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Biomedical subjects

J K Zhu

Publications and source records attributed to J K Zhu.

At least 19 recordsLinked to original sources
PubMed2004

SOS3 (salt overly sensitive 3) from Arabidopsis thaliana: expression, purification, crystallization and preliminary X-ray analysis.

The salt-tolerance gene SOS3 (salt overly sensitive 3) of Arabidopsis thaliana encodes a calcium-binding protein that is able to sense the cytosolic calcium signal elicited by salt stress. SOS3 activates the SOS2 protein kinase, which activates various ion transporters. SOS3 was cloned into a plasmid and expressed in Escherichia coli, allowing purification of the protein to homogeneity. Two crystals with different additive contents were grown. Both diffract to 3.2 A resolution and belong to space group I4(1), with unit-cell parameters a = 93.65, c = 80.08 A and a = 91.79, c = 85.78 A, respectively. A promising molecular-replacement solution has been found using neuronal calcium-sensor 1 as the search model. Interestingly, no solution was found using AtCBL2 (A. thaliana calcineurin B-like protein) structure as a search model, although this protein belongs to the same family and displays 50% sequence identity.

Arabidopsis↗
PubMed2001

AtHKT1 is a salt tolerance determinant that controls Na(+) entry into plant roots.

Two Arabidopsis thaliana extragenic mutations that suppress NaCl hypersensitivity of the sos3-1 mutant were identified in a screen of a T-DNA insertion population in the genetic background of Col-0 gl1 sos3-1. Analysis of the genome sequence in the region flanking the T-DNA left border indicated that sos3-1 hkt1-1 and sos3-1 hkt1-2 plants have allelic mutations in AtHKT1. AtHKT1 mRNA is more abundant in roots than shoots of wild-type plants but is not detected in plants of either mutant, indicating that this gene is inactivated by the mutations. hkt1-1 and hkt1-2 mutations can suppress to an equivalent extent the Na(+) sensitivity of sos3-1 seedlings and reduce the intracellular accumulation of this cytotoxic ion. Moreover, sos3-1 hkt1-1 and sos3-1 hkt1-2 seedlings are able to maintain [K(+)](int) in medium supplemented with NaCl and exhibit a substantially higher intracellular ratio of K(+)/Na(+) than the sos3-1 mutant. Furthermore, the hkt1 mutations abrogate the growth inhibition of the sos3-1 mutant that is caused by K(+) deficiency on culture medium with low Ca(2+) (0.15 mM) and <200 microM K(+). Interestingly, the capacity of hkt1 mutations to suppress the Na(+) hypersensitivity of the sos3-1 mutant is reduced substantially when seedlings are grown in medium with low Ca(2+) (0.15 mM). These results indicate that AtHKT1 is a salt tolerance determinant that controls Na(+) entry and high affinity K(+) uptake. The hkt1 mutations have revealed the existence of another Na(+) influx system(s) whose activity is reduced by high [Ca(2+)](ext).

Alleles↗
PubMed2001

FIERY1 encoding an inositol polyphosphate 1-phosphatase is a negative regulator of abscisic acid and stress signaling in Arabidopsis.

The plant hormone abscisic acid (ABA) plays a wide range of important roles in plant growth and development, including embryogenesis, seed dormancy, root and shoot growth, transpiration, and stress tolerance. ABA and various abiotic stresses also activate the expression of numerous plant genes through undefined signaling pathways. To gain insight into ABA and stress signal transduction, we conducted a genetic screen based on ABA- and stress-inducible gene transcription. Here we report the identification of an Arabidopsis mutation, fiery1 (fry1), which results in super-induction of ABA- and stress-responsive genes. Seed germination and postembryonic development of fry1 are more sensitive to ABA or stress inhibition. The mutant plants are also compromised in tolerance to freezing, drought, and salt stresses. Map-based cloning revealed that FRY1 encodes an inositol polyphosphate 1-phosphatase, which functions in the catabolism of inositol 1, 4, 5-trisphosphate (IP(3)). Upon ABA treatment, fry1 mutant plants accumulated more IP(3) than did the wild-type plants. These results provide the first genetic evidence indicating that phosphoinositols mediate ABA and stress signal transduction in plants and their turnover is critical for attenuating ABA and stress signaling.

