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The development of neuronal excitability involves the coordinated expression of different voltage-gated ion channels. We have characterized the expression of two sensory neuron-specific tetrodotoxin-resistant sodium channel alpha subunits, Na(v)1. (SNS/PN3) and Na(v)1.9 (SNS2/NaN), in developing rat lumbar dorsal root ganglia (DRGs). Expression of both Na(v)1.8 and Na(v)1.9 increases with age, beginning at embryonic day (E) 15 and E17, respectively, and reaching adult levels by postnatal day 7. Their distribution is restricted mainly to those subpopulations of primary sensory neurons in developing and adult DRGs that give rise to unmyelinated C-fibers (neurofilament 200 negative). Na(v)1.8 is expressed in a higher proportion of neuronal profiles than Na(v)1.9 at all stages during development, as in the adult. At E17, almost all Na(v)1.8-expressing neurons also express the high-affinity NGF receptor TrkA, and only a small proportion bind to IB4, a marker for c-ret-expressing (glial-derived neurotrophic factor-responsive) neurons. Because IB4 binding neurons differentiate from TrkA neurons in the postnatal period, the proportion of Na(v)1.8 cells that bind to IB4 increases, in parallel with a decrease in the proportion of Na(v)1.8-TrkA co-expressing cells. In contrast, an equal number of Na(v)1.9 cells bind IB4 and TrkA in embryonic life. The differential expression of Na(v)1.8 and Na(v)1.9 in late embryonic development, with their distinctive kinetic properties, may contribute to the development of spontaneous and stimulus-evoked excitability in small diameter primary sensory neurons in the perinatal period and the activity-dependent changes in differentiation they produce.

Sensory neurones co-express voltage-gated sodium channels that mediate TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) currents, which may contribute to chronic pain after nerve injury. We previously demonstrated that TTX-R channels were decreased acutely in human sensory cell bodies after central axotomy, but accumulated in nerve terminals after peripheral axotomy. We have now studied the TTX-S channels PN1 and Brain III, using specific antibodies for immunohistochemistry, in dorsal root ganglia (DRG) from 10 patients with traumatic central axotomy, nerves from 16 patients with peripheral axotomy, and controls. PN1 showed temporal changes similar to the TTX-R channels in sensory cell bodies of injured DRG. In contrast, Brain III was found only in injured nerves (not control nerves, or control/central axotomy DRG). PNI and Brain III are distinct targets for novel analgesics.

Voltage-gated sodium channels consist of a pore-containing alpha-subunit and one or more auxiliary beta-subunits, which may modulate channel function. We previously demonstrated that sodium channel SNS/PN3 alpha-subunits were decreased in human sensory cell bodies after spinal root avulsion injury, and accumulated at injured nerve terminals in pain states. Using specific antibodies for immunohistochemistry, we have now detected sodium channel beta1 and beta2 subunits in sensory cell bodies within control human postmortem sensory ganglia (78% of small/medium (< or = 50 microm) and 68% of large (> or = 50 microm) cells); their changes in cervical sensory ganglia after avulsion injury paralleled those described for SNS/PN3 alpha-subunits. Our results suggest that alpha- and beta-subunits share common regulatory mechanisms, but present distinct targets for novel analgesics.

The voltage-gated 'glial' sodium channel NaG belongs to a distinct molecular class within the multi-gene family of mammalian sodium channels. Originally found in central and peripheral glia, NaG has since been detected in neurons in rat dorsal root ganglia (DRG) and may play a role in Schwann cell-axon interactions. We have studied the presence of NaG-like immunoreactivity in the intact and injured human peripheral nervous system using a specific affinity-purified antibody. Nerve fibres in normal and injured peripheral nerves and normal skin exhibited intense NaG-immunoreactivity. Numerous NaG-immunoreactive nerve fibres surrounded neuronal cell bodies within postmortem control DRG, and in DRG avulsed from the spinal cord (i.e. after traumatic central axotomy). There were no significant differences in the pattern of NaG immunostaining between control and avulsed DRG, or with delay after injury. Generally, the neuronal cell bodies were only very weakly immunoreactive to NaG, indicating that the NaG immunoreactivity was predominantly in Schwann cells/myelin. In accord, we demonstrated NaG immunostaining in cultured human and rat Schwann cells, and in distal nerve after wallerian degeneration. NaG thus appears to be a useful new marker for Schwann cells in the human PNS, and a role in neuropathy deserves investigation.

The sodium channels SNS/PN3 and NaN/SNS2 are regulated by the neurotrophic factors-nerve growth factor (NGF) and glial-derived neurotrophic factor (GDNF), and may play an important role in the development of pain after nerve injury or inflammation. These key molecules have been studied in an amputated causalgic finger and control tissues by immunohistochemistry. There was a marked increase in the number and intensity of SNS/PN3-immunoreactive nerve terminals in the affected finger, while GDNF-immunoreactivity was not observed, in contrast to controls. No differences were observed for NGF, trk A, NT-3 or NaN/SNS2-immunoreactivity. While further studies are required, these findings suggest that accumulation of SNS/PN3 and/or loss of GDNF may contribute to pain in causalgia, and that selective blockers of SNS/PN3 and/or rhGDNF may provide effective novel treatments., (Copyright 2001 European Federation of Chapters of the International Association for the study of Pain.)

The adverse effects of combination chemotherapy with paclitaxel and carboplatin, including neurotoxicity, arthralgia and muscle pain, were evaluated in 21 patients (30 courses) using questionnaires of the Gynecologic Oncology Chemotherapy Joint Research Group. The scores of pain and numbness peaked from the third to fourth day of treatment. Although the pain score improved subsequently, the numbness score persisted at a high level. Compared to the first and second courses, the peak pain score was higher and persisted for a longer duration in the fifth and sixth courses. Using the questionnaires, we were able to recognize a high incidence of numbness and pain in patients on combination chemotherapy with paclitaxel and carboplatin, and identify the degree and temporal changes of the adverse effects. Our results suggest that the questionnaires used in this study are clinically useful for evaluating the degree and clinical course of pain and numbness in anticancer chemotherapy.

