Mutant beta-spectrin 4 causes auditory and motor neuropathies in quivering mice.

The autosomal recessive mouse mutation quivering (qv), which arose spontaneously in 1953, produces progressive ataxia with hind limb paralysis, deafness and tremor. Six additional spontaneous alleles, qvJ, qv2J, qv3J, qv4J, qvlnd and qvlnd2J, have been identified. Ear twitch responses (Preyer's reflex) to sound are absent in homozygous qv/qv mice, although cochlear morphology seems normal and cochlear potentials recorded at the round window are no different from those of control mice. However, responses from brainstem auditory nuclei show abnormal transmission of auditory information, indicating that, in contrast to the many known mutations causing deafness originating in the cochlea, deafness in qv is central in origin. Here we report that quivering mice carry loss-of-function mutations in the mouse beta-spectrin 4 gene (Spnb4) that cause alterations in ion channel localization in myelinated nerves; this provides a rationale for the auditory and motor neuropathies of these mice.

TARDBP in amyotrophic lateral sclerosis: identification of a novel variant but absence of copy number variation.

BACKGROUND: Mutations in the gene encoding TDP-43 have been identified in both familial and sporadic amyotrophic lateral sclerosis (ALS). METHODS: A mutation screen and copy number analysis in a motor neuron disease clinic cohort was conducted to characterise the genetic contribution of TARDBP. RESULTS: A novel missense mutation in a highly conserved region of TDP-43 was identified in a patient with sporadic ALS. The mutation is in close vicinity to previously identified changes. Copy number variation abnormalities were not detected. CONCLUSIONS: The findings stress the importance of TDP-43 in the pathogenesis of sporadic ALS.

Candidate screening of the bovine and feline spinal muscular atrophy genes reveals no evidence for involvement in human motor neuron disorders.

Spinal muscular atrophy (SMA) is a devastating neuromuscular disease characterised by progressive loss of spinal motor neurons. Mutations in the genes underlying spontaneous bovine and feline models of SMA have recently been described. The clinical and pathological features of these disorders are similar to human forms of SMA making both genes excellent candidates in patients with motor neuron loss of no known aetiology. Here we report that a screen for mutations in coding regions and splice sites of the LIX1 and FVT1 genes in a cohort of 96 non-5q SMA patients and 119 familial and sporadic Amyotrophic Lateral Sclerosis patients identified no obvious pathogenic changes. This study indicates that mutations in these genes do not contribute significantly to the cause of motor neuron diseases in the human population.

Plant-pathogenic bacteria as biological weapons - real threats?

At present, much attention is being given to the potential of plant pathogens, including plant-pathogenic bacteria, as biological weapons/bioterror weapons. These two terms are sometimes used interchangeably and there is need for care in their application. It has been claimed that clandestine introduction of certain plant-pathogenic bacteria could cause such crop losses as to impact so significantly on a national economy and thus constitute a threat to national security. As a separate outcome, it is suggested that they could cause serious public alarm, perhaps constituting a source of terror. Legislation is now in place to regulate selected plant-pathogenic bacteria as potential weapons. However, we consider it highly doubtful that any plant-pathogenic bacterium has the requisite capabilities to justify such a classification. Even if they were so capable, the differentiation of pathogens into a special category with regulations that are even more restrictive than those currently applied in quarantine legislation of most jurisdictions offers no obvious benefit. Moreover, we believe that such regulations are disadvantageous insofar as they limit research on precisely those pathogens most in need of study. Whereas some human and animal pathogens may have potential as biological or bioterror weapons, we conclude that it is unlikely that any plant-pathogenic bacterium realistically falls into this category.

Detection of Xanthomonas fragariae and presumptive detection of Xanthomonas arboricola pv. fragariae, from strawberry leaves, by real-time PCR.