Abscisic Acid↗
PubMed2001

The Arabidopsis HOS1 gene negatively regulates cold signal transduction and encodes a RING finger protein that displays cold-regulated nucleo--cytoplasmic partitioning.

Low temperature is one of the most important environmental stimuli that control gene transcription programs and development in plants. In Arabidopsis thaliana, the HOS1 locus is a key negative regulator of low temperature-responsive gene transcription. The recessive hos1 mutation causes enhanced induction of the CBF transcription factors by low temperature as well as of their downstream cold-responsive genes. The hos1 mutant plants flower early, and this correlates with a low level of Flowering Locus C gene expression. The HOS1 gene was isolated through positional cloning. HOS1 encodes a novel protein with a RING finger motif near the amino terminus. HOS1 is ubiquitously expressed in all plant tissues. HOS1--GFP translational fusion studies reveal that HOS1 protein resides in the cytoplasm at normal growth temperatures. However, in response to low temperature treatments, HOS1 accumulates in the nucleus. Ectopic expression of HOS1 in wild-type plants causes cosuppression of HOS1 expression and mimics the hos1 mutant phenotypes.

Acclimatization↗
PubMed2001

Plant salt tolerance.

Soil salinity is a major abiotic stress in plant agriculture worldwide. This has led to research into salt tolerance with the aim of improving crop plants. However, salt tolerance might have much wider implications because transgenic salt-tolerant plants often also tolerate other stresses including chilling, freezing, heat and drought. Unfortunately, suitable genetic model systems have been hard to find. A recently discovered halophytic plant species, Thellungiella halophila, now promises to help in the detection of new tolerance determinants and operating pathways in a model system that is not limited to Arabidopsis traits or ecotype variations.

Adaptation, Physiological↗
PubMed2001

Cell signaling under salt, water and cold stresses.

Forward genetics and biochemical approaches to studying plant responses to salt, water and cold stresses began to bear fruit recently. Analysis of salt overly sensitive (sos) Arabidopsis mutants revealed a novel calcium-regulated protein kinase pathway for response to the ionic aspect of salt stress. In-gel kinase assays identified several SOS-independent protein kinases that are either activated specifically by osmotic stress or by multiple abiotic and biotic stresses. Molecular analysis revealed a transcriptional cascade in cold-regulated gene expression.

Adaptation, Physiological↗
PubMed2001

Modulation of abscisic acid signal transduction and biosynthesis by an Sm-like protein in Arabidopsis.

The phytohormone abscisic acid (ABA) regulates plant growth and development as well as stress tolerance. The Arabidopsis sad1 (supersensitive to ABA and drought) mutation increases plant sensitivity to drought stress and ABA in seed germination, root growth, and the expression of some stress-responsive genes. sad1 plants are also defective in the positive feedback regulation of ABA biosynthesis genes by ABA and are impaired in drought stress induction of ABA biosynthesis. SAD1 encodes a polypeptide similar to multifunctional Sm-like snRNP proteins that are required for mRNA splicing, export, and degradation. These results suggest a critical role for mRNA metabolism in the control of ABA signaling as well as in the regulation of ABA homeostasis.

Abscisic Acid↗
PubMed2001

The Arabidopsis LOS5/ABA3 locus encodes a molybdenum cofactor sulfurase and modulates cold stress- and osmotic stress-responsive gene expression.