The zebrafish is an important vertebrate model for the mutational analysis of genes effecting developmental processes. Understanding the relationship between zebrafish genes and mutations with those of humans will require understanding the syntenic correspondence between the zebrafish and human genomes. High throughput gene and EST mapping projects in zebrafish are now facilitating this goal. Map positions for 523 zebrafish genes and ESTs with predicted human orthologs reveal extensive contiguous blocks of synteny between the zebrafish and human genomes. Eighty percent of genes and ESTs analyzed belong to conserved synteny groups (two or more genes linked in both zebrafish and human) and 56% of all genes analyzed fall in 118 homology segments (uninterrupted segments containing two or more contiguous genes or ESTs with conserved map order between the zebrafish and human genomes). This work now provides a syntenic relationship to the human genome for the majority of the zebrafish genome.

Reelin is a key mediator of ordered neuronal alignment in the brain. Here, we demonstrate that Reelin molecules assemble with each other to form a huge protein complex both in vitro and in vivo. The Reelin-Reelin interaction clearly is inhibited by the function-blocking anti-Reelin antibody, CR-50, at a concentration known to inhibit Reelin function. This assembly is mediated by electrostatic interaction of the CR-50 epitope region. Recombinant CR-50 epitope fragments spontaneously constitute a soluble, string-like homopolymer with a regularly repeated structure composed of more than 40 monomers. Mutated Reelin, which lacks the CR-50 epitope region, cannot form a homopolymer and fails to induce efficient tyrosine phosphorylation of Disabled 1 (Dab1), which should occur to transduce the Reelin signal. These data suggest that Reelin exerts its biological function by composing a large protein assembly driven by the CR-50 epitope region, proposing a novel model of the Reelin signaling in neurodevelopment.

The differential distribution of two tetrodotoxin resistant (TTXr) voltage-gated sodium channels SNS (PN3) and SNS2 (NaN) in rat primary sensory neurons has been investigated. Both channels are sensory neuron specific with SNS2 restricted entirely to those small dorsal root ganglion (DRG) cells with unmyelinated axons (C-fibers). SNS, in contrast, is expressed both in small C-fiber DRG cells and in 10% of cells with myelinated axons (A-fibers). All SNS expressing A-fiber cells are Trk-A positive and many express the vanilloid-like receptor VRL1. About half of C-fiber DRG neurons express either SNS or SNS2, and in most, the channels are colocalized. SNS and SNS2 are found both in NGF-responsive and GDNF-responsive C-fibers and many of these cells also express the capsaicin receptor VR1. A very small proportion of small DRG cells express either only SNS or only SNS2. At least four different classes of A- and C-fiber DRG neurons exist, therefore, with respect to expression of these sodium channels.

The tetrodotoxin-resistant (TTX-R) voltage-gated sodium channel SNS/PN3 and the newly discovered NaN/SNS2 are expressed in sensory neurones, particularly in nociceptors. Using specific antibodies, we have studied, for the first time in humans, the presence of SNS/PN3 and NaN/SNS2 in peripheral nerves, including tissues from patients with chronic neurogenic pain. In brachial plexus injury patients, there was an acute decrease of SNS/PN3- and NaN/SNS2-like immunoreactivity in sensory cell bodies of cervical dorsal root ganglia (DRG) whose central axons had been avulsed from spinal cord, with gradual return of the immunoreactivity to control levels over months. In contrast, there was increased intensity of immunoreactivity to both channels in some peripheral nerve fibers just proximal to the site of injury in brachial plexus trunks, and in neuromas. These findings suggest that the expression of these sodium channels in neuronal cell bodies is reduced after spinal cord root avulsion injury in man, but that pre-synthesized channel proteins may undergo translocation with accumulation at sites of nerve injury, as in animal models of peripheral axotomy. The latter may contribute to positive symptoms, as our patients all showed a positive Tinel's sign. Nerve terminals in distal limb neuromas and skin from patients with chronic local hyperalgesia and allodynia all showed marked increases of SNS/PN3-immunoreactive fibers, but little or no NaN/SNS2-immunoreactivity, suggesting that the former may be related to the persistent hypersensitive state. Axonal immunoreactivity to both channels was similar to control nerves in sural nerve biopsies in a selection of neuropathies, irrespective of nerve inflammation, demyelination or spontaneous pain, including a patient with congenital insensitivity to pain. Our studies suggest that the best target for SNS/PN3 blocking agents is likely to be chronic local hypersensitivity.

Immunocytochemical and morphometric techniques were used to quantify the distribution of cyclooxygenase (cox)-containing neurons in rat L5 dorsal root ganglia (DRG). Cox-1 immunolabelling was almost exclusively restricted to small diameter DRG neurons (< 1000 microm2), and was extensively colocalized with calcitonin gene-related peptide (CGRP) and isolectin B4 (IB4). Cox-1 was present in 65% and 70% of CGRP- and IB4-labelled neurons, respectively. Cox-1 labelling was also found in neurons expressing the sensory neuron-specific (SNS) Na+ channel. Cox-2 labelling was absent in DRG from normal rats. In the Freund's adjuvant model of monoarthritis, the proportion of cox-1-positive DRG neurons was unchanged and no neurons were found to be labelled for cox-2. In primary tissue culture, cox-1 immunolabelling persisted in vitro for up to 9 days and was present in morphologically identical neurons. The selective expression of cox-1 in peripheral ganglia was confirmed by the small number of nodose ganglion neurons and superior cervical ganglion (SCG) neurons labelled for cox-1. These data suggest that cox-1 is a marker for a subpopulation of putative nociceptive neurons in vitro and in vivo, and suggests that the prostaglandins synthesized by these neurons may be important for nociceptor function. These data may have important implications for the mode and mechanism of action of non-steroidal anti-inflammatory drugs (NSAIDs).

