Biomedical subjects

B J Lee

Publications and source records attributed to B J Lee.

At least 19 recordsLinked to original sources

PubMed2006

Prenatal exposure to thalidomide, altered vasculogenesis, and CNS malformations.

Malformations of cortical development (MCD) result from abnormal neuronal positioning during corticogenesis. MCD are believed to be the morphological and perhaps physiological bases of several neurological diseases, spanning from mental retardation to autism and epilepsy. In view of the fact that during development, an appropriate blood supply is necessary to drive organogenesis in other organs, we hypothesized that vasculogenesis plays an important role in brain development and that E15 exposure in rats to the angiogenesis inhibitor thalidomide would cause postnatal MCD. Our results demonstrate that thalidomide inhibits angiogenesis in vitro at concentrations that result in significant morphological alterations in cortical and hippocampal regions of rats prenatally exposed to this vasculotoxin. Abnormal neuronal development was associated with vascular malformations and a leaky blood-brain barrier. Protein extravasation and uptake of fluorescent albumin by neurons, but not glia, was commonly associated with abnormal cortical development. Neuronal hyperexcitability was also a hallmark of these abnormal cortical regions. Our results suggest that prenatal vasculogenesis is required to support normal neuronal migration and maturation. Altering this process leads to failure of normal cerebrovascular development and may have a profound implication for CNS maturation.

Action Potentials↗

PubMed2006

Sexual behaviour induces the expression of activity-regulated cytoskeletal protein and modifies neuronal morphology in the female rat ventromedial hypothalamus.

Female sexual behaviour activates a distributed network within the brain, including the ventrolateral subdivision of the hypothalamic ventromedial nucleus (vlVMH), as demonstrated by behavioural studies performed in conjunction with the neuroanatomical analysis of immediate early gene (IEG) expression. However, it has been difficult to interpret mating-induced IEG expression because the precise function of many IEGs remains poorly defined. One possible function for genomic activation of the vlVMH during mating behaviour is to establish synaptic remodelling. The present experiments tested the hypothesis that sexual behaviour rapidly induces the expression of a structural protein associated with synaptic plasticity and ultimately causes morphological changes in the vlVMH. First, the expression of activity-regulated cytoskeletal protein (Arc), an IEG associated with neural plasticity, was assayed immunohistochemically in females after approximately 1 h of mating. The number of Arc-labelled neurones in the vlVMH was greater in mated females compared to unmated controls. Second, VMH neurones were biolistically labelled for morphological measurements, including soma size, dendrite number and length and dendritic spine density. Dendritic spine density in the vlVMH was significantly reduced 5 days after mating in experienced females compared to sexually naïve females. There were no differences between these groups in soma size, dendrite length or dendrite number. Collectively, these studies suggest that mating behaviour produces short-term changes in structural proteins and long-term, selective changes in dendrite morphology, which then may influence future behaviours and/or physiology.

Animals↗

PubMed2006

Measurement and modelling of ordinary heterotrophic organism active biomass concentrations in anoxic/aerobic activated sludge mixed liquor.

Ordinary heterotrophic organism (OHO) active biomass (ZBH) is a key parameter in models for activated sludge systems, which defines quantitatively the kinetic rates of relevant processes. However, ZBH has not been measured directly with consistent success: a simple respirometric batch test has provided varying correspondence between measured and theoretical concentrations. In this paper, the batch test is applied to mixed liquors drawn from well defined anoxic/aerobic parent systems at 10 and 20 d sludge ages, with consistent but poor correspondence between measured and theoretical values. In contrast, aerobic digestion batch tests on the same mixed liquors give good correspondences. It is concluded that the differences between theoretical and batch test measured values are due to the batch test method itself and its interpretation. It is found that the batch test conditions (particularly the substrate/ZBH ratio) influence the kinetic constants derived from the data, and hence the ZBH estimate. Two kinetic models with two competing OHO populations, a fast and a slow grower, are developed and applied to the batch tests and parent systems. The first model is based on kinetic selection only, while the second includes additional metabolic selection. Both models can account for the observations in the batch tests, but the second provides greater consistency between simulations of the parent systems and batch tests.

