine-induced intoxication: an in vitro model for the characterization of anti-ischemic compounds?

Due to the complexity of ischemia-induced cellular dysfunction many different pharmacological approaches have been tested to improve cellular ischemia resistance. However, despite the importance of [Na+]i for ischemia-induced dysfunction, only very few studies have investigated the contribution of the Na+ channel to ischemia-induced failure of intracellular ion homeostasis. Since an activation of Na+ channels by veratridine also results in a failure of intracellular ion homeostasis, the veratridine- and ischemia-induced alterations of cellular function were compared. Moreover, despite the difference in the electrophysiological changes induced by veratridine and ischemia, the possible involvement of a slowly inactivating, less selective Na+ channel in both veratridine- and ischemia-induced cellular dysfunction is discussed. As a conclusion it is suggested that veratridine intoxication could be a helpful in vitro method for the characterization of putative anti-ischemic compounds.


Pardaxin-stimulated calcium uptake in PC12 cells is blocked by cadmium and is not mediated by L-type calcium channels.

Pardaxin is an excitatory neurotoxin which triggers neurotransmitter release as a result of voltage-dependent pore formation within the neuronal membrane. We have used several pharmacological manipulations of calcium influx to characterize pardaxin pore activity in PC12 cells in culture. Pardaxin stimulates the uptake of radioactive calcium into PC12 cells in a dose dependent fashion (ED50 of 0.4 microM). This stimulation is partially inhibited by nifedipine, a blocker of L-type calcium channels. Effective blockade of pardaxin stimulation was produced by the inorganic calcium channel blockers cadmium (IC50 of 10 microM) and nickel (2 mM). Homologous down regulation of L-calcium channels by the agonist Bay K-8644, inhibited the subsequent stimulation of calcium uptake by this drug, but not by pardaxin. A fluorometric analysis of pardaxin pore formation in unilamellar large liposomes indicates pardaxin pores are blocked by cadmium (10-200 microM). These data distinguish between pardaxin pores and L-type calcium channels in PC12 cells. We suggest pardaxin as a pharmacological ionophore tool to modulate neuronal calcium homeostasis and neurotransmitter release.


Predictive markers for hepatitis C antibody ELISA specificity in Australian blood donors.

The hepatitis C antibody reactivity rate in 91,748 blood donors tested using the ORTHO HCV C-100 ELISA system was 0.51%. Specificity of ELISA positive reactions was measured using a recombinant immunoblot assay (RIBA). The aim of this study was to identify markers in ELISA positive donors which were predictive of a RIBA positive result. Samples from 430 ELISA positive donors were tested by the first generation RIBA, RIBA-1, which incorporates two HCV peptides C-100 and 5-1-1. Fifty-five per cent (236) were positive and 19% (83) indeterminate. Multivariate analysis of gender, age, HCV ELISA OD ratio, alanine aminotransferase (ALT) status and hepatitis B core antibody (anti-HBc) status identified age, magnitude of HCV ELISA OD ratio and anti-HBc status as the only independent predictors of a positive RIBA-1 result. The relative odds of being RIBA-1 positive were 4.6-fold (95% CI 1.3-16.4) higher among donors aged 25-34 years compared with donors less than 25 or greater than 44; 6.1-fold (2.1-17.9) higher if the donor was anti-HBc positive and 273.4-fold (30.9-2417) higher if the HCV ELISA OD ratio was greater than 5.98 compared to those with a ratio less than 1.77. Seventy-eight of the 83 RIBA-1 indeterminates were tested on the second generation RIBA, RIBA-2, which includes two additional HCV peptide, C22 and C33c. Thirty-one per cent (24) were positive and 41% (32) were negative.(ABSTRACT TRUNCATED AT 250 WORDS)


Specificity and sensitivity of first and second generation anti-HCV ELISA in a low prevalence population.

Specificity and sensitivity of the second generation enzyme immunosorbent assay for hepatitis C antibodies were calculated from data on 208,554 blood donors screened with either the first or the second generation tests (ELISA I, ELISA II) or both (4,639 donors). The second generation ELISA was no more specific than the first generation test in this low-prevalence blood donor population (specificity 99.7% vs 99.6%). Most of the donors (93.5%) who were found to be reactive using ELISA II were negative or indeterminate according to a supplemental test, the second generation recombinant immunoblot assay (RIBA II). The second generation ELISA was found to be more sensitive than the first. It identified all 16 donors who were positive according to RIBA II during screening with ELISA I. The latter found only 13 out of 30 donors (43%) who were positive according to RIBA II during screening with ELISA II. If the number of RIBA II positive donors found during the first year of screening with ELISA I (74, 0.024%), and the number of RIBA II positive but ELISA I negative donors found during screening with ELISA II, are taken into consideration it can be estimated that the added sensitivity due to using ELISA II is about 40%.


