Biotechnology and economic development: the winning formula.
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Biotechnology↗
Biomedical subjects
W J Roberts
Publications and source records attributed to W J Roberts.
Interactions of the A1 heterogeneous nuclear ribonucleoprotein and its proteolytic derivative, UP1, with RNA and DNA: evidence for multiple RNA binding domains and salt-dependent binding mode transitions.
The 319-residue A1 heterogeneous nuclear ribonucleoprotein is the best studied of the group of major or core mammalian hnRNP proteins that bind pre-mRNA immediately following transcription. Circular dichroism studies suggest that binding of A1 and its proteolytic fragment, UP1 (residues 1-195), to nucleic acids results in an unstacking of the bases of poly(A). On the basis of poly[d(A-T)] and poly[r(A-U)] melting studies, both A1 and UP1 are helix-destabilizing proteins. Titrations of A1 and UP1 with poly(A), poly(U), and poly[d(T)] suggest that these two proteins do not bind with significant base specificity. A previous study indicated that A1, which contains a glycine-rich COOH terminus (residues 196-319) not present in UP1, binds cooperatively to polynucleotides while UP1 does not [Cobianchi et al. (1988) J. Biol. Chem. 263, 1063-1071]. Here we confirm this latter finding and demonstrate that the cooperativity parameter for A1 binding, which has a value of about 35 for binding to both single-stranded RNA and DNA, is insensitive to the NaCl concentration at least up to 0.4 M. In contrast to the cooperativity parameter, the occluded site size for A1 binding to RNA is salt dependent and increases from about 14 to 28 upon increasing the NaCl concentration from 25 to 250 mM. This variation in site size is best explained by assuming that A1 can interact with nucleic acids via at least two different binding modes. Both A1 and UP1 have higher affinity for single-stranded as opposed to double-stranded nucleic acids and bind preferentially to single-stranded RNA as compared to DNA. Comparative studies on the binding of A1 versus UP1 to poly[r(epsilon A)] demonstrate that in addition to cooperative protein/protein interactions, the glycine-rich COOH-terminal domain of A1 is also directly involved in protein/nucleic acid interactions.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
A novel function for zinc(II) in a nucleic acid-binding protein. Contribution of zinc(II) toward the cooperativity of bacteriophage T4 gene 32 protein binding.
The contribution of Zn(II) toward the binding of bacteriophage T4 gene 32 single-stranded nucleic acid-binding protein (gp32) has been examined by the use of two independent approaches. Studies carried out with successively longer oligonucleotides which have the general structure p(dT)n, where n is equal to 8, 16, 24, or 32 nucleotides, suggest that removal of Zn(II) decreases the cooperativity of binding by as much as 30-fold. Hence, whereas apo-gp32 and native gp32 have similar apparent affinities for the single-site lattice p(dT)8, native gp32 has an approximately 10-fold higher affinity compared to apo-gp32 for a two-site lattice, such as p(dT)16. In contrast to native gp32, where full cooperativity (in terms of the strength of a single gp32-gp32 interaction) is reached with only a two-site lattice, the cooperativity of apo-gp32 binding appears to increase approximately 4-fold upon going from a two- to a four-site lattice such as p(dT)32. The conclusion reached from these oligonucleotide studies agrees well with a series of titrations with polyribo(ethenoadenylic) acid, in 0.275-0.40 M NaCl. These latter studies indicate that the 6-38-fold higher affinity of native gp32 as compared to apo-gp32 for polyribo(ethenoadenylic) acid results primarily from the higher cooperativity of binding of native gp32. By stabilizing a specific subdomain within gp32 that is essential along with the NH2-terminal domain (residues 1-9), Zn(II) contributes from 20 to 50% of the free energy of cooperative gp32-gp32 interactions that occur along a polynucleotide lattice.
Amino Acid Sequence↗
Purification and functional characterization of adenovirus ts111A DNA-binding protein. Fluorescence studies of protein-nucleic acid binding.
