Ravindra K. Hajela

Cloning of a chitinase-like cDNA (hs2), its transfer to creeping bentgrass (Agrostis palustris Huds.) and development of brown patch (Rhizoctonia solani) disease resistant transgenic lines

Abstract We isolated a cDNA clone ( hs2 ) encoding a chitinase-like protein from a triploid Dutch elm disease resistant American elm ( Ulmus americana NPS-3-487). Amino acid sequence of this chitinase-like protein showed 65–78% homology to other plant chitinases. A plasmid KYLX71-pHS2 was constructed using the hs2 sequence under the control of cauliflower mosaic virus 35S promoter and nos terminator. This construct was used to genetically engineer creeping bentgrass ( Agrostis palustris Huds.) via Biolistic® PDS-100/He system. Plasmid JS101 containing the bar herbicide resistance selectable marker gene regulated by the rice actin 1 (Act1) promoter was used as a selectable co-transformation marker. Transgenic plants were produced with a 21.1% transformation frequency on the basis of bialophos selection. The co-transformation frequency for hs2 was 5.68%. Linked transgenes ( bar and mtld ) were co-integrated with a frequency of 100% as expected and 21.3% of the transgenic plants showed integration of all three genes. Southern blot analysis of transformants showed that the hs2 transgene copy numbers ranged from 1 to 5. Northern blot hybridization confirmed transcription of the transgenes, and western blot determined the HS2 protein expression. No correlation was found between gene copy number and the level of gene expression. Disease resistance profiles of five independent transgenic lines in greenhouse trials revealed that two lines (711 and 9603) were significantly ( P Rhizoctonia solani , the causative agent of brown patch disease.

Acetylcholine Release at Neuromuscular Junctions of Adult Tottering Mice Is Controlled by N-(Cav2.2) and R-Type (Cav2.3) but Not L-Type (Cav1.2) Ca2+ Channels

The mutation in the α1A subunit gene of the P/Q-type (Cav2.1) Ca2+ channel present in tottering (tg) mice causes ataxia and motor seizures that resemble absence epilepsy in humans. P/Q-type Ca2+channels are primarily involved in acetylcholine (ACh) release at mammalian neuromuscular junctions. Unmasking of L-type (Cav1.1–1.2) Ca2+ channels occurs in cerebellar Purkinje cells of tg mice. However, whether L-type Ca2+ channels are also up-regulated at neuromuscular junctions of tg mice is unknown. We characterized thoroughly the pharmacological sensitivity of the Ca2+ channels, which control ACh release at adult tg neuromuscular junctions. Block of N- and R-type (Cav2.2–2.3), but not L-type Ca2+ channels, significantly reduced quantal content of end-plate potentials in tg preparations. Neither resting nor KCl-evoked miniature end-plate potential frequency differed significantly between tg and wild type (WT). Immunolabeling of Ca2+ channel subunits α1A, α1B, α1C, and α1E revealed an apparent increase of α1B, and α1E staining, at tg but not WT neuromuscular junctions. This presumably compensates for the deficit of P/Q-type Ca2+channels, which localized presynaptically at WT neuromuscular junctions. No α1C subunits juxtaposed with pre- or postsynaptic markers at either WT or tg neuromuscular junctions. Thus, in adult tg mice, immunocytochemical and electrophysiological data indicate that N- and R-type channels both assume control of ACh release at motor nerve terminals. Recruitment of alternate subtypes of Ca2+ channels to control transmitter release seems to represent a commonly occurring method of neuronal plasticity. However, it is unclear which conditions underlie recruitment of Cav2 as opposed to Cav1-type Ca2+ channels.

Exposure to an Environmental Neurotoxicant Hastens the Onset of Amyotrophic Lateral Sclerosis-Like Phenotype in Human Cu2+/Zn2+ Superoxide Dismutase 1 G93A Mice: Glutamate-Mediated Excitotoxicity