Transmission of HCV infection by RIBA indeterminate and positive blood units.

A retrospective study was carried out on the recipients of 73 units of blood from 53 donors found reactive for anti-HCV. The donors were screened with anti-HCV enzyme-linked immunosorbent assay (ELISA C-100) and reactivity was confirmed with the first generation recombinant immunoblot assay (RIBA I). Fifty-two patients were recipients of blood from donors reacting as RIBA I 'indeterminate' and 21 of blood from RIBA I 'positive' donors. Only three recipients (5.8%) from 'indeterminate' donors were anti-HCV positive indicating that such donors are very seldom infectious. Eleven (52.4%) recipients from 'positive' donors had antibodies to HCV, indicating that not all RIBA-positive donors are necessarily infectious. Pretransfusion samples of the seropositive recipients were unavailable. All samples were analyzed with the first generation ELISA and with either the second-generation ELISA or RIBA (RIBA II) in order to evaluate test sensitivity. RIBA II was more sensitive than RIB I. One RIBA I indeterminate donor was positive by RIBA II. His recipient had antibodies to HCV. Twelve RIBA I indeterminate and three RIBA I positive donors were negative by RIBA II. All their recipients were anti-HCV negative. The second-generation ELISA was also shown to be more sensitive than ELISA C-100. The second-generation ELISA detected six confirmed anti-HCV positive recipients who were negative by ELISA C-100.


Integration, expression and germ-line transmission of foreign growth hormone genes in medaka (Oryzias latipes).

Gene constructs consisting of human growth hormone (hGH) gene driven by promoter/regulatory sequence of mouse metallothionein (mMT), viral thymidine kinase (vTK), rat cholecystokinin (rCCK), or chicken beta-actin (cBA) gene were injected into the cytoplasm of fertilized medaka eggs via the micropyle. More than 49% of the injected embryos survived at hatching. Up to 26% of the survivors showed integration of the introduced gene construct, as determined by polymerase chain reaction analysis and subsequent confirmation by Southern blot hybridization of the genomic DNA. A significant fraction of F1 progeny, derived from crosses between transgenic founders and the nontransgenic individuals, inherited the transgene. Expression of hGH gene was also observed in some of the P1 founders and F1 transgenic progeny carrying mMT-hCG or cBA-hGH gene. Furthermore, the growth performance of the P1 mMT-hGH and cBA-hGH transgenic founders and F1 cBA-hGH F1 transgenic progeny was significantly greater than their full sibling, nontransgenic individuals. In addition to the microinjection experiment, a gene construct containing the long-terminal repeat (LTR) sequence of avian Rous sarcoma virus (RSV) and rainbow trout (rt) GH2 cDNA was introduced into embryos of medaka by electroporation using an exponential decay electroporator. Approximately 70% of the electroporated embryos survived at hatching, and 20% of the survived individuals integrated RSVLTR-rtGH2 cDNA into their genomes. These two techniques will greatly enhance the ability to study regulation of gene expression in transgenic animals during differentiation and development.


Viruses, chemotherapy and immunity.

An increasing number of antiviral agents are presently in various stages of development and testing, and an increasing number have recently been licensed for use in humans. These drugs have been used extensively to treat viral infections in immunocompromised individuals, and these studies indicate that for many antiviral agents the response to therapy is highly dependent on the integrity of the underlying host immune response. In particular, the response to zidovudine, acyclovir and ganciclovir in persons with HIV-1 infection is highly dependent upon CD4 number, which can be considered a surrogate marker for the state of host immune function in these subjects. Responses to interferons likewise can be shown to depend on the host immune response, with responses due to both direct antiviral effects of this agent as well as immunomodulatory effects mediated through interferon-induced upregulation of HLA molecule expression. The interdependence of host immunity with antiviral efficacy is underscored by the increased antiviral drug resistance in persons with advanced degrees of chronic immunosuppression, related to the higher level of viral replication and viraemia which occurs in the absence of an effective host immune response. Further definition of the precise mechanisms of these interactions should facilitate the rational design of antiviral agents and immunomodulatory therapies to improve treatment of viral infections.


Immunohistochemical study on antigenic phenotype of Langerhans cell histiocytosis.

Immunohistochemical study on 26 cases of Langerhans cell histiocytosis (LCH) using several leukocyte antibodies in addition to traditionally used markers (S-100 protein and peanut agglutinin) revealed that the proliferating cells of LCH expressed UCHL1, MT1 as well as classically known positivity for S-100 protein, HLA-DR and peanut agglutinin but were negative for OPD4. In comparison to S-100 protein peanut agglutinin (PNA) using a two stage method produced weaker staining and positively stained cells were sparse. Also in this study, a small proportion of proliferating cells in LCH was observed to be reactive for both myeloid/macrophage antigens (KPI, MAC 387 and lysozyme) and Langerhans cell marker (S-100 protein), verifying the existence of a hybrid form of histiocytes.


