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With the goal of developing a library synthesis of tetrahydroquinoline-derived natural-product-like small molecules, a practical synthesis of enantiomerically pure tetrahydroquinoline scaffold was achieved. An asymmetric aminohydroxylation reaction was the key step in this strategy. This scaffold was further immobilized onto the solid support for the library generation. The library was obtained from three diversity sites:  (i) acylation of the hydroxyl group (R1), (ii) coupling of the Fmoc-protected amino acid to the amino group (R2), and (iii) amidation of the N-terminal amine group (R3).

Recent structural analysis of the peptide-MHC complex reveals that an antigenic peptide binds to MHC in only one conformation and that side chains anchoring in the binding pocket would not contact TCR. The identification of all the MHC-anchoring residues on an antigenic peptide is a prerequisite to understand how a given peptide interacts with the TCR. In a combination of binding analysis and model simulation, model peptide lambda repressor cl 16-26 was shown to bind to I-Ek through four anchor residues (Leu18, IIe21, Glu23 and Lys26), a pattern found in many I-Ek-binding peptides. TCR reactivity analysis clearly indicates a great variation in the interaction with cl 16-26 by T cells generated from different strains of I-Ek-bearing mice. Most of the T cell generated from A/J mice reacted with the central regions of cl 16-26, while there is a great diversity on the recognition of cl 16-26 by T cells from C3H and B10.BR mice. Despite the diverse interactions with antigenic peptide by these T cells, most TCR-E-k contacts are limited to the central region of the I-Ek beta-chain. T cells recognizing only the N-terminal part of cl 16-26 were found to contact I-Ek at nearly the same residues as T cells interacting with the C-terminal of cl 16-26. TCR-I-Ek recognition was apparently independent of TCR-cl 16-26 contact. The discordant TCR-peptide and TCR-MHC interaction may represent a unique feature of TCR recognition.

The localization of the central cough mechanism was studied by electrically stimulating the lower brainstem in cats lightly anesthetized with pentobarbital sodium or in unanesthetized midcollicular decerebrate preparations. Cough responses were recorded with the aid of a microphone. The cough responsive region was concentrated in an area dorsomedial to the trigeminal tract and nucleus. The sites of action of antitussive agents (dextromethorphan, codeine, clonazepam, diazepam and caramiphen) were studied on the centrally induced cough responses. Each of these drugs was administered intravenously to determine the dorsal levels for cough suppression. In other series, the threshold dose via the intravertebral route was determined first. Because the agent necessary to prevent cough via this route was so small in amount, recovery usually occurred in 20 to 90 minutes. Then the same agent was given intravenously in an attempt to obtain an effective dose which was close to the minimum dose for blocking the cough. The mean effective doses of these agents to abolish the cough via the vertebral artery were only about 1/20 of those required via intravenous injection. The findings suggest that these agents act centrally to suppress the cough responses. Clonazepam was found to be the most potent antitussive among these agents, the mean effective dose being about 1/35 of that of codeine. The antitussive potency of benzodiazepines is not well correlated with their muscle relaxant activity. For instance, clonazepam and diazepam have the same potency in depressing polysynaptic spinal reflexes, whereas the former is 6 times more potent than diazepam as an antitussive. This finding indicates that clonazepam has a high specificity as an antitussive.

We investigated whether the L2/HNK-1 carbohydrate epitope, expressed by two unusual glycolipids and several neural adhesion molecules, including L1, neural cell adhesion molecule, J1, and the myelin-associated glycoprotein, is involved in adhesion. Monoclonal L2 antibodies, the L2/HNK-1-reactive, sulfate-3-glucuronyl residue carrying glycolipids (L2 glycolipid) and a tetrasaccharide derived from the L2 glycolipid (L2 tetrasaccharide) were added to microexplant cultures of early postnatal mouse cerebellum, and cell migration and process extension were monitored. On the substrate poly-D-lysine, Fab fragments of L2 antibodies, L2 glycolipid, and L2 tetrasaccharide inhibited outgrowth of astrocytic processes and migration of cell bodies, but only L2 glycolipid and L2 tetrasaccharide reduced neurite outgrowth. On laminin, L2 antibodies, L2 glycolipid, and L2 tetrasaccharide inhibited outgrowth of astrocytic processes. Additionally, L2 glycolipid and L2 tetrasaccharide inhibited cell migration and neurite outgrowth. Several negatively charged glycolipids, lipids, and saccharides were tested for control and found to have no effect on outgrowth patterns, except for sulfatide and heparin, which modified outgrowth patterns in a similar fashion as L2 glycolipid and L2 tetrasaccharide. On astrocytes none of the tested compounds interfered with explant outgrowth. In short-term adhesion assays L2 glycolipid, sulfatide, and heparin inhibited adhesion of neural cells to laminin. L2 glycolipid and sulfatide interfered with neuron to astrocyte and astrocyte to astrocyte adhesion, but not with neuron-neuron adhesion. The most straightforward interpretation of these observations is that the L2/HNK-1 carbohydrate and the sulfated carbohydrates, sulfatide and heparin, act as ligands in cell adhesion.