Real-time (TaqMan) PCR assays were developed to detect the strawberry angular leaf spot pathogen Xanthomonas fragariae (Xf) and the strawberry bacterial blight pathogen Xanthomonas arboricola pv. fragariae (Xaf). The Xf PCR (Xf gyrB) was designed within regions of the gyraseB gene, unique to Xf, after generating gyraseB DNA sequence data from Xf and other closely related strains. The Xaf PCR (Xaf pep) was designed within regions of the pep prolyl endopeptidase gene that were unique to Xaf, after generating pep DNA sequence data from Xf and Xaf strains. The Xf gyrB PCR detected only Xf strains amongst a panel of 20 Xanthomonas-related spp. and pathovars. The Xaf pep PCR assay detected all Xaf strains tested plus two other (of three tested) X. arboricola pathovars. An existing genomic DNA extraction protocol was modified to facilitate detection of both pathogens to 10(3) cells per strawberry leaf disc.

Adenovirus-mediated p53 gene therapy in osteosarcoma cell lines: sensitization to cisplatin and doxorubicin.

The poor prognosis for patients with metastatic osteosarcoma (OS) indicates that new therapeutic options should be explored. Studies with adenoviral-mediated p53 gene transfer have been conducted in many cancer types including cervical, ovarian, prostatic and head and neck tumors. However, limited work has been carried out with pediatric cancers, including OS. Using three viral constructs containing cDNA for wild-type p53, mutant p53 (Cys135Ser) and lacZ, we studied the effect of adenoviral-mediated gene therapy in four OS cell lines: Saos-2 (p53-/-), HOS (R156P), KHOS/NP (R156P) and MNNG (R156P, F270L). We demonstrated that the virus efficiently enters the cells using the beta-galactosidase assay. Using the MTT assay, we have shown a dose-dependent decrease in cell viability 72 h post-treatment that occurs with Ad-wtp53 but not with Ad-mutp53. We have also shown that treatment with Ad-wtp53 significantly increases sensitivity of the cell lines to cisplatin and doxorubicin, chemotherapeutic agents commonly used in the treatment of OS. Our results indicate that restoration of wt p53 function in OS cells provides a basis for novel approaches to treatment of this disease.

Phosphatidylserine-dependent adhesion of T cells to endothelial cells.

Phosphatidylserine (PS) was exposed at the surface of human umbilical vein endothelial cells (HUVECs) and cultured cell lines by agonists that increase cytosolic Ca(2+), and factors governing the adhesion of T cells to the treated cells were investigated. Thrombin, ionophore A23187 and the Ca(2+)-ATPase inhibitor 2, 5-di-tert-butyl-1,4-benzohydroquinone each induced a PS-dependent adhesion of Jurkat T cells. A23187, which was the most effective agonist in releasing PS-bearing microvesicles, was the least effective in inducing the PS-dependent adhesion of Jurkat cells. Treatment of ECV304 and EA.hy926 cells with EGTA, followed by a return to normal medium, resulted in an influx of Ca(2+) and an increase in adhering Jurkat cells. Oxidised low-density lipoprotein induced a procoagulant response in cultured ECV304 cells and increased the number of adhering Jurkat cells, but adhesion was not inhibited by pretreating ECV304 cells with annexin V. PS was not significantly exposed on untreated Jurkat cells, as determined by flow cytometry with annexin V-FITC. However, after adhesion to thrombin-treated ECV304 cells for 10 min followed by detachment in 1 mM EDTA, there was a marked exposure of PS on the Jurkat cells. Binding of annexin V-FITC to the detached cells was inhibited by pretreating them with unlabelled annexin V. Contact with thrombin-treated ECV304 cells thus induced the exposure of PS on Jurkat cells and, as Jurkat cells were unable to adhere to thrombin-treated ECV304 cells in the presence of EGTA, the adhesion of the two cell types may involve a Ca(2+) bridge between PS on both cell surfaces. The number of T cells from normal, human peripheral blood that adhered to ECV304 cells was not increased by treating the latter with thrombin. However, findings made with several T cell lines were generally, but not completely, consistent with the possibility that adhesion to surface PS on endothelial cells may be a feature of T cells that express both CD4(+) and CD8(+) antigens. Possible implications for PS-dependent adhesion of T cells to endothelial cells in metastasis, and early in atherogenesis, are discussed.

Nuclear and cytosolic calcium changes in osteoclasts stimulated with ATP and integrin-binding peptide.