The effect of substituting Pro25, located in the alpha-helical region of the cystatin A structure, with Ser has been studied. The structures of wild type and P25S cystatin A were determined by multidimensional NMR spectroscopy under comparable conditions. These two structures were virtually identical, and the alpha-helix between Glu15-Lys30 exists with uninterrupted continuity, with a slight bend at residue 25. In order to characterize the possible substitution effects of Pro25 with Ser on the alpha-helix, the chemical shifts of the amide nitrogens and protons, the generalized order parameters obtained by the analyses of the 15N-1H relaxation data, the amide proton exchange rates, and the NOE networks among the alpha-helical and surrounding residues were carefully compared. None of these parameters indicated any significant static or dynamic structural differences between the alpha-helical regions of the wild-type and P25S cystatin A proteins. We therefore conclude that our previous structure of the wild-type cystatin A, in which the alpha-helix exhibited a sharp kink at Pro25, must be revised. The asymmetric distribution of hydrophobic interactions between the side-chain residues of the alpha-helix and the rolled beta-sheet surface, as revealed by NOEs, may be responsible for the slight bend of the alpha-helix in both variants and for the destabilized hydrogen bonding of the alpha-helical residues that follow Pro25/Ser25, as evidenced by increased amide exchange rates.

Using in situ hybridization and immunochemical methods, we have observed an increase in the expression of SNS/PN3 sodium channel mRNA and protein in cerebellar Purkinje cells of the taiep rat. These changes are present in taiep rats at 12 months of age, following loss of myelin, but not at one month, prior to loss of myelin. Increased SNS/PN3 expression is not associated with aging per se, because it was not observed in control rats at 12 months of age. These results suggest that altered sodium channel expression in Purkinje cells may contribute to the ataxia that occurs in taiep rats.

A mutant lysozyme where R14 and H15 are deleted together has higher activity and a similar binding ability to an inhibitor, trimer of N-acetylglucosamine ((NAG)3), compared with wild-type lysozyme. Since this has been attributed to intrinsic protein dynamic properties, we investigated the relationship between the activity and the internal motions of proteins. Backbone dynamics of the free and the complex forms with the (NAG)3 have been studied by measurement of the 15N T1 and T2 relaxation rates and NOE determinations at 600 MHz. Analysis of the data using the model-free formalism showed that the generalized order parameters (S2) were almost the same in wild-type and mutant lysozyme in unbound state, indicating that the mutation had little effect on the global internal motions. On the other hand, in the presence of (NAG)3, although some signals located around the active site were broadened or decreased in intensity because of strong perturbation by (NAG)3, there were several residues that showed increased or decreased backbone S2 in the complexed lysozymes. A comparison of the internal motions of the wild-type and mutant complexes showed a number of distinct dynamic differences between them. In particular, many residues located at or near active-site regions (turn 1, strand 2, turn 2 and long loop), displayed greater backbone dynamics reflecting the order parameter in mutant complex relative to mutant free. Furthermore, the Rex values at the loop C-D region, which was considered to be important for enzymatic activity, significantly increased. From these results, it was suggested that variations in the dynamics of these regions may play an important role in the enzyme activity., (Copyright 1999 Academic Press.)

Fibroblast growth factors (FGFs) bind to extracellular matrices, especially heparin-like carbohydrates of heparan-sulfate proteoglycans which stabilize FGFs to protect against inactivation by heat, acid, proteolysis and oxidation. Moreover, binding of FGFs to cell surface proteoglycans promotes to form oligomers, which is essential for receptor oligomerization and activation. In the present study, we determined the solution structure of acidic FGF using a series of triple resonance multi-dimensional NMR experiments and simulated annealing calculations. Furthermore, we prepared the sample complexed with a heparin-derived hexasaccharide which is a minimum unit for aFGF binding. From the chemical shift differences between free aFGF and aFGF-heparin complex, we concluded that the major heparin binding site was located on the regions 110-131 and 17-21. The binding sites are quite similar to those observed for bFGF-heparin hexasaccharide complex, showing that both FGFs recognize heparin-oligosaccharides in a similar manner.

Streptomyces metalloproteinase inhibitor (SMPI) is the only inhibitor to show "standard mechanism inhibition" against metalloproteinases. SMPI is a globular protein with an exposed loop containing the reactive site, C64-V65. To analyze the importance of basic residues in the reactive site loop of SMPI, mutants were constructed for R60, K61, and R66 (R60A, K61A, R66A, R60/K61A, 60/61/66A, and 60/61/66E). The mutants involving only R60, K61, and R60/K61 residues, respectively, showed strong inhibitory activity and were stable against enzyme activity. Both the triple mutants showed very weak inhibitory activity and underwent rapid degradation. The addition of basic residues to the loop (V62R and T63R) did not cause any further increase in inhibitory activity. These results suggest that basic residues in the reactive site loop play some role in maintaining a stable enzyme-inhibitor complex. The R66 mutant showed reduced activity and was rapidly degraded by enzymes. It was concluded that R66 is essential for maintaining a strong hydrophobic interaction with the S1' hydrophobic pocket of the enzyme. To investigate the roles of the disulfide bridge and the P68 residue near the reactive site, C64/69S and P68T mutants were constructed. These mutants showed very weak inhibitory activity and were rapidly degraded by enzymes. These results suggest that the disulfide bridge and P68 residue are very essential for SMPI to function as an inhibitor.