Regulation of plasminogen activation in rat cell lines.

The regulation of plasminogen activators (PA) and their inhibitors (PAI) in the rat cell lines: HTC and L2 was studied. HTC plasminogen activator inhibitor type 1 (PAI-1) production was stimulated by dexamethasone, serum factors and insulin; that of tissue-type plasminogen activator (tPA) by cAMP raising agents. Retinoic acid, butyrate, phorbol ester and endotoxin did not affect net PA/PAI activity elaborated by HTC. L2 cells produced tPA, which production was stimulated by retinoic acid, phorbol myristate acetate, butyrate and cAMP; serum factors blunted their response, whereas in the synthetic serum substituting medium Ultraculture and with cocktail Ultroser the action of tPA stimulators was enhanced.


[Efficiency of inhibitors (phenylethanol, nalidixic acid, sodium azide)in the isolation of strictly anaerobic bacteria from a polymicrobial specimen].

The efficacy of inhibitors (PEA, Ac Nal, Az de Na) in the isolation of strict anaerobes in polymicrobial sample. The efficacy of three inhibitors to select strict anaerobic bacteria in the polymicrobial sample had been studied. First step: The most frequent anaerobes encountered in the infections are isolated in the agar Columbia containing the different inhibitors. This step allowed us to check the inhibition of the germ we have to isolate. Next step: polymicrobial mixtures were made. The composition of which is very similar to the samples we receive in the laboratory. The swarming Proteus is the facultative anaerobic germ which gives us difficulties when isolating strict anaerobic bacteria. Then, the different mixtures were isolated separately in the agar in which the inhibitors were added. The plates containing Azide of Na and PEA gave us the best results.


[Optimization of culture conditions for the production of Salmonella typhi O and H antigenic suspensions].

A Behring's production protocol of antigenic suspensions O and H of Salmonella typhi has been studied. The object of this study has been the Search of optimal conditions of cultivation and their exploitation on fermentor. A fermentation using the I.P.A culture medium has been realized to compare its results with that obtained using Behring's culture medium (B.C.M). The optimal cultivation conditions obtained are a waving rate of 400 tr/mn, a pH of 7.6 and an important air flow. Relating to nutritious constituents, the adequate glucose concentration is about 8g/1 and it seems better to replace meat extract by yeast extract. Comparatively to I.P.A culture medium, one of the most important advantage of Behring's culture medium modified reside in the conservation of the antigenicity of Salmonella typhi.


[Epidemiology of salmonella infections in Algeria. Evolution of the salmonella serovars isolated from 1986 to 1990].

The serovars evolution of 3340 Salmonella strains isolated from 1986 to 1990 in Algeria have been studied. Among Salmonella responsible for typhoid-paratyphoid fevers, 98% are S. typhi; the prevalence of this serovar is observed every year. Among the other Salmonella, 50 different serovars have been isolated from 1986 to 1990. The predominant serovars change every year but 98% out of the total belong to 8 groups of Kauffmann White scheme; the most frequent are: groups O: 6, 7-O: 8-O: 4 and O: 9.


Macroscopic and microscopic imaging of stable iodine (127I) in the thyroid.

The purpose of this review is to describe the two imaging methods which allow direct estimation of stable iodine (127I) within thyroid gland either in vivo by X-ray fluorescence or in vitro by secondary ion mass spectrometry (SISM) microscopy. X-ray fluorescence of thyroid is mainly used for the estimation of thyroid iodine content (TIC) in human pathophysiology. Although its measurement has little revelance for routine explorations of thyroid function, this is a valuable method for understanding complex pathophysiological conditions such as hyperthyroidism without radioactive uptake, the fate of irradiated glands or the thyroid adaptation to iodine overload. On the other hand, SIMS microscopy which combines the phenomena of secondary ion emission and mass spectrometry, makes possible the quantitative mapping of 127I on tissue section. This technique is able to characterize the functional activity of thyroid tissue without prior administration of radioiodine by measuring 127I concentration within the thyroid follicles. Furthermore, SIMS microscopy can be used to determine the extent to which exogeneous iodine affects the regulation of iodine within the thyroid follicles. Both methods were used to evaluate the quantitative changes in thyroid 127I induced by amiodarone overload. TIC measurements shows that hyperthyroidism occurred only in patients who increased their iodine stores, while the patients who developed hypothyroidism had low iodine stores. These data demonstrate that the mechanisms by which subjects who become hypothyroid under amiodarone iodine overload differ from those of subjects who remain euthyroid or become hyperthyroid: iodine organification is blocked in hypothyroid patients and increased in the euthyroid and hyperthyroid patients. The SIMS microscopy data obtained in mice demonstrated that the thyroid response to amiodarone is related to dietary iodine intake leading to an increase in iodine stores in iodine deficient mice and a decrease in iodine supplemented mice. These results could explain that hyperthyroidism with high thyroid iodine content occurred in areas with low thyroid iodine intake and hypothyroidism with low thyroid iodine content in areas with a supplemented iodine diet.


