Biomedical subjects

E Han

Publications and source records attributed to E Han.

At least 19 recordsLinked to original sources

PubMed2001

Origin pairing ('handcuffing') as a mode of negative control of P1 plasmid copy number.

In one family of bacterial plasmids, multiple initiator binding sites, called iterons, are used for initiation of plasmid replication as well as for the control of plasmid copy number. Iterons can also pair in vitro via the bound initiators. This pairing, called handcuffing, has been suggested to cause steric hindrance to initiation and thereby control the copy number. To test this hypothesis, we have compared copy numbers of isogenic miniP1 plasmid monomer and dimer. The dimer copy number was only one-quarter that of the monomer, suggesting that the higher local concentration of origins in the dimer facilitated their pairing. Physical evidence consistent with iteron-mediated pairing of origins preferentially in the dimer was obtained in vivo. Thus, origin handcuffing can be a mechanism to control P1 plasmid replication.

DNA Replication↗

PubMed2001

Array-based gene expression profiling to study aging.

With recent sequencing of the genome and development of high-density array technology, it is now possible to assess global gene expression in cells/tissues by a technique that is sensitive, quantitative, and rapid. Gene expression array technology is extremely useful in studying a complex, multigenetic process, such as aging, where one needs to understand the interaction of a large number of genes. Although the technology holds great promise, it is novel and not yet well-established and there are no widely-accepted standards to guide investigators in the analysis and interpretation of the data obtained. Gene expression array analysis requires strong biostatistical support to minimize false-positives and maximize true-positives in candidate gene identification. It also requires independent validation of the array measurements using other detection methods. Confirmation that differentially expressed (transcribed) genes are reflected by differential expression at the protein level will ultimately be an important measurement. In this review, we focus on the three steps necessary for aging studies when using the gene expression array technology: (1) array hybridization; (2) biostatistical analysis; and (3) array result confirmation. Genes identified by several investigators for their age-associated change using the gene expression array systems are also discussed.

Aging↗

PubMed2001

Differences in tissue-specific and embryonic expression of mouse Ceacam1 and Ceacam2 genes.

The intercellular adhesion molecule CEACAM1, also known as C-CAM1 (where CAM is cell-adhesion molecule), can function as a tumour suppressor in several carcinomas, including those of the prostate, breast, bladder and colon. This suggests that CEACAM1 may play an important role in the regulation of normal cell growth and differentiation. However, there is no direct evidence to support this putative function of CEACAM1. To elucidate its physiological function by targeted gene deletion, we isolated the Ceacam genes from a mouse 129 Sv/Ev library. Although there is only one Ceacam1 gene in humans and one in rats, two homologous genes (Ceacam1 and Ceacam2) have been identified in the mouse. Our sequence analysis revealed that the genes encoded nine exons and spanned approx. 16-17 kb (Ceacam1) and 25 kb (Ceacam2). The genes were highly similar (79.6%). The major differences in the protein-coding regions were located in exons 2, 5 and 6 (76.9%, 87.0% and 78.5% similarity respectively). In addition, introns 2, 5 and 7 were also significantly different, being 29.7%, 59.8% and 64.5% similar respectively. While most of these differences were due to nucleotide substitutions, two insertions of 418 and 5849 bp occurred in intron 2 of Ceacam2, and another two insertions of 1384 and 197 bp occurred in introns 5 and 7 respectively. To determine whether functional redundancy exists between Ceacam1 and Ceacam2, we examined their expression in 16 mouse tissues by using semi-quantitative reverse transcription-PCR. As in human and rat, in the mouse Ceacam1 mRNA was highly abundant in the liver, small intestine, prostate and spleen. In contrast, Ceacam2 mRNA was only detected in kidney, testis and, to a lesser extent, spleen. Reverse transcription-PCR using testis RNA indicated that Ceacam2 in the testis is an alternatively spliced form containing only exons 1, 2, 5, 6, 8 and 9. In the mouse embryo, Ceacam1 mRNA was detected at day 8.5, disappeared between days 9.5 and 12.5, and re-appeared at day 19. On the other hand, no Ceacam2 mRNA was detected throughout embryonic development. The different tissue expression patterns and regulation during embryonic development suggest that the CEACAM1 and CEACAM2 proteins, although highly similar, may have different functions both during mouse development and in adulthood.