The adenovirus single-stranded DNA (ssDNA)-binding protein (DBP) is necessary for the elongation step in viral DNA replication. In an attempt to characterize the putative ssDNA-binding domain of the DBP, we purified and characterized the Ad2ts111A DBP, which contains a glycine-to-valine substitution at amino acid 280. This mutation is adjacent to that in the previously studied Ad2+ND1ts23. Ad2+ND1ts23 exhibits a temperature-sensitive defect in DNA replication, and its DBP has previously been shown to bind ssDNA with reduced affinity. Ad2ts111A DBP, like Ad2+ND1ts23, does not support adenovirus DNA replication in vitro at elevated temperatures. However, the Ad2ts111A DBP binds ssDNA more tightly than does Ad2+ND1ts23 and is not temperature sensitive in this function. To determine the nucleic acid-binding properties of DBP, we applied spectrofluorometric techniques, which had not been used previously to study adenovirus DBP. Using the homopolynucleotide poly(1,N6)-ethenoadenylic acid (poly(r epsilon A], we have determined that the binding site size is approximately 16 nucleotides. In 20 mM NaCl, the Ad2wt, Ad2ts111A, and Ad2+ND1ts23 DBP proteins all bound stoichiometrically to poly(r epsilon A) with overall apparent affinities above 108 M-1. Based on titrations carried out at higher salt concentrations, however, the stability of these complexes did appear to increase in the order Ad2+ND1ts23 less than Ad2ts111A less than Ad2wt. By these techniques, we have confirmed also that the DBP of another temperature-sensitive mutant, H5ts107, like the Ad2ts111A DBP, retains its ability to bind ssDNA even at a restrictive temperature utilizing the salt concentration compatible with adenovirus DNA replication in vitro. The H5ts107 DBP, which contains an amino acid substitution at position 413, is defective for in vitro replication at nonpermissive temperature but is not temperature sensitive for binding to ssDNA. In summary, our results indicate that the replication defects of the Ad2ts111A are similar to those of H5ts107 and cannot be attributed to defective, nonspecific ssDNA binding by the DBP. It appears that ssDNA binding by itself is not sufficient to account for the role of DBP in adenovirus DNA replication.
Adenoviridae↗
p10 single-stranded nucleic acid binding protein from murine leukemia virus binds metal ions via the peptide sequence Cys26-X2-Cys29-X4-His34-X4-Cys39.
The RNA binding protein of 56 residues encoded by the extreme 3' region of the gag gene of Rauscher murine leukemia virus (MuLV) has been chemically synthesized by a solid-phase synthesis approach. Since the peptide contains a Cys26-X2-Cys29-X4-His34-X2-Cys39 sequence that is shared by all retroviral gag polyproteins which has been proposed to be a metal binding region, it was of considerable interest to examine the metal binding properties of the complete p10 protein. As postulated, p10 binds the metal ions Cd(II), Co(II), and Zn(II). The Co(II) protein shows a set of d-d absorption bands typical of a tetrahedral Co(II) complex at 695 (epsilon = 565 M-1 cm-1), 642 (epsilon = 655 M-1 cm-1), and 615 nm (epsilon = 510 M-1 cm-1) and two intense bands at 349 (epsilon = 2460 M-1 cm-1) and 314 nm (epsilon = 4240 M-1 cm-1) typical of Co(II)----(-)S- charge transfer. The ultraviolet absorption spectrum also indicates Cd(II) binding by the appearance of a Cd(II)----(-)S- charge-transfer band at 255 nm. The 113Cd NMR spectrum of 113Cd(II)-p10 reveals one signal at delta = 648 ppm. This chemical shift correlates well with that predicted for ligation of 113Cd(II) to three -S- from the three Cys residues of p10. The chemical shift of 113Cd(II)-p10 changes by only 4 ppm upon binding of d(pA)6, indicating that the chelate complex is little changed by oligonucleotide binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Site-specific methylation of a strategic lysyl residue in aspartate aminotransferase.