To survive in a mechanically active environment, cells must adapt to variations of applied membrane tension. A collagen-coated magnetic bead model was used to apply forces directly to the actin cytoskeleton through integrin receptors. We demonstrate here that by a calcium-dependent mechanism, human fibroblasts reinforce locally their connection with extracellular adhesion sites by inducing actin assembly and by recruiting actin-binding protein 280 (ABP-280) into cortical adhesion complexes. ABP-280 was phosphorylated on serine residues as a result of force application. This phosphorylation and the force-induced actin reorganization were largely abrogated by inhibitors of protein kinase C. In a human melanoma cell line that does not express ABP-280, actin accumulation could not be induced by force, whereas in stable transfectants expressing ABP-280, force-induced actin accumulation was similar to human fibroblasts. Cortical actin assembly played a role in regulating the activity of stretch-activated, calcium-permeable channels (SAC) since sustained force application desensitized SAC to subsequent force applications, and the decrease in stretch sensitivity was reversed after treatment with cytochalasin D. ABP-280-deficient cells showed a > 90% increase in cell death compared with ABP-280 +ve cells after force application. We conclude that ABP-280 plays an important role in mechanoprotection by reinforcing the membrane cortex and desensitizing SACs.

Sulfoglucuronyl carbohydrate (SGC) linked to the terminal moiety of neolacto-oligosaccharides is expressed in several glycoproteins of the immunoglobulin superfamily involved in neural cell-cell recognition as well as in two sulfoglucuronylglycolipids (SGGLs) of the nervous system. SGGLs and SGC-containing glycoproteins are temporally and spatially regulated during development of the nervous system. In the cerebellum, the expression of SGC, particularly that of SGGLs, is biphasic. Several studies have suggested that the initial rise and decline in the levels of SGGLs and SGC-containing proteins correlated with the migration of granule neurons from the external granule cell layer to the internal granule cell layer and their subsequent maturation, whereas the later rise and continued expression of SGGLs in the adult was associated with their localization in the Purkinje neurons and their dendrites in the molecular layer. Here it is shown by immunocytochemical methods that the expression of SGC declined progressively in granule neurons isolated from cerebella of increasing age. The decline in the expression of SGC in granule neurons was also shown with time in culture. These results correlated with the previously shown declining activity of the regulatory enzyme lactosylceramide N-acetylglucosaminyltransferase (GlcNAc-Tr) with age in vivo and in isolated granule neurons in culture. GlcNAc-Tr synthesizes a key precursor, lactotriosylceramide, involved in the biosynthesis of SGGL-1. The down-regulated synthesis of SGGLs in the mature granule neurons was shown by immunocytochemical and biochemical methods to be restored when a precursor, glucuronylneolactotetraosylceramide (GGL-1), which is beyond the GlcNAc-Tr step, was exogenously provided to these cells. The biological effect of such restoration of the synthesis of SGGLs in the mature granule neurons leads to cell aggregation and enhanced proliferation of neurites, amounting to dedifferentiation.

The presence of free fatty acids in liposome model membranes sensitizes these membranes to the action of the imidazole antifungals, clotrimazole, micronazole, and sulconazole. Unsaturation of the fatty acids is an important variable; the effect of linoleic and oleic acids is much greater than that of stearic acid. The imidazoles differ somewhat in action, with clotrimazole potency greatest both on membranes with and without fatty acids. Sulconazole has very little activity on membranes without fatty acids even at the highest concentrations tested. The data are discussed with reference to the susceptibility of various cells to the imidazoles and the specificity of imidazole action. A modification of the enzymatic method generally used for assay of marker glucose with liposome systems is also presented.

