Lower airway smooth muscle contraction induced by Ptychodiscus brevis (red tide) toxin

Ptychodiscus brevis toxin (PBTX) is produced by the organism Ptychodiscus brevis. This toxin causes a phenomenon that has come to be known as Florida red tide. It also stimulates neuronal sodium channels, resulting in activation of the cholinergic and adrenergic nerve endings of the autonomic nervous system in upper airway smooth muscle and rat vas deferens, respectively, as previously reported. It is the cholinergic stimulating action that has been implicated as a possible "triggering" event in bronchial asthma. This article concerns the investigation of whether PBTX may also affect lower airways and by what mechanism any contractile response to PBTX in lower airways may be induced. PBTX was found to elicit contractions in isolated canine lower airway smooth muscle. The threshold concentration was 0.15 microgram/ml, the peak response occurred at 6.0 micrograms/ml, and the concentration causing half-maximal response of the group was 0.57 microgram/ml. Pharmacologic analysis demonstrated that atropine (10(-6) mol/L) blocked the response to both PBTX and acetylcholine, tetrodotoxin (10(-7) mol/L) blocked the response to PBTX but not to acetylcholine, and verapamil (10(-5) mol/L) blocked the response to PBTX and reduced the response to acetylcholine. Four consecutive contractile responses to PBTX (3 micrograms/ml) produced rapid tachyphylaxis. The fourth contraction was 60% less than the initial response. A fifth response to PBTX after exposure to indomethacin (2.8 X 10(-6) mol/L) was increased and resulted in a contraction that was only 25% less than the initial response. The exogenous addition of prostaglandins (PG), PGE1 and PGE2, to indomethacin-treated lower airway strips selectively suppressed the contractile response to PBTX. Other PGs tested (PGA2, PGB2, PGD2, PGF2 alpha and PGI2) did not affect the PBTX response. These results indicate that PBTX produces spasm in lower airway smooth muscle and that it does this by stimulation of sodium channels in the cholinergic nerve fibers. The results also demonstrate a rapid reduction in the contractile response to PBTX. The results also demonstrate that the reduction is mediated by PGs of the E series.     

An octopus toxin, maculotoxin, selectively blocks sodium current in squid axons.

1. A low molecular weight, stable, cationic neurotoxin (maculotoxin, MTX) extracted from the posterior salivary glands of the octopus Hapalochlaena maculosa, blocked sodium current in voltage-clamped squid axons without affecting potassium current. 2. The effectiveness of MTX was increased by repetitive, brief, depolarizing pulses but not by a single prolonged depolarization. 3. The potency of MTX decreased at pHs from 8 to 9. Effectiveness could be restored be restored by lowering the pH to 7-1 again. It was concluded that MTX is active in its cationic form. 4. MTX affected sodium conductance kinetics, slowing the turn-on of sodium current. This effect was most noticeable with small deploarizations but became progressively less with larger depolarizations. Neither the turn-off of sodium current nor sodium inactivation kinetics were affected by the toxin. 5. MTX inhibited sodium current without inhibiting sodium gating current. 6. The effectiveness of MTX was not detectably changed when calcium concentration was varied from 50 to 10 mM, or sodium concentration was varied from 225 to 750 mM.     

Kinetics of sodium activation in giant axons of squid (Doryteuthis bleekeri)

The variation with time of the rising phase of the sodium conductance in squid giant axons was fitted to the Xth power of running integrals of the colchicine-sensitive components of the asymmetry currents, where the running integrals were experimentally obtained. We found that excellent fitting was obtained for all of the potentials examined from -60 to +60 mV, when we put the value of X equal to 3 at potentials above 10 mV and equal to 5 below -20 mV, and it varied continuously from 3 to 5 when pulse potentials changed from 10 to -25 mV. Further, it was found that the voltage dependence of the peak sodium currents was fitted to the third power of charges carried by the colchicine-sensitive component of the asymmetry current by the moment when the sodium current attained its peak. These results agree well with the expectation by Hodgkin and Huxley [(1952) J. Physiol., Lond. 117, 500-544] although the gating kinetics differ from the Hodgkin-Huxley prediction [Matsumoto, Ichikawa and Tasaki (1983) J. Membr. Biol. 77, 93-99].     

