Reciprocal regulation of nuclear factor kappa B and its inhibitor ZAS3 after peripheral nerve injury

BACKGROUND: NF-κB binds to the κB motif to regulate transcription of genes involved in growth, immunity and inflammation, and plays a pivotal role in the production of pro-inflammatory cytokines after nerve injuries. The zinc finger protein ZAS3 also binds to the κB or similar motif. In addition to competition for common DNA sites, in vitro experiments have shown that ZAS3 can inhibit NF-κB via the association with TRAF2 to inhibit the nuclear translocation of NF-κB. However, the physiological significance of the ZAS3-mediated inhibition of NF-κB has not been demonstrated. The purpose of this study is to characterize ZAS3 proteins in nervous tissues and to use spinal nerve ligation, a neuropathic pain model, to demonstrate a functional relationship between ZAS3 and NF-κB. RESULTS: Immunohistochemical experiments show that ZAS3 is expressed in specific regions of the central and peripheral nervous system. Abundant ZAS3 expression is found in the trigeminal ganglion, hippocampal formation, dorsal root ganglia, and motoneurons. Low levels of ZAS3 expressions are also found in the cerebral cortex and in the grey matter of the spinal cord. In those nervous tissues, ZAS3 is expressed mainly in the cell bodies of neurons and astrocytes. Together with results of Western blot analyses, the data suggest that ZAS3 protein isoforms with differential cellular distribution are produced in a cell-specific manner. Further, neuropathic pain confirmed by persistent mechanical allodynia was manifested in rats seven days after L5 and L6 lumbar spinal nerve ligation. Changes in gene expression, including a decrease in ZAS3 and an increase in the p65 subunit of NF-κB were observed in dorsal root ganglion ipsilateral to the ligation when compared to the contralateral side. CONCLUSION: ZAS3 is expressed in nervous tissues involved in cognitive function and pain modulation. The down-regulation of ZAS3 after peripheral nerve injury may lead to activation of NF-κB, allowing Wallerian regeneration and induction of NF-κB-dependent gene expression, including pro-inflammatory cytokines. We propose that reciprocal changes in the expression of ZAS3 and NF-κB might generate neuropathic pain after peripheral nerve injury

Preferential protection of domains ii and iii of bacillus thuringiensis cry1aa toxin by brush border membrane vesicles

Título español: Protección preferencial de los dominios II y III de la toxina Cry1Aa de Bacillus thuringiensis en Vesículas de Membrana de Borde de Cepillo Abstract The surface exposed Leucine 371 on loop 2 of domain II, in Cry1Aa toxin, was mutated to Lysine to generate the trypsin-sensitive mutant, L371K. Upon trypsin digestion L371K is cleaved into approximately 37 and 26 kDa fragments. These are separable on SDS-PAGE, but remain as a single molecule of 65 kDa upon purification by liquid chromatography. The larger fragment is domain I and a portion of domain II (amino acid residues 1 to 371). The smaller 26-kDa polypeptide is the remainder of domain II and domain III (amino acids 372 to 609). When the mutant toxin was treated with high dose of M. sexta gut juice both fragments were degraded. However, when incubated with M. sexta BBMV, the 26 kDa fragment (domains II and III) was preferentially protected from gut juice proteases. As previously reported, wild type Cry1Aa toxin was also protected against degradation by gut juice proteases when incubated with M. sexta BBMV. On the contrary, when mouse BBMV was added to the reaction mixture neither Cry1Aa nor L371K toxins showed resistance to M. sexta gut juice proteases and were degraded. Since the whole Cry1Aa toxin and most of the domain II and domain III of L371K are protected from proteases in the presence of BBMV of the target insect, we suggest that the insertion of the toxin into the membrane is complex and involves all three domains. Key words: Bacillus thuringiensis, site directed mutagenesis, -endotoxin. Resumen La superficie de la toxina Cry1Aa, en el asa 2 del dominio II contiene expuesta la leucina 371, la cual fue modificada a lisina produciendo una mutante sensible a la tripsina, L371K. Esta mutante produce dos fragmentos de 37 y 26 kDa por acción de la tripsina que son separables por SDS-PAGE, pero que a la purificación por cromatografía líquida se mantienen como una sola molécula de 65 kDa. El fragmento grande contiene al dominio I y una parte del dominio II (aminoácidos 1 al 371). El polipéptido de 26 kDa contiene la parte restante del dominio II y dominio III (aminoácidos 372 al 609). Cuando la toxina mutante fue tratada con dosis altas de jugo intestinal de Manduca sexta, ambos fragmentos fueron degradados. Sin embargo, cuando fueron incubados en VMBC de M. sexta, el fragmento de 26 kDa fue protegido preferencialmente de las proteasas intestinales. Como se ha reportado, la toxina silvestre Cry1Aa también es protegida de la degradación de las proteasas cuando es incubada en VMBC de M. sexta. Sin embargo, cuando se adicionó VMBC de ratón a la mezcla de reacción, ni la toxina Cry1Aa ni la mutante L371K mostraron resistencia a las proteasas y fueron degradadas. Dado que la toxina completa de Cry1Aa y casi todo de los dominios II y III de L371K están protegidos de proteasas en presencia de VMBC del insecto, este estudio sugiere que la inserción de la toxina en la membrana involucra los tres dominios. Palabras clave: Bacillus thuringiensis, mutagénesis sitio dirigida, - endotoxin

