ABSTRACT
The most common side effects of transdermal patches are mechanical reactions caused by applying or removing the transdermal patch, or by excessive perspiration under the patch. Allergic contact dermatitis (type IV allergic reaction) is the most commonly occurring hypersensitivity reaction and can be caused by the active substance or by excipients. Type I allergic reactions such as urticaria, bronchospasm and angioedema are rare and usually caused by the active substance in the patch. Allergy testing to determine the allergen is indicated following a type I allergic reaction, or after a type IV allergic reaction that requires an alternative for the patch. It is important to document hypersensitivity reactions in the electronic patient records in order to prevent a hypersensitivity reaction in the future.
Subject(s)
Dermatitis, Allergic Contact/etiology , Excipients/adverse effects , Transdermal Patch/adverse effects , Angioedema/etiology , Dermatitis, Allergic Contact/diagnosis , Female , Humans , Male , Skin Tests , Urticaria/etiologyABSTRACT
Globe artichoke (Cynara cardunculus var. scolymus) belongs to the Asteraceae family, in which one of the most biologically significant class of secondary metabolites are sesquiterpene lactones (STLs). In globe artichoke the principal STL is the cynaropicrin, which contributes to approximately 80% of its characteristic bitter taste. Cynaropicrin content was assessed in globe artichoke tissues and was observed to accumulate in leaves of different developmental stages. In the receptacle, a progressive decrease was observed during inflorescence development, while the STL could not be detected in the inflorescence bracts. Almost undetectable amounts were found in the roots and inflorescence stems at the commercial stage. Cynaropicrin content was found to correlate with expression of genes encoding CcGAS, CcGAO and CcCOS, which are involved in the STL biosynthesis. A more detailed study of leaf material revealed that cynaropicrin predominantly accumulates in the trichomes, and not in the apoplastic cavity fluids. Analysis of the promoter regions of CcGAO and CcCOS revealed the presence of L1-box motifs, which confers trichome-specific expression in Arabidopsis, suggesting that cynaropicrin is not only stored but also synthesized in trichomes. A transient expression of GFP fusion proteins was performed in Nicotiana benthamiana plants: the CcGAS fluorescence signal was located in the cytoplasm while the CcGAO and CcCOS localized to the endoplasmatic reticulum.
Subject(s)
Cynara scolymus/genetics , Gene Expression Regulation, Plant , Lactones/metabolism , Plant Proteins/genetics , Sesquiterpenes/metabolism , Cynara scolymus/enzymology , Microscopy, Confocal , Microscopy, Fluorescence , Plant Proteins/metabolism , Tissue DistributionABSTRACT
Steroidal glycoalkaloids (SGAs) such as α-solanine found in solanaceous food plants--as, for example, potato--are antinutritional factors for humans. Comparative coexpression analysis between tomato and potato coupled with chemical profiling revealed an array of 10 genes that partake in SGA biosynthesis. We discovered that six of them exist as a cluster on chromosome 7, whereas an additional two are adjacent in a duplicated genomic region on chromosome 12. Following systematic functional analysis, we suggest a revised SGA biosynthetic pathway starting from cholesterol up to the tetrasaccharide moiety linked to the tomato SGA aglycone. Silencing GLYCOALKALOID METABOLISM 4 prevented accumulation of SGAs in potato tubers and tomato fruit. This may provide a means for removal of unsafe, antinutritional substances present in these widely used food crops.