To understand low temperature and osmotic stress signaling in plants, we isolated and characterized two allelic Arabidopsis mutants, los5-1 and los5-2, which are impaired in gene induction by cold and osmotic stresses. Expression of RD29A-LUC (the firefly luciferase reporter gene under the control of the stress-responsive RD29A promoter) in response to cold and salt/drought is reduced in the los5 mutants, but the response to abscisic acid (ABA) remains unaltered. RNA gel blot analysis indicates that the los5 mutation reduces the induction of several stress-responsive genes by cold and severely diminishes or even completely blocks the induction of RD29A, COR15, COR47, RD22, and P5CS by osmotic stresses. los5 mutant plants are compromised in their tolerance to freezing, salt, or drought stress. los5 plants are ABA deficient, as indicated by increased transpirational water loss and reduced accumulation of ABA under drought stress in the mutant. A comparison with another ABA-deficient mutant, aba1, reveals that the impaired low-temperature gene regulation is specific to the los5 mutation. Genetic tests suggest that los5 is allelic to aba3. Map-based cloning reveals that LOS5/ABA3 encodes a molybdenum cofactor (MoCo) sulfurase. MoCo sulfurase catalyzes the generation of the sulfurylated form of MoCo, a cofactor required by aldehyde oxidase that functions in the last step of ABA biosynthesis in plants. The LOS5/ABA3 gene is expressed ubiquitously in different plant parts, and the expression level increases in response to drought, salt, or ABA treatment. Our results show that LOS5/ABA3 is a key regulator of ABA biosynthesis, stress-responsive gene expression, and stress tolerance.

Abscisic Acid↗
PubMed2001

Molecular characterization of functional domains in the protein kinase SOS2 that is required for plant salt tolerance.

The SOS3 (for SALT OVERLY SENSITIVE3) calcium binding protein and SOS2 protein kinase are required for sodium and potassium ion homeostasis and salt tolerance in Arabidopsis. We have shown previously that SOS3 interacts with and activates the SOS2 protein kinase. We report here the identification of a SOS3 binding motif in SOS2 that also serves as the kinase autoinhibitory domain. Yeast two-hybrid assays as well as in vitro binding assays revealed a 21-amino acid motif in the regulatory domain of SOS2 that is necessary and sufficient for interaction with SOS3. Database searches revealed a large family of SOS2-like protein kinases containing such a SOS3 binding motif. Using a yeast two-hybrid system, we show that these SOS2-like kinases interact with members of the SOS3 family of calcium binding proteins. Two-hybrid assays also revealed interaction between the N-terminal kinase domain and the C-terminal regulatory domain within SOS2, suggesting that the regulatory domain may inhibit kinase activity by blocking substrate access to the catalytic site. Removal of the regulatory domain of SOS2, including the SOS3 binding motif, resulted in constitutive activation of the protein kinase, indicating that the SOS3 binding motif can serve as a kinase autoinhibitory domain. Constitutively active SOS2 that is SOS3 independent also was produced by changing Thr(168) to Asp in the activation loop of the SOS2 kinase domain. Combining the Thr(168)-to-Asp mutation with the autoinhibitory domain deletion created a superactive SOS2 kinase. These results provide insights into regulation of the kinase activities of SOS2 and the SOS2 family of protein kinases.

Adaptation, Physiological↗
PubMed2001

Effects of dexamethasone on Flt3 receptor expression and proliferation induced by recombinant human Flt3 ligand in malignant hematopoietic cells.

AIM: To investigate the effect of dexamethasone (DXM) on the expression of Flt3 receptor and the proliferation mediated by recombinant human Flt3 ligand (rhFL) in leukemia cells. METHODS: Eighteen malignant hematopoietic cell lines and 10 leukemia blasts from leukemia patients were examined by flow cytometry for the expression of Flt3 receptor before and after incubation with DXM 0.1 micromol/L for 24 h. The effect of DXM on the proliferation of malignant hematopoietic cells was measured by MTT assay. RESULTS: (1) Expression of the Flt3 receptor in malignant hematopoietic cell lines and leukemia blasts was widespread and extremely heterogeneous; (2) The presence of receptor on the surface of malignant hematopoietic cell didn't necessarily imply a significant ligand-induced response, at least in terms of proliferation. Conversely, some Flt3 receptor-negative malignant hematopoietic cells responded to rhFL; (3) DXM down-regulated the expression of Flt3 receptor and inhibited the proliferation induced by rhFL in some malignant hematopoietic cell lines and fresh leukemia cells. CONCLUSION: DXM may down-regulate the expression of Flt3 receptor on the surface of malignant hematopoietic cells and inhibit the proliferation induced by rhFL. A combination of rhFL and DXM may serve to control hematopoietic defects in malignant hematopoietic diseases.