Aerobiosis↗

PubMed2004

Role of C-terminal heptapeptide in pore-forming activity of antimicrobial agent, gaegurin 4.

Gaegurin 4 (GGN4) is an antimicrobial peptide of 37 amino acids isolated from the skin of a frog, Rana rugosa. GGN4 has a disulfide bond between the residues 31 and 37, which is highly conserved among the antimicrobial peptides isolated from skin of the genus, Rana. However, the role of this C-terminal heptapeptide motif is not well understood. In this work, we compared the membrane effects of the full-length GGN4 (C37) and GGN4 1-30 (C30), which is devoid of the C-terminal seven amino acids to elucidate the function of the C-terminal motif. C37 induced significantly larger membrane conductance (>10x) in the model lipid bilayers formed with acidic and neutral phospholipids and larger K+ efflux from gram-positive (>30x) and gram-negative bacteria. However, the pores induced by C37 and C30 were not different in their permeability to K+ over Cl- (permeability ratio of K+ to Cl- = 4.8-7.1). In addition, the pore-forming effect of C37 or C30 in acidic membranes was not different from that in neutral membranes. Furthermore, C37-induced K+ efflux was not significantly decreased by the reducing agent, dithiothreitol. The results indicate that C-terminal heptapeptide sequence plays an important role in maintaining the high pore-forming activity of GGN4, but does not participate in forming GGN4-induced pore structure. The disulfide bond in this region does not appear critical for such high ionophoric activity of GGN4.

Amino Acid Motifs↗

PubMed2002

High prevalence of current asthma and active smoking effect among the elderly.

BACKGROUND: Although asthma is a common cause of morbidity in adults, relatively few objectively measured population studies of asthma prevalence in adult populations have been conducted. OBJECTIVE: To evaluate the prevalence of asthma, based on both a questionnaire and methacholine bronchial provocation test, and to determine the risk factors of asthma prevalence in an adult population. METHODS: A total of 2,467 adults, who were randomly selected from metropolitan urban, non-metropolitan urban and rural areas, responded to the modified ISAAC questionnaire, and underwent methacholine bronchial provocation tests and skin prick tests to locally common aeroallergens. RESULTS: The prevalence of current asthma based on the questionnaire and the methacholine challenge was 2.0% in adults younger than 40, 3.8% in 40- to 54-year-olds, 7.7% in 55- to 64-year-olds and 12.7% in those aged 65 or higher. For subjects of 55-64 years, active smoking was found to be significantly related with the prevalence of current asthma and bronchial hyper-responsiveness, although smoking was positively associated with percentage predictive value of forced expiratory volume of 1 s (FEV1). CONCLUSION: The prevalence of current asthma is common among the elderly, and active smoking may play an important role in the development of asthma and bronchial hyper-responsiveness among the elderly.

Adult↗

PubMed2001

Cloning and expression of cryptochrome2 cDNA in the rat.

Cryptochromes (CRY) are blue-light photoreceptors that regulate the circadian rhythm in animals and plants. In mammals, two types of CRY are involved in the regulation of circadian rhythm, but rat cryptochromes have not yet been identified. Therefore, we isolated and characterized cry2 cDNA from the rat brain. The cloned rat cry2 cDNA consists of 2,131 nucleotides and has a single open-reading frame that encodes the rat CRY2 of 594 amino acids with start and stop codons. The deduced amino acid sequence of the rat CRY2 was 97% identical with that of mice and 93% with humans, but it showed a relatively low identity of 64% with that of zebrafish. It also exhibited a high homology (about 70%) with CRY1 of mice and humans. A Northern blot analysis showed that rat cry2 was expressed in all of the tissues examined. Rat cry2 was expressed at a relatively higher level in peripheral tissues than in the brain. In situ hybridization in the whole brain indicated that the strong signal of cry2 mRNA is mainly present in the suprachiasmatic nucleus (SCN) region, but very weak in other brain regions. Therefore, present results indicate that rat cry2 may function in circadian photoreception in the rat brain.

Amino Acid Sequence↗

PubMed2001

Low-density cDNA array-coupled to PCR differential display identifies new estrogen-responsive genes during the postnatal differentiation of the rat hypothalamus.