Effect of a single oral dose of L-thyroxine (150 micrograms) on serum thyroid hormone and TSH concentrations in clinically euthyroid goitrous patients.

Serum TT4, FT4, TT3, FT3 and TSH, concentrations were measured before and 0.5, 1, 2, 3, 4, 5 hours after a single oral dose of L-T4 (150 micrograms) in 21 clinically euthyroid goitrous patients on a long-term therapy program. The mean basal TT4 concentration, 8.85 +/- 0.52 micrograms/dl (mean +/- se), increased significantly at the second hour (10.04 +/- 0.52 micrograms/dl, p < 0.001) and reached the highest value at the third hour (10.35 +/- 0.55 microgram/dl, p < 0.001). Subsequently the mean serum TT4 concentration remained elevated until the fifth hour (10.20 + 0.60 microgram/dl, p < 0.001). The mean basal value of FT4 concentration, 8.66 +/- 0.40 pg/ml, increased significantly at 30 minutes (9.19 +/- 0.40 pg/ml, p < 0.01) and reached the highest value at the third hour (11.81 +/- 0.56 pg/ml, p < 0.001). The mean basal serum FT3 concentration, 3.56 +/- 0.10 pg/ml, increased significantly 30 minutes (3.72 +/- 0.11 pg/ml p < 0.05) after L-T4 administration; the mean serum FT3 concentration was significantly higher than the basal level and 1 and 2 hours as well, and reached the highest value at the third hour (3.86 +/- 0.09 pg/ml). The mean serum TT3 concentration did not show any significant change at any time. The mean basal TSH value (2.73 +/- 0.42 microUI/ml) declined progressively. At 30 minutes it decreased significantly (2.29 +/- 0.38 microUI/ml, p < 0.01) and reached the lowest value at the fifth hour (1.39 +/- 0.26 microUI/ml, p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)


Age influence on clinical features in hospitalized thyroid patients: dissimilarity between clinical and laboratory findings in adulthood. A retrospective study.

To evaluate the influence of age on clinical findings of thyroid disease, the records of 209 consecutive hospitalized patients with hyperthyroidism (n = 109) or hypothyroidism (n = 100) over a ten year period (1979 to 1988) were retrospectively reviewed. Patients included 51 males and 158 females, aged 21-99 years. Control euthyroid subjects (n = 70) were selected in the same age range and for similar non-thyroid illness as patients. Patients and controls were sorted by age decade of life. For each group a clinical index was calculated on the basis of 10 symptoms and 10 signs most frequently observed in thyroid disease. With increasing age, the occurrence rate of the clinical manifestations of thyroid disease decreased in patients while it increased in controls. This opposite evolution was less marked in hypothyroidism than in hyperthyroidism and was independent of biological parameters which remained stable whatever the age of patients and controls. Specific clinical features were identified for hyper- and hypothyroidism but none of them had sensitivity higher than 60% except goiter in hyperthyroid patients aged 61-80. In conclusion, a clinical diagnosis of thyroid disease is difficult in aged subjects because there are only a few specific features and their frequency is low. Therefore, in subjects over 60 years, we advocate performance of thyroid biochemical screening.


Hyalinizing trabecular adenoma.

A case is presented of hyalinizing trabecular adenoma in a 54-year-old Japanese female. The patient underwent a hemithyroidectomy for the diagnosis of thyroid carcinoma. An intraoperative frozen section diagnosis was medullary carcinoma. Hematoxylin and eosin stained permanent sections from the surgical specimen, a well encapsulated 3.0 x 2.0 x 1 cm nodule, also led us to suspect medullary carcinoma, but immunohistochemical staining for thyrocalcitonin, CEA, and thyroglobulin showed that it was a hyalinizing trabecular adenoma.


Recurrent laryngeal nerve palsy after thyroid and parathyroid surgery. Experience from Lund University.

All cases undergoing neck operation for thyroid or parathyroid disease at Lund University Hospital, Lund, over a 7 year period were examined with indirect laryngoscopy pre- and postoperatively for the determination of the true incidence of operatively induced palsy of the recurrent laryngeal nerves. A total of 1.048 patients were operated; 810 females and 238 males. At 439 operations, bilateral neck exploration was performed; hence, 1.487 nerves were at risk for complications. It was found that immediate postoperative recurrent laryngeal nerve palsy was induced in 72 patients (= 4.8% of nerves at risk). In 44 of these patients, the palsy was temporary and disappeared within 6 months. Therefore, a total of 28 patients developed permanent nerve palsy postoperatively. In 5 of these operations, the nerve was intentionally divided, yielding a true incidence of permanent accidental postoperative nerve palsy of 1.6% of nerves at risk.