Mice expressing the human Cu2+/Zn2+ superoxide dismutase 1 (hSOD1) gene mutation (hSOD1G93A; G93A) were exposed to methylmercury (MeHg) at concentrations that did not cause overt motor dysfunction. We hypothesized that low concentrations of MeHg could hasten development of the amyotrophic lateral sclerosis (ALS)-like phenotype in G93A mice. MeHg (1 or 3 ppm/day in drinking water) concentration-dependently accelerated the onset of rotarod failure in G93A, but not wild-type, mice. At the time of rotarod failure, MeHg increased Fluo-4 fluorescence (free intracellular calcium concentration [Ca2+]i) in soma of brainstem-hypoglossal nucleus. These motor neurons control intrinsic and some extrinsic tongue function and exhibit vulnerability in bulbar-onset ALS. The α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)/kainic acid receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione reduced [Ca2+]i in all G93A mice, irrespective of MeHg treatment. N-acetyl spermine, which antagonizes Ca2+-permeable AMPA receptors, further reduced [Ca2+]i more effectively in MeHg-treated than untreated G93A mice, suggesting that MeHg-treated mice have a greater Ca2+-permeable AMPA receptor contribution. The non-Ca2+ divalent cation chelator N,N,N′,N′-tetrakis(pyridylmethyl)ethylenediamine reduced Fluo-4 fluorescence in all G93A mice; FluoZin-(Zn2+ indicator) fluorescence was increased in all MeHg-treated mice. Thus in G93A mice Zn2+ apparently contributed measurably to the MeHg-induced effect. This is the initial demonstration of accelerated onset of ALS-like phenotype in a genetically susceptible organism by exposure to low concentrations of an environmental neurotoxicant. Increased [Ca2+]i induced by the G93A-MeHg interaction apparently was associated with Ca2+-permeable AMPA receptors and may contribute to the hastened development of ALS-like phenotypes by subjecting motor neurons to excessive elevation of [Ca2+]i, leading to excitotoxic cell death.

Apoptosis in microencapsulated juvenile rabbit chondrocytes induced by ofloxacin : Role played by β1 -integrin receptor

Quinolone(s) (QNs) is widely used in infection therapy due to its good antimicrobial characteristics. However, QNs-induced arthropathy of immature animals has led to restrictions on the therapeutic use of these antimicrobial agents. The exact mechanism(s) of QNs-induced chondrotoxicity remain unknown. In the present study, we investigated the possible mechanism of ofloxacin (one typical QNs)-induced injuries of chondrocytes. Juvenile rabbit joint chondrocytes cultured in alginate microspheres were incubated with ofloxacin at concentrations of 0, 2, 5, 10, 20, and 40 μg/ml for up to 96 h. Concentration of 10 μg/ml ofloxacin induced apoptosis of chondrocyte with visible apoptotic signs, including degradation of poly(ADP-ribose) polymerase, caspase-3 activation, and DNA ladder formation. Furthermore, extracellular signal-regulated kinase 1/2 (phospho-ERK1/2) and growth factor receptor-bound protein 2 (Grb2) were significantly reduced, and similar changes were also observed in the β1-integrin receptor as assessed by immunoblotting. However, the mRNA level of β1-integrin obtained from reverse transcription-polymerase chain reaction remained unchanged. Results of β1-integrin immunoprecipitation have also shown that β1-integrin did not interact with activated intracellular signaling proteins. In addition, ofloxacin did not induce apoptosis and decrease β1-integrin expression in chondrocytes supplemented with Mg2+, and the ofloxacin-induced apoptosis was caspase-8-dependent, inhibition of which did not affect the expression mode of phospho-ERK1/2 and β1-integrin. Our results demonstrate that ofloxacin affects β1-integrin receptor functions and the ERK mitogen-activated protein kinase signaling pathway, causing caspase-8-dependent apoptosis after exposure of 48 h.

Comparative Effects of Methylmercury and Hg2+ on Human Neuronal N- and R-Type High-Voltage Activated Calcium Channels Transiently Expressed in Human Embryonic Kidney 293 Cells

