Biomedical subjects

J Lu

Publications and source records attributed to J Lu.

At least 19 recordsLinked to original sources

PubMed1998

cDNA cloning reveals two mouse beta5 integrin transcripts distinct in cytoplasmic domains as a result of alternative splicing.

The integrin beta5 subunit has only been found to form a heterodimer with subunit alphav which acts as a vitronectin receptor. Integrin alphavbeta5 has been implicated in cell migration and growth factor-induced angiogenesis. In the present study, a mouse liver cDNA library was screened using a human beta5 cDNA fragment obtained by reverse transcriptase PCR (RT-PCR). Three of the clones (MB5, MB15 and MB17) overlapped to give an open reading frame, called beta5A, which is homologous to the human beta5 subunit. The sequence of another clone (MB26), called beta5B, was identical with beta5A, except for a deletion of 29 bp near the 3' end of the open reading frame. The 29 bp deletion resulted in an open-reading-frame shift and a completely different C-terminal sequence in beta5B. beta5A and beta5B were shown, by RT-PCR, to be co-expressed in most mouse tissues tested, although beta5B mRNA was detected at much lower levels than beta5A. beta5A and beta5B mRNAs were also detected in the mouse monocytic cell line, J774, and in isolated mouse peritoneal macrophages. Adhesion of peritoneal macrophages has been shown to up-regulate the expression of both beta5A and beta5B mRNAs. The 29 bp sequence begins with a putative intron-splicing donor site (GTGAT...). A 3' fragment of the mouse integrin beta5 gene was cloned by PCR and sequenced showing that the 29 bp sequence was also immediately followed by an intron. Therefore, the 29 bp sequence was apparently expressed as part of the beta5A mRNA but was spliced out as part of the downstream intron in beta5B. Since the cytoplasmic domains of the integrin beta subunits are important in cytoskeleton attachment and signalling, the two alternatively spliced beta5 isoforms may have distinct roles in cell adhesion and other cellular functions.

Alternative Splicing↗

PubMed1998

Disruption of cellular translational control by a viral truncated eukaryotic translation initiation factor 2alpha kinase homolog.

Phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha) is a common cellular mechanism to limit protein synthesis in stress conditions. Baculovirus PK2, which resembles the C-terminal half of a protein kinase domain, was found to inhibit both human and yeast eIF2alpha kinases. Insect cells infected with wild-type, but not pk2-deleted, baculovirus exhibited reduced eIF2alpha phosphorylation and increased translational activity. The negative regulatory effect of human protein kinase RNA-regulated (PKR), an eIF2alpha kinase, on virus production was counteracted by PK2, indicating that baculoviruses have evolved a unique strategy for disrupting a host stress response. PK2 was found in complex with PKR and blocked kinase autophosphorylation in vivo, suggesting a mechanism of kinase inhibition mediated by interaction between truncated and intact kinase domains.

Animals↗

PubMed1998

Origin, nature, and some functional considerations of intraventricular macrophages, with special reference to the epiplexus cells.

Intraventricular macrophages encompass the supraependymal, free-floating, and epiplexus (Kolmer) cells; the supraependymal cells lie in close apposition to the ventricular ependyma, the epiplexus cells are closely associated with the choroid plexus epithelium, and the free-floating cells are at a variable distance from the epithelial surface. Although the three cell types are regarded as one cellular entity, the epiplexus cells preponderate. On scanning electron microscopy, the epiplexus cells display diverse morphological forms, ranging from round to bipolar to stellate, and bear a variable number of cytoplasmic processes. Transmission electron microscopy shows the presence of large numbers of lysosomes. The phagocytic nature of epiplexus cells is shown by their intense staining for nonspecific esterase and active uptake of tracers, e.g., horseradish peroxidase and rhodamine isothiocynate, administered intravenously or intraperitoneally. The mode of entry of these tracers in the cerebral ventricles is by way of transepithelial transport. In rats, the population of intraventricular macrophages increases steadily after birth until 17 days of age; thereafter, their cell population remains relatively unchanged. The early upsurge is attributed to proliferation of residential cells and/or influx of circulating monocytes/stromal macrophages through the process of "emperipolesis." The immunophenotypic features of intraventricular macrophages are consistent with other mononuclear phagocytes being immunoreactive for OX-42, OX-18, OX-6, and OX-1 and ED1 for the detection of CR3 receptors, MHC class I and II antigens, leucocyte common antigen, and macrophage antigen, respectively. The expression of these antigens is noticeably enhanced following the injection of lipopolysaccharide (LPS) into postnatal rats. Remarkably, the intraventricular macrophages are induced to express MHC class II (Ia) antigen after LPS or interferon-gamma injections. Furthermore, the expression of transferrin receptors as detected with OX-26 is also upregulated after these treatments. Epiplexus cells are also elicited to display a de novo expression of nitric oxide synthase-like immunoreactivity following intracerebral injection of LPS. They also respond vigorously to a single nonpenetrative blast. Results of our series of studies suggest that, besides their primary function as scavenger cells, the intraventricular macrophages partake in possible immunological responses and iron regulation in the ventricular system or the brain as a whole.