Cytosolic calcium modulates the activity of osteoclasts, large multinucleate cells that resorb bone. Nuclear events, such as gene transcription, are also calcium-regulated in these cells, and fluorescence imaging has suggested that calcium signals produced by some stimuli are specifically targeted to, or amplified within, osteoclast nuclei. We used two alternative techniques of dye loading to examine the changes of intracellular calcium induced in rat osteoclasts by three stimuli. Osteoclasts loaded with the calcium indicator Fura-2 by the acetoxymethyl (AM) ester technique appeared to display marked nuclear calcium amplification. During stimulation with integrin-binding peptides, ATP, or high extracellular calcium, fluorescence ratios recorded from the nuclei rose higher than did ratios recorded from extranuclear regions. In contrast, nuclear calcium amplification was not observed after AM loading in the presence of the anion transport inhibitor sulfinpyrazone, nor in osteoclasts injected with Fura-2 conjugated to a high MW dextran. In these cells, nuclear fluorescence ratios were equal to the extranuclear values at all times: upon stimulation by an agonist, the nuclear and cytosolic calcium concentrations increased by the same amount. The calcium changes seen in stimulated osteoclasts can no longer be taken as evidence for the general validity of the phenomenon of nuclear calcium amplification.

Simple staining for heat-fixed brain sections after topographical pH analysis or autoradiographic studies.

Powerful topographic techniques are now available, among which autoradiographic and fluorescent mapping are the most prevalent. These techniques produce images that usually do not correlate with brain anatomy; subsequent staining is required to allow a precise association between the parameter(s) investigated and brain structures. A simple staining procedure is described, compatible with heat-fixed brain sections, and that was found particularly valuable in association with topographical tissue pH studies using the fluorescent pH indicator umbelliferone.

The mechanism of action of alpha 2-adrenoceptors in human isolated subcutaneous resistance arteries.

1. The effect of noradrenaline and the selective alpha 2-adrenoceptor agonist, azepexole, on tone and intracellular Ca2+ ([Ca2+]i) was examined in human isolated subcutaneous resistance arteries. Isolated arteries were mounted on an isometric myograph and loaded with the Ca2+ indicator, fura-2, for simultaneous measurement of force and [Ca2+]i. 2. High potassium solution (KPSS), noradrenaline and azepexole increased [Ca2+]i and contracted subcutaneous arteries in physiological saline. When extracellular Ca2+ was removed and the calcium chelator, BAPTA, added to the physiological saline (PSSo), responses to noradrenaline were transient and reduced, and responses to azepexole were markedly inhibited. 3. Ryanodine, an agent which interferes with Ca2+ release from intracellular stores, had little effect on contractile responses to KPSS, noradrenaline or azepexole in physiological saline. The response to caffeine in physiological saline was inhibited by ryanodine. In PSSo, ryanodine partially inhibited contractile responses to noradrenaline and azepexole, and completely abolished the response to caffeine. 4. Noradrenaline and azepexole both significantly increased maximum force achieved by cumulative addition of Ca2+ to a Ca(2+)-free depolarizing solution and shifted the calculated relationship between [Ca2+]i and force to the left, suggesting these agents increase the sensitivity of the contractile apparatus to [Ca2+]i. 5. (-)-202 791, a dihydropyridine antagonist of voltage-operated calcium channels partially inhibited both the contractile response and the rise in [Ca2+]i induced by azepexole. Pre-treatment of arteries with pertussis toxin inhibited responses to azepexole, but had no significant effect on tone induced by KPSS or noradrenaline. ETYA, an inhibitor of phospholipase A2, lipoxygenase and cyclo-oxygenase, had no effect on azepexole-induced contraction in the presence of N omega nitro-L-arginine methyl ester.6. Azepexole, a selective alpha2-adrenoceptor agonist, contracts human subcutaneous resistance arteries by a mechanism largely dependent on the influx of extracellular Ca2", probably through voltage-operated calcium channels. This action involves a pertussis toxin-sensitive G protein, possibly Gi.

Neurally evoked responses of human isolated resistance arteries are mediated by both alpha 1- and alpha 2-adrenoceptors.