Expression cDNA clones of α1B-1 or α1E-3 subunits coding for human neuronal N-(Cav2.2) or R-subtype (Cav2.3) Ca2+ channels, respectively, was combined with α2-bδ and β3-a Ca2+ channel subunits, and transfected into human embryonic kidney cells for transient expression to determine whether specific types of neuronal voltage-sensitive Ca2+ channels are affected differentially by methylmercury (MeHg) and Hg2+. For both Ca2+ channel subtypes, MeHg (0.125-5.0 μM) or Hg2+ (0.1-5 μM) caused a time- and concentration-dependent reduction of current. MeHg caused an initial, rapid component and a subsequent more gradual component of inhibition. The rapid component of block was completed between 100 and 150 s after beginning treatment. At 0.125 to 1.25 μM, MeHg caused a more gradual decline in current. Apparent IC50 values were 1.3 and 1.1 μM for MeHg, and 2.2 and 0.7 μM for Hg2+ on N- and R-types, respectively. For N-type current, effects of Hg2+ were initially greater on the peak current than on the sustained current remaining at the end of a test pulse; subsequently, Hg2+ blocked both components of current. For R-type current, Hg2+ affected peak and sustained current approximately equally. Kinetics of inactivation also seemed to be affected by Hg2+ in cells expressing N-type but not R-type current. Washing with MeHg-free solution could not reverse effects of MeHg on either type of current. The effect of Hg2+ on N- but not R-type current was partially reversed by Hg2+-free wash solution. Therefore, different types of Ca2+ channels have differential susceptibility to neurotoxic mercurials even when expressed in the same cell type.

Fluid flow-induced increase in inward Ba2+ current expressed in HEK293 cells transiently transfected with human neuronal L-type Ca2+ channels☆

Mechanical forces can alter the gating of several kinds of ion channels in many types of cells, but the mechanisms underlying the mechanosensitivity are not clearly understood. To date, there are very few reports on mechanosensitivity of Ca2+ channels, particularly neuronal Ca2+ channels. We examined the mechanical sensitivity of human recombinant L-type Ca2+ channels in response to fluid flow. Neuronal L-type Ca2+ channels (Ca(v) 1.2) were expressed transiently in HEK293 cells using expression cDNA clones of human alpha1C, alpha2delta, and beta subunits along with green fluorescent protein (GFP) as a reporter protein. Current (I(Ba)) through these heterologously-expressed channels was measured using whole cell recording technique with 20 mM Ba2+ as charge carrier. Transfected cells were exposed to a constant, increased fluid flow from a separate pipette during current recording. The L-type I(Ba) was found to be very sensitive to the flow-induced shear forces. Peak current amplitude increased by as much as approximately 50% during fluid flow as compared to that in the absence of fluid pressure. However, no change was observed in the amplitude of the average current during the final 5 ms of the 150-ms voltage step. Current amplitude promptly returned to normal control levels upon stopping fluid flow. The current-voltage relationship was not altered by fluid flow. The flow-induced increase in current amplitude exhibited an apparent shift in steady-state inactivation toward more negative potentials; inactivation was faster but was not voltage dependent. Activation was slightly faster under flow. Thus, increased mechanical tension associated with fluid flow can alter the fundamental properties of voltage-gated Ca2+ channels, even for channels which might not normally be exposed to fluid flow shear forces in their native environment.

Differential Effects of Methylmercury on γ-Aminobutyric Acid Type A Receptor Currents in Rat Cerebellar Granule and Cerebral Cortical Neurons in Culture

Cerebellar granule cells are particularly sensitive to inhibition by methylmercury (MeHg) on GABAA receptor function. This is manifested as a more rapid block of inhibitory postsynaptic currents/inhibitory postsynaptic potentials than for Purkinje cells. The underlying mechanism(s) for differential sensitivity of GABAergic transmission to MeHg in cerebellar neurons is unknown. Differential expression of α6 subunit-containing GABAA receptors in cerebellar granule and Purkinje neurons could partially explain this. GABA-evoked currents (IGABA) were recorded in response to MeHg in α6 subunit-containing cerebellar granule cells and α6 subunit-deficient cerebral cortical cells in culture. Cortical cells were substituted for Purkinje cells, which do not express α6 subunits. They express the same α1-containing GABAA receptor as Purkinje cells but lack characteristics that enhance Purkinje cell resistance to MeHg. IGABA were obtained using whole-cell recording and symmetrical [Cl–]. MeHg reduced IGABA to complete block in both cell types in a time- and concentration-dependent manner. This effect was faster in granule cells than cortical cells. Effects of MeHg on IGABA were recorded in granule cells at various developmental stages (days in vitro 4, 6, and 8) to alter the expression level of α6 subunit-containing GABAA receptors. Effects of MeHg on IGABA were similar in cells at all days. In human embryonic kidney 293 cells expressing either α6 or α1 subunit-containing GABAA receptors, time to block of IGABA by MeHg was comparable. Thus, the presence of the α6 subunit alone may not underlie the differential effects of MeHg on IGABA observed in cerebellar granule and cortical neurons; other factors are likely to be involved as well.