To identify estrogen (E)-responsive genes that may play important roles in the sexual differentiation and maturation of the neuroendocrine hypothalamus, we used mRNA differential display PCR to analyze hypothalamic RNA derived from estrogen-sterilized rats (ESRs). Neonatal rats were s.c.-injected with 100 microg of 17 beta-estradiol-benzoate (EB) for 5 days. Approximately 300 out of more than 2000 RNAs examined displayed a differential expression pattern between hypothalami of the ESR females compared to their 60-day-old controls. EB-dependent expression of these genes was further analyzed by low-density cDNA array using cDNA probe sets reverse-transcribed from the same groups; 98 genes were confirmed to be differentially expressed. We selected 41 clones that showed higher density differences between the two probe sets than mean density difference in control cyclophilin cDNA blots in the cDNA array. After being cloned into pGEM-T vectors, their sequences were analyzed. Homology searches identified four genes as a protein kinase C (PKC)-binding protein, NELL2 (clone 6-1), a thyroid nuclear factor, TTF-1 (9-1), Munc18-1 (17-6), and leuserpin-2 (18-5). The other 22 genes were similar to reported genes or cDNAs such as mouse kinesin-associated protein 3 (KAP3, 8b), mouse IgE binding lectin (15-1), normalized rat brain cDNA (5-1), rat cDNA (8-1) and rat embryonic cDNA (17-1). Fifteen clones such as clone 7-3 showed no match in the GenBank Database. Further characterization of eight clones (17-1, 7-3, 8-1, 5-1, NELL2, KAP3 homolog, IgE binding lectin homolog, and TTF-1) showed that their expression in the adult female rat hypothalamus is sensitive to neonatal treatment with EB. They showed brain-specific expression and moreover, showed an increase in their mRNA level before the initiation of puberty. Some of them showed gender differences in their different postnatal expression pattern. We speculate that further study will demonstrate that many of the E-regulated genes identified in the present study play important roles in the regulation of the sexual differentiation and E-dependent maturation of the hypothalamus.

Animals↗

PubMed2001

The MED-7 transcriptional mediator encoded by let-49 is required for gonad and germ cell development in Caenorhabditis elegans.

Transcription mediators are evolutionarily conserved from yeast to human. We previously reported the specific in vivo roles of mediators during development. Transcriptional mediators including med-6, med-7, and med-10 were shown to be involved in the regulated transcription of specific genes, but not in the transcription of ubiquitous genes. In this report we have identified and characterized the Caenorhabditis elegans med-7 gene. A genetic mutation in the med-7 gene was identified by comparing genetic and physical maps and determining the molecular lesion. let-49 was found to have a nonsense mutation in the coding region of the med-7 gene. The identification of let-49 as the med-7 gene was confirmed by rescue experiments. The phenotype of the let-49 mutation indicated that the med-7 gene is required for normal postembryonic development. RNAi experiments showed that med-7 is also involved in embryogenesis and the gonad and germ cell development.

Animals↗

PubMed2001

Structural study of novel antimicrobial peptides, nigrocins, isolated from Rana nigromaculata.

Novel cationic antimicrobial peptides, named nigrocin 1 and 2, were isolated from the skin of Rana nigromaculata and their amino acid sequences were determined. These peptides manifested a broad spectrum of antimicrobial activity against various microorganisms with different specificity. By primary structural analysis, it was revealed that nigrocin 1 has high sequence homology with brevinin 2 but nigrocin 2 has low sequence homology with any other known antimicrobial peptides. To investigate the structure-activity relationship of nigrocin 2, which has a unique primary structure, circular dichroism (CD) and homonuclear nuclear magnetic resonance spectroscopy (NMR) studies were performed. CD investigation revealed that nigrocin 2 adopts mainly an alpha-helical structure in trifluoroethanol (TFE)/H(2)O solution, sodium dodecyl sulfate (SDS) micelles, and dodecylphosphocholine micelles. The solution structures of nigrocin 2 in TFE/H(2)O (1:1, v/v) solution and in SDS micelles were determined by homonuclear NMR. Nigrocin 2 consists of a typical amphipathic alpha-helix spanning residues 3-18 in both 50% TFE solution and SDS micelles. From the structural comparison of nigrocin 2 with other known antimicrobial peptides, nigrocin 2 could be classified into the family of antimicrobial peptides containing a single linear amphipathic alpha-helix that potentially disrupts membrane integrity, which would result in cell death.