Subject(s)
Crops, Agricultural/genetics , Multigene Family , Nutritive Value/genetics , Solanaceous Alkaloids/biosynthesis , Solanaceous Alkaloids/genetics , Solanum lycopersicum/genetics , Solanum tuberosum/genetics , Gene Expression Profiling , Gene Expression Regulation, Plant , Gene Silencing , Genes, Plant , Solanaceous Alkaloids/toxicityABSTRACT
Regulation of the intracellular calcium concentration ([Ca(2+)](i)) is of critical importance for synaptic function. Therefore, neurons buffer [Ca(2+)](i) using intracellular Ca(2+)-binding proteins (CaBPs). Previous evidence suggests that Calbindin-D(28K) (CB), an abundantly expressed endogenous fast CaBP, plays an important role in neuronal survival, motor coordination, spatial learning paradigms and some forms of synaptic plasticity. In the present study, the role of CB in synaptic transmission and plasticity was further investigated using extracellular recordings of synaptic activity in cell- and dendritic layers of dentate gyrus (DG) and CA1 area in hippocampal slices from wild-type, heterozygous and homozygous CB knockout mice. The results demonstrate a consistent failure to maintain long-term potentiation (LTP) in hippocampal DG and CA1 area of knockout mice. Compared to wild-type mice, the paired-pulse ratio of EPSPs recorded in DG is significantly lower in slices from knockout mice, whereas it is significantly higher in CA1 area. The amplitude of the population spike recorded in CA1 area of wild-type mice steadily increases following tetanic stimulation, whereas it steadily decreases in knockout mice. The combined results demonstrate that the absence of CB results in an impairment of LTP maintenance in both hippocampal DG and CA1 area, whereas paired-pulse facilitation and cellular excitability in CA1 area are differentially affected. These results support the role of CB as a critical determinant for several forms of synaptic plasticity in hippocampal DG and CA1 area. It is hypothesized that CB functions as a postsynaptic Ca(2+) buffer as well as a presynaptic Ca(2+) sensor.
Subject(s)
CA1 Region, Hippocampal/physiology , Dentate Gyrus/physiology , Neuronal Plasticity/physiology , Neurons/physiology , S100 Calcium Binding Protein G/genetics , Synaptic Transmission/physiology , Animals , Calbindin 1 , Calbindins , Electric Stimulation , Long-Term Potentiation/physiology , Mice , Mice, Knockout , Synapses/physiologyABSTRACT
Vagus nerve stimulation (VNS) has become an established therapy for difficult-to-treat epilepsy during the past 20 years. The vagus nerve provides a unique entrance to the brain. Electrical stimulation of this structure in the cervical region allows direct modulative access to subcortical brain areas, requiring only minimally invasive surgery with low risks involved. VNS therapy has shown to reduce epileptic seizures both in number and severity in a group of patients not responding to antiepileptic drugs. The effects are accompanied by an atypical set of central side effects. After the success of the VNS therapy with epilepsy, the technique has been applied to a wide variety of disorders, ranging from major depressive disorder to Alzheimer's disease. The results of several of these are promising. In this review, the results as well as the rationale for the different applications of VNS are discussed.
Subject(s)
Vagus Nerve Stimulation , Animals , Humans , Vagus Nerve Stimulation/adverse effects , Vagus Nerve Stimulation/instrumentation , Vagus Nerve Stimulation/methodsABSTRACT
Calbindin-D(28K) is suggested to play a postsynaptic role in neurotransmission and in the regulation of the intracellular Ca(2+) concentration. However, it is still unclear whether calbindin-D(28K) has a role in the regulation of exocytosis, either as Ca(2+) buffer or as Ca(2+) sensor. Amperometric recordings of catecholamine exocytosis from wild-type and calbindin-D(28K) knockout mouse chromaffin cells reveal a strong reduction in the number of released vesicles, as well as in the amount of neurotransmitter released per fusion event in knockout cells. However, Ca(2+) current recordings and Ca(2+) imaging experiments, including video-rate confocal laser scanning microscopy, revealed that the intracellular Ca(2+) dynamics are remarkably similar in wild-type and knockout cells. The combined results demonstrate that calbindin-D(28K) plays an important and dual role in exocytosis, affecting both release frequency and quantal size, apparently without strong effects on intracellular Ca(2+) dynamics. Consequently, the possibility that calbindin-D(28K) functions not only as a Ca(2+) buffer but also as a modulator of vesicular catecholamine release is discussed.