Distinct cell-surface glycoconjugates are expressed on specific subsets of dorsal root ganglion (DRG) neurons and DRG terminals projecting to the superficial dorsal horn of rat spinal cord (Dodd, J., and Jessell, T. M. (1985) J. Neurosci. 5, 3278–3294). Carbohydrate antigens detected by monoclonal antibodies (mAbs) TC6, KH10, and LD2 are restricted to about 20% of DRG neurons projecting to lamina IIB (dorsal), whereas antigens recognized by mAb LA4 are expressed by about 50% of DRG neurons projecting to lamina IIB (ventral). These mAbs were generated against rat pancreatic acinar cell line AR4-2J antigens. The glycolipid antigens in AR4-2J cells reacting with these mAbs have been structurally characterized by sequential hydrolysis with various exoglycosidases, immunochemical tests, linkage analysis of permethylated alditol acetates, capillary gas liquid chromatography-mass spectrometry, mass spectrometry of permethylated compounds, and by fast atom bombardment mass spectrometry of the native antigens. The structure of the major antigen (IA) in AR4-2J cells was determined to be: The asialo derivative of IA and the novel disialo form of IA (Gal α 1→3(Fuc α 1→2)→GD1b) have been also identified. The DRG neurons contained only the neutral glycolipid, asialo form of IA. All these antigens reacted equivalently in the high performance thin layer chromatography-immuno overlay assay with the TC6, LD2, and LA4 mAbs. The molecular specificity of the three mAbs was determined by rection with a variety of possible antigens and appears to be the same. All three mAbs required terminal Gal α 1→3(Fuc α 1→2)Gal β 1→3GalNAc (or 4GlcNAc) for full reactivity. Only partial reactivity was observed with compounds in which α-Fuc was removed. The observed restricted reactivity of mAbs TC6, LD2, and LA4 in subsets of DRG neurons and in ventral and dorsal areas of lamina IIB may be due to different topographical expression of the antigen in the neuronal membrane.

In the adult cerebellum, sulfoglucuronyl glycolipids (SGGLs) are specifically localized in Purkinje cells and their dendrites in the molecular layer. Other major cell types such as granule neurons and glial cells lack SGGLs. To explain the cell specific localization and the known biphasic expression of SGGLs, enzymic activities of four glycosyltransferases involved in the biosynthesis of SGGLs were studied in murine cerebellar mutants, in distinct cellular layers of rat cerebellum, and in isolated granule neurons during development. The enzymes studied were lactosylceramide: N-acetylglucosaminyl transferase (GlcNAc-Tr), lactotriaosylceramide:galactosyltransferase, neolactotetraosylceramide:glucuronyltransferase, and glucuronylglycolipid:sulfotransferase. In the cerebellum of Purkinje cell-deficient mutants, such as (pcd/pcd) and lurcher (Lc/+) where Purkinje cells are lost, GlcNAc-Tr was absent, but the other three glycosyltransferase were not severely affected. This indicated that the latter three enzymes were localized in other cell types, such as in mature granule neurons and glial cells, in addition to that in Purkinje cells, and the lack of SGGLs in these mutants was due to absence of GlcNAc-Tr. Analyses of the enzymes in the specific micro-dissected cellular layers also showed that Purkinje cell layer and molecular layer (where Purkinje cell dendrites are localized) contained all four enzymes. However, granule neurons and glial cells in the white matter lacked GlcNAc-Tr, but expressed the other three enzymes. It was concluded that the absence of SGGLs in adult granule neurons and glial cells was due to specific deficiency of the GlcNAc-Tr. Although adult granule neurons lacked GlcNAc-Tr and therefore SGGLs, isolated granule neurons from the neonatal cerebellum contained all four enzymes necessary for the synthesis of SGGLs. With development, the activity of GlcNAc-Tr in the isolated granule neurons declined but the other enzymes were not as affected, indicating that immature granule neurons were capable of synthesizing SGGLs and with maturation the synthesis was down-regulated. This also explains the biphasic expression of SGGLs in the developing cerebellum.

A theory is developed for predicting the limiting transmission loss of sound through pipe wall. The approach is general to allow an evaluation of the effects of the internal and external fluids as well as the basic physical characteristics of the cylinder. The effects of internal static pressure and the presence of a uniforn flow are also discussed. Results of an experimental program are presented which verify the theory for standard commercial pipes.