Animals↗

PubMed1998

Human L-ficolin: plasma levels, sugar specificity, and assignment of its lectin activity to the fibrinogen-like (FBG) domain.

Ficolins are characterised by the presence of collagen-like and fibrinogen-like (FBG) sequences. Human L-ficolin is synthesised in the liver and secreted into blood circulation. In previous studies, it was shown to bind to N-acetyl-D-glucosamine (GlcNAc). In the present study, its detailed sugar specificity and binding site have been investigated. It was found to bind to GlcNAc and GalNAc (N-acetyl-D-galactosamine) while showing no significant affinity for the precursor sugars. The structure in these molecules which is recognised by L.-ficolin has been deduced to include an amide (-CO-NH-) or similar group. L-Ficolin was digested with collagenase and the collagenase resistant FBG domain was shown to bind to GlcNAc. Its levels in adult and cord blood-derived human plasma were also determined and showed that adult plasma contains approximately three times more L-ficolin than that of newborn babies.

Adult↗

PubMed1998

Anatomic considerations of C2 nerve root ganglion.

STUDY DESIGN: Dissection and observation of the dorsal root ganglion of the second cervical spinal nerve bilaterally. OBJECTIVES: To determine the position of the C2 dorsal root ganglion and the heights of the C2 ganglion and its corresponding foramen. SUMMARY OF BACKGROUND DATA: The dorsal root ganglion has been well studied in the middle and lower cervical spine and in the lumbar spine. In no previous study are the position and height of the C2 dorsal root ganglion and its corresponding foramen described. METHODS: Fifteen cadaveric cervical spines were exposed posteriorly. The C2 nerve roots and ganglia were dissected between the arch of the atlas and the lamina of the axis. The heights of the C2 ganglion and foramen were measured. The location of the C2 ganglion was also macroscopically observed. RESULTS: The heights of the C2 ganglion and foramen are 5.7 +/- 0.8 mm and 7.7 +/- 1.2 mm, respectively. The C2 ganglion occupies 76% of the foramen height. All C2 dorsal root ganglia are confined within foramens between the arch of the atlas and the lamina of the axis. CONCLUSIONS: The C2 dorsal dorsal root ganglia are all proximally placed and occupy most of the foramen height, which may render the C2 ganglion vulnerable to entrapment.

Cadaver↗

PubMed1998

Structure-function study of the extracellular domain of the human IFN-alpha receptor (hIFNAR1) using blocking monoclonal antibodies: the role of domains 1 and 2.

We have performed a structure-function analysis of extracellular domain regions of the human IFN-alpha receptor (hIFNAR1) using mAbs generated by immunizing mice with a soluble hIFNAR1-IgG. Five mAbs described in this study recognize different epitopes as determined by a competitive binding ELISA and by alanine substitution mutant analyses of the hIFNAR1-IgG. Two mAbs, 2E1 and 4A7, are able to block IFN-stimulated gene factor 3 (ISGF3) formation and inhibit the antiviral cytopathic effect induced by several IFN-alpha (IFN-alpha 2/1, -alpha 1, -alpha 2, -alpha 5, and -alpha 8). None of these anti-IFNAR1 mAbs were able to block activity of IFN-beta. mAb 4A7 binds to a domain 1-hIFNAR1-IgG but not to a domain 2-hIFNAR1-IgG, which suggests that its binding region is located in domain 1. The binding of the most potent blocking mAb, 2E1, requires the presence of domain 1 and domain 2. The most critical residue for 2E1 binding is a lysine residue at position 249, which is in domain 2. These findings suggest that both domain 1 and domain 2 are necessary to form a functional receptor and that a region in domain 2 is important. IFN-beta recognizes regions of the hIFNAR complex that are distinct from those important for the IFN-alpha.