Thyroid stimulating immunoglobulins are not influenced by smoking in healthy subjects.

In view of the increasing suggestion of an effect of cigarette smoking on thyroid disease and in particular Graves' ophtalmophathy we investigated thyroid stimulating immunoglobulins in relation to smoking habits in healthy subjects. No difference between smokers and non-smokers could be demonstrated, therefore, our study does not support the previously suggested altered immunologic surveillance of the thyroid in cigarette smokers.


Is standard 555 MBq 131I-therapy of hyperthyroidism ablative?

The effect of a standard 555 MBq 131I dose in ablating the thyroid gland was investigated in 116 consecutive hyperthyroid patients. Fifty-one had Graves' disease, 50 a multinodular toxic goitre and 15 had a solitary toxic nodule. 555 MBq 131I was given regardless of size or type of the gland and severity of the disease. Within one year after this dose hypothyroidism was induced in 41% of patients with Graves' disease, but in only 13% with a solitary toxic adenoma, and 6% with a multinodular gland. Forty-eight percent of the patients with a multinodular gland, 33% with Graves' disease and 13% with a solitary toxic nodule were still hyperthyroid. Since this so called ablative treatment only accomplishes hypothyroidism in 26/116 (23%) of our patients and results seem unpredictable 131I treatment adjusted according to gland size and type aiming at achieving euthyroidism could be contemplated.


Inadequacy of the WHO classification of the thyroid gland.

In order to evaluate the reproducibility of the WHO classification of goitre, the observer variation was estimated as kappa coefficients. Three observers independently inspected and palpated the thyroid gland of 53 patients twice and assessed the thyroid according to the five grades of the WHO classification. The thyroid volume was also measured by ultrasonography. We found kappa values from 0.15 to 0.70 in the interobserver study, and from 0.02 to 0.89 in the intraobserver study. Considerable overlap between the five grades was demonstrated when the assessments were related to volume estimated by ultrasonography. Description of the thyroid gland according to the WHO classification is inaccurate and not reproducible and is therefore of limited value.


Full-blown hypothyroidism associated with vitiligo and acropachy. Report of one case.

During the first two years after the onset of hypothyroidism, a patient with spontaneous primary myxedema developed thyroid acropachy. Fifteen years before the diagnosis of thyroid disease, he had patchy vitiligo of the face. There were high serum levels of antibodies against thyroglobulin (anti-Tg) and microsomal antigen (anti-M) were present, while the serum levels of antibodies against the second antigen of the colloid and the cell-surface antigen fell within the normal range. Moreover, antibodies to gastric parietal cells, adrenocortical cells or pancreatic islets in the serum were not present. Concerning the immuno-genetic pattern of our patient, his HLA system did not appear to confirm the well documented prevalence in whites with autoimmune disorders of an antigen specificity positive for the locus DR3.


Long term therapy with a new liquid L-thyroxine preparation: bioequivalence with L-T4 tablets.

Seventy-four euthyroid patients with nodular goiter (55) or primary hypothyroidism (19) were selected for long term treatment with a new preparation containing L-T4 in solution (Tiroxen, Laboratori Baldacci, Pisa, Italy). Each patient underwent, before or after receiving the L-T4 in solution, long term treatment with L-T4 in tablet form at the same dose. The serum concentrations of TSH, TT4, TT3, FT4 and FT3 were measured basally and during therapy with each of the two L-T4 preparations (liquid and tablet). In the golter group, mean serum TSH concentration was 1.4 microUI/ml basally, while it was 0.47 microUI/ml following both L-T4 tablet therapy and L-T4 solution administration. Mean basal TSH value was significantly different from the two values on the therapy (p < 0.001 in each instance). Mean basal serum TT4 concentration was 8.2 +/- 0.25 microgram/dl basally while it was 9.9 +/- 0.28 microgram/dl (p < 0.001) on L-T4 tablet therapy and 9.7 +/- 0.26 (p < 0.001) on L-T4 solution administration. Mean basal serum concentration of TT3, FT4, FT3 was not significantly different from the value on the therapy, either with L-T4 tablet or with L-T4 solution. In the hypothyroid patients the high mean basal serum TSH concentration (23.6 microUI/ml) returned to normal similar values on L-T4 tablet therapy (0.96 microUI/ml; p < 0.01) and on L-T4 solution administration (1.24 microUI/ml; p < 0.01). The serum TSH concentration value during L-T4 therapy varied from unmeasurable level to 3.5 microUI/ml during the tablet administration and to 4.8 microUI/ml during the solution administration.(ABSTRACT TRUNCATED AT 250 WORDS)


Relation between thyroid status and ferritin metabolism in rats.