Antineoplastic Agents, Hormonal↗
PubMed2001

[Expression and purification of recombinant human interleukin-11 in Pichia pastoris].

OBJECTIVE: To express recombinant human interleukin-11 (rhIL-11) in methylotropic yeast Pichia pastoris. METHODS: By designing and synthesizing an artificial gene for IL-11, the expression vector pPICZ alpha-A-IL-11 was constructed and introduced into Pichia pastoris by linearized electroporation. The rhIL-11 protein was identified by ELISA and SDS-PAGE analysis. The bioactivity was analyzed by B9-11 cell line. A combination of liquid chromatography was developed to purify the rhIL-11 from ferment supernatant. RESULTS: The nucleotide sequence analysis indicated that the sequence of cloned artificial IL-11 gene accorded with that of designed; the secreted yield of rhIL-11 by yeast Pichia pastoris KM71-2424 in flask reached 60 mg/L. The biological activity of IL-11 in yeast supernatant and E. coli standard determined by B9-11 was 5.5 x 10(7) U/mg and 2.2 x 10(7) U/mg respectively. The rhIL-11 was purified to electrophoretic purity by a combination of liquid chromatography. CONCLUSION: The human IL-11 artificial gene was obtained and successfully expressed in the Pichia pastoris(KM71-2424). The biological activity of IL-11 in yeast supernatant was significantly higher than that of E. coli standard. The rhIL-11 was purified to electrophoretic purity.

Electrophoresis, Polyacrylamide Gel↗
PubMed2000

The Arabidopsis thaliana salt tolerance gene SOS1 encodes a putative Na+/H+ antiporter.

In Arabidopsis thaliana, the SOS1 (Salt Overly Sensitive 1) locus is essential for Na(+) and K(+) homeostasis, and sos1 mutations render plants more sensitive to growth inhibition by high Na(+) and low K(+) environments. SOS1 is cloned and predicted to encode a 127-kDa protein with 12 transmembrane domains in the N-terminal part and a long hydrophilic cytoplasmic tail in the C-terminal part. The transmembrane region of SOS1 has significant sequence similarities to plasma membrane Na(+)/H(+) antiporters from bacteria and fungi. Sequence analysis of various sos1 mutant alleles reveals several residues and regions in the transmembrane as well as the tail parts that are critical for SOS1 function in plant salt tolerance. SOS1 gene expression in plants is up-regulated in response to NaCl stress. This up-regulation is abated in sos3 or sos2 mutant plants, suggesting that it is controlled by the SOS3/SOS2 regulatory pathway.

Alleles↗
PubMed2000

The Arabidopsis thaliana SOS2 gene encodes a protein kinase that is required for salt tolerance.

In Arabidopsis thaliana, the Salt Overly Sensitive 2 (SOS2) gene is required for intracellular Na(+) and K(+) homeostasis. Mutations in SOS2 cause Na(+) and K(+) imbalance and render plants more sensitive toward growth inhibition by high Na(+) and low K(+) environments. We isolated the SOS2 gene through positional cloning. SOS2 is predicted to encode a serine/threonine type protein kinase with an N-terminal catalytic domain similar to that of the yeast SNF1 kinase. Sequence analyses of sos2 mutant alleles reveal that both the N-terminal catalytic domain and the C-terminal regulatory domain of SOS2 are functionally essential. The steady-state level of SOS2 transcript is up-regulated by salt stress in the root. Autophosphorylation assays show that SOS2 is an active protein kinase. In the recessive sos2-5 allele, a conserved glycine residue in the kinase catalytic domain is changed to glutamate. This mutation abolishes SOS2 autophosphorylation, indicating that SOS2 protein kinase activity is required for salt tolerance.

Adaptation, Physiological↗
PubMed2000

The Arabidopsis SOS2 protein kinase physically interacts with and is activated by the calcium-binding protein SOS3.