Conditions for reductive methylation of amine groups in proteins using formaldehyde and cyanoborohydride can be chosen to modify selectively the active site lysyl residue of aspartate aminotransferase among the 19 lysyl residues in each subunit of this protein. Apoenzyme must be treated, under mildly acidic conditions (pH = 6), at a relatively low molar ratio of formaldehyde to protein (40:1); and, upon reduction with sodium cyanoborohydride, 85% of the formaldehyde is incorporated at Lysine 258 and 15% at the amino-terminal alanyl residue. The modified protein, characterized after tryptic hydrolysis, separation of the peptides by high performance liquid chromatography procedures and subsequent amino acid analysis, shows that lysine 258 is preferentially modified as a dimethylated derivative. Modified apoenzyme can accept and tightly bind added coenzyme pyridoxal phosphate, as measured by circular dichroism procedures. The methylated enzyme is essentially catalytically inactive when measured by standard enzymatic assays. On the other hand, addition of the substrate, glutamate, produces the characteristic absorption spectral shifts for conversion of the active site-bound pyridoxal form of the coenzyme (absorbance at 400 nm) to its pyridoxamine form (absorbance at 330 nm). Such a half-transamination-like process occurs as in native enzyme, albeit at several orders of magnitude lower rate. This event takes place even though the characteristic internal holoenzyme Schiff's base between Lys-258 and aldehyde of bound pyridoxal phosphate does not exist in methylated, reconstituted holoenzyme. It is concluded that this chemically transformed enzyme can undergo a half-transamination reaction with conversion of active site-bound coenzyme from a pyridoxal to a pyridoxamine form, even when overall catalytic turnover transamination cannot be detected.
Amino Acid Sequence↗
Clustering of primary afferent fibers in peripheral nerve fascicles by sensory modality.
The spatial organization of cutaneous afferent fibers in the cat saphenous nerve was studied by recording from functionally identified units in split filaments. It was found that within each fascicle, fibers tend to be clustered together with others of like modality; they are not randomly distributed. These results suggest that the sensory modality of primary afferent neurons is determined prior to their innervation of the skin.
Adaptation, Physiological↗
Sympathetic activation of unmyelinated mechanoreceptors in cat skin.
In single unit recordings from the saphenous nerve in anesthetized cats, afferent units were functionally identified as unmyelinated (C) mechanoreceptors. All units responded to stimulation of the sympathetic trunk with a repetitive discharge which was maintained throughout the period of stimulation. This discharge was asynchronous with respect to efferent activity and was unaffected by brief occlusion of the arterial blood supply. The mechanism underlying the strong excitatory action of the sympathetic system on these afferents is not known although changes in blood flow and temperature can be ruled out. It is suggested that the primary function of these afferents may be to signal conditions within the skin for regulatory purposes rather than to encode external mechanical events.
Animals↗
Sympathetically induced changes in the responses of slowly adapting type I receptors in cat skin.
The effects of sympathetic efferent activity on slowly adapting Type I receptors in the hairy skin of cats were studied by recording from single afferent units in the saphenous nerve. Stimulation of the sympathetic trunk at 10 Hz had predominantly excitatory effects, which were seen in some units as the development of a background discharge in the absence of overt mechanical stimulation, or in most other units as a reduction in the threshold for mechanical activation. These effects generally persisted throughout the 3-min period of sympathetic stimulation (SS). The percentage of afferent units that began to discharge during SS was significantly greater in female cats than in males (53% vs. 19%). An increase in the force exerted by the skin on the stimulus probe was also observed during SS. Several tests were conducted to assess possible neurochemical and mechanical mechanisms of action. Administration of the alpha-adrenergic blocking agent phentolamine produced a marked reduction in the sympathetic effects. However, histochemical analysis of sections from the touch domes showed no catecholamine fluorescence near the sensory fibers. Cessation of local blood flow just prior to SS, produced by occlusion of the descending aorta, had no apparent effect on the sympathetically induced changes in afferent activity. It was concluded that sympathetic activity has an excitatory action on most Type I afferents in the cat. Because this sympathetic action is neither replicated nor altered by aortic occlusion, it appears not to be mediated by changes in blood flow. It also appears not to be mediated by direct neurotransmitter action on the sensory receptor, because no catecholamine fluorescence was observed, yet the action was blocked by an alpha-adrenergic blocker. It is likely, therefore, that this sympathetic action is mediated by some unidentified mechanical response within the skin.