Adenosine Triphosphatases↗

PubMed2001

Purification and characterization of human cell--cell adhesion molecule 1 (C-CAM1) expressed in insect cells.

The cell--cell adhesion molecule 1 (C-CAM1) plays an important role as a tumor suppressor for prostate cancer. Decreased expression of C-CAM1 was detected in prostate, breast, and colon carcinoma. Reexpression of C-CAM1 in prostate and breast cancer cell lines was able to suppress tumorigenicity in vivo. These observations suggest that C-CAM1 may be used as a marker for cancer detection or diagnosis. To generate monoclonal antibodies specific to C-CAM1, we have overexpressed full-length human C-CAM1 in Sf9 cells using a baculovirus expression system. The protein was purified 104-fold using nickel affinity chromatography. About 0.4 mg purified C-CAM1 was obtained from 200 mg of infected cells. When the purified protein was digested with peptidyl-N-glycosidase, the apparent mobility of the protein on SDS--PAGE changed from 90 to 58 kDa, which is close to the molecular weight predicted from the cloned cDNA sequence. This observation suggests that C-CAM1 was glycosylated on asparagine residues when expressed in Sf9 cells. Western blotting and internal protein sequencing analysis confirmed that the purified protein is human C-CAM1. Biochemical and functional assays indicate that this protein expressed in Sf9 cells displays characteristics similar to those of native protein, including adhesion function and glycosylation modification. Using this protocol, sufficient quantity of this protein can be produced with purity suitable for monoclonal antibody generation and biochemical study.

Adenosine Triphosphatases↗

PubMed2001

Desiccation resistance in pre-diapause, diapause and post-diapause larvae of Choristoneura fumiferana (Lepidoptera: Tortricidae).

Desiccation resistance was examined in pre-diapause, diapause and post-diapause larvae of the spruce budworm, Choristoneura fumiferana (Clemens), in terms of passive water evaporation under three desiccation conditions: freeze-drying, desiccant-drying at 2 degrees C and desiccant-drying at 18 degrees C. Diapausing second instar larvae and post-diapause non-feeding second instar larvae showed strongest desiccation resistance: a significant amount of water was retained after repeated drying under desiccating conditions, while pre-diapause first instar larvae and post-diapause feeding second instar larvae lost almost all their water content after one or two drying cycles. A hibernaculum covering had no effect on water evaporation. While dead larvae tended to lose significantly more water than their living counterparts, particularly among first instar larvae, such an impact was much weaker among diapausing second instar larvae. Desiccation resistance was lost when post-diapause second instar larvae were allowed access to water while the level of desiccation resistance was maintained or enhanced when the larvae did not have access to water. These results are discussed in the context of overwintering ecology of the species and possible mechanisms for the desiccation resistance are also discussed.

Animals↗

PubMed2000

cDNA expression arrays reveal incomplete reversal of age-related changes in gene expression by calorie restriction.

Calorie restriction (CR) extends life span and retards many age-related cellular and molecular changes in laboratory rodents. However, neither the breadth of its effects, its underlying mechanisms, nor the limits of its action is fully understood. Expression levels of 588 genes in livers from 3- and 24-month-old ad libitum-fed (AL), and 24-month-old CR (60% of AL intake) male C57BL/6J mice (four per group) were measured. Six genes met the statistical criteria for differential expression in old AL compared to young AL mice. Only one of these age-related changes was attenuated by CR. Four additional gene products, that did not change with age in AL mice, were differentially expressed in old CR compared to old AL mice. Northern and RT-PCR analyses confirmed differential expression of four of the six candidate genes identified by the array results. Many of the identified genes have not previously been reported to be affected by CR or aging. Some of the age-related changes in gene expression are consistent with an increased vulnerability of the aged liver to carcinogenic or other insults, with only partial protection against insult by CR. Incomplete reversal by CR of age-related changes in gene expression provides a potentially important path for probing the limits of CR action. These results also show the importance of independent confirmation in expression array profiling of age-related changes in gene expression.