Bleeding disorders in the tribe: result of consanguineous in breeding

<p>Abstract</p> <p>Objective</p> <p>To determine the frequency and clinical features of bleeding disorders in the tribe as a result of consanguineous marriages.</p> <p>Design</p> <p>Cross Sectional Study</p> <p>Introduction</p> <p>Countries in which consanguinity is a normal practice, these rare autosomal recessive disorders run in close families and tribes. Here we describe a family, living in village Ali Murad Chandio, District Badin, labeled as haemophilia.</p> <p>Patients & Methods</p> <p>Our team visited the village & developed the pedigree of the whole extended family, up to seven generations. Performa was filled by incorporating patients, family history of bleeding, signs & symptoms, and bleeding from any site. From them 144 individuals were screened with CBC, bleeding time, platelet aggregation studies & RiCoF. While for PT, APTT, VWF assay and Factor VIII assay, samples were kept frozen at -70 degrees C until tested.</p> <p>Results</p> <p>The family tree of the seven generations comprises of 533 individuals, 63 subjects died over a period of 20 years and 470 were alive. Out of all those 144 subjects were selected on the basis of the bleeding history. Among them 98(68.1%) were diagnosed to have a bleeding disorder; 44.9% patients were male and 55.1% patients were female. Median age of all the patients was 20.81, range (4 months- 80 yrs). The results of bleeding have shown that majority had gum bleeding, epistaxis and menorrhagia. Most common bleeding disorder was Von Willebrand disease and Platelet functional disorders.</p> <p>Conclusion</p> <p>Consanguineous marriages keep all the beneficial and adversely affecting recessive genes within the family; in homozygous states. These genes express themselves and result in life threatening diseases. Awareness, education & genetic counseling will be needed to prevent the spread of such common occurrence of these bleeding disorders in the community.</p

Preferential Protection of Domains II and III of Bacillus thuringiensis Cry1Aa Toxin by Brush Border Membrane Vesicles

The surface exposed Leucine 371 on loop 2 of domain II, in Cry1Aa toxin, was mutated to Lysine to generate the trypsin-sensitive mutant, L371K. Upon trypsin digestion L371K is cleaved into approximately 37 and 26 kDa fragments. These are separable on SDS-PAGE, but remain as a single molecule of 65 kDa upon purification by liquid chromatography. The larger fragment is domain I and a portion of domain II (amino acid residues 1 to 371). The smaller 26-kDa polypeptide is the remainder of domain II and domain III (amino acids 372 to 609). When the mutant toxin was treated with high dose of M. sexta gut juice both fragments were degraded. However, when incubated with M. sexta BBMV, the 26 kDa fragment (domains II and III) was preferentially protected from gut juice proteases. As previously reported, wild type Cry1Aa toxin was also protected against degradation by gut juice proteases when incubated with M. sexta BBMV. On the contrary, when mouse BBMV was added to the reaction mixture neither Cry1Aa nor L371K toxins showed resistance to M. sexta gut juice proteases and were degraded. Since the whole Cry1Aa toxin and most of the domain II and domain III of L371K are protected from proteases in the presence of BBMV of the target insect, we suggest that the insertion of the toxin into the membrane is complex and involves all three domains.La superficie de la toxina Cry1Aa, en el asa 2 del dominio II contiene expuesta la leucina 371, la cual fue modificada a lisina produciendo una mutante sensible a la tripsina, L371K. Esta mutante produce dos fragmentos de 37 y 26 kDa por acción de la tripsina que son separables por SDS-PAGE, pero que a la purificación por cromatografía líquida se mantienen como una sola molécula de 65 kDa. El fragmento grande contiene al dominio I y una parte del dominio II (aminoácidos 1 al 371). El polipéptido de 26 kDa contiene la parte restante del dominio II y dominio III (aminoácidos 372 al 609). Cuando la toxina mutante fue tratada con dosis altas de jugo intestinal de Manduca sexta, ambos fragmentos fueron degradados. Sin embargo, cuando fueron incubados en VMBC de M. sexta, el fragmento de 26 kDa fue protegido preferencialmente de las proteasas intestinales. Como se ha reportado, la toxina silvestre Cry1Aa también es protegida de la degradación de las proteasas cuando es incubada en VMBC de M. sexta. Sin embargo, cuando se adicionó VMBC de ratón a la mezcla de reacción, ni la toxina Cry1Aa ni la mutante L371K mostraron resistencia a las proteasas y fueron degradadas. Dado que la toxina completa de Cry1Aa y casi todo de los dominios II y III de L371K están protegidos de proteasas en presencia de VMBC del insecto, este estudio sugiere que la inserción de la toxina en la membrana involucra los tres dominios