Adaptation, Physiological↗
Sympathetic activation of A-delta nociceptors.
Primary afferent units in the saphenous nerve of cats, functionally identified as A-delta myelinated nociceptors, were tested for their responses to stimulation of the sympathetic trunk. The units were subdivided functionally into A-mechano-heat receptors (AMHs), which respond to both noxious heat and pressure, and high-threshold mechanoreceptors (HTMs), which respond only to pressure. No units of either subdivision were activated by sympathetic stimulation (SS) prior to noxious heating of their receptive fields. However, six of the seven AMH units with the highest mechanical thresholds (greater than 5 g von Frey) were activated by SS alone (10 Hz) after they had been sensitized by noxious heating of their receptive fields. Sensitized AMH units with lower mechanical thresholds (less than 5 g) were generally not activated by SS alone (1 of 22 units), and their responses to warming of their receptive fields were not altered by SS. The excitatory sympathetic action on AMH units was abolished by alpha- but not beta-andrenergic blockade in the two units tested. HTMs were unresponsive to SS even after repeated noxious heating of their receptive fields (15 units tested). The results of this study indicate that relatively high rates of sympathetic efferent activity (10 Hz) can induce firing in a small population of AMH receptors in damaged skin, specifically those units with high mechanical thresholds. This sympathetically evoked activity might trigger or exacerbate pain associated with skin damage; however, functional conclusions are difficult to draw, because of the scarcity of such units and the fact that the responses in some were brief and of low firing rates.
Animals↗
Histochemical evidence for sympathetic innervation of hair receptor afferents in cat skin.
The goal of the present study was to demonstrate histochemically fine catecholamine-containing sympathetic nerve fibers which might be associated with hair receptor afferent fibers in cat skin. Positive results would be consistent with the hypothesis that the sympathetically mediated desensitization of hair afferents, observed in this an earlier studies, is mediated by direct neurotransmitter release onto the afferent fibers. Activity in single primary afferent fibers was recorded electrophysiologically during mechanical stimulation of hairs and electrical stimulation of the lumbar sympathetic trunk in anesthetized cats. The mechanical threshold for activation was seen to increase markedly during low-frequency sympathetic stimulation. Fresh-frozen sections of skin from cat hindleg were treated with glyoxylic acid for detection of fluorescent catecholamines. Fluorescent fibers were observed in association with hair follicles as well as with arterioles and arrector-pili muscles. Subsequent silver staining of the identical skin sections revealed that the fine catecholamine-containing fibers located near hair follicles were closely associated with hair receptor afferent fibers. The results of hair receptor sensitivity is mediated by direct neurotransmitter release onto the afferent fibers.
Adrenergic Fibers↗
Efficacy of rubella vaccination.
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Adolescent↗
Climbing fiber responses of cerebellar Purkinje cells to passive movement of the cat forepaw.
The activity of cerebellar Purkinje cells during controlled and passive movement of the forepaw was studied in the cat. Burst responses characteristic of activation by climbing fibers were observed in Purkinje cells in lobules Vb and Vc of the cerebellar vermis and paravermis. The climbing fiber responses followed the onset of a movement with a latency ranging from 20 to 60 msec depending upon movement type and amplitude. Responsive Purkinje cells were localized in a well defined parasagittal strip very near the paravermal vein in lobules Vb and Vc. Cells within the responsive strip responded with identical response probabilities and latencies for any particular type of movement presentation. Responses were independent of starting paw position and direction of movement. Climbing fiber responses could be evoked by extremely small movements with most cells responding to displacements of 50 mum. The latencies and probabilities for climbing fiber responses were inversely related to movement amplitude with latencies as long as 80 msec for very small displacements.