Aging↗

PubMed2000

Distinction among neuronal subtypes of voltage-activated sodium channels by mu-conotoxin PIIIA.

The functional properties of most sodium channels are too similar to permit identification of specific sodium channel types underlying macroscopic current. Such discrimination would be particularly advantageous in the nervous system in which different sodium channel family isoforms are coexpressed in the same cell. To test whether members of the mu-conotoxin family can discriminate among known neuronal sodium channel types, we examined six toxins for their ability to block different types of heterologously expressed sodium channels. PIIIA mu-conotoxin blocked rat brain type II/IIA (rBII/IIA) and skeletal muscle sodium current at concentrations that resulted in only slight inhibition of rat peripheral nerve (rPN1) sodium current. Recordings from variant lines of PC12 cells, which selectively express either rBII/IIA or rPN1 channel subtypes, verified that the differential block by PIIIA also applied to native sodium current. The sensitivity to block by PIIIA toxin was then used to discriminate between rBII/IIA and rPN1 sodium currents in NGF-treated PC12 cells in which both mRNAs are induced. During the first 24 hr of NGF-treatment, PN1 sodium channels accounted for over 90% of the sodium current. However, over the ensuing 48 hr period, a sharp rise in the proportion of rBII/IIA sodium current occurred, confirming the idea, based on previous mRNA measurements, that two distinct sodium channel types appear sequentially during neuronal differentiation of PC12 cells.

Action Potentials↗

PubMed2000

Role of the ribosome in sequence-specific regulation of membrane targeting and translocation of P-glycoprotein signal-anchor transmembrane segments.

It is thought that the topology of a polytopic protein is generated by sequential translocation and membrane integration of independent signal-anchor and stop-transfer sequences. Two well-characterized cell-free systems (rabbit reticulocyte lysate and wheat germ extract) have been widely used to study the biogenesis of secretory and membrane proteins, but different results have been observed with proteins expressed in these two different systems. For example, different topologies of P-glycoprotein (Pgp) were observed in the two systems and the cause was thought to be the source of ribosomes. To understand how the ribosome is involved in dictating membrane translocation and orientation of polytopic proteins, individual signal-anchor sequences of Pgp were dissected and examined for their membrane targeting and translocation in a combined system of wheat germ ribosomes (WGR) and rabbit reticulocyte lysate (RRL). Addition of wheat germ ribosomes to the rabbit reticulocyte lysate translation system can enhance, reduce, or have no effect on the membrane targeting and translocation of individual Pgp signal-anchor sequences, and these effects appear to be determined by the amino acid residues flanking each signal-anchor. Ribosomes regulate the membrane targeting and translocation of Pgp signal-anchors in a polytopic form differently from the same signal-anchors in isolation. Furthermore, we demonstrated that ribosomes regulate the membrane targeting and translocation of each signal-anchor cotranslationally and that this activity of ribosomes is associated with the 60S subunit. Based on this and previous studies, we propose a mechanism by which ribosomes dynamically dictate the membrane targeting and translocation of nascent polytopic membrane proteins.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Dissection of de novo membrane insertion activities of internal transmembrane segments of ATP-binding-cassette transporters: toward understanding topological rules for membrane assembly of polytopic membrane proteins.