Preferential Protection of Domains II and III of Bacillus thuringiensis Cry1Aa Toxin by Brush Border Membrane Vesicles

Título español: Protección preferencial de los dominios II y III de la toxina Cry1Aa de Bacillus thuringiensis en Vesículas de Membrana de Borde de Cepillo Abstract The surface exposed Leucine 371 on loop 2 of domain II, in Cry1Aa toxin, was mutated to Lysine to generate the trypsin-sensitive mutant, L371K. Upon trypsin digestion L371K is cleaved into approximately 37 and 26 kDa fragments. These are separable on SDS-PAGE, but remain as a single molecule of 65 kDa upon purification by liquid chromatography. The larger fragment is domain I and a portion of domain II (amino acid residues 1 to 371). The smaller 26-kDa polypeptide is the remainder of domain II and domain III (amino acids 372 to 609). When the mutant toxin was treated with high dose of M. sexta gut juice both fragments were degraded. However, when incubated with M. sexta BBMV, the 26 kDa fragment (domains II and III) was preferentially protected from gut juice proteases. As previously reported, wild type Cry1Aa toxin was also protected against degradation by gut juice proteases when incubated with M. sexta BBMV. On the contrary, when mouse BBMV was added to the reaction mixture neither Cry1Aa nor L371K toxins showed resistance to M. sexta gut juice proteases and were degraded. Since the whole Cry1Aa toxin and most of the domain II and domain III of L371K are protected from proteases in the presence of BBMV of the target insect, we suggest that the insertion of the toxin into the membrane is complex and involves all three domains. Key words: Bacillus thuringiensis, site directed mutagenesis, -endotoxin. Resumen La superficie de la toxina Cry1Aa, en el asa 2 del dominio II contiene expuesta la leucina 371, la cual fue modificada a lisina produciendo una mutante sensible a la tripsina, L371K. Esta mutante produce dos fragmentos de 37 y 26 kDa por acción de la tripsina que son separables por SDS-PAGE, pero que a la purificación por cromatografía líquida se mantienen como una sola molécula de 65 kDa. El fragmento grande contiene al dominio I y una parte del dominio II (aminoácidos 1 al 371). El polipéptido de 26 kDa contiene la parte restante del dominio II y dominio III (aminoácidos 372 al 609). Cuando la toxina mutante fue tratada con dosis altas de jugo intestinal de Manduca sexta, ambos fragmentos fueron degradados. Sin embargo, cuando fueron incubados en VMBC de M. sexta, el fragmento de 26 kDa fue protegido preferencialmente de las proteasas intestinales. Como se ha reportado, la toxina silvestre Cry1Aa también es protegida de la degradación de las proteasas cuando es incubada en VMBC de M. sexta. Sin embargo, cuando se adicionó VMBC de ratón a la mezcla de reacción, ni la toxina Cry1Aa ni la mutante L371K mostraron resistencia a las proteasas y fueron degradadas. Dado que la toxina completa de Cry1Aa y casi todo de los dominios II y III de L371K están protegidos de proteasas en presencia de VMBC del insecto, este estudio sugiere que la inserción de la toxina en la membrana involucra los tres dominios. Palabras clave: Bacillus thuringiensis, mutagénesis sitio dirigida, - endotoxin