It was previously reported that monoclonal IgM from two patients with gammopathy and neuropathy showed similar specificity by reacting with the same group of unidentified minor components in the ganglioside fractions of human nervous tissues (Ilyas, A. A., Quarles, R. H., Dalakas, M. C., and Brady, R. O. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6697-6700). Enzymatic degradation, ion-exchange chromatography, and immunostaining of purified ganglioside standards on thin-layer chromatograms have now revealed that the antigenic glycolipids recognized by the IgM from these patients are gangliosides GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-4Glc beta 1-1Cer(GM2), GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer (IV4GalNAcGM1b), and GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-3GalNAc beta 1-4 beta Gal(3-2 alpha NeuAc)beta 1-4Glc beta 1-1-Cer (IV4GalNAcGD1a). The monoclonal IgM appears to be reacting with the terminal [GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-] moiety shared by these three gangliosides and is a useful probe for detecting small amounts of GM2, IV4GalNAcGM1b, IV4GalNAcGD1a, and other gangliosides with the same terminal sugar configuration in tissues. Species distribution studies using the antibody revealed that GM2 is present in the brains and nerves of all species examined, while IV4GalNAcGM1b and IV4GalNAcGD1a exhibit some striking species specificity. GM2, but not IV4GalNAcGD1a, is enriched in purified myelin from human brain.

Novel sulfated glucuronic acid-containing glycolipids have been identified in the nervous system. These glycolipids are highly antigenic and share antigenic determinants with several nervous system glycoproteins, such as neural cell adhesion molecules, myelin-associated glycoprotein, and ependymins. The structure of the major antigenic glycolipid from human peripheral nerve was determined by chemical and enzymatic degradation, incorporation studies, sugar analysis after permethylation, pertrimethylsilylation, and gas liquid chromatography-mass spectrometry techniques as well as fast atom bombardment-mass spectrometry of the native antigen. The following structure was established for the major antigenic glycolipid. sulfate-3-GlcA beta(1---3)Gal beta(1----4)GlcNAc beta(1----3)Gal beta(1----4)Glc beta(1----1)-ceramide. The major fatty acids in the ceramide were 18:0, 18:1, 24:0, and 24:1, with C18-sphingenine as the long chain base.

Acute administration of deanol-p-acetamidobenzoate (Deaner; deanol) has been reported to elevate brain choline (CH) and acetylcholine (ACh) levels. We have developed a specific and sensitive gas chromatographic assay to measure deanol levels in tissue and have applied this assay to our studies of the effect of acute deanol administration on deanol, ACh and Ch levels in rodent brains. Details of the method are described in this text. This procedure is quantitative and yields reproducible results over a wide range of deanol concentrations (0.30-200 nmol). Seven endogenous and pharmacological parameters have been studied using this procedure. In control rodent brain, liver, heart, lung and plasma, we detected no free endogenous deanol (less than 1 nmol/g). After deanol administration, we were able to detect deanol in tissue and have attempted to determine a relationship between these levels and values of ACh in the same tissue. Regardless of deanol pretreatment time (1-30 minutes) or doses (33.3-3000 mg/kg i.p.) used, we detected no increase in mouse whole brain ACh levels. Likewise, there was no detectable elevation in ACh levels in rat whole brain, cortex, striatum or hippocampus after a 15-minute pretreatment with 550 mg/kg of deanol (i.p.). The only elevation in ACh levels which we detected occurred selectively in the striatum of mice pretreated with a massive dose (900 mg/kg i.p.) of deanol for 30 minutes. This selective increase in striatal ACh levels oculd not, however, be related to levels of deanol in the striatum because there was no greater accumulation of deanol in the striatum than in other brain areas tested or in whole brain. These data do not confirm the results of other investigators who reported elevations in whole brain or striatal ACh levels after acute administration of lower doses of deanol. The data emphasize the need for further investigation into the mode of action of deanol and question its suggested role as an immediate precursor of ACh synthesis in the central nervous system.

The effects of caffeine and d-amphetamine sulfate were evaluated upon single unit activity of medial thalamus in chloral hydrate anesthetized rats. Single unit activity recorded extracellularly with platinum-iridium microelectrodes. It was found that caffeine, 0.1 to 0.5 mg/kg i.v., markedly suppressed and that amphetamine, 0.1 to 1 mg/kg i.v., markedly augmented the spontaneous firing rates of medial thalamic neurons. It was also demonstrated that amphetamine was capable of activating the medial thalamic neuronal activity while it was suppressed by caffeine. On the other hand, while the firing rate was increased by a previous amphetamine injection, administration of caffeine suppressed the amphetamine-induced augmentation of neuronal activity. The data imply that these two central nervous system (CNS) stimulants, caffeine and amphetamine, have different mechanisms of action at least at the medial thalamic site. Moreover, since medial thalamus participates in the recruitment phenomenon and exerts a stabilizing action on the cerebral cortex, our findings that caffeine suppressed this brain area suggest that the medial thalamus may be an important site of action for the arousal induced by caffeine. In addition, the demonstration of inhibitory effects of caffeine on the medial thalamus in this study and excitatory effects on brain stem reticular formation from our previous studies indicate that caffeine has differential actions on different brain areas. This observation broadens the generally held belief that caffeine excites at all levels of the CNS to specifically include inhibition of inhibitory influences in at least one functional system of the brain.