Subject(s)
Adrenal Medulla/metabolism , Calcium Signaling/physiology , Catecholamines/metabolism , Chromaffin Cells/metabolism , Cytoplasmic Vesicles/metabolism , S100 Calcium Binding Protein G/physiology , Adrenal Medulla/ultrastructure , Animals , Calbindin 1 , Calbindins , Calcium/metabolism , Cells, Cultured , Chromaffin Cells/ultrastructure , Cytoplasmic Vesicles/ultrastructure , Exocytosis/genetics , Female , Immunohistochemistry , Intracellular Fluid/metabolism , Male , Membrane Fusion/genetics , Membrane Potentials/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Confocal , S100 Calcium Binding Protein G/genetics , Synaptic Transmission/genetics , Synaptic Vesicles/metabolism , Synaptic Vesicles/ultrastructureABSTRACT
In this study, we investigated the effects of the local anesthetic n-butyl-p-aminobenzoate (BAB) on the delayed rectifier potassium current of cultured dorsal root ganglion (DRG) neurons using the patch-clamp technique. The majority of the K(+) current of small DRG neurons rapidly activates and slowly inactivates at depolarized voltages. BAB inhibited the whole-cell K(+) current of these neurons with an IC(50) value of 228 microM. Dendrotoxin K (DTX(K)), a specific inhibitor of Kv1.1, reduced the DRG K(+) current at +20 mV by 34%, consistent with an important contribution of channels incorporating the Kv1.1 subunit to the delayed rectifier current. To further investigate the mechanism of BAB inhibition, we examined its effect on Kv1.1 channels heterologously expressed in mammalian tsA201 cells. BAB inhibits the Kv1.1 channels with an IC(50) value of 238 microM, similar to what was observed for the native DRG current. BAB accelerates the opening and closing of Kv1.1, but does not alter the midpoint of steady-state activation. BAB seems to inhibit Kv1.1 by stabilizing closed conformations of the channel. Coexpression with the Kv beta 1 subunit induces rapid inactivation and reduces the BAB sensitivity of Kv1.1. Comparison of the heterologously expressed Kv1.1 and native DRG currents indicates that the Kv beta 1 subunit does not modulate the gating of the DTX(K)-sensitive Kv1.1 channels of DRG neurons. Inhibition of the delayed rectifier current of these neurons may contribute to the long-duration anesthesia attained during the epidural administration of BAB.
Subject(s)
Anesthetics, Local/pharmacology , Benzocaine/analogs & derivatives , Benzocaine/pharmacology , Ganglia, Spinal/drug effects , Neurons/metabolism , Pain/drug therapy , Potassium Channel Blockers/pharmacology , Potassium Channels, Voltage-Gated , Potassium Channels/physiology , Action Potentials/drug effects , Action Potentials/physiology , Anesthetics, Local/therapeutic use , Animals , Cells, Cultured , Chronic Disease , Ganglia, Spinal/physiology , Humans , In Vitro Techniques , Kv1.1 Potassium Channel , Mice , Neurons/drug effects , Neurons/physiology , Potassium Channel Blockers/therapeutic useABSTRACT
The mustard trypsin inhibitor MTI-2 is a potential tool in the study of interactions between pest insects and plants. It can be applied to study the adaptations of digestive proteases in pest insects. Phage display allows a rapid and exhaustive system for the selection of heterologous protein variants with novel specificities. Here we describe a bacteriophage expression system which permits functional expression of MTI-2 variants. Active and inactive mutants of MTI-2 are constructed and displayed on phage. These are used to demonstrate that an active variant can be selected from a background of 10,000 inactive mutants in four rounds of selection and amplification.
Subject(s)
Mustard Plant/genetics , Plant Proteins/genetics , Plants, Medicinal , Trypsin Inhibitors/genetics , Animals , Base Sequence , DNA Primers/genetics , Escherichia coli/genetics , Gene Expression , Genetic Variation , Peptide Library , Pichia/genetics , Plant Proteins/metabolism , Protein Engineering , Trypsin Inhibitors/metabolismABSTRACT
Potato proteinase inhibitor II (PI-2) is composed of two sequence repeats. It contains two reactive site domains. We developed an improved protocol for the production of PI-2 using the yeast Pichia pastoris as the expression host. We then assessed the role of its two reactive sites in the inhibition of trypsin and chymotrypsin by mutating each of the two reactive sites in various ways. From these studies it appears that the second reactive site strongly inhibits both trypsin (Ki = 0.4 nM) and chymotrypsin (Ki = 0.9 nM), and is quite robust towards mutations at positions P2 or P1'. In contrast, the first reactive site inhibits only chymotrypsin (Ki = 2 nM), and this activity is very sensitive to mutations. Remarkably, replacing the reactive site amino acids of domain I with those of domain II did not result in inhibitory activities similar to domain II. The fitness for protein engineering of each domain is discussed.