1. Human subcutaneous resistance arteries (internal diameter 113-626 microns) were mounted in an isometric myograph. Electrical field stimulation was applied either continuously in the form of a frequency-response curve or intermittently at 16 Hz. The magnitude of the maximum contraction induced by continuous stimulation expressed as a percentage of the response to a supramaximal concentration of noradrenaline (10 microM) was highly variable but unrelated to vessel calibre. Contractile responses to both continuous and intermittent stimulation were abolished by 1 microM tetrodotoxin. 2. Prazosin (100 nM and 1 microM, alpha 1-adrenoceptor antagonist) inhibited responses to continuous stimulation over a range of frequencies (2-8 Hz). The response to continuous stimulation at 8 Hz was inhibited by 78 +/- 6% by 1 microM prazosin. Rauwolscine (100 nM, alpha 2-adrenoceptor antagonist) had a smaller effect on contractions induced by continuous stimulation. Rauwolscine inhibited the response at 8 Hz by 36 +/- 6%. Rauwolscine at a higher concentration (1 microM) caused further inhibition of the response to continuous stimulation. Prazosin and rauwolscine in combination almost completely inhibited the response to continuous stimulation at concentrations of 1 microM. 3. Prazosin and rauwolscine inhibited responses to intermittent stimulation in a concentration-dependent manner. The IC50 for this action of prazosin was 3.7 +/- 1.6 nM and the maximum inhibition induced by 100 nM prazosin was 78 +/- 6%. The IC50 of rauwolscine was 12.0 +/- 1.3 nM and 100 nM rauwolscine caused a 86 +/- 7% reduction in the response to intermittent stimulation.Prazosin and rauwolscine in combination (each at 100 nM) caused marked inhibition of the response to intermittent stimulation leaving only 7.0 +/- 2.6% of the response.4. These data suggest that neurally released noradrenaline evokes contractions of human resistance arteries by activation of both alpha 1,- and alpha 2-adrenoceptors postjunctionally.

A new form of arthropod phenoloxidase is abundant in venom of the parasitoid wasp Pimpla hypochondriaca.

We have recently identified phenoloxidase (PO) activity among several biologically active factors in venom from the parasitoid wasp Pimpla hypochondriaca. We have now isolated three genes, designated POI, POII and POIII, from a cDNA library made from venom-producing glands and found that their products are related to pro-phenoloxidases (PPOs), which are expressed as proenzymes in haemocytes and which mediate immune processes in arthropods. This is the first report of PO as a venom constituent. Amino acid sequence comparisons between the three Pimpla POs and PPOs revealed several notable differences, including the absence of sequences which specify the site of proteolytic activation in insect PPOs and the unprecedented occurrence of signal peptide sequences. NH(2)-terminal amino acid analysis of PO purified from venom yielded a peptide sequence matching the predicted mature NH(2) termini of POI and POII, confirming the authenticity of the signal peptide and indicating that proteolytic processing, other than to remove the signal peptide, does not occur in the wasp. Expression of POI, analysed by Northern hybridization, was approximately uniform from the time of adult emergence to day 6 post-emergence, after which it declined. A novel means of host immune suppression, mediated by the unregulated activity of venom PO in the haemocoel, is proposed.

Purification of pimplin, a paralytic heterodimeric polypeptide from venom of the parasitoid wasp Pimpla hypochondriaca, and cloning of the cDNA encoding one of the subunits.

We have previously detected a paralytic factor in gel filtration-separated venom from the endoparasitoid wasp Pimpla hypochondriaca which is active against the fly Musca domestica. Now we have further purified this factor, which we have called pimplin, by reverse phase chromatography, and established using SDS-PAGE that it has a molecular mass of approximately 22 kDa. A 40 ng dose of pimplin administered to adult M. domestica by intrahaemocoelic injection was sufficient to kill all flies tested. Treatment of pimplin with beta-mercaptoethanol prior to SDS-PAGE analysis resulted in the appearance of two polypeptides of approximately 15 and 6 kDa, indicating that pimplin is a heterodimer whose polypeptides are linked through a disulphide bond. Subunit masses of 10.544 and 6.318 kDa were determined using MALDI-TOF analysis indicating that the larger subunit migrates anomalously in SDS-PAGE. Using an oligonucleotide probe designed from N-terminal sequence obtained for the 15 kDa polypeptide, we have isolated a cDNA (pim1) encoding this larger pimplin subunit. The N-terminal amino acid sequence of pim1 occurred 28 residues beyond a predicted signal peptide cleavage site, indicating that pim1 is synthesised as a pre-propolypeptide which is secreted and proteolytically cleaved to yield the mature polypeptide stored in the venom sac. Beginning at the fourth residue of the mature pim1 venom polypeptide is a stretch of 46 residues consisting of alternating prolines, the significance of which is discussed in terms of possible host processing.

