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Biomedical subjects

P Jin

Publications and source records attributed to P Jin.

At least 19 recordsLinked to original sources
PubMed2001

Fragile X mental retardation protein targets G quartet mRNAs important for neuronal function.

Loss of fragile X mental retardation protein (FMRP) function causes the fragile X mental retardation syndrome. FMRP harbors three RNA binding domains, associates with polysomes, and is thought to regulate mRNA translation and/or localization, but the RNAs to which it binds are unknown. We have used RNA selection to demonstrate that the FMRP RGG box binds intramolecular G quartets. This data allowed us to identify mRNAs encoding proteins involved in synaptic or developmental neurobiology that harbor FMRP binding elements. The majority of these mRNAs have an altered polysome association in fragile X patient cells. These data demonstrate that G quartets serve as physiologically relevant targets for FMRP and identify mRNAs whose dysregulation may underlie human mental retardation.

Base Sequence↗
PubMed2001

Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome.

Fragile X syndrome results from the absence of the RNA binding FMR protein. Here, mRNA was coimmunoprecipitated with the FMRP ribonucleoprotein complex and used to interrogate microarrays. We identified 432 associated mRNAs from mouse brain. Quantitative RT-PCR confirmed some to be >60-fold enriched in the immunoprecipitant. In parallel studies, mRNAs from polyribosomes of fragile X cells were used to probe microarrays. Despite equivalent cytoplasmic abundance, 251 mRNAs had an abnormal polyribosome profile in the absence of FMRP. Although this represents <2% of the total messages, 50% of the coimmunoprecipitated mRNAs with expressed human orthologs were found in this group. Nearly 70% of those transcripts found in both studies contain a G quartet structure, demonstrated as an in vitro FMRP target. We conclude that translational dysregulation of mRNAs normally associated with FMRP may be the proximal cause of fragile X syndrome, and we identify candidate genes relevant to this phenotype.

Amino Acid Sequence↗
PubMed2001

In vivo roles of RORalpha and Sp4 in the regulation of murine prosaposin gene.

Prosaposin has a central role in intracellular glycosphingolipid catabolism and also has extracellular functions. This locus is regulated temporally and spatially. The highest mRNA expression occurs in the central nervous system (CNS) and reproductive system. In vitro, the CNS-expressed proteins Sp4 and RORalpha bind to Sp1 and RORE sites within a 310-bp fragment directly upstream of the transcription start site. These transcription factors exhibit negative cooperativity in vitro for prosaposin expression. Mice deficient in RORalpha and Sp4 (Staggerer [Sg(-/-)] and Sp4 knockout [Sp4 KO], respectively) containing selected prosaposin promoter deletion transgenes were used in comparative expression studies to evaluate this negative cooperativity in vivo. Constructs containing the RORE or Sp1/U cluster alone were independently stimulatory. Deletion of the Sp1/U site led to a decrease in reporter activity only in the cerebellum of Sg(-/-) mice. The deletion of RORE and Sp1/U sites did alter the increase of reporter activity in the brain and eye, but not in the spinal cord, of Sg(-/-) mice. These results indicate that Sp4 and RORalpha play minor and major roles, respectively, in regional expression of the prosaposin locus in the brain, whereas expression in the spinal cord is independent of RORalpha.

Animals↗
PubMed2001

Reciprocal activation by cyclin-dependent kinases 2 and 7 is directed by substrate specificity determinants outside the T loop.

Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of the metazoan CDK-activating kinase (CAK), which activates CDKs, such as CDC2 and CDK2, through phosphorylation of a conserved threonine residue in the T loop. Full activation of CDK7 requires association with a positive regulatory subunit, cyclin H, and phosphorylation of a conserved threonine residue at position 170 in its own T loop. We show that threonine-170 of CDK7 is phosphorylated in vitro by its targets, CDC2 and CDK2, which also phosphorylate serine-164 in the CDK7 T loop, a site that perfectly matches their consensus phosphorylation site. In contrast, neither CDK4 nor CDK7 itself can phosphorylate the CDK7 T loop in vitro. The ability of CDC2 or CDK2 and CDK7 to phosphorylate each other but not themselves implies that each kinase can discriminate among closely related sequences and can recognize a substrate site that diverges from its usual preferred site. To understand the basis for this paradoxical substrate specificity, we constructed a chimeric CDK with the T loop of CDK7 grafted onto the body of CDK2. Surprisingly, the hybrid enzyme, CDK2-7, was efficiently activated in cyclin A-dependent fashion by CDK7 but not at all by CDK2. CDK2-7, moreover, phosphorylated wild-type CDK7 but not CDK2. Our results suggest that the primary amino acid sequence of the T loop plays only a minor role, if any, in determining the specificity of cyclin-dependent CAKs for their CDK substrates and that protein-protein interactions involving sequences outside the T loop can influence substrate specificity both positively and negatively.