Caracterización de una delta endotoxina mutante de Bacillus thuringiensis con estabilidad y toxicidad aumentadas

<p class="MsoNormal" style="line-height: normal; margin: 0cm 0cm 0pt; tab-stops: 184.3pt;"><span style="font-size: small;"><strong style="mso-bidi-font-weight: normal;"><span style="font-family: " lang="EN-US">Título en ingles: Characterization of a Mutant <em style="mso-bidi-font-style: normal;">Bacillus</em> <em style="mso-bidi-font-style: normal;">thuringiensis</em> </span></strong><strong style="mso-bidi-font-weight: normal;"><span style="font-family: Symbol; mso-bidi-font-size: 12.0pt;">d-</span></strong><strong style="mso-bidi-font-weight: normal;"><span style="font-family: " lang="EN-US">endotoxin With Enhanced Stability and Toxicity</span></strong></span></p><p class="MsoNormal" style="line-height: normal; margin: 0cm 0cm 0pt; tab-stops: 184.3pt;"><strong style="mso-bidi-font-weight: normal;"><span style="font-family: " lang="EN-US"><span style="font-size: small;"> </span></span></strong></p><p class="MsoNormal" style="text-align: justify; line-height: normal; margin: 0cm 0cm 0pt;"><strong style="mso-bidi-font-weight: normal;"><span style="font-family: " lang="EN-US"><span style="font-size: small;">Summary</span></span></strong></p><p class="MsoNormal" style="text-align: justify; line-height: normal; margin: 0cm 0cm 0pt;"><span style="font-size: small;"><span style="font-family: " lang="EN-US">The centrally located </span><span style="font-family: Symbol; mso-bidi-font-size: 12.0pt;">a-</span><span style="font-family: " lang="EN-US">helix 5 of <em style="mso-bidi-font-style: normal;">Bacillus thuringiensis</em> </span><span style="font-family: Symbol; mso-bidi-font-size: 12.0pt;">d-</span><span style="font-family: " lang="EN-US">endotoxins is critical for insect toxicity through ion-channel formation. We analyzed the role of the highly conserved residue Histidine 168 (H168) using molecular biology, electrophysiology and biophysical techniques. Toxin H168R was ~3-fold more toxic than the wild type (wt) protein whereas H168Q was 3 times less toxic against <em style="mso-bidi-font-style: normal;">Manduca sexta</em>. Spectroscopic analysis revealed that the H168Q and H168R mutations did not produce gross structural alterations, and that H168R (T_{<span style="position: relative; top: 2pt; mso-text-raise: -2.0pt;">m</span>}= 59 °C) was more stable than H168Q (T_{<span style="position: relative; top: 2pt; mso-text-raise: -2.0pt;">m</span>}= 57.5 °C) or than the wt (T_{<span style="position: relative; top: 2pt; mso-text-raise: -2.0pt;">m</span>}= 56 °C) toxins. These three toxins had similar binding affinities for larval midgut vesicles (K_{<span style="position: relative; top: 2pt; mso-text-raise: -2.0pt;">com</span>}) suggesting that the differences in toxicity did not result from changes in initial receptor binding. Dissociation binding assays and voltage clamping analysis suggest that the reduced toxicity of the H168Q toxin may result from reduced insertion and/or ion channel formation. In contrast, the H168R toxin had a greater inhibition of the short circuit current than the wt toxin and an increased rate of irreversible binding (k_obs), consistent with its lower LC_{<span style="position: relative; top: 2pt; mso-text-raise: -2.0pt;">50</span>}<span style="mso-spacerun: yes;"> </span>value.<span style="mso-spacerun: yes;">  </span>Molecular modeling analysis suggested that both the H168Q and H168R toxins could form additional hydrogen bonds that could account for their greater thermal stability. In addition to this, it is likely that H168R has an extra positive charge exposed to the surface which could increase its rate of insertion into susceptible membranes.