Analysis of venom constituents from the parasitoid wasp Pimpla hypochondriaca and cloning of a cDNA encoding a venom protein.

Venom from Pimpla hypochondriaca, an endoparasitoid of pupae, was size-fractionated using gel filtration chromatography and analysed by SDS-PAGE in the presence and absence of reducing agent. A complex mixture of more than 20 venom constituents was identified which ranged in M(r) between approximately 5 and 100 kDa. Venom from a wide range of size fractions inhibited the motility of larval haemocytes and prevented the formation of cell aggregates when analysed in vitro, indicating that anti-haemocytic activity is mediated by multiple venom components. Sephadex A25 beads injected into the haemocoel of pupae were encapsulated within 24h. This reaction was abolished when the pupae were injected with 30 microg of venom protein, equivalent to one-fifth of a venom sac, 1h prior to implantation of the beads, confirming that venom suppresses encapsulation in pupae. Using random 5' end sequencing of a P. hypochondriaca venom gland cDNA library, we have isolated a cDNA encoding a 25.3 kDa protein containing a signal peptide and having sequence similarity to serine proteases. The N-terminal sequence of six residues from two venom proteins of 28 and 30 kDa was the same and identical to amino acids encoded by the cDNA, confirming that two mass forms of the protein are secreted into the venom sac. The N-terminal sequence of both venom proteins began nine residues towards the C terminus following the predicted signal sequence cleavage site, suggesting that the proteins are proteolytically processed before or during storage in the venom sac. The general applicability of using random 5' sequencing to identify cDNAs encoding secretory products is discussed.

A nuclear location for Ca2+-activated adenylyl cyclases I and III in neurones.

Calcium/calmodulin-sensitive adenylyl cyclases are increasingly recognised as important factors in memory formation and synaptic plasticity. We have examined the distributions of adenylyl cyclases types I, III, and VIII within rat primary sensory neurons. Immunofluorescence revealed distinct staining for adenylyl cyclases type I and III, but not adenylyl cyclase type VIII, within the cell nucleus. Western blots suggest that a processed form of adenylyl cyclase type III may be found within primary neurons and PC12 cells as a 70-kDa protein. We propose that the observed nuclear adenylyl cyclases are soluble forms of the cyclases.

A critical analysis of the use of radiation inactivation to measure the mass of protein.

Measurements are presented of the radiation inactivation of four enzymes exposed to a 6 MeV proton beam. It has long been thought that the measurement of the susceptibility of an enzyme to ionizing radiation can be used to determine its molecular mass. Results are frequently interpreted using the empirical analysis of Kempner and Macey (Biochim. Biophys. Acta 163, 188-203, 1963). We examine this analysis and discuss the validity and limitations of the assumptions on which it is based. Our results indicate that the specific biochemical properties of each enzyme make a significant contribution to its radiation sensitivity.

A postmortem study of the mediodorsal nucleus of the thalamus in schizophrenia.

Four studies have reported that the mediodorsal nucleus of the thalamus (MD) is smaller and contains fewer neurons in schizophrenia. The MD is a key node in a circuit proposed to be dysfunctional in the disorder. However, one study did not find a MD volume loss in schizophrenia, and all the studies to date are relatively small. Given the importance of establishing unequivocally the presence of MD pathology, we have carried out a study of the volume and number of neurons in the left and right MD in 21 patients with schizophrenia and 27 healthy comparison subjects. We also measured the size of MD neurons, and estimated total thalamic volume. We found no difference in the volume of the MD, the number of MD neurons, or the size of MD neurons in either hemisphere in schizophrenia. Neither was total thalamic volume altered. There are no obvious methodological or clinical factors to explain our failure to replicate the finding of MD involvement in schizophrenia. Hence our negative observations, in the largest sample yet investigated, cast doubt on the robustness and/or the generalisability of MD neuropathology in schizophrenia.