Detection of extracellular toxin(s) produced by Vibrio vulnificus.

Conditions are described for the production, in high titers, a heat-labile, antigenic, extracellular toxin(s) by Vibrio vulnificus, a recently recognized human pathogen. Bacteriologically sterile culture filtrate preparations obtained from mid-logarithmic-phase cultures of the bacterium possessed cytolytic activity against mammalian erythrocytes, cytotoxic activity for Chinese hamster ovary cells, vascular permeability factor activity in guinea pig skin, and lethal activity for mice. The specific activity of toxin preparations from cultures of a virulent strain of the bacterium was ca. 25-fold more than that of toxin preparations obtained from cultures of a weakly virulent strain. The four toxic activities were inseparable by gel filtration with Sephadex G-100; however, two components, which had markedly different elution behavior but which possessed the four activities mentioned above, were obtained. The major (ca. 88% of the recovered activity) and minor components had apparent molecular weights of ca. 38,500 and greater than 150,000, respectively.     

Ciguatera toxin in Cheilinus rhodochrous (Po'ou wrasse)

Cheilinus rhodochrous (po'ou, wrasse) was examined by various assays for the presence of toxins associated with ciguatera fish poisoning. These assays were a stick enzyme immunoassay (S‐EIA); and extraction, chromatography on silica gel, and examination of the fractions by stick enzyme immunoassay, mouse toxicity assay, and guinea pig atrial assay. The active toxic principles were found in the 10% (v/v) and 50% (v/v) methanol/chloroform eluates of viscera and flesh, and had characteristics similar to ciguatoxins found in carnivores.     

Cecal toxin(s) from guinea pigs with clindamycin-associated colitis, neutralized by Clostridium sordellii antitoxin.

The cecal contents of guinea pigs with clindamycin-associated colitis contained a heat-labile toxin. This toxin was lethal for guinea pigs and mice, produced vascular permeability in the skin of rabbits, and was cytotoxic in tissue culture. The lethality in mice, vascular permeability in rabbit skin, and cytotoxicity in tissue culture monolayers were neutralized by Clostridium sordellii antitoxin.     

Chromium (III) produces distinct type of cell death in cultured cells

Chromium acetate hydroxide is a chemical form of trivalent chromium (Cr III) originating from industrial processes. Although considered relatively nontoxic, upon longer treatment intervals it may cause irreversible cellular damage culminating in cell death. In our present work we have attempted to analyze some of the mechanisms whereby this chemical inflicts damage while focusing on the final observed endpoints. We report that 1 mM chromium acetate hydroxide is during weeks lasting treatment capable of injuring the plasma membrane of Hep-2 cells, which in turn becomes permeable to Cr (III) ions. Analyses of several markers of cellular damage; i.e. mitochondrial activity, nuclear integrity and oxidative stress have shown that tested compound interacts directly with subcellular organelles and upon tested concentration and time induces distinct type of cell death bearing features of apoptosis and necrosis.     

Ca2+-activated K+ (BK) channel inactivation contributes to spike broadening during repetitive firing in the rat lateral amygdala

In many neurons, trains of action potentials show frequency-dependent broadening. This broadening results from the voltage-dependent inactivation of K⁺ currents that contribute to action potential repolarisation. In different neuronal cell types these K⁺ currents have been shown to be either slowly inactivating delayed rectifier type currents or rapidly inactivating A-type voltage-gated K⁺ currents. Recent findings show that inactivation of a Ca²⁺-dependent K⁺ current, mediated by large conductance BK-type channels, also contributes to spike broadening. Here, using whole-cell recordings in acute slices, we examine spike broadening in lateral amygdala projection neurons. Spike broadening is frequency dependent and is reversed by brief hyperpolarisations. This broadening is reduced by blockade of voltage-gated Ca²⁺ channels and BK channels. In contrast, broadening is not blocked by high concentrations of 4-aminopyridine (4-AP) or α-dendrotoxin. We conclude that while inactivation of BK-type Ca²⁺-activated K⁺ channels contributes to spike broadening in lateral amygdala neurons, inactivation of another as yet unidentified outward current also plays a role.     