The membrane assembly of polytopic membrane proteins is a complicated process. Using Chinese hamster P-glycoprotein (Pgp) as a model protein, we investigated this process previously and found that Pgp expresses more than one topology. One of the variations occurs at the transmembrane (TM) domain including TM3 and TM4: TM4 inserts into membranes in an N(in)-C(out) rather than the predicted N(out)-C(in) orientation, and TM3 is in cytoplasm rather than the predicted N(in)-C(out) orientation in the membrane. It is possible that TM4 has a strong activity to initiate the N(in)-C(out) membrane insertion, leaving TM3 out of the membrane. Here, we tested this hypothesis by expressing TM3 and TM4 in isolated conditions. Our results show that TM3 of Pgp does not have de novo N(in)-C(out) membrane insertion activity whereas TM4 initiates the N(in)-C(out) membrane insertion regardless of the presence of TM3. In contrast, TM3 and TM4 of another polytopic membrane protein, cystic fibrosis transmembrane conductance regulator (CFTR), have a similar level of de novo Nin-Cout membrane insertion activity and TM4 of CFTR functions only as a stop-transfer sequence in the presence of TM3. Based on these findings, we propose that 1) the membrane insertion of TM3 and TM4 of Pgp does not follow the sequential model, which predicts that TM3 initiates N(in)-C(out) membrane insertion whereas TM4 stops the insertion event; and 2) "leaving one TM segment out of the membrane" may be an important folding mechanism for polytopic membrane proteins, and it is regulated by the N(in)-C(out) membrane insertion activities of the TM segments.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

[Inhibition of beta-amyloid precursor protein gene expression by haifengteng].

OBJECTIVE: To study the inhibition of beta-amyloid precursor protein(beta-APP) gene expression by Haifengteng. METHOD: Northern Blot analysis was used to investigate the level of beta-APP mRNA, and cell viability, LDH-release and colony formation assay were used to study the effect of Hairengteng of neurons. RESULT: Haifengteng inhibited the expression of beta-APP gene selectively without causing cell injuries. CONCLUSION: beta-APP is closely related to Alzheimer's disease. The findings in this study could be a basis for Haifengteng to have therapeutic potentials in treating Alzheimer's disease.

Amyloid beta-Protein Precursor↗

PubMed1997

Role of ribosomes in reinitiation of membrane insertion of internal transmembrane segments in a polytopic membrane protein.

The topogenesis of membrane proteins with a single transmembrane (TM) segment is well understood. However, understanding the topogenesis and membrane assembly of membrane proteins with multiple TM segments (polytopic) is still incomplete. Recently, several studies on P-glycoprotein (Pgp) suggested that the topogenesis of polytopic membrane proteins is likely more complicated than anticipated. While studying the mechanism by which Pgp topogenesis is determined, we unexpectedly found that ribosomes or proteins associated with ribosomes are involved in regulating the membrane insertion and folding of Pgp during its translation. We discovered that when Pgp was translated by wheat germ ribosomes in vitro, TM3 could not reinitiate the insertion of the protein into microsomal membranes following the membrane insertion of TM1 and TM2. In contrast, TM3 could reinitiate membrane insertion when the protein was translated by rabbit reticulocyte ribosomes. These findings suggest that ribosomes or proteins associated with ribosomes play an important role in membrane insertion and folding of TM segments of Pgp and that rabbit reticulocyte and wheat germ ribosomes may use different mechanisms to control the membrane insertion of the same nascent peptide. We propose that ribosomes or proteins associated with ribosomes help reinitiate insertion of internal TM segments into the membrane by dissociation and reassociation with the protein-conducting channel in ER membranes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

DNA-dependent RNA polymerase from Enterobacter cloacae is closely related to Escherichia coli.

The RNA polymerase holoenzyme (RNAP) of Enterobacter cloacae was purified by gel filtration and heparin affinity chromatography and shown to consist of four subunits (beta', beta, alpha and sigma) of 156, 151, 45 and 82 kDa, as measured by SDS-PAGE. The 82 kDa protein was shown to be related to the Escherichia coli primary sigma factor by western blot analysis with polyclonal antisera raised against purified E. coli sigma 70. Functional reconstitution of E. cloacae core enzyme with purified E. coli sigma 70 showed that E. cloacae and E. coli sigma factors are closely related. The RNAP of E. cloacae initiated transcription from the tac promoter with an efficiency similar to that of E. coli. Measuring promoter-specific transcription, the dependence of holoenzyme activity on salt, divalent cation and temperature was also similar to that of the E. coli RNAP. We also showed that the transcriptional inhibitor, rifampicin, inhibits the enzyme activity of the purified RNAP of E. cloacae and E. coli at similar concentrations. We conclude, based on these data, that the RNAP of E. cloacae and E. coli, both enterobacteria, are closely related.