Amino Acid Sequence↗

PubMed2001

Binding aspects of baicalein to HIV-1 integrase.

Human immunodeficiency virus type 1 (HIV-1) integrase is an essential enzyme in the life cycle of the virus. It is responsible for catalyzing the insertion of the viral genome into the host cell chromosome. This integrase is an attractive target for the design of a HIV antiviral drug, because integrase has no human counterpart. In order to know the interaction mode of HIV-1 integrase with its inhibitor, we investigated the effect of the inhibitor, baicalein, on the conformation of the HIV-1 integrase catalytic domain [IN-(50-212/F185K)] using fluorescence and circular dichroism (CD) spectroscopy. We found that baicalein binds to the hydrophobic region of the HIV-1 integrase catalytic core domain. This binding of baicalein induces the conformational change of the enzyme. We also found that the binding ratio of baicalein to the HIV-1 integrase catalytic domain is 2:1.

Circular Dichroism↗

PubMed2001

Selenium metabolism in Drosophila: selenoproteins, selenoprotein mRNA expression, fertility, and mortality.

Selenocysteine is a rare amino acid in protein that is encoded by UGA with the requirement of a downstream mRNA stem-loop structure, the selenocysteine insertion sequence element. To detect selenoproteins in Drosophila, the entire genome was analyzed with a novel program that searches for selenocysteine insertion sequence elements, followed by selenoprotein gene signature analyses. This computational screen and subsequent metabolic labeling with (75)Se and characterization of selenoprotein mRNA expression resulted in identification of three selenoproteins: selenophosphate synthetase 2 and novel G-rich and BthD selenoproteins that had no homology to known proteins. To assess a biological role for these proteins, a simple chemically defined medium that supports growth of adult Drosophila and requires selenium supplementation for optimal survival was devised. Flies survived on this medium supplemented with 10(-8) to 10(-6) m selenium or on the commonly used yeast-based complete medium at about twice the rate as those on a medium without selenium or with >10(-6) m selenium. This effect correlated with changes in selenoprotein mRNA expression. The number of eggs laid by Drosophila was reduced approximately in half in the chemically defined medium compared with the same medium supplemented with selenium. The data provide evidence that dietary selenium deficiency shortens, while supplementation of the diet with selenium normalizes the Drosophila life span by a process that may involve the newly identified selenoproteins.

Algorithms↗

PubMed2001

Stimulation via CD40 can substitute for CD4 T cell function in preventing reactivation of a latent herpesvirus.

Reactivation of latent herpesviruses is a particular problem in immunocompromised individuals, such as AIDS patients, who lack effective CD4 T helper cell function. An important question is whether residual immune defenses can be mobilized to combat such opportunistic infections, in the absence of CD4 T cells. In the present study, we used a mouse model of opportunistic infection to determine whether stimulation via CD40 could substitute for CD4 T cell function in preventing reactivation of a latent herpesvirus. Treatment with an agonistic antibody to CD40 was highly effective in preventing reactivation of latent murine gammaherpesvirus (MHV-68) in the lungs of CD4 T cell-deficient mice. CD8(+) T cells were essential for this effect, whereas virus-specific serum antibody was undetectable and IFN-gamma production was unchanged. This demonstration that immunostimulation via CD40 can replace CD4 T cell help in controlling latent virus in vivo has potential implications for the development of novel therapeutic agents to prevent viral reactivation in immunocompromised patients.

3T3 Cells↗

PubMed2001

Neurological diseases and viral dynamics in the brains of neonatally borna disease virus-infected gerbils.