The Arabidopsis thaliana SOS2 and SOS3 genes are required for intracellular Na(+) and K(+) homeostasis and plant tolerance to high Na(+) and low K(+) environments. SOS3 is an EF hand type calcium-binding protein having sequence similarities with animal neuronal calcium sensors and the yeast calcineurin B. SOS2 is a serine/threonine protein kinase in the SNF1/AMPK family. We report here that SOS3 physically interacts with and activates SOS2 protein kinase. Genetically, sos2sos3 double mutant analysis indicates that SOS2 and SOS3 function in the same pathway. Biochemically, SOS2 interacts with SOS3 in the yeast two-hybrid system and in vitro binding assays. The interaction is mediated by the C-terminal regulatory domain of SOS2. SOS3 activates SOS2 protein kinase activity in a Ca(2+)-dependent manner. Therefore, SOS3 and SOS2 define a novel regulatory pathway important for the control of intracellular ion homeostasis and salt tolerance in plants.

Amino Acid Sequence↗
PubMed2000

SOS3 function in plant salt tolerance requires N-myristoylation and calcium binding.

The salt tolerance gene SOS3 (for salt overly sensitive3) of Arabidopsis is predicted to encode a calcium binding protein with an N-myristoylation signature sequence. Here, we examine the myristoylation and calcium binding properties of SOS3 and their functional significance in plant tolerance to salt. Treatment of young Arabidopsis seedlings with the myristoylation inhibitor 2-hydroxymyristic acid caused the swelling of root tips, mimicking the phenotype of the salt-hypersensitive mutant sos3-1. In vitro translation assays with reticulocyte showed that the SOS3 protein was myristoylated. Targeted mutagenesis of the N-terminal glycine-2 to alanine prevented the myristoylation of SOS3. The functional significance of SOS3 myristoylation was examined by expressing the wild-type myristoylated SOS3 and the mutated nonmyristoylated SOS3 in the sos3-1 mutant. Expression of the myristoylated but not the nonmyristoylated SOS3 complemented the salt-hypersensitive phenotype of sos3-1 plants. No significant difference in membrane association was observed between the myristoylated and nonmyristoylated SOS3. Gel mobility shift and (45)Ca(2)+ overlay assays demonstrated that SOS3 is a unique calcium binding protein and that the sos3-1 mutation substantially reduced the capacity of SOS3 to bind calcium. The resulting mutant SOS3 protein was not able to interact with the SOS2 protein kinase and was less capable of activating it. Together, these results strongly suggest that both N-myristoylation and calcium binding are required for SOS3 function in plant salt tolerance.

Adaptation, Physiological↗
PubMed2000

[Extraction and purification of Schwann cells cytoplasmic neurotrophic proteins with high pressure liquid chromatography and study on its neurobiological activities].

OBJECTIVE: To purify and study Schwann cells cytoplasmic neurotrophic protein. METHODS: The dissociated SC taken from 300 newborn rats sciatic nerves were cultured, collected, ultrasonicated and ultraspeed centrifuged. The supernates were ultrafiltrated and concentrated by using ultrafiltration units with PM10, PM30, PM50 ultrafiltration membranes. The ultrafiltrated-concentrated solution with the protein molecular weight 10-30 ku, 30-50 ku and > 50 ku were collected respectively. The dissociated spinal cord motoneurons of 14 days embryonic rats were cultured with serum-free conditional medium and the additional SC cytoplasmic proteins were added into the medium. The results showed that the 10-30 ku and > 50 ku SC cytoplasmic proteins were able to maintain the survival of motoneurons for 24 hours. Then the 26 ku and 58 ku proteins were further extracted and purified from SC cytoplasm by high pressure liquid chromatography, and their neurobiological activities were studied. RESULTS: The 26 ku and 58 ku Schwann cell's cytoplasmic proteins were able to maintain the survival of motoneurons cultured in the serum-free medium for 48 hours. The highest biological activity concentration is 20 ng per well. CONCLUSION: Schwann cells cytoplasm contains motoneuron neurotrophic proteins with molecular weight 26 ku and 58 ku.

Animals↗