Subject(s)
Plant Proteins/metabolism , Protease Inhibitors , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA , Molecular Sequence Data , Pichia/genetics , Plant Proteins/chemistry , Plant Proteins/genetics , Sequence Homology, Amino Acid , Solanum tuberosum/metabolism , Trypsin/metabolismABSTRACT
Ischemia-induced depolarizations may play a key role in the development of cerebral ischemic injury. Our goal was to assess the relationship between tissue depolarizations and tissue damage in focal ischemia. We performed multi-electrode cortical direct current (DC) potential recording and, subsequently, diffusion-weighted and T(2)-weighted magnetic resonance imaging (MRI) in rats after i) cortical application of KCl, and ii) permanent and transient middle cerebral artery (MCA)-occlusion in rats. Cortical KCl application induced 10.0+/-2.2 transient negative DC potential shifts per h on the ipsilateral hemisphere (i.e. cortical spreading depressions) (n=4). During 6 h of permanent MCA-occlusion (n=9) 1-10 DC potential shifts were observed, dependent on the brain location. Anoxic depolarization developed in the ischemic core. Outside ischemic areas DC potential shifts resembled cortical spreading depressions. Depolarizations in cortical ischemic borderzones were also transient, but generally long-lasting. Reperfusion induced 1 (n=5) or 3 h (n=6) after MCA-occlusion resulted in repolarization in 2.9+/-1.5 min. Ischemic lesion volumes after 7 h, calculated from diffusion-weighted and T(2)-weighted MR images, correlated significantly with total depolarization time in cortical perifocal zones (R=0.741, p<0.05), but not with the number of depolarizations. The extent of ischemic damage, as measured from alterations in the water diffusion coefficient and T(2), was also significantly related to the total time of depolarization (R=0.762 and 0.738, respectively, p<0.01). We conclude that early ischemic tissue injury is related to the total duration of tissue depolarization and not to the frequency of depolarizations.
Subject(s)
Brain Ischemia/pathology , Brain Ischemia/physiopathology , Brain/pathology , Brain/physiopathology , Animals , Brain/drug effects , Brain Ischemia/diagnosis , Cortical Spreading Depression , Electrophysiology , Ischemic Attack, Transient/diagnosis , Ischemic Attack, Transient/pathology , Ischemic Attack, Transient/physiopathology , Magnetic Resonance Imaging , Male , Potassium Chloride/pharmacology , Rats , Rats, Wistar , Reperfusion Injury/pathology , Reperfusion Injury/physiopathologyABSTRACT
Phage display is a powerful tool with which to adapt the specificity of protease inhibitors. To this end, a library of variants of the potato protease inhibitor PI2 was introduced in a canonical phagemid vector. Although PI2 is a natural trypsin inhibitor, we were unable to select trypsin-binding variants from the library. Instead, only mutants carrying deletions or amber stop codons were found. Bacteria carrying these mutations had a much faster growth rate than those carrying the wt PI2-encoding gene, even when the promoter was repressed. To overcome these problems, two new phagemid vectors for g3-mediated phage display were constructed. The first vector has a lower plasmid copy number, as compared to the canonical vector. Bacteria harboring this new vector are much less affected by the presence of the PI2-g3 fusion gene, which appears from a markedly reduced growth retardation. A second vector was equipped with the promoter of the Escherichia coli psp operon, instead of the lac promoter, to control the PI2-g3 gene fusion expression. The psp promoter is induced upon helper phage infection. A phagemid vector with this promoter controlling a PI2-g3 gene fusion did not affect the viability of the host. Furthermore, both new vectors were shown to produce phage particles that display the inhibitor protein and were therefore considered suitable for phage display. The inhibitor library was introduced in both new vectors. Trypsin-binding phages with inhibitory sequences were selected, instead of sequences with stop codons or deletions. This demonstrates the usefulness of these new vectors for phage display of proteins that affect the viability of E. coli.