Borna disease virus (BDV) is a noncytolytic, neurotropic RNA virus that causes a chronic neurological disease in a wide variety of animal species. To develop a better understanding of the correlation between neurological disorders caused by BDV infection and virus distribution in the brain, we investigated viral dynamics in the central nervous system (CNS) of neonatally BDV-infected gerbils during the late stage of infection. Despite the severe symptoms and aggressive proliferation of BDV in the infected gerbils, no apparent neuroanatomical abnormalities or neuronal cell loss was observed in the infected gerbil brain. Furthermore, no or only minimal infiltration was observed in the infected gerbil brain. By in situ hybridization and real-time PCR analyses, we demonstrated that the predominant area of expression of BDV mRNA, as well as the protein, was shifted in the brain in association with progression of disease. In nondiseased gerbils, the virus replication was predominantly detected in the cerebral cortex and hippocampus of the CNS. On the other hand, diseased animals showed a high level of expression in the lower brain stem and cerebellum, especially in Purkinje cell neurons. These observations suggested that significant replication of the virus in specific areas of the CNS is critical for development of the neurological disorders in BDV-infected neonatal gerbils.

Animals↗

PubMed2001

1-Methylguanosine in place of Y base at position 37 in phenylalanine tRNA is responsible for its shiftiness in retroviral ribosomal frameshifting.

Many mammalian retroviruses express their protease and polymerase by ribosomal frameshifting. It was originally proposed that a specialized shifty tRNA promotes the frameshift event. We previously observed that phenylalanine tRNA(Phe) lacking the highly modified wybutoxosine (Y) base on the 3' side of its anticodon stimulated frameshifting, demonstrating that this tRNA is shifty. We now report the shifty tRNA(Phe) contains 1-methylguanosine (m(1)G) in place of Y and that the m(1)G form from rabbit reticulocytes stimulates frameshifting more efficiently than its m(1)G-containing counterpart from mouse neuroblastoma cells. The latter tRNA contains unmodified C and G nucleosides at positions 32 and 34, respectively, while the former tRNA contains the analogous 2'-O-methylated nucleosides at these positions. The data suggest that not only does the loss of a highly modified base from the 3' side of the anticodon render tRNA(Phe) shifty, but the modification status of the entire anticodon loop contributes to the degree of shiftiness. Possible biological consequences of these findings are discussed.

Animals↗

PubMed2001

TTF-1, a homeodomain gene required for diencephalic morphogenesis, is postnatally expressed in the neuroendocrine brain in a developmentally regulated and cell-specific fashion.

TTF-1 is a member of the Nkx family of homeodomain genes required for morphogenesis of the hypothalamus. Whether TTF-1, or other Nkx genes, contributes to regulating differentiated hypothalamic functions is not known. We now report that postnatal hypothalamic TTF-1 expression is developmentally regulated and associated with the neuroendocrine process of female sexual development. Lesions of the hypothalamus that cause sexual precocity transiently activate neuronal TTF-1 expression near the lesion site. In intact animals, hypothalamic TTF-1 mRNA content also increases transiently, preceding the initiation of puberty. Postnatal expression of the TTF-1 gene was limited to subsets of hypothalamic neurons, including LHRH neurons, which control sexual maturation, and preproenkephalinergic neurons of the lateroventromedial nucleus of the basal hypothalamus, which restrain sexual maturation and facilitate reproductive behavior. TTF-1 mRNA was also detected in astrocytes of the median eminence and ependymal/subependymal cells of the third ventricle, where it colocalized with erbB-2, a receptor involved in facilitating sexual development. TTF-1 binds to and transactivates the erbB-2 and LHRH promoters, but represses transcription of the preproenkephalin gene. The singular increase in hypothalamic TTF-1 gene expression that precedes the initiation of puberty, its highly specific pattern of cellular expression, and its transcriptional actions on genes directly involved in neuroendocrine reproductive regulation suggest that TTF-1 may represent one of the controlling factors that set in motion early events underlying the central activation of mammalian puberty.

Aging↗

PubMed2001

Solution structure of the SL1 RNA of the M1 double-stranded RNA virus of Saccharomyces cerevisiae.