Chromatography, Affinity↗

PubMed1996

VH gene usage is multiple myeloma: complete absence of the VH4.21 (VH4-34) gene.

The immunoglobin heavy chain variable region (VH) gene usage in multiple myeloma (MM) has not been reported, although a few studies have incidentally identified the VH gene rearranged in small cohorts of MM patients. We used a reverse transcriptase-polymerase chain reaction based technique to analyze the VH gene usage in MM. The VH sequences were obtained after amplification of bone marrow cDNA using the seven VH family-specific and constant region primers. The VH sequences of 72 patients were successfully identified. The frequency of VH family usage in decreasing order was VH3>VH4>VH1>VH5>VH2>VH6>VH7 and corresponded to the functional germline complexity of the VH families. Individual VH genes (VH1-69, VH3-9, VH3-23, and VH3-30) were overrepresented in our cohort of MM patients; some VH genes [VH3-49, VH3-53, and VH4.21 (VH4-34)], which are rearranged with increased frequency in normal circulating B cells, autoimmune diseases, and other B-cell malignancies, were not detected in any MM patient. Compared with germline sequences, an average of 8.8% (range, 2.7% to 16.5%) of the nucleotides had evidence of mutation within each VH sequence. Based on these results, we conclude that (1) the VH gene usage in MM is unique compared with other malignant and nonmalignant B-cell populations, (2) the physiologic process of clonal deletion functions to remove clones that have rearranged VH genes (VH4.21) capable of expressing antibodies, which recognize self-antigens, and (3) the complete lack of VH4.21 gene rearrangement may help to partially explain the paucity of autoimmune phenomena in MM.

Base Sequence↗

PubMed1993

Neuronal beta-amyloid precursor protein gene expression: regulation by aurintricarboxylic acid.

beta-Amyloid precursor protein (beta-APP) and its derivative, amyloid beta-protein (beta-A4), may cause death of differentiated neurons and aurintricarboxylic acid (ATA), a metabolic inhibitor, improves neuronal survival. Therefore, we studied the effect of ATA on neuronal beta-APP gene expression. ATA decreased beta-APP mRNA levels by increasing its degradation, without changing the rate of transcription. ATA decreased both steady state and interleukin-1 (IL1)-induced increase in beta-APP mRNA levels. These effects of ATA were associated with rounding of cells suggestive of decreased cell adhesion or neurite retraction that was completely reversible when ATA was removed. However, beta-APP mRNA levels continued to remain suppressed in neurons that were actively regrowing neurites following discontinuation of ATA. In studies carried out upto 24 h, ATA did not damage cells as determined by Trypan blue exclusion, lactate dehydrogenase (LDH)-release and transmission electron microscopy. The findings suggest that constitutive or steady state levels of beta-APP mRNA may not be essential for the survival and growth of neurons and that ATA suppresses beta-APP expression without causing cell damage. These observations may be a basis for studying whether ATA or a related compound could beneficially regulate beta-APP levels in vivo.

Amyloid beta-Protein Precursor↗

PubMed1993

Identification of cyclic nucleotide-regulated phosphoproteins, including phosducin, in motile rod inner-outer segments of teleosts.