Rats were made hypo and 'hyperthyroid' with propylthiouracil (PTU) and L-Thyroxine (L-T) respectively. The hypo and hyperthyroid status in these rats were confirmed by serum level of T4 and T3. Liver iron was significantly increased in both the hypo and hyperthyroid animals. However, liver ferritin synthesis rate was reduced by 36% in hypothyroid rats, and elevated by 38% in hyperthyroid ones. A similar trend was seen in liver ferritin concentration. Further, serum transaminases were elevated only in animals of the hyperthyroid group. It appears from the present data that ferritin metabolism is influenced by thyroid hormone as well as by iron. Thus, the raised serum ferritin in hyperthyroid patients may be partially attributed to increased ferritin synthesis in the liver and its possible leakage into circulation.


Prevalence of thyroid abnormalities in patients with rheumatoid arthritis.

The prevalence of thyroid abnormalities was determined in 131 patients: group I = rheumatoid arthritis, 68 patients, group II = systemic immunological diseases (IIa Sjögren's syndrome, n = 6; IIb other rheumatic autoimmune disease, n = 17), group III = other rheumatic diseases n = 13 and control group (n = 27). Thyroid abnormalities (hypo, hyperthyroidism, nodular goiter) were frequent: 33.8% in group I, 100% in group IIa, 11.7% in group IIb. Hypothyroidism was present in 19.1% (group I), 50% (group IIa). 6% (group IIb). Autoimmune thyroiditis was found in 16.2% in group I, 100% in group IIa, 11.7% in group III. Thyroid diseases are frequent in patients with rheumatoid arthritis. Therefore thyroid tests might be performed in patients with rheumatoid arthritis.


Scanning tunnelling microscopy observations of biomolecules on layered materials.

Scanning tunnelling microscopy (STM) has been performed on the reverse transcriptases of the human immunodeficiency virus (HIV-1) and the moloney murine leukaemia virus (MuLV). The biological molecules are adsorbed on n-type semiconducting MoTe2. The p66 (66 kD) subunit of the RT of HIV-1 is imaged by STM. Both STM and processed transmission electron microscopy (TEM) data show a spherical and horseshoe-like shape of external diameter ca. 65 A, depending on the angle of observation. The STM results show a larger diameter which is related to the curvature radius of the tip of the probing needle. The RTs of HIV-1 and MuLV exhibit a circular hole of ca. 20 A diameter in accordance with structure predictions and functioning considerations. The surface-molecule interaction is discussed in terms of the electronic properties of the semiconductor surface including the influence of small defect sites at the layered crystal surface.


Location of the granulocyte-specific antigen LAN on the Fc-receptor III.

This report investigates the location of the binding site of a recently described granulocyte-specific alloantibody, designated LAN, which causes alloimmune neonatal neutropenia. The LAN antigen is shown to be situated on the Fc-receptor III (FcRIII).


A controlled trial of intra-operative autologous transfusion in cardiothoracic surgery measuring effect on transfusion requirements and clinical outcome.

We carried out a prospective, controlled trial of intra-operative autologous transfusion (IOAT) in cardiac surgery using the Haemonetics Cellsaver 4, to determine the effects on transfusion requirements and early clinical outcome. Intra-operative autologous transfusion in unselected patients resulted in a reduction in the use of red cells in patients undergoing first-time operations (IOAT median 3 units, controls median 4 units, P = 0.0023), with no difference in the use of other blood products. Post-operative haemoglobin was higher in IOAT patients (IOAT 11.6 g/dl +/- 1.1 versus controls 11.2 g/dl +/- 0.98, P < 0.001). There is therefore the potential for a further reduction in homologous blood use in the IOAT group. There was no difference in early clinical outcome in the two groups; in particular the incidence of coagulopathies was not influenced by IOAT. The routine use of IOAT would add substantially to the cost of these operations. The decision to use it must therefore be based on an assessment of the value of the reduction in risk to the patient achieved by a small reduction in homologous donor exposures.


U.K. multicentre study on blood donors for surrogate markers of non-A non-B hepatitis. Part I: Alanine transferase and anti-HBc testing.

Blood samples from 9,215 blood donors in three U.K. centres (North London, Bristol and Manchester) were tested for their alanine aminotransferase (ALT) level and the presence of anti-HBc and anti-HCV. This paper presents the results of the ALT and anti-HBc tests. The prevalence of ALT > 45 IU/l was 3.1% overall (North London 3.06%, Bristol 4.56% and Manchester 1.97%). Manchester results were skewed by the methodology used for ALT measurement, highlighting the need for standard test methods. Anti-HBc was detected using the Wellcome enzyme-immunosorbent assay (EIA) and confirmatory testing was performed using a radioimmunoassay (RIA) and the Corecell haemagglutination assay. Repeat reactive rates were 0.9, 0.79 and 0.94% for North London, Bristol and Manchester, respectively, with an overall rate of 0.9%. The confirmed positive rate was 0.73, 0.53 and 0.65% for the three centres with an overall rate of 0.63%. Donors with an ALT > 45 IU/l, or with confirmed anti-HBc, were interviewed with a medical questionnaire for risk factors. The major contributing factors in donors with a raised ALT were alcohol consumption and obesity.