The 20-nucleotide SL1 VBS RNA, 5'-GGAGACGC[GAUUC]GCGCUCC (bulged A underlined and loop bases in brackets), plays a crucial role in viral particle binding to the plus strand and packaging of the RNA. Its structure was determined by NMR spectroscopy. Structure calculations gave a precisely defined structure, with an average pairwise root mean square deviation (RMSD) of 1.28 A for the entire molecule, 0.57 A for the loop region (C8-G14), and 0.46 A for the bulge region (G4-G7, C15-C17). Base stacking continues for three nucleotides on the 5' side of the loop. The final structure contains a single hydrogen bond involving the guanine imino proton and the carbonyl O(2) of the cytosine between the nucleotides on the 5' and 3' ends of the loop, although they do not form a Watson-Crick base pair. All three pyrimidine bases in the loop point toward the major groove, which implies that Cap-Pol protein may recognize the major groove of the SL1 loop region. The bulged A5 residue is stacked in the stem, but nuclear Overhauser enhancements (NOEs) suggest that A5 spends part of the time in the bulged-out conformation. The rigid conformation of the upper stem and loop regions may allow the SL1 VBS RNA to interact with Cap-Pol protein without drastically changing its own conformation.

Base Sequence↗

PubMed2001

Differences in sensitization rates to outdoor aeroallergens, especially citrus red mite (Panonychus citri), between urban and rural children.

BACKGROUND: A recent investigation has suggested that citrus red mite (Panonychus citri, CRM) is the most important allergen in citrus-cultivating farmers with asthma and allergic rhinitis. OBJECTIVE: A cross-sectional survey was performed to evaluate the prevalence of asthma and chronic rhinitis symptoms and sensitization to common indoor and outdoor aeroallergens, including CRM and Japanese cedar pollen, in rural and urban Korean children. METHODS: A total of 2,055 children (1,055 subjects living in rural areas with citrus farms and 1,000 controls in urban areas without citrus farms) were enrolled. They were evaluated by a questionnaire, and by skin prick tests with 13 common indoor and outdoor aeroallergens, including CRM and Japanese cedar pollen. RESULTS: The prevalence of wheezing and chronic rhinitis symptoms during the last 12 months was 8.3% and 35.7% in the rural children and 10.5% and 22.4% in the control group. The most common sensitizing allergens in order of decreasing frequency were Dermatophagoides pteronyssinus (26.6%), Dermatoplagoides farinae (22.7%), CRM (14.2%), cockroach (11.3%), and Japanese cedar pollen (9.7%) among the rural children, but the sensitization rates to CRM and Japanese cedar pollen were 1.3% and 0.2% among the control children, respectively. The prevalence of wheeze during the last 12 months was not different between rural children with sensitization to CRM or Japanese cedar pollen and those without sensitization (5.4% vs 6.1%; 6.9% vs 5.9%). However, the prevalence of chronic rhinitis during the last 12 months was higher among those with sensitization to CRM or to Japanese cedar pollen than among those without sensitization (40.8% vs 34.4%; 51.5% vs 33.5%). CONCLUSIONS: CRM is a common sensitizing allergen in rural children, and the sensitization rates to outdoor aeroallergens, especially CRM and Japanese cedar pollen, are very different between children from rural and urban areas in Korea.

Adolescent↗

PubMed2001

Simultaneous determination of multiple transcripts and splice variants of a primary transcript using ribonuclease protection assays.

The ribonuclease (RNase) protection assay (RPA) is an extremely sensitive technique used to determine specific mRNAs from cell and tissue extracts. The present protocol presents detailed procedures for a conventional RPA using antisense RNA probes purified with a Fullengther apparatus. The Fullengther has the advantage of being a relatively quick and safe procedure compared to more conventional methods for purification of full-length RNA probes. Using this protocol, we sought to simultaneously determine multiple mRNA species, including splice variants of the type I receptor (PAC(1)) of pituitary adenylate cyclase-activating polypeptide (PACAP), an important mediator in the regulation of luteinizing hormone-releasing hormone (LHRH) synthesis by ovarian steroids such as progesterone [7]. PAC(1) has more than eight splice variants. We have been able to discriminate the hop1 variant from other splice variants. To improve our understanding of the regulation mechanism of genes that are related to each other, such as LHRH and PACAP, it is most important to simultaneously determine genes that are involved in the same physiological areas of regulation. Using only 5 microg of total RNA sample from a single rat preoptic area, we simultaneously determined five different transcripts, including four rare mRNA species such as LHRH, PACAP, and hop1 variant and other splice variants of PAC(1), as well as the internal control of cyclophilin mRNA. This protocol provides a method for the simultaneous determination of multiple transcripts using the RPA.

Alternative Splicing↗