The Proteins Synaptotagmin and Syntaxin Are Not General Targets of Lambert-Eaton Myasthenic Syndrome Autoantibody†

Abstract: Lambert‐Eaton myasthenic syndrome (LEMS) is an autoimmune neuromuscular disease in which impairment of Ca2+ entry into the nerve ending and consequent impaired release of acetylcholine (ACh) results in muscle weakness. The identity of the primary antigenic target molecule(s) of the autoantibodies is uncertain. Electrophysiological studies and 45Ca2+ uptake studies implicate a direct effect on the Ca2+ channel complex at the motor nerve terminal. Some recent studies, however, suggest a more indirect interference caused by binding of autoantibodies to synaptotagmin or syntaxin, molecules presumed to be involved in docking and/or coupling the synaptic vesicles to the Ca2+ channels in the active zone for vesicle exocytosis and transmitter release. Western blot analyses of rat and human brain membrane proteins and pure recombinant synaptotagmin and syntaxin were used to examine directly the targets of LEMS autoantibodies and determine specifically whether or not synaptotagmin and/or syntaxin were general targets in LEMS. IgG from 14 patients with LEMS was used to probe western blots of gels containing synaptotagmin, syntaxin, rat synaptosomal proteins, and human brain membrane proteins. Several similar immunoreactive bands were observed using both rat and human brain membranes. These include high‐molecular‐weight protein bands whose size would be consistent with being components of Ca2+ channels. No reactive component was observed against either syntaxin or synaptotagmin in IgG of the 14 LEMS patients. However, both human and rat brain membranes contain proteins recognized by antibodies directed against synaptotagmin or syntaxin, indicating their immunologic relatedness and evolutionary conservation. These results suggest that large‐molecular‐weight proteins consistent with being Ca2+ channel subunits rather than syntaxin and synaptotagmin are general targets of LEMS autoantibodies.

Differential Effects of Methylmercury on GABAA Receptor Currents in Rat Cerebellar Granule and Cerebral Cortical Neurons in Culture

Cerebellar granule cells are particularly sensitive to inhibition by methylmercury (MeHg) on GABAA receptor function. This is manifested as a more rapid block of inhibitory postsynaptic currents/inhibitory postsynaptic potentials than for Purkinje cells. The underlying mechanism(s) for differential sensitivity of GABAergic transmission to MeHg in cerebellar neurons is unknown. Differential expression of α6 subunit-containing GABAA receptors in cerebellar granule and Purkinje neurons could partially explain this. GABA-evoked currents (IGABA) were recorded in response to MeHg in α6 subunit-containing cerebellar granule cells and α6 subunit-deficient cerebral cortical cells in culture. Cortical cells were substituted for Purkinje cells, which do not express α6 subunits. They express the same α1-containing GABAA receptor as Purkinje cells but lack characteristics that enhance Purkinje cell resistance to MeHg. IGABA were obtained using whole-cell recording and symmetrical [Cl–]. MeHg reduced IGABA to complete block in both cell types in a time- and concentration-dependent manner. This effect was faster in granule cells than cortical cells. Effects of MeHg on IGABA were recorded in granule cells at various developmental stages (days in vitro 4, 6, and 8) to alter the expression level of α6 subunit-containing GABAA receptors. Effects of MeHg on IGABA were similar in cells at all days. In human embryonic kidney 293 cells expressing either α6 or α1 subunit-containing GABAA receptors, time to block of IGABA by MeHg was comparable. Thus, the presence of the α6 subunit alone may not underlie the differential effects of MeHg on IGABA observed in cerebellar granule and cortical neurons; other factors are likely to be involved as well.

Genetic Transformation in Ulmus Species (Elms)

American elm (Ulmus americana) is believed to be the most aesthetically pleasing tree in the North American landscape. Its height, symmetrical crown, and arching vase shape made it a favorite of arboriculturists and nurserymen, and it was planted in great numbers. Prior to its devastation by Dutch elm disease (DED), the American elm was also prized by American and Canadian timber industries. Since the arrival of DED to North America, millions of American elms have been lost.