An 8.6 kDa protein, which the authors call a modifin, has been purified from Methylococcus capsulatus (Bath) and has been shown to alter the substrate specificity and kinetics of NAD+-linked formaldehyde dehydrogenase (FDH) isolated from the same organism. Purification methods for both the modifin and FDH are presented which reliably produced pure protein for further analysis. Analysis of the molecular mass and N-terminal sequence of both FDH and the modifin indicate that they are unique proteins and show no similarity to alcohol or aldehyde dehydrogenase enzymes isolated from methylotrophic bacteria. Substrate specificity studies demonstrated that FDH oxidized formaldehyde exclusively in the presence of the modifin; a diverse range of aldehydes and alcohols were oxidized by FDH in the absence of the modifin. No formaldehyde oxidation was detected in the absence of the modifin. Attempts to replace the modifin with glutathione or high concentrations of methanol to stimulate formaldehyde oxidation failed. With acetaldehyde as substrate, FDH showed standard Michaelis-Menten kinetics; interaction of FDH with the modifin using formaldehyde as substrate altered the kinetics of the reaction to sigmoidal. Kinetic analysis during turnover experiments indicated that the FDH may be associated with bound formaldehyde following enzyme isolation and that NAD may also be associated with the enzyme but in a form that is less tightly bound than found with the methanol dehydrogenase from Bacillus methanolicus. Data are presented which indicate that the modifin may play an important role in regulating formaldehyde concentration in vivo.

The Streptomyces metalloproteinase inhibitor, SMPI, isolated from Streptomyces nigrescens TK-23, is a proteinaceous metalloproteinase inhibitor, and consists of 102 amino acid residues with two disulfide bridges. SMPI specifically inhibits metalloproteinases such as thermolysin. In the present work, the solution structure of SMPI was determined on the basis of 1536 nuclear Overhauser enhancement derived distance restraints and 52 dihedral angle restraints obtained from three-bond spin coupling constants. The final ensemble of 20 NMR structures overlaid onto their mean coordinate with backbone (N, Calpha, C') r.m.s.d. values of 0. 45(+/-0.11) A and 0.57(+/-0.18) A for residues 6 to 99 and the entire 102 residues, respectively. SMPI is essentially composed of two beta-sheets, each consisting of four antiparallel beta-strands. The structure can be considered as two Greek key motifs with 2-fold internal symmetry, a Greek key beta-barrel. One unique structural feature found in SMPI is in its extension between the first and second strands of the second Greek key motif. Interestingly, this extended segment is known to be involved in the inhibitory activity of SMPI. In the absence of sequence similarity, the SMPI structure shows clear similarity to both domains of the eye lens crystallins, both domains of the calcium sensor protein-S, as well as the single-domain yeast killer toxin. The yeast killer toxin structure was thought to be a precursor of the two-domain beta gamma-crystallin proteins, because of its structural similarity to each domain of the beta gamma-crystallins. SMPI thus provides another example of a single-domain protein structure that corresponds to the ancestral fold from which the two-domain proteins in the beta gamma-crystallin superfamily are believed to have evolved., (Copyright 1998 Academic Press)

SMPI is a proteinaceous microbial metalloproteinase inhibitor that was isolated from Streptomyces nigrescens TK-23 in 1979. SMPI is known to selectively inhibit the metalloproteinases in the gluzincin family, according to the Rawling and Barrett classification. There has been no report on the interaction of a metalloproteinase in the family of gluzincins with its specific proteinaceous inhibitor. We have solved the solution structure of SMPI by NMR. Here, we report the binding mode of SMPI to thermolysin, based on the model complex structure generated using our high-resolution NMR structure of SMPI and the crystal structure of thermolysin. The obtained complex model shows that the extruded loop of SMPI, with the scissile bond Cys64-Val65, is complementary in shape to the active cleft of thermolysin. In the complex, the Cys64 (P1) carbonyl oxygen atom can form a tetrahedral coordination to the active zinc in thermolysin, and simultaneously, the methyl groups of Val65 (P1') are closely located in the hydrophobic S1' pocket in thermolysin. From the electrostatic potential surface calculation, the active loop of SMPI and the active cleft in thermolysin have been shown to be complementary in the surface charge distribution, resulting in the stabilization of the complex. The apparently large active loop is less flexible, but maintains a conformation in the nano- to picosecond time-scale, as elucidated from the 15N spin relaxation analysis. This is a quite different structural feature of SMPI from the flexible binding loop generally found in the serine proteinase inhibitors, such as SSI and eglin c, and can be related to the narrow specificity of SMPI. The present study provides the first insight into the interaction between a proteinaceous inhibitor and a gluzincin metalloproteinase., (Copyright 1998 Academic Press.)

The heat shock protein (HSP) 27 is constitutively expressed at low levels in medium-sized lumbar dorsal root ganglion (DRG) cells in adult rats. Transection of the sciatic nerve results in a ninefold upregulation of HSP27 mRNA and protein in axotomized neurons in the ipsilateral DRG at 48 hr, without equivalent changes in the mRNAs encoding HSP56, HSP60, HSP70, and HSP90. Dorsal rhizotomy, injuring the central axon of the DRG neuron, does not upregulate HSP27 mRNA levels. After peripheral axotomy, HSP27 mRNA and protein are present in small, medium, and large DRG neurons, and HSP27 protein is transported anterogradely, accumulating in the dorsal horn and dorsal columns of the spinal cord, where it persists for several months. Axotomized motor neurons also upregulate HSP27. Only a minority of cultured adult DRG neurons are HSP27-immunoreactive soon after dissociation, but all express HSP27 after 24 hr in culture with prominent label throughout the neuron, including the growth cone. HSP27 differs from most axonal injury-regulated and growth-associated genes, which are typically present at high levels in early development and downregulated on innervation of their targets, in that its mRNA is first detectable in the DRG late in development and only approaches adult levels by postnatal day 21. In non-neuronal cells, HSP27 has been shown to be involved both in actin filament dynamics and in protection against necrotic and apoptotic cell death. Therefore, its upregulation after adult peripheral nerve injury may both promote survival of the injured neurons and contribute to alterations in the cytoskeleton associated with axonal growth.