Potentiation by phenolphthalein of the responses of guinea-pig ileum and rat stomach strip to PGE2 and other agonists.

Phenolphthalein was examined for its effect on the activity of isolated muscle (guinea-pig ileum and colon, rat stomach), and on the tissue responses to PGE2, histamine and 5-HT. The ileal circular muscle and both muscle layers of the colon were unaffected by phenolphthalein. In contrast, the laxative potentiated the responses of the longitudinal muscle of guinea-pig isolated ileum and the rat stomach strip to the agonists, particularly PGE2. This potentiation was reduced by indomethacin in vivo, but mepyramine or methysergide had little or no effect. Augmentation of muscle activity by phenolphthalein, particularly in the response to PGE2, may contribute to the laxative effect.

Isolation of single vascular smooth muscle cells from human omental resistance arteries.

Small arteries less than 500 microns make a significant contribution to peripheral vascular resistance. Single vascular smooth muscle cells were isolated from human omental resistance arteries by enzymatic dispersion. The method yielded relaxed vascular smooth muscle cells approximately 120 microns long and 7 microns wide. The cells were studied using whole cell voltage clamp techniques and a number of passive and active membrane properties were described.

Studies of the lethargic (lh/lh) mouse model of absence seizures: regulatory mechanisms and identification of the lh gene.

To understand the cellular and molecular mechanisms that underlie generalized absence seizures sufficiently well to design rational, efficacious new therapies for patients, it is necessary to turn to animal models to gain insights into these mechanisms. The lethargic (lh/lh) mutant mouse expresses spontaneous absence seizures that share behavioral, electrographic, and anticonvulsant profiles with absence seizures in patients. This validates its use to study the mechanisms that underlie absence seizures. This chapter discusses two scientific approaches that involve the use of lh/lh mice. The first part of the chapter discusses neurobiologic approaches used to investigate critical mechanisms that regulate the synchronized burst firing within the thalamocortical network that generates absence seizures. Two of these critical mechanisms have been studied in detail with lh/lh mice. The first critical mechanism involves the required activation of gamma-aminobutyric acid B (GABAB) receptors to generate absence seizures. Because the numbers of GABAB receptors are increased in thalamocortical populations among lh/lh mice compared with littermates without epilepsy, these receptors appear to play a pathophysiologic role in the expression of absence seizures among lh/lh mice. Moreover, there may be a role for GABAB receptors in the generation of absence seizures among humans, because administration of compounds that activate GABAB receptors can produce absence seizures among humans. These findings suggest that GABAB receptor antagonists may represent a new class of antiabsence compounds that will be efficacious against absence seizures among patients. A second critical mechanism that regulates generation of absence seizures involves GABAA receptors in the nucleus reticularis thalami (NRT), a nucleus that sends GABA-ergic afferents to thalamic relay nuclei. Activation of GABAA receptors in the NRT appears to suppress the generation of absence seizures among lh/lh mice and in other models. Moreover, clonazepam may exert its antiabsence actions through this mechanism. Together, these findings suggest that compounds that selectively activate GABAA receptor isoforms expressed in NRT may represent a class of antiabsence drugs that could have fewer side effects than compounds currently used to treat patients. The second part of the chapter discusses a molecular genetic approach to delineation of the mechanisms that underlie absence seizures. Absence seizures among lh/lh mice are caused by a single-gene defect on chromosome 2. If positional cloning and gene isolation techniques are successful, it will be possible to identify the lh disease gene. Subsequent studies of the lh gene product should greatly increase not only our understanding of the pathophysiologic basis for absence seizures among lh/lh mice but also our ability to seek similar mutations in homologous genes in human families that express absence seizures. Accordingly, strategies and progress in cloning and identifying the lh disease gene are presented.