Systemic mycosis in Scottish red deer (Cervus elaphus)

The pathological features of 10 cases of systemic infection of young red deer by fungi morphologically identified as phycomycetes are described. The most frequently affected organs were the kidneys, followed by liver and central nervous system. The fungi stimulated an acute pyogenic inflammatory reaction and a phagocytic giant cell response. Vasculitis with thrombosis and spread of fungi to the surrounding tissue was a common finding. Culture was attempted in 4 cases; one yielded Absidia, 2 Aspergillus and the other an unidentified fungal growth. Analysis of the histories suggests that, in nine of the ten cases, stressful husbandry may have been a predisposing factor.     

SIV/HIV Nef recombinant virus (SHIVnef) produces simian AIDS in rhesus macaques

The simian immunodeficiency virus (SIV) nef gene is an important determinant of viral load and acquired immunodeficiency syndrome (AIDS) in macaques. A role(s) for the HIV-1 nef gene in infection and pathogenesis was investigated by constructing recombinant viruses in which the nef gene of the pathogenic molecular clone SIVmac239 nef was replaced with either HIV-1sf2nef or HIV-1sf33nef. These chimeras, designated SHIV-2nef and SHIV-33nef, expressed HIV-1 Nef protein and replicated efficiently in cultures of rhesus macaque lymphoid cells. In two SHIV-2nef-infected juvenile rhesus macaques and in one of two SHIV-33nef-infected juvenile macaques, virus loads remained at low levels in both peripheral blood and lymph nodes in acute and chronic phases of infection (for >83 weeks). In striking contrast, the second SHIV-33nef-infected macaque showed high virus loads during the chronic stage of infection (after 24 weeks). CD4+ T-cell numbers declined dramatically in this latter animal, which developed simian AIDS (SAIDS) at 47-53 weeks after inoculation; virus was recovered at necropsy at 53 weeks and designated SHIV-33Anef. Sequence analysis of the HIV-1sf33 nef gene in SHIV-33Anef revealed four consistent amino acid changes acquired during passage in vivo. Interestingly, one of these consensus mutations generated a tyr-x-x-leu (Y-X-X-L) motif in the HIV-1sf33 Nef protein. This motif is characteristic of certain endocytic targeting sequences and also resembles a src-homology region-2 (SH-2) motif found in many cellular signaling proteins. Four additional macaques infected with SHIV-33Anef contained high virus loads, and three of these animals progressed to fatal SAIDS. Several of the consensus amino acid changes in Nef, including Y-X-X-L motif, were retained in these recipient animals exhibiting high virus load and disease. In summary, these findings indicate that the SHIV-33Anef chimera is pathogenic in rhesus macaques and that this approach, i.e., construction of chimeric viruses, will be important for analyzing the function(s) of HIV-1 nef genes in immunodeficiency in vivo, testing antiviral therapies aimed at inhibiting AIDS, and investigating adaptation of this HIV-1 accessory gene to the macaque host.     

Inverted repetitive sequences in human papilloma virus 1 (HPV-1) DNA.

Two pairs of inverted repetitive (palindromic) sequences (IR 1a/b and IR 2a/b) were detected electron microscopically within the DNA of human papilloma virus 1 (HPV-1) after cleavage by BamHI or EcoRI endonucleases followed by denaturation and a short period of reassociation. The localization of these sequences within the genome was determined. After separation and isolation of the single HPV-1 DNA strands, IR 1a/b and IR 2a/b were identified by polyacrylamide-gel electrophoresis after prior digestion of the nonhomologous regions with S1 nuclease. The molecular weights of the palindromic sequences were determined to be 0.11 × 10⁶ and 0.10 × 10⁶.     

Altered red cell osmotic fragility in Huntington disease (HD)

We have studied in fresh and 24-hour incubated samples the osmotic fragility of erythrocytes from 13 individuals with Huntington disease (HD) and 22 at-risk, asymptomatic individuals. Five older at-risk, asymptomatic individuals and six Alzheimer disease individuals were also studied. Results suggest that osmotic fragility of red cells from HD individuals in significantly decreased in fresh (P less than 0.0001) and incubated (P less than 0.0001) samples. At-risk individuals appear to fall into two groups: 1) those with normal osmotic fragility (n =10), and 2) those with decreased osmotic fragility (n = 12). Fragility in older at-risk persons and those with Alzheimer disease were within normal limits. These data suggest that red cell osmotic fragility measurement may be useful to identify at-risk persons with an HD gene; however, longitudinal follow-up will be required to confirm the predictive power of this observation. These data suggest additional support for focusing on the erythrocyte in investigating the molecular pathogenesis of HD.     