[Influence of 5.7-dihydroxytryptamine on electro-acupuncture analgesia and substance P level in central nervous system of the arthralgic rats].

The relation between electroacupuncture (EA) analgesia (A) and substance P (SP) level in the brain stem (BS) and lumbar spinal cord (LSC) of arthralgic rats was investigated. The rats were divided into three groups: 1)5.7-dihydroxytryptamine (5.7-DHT) + EA, 2) vehicle (V) + EA, and 3)5.7-DHT. All the animals were induced arthralgesia by injecting Freund's adjuvant 7 days after cisterna injection of 5.7-DHT or vehicle. The SP level in the BS and LSC was determined by RIA. The results indicated that in V + EA group the EA could prolong tail flick latency by 39.6%, but in other two groups did not. The SP level in LSC of V + EA group (179.1 +/- 11.5 pmol/g) was higher than that in the 5.7-DHT + EA (135.9 +/- 9.3pmol/g) and 5.7-DHT (125.8 +/- 10.0 pmol/g) groups. It suggested that both EA and arthralgia could activate the descending 5-HTergic inhibitory system to inhibit the release of SP in LSC. When the 5-HTergic system was destroyed by 5.7-DHT, the EAA was attenuated, and the SP level in LSC was lowered due to its release was decreased by EA and arthralgia.


[Ultrastructure identification of raphe-spinal serotonergic pain modulating system in rats].

5,6-dihydroxytryptamine was microinjected into the nucleus raphe magus (NRM) of rats. The sections of upper cervical segments were processed for immunoreactive substance P (SP). The degenerated axon terminals and immunoreactive fibers were identified by electron microscopy. The results show that the degenerated axon terminals and immunoreactive positive dendrites and axons were found in the laminae I and II of the dorsal horn. The electron-dense axon terminals were in contact with unlabelled dendrites, some also in contract with immunoreactive dendrites. In lamina II, one of degenerated axon terminals as a center was contacted by several unlabelled axonal boutons. SP labelled terminals were synapsed with unlabelled dendrites and dendritic spine; in addition, unlabelled axo-axonal synapse was found in lamina II. These results suggest that 5-HT axon terminals from NRM directly innervate SP and non-SP neurons in the laminae I and II.


[Effect electroacupuncture on the neurogenic inflammation].

The neurogenic inflammation caused by antidromic stimulation of the saphenous nerve was taken as an index of peripheral release of substance P. Electroacupuncture could reduce the plasma extravasation from the neurogenic inflammation by 69.7%, but electroacupuncture per se did not cause obvious plasma extravasation. In rats pretreated with capsaicin the plasma extravasation could be markedly reduced, in consistent with the reduction of substance P-like immunoreactivity in dorsal horn. It is referred that electroacupuncture might block the conveying the signals induced by nerve stimulation along the C-fibers and the axo-axonal reflex, leading to the reduction of peripheral release of substance P. Besides the mediation by different central structures, acupuncture might have direct effects on regulating peripherally the release of some inflammatory and painful mediators.


Principles of flap prefabrication.

Clinical flap prefabrication can be classified according to the basic technique of plastic surgery used for the prefabrication. There are currently three methods: (1) delay or expansion; (2) grafting; and (3) vascular induction by staged transfer. Illustrative cases are given to point out the advantages and indications for each method. A fourth, still experimental, method is based on cell biology advances that are looming on the horizon and may have revolutionary future clinical applications.


Thrombosis and antithrombotic therapy in microvascular surgery.

Thrombosis in microsurgery can either totally or partially reduce the perfusion to the free tissue transfer. Total occlusion normally occurs at or near the microvascular anastomosis, while microscopic vasospasm in the distal microcirculation can account for malperfusion and wound healing problems. The mechanisms of the anastomotic and microcirculatory responses to thrombosis are discussed. Drug therapy for each risk zone is identified, and a rationale for therapy is given.


The effect of enalapril on cardiac arrhythmias in patients with congestive heart failure.