Amino Acid Motifs↗
PubMed2001

Glucose sensing based on interdigitated array microelectrode.

A micro glucose sensor consisting of an interdigitated array gold microelectrode was developed. The interdigitated array structure, which has 10 microns band width and 10 microns band gap, was fabricated in a small region (2.5 x 5 mm2) on a quartz substrate. Glucose oxidase was chemically fixed onto the electrode surface through self-assembled monolayer of 11-mercaptoundecanoic acid; ferroceneacetic acid was used as electron mediator. Electrochemical properties of the glucose oxidase-immobilized microelectrode were investigated by cyclic voltammogram measurements. Results confirmed that the reductive ferroceneacetic acid generated at counter electrode diffuses through a narrow band gap (10 microns) and can reach the working electrode surface.

Acetic Acid↗
PubMed2000

Prosaposin: promoter analysis and central-nervous-system-preferential elements for expression in vivo.

The expression of prosaposin is temporally and spatially regulated at the transcriptional and post-translational levels. In vitro, the mouse prosaposin promoter contains functional RORE [retinoic acid-receptor-related orphan receptor alpha subunit (RORalpha)-binding element], Sp1 and U (unknown) sites within 310 bp directly 5' to the transcription start site and additional elements within 2400 bp 5' to the transcription start site. To elucidate promoter regions important to tissue-preferential expression in vivo, transgenic mice were created with 5'-flanking deletions of the prosaposin gene fused to a luciferase reporter. Nearly exclusive expression was observed in cerebrum, cerebellum and eyes of adult transgenic mice containing constructs with 234-310 bp of 5'-flanking DNA. This central nervous system (CNS) expression was due to the presence of RORE and overlapping Sp1 sites in this region. Internal deletion of RORE and the Sp1 cluster from the longer constructs with 2400 bp of 5'-flanking DNA significantly diminished expression in the CNS. The appearance of substantial visceral tissue (e.g. liver, spleen, lung, kidney, thymus and heart) expression was obtained with transgenic mice bearing constructs with 742-2400 bp of 5'-flanking DNA. The cellular localization of luciferase reporter-gene expression from these constructs corresponded closely with that for prosaposin. These results define important CNS and visceral regulatory regions in the promoter in vivo and may be sufficient to account for the majority of prosaposin's tissue-preferential expression.

Animals↗
PubMed2000

Understanding the molecular basis of fragile X syndrome.

Fragile X syndrome, a common form of inherited mental retardation, is mainly caused by massive expansion of CGG triplet repeats located in the 5'-untranslated region of the fragile X mental retardation-1 ( FMR1 ) gene. In patients with fragile X syndrome, the expanded CGG triplet repeats are hypermethylated and the expression of the FMR1 gene is repressed, which leads to the absence of FMR1 protein (FMRP) and subsequent mental retardation. FMRP is an RNA-binding protein that shuttles between the nucleus and cytoplasm. This protein has been implicated in protein translation as it is found associated with polyribosomes and the rough endoplasmic reticulum. We discuss here the recent progress made towards understanding the molecular mechanism of CGG repeat expansion and physiological function(s) of FMRP. These studies will not only help to illuminate the molecular basis of the general class of human diseases with trinucleotide repeat expansion but also provide an avenue to understand aspects of human cognition and intelligence.

DNA Methylation↗
PubMed2000

The maize CR4 receptor-like kinase mediates a growth factor-like differentiation response.

The maize crinkly4 (cr4) gene encodes a predicted receptor kinase that is required for the normal differentiation of leaf epidermis (Becraft et al., 1996). Here we confirm that the presumptive cytoplasmic domain is capable of autophosphorylation on serine and threonine residues. The cr4 transcript is most abundant in young developing tissues of the shoot but was not detected in the root. Phenotypic analysis of strong mutant alleles indicates that cr4 functions throughout shoot development to control the differentiation of internal as well as epidermal tissues. However, epidermal cells appear most sensitive to perturbations in CR4 signaling because epidermal defects are more pronounced than internal cell defects. Epidermal cells show irregularities in shape, cell wall thickness and structure, cuticle formation, and vesicle trafficking. Some plants show tumor-like proliferations of cells on the leaves. Thus CR4 regulates an array of developmental responses including cell proliferation, fate, pattern, and differentiation suggesting a function analogous to growth factor responses in animals.

Alleles↗
PubMed2000

[An analysis of precision and accuracy of the phonocardiogram exercise test].