A novel point mutation in the mitochondrial tRNA(Trp) gene produces a neurogastrointestinal syndrome

We report a novel, heteroplasmic point mutation in the mitochondrial tRNA for tryptophan at position 5532. The mutation was present in all the tissues studied and segregated with the biochemical defect, with higher levels of mutation present in cytochrome c oxidase-deficient muscle fibres. The patient manifested a neurogastrointestinal syndrome with features including failure to thrive, psychomotor retardation, ophthalmoplegia, sensorineural deafness and encephalopathy together with vomiting, diarrhoea and colitis.     

Excitatory and inhibitory after-effects after repetitive magnetic transcranial stimulation (rTMS) in normal subjects

We investigated the post-train effects of repetitive transcranial magnetic stimulation (rTMS) on motor evoked potential (MEP) size and cortical silent period (SP) duration. rTMS was delivered over the primary motor cortex in trains of 5, 10, 20, 40 and 60 stimuli in normal subjects at rest and in trains of 5, 10 and 20 stimuli during voluntary muscle contraction. The intensity of stimulation was 120% of resting motor threshold. Test MEPs were delivered at different interstimulus intervals after rTMS ended. At rest, 5 Hz trains produced an increase in the MEP size that persisted after the end of the trains. Trains of 5 stimuli produced after-effects that persisted for 0.5 s, whereas trains of 40 and 60 stimuli produced a facilitation that lasted for several seconds. 5 Hz-rTMS delivered during muscle contraction increased the SP duration during stimulation but the increase persisted for only 1 s after the train ended. The present experiments show that the after-effects of rTMS on MEP amplitude and SP duration have different time-courses. rTMS probably elicits its after-effects on excitatory and inhibitory cortical elements through different physiological mechanisms.     

Presumptive adenoviral bronchiolitis in a red deer (Cervus elaphus)

A case of fatal presumptive adenoviral bronchiolitis in a 1-week-old red deer (Cervus elaphus) fawn is described. Retrospective diagnosis of the adenoviral infection was made by electron microscopy on lung tissue that had been embedded in paraffin wax.     

Regeneration of axons in the optic nerve of the adult Browman-Wyse (BW) mutant rat

Summary We have studied the regeneration of axons in the optic nerves of the BW rat in which both oligodendrocytes and CNS myelin are absent from a variable length of the proximal (retinal) end of the nerve. In the optic nerves of some of these animals, Schwann cells are present. Axons failed to regenerate in the exclusively astrocytic environment of the unmyelinated segment of BW optic nerves but readily regrew in the presence of Schwann cells even across the junctional zone and into the myelin debris filled distal segment. In the latter animals, the essential condition for regeneration was that the lesion was sited in a region of the nerve in which Schwann cells were resident. Regenerating fibres appeared to be sequestered within Schwann cell tubes although fibres traversed the neuropil intervening between the ends of discontinuous bundles of Schwann cell tubes, in both the proximal unmyelinated and myelin debris laden distal segments of the BW optic nerve. Regenerating axons never grew beyond the distal point of termination of the tubes. These observations demonstrate that central myelin is not an absolute requirement for regenerative failure, and that important contributing factors might include inhibition of astrocytes and/or absence of trophic factors. Regeneration presumably occurs in the BW optic nerve because trophic molecules are provided by resident Schwann cells, even in the presence of central myelin, oligodendrocytes and astrocytes. All the above experimental BW animals also have Schwann cells in their retinae which myelinate retinal ganglion cell axons in the fibre layer. Control animals comprised normal Long Evans Hooded rats, BW rats in which both retina and optic nerve were normal, and BW rats with Schwann cells in the retina but with normal, i.e. CNS myelinated, optic nerves. Regeneration was not observed in any of the control groups, demonstrating that, although the presence of Schwann cells in the retina may enhance the survival of retinal ganglion cells after crush, concomitant regrowth of axons cut in the optic nerve does not take place.