The purpose of the study was to evaluate the effect of enalapril on the frequency of ventricular premature beats in patients with congestive heart failure. The study group consisted of 30 patients with a mean age of 47 +/- 0.6 years with chronic congestive heart failure (NYHA classes III and IV) due to primary dilated cardiomyopathy and cardiomyopathy in the course of ischaemic heart disease. Initial therapy with digitalis and diuretics was supplemented with enalapril at a dose of 5-20 mg daily. Initially and at three months after enalapril, the following parameters were evaluated: NYHA functional class, the presence of premature ventricular beats in 24-hour ECG recording and left ventricular function by echocardiography. The scheduled therapy was completed by 23 patients; in 5 patients, the intake was discontinued because of hypotension, one patient died after 14 days due to worsening heart failure, and one patient was submitted for pacemaker implantation. Clinical improvement manifesting itself by a shift to lower NYHA classes was found in 20 patients. A reduced number of premature ventricular beats was observed in 52% of the patients. Termination of cardiac arrhythmias, especially of complex beats, was parallel to the circulatory improvement.


Anti-hypertensive treatment with magnesium-aspartate-dichloride and its influence on peripheral serotonin metabolism in man: a subacute study.

Dichlormagnesium-aspartate-hydrochloride was given to 12 patients with mild hypertension in antihypertensive indication at a dose of 10.5 mmol Mg2+/day for 3 months. Blood pressure normalized (from 161.7 +/- 3.4/95.8 +/- 0.8 mmHg to 140.4 +/- 4.0/81.7 +/- 0.9 mmHg, after the third month (p < 0.01). While no changes in Mg2+ concentration in serum were observed in patients with normomagnesaemia, in hypomagnesaemic patients vS-Mg level normalized. Renal excretion of Mg2+ increased: from 3.41 +/- 0.36 before to 5.7 +/- 0.57 mmol Mg2+/24 h after treatment, p < 0.01. Mean plasma serotonin (5HT) concentration showed no changes, although a trend towards an increase in platelet 5HT content was observed. Elevated pre-treatment plasma 5-hydroxyindole acetic acid (5HIAA) concentrations normalized (from 137.29 +/- 20.3 to 78.96 +/- 31.64 nmol/l after the third month, p < 0.05). These findings point to a platelet-stabilizing effect of Mg2+. Fractional excretion of 5HIAA increased (from 1.42 +/- 0.27 to 5.4 +/- 1.22 after treatment, p < 0.01) while mean urinary 5HIAA excretion remained unchanged. It is deduced that a) total body 5HT and 5HIAA production was not affected; b) a long-term supplementation of Mg2+ stimulates the transport of 5HIAA in proximal tubules and, probably, intrarenal 5HIAA synthesis. A functional block in 5HT metabolism under Mg2+ treatment is anticipated. Thus, Mg2+ supplementation has renal and extrarenal effects that are important in treating hypertension and its complications.


[An experimental study model of human neo-angiogenesis. Heterotopic graft of the cornea in nude mice].

Two grafting methods were devised to transplant heterotopically human corneas to nude mice. Half of them were secured on the mesenterium, enclosed in a small intestine ring; the others were stitched to the anterior abdominal muscles after removal of a muscular disc. We used theses procedures to implant 13 human corneas: 7 according to the first technique and 6 tho the second one. Most of the corneas observed from Day 3 to Day 90 remained transparent along the whole grafting period. The grafts were surgically removed from Day 3 to Day 90 and submitted to an histological examination. It was not observed any morphological changes, versus normal human cornea and especially none neovascularization. These observations are fully in agreement with the persistent transparency of the corneas. With this model, many experimental studies in vivo on human corneas can be planed and conducted without any ethical restriction.


[A new method for transurethral prostatic commissurotomy].

Based on the principles of Shafik's prostatic commissurotomy, we have developed a new transurethral prostatic commissurotomy, which is characterized by cutting, layer by layer and section by section, the urethral mucosa, the bladder muscle layer and the prostate tissue within combined part of the urethra and prostate and by cutting the prostatic capsule with electrocoagulation. The comparison of clinical objective indexes has proved that the method is applicable to high risk patients with prostate hyperplasia and neck contracture leading to urethral obstruction after TURP.


Expression of ABO blood-group antigenic determinants identified by monoclonal antibodies B7H4 and D1E8 in normal human tissues and organs.

Human blood group antigens are genetically determined structures of undefined nature found not only in red blood cells but also in various tissues. Monoclonal antibodies against A and B human blood group antigens (B7H4 and D1E8) were obtained. Their tissue compatibility was tested in 11 different normal human organs taken from people with A1 and B blood group phenotypes. The antigen-antibody reaction was visualized using the indirect immunoperoxidase technique. The results of the study showed that the antibodies B7H4 and D1E8 react specifically with the tissue blood group antigens in healthy subjects. The findings obtained in most of the tested organs were in accord with those described by other researchers. Differences were found only in the stomach, urothelium, the lungs and the thyroid gland. A possible explanation of the results is the difference in the epitopes recognized by the monoclonal antibodies used by us and the blood group antibodies applied by other researchers. It is suggested that the monoclonal antibodies against the human blood group antigens A and B obtained by us could be used to establish suspected modification of ABH-antigens in the process of oncogenesis.