Phonocardiogram exercise testing (PCGET) is a recently developed method to evaluate cardiac contractility and the cardiac reserve of patients with heart disease and of healthy subjects. In order to test the reliability of PCGET method, the present author conducted a study on its precision and accuracy. Thirty volunteers underwent PCGET. When different examiners measured the S1 amplitude in the same cardiac cycle of the same subject, the data obtained by examiner A were: x +/- s = 5.05 +/- 0.0451; the data obtained by examiner B were: x +/- s = 4.95 +/- 0.0346, F = 1.699, P > 0.05. When different examiners measured the same cardiac cycle of the same subject, the data obtained by examiner A were: x +/- s = 0.789 +/- 0.0018; the data obtained by examiner B were: x +/- s = 0.787 +/- 0.0017, F = 1.167, P > 0.05. The results suggest that PCGET is a nonivasive, convenient, and inexpensive technique to quantitatively evaluate cardiac reserve for abnormal or normal persons.

Coronary Circulation↗
PubMed1999

Structural features and assembly of the soluble overexpressed PsaD subunit of photosystem I.

PsaD is a peripheral protein on the reducing side of photosystem I (PS I). We expressed the psaD gene from the thermophilic cyanobacterium Mastigocladus laminosus in Escherichia coli and obtained a soluble protein with a polyhistidine tag at the carboxyl terminus. The soluble PsaD protein was purified by Ni-affinity chromatography and had a mass of 16716 Da by MALDI-TOF. The N-terminal amino acid sequence of the overexpressed PsaD matched the N-terminal sequence of the native PsaD from M. laminosus. The soluble PsaD could assemble into the PsaD-less PS I. As determined by isothermal titration calorimetry, PsaD bound to PS I with 1.0 binding site per PS I, the binding constant of 7.7x10(6) M-1, and the enthalpy change of -93.6 kJ mol-1. This is the first time that the binding constant and binding heat have been determined in the assembly of any photosynthetic membrane protein. To identify the surface-exposed domains, purified PS I complexes and overexpressed PsaD were treated with N-hydroxysuccinimidobiotin (NHS-biotin) and biotin-maleimide, and the biotinylated residues were mapped. The Cys66, Lys21, Arg118 and Arg119 residues were exposed on the surface of soluble PsaD whereas the Lys129 and Lys131 residues were not exposed on the surface. Consistent with the X-ray crystallographic studies on PS I, circular dichroism spectroscopy revealed that PsaD contains a small proportion of alpha-helical conformation.

Amino Acid Sequence↗
PubMed1999

The spreading of six comprehensive medical books of the Ming dynasty to Japan and their influence.

Plenty of medical books were spread to Japan from China in the Ming dynasty. The most influential ones include the six comprehensive books, viz, "Yi Shu Da Quan", "Yu Ji Wei Yi", "Yi Xue Zheng Chuan", "Yi Xue Men", "Wan Bing Hui Chun" and "Nan Bei Jing Yan Yi Fang Da Quan", all spread quite fast. To begin with, they promoted the reprinting of Chinese medical books in Japan in large amount since the 16th century, providing convenient textbooks for Japanese medical teaching. Finally, they help to develop the scholastic thought of "Dao San" in theory and practice. As a result, they also helped to rejuvenate Japanese medicine.

China↗
PubMed1998

Presynaptic calcium/calmodulin-dependent protein kinase II regulates habituation of a simple reflex in adult Drosophila.

On repetitive stimulation, the strength of a reflex controlling leg position in Drosophila decreased, and this response decrement conformed to the parametric features of habituation. To study the presynaptic function of CaMKII in this nonassociative form of learning, we used a P[Gal4] insertion line to target the expression of mutant forms of CaMKII to the sensory neurons controlling the reflex. Targeted expression of a calcium-independent CaMKII construct (T287D) in the sensory neurons eliminated habituation. Targeted expression of a mutant CaMKII incapable of achieving calcium independence (T287A) reduced the initial reflex response, but a strong facilitation then occurred, and this eliminated most of the habituation. Finally, when a CaMKII inhibitory peptide (ala) was expressed in sensory neurons, the initial response was reduced, followed by facilitation. These results suggest that basal CaMKII levels in the presynaptic neurons set the response level and dynamics of the entire neural circuit.

Animals↗
PubMed1998

Isolation and characterization of the human prosaposin promoter.