Fourier transform ion cyclotron resonance mass spectra of 13C,15N-doubly depleted cystatin A M65L, produced by Escherichia coli grown on 99.9% [12C]glucose and 99.99% [14N]ammonium sulfate, showed salient monoisotopic peaks composed of 12C and 14N. Collision-induced dissociation spectra were obtained by increasing the capillary-skimmer potential for the electrospray ionization and by extending the trapping time in a radio frequency-only hexapole ion guide. Fragment ions in the spectra could be readily assigned to the amino acid sequence, owing to their markedly improved resolution and sensitivity as compared to those with the natural isotopic composition. Detailed analyses of the fragmentation patterns, facilitated by the use of 13C,15N-doubly depleted proteins, enabled the assignment of approximately 180 fragment ions to the sequence, while natural isotopic cystatin A allowed the assignment of approximately 110 fragment ions. Interestingly, no fragmentation was detected between residues 50-61 and 62-67, which are stretches known to be involved in the antiparallel beta-sheet at the center of the protein.

Nearly complete 1H, 13C and 15N NMR assignments have been obtained for a doubly labeled 14-base pair DNA duplex in solution both in the free state and complexed with the uniformly 15N-labeled Antennapedia homeodomain. The DNA was either fully 13C, 15N-labeled or contained uniformly 13C, 15N-labeled nucleotides only at those positions which form the protein-DNA interface in the previously determined NMR solution structure of the Antennapedia homeodomain-DNA complex. The resonance assignments were obtained in three steps: (i) identification of the deoxyribose spin systems via scalar couplings using 2D and 3D HCCH-COSY and soft-relayed HCCH-COSY; (ii) sequential assignment of the nucleotides via 1H-1H NOEs observed in 3D 13C-resolved NOESY; and (iii) assignment of the imino and amino groups via 1H-1H NOEs and 15N-1H correlation spectroscopy. The assignment of the duplex in the 17 kDa protein-DNA complex was greatly facilitated by the fact that 1H signals of the protein were filtered out in 13C-resolved spectroscopy and by the excellent carbon chemical shift dispersion of the DNA duplex. Comparison of corresponding 13C chemical shifts of the free and the protein-bound DNA indicates conformational changes in the DNA upon complex formation.

Nearly complete (1)H, (13)C and(15) N NMR assignments have been obtained for a doubly labeled 14-base pair DNA duplex in solution both in the free state and complexed with the uniformly (15)N-labeled Antennapedia homeodomain. The DNA was either fully (13)C,(15)N-labeled or contained uniformly (13)C, (15)N-labeled nucleotides only at those positions which form the protein-DNA interface in the previously determined NMR solution structure of the Antennapedia homeodomain-DNA complex. The resonance assignments were obtained in three steps: (i) identification of the deoxyribose spin systems via scalar couplings using 2D and 3D HCCH-COSY and soft-relayed HCCH-COSY; (ii) sequential assignment of the nucleotides via(1) H-(1)H NOEs observed in 3D(13) C-resolved NOESY; and (iii) assignment of the imino and amino groups via (1)H-(1)H NOEs and(15) N-(1)H correlation spectroscopy. The assignment of the duplex in the 17 kDa protein-DNA complex was greatly facilitated by the fact that (1)H signals of the protein were filtered out in (13)C-resolved spectroscopy and by the excellent carbon chemical shift dispersion of the DNA duplex. Comparison of corresponding (13)C chemical shifts of the free and the protein-bound DNA indicates conformational changes in the DNA upon complex formation.

This paper describes a research study to investigate the efficacy of peppermint oil as a treatment for postoperative nausea. It uses a three-condition experimental design using statistical analysis to compare groups. The Kruskal-Wallis test was used to establish significance and the Mann-Whitney test to differentiate significance between the groups. The control, placebo and experimental groups of gynaecological patients were compared, using variables known to affect postoperative nausea. They were found to be homogeneous for the purposes of the study. A statistically significant differences was demonstrated on the day of operation, using the Kruskal-Wallis test, P = 0.0487. Using the Mann-Whitney test the difference was shown to be between the placebo and experimental group (U = 3; P = 0.02). The experimental group also required less traditional antiemetics and received more opioid analgesia postoperatively. The total cost of the treatment was 48 pence per person.

We report here the successful scale up of transient recombinant protein expression to litre scale using Semliki Forest Virus System. The expression of bacterial β-galactosidase was initially compared in BHK and CHO cells and the conditions for optimal infection of BHK cells were identified. 10% FCS in a medium at pH 6.9 and infection in small volumes were found to be optimal. A high MOI results in an increased recombinant protein yield. Stirring does not affect the infection process. Finally we applied these optimal conditions to the production of a microsomal enzyme, human cyclooxygenase-2 in suspension spinners. Five independant productions at the 1 litre scale yielded reproducible substantial amounts of recombinant protein (16 mg microsomal protein 10(9) cells(-1)) with an average specific activity of 3942 ± 765 pg PGE(2) μg(-1) microsomal protein 5 min(-1).