<strong style="mso-bidi-font-weight: normal;"></strong></span></span></p><p class="MsoNormal" style="text-align: justify; line-height: normal; margin: 0cm 0cm 0pt;"><span style="font-family: " lang="EN-US"><span style="font-size: small;"> </span></span></p><p class="MsoNormal" style="text-align: justify; line-height: normal; margin: 0cm 0cm 0pt;"><span style="font-size: small;"><strong style="mso-bidi-font-weight: normal;"><span style="font-family: " lang="EN-US">Key words:</span></strong><span style="font-family: " lang="EN-US"> </span><span style="font-family: Symbol; mso-bidi-font-size: 12.0pt;">a</span><span style="font-family: " lang="EN-US">-helix 5; Circular dichroism; molecular modeling; site-directed mutagenesis; <span style="mso-spacerun: yes;"> </span>thermal stability; <em style="mso-bidi-font-style: normal;">Bacillus thuringiensis</em></span></span></p><p class="MsoNormal" style="text-align: justify; line-height: normal; margin: 0cm 0cm 0pt;"><strong style="mso-bidi-font-weight: normal;"><span style="font-family: " lang="EN-US"><span style="font-size: small;"> </span></span></strong></p><p class="MsoNormal" style="text-align: justify; line-height: normal; margin: 0cm 0cm 0pt;"><strong style="mso-bidi-font-weight: normal;"><span style="font-family: "><span style="font-size: small;">Resumen</span></span></strong></p><p class="MsoNormal" style="text-align: justify; line-height: normal; margin: 0cm 0cm 0pt;"><span style="font-size: small;"><span style="font-family: ">La </span><span style="font-family: Symbol; mso-bidi-font-size: 12.0pt;">a</span><span style="font-family: ">-Hélice 5 del domino I de las </span><span style="font-family: Symbol; mso-bidi-font-size: 12.0pt;">d-</span><span style="font-family: ">endotoxinas de <em style="mso-bidi-font-style: normal;">Bacillus thuringiensis,</em> es crítica para la toxicidad de las toxinas contra insectos al participar en la formación de canales iónicos. La participación en la función tóxica del residuo Histidina 168 (H168) –el cual es altamente conservado– fue estudiada mediante técnicas de biología molecular, electrofisiología y biofísica. La toxina mutante H168R fue ~ 3 veces más tóxica que la toxina silvestre (ts) en <em style="mso-bidi-font-style: normal;">Manduca sexta</em>, mientras que H168Q fue 3 veces menos tóxica. Los análisis espectroscópicos indicaron que las mutaciones no producen alteraciones estructurales significativas y que la toxina H168R (T_{<span style="position: relative; top: 2pt; mso-text-raise: -2.0pt;">m</span>}= 59 °C) es más estable que las toxinas H168Q (T_{<span style="position: relative; top: 2pt; mso-text-raise: -2.0pt;">m</span>}= 57.5 °C) y wt (T_{<span style="position: relative; top: 2pt; mso-text-raise: -2.0pt;">m</span>}= 56 °C). Las tres toxinas exhibieron uniones de afinidad similares (K_com) en vesículas de intestino de larvas de insecto, indicando que las diferencias en la toxicidad no se deben a cambios en la unión inicial al receptor. Los ensayos de unión/disociación y fijación de voltaje mostraron que la reducción de la toxicidad de la toxina H168Q se puede atribuir a una disminución en la inserción y/o en la formación de canales iónicos. De otro lado, H168R mostró una inhibición a la corriente de corto circuito mayor que la ts y un aumento en unión irreversible (k_obs), lo cual es consistente con un menor valor de CL_{<span style="position: relative; top: 2pt; mso-text-raise: -2.0pt;">50</span>}. La modelación molecular sugiere que H168Q y H168R forman puentes de hidrógeno adicionales, lo que les confiere mayor estabilidad térmica. Adicionalmente, es probable que H168R tenga una carga positiva extra expuesta en la superficie, lo cual aumentaría su tasa de inserción en membranas susceptibles. <span style="mso-spacerun: yes;"> </span><strong style="mso-bidi-font-weight: normal;"></strong></span></span></p><p class="MsoNormal" style="text-align: justify; line-height: normal; margin: 0cm 0cm 0pt;"><strong style="mso-bidi-font-weight: normal;"><span style="font-family: "><span style="font-size: small;"> </span></span></strong></p><p class="MsoNormal" style="text-align: justify; line-height: normal; margin: 0cm 0cm 0pt;"><span style="font-size: small;"><strong style="mso-bidi-font-weight: normal;"><span style="font-family: ">Palabras clave:</span></strong><span style="font-family: "> </span><span style="font-family: Symbol; mso-bidi-font-size: 12.0pt;">a</span><span style="font-family: ">-hélice 5; dicroísmo circular; modelamiento molecular; mutagénesis sitio dirigida; estabilidad térmica; <em style="mso-bidi-font-style: normal;">Bacillus thuringiensis</em></span></span></p