Animals↗
Effects from the vestibulospinal tract on transmission from primary afferents to ventral spino-cerebellar tract neurones.
Convergence of vestibulospinal and segmental effects onto spinal interneurones which project to the ventral spino-cerebellar tract (VSCT) neurones has been studied by intracellular recording in VSCT cells. The disynaptic Ia IPSPs evoked in a group of VSCT neurones from the quadriceps nerve are monosynaptically facilitated by the vestibulospinal tract while there was no facilitation of Ia IPSP evoked from a flexor nerve. These results support the view that Ia inhibition to VSCT cells and motoneurones is mediated by common interneurones. The disynaptic inhibition evoked in other VSCT cells from the vestibulospinal tract is facilitated by volleys in the contralateral flexor reflex afferents (FRA) or bilaterally from the FRA. It is postulated that these actions are mediated by collaterals of the interneurones responsible for the analogous effects in motoneurones. Findings are reported suggesting that the monosynaptic vestibulospinal EPSP in VSCT cells in most cases is collateral to the excitatory input to the last order interneurones of reflex pathways from the FRA to motoneurones and only exceptionally to the corresponding input to Ia inhibitory interneurones. In many VSCT cells the vestibulospinal tract evoked disynaptic EPSPs which are facilitated from the FRA; the functional significance of this action is uncertain. The results are consistent with the hypothesis that VSCT neurones signal information on interneuronal transmission to motoneurones.
Afferent Pathways↗
Transfer characteristics between EMG activity and muscle tension under isometric conditions in man.
The relationship between motor unit activity and a voluntarily produced, sinusoidally modulated isometric tension was evaluated as a function of the modulation frequency. These date are reported in terms of the gain and phase difference of the motor activity (input) and tension (output) relationship, the gain being the logarithmic ratio of the amplitudes of the output and input sinusoids. It was found that an increase in the modulation amplitude of the motor unit activity was required to produce the same amount of modulation of the output tension as the modulation frequency was increased. For example, the modulation amplitude of the activity is about twice as much at 2 Hz as at 0.25 Hz and about 4 times as much at 5 Hz. It was also found that the maximum tension which could be produced voluntarily during brief jerks at 5 Hz was the same as the maximum sustained tension which could be attained. This latter finding emphasizes the importance of recruitment and especially synchronization of motor unit activity to the gradation of output tension.
Action Potentials↗
Effects on the ventral spinocerebellar tract neurones from Deiters' nucleus and the medial longitudinal fascicle in the cat.
Effects from the vestibulospinal tract (VST) and from fibres descending in the medial longitudinal fascicle (MLF) on the cells of origin of the ventral spinocerebellar tract (VSCT) have been studied with intracellular recording. Out of 110 VSCT neurones, the VST evoked monosynaptic EPSPs in 27, di- or polysynaptic EPSPs in 56 and disynaptic IPSPs in 26. In 93 tested VSCT cells, MLF stimulation evoked monosynaptic EPSPs in 26, monosynaptic IPSPs in 2, di- or polysynaptic EPSPs in 25 and disynaptic IPSPs in 21. Convergence of monosynaptic EPSPs from VST and MLF was found in a small proportion of cells whereas the two descending pathways evoked reciprocal effects in another small group of neurones. Convergence of monosynaptic EPSPs from VST or MLF and from group I afferents was also modest. In 9 VSCT neurones there was convergence of monosynaptic excitation and disynaptic inhibition from the vestibulospinal tract and the same pattern from MLF was recorded in 9 neurones. The results are discussed in view of the hypothesis that VSCT neurones carry information on the interneuronal ttransmission in the spinal cord.
Action Potentials↗
A long propriospinal system with direct effect on motoneurones and on interneurones in the cat lumbosacral cord.
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Animals↗
Analysis of threshold currents during microstimulation of fibres in the spinal cord.
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Action Potentials↗