Subject(s)
Bacterial Proteins/genetics , Bacteriophages/genetics , Escherichia coli/genetics , Genetic Vectors/genetics , Heat-Shock Proteins/genetics , Promoter Regions, Genetic , Protease Inhibitors/metabolism , Amino Acid Sequence , Bacteria/genetics , Bacteria/growth & development , Bacteria/virology , Base Sequence , Binding, Competitive , Capsid Proteins , Cloning, Molecular , DNA-Binding Proteins/genetics , Endopeptidases/metabolism , Gene Library , Genetic Variation , Inovirus/genetics , Plasmids/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Trypsin/metabolism , Viral Fusion Proteins/genetics , Virus AssemblyABSTRACT
We present a secondary structure model for the first 860 nucleotides of Q beta RNA. The model is supported by phylogenetic comparison, nuclease S1 structure probing and computer prediction using energy minimization and a Monte Carlo approach. To provide the necessary data for the comparative analysis we have sequenced the single-stranded RNA coliphages MX1, M11 and NL95. Together with the known sequences of Q beta and SP, this yields five sequences with sufficient sequence diversity to be useful for the analysis. The part of the Q beta genome examined contains the 60 nucleotide 5' untranslated region and the first 800 nucleotide of the maturation protein gene. The RNA adopts a highly ordered structure in which all hairpins are held in place by a network of long-distance interactions, which form three-way and four-way junctions. Only the 5'-terminal hairpin is unrestrained, while connected by a few single-stranded nucleotides to the body of the RNA. The start region of the A-protein gene, which is part of the network of long-distance interactions, is base-paired to three non-contiguous downstream sequences. As a result, translation is expected to be progressively quenched when the length of the nascent chains increases. This feature explains the previous observation that A-protein synthesis on Q beta RNA can start only on short nascent strands. Translational control of the A protein in the distantly related phage MS2 was recently shown to be controlled by the kinetics of RNA folding. This basic difference and its possible biological purpose can be explained by the different RNA folding pathways in Q beta and MS2. Interestingly, due to the presence of G-U pairs, structure prediction for the minus strand differs in some aspects from that for the plus strand. More specifically, there is a minus-strand specific, long-distance interaction bordering the minus-strand equivalent of the 5'-terminal hairpin. This interaction extends at the expense of the lower part of the terminal helix, thereby exposing the terminal C residues at which replication starts. This long-distance interaction, which was recently shown to be required for minus-strand replication, is strongly supported by our comparative data.
Subject(s)
Allolevivirus/chemistry , Models, Molecular , Nucleic Acid Conformation , RNA, Viral/chemistry , Viral Proteins/biosynthesis , Allolevivirus/genetics , Allolevivirus/metabolism , Base Sequence , Molecular Sequence Data , RNA, Viral/genetics , Sequence Homology, Nucleic Acid , Thermodynamics , Viral Proteins/geneticsABSTRACT
F-specific RNA phages can be used as model organisms for enteric viruses to monitor the effectiveness of sewage treatment, and to assess the potential contamination of surface water with these viruses. In this paper a method is described which identifies RNA phages quantitatively by a plaque hybridization assay. Oligonucleotide probes were developed that can assign phages to their phylogenetic subgroups. Such a distinction is important, since some subgroups preferentially occur in sewage of human origin, while others tend to be associated with animal wastewater. The method has been tested on a large number of isolates and represents an improvement in time and reliability over the previously used serological classification.
Subject(s)
Nucleic Acid Hybridization , RNA Phages/isolation & purification , RNA, Viral , Base Sequence , Filtration , Molecular Sequence Data , Oligonucleotide Probes , RNA Phages/classification , RNA Phages/genetics , Reproducibility of Results , Sensitivity and Specificity , Water MicrobiologyABSTRACT
Binding of high-density lipophorin (HDLp) to a plasma membrane preparation of locust flight muscle tissue was studied using a radiolabelled ligand binding assay and ligand blotting techniques. Analysis at 33 degrees C of the concentration-dependent total binding of tritium-labelled HDLp ([3H]HDLp) to the membrane preparation revealed the presence of a single specific binding site with an equilibrium dissociation constant of Kd = 9 (+/- 2) X 10(-7) M and a maximal binding capacity of 84 (+/- 10) ng X (micrograms protein)-1. Unlabelled HDLp as well as unlabelled low-density lipophorin (LDLp) competed with [3H]HDLp for binding to the identified binding site. In addition, ligand blotting demonstrated that both HDLp and LDLp bind specifically to a 30-kDa protein in the plasma membrane preparation, suggesting the involvement of this protein in the binding of lipophorins to the isolated membranes. A possible relationship between the identified binding of lipophorins and the observed co-purification of lipophorin lipase activity with the plasma membranes is discussed.