A method to prepare 5'-monodeuterated nucleosides with various S/R-ratios is described. 5-Oxopentose derivatives synthesized from glucose were converted into 5-monodeuterated pentose derivatives by LiAID4 in the presence of various ligands. The stereoselectivities of the deuteration reactions were investigated under a variety of conditions, and the S/R-ratios of the 5-monodeuterated pentoses varied from 4 : 1 to 1 : 7.4. By mixing these 5-monodeuterated pentose derivatives, we have successfully synthesized thymidine with a defined S/R-ratio at C5'.

A systematic synthesis of pyrimidine nucleosides with 13C-labels only at specific atoms in both the base and sugar moieties has been developed in order to obtain conformational information about the glycosidic bond through measurement of the accurate vicinal coupling constants between H1' and C2/C6. For this purpose, 13C labels at three positions, namely C1', C2, and C6, are essential. We have synthesized selectively multiply labeled nucleosides, such as [2, 1'-13C2]-thymidine, [2, 1'-13C2]-2'-deoxycytidine, [6, 1'-13C2]-thymidine, and [6, 1'-13C2]-2'-deoxycytidine. These nucleosides will be useful to determine the relative orientation of the base and sugar moieties.

(2'S,5'S)-(1',2',3',4',5'-13C5;2',5'-2H2-thymidine, prepared via labeled ribosyl thymidine as described in the accompanying paper, has been successfully converted into isotopically labeled dA, dG, and dC using enzymatic transdeoxyribosylation reactions.

To analyze direct effects of estrogen on gene expressions of inhibin subunits in vitro, the mRNA levels of inhibin alpha- and beta A-subunits were measured in rat granulosa cells cultured with FSH or estradiol. Through the culture process of the granulosa cells with serum-free medium, both the alpha- and beta A subunit mRNAs decreased, and were partially increased again by adding FSH to the culture medium. To examine whether these FSH effects are mediated via estrogen production, estradiol or tamoxifen was added to the cultured granulosa cells. After 36-h culture with estradiol, the inhibin alpha-subunit decreased but the inhibin beta A-subunit increased, in a dose-responsive manner. Tamoxifen showed completely opposite effects to estradiol, and a combination of estradiol and tamoxifen resulted in similar levels of inhibin-alpha and -beta A mRNAs to the control. These results indicate that estrogen would by a certain pathway affect gene expressions of the inhibin subunits in the rat granulosa cells, and may regulate the production of inhibin and activin through the paracrine system.

The management and nursing care of patients experiencing postoperative nausea and vomiting (PONV) are discussed, together with the various types of pharmacological and complementary approaches to treatment. These treatments are examined in relation to the causes of PONV and a scientific approach to the use of antiemetics which links causation and treatment is suggested. It is argued that by adopting this approach PONV could be minimized, drug bills reduced, patient choice encouraged and postoperative recovery enhanced.

There is a vast amount of research available relating to postoperative nausea and vomiting (PONV). This research has been analyzed and summarized in a series of two articles which draw out the main factors influencing the incidence and affecting the treatment of the condition. This, the first article, will describe the physiological processes that result in PONV. An analysis will then follow of the many factors that contribute to the complex multifactorial aetiology of PONV. These factors include personal, preoperative, intraoperative and postoperative variables which affect differing neurotransmitter centres in the chemoreceptor trigger zone (CTZ) which is situated in the brainstem. These variables will then be related to the appropriate neurotransmitter centre allowing a greater understanding of the most likely physiological pathways involved in specific cases of PONV. The second article will explore the management and treatment of PONV.

The solution structure of a human cystatin A variant, cystatin A2-98 M65L, which maintains the full inhibitory activity of the wild-type protein, was determined at pH 3.8 by sD/3D heteronuclear double- and triple-resonance NMR spectroscopy. The structure is based on a total of 1343 experimental restraints, comprising 1139 distance, 154 phi and chi 1 torsion angle restraints, and 50 distance constraints for 25 backbone hydrogen bonds. A total of 15 structures was calculated using the YASAP protocol with X-PLOR, and the atomic rms distribution about the mean coordinate positions for residues 8-93 was 0.55 +/- 0.10 A for the backbone atoms and 1.05 +/- 0.11 A for all heavy atoms. The structure consists of five antiparallel beta-sheets and two short alpha-helices. Comparison with the X-ray structure of cystatin B in the papain complex shows that the conformation of the first binding loop is quite similar to that of cystatin A, with an rms deviation of 0.78 A for the backbone atoms in the 43-53 region (cystatin A numbering). The second binding loop, however, is significantly different in the two structures, with an rms deviation greater than 2 A. There are some other significant differences, especially for the N-terminal and alpha-helix regions. The overall structure of cystatin A is also compared with the recently reported NMR structure of the wild-type cystatin A (stefin A) at pH 5.5 (Martin et al., 1995) and reveals the following features. that differ in our structure from the previous one: (1) the N-terminal segment, which was unstructured in the previous report, folds over in close vicinity to the C-terminus, as revealed by the distinctive NOEs between those segments; (2) two discrete short alpha-helices linked by a type II reverse turn were found, instead of the continuous single alpha-helix with a slight kink shown in the previous structure; (3) the second binding loop, which was not well converged in the previous study at pH 5.5, is determined very well in our structure. The effect of the N-terminal truncation on the cystatin A structure was examined by comparing the 1H-15N HSQC spectrum of cystatin A2-98 with that of the cystatin A5-98 variant, which lacks the anti-papain activity, revealing significant chemical shift differences in the residual N-terminal segment and the first binding loop, together with small shifts in the other parts.(ABSTRACT TRUNCATED AT 400 WORDS)