In teleost retinas, rods elongate in the light and shorten in the dark. Rod motility is mediated by the actin cytoskeleton of the inner segment and is regulated by cyclic AMP- or cyclic GMP-stimulated phosphorylation of target proteins. In this study, we have identified the target proteins of cyclic nucleotide-dependent kinases in rods, using preparations of isolated, motile rod inner-outer segments (RIS-ROS). Five proteins found in Percoll-purified RIS-ROS were phosphorylated in the presence of cAMP (> 10 nM), cGMP (> or = 10 microM) and exogenous catalytic subunit of cAMP-dependent protein kinase (PKA). The PKA inhibitor, PKI, blocked stimulation of phosphorylation by both cAMP and cGMP. Three cAMP-stimulated phosphoproteins were detected in cytoskeletal fractions of light- and dark-adapted RIS-ROS. One of these, PP33, appears to be a fish homologue of mammalian phosducin, based on immunolabeling by two different antibodies against mammalian phosducin and on electrophoretic characteristics in 2-D gels. Two additional phosducin immunoreactive bands were detected in Western blots. One, at 35 kDa, comigrated with a second cAMP-stimulated RIS-ROS phosphoprotein, PP35, which was also detected in the cytoskeleton. The other, at 37 kDa, was present in whole teleost retinas but not in purified RIS-ROS. Our results suggest that the effects of both cAMP and cGMP on teleost rod motility are mediated through PKA modulation of target phosphoproteins. These phosphoproteins include a cytoskeleton-associated phosducin homologue.

Animals↗

PubMed1993

Neuronal death, cytoplasmic calcium and internucleosomal DNA fragmentation: evidence for DNA fragments being released from cells.

Neuronal death, secondary to endogenous agents such as glutamate, may involve changes in cytoplasmic calcium. Besides its well recognized role as a second messenger mediating cellular response, calcium is necessary for the activation of endonuclease(s), resulting in DNA fragmentation and cell death. Therefore, we investigated the relationship between changes in cytoplasmic calcium, DNA fragmentation and neuronal death, using PC12 and NCB-20 cell lines. The calcium ionophore, A23187, caused a dose-dependent increase in cytoplasmic calcium, loss of cell viability, increased lactate dehydrogenase (LDH)-release, and DNA fragmentation. DNA fragments, typical of internucleosomal digestion of genomic DNA, characteristic of endonuclease(s) activation, were consistently detected in the incubating medium. Release of DNA fragments into the medium was seen with A23187 in concentrations as low as 10 nM, and within an hour of treatment. Furthermore, calcium added to preparations of PC12 nuclei also produced DNA fragmentation, although, less pronounced than when intact cells were treated with A23187. The findings indicate that A23187-induced neuronal death involves the activation of endonuclease(s). The role of cytoplasmic calcium in this process is supported by evidence that A23187 selectively mobilizes cytoplasmic calcium, and that calcium can directly activate endonuclease(s) in nuclear preparations.

Animals↗

PubMed1992

Amyloid beta-protein fragment 25-35 causes activation of cytoplasmic calcium in neurons.

The cellular mechanism by which beta-amyloid has its effect on neurons is unknown. Based on observations that endogenous neurotoxins, such as glutamate and platelet activating factor (PAF), cause activation of cytoplasmic calcium, we tested if this was true with beta-amyloid. Using nerve growth factor-treated PC12 cells, we noted that the active beta-amyloid fragment, containing residues 25 to 35, caused a specific and dose-dependent increase in intracellular calcium due to an influx of extracellular calcium.

Aequorin↗

PubMed1992

Platelet activity and stroke severity.

Although platelets constitute the major component of a thrombus, its role in determining the clinical severity of thrombotic stroke is unknown. Therefore, we investigated the relationship between platelet ionized calcium ([Ca2+i]), a measure of platelet activity and presumably proneness to thrombosis, and clinical stroke severity in 45 consecutively studied acute ischemic stroke patients. Even though there was no correlation between the clinical neurological scores and the levels of baseline and activated platelet [Ca2+i], stroke was less severe in patients who had been taking aspirin at the time of stroke onset. These results raise several important questions: (a) is the extent of platelet activation a reflection of thrombus volume, (b) does the clinical severity of neurological deficit reflect the causative thrombus volume, and (c) whether the beneficial effect of aspirin in stroke prophylaxis is through its inhibition of platelets alone.

Aged↗