Prosaposin is a multifunctional protein that encodes four glycoproteins, named saposins A, B, C and D. They participate in the catabolism of glycosphingolipids in lysosomes. When secreted, intact prosaposin may function as a neuritogenic factor. Human and mouse prosaposin displayed similar temporal and spatial regulation of expression. To gain insight into the transcriptional regulation of this locus, the 5' region was characterized from the human prosaposin gene. The putative human promoter was shown to be TATA-less, i.e. it belonged to the TATA-less housekeeping gene family. The transcription initiation sites were localized to -23, -27, -31 and -83bp 5' to ATG, compared to -87 and -94bp in the mouse. In SK-N-SH neuroblastoma cells, positive regulatory elements were detected -343 to -813bp upstream of ATG. A negative regulatory region existed between -813 and -2500bp using SK-N-SH, H441 and NS20Y cells. EMSA and DNA-footprint analysis showed that Sp1 and Sp3 are involved in human prosaposin gene regulation. Compared to the mouse promoter, the human promoter is missing a Sp1 cluster within a 310-bp upstream segment, and has AP-1, Oct-1 and two RORalpha sites that are protected from DNaseI by selected nuclear extracts.

Animals↗
PubMed1998

Role of Sp proteins and RORalpha in transcription regulation of murine prosaposin.

Prosaposin is the precursor of four low molecular weight sphingolipid-activating proteins (SAPs) or saposins. These four proteins function as intracellular activators of several lysosomal enzymes involved in the degradation of glycosphingolipids, and prosaposin itself has neurite outgrowth effects. Expression of prosaposin is regulated in a temporal and spatial manner with expression in specific brain neurons and visceral cell types. Here a major regulatory fragment was characterized within 310 bp 5' to the transcription start site. Using electrophoretic mobility shift assay (EMSA) and DNA footprinting, members of the Sp family (Sp1, Sp3, and Sp4), the orphan nuclear receptor (RORalpha), and an unknown transcription factor (U; TGGGGGAG) were shown to bind to this region. To evaluate the role of such transcription factor binding sites for this locus, a series of mutant constructs was generated within this region, and their function was evaluated in cultured NS20Y neuroblastoma cells. A 3' Sp1 site, a 5' Sp1/U cluster and the RORalpha binding sites were functional. The data are consistent with a model in which the factors that bind to the Sp1/U cluster and RORE site interact negatively to diminish promoter activity to a background level that is determined primarily by the 3' Sp1 site. These interactions depend on the tissue-specific repertoire of transcription factors leading to differential expression of this locus.

Animals↗
PubMed1998

Nuclear localization of cyclin B1 controls mitotic entry after DNA damage.

Mitosis in human cells is initiated by the protein kinase Cdc2-cyclin B1, which is activated at the end of G2 by dephosphorylation of two inhibitory residues, Thr14 and Tyr15. The G2 arrest that occurs after DNA damage is due in part to stabilization of phosphorylation at these sites. We explored the possibility that entry into mitosis is also regulated by the subcellular location of Cdc2-cyclin B1, which is suddenly imported into the nucleus at the end of G2. We measured the timing of mitosis in HeLa cells expressing a constitutively nuclear cyclin B1 mutant. Parallel studies were performed with cells expressing Cdc2AF, a Cdc2 mutant that cannot be phosphorylated at inhibitory sites. Whereas nuclear cyclin B1 and Cdc2AF each had little effect under normal growth conditions, together they induced a striking premature mitotic phenotype. Nuclear targeting of cyclin B1 was particularly effective in cells arrested in G2 by DNA damage, where it greatly reduced the damage-induced G2 arrest. Expression of nuclear cyclin B1 and Cdc2AF also resulted in significant defects in the exit from mitosis. Thus, nuclear targeting of cyclin B1 and dephosphorylation of Cdc2 both contribute to the control of mitotic entry and exit in human cells.

Adenoviridae↗
PubMed1998

Cognitive Load and Learner Expertise: Split-Attention and Redundancy Effects in Reading with Explanatory Notes

Five experiments were conducted to examine the effects of cognitive load management using explanatory notes in reading passages for readers with different levels of expertise. Experiment 1 found that explanatory notes improved 5th-grade, first-language learners' comprehension (high-level processing) but not vocabulary learning (low-level processing). Experiment 2 found that vocabulary definitions integrated within a passage (integrated format) enhanced 5th graders' comprehension compared to a separate vocabulary list (separated format) but reduced vocabulary learning. Experiment 3, using adult readers, found that an integrated format reduced comprehension but enhanced vocabulary learning. Experiment 4 used low-ability 8th-grade learners of English as a second language (ESL) and found an effect similar to the 5th graders in Experiment 2. Experiment 5 showed that the effect for high-ability ESL learners was similar to the adults in Experiment 3. We argue that the efficiency of instruction depends on the extent to which it imposes an extraneous cognitive load. The same presentation format may facilitate performance or interfere with performance either through split-attention or redundancy effects, depending on learners' expertise. Copyright 1998 Academic Press.

Journal Article↗