A series of NH2-terminal truncated forms of human cysteine proteinase inhibitor, cystatin A, was prepared by genetic engineering using Escherichia coli harboring mutated genes. Each variant of cystatin A was efficiently expressed as a fused protein with porcine adenylate kinase and released by CNBr degradation after exchange of the sole inner Met to Leu. The mutant cystatin A lacking an amino-terminal Met residue (called standard variant starting from Ile2, CystA2-98(M65L) showed the same inhibitory activity as authentic one isolated from human epidermis. Two-residue truncation scarcely influenced the activity, but further truncations deleting Pro3 and beyond conservative Gly4 and Gly5 caused a remarkable decrease of their inhibitory activity. But little effect was observed by a substitution of Pro3 with Leu. The loss of the activity by amino-terminal truncation was compensated slightly by engineered substitution of Gly75 with His on a second loop. In the two-dimensional 15N-1H HSQC NMR spectrum, four-residue truncation was found to cause changes in the chemical shifts of Val47 and Val48, which locate on a first loop and consist of a conservative QVVAG sequence. Furthermore, the truncation led to a change in fluorescence spectroscopic behavior of Trp75, which was introduced as a probe on the second loop. Fluorescence intensity of the Trp of the truncated (5-98) form was more affected by heating than the active standard variant. Conversely, fluorescence of Trp75 in 2-98 form was more quenched by acrylamide than the 5-98 variant. Thus, the amino-terminal region of cystatin A is essential for the expression of its inhibitory activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The expression system for human recombinant cystatin A has already been established to be a fusion protein with porcine adenylate kinase in Escherichia coli [Kaji et al. (1990) Biol. Chem. Hoppe-Seyler 371, Suppl., 145-150]. After cyanogen bromide cleavage of the fused protein expressed in E. coli, the cystatin portion could be readily isolated. The inhibitory activity of the obtained variant (Cyst A (2-98)) was found to be almost identical with that of the wild type, and thereafter a mutation was introduced into this variant (Ctst A(2-98)), called the standard variant. To elucidate the role of the Gly-4 residue, which is completely conserved in all cystatin species, this residue was substituted with 17 other amino acids by means of cassette mutagenesis. Thus 17 variants (Cyst A(2-98)[G4X]) obtained were examined as to their inhibitory activity towards papain. As the side chain of the substituted amino acid residue became more bulky, the inhibitory activity of the variant markedly decreased. Variants whose side chains were bulkier than a Val residue showed almost no inhibitory effect towards papain. Consequently, it was deduced that the large side chain of a substituted amino acid may cause steric hindrance, which may be responsible for the decrease in inhibitory activity. Thus, we could conclude that the 4th (Gly) residue on cystatin A must be small, because amino acids which existed on the N-terminal side of this residue could interact with a papain molecule.

Oxidative stress and protein glycation are closely related processes that may contribute to the development of complications in diabetes mellitus. Treatment with antioxidants could protect against these processes at a biochemical level, and we have therefore investigated the effects of ascorbate and desferrioxamine treatment in the streptozotocin diabetic rat. Diabetic animals were given ascorbate 1 g/l in drinking water or desferrioxamine 6 mg/kg/day by subcutaneous injection and were killed after 6 weeks. In diabetic animals, oxidative stress was increased as shown by increased levels of conjugated dienes (CD) in plasma and malondialdehyde (MDA) in plasma, erythrocyte membranes, and urine. In addition, there was depletion of the nutritional antioxidants ascorbate, alpha-tocopherol, and retinol. Insulin treatment returned all of these parameters to normal. Ascorbate supplementation or desferrioxamine treatment alone failed to reduce oxidative stress, but a combination of both interventions restored MDA, CD, and antioxidant vitamins to control values. Both ascorbate and desferrioxamine also reduced HbA1c and glycated albumin levels. Treatment with antioxidants can reduce both oxidative stress and protein glycation and may help to reduce the risk of developing diabetic complications. However, ascorbate can have both prooxidant and antioxidant effects in vivo, and its use in pharmacological doses should be approached with caution.

We analyzed the lipids in the tissues of a patient with an atypical form of Farber disease who developed several clinical symptoms not seen in patients with typical Farber disease (acid ceramidase deficiency). Lipids were extracted from formalin-fixed brain, liver and kidney and purified by ion exchange and silica gel column chromatographies and further by high-performance liquid chromatography on a silica gel column. We performed structural and quantitative analyses of three lipids named lipids X, Y and Z. Lipid X accumulated in the liver but not in the brain. Accumulation of lipids Y and Z was observed in liver and kidney. The content of lipid Y in the patients liver was more than ten times that in a control. The structures of lipids X, Y and Z were confirmed by means of 1H-nuclear magnetic resonance spectroscopy, fast atom bombardment mass spectrometry, infrared absorption spectroscopy, and component analysis involving gas liquid chromatography and gas chromatography-mass spectrometry. The structures of lipids X, Y and Z were identified as those of ceramide, sulfatide and GM3 ganglioside, respectively. These results suggest two possibilities. One is that the accumulation of glycolipids such as sulfatide and GM3 ganglioside is a secondary event produced by the accumulation of ceramide due to ceramidase deficiency. The other is that the accumulation of glycolipids other than ceramide is due to a deficiency of sphingolipid activator proteins which may affect the degradation of sulfatide and GM3 ganglioside as well as ceramide.

Stereospecific deuteration of the C2' methylenes of 2'-deoxynucleosides together with 13C label has been found to open up various applications for structural studies of DNA oligomers in solution. Major problems in analyzing the structure and dynamics of larger DNA oligomers by NMR are associated with geminal proton pairs attached to the C2' and C5' of sugar moieties. We have employed, with a minor modification, existing synthetic routes to prepare stereospecific deuteration of the C2' methylene to prepare 13C/2H-doubly labeled nucleosides. For example, [ul-13C/15N] adenosine, which was prepared by microbial fermentation using [13C6]-glucose and [15N]-ammonium salt as precursors, was derived into (2'R)- and (2'S)-[ul-13C/15N;2'-2H1]-2'-deoxy-adenosines. Each of these multiply labeled nucleosides was then incorporated into a DNA dodecamer, 5'-d(CGCG AATTCGCG)-3', which was examined by various NMR techniques in order to evaluate the precision and accuracy of the NMR parameters obtained for the labeled moieties.