Problems concerned with the propagation of weak planar shock waves in a nonuniform, nonequilibrium gas is theoretically investigated. The medium under consideration is a diatomic thermally perfect gas with excited vibrational energy and is initially inhomogeneous with exponential density and temperature distributions. The systematic characteristic perturbation scheme is employed to render a first-order frozen shock expression. It is shown quantitatively that combined effects of nonequilibrium, nonlinearity, and stratification govern the nature of the shock wave propagation. The uniform gas limit of present theory agrees with previously known results of shock wave propagation in a general relaxing fluid. Numerical examples illustrate the variation of frozen shock strength and speed due to different magnitudes of relaxation rates and inhomogeneity. The interesting competition phenomenon between nonequilibrium effects and nonuniform effects on shock wave propagation is examined.

Several antitussive agents were assessed for their cough-suppressant activity. Cough responses were obtained by electrically stimulating the lower brainsten., in cats lightly anesthetized with sodium pentobarbital or in unanesthetized midcollicular decerebrate preparations. Cough sounds were recorded with the aid of a microphone. The cough reactive region was concentrated in an area dorsomedial to the trigeminal tract and nucleus. The potency of these antitussive (dextromethorphan, RO 21-47900-001, codeine, clonazepam, diazepam and caramiphen) were determined by studying their effect on the centrally induced cough responses. Each of these agents was administered in graded doses intravenously to determine the minimal effective doses for suppressing the cough responses. They are 0.57, 2.55, 1.71, 0.048, 0.28 and 3.18 mg/kg for the above listed drugs. The results indicate that clonazepam was found to be the most potent antitussive among these agents, the mean effective dose being about 1/35 of that of codeine. The antitussive potency of benzodiazepines is not well correlated with their muscle relaxant activity. For instance, clonazepam and diazepam have the same potency in depressing polysynaptic spinal reflexes, whereas the former is six times more potent than diazepam as an antitussive. This finding indicates that clonazepam has a high specificity as an antitussive.

The van Hove self-correlation function in a hard-disk fluid is analyzed using the Lorentz-Enskog kinetic equation and the kinetic model method of solution. Numerical convergence of the model solutions is demonstrated and accurate model results are used to interpret molecular dynamics simulation data at finite wave numbers. It is found that at about 60% of freezing density the error in the Enskog theory can be mainly attributed to an underestimate of the effective self-diffusion coefficient, but at 90% freezing density a theory which treats correlated collisions is needed to describe the width behavior of the singleparticle density fluctuation spectrum.

Electron microscopy reveals that, in Bdellovibrioinfection, after the formation of a passage pore in the host cell wall, the differentiated parasite penetration pole is associated with the host protoplast. This firm contact persists throughout the parasite penetration and after this process is completed. In penetrated hosts this contact is also apparent by phase microscopy. The association between the walls of the parasite and the host at the passage pore, on the other hand, is transient. Bdellovibriodo not penetrate hosts whose protoplast and cell walls are separated by plasmolysis, or in which the membrane-wall relationship is affected by low turgor pressure. It is concluded, therefore, that for penetration to occur it is essential that the host protoplast be within reach of the parasite, so that a firm contact can be established between them. A penetration mechanism is proposed that is effected by forces generated by fluxes of water and solutes due to structural changes in the infected host envelope. These forces cause a differential expansion of the host protoplast and cell wall and their separation from each other around the entry site, while the parasite remains firmly anchored to the host protoplast. Consequently, the parasite ends up enclosed in the expanded host periplasm. The actual entry, therefore, is a passive act of the parasite.

Summary The method of strained coordinates is extended to expand the dependent variables as well as independent coordinates of a nonlinear hyperbolic system in asymptotic series of several parameters. The perturbation parameters may be of a different nature but are required to be intrinsically independent of each other. The method is found to be particularly useful for treating problems with several relevant parameters being of the same order of one another. The illustrative example discussed is a nonlinear supersonic nonequilibrium flow over a wedge where the nonlinear effect in the flow becomes of the same order as the nonequilibrium effect. A second-order theory is developed to provide a description of the near-field flow pattern and the expression of the front frozen shock wave attached to the nose of the wedge.