In order to distinguish between NMR signals for pro-R and pro-S protons of the 5'-methylene group, DNA oligomers containing [5'-2H]-2'-deoxynucleosides were synthesized. As the deuterium labeled nucleosides were mixtures of [(5'S)-2H]- and [(5'R)-2H]-isotopomers approximately in a ratio of 2:1, the signals du to pro-R and pro-S protons were assigned undoubt-edly.

The three-dimensional structure of erabutoxin b, a short-chain neurotoxic peptide purified from the venom of the sea snake Laticauda semifasciata, was determined in aqueous solution by two-dimensional proton nuclear magnetic resonance and simulated annealing-based calculations. On the basis of 883 assigned nuclear Overhauser effect (NOE) connectivities, 676 final distance constraints were derived and used together with 38 torsion angle (phi, chi 1) constraints, four distance constraints derived from disulfide bridges and 30 distance constraints derived from hydrogen bonds. A total of 14 converged structures were obtained from 50 runs of calculations. The atomic root-mean-square difference about the mean coordinate positions (excluding the residues 18 to 22) is 0.60 A for backbone atoms (N, C alpha and C'). The protein consists of a core region from which three finger-like loops emerge outwards. It includes a short, two-stranded antiparallel beta-sheet of residues 2 to 5 and 13 to 16, a three-stranded antiparallel beta-sheet involving residues 23 to 30, 35 to 41 and 50 to 56, and four disulfide bridges in the core region. Comparison with two crystal structures of erabutoxin b at 1.4 A and 1.7 A resolution indicated that the solution and the crystal structures were very similar, but less defined regions were observed at the localized region of the tip of the central loop and the outside of the third loop in solution. Other short-chain alpha-neurotoxins showed structural characteristics similar to those of erabutoxin b.

[13C5]-2-Deoxy-D-ribose, synthesized from [13C6]-D-glucose (98% 13C), was coupled with thymine to give [1',2',3',4',5'-13C5]-thymidine (T) in an 18% overall yield. The thymidine was converted to the 3'-phosphoramidite derivative and was then incorporated into a dodecamer 5'-d(CGCGAATTCGCG)-3' by solid-phase DNA synthesis. Preparation of 0.24 mumole of the labeled dodecamer, which is sufficient for a single NMR sample, consumed only 25 mg of glucose. By virtue of the 13C labels, all of the 1H-1H vicinal coupling constants in the sugar moieties were accurately determined by HCCH-E.COSY.

The human cholecystokinin B (CCKB) receptor has been isolated from a human temporal cortex cDNA library. Transient transfection of the receptor into COS-M6 cells resulted in high specific binding of 125I-sulphated CCK-8 labelled with Bolton and Hunter Reagent (KD = 31 pM). Competition experiments yielded the expected CCKB receptor ligand binding profile for agonists and antagonists. Similar results were obtained in human small cell lung carcinoma cells, which express an endogenous CCKB receptor. Extensive functional characterization of the receptor was performed in stably transfected HeLa cells using intracellular calcium imaging and microphysiometry techniques. Molecular analysis of the human CCKB receptor using Southern blotting of genomic DNA suggests the presence of a single gene for the CCKB receptor with no closely related homologues. This was confirmed by the polymerase chain reaction cloning of identical receptor coding sequences from human small cell lung carcinoma cells and human gastric enterochromaffin-like cell-oma (ECLoma) tissue.

The OmpR protein of Escherichia coli is a positive regulator involved in the activation of expression of ompC and ompF genes encoding the major outer membrane protein OmpC and OmpF, respectively. The C-terminal half domain of OmpR (OmpR-C), which is responsible for DNA-binding, has been crystallized using the hanging drop vapour diffusion method. X-ray studies show that the crystals belong to the trigonal space group P3(1)21 (or P3(2)21) with a = b = 60.4 A, c = 58.8 A and gamma = 120 degrees. The asymmetric unit contains one molecule. The crystals diffract to at least 3 A resolution and are suitable for X-ray structure analysis.

Oligosaccharides were released from the gangliosides of a human rectal adenocarcinoma with endoglycoceramidase (Rhodococcus sp. G-74-2) and then purified by affinity chromatography on a column of an immobilized monoclonal antibody, MSW 113. Structural studies, involving 600-MHz 1H NMR spectrometry, indicated the structures of these compounds to be as follows. [formula: see text] Three of these oligosaccharides, 2, 3, and 4, are novel as to ganglioside sugar chains and contain both lacto series types 1 and 2 chains. Oligosaccharides 3 and 4 are unique in that they contain the sialyl-Le(a)-X structure in linear and branched structures. Gangliosides with the oligosaccharide structures presented above might be new potential tumor markers.

We examined associations between maternal-fetal attachment and the pregnant adolescent's perceived relationships with her mother and the baby's father. Questionnaires were administered in health department prenatal clinics to a sample of 61 Mexican-American, African-American, and Caucasian adolescents at 13 to 40 weeks' gestation. Each woman complete Cranley's maternal-fetal attachment scale (MFAS), an investigator-developed relationship with mother scale and a survey instrument that assessed living situation, perceptions of the relationship with the baby's father, and standard demographic variables. Scores on the MFAS significantly correlated with the adolescent's perceived close relationships with her mother and the baby's father, frequency of contact with the baby's father, gestation, and marital status. Mexican-American adolescents scored significantly lower on the MFAS than Caucasian adolescents. The scale needs further testing in multiethnic groups of adults and adolescents.