Alanine↗

PubMed1998

Capsulin: a novel bHLH transcription factor expressed in epicardial progenitors and mesenchyme of visceral organs.

Members of the basic helix-loop-helix (bHLH) family of transcription factors have been shown to control development and differentiation of a variety of cell types. We describe a novel bHLH protein, called capsulin, which is expressed specifically in mesodermally-derived cells that surround the epithelium of the developing gastrointestinal, genitourinary and respiratory systems during mouse embryogenesis. Capsulin transcripts also mark the spiral septum of the heart and progenitor cells that give rise to the pericardium and coronary arteries. Capsulin shares high homology with a recently identified bHLH protein from Drosophila, called bHLH102C, which is expressed in visceral muscle cells that surround the midgut. Capsulin binds a specific E-box consensus sequence (CANNTG) as a heterodimer with the widely-expressed bHLH protein E12, but it does not activate transcription through that sequence on its own. Its restricted expression pattern and DNA binding activity suggest that capsulin regulates gene expression in specific subtypes of visceral mesodermal cells involved in organogenesis and in precursor cells that contribute to the pericardium, coronary arteries and regions of the heart.

Amino Acid Sequence↗

PubMed1998

Pathogenic characterization of 1-methyl-1-nitrosourea-induced mammary carcinomas in the rat.

The induction of mammary carcinogenesis in the rat by 1-methyl-1-nitrosourea (MNU) is widely used in experimental breast cancer research. In the experiments reported, the Ha-ras codon 12 (ras12) mutation (GGA-->GAA) was used as a molecular marker to address issues of the clonality of carcinomas induced, pathogenetic independence among multiple carcinomas within the same animal and topographic distribution of mutant ras12 carcinomas in different mammary gland chains. In order to determine whether the frequently observed morphologically distinguishable lobules within carcinomas originate from the coalescence of independent lesions or whether cancerous cells within a carcinoma share a common origin, 44 randomly selected MNU-induced mammary carcinomas were genotyped for two to four lobules each for the ras12 mutation. A total of 43 carcinomas out of 44 (97.7%) had concordant ras12 genotypes among the multiple sites within each tumor, which is consistent with the latter possibility. Next, it was observed that as carcinoma multiplicity increased, the discordance rate of ras12 genotypes among multiple carcinomas within the same animal increased in a manner that was in excellent agreement with the expected discordance rate based on an assumption of no pathogenetic association among carcinomas. Furthermore, a significant difference was observed in the occurrence of mutant ras12 carcinomas between the cervical-thoracic and the abdominal-inguinal mammary glands in that three times as many carcinomas were mutant in the former as in the latter glands, whereas the occurrence of wild-type carcinomas was approximately the same in both regions. Taken together, the data are consistent with (i) carcinomas induced by MNU and detected by palpation are monoclonal in origin, (ii) independently-initiated cells emerge as distinct mammary carcinomas in the same animal, and (iii) the anatomical location of the gland may affect the prevalence of mammary carcinomas that harbor a mutant ras12.

Animals↗

PubMed1998

Changes in murine jejunal morphology evoked by the bacterial superantigen Staphylococcus aureus enterotoxin B are mediated by CD4+ T cells.

Bacterial superantigens (SAgs) are potent T-cell stimuli that have been implicated in the pathophysiology of autoimmune and inflammatory disease. We used Staphylococcus aureus enterotoxin B (SEB) as a model SAg to assess the effects of SAg exposure on gut form and cellularity. BALB/c, SCID (lacking T cells) and T-cell-reconstituted SCID mice were treated with SEB (5 or 100 microg intraperitoneally), and segments of the mid-jejunum were removed 4, 12, or 48 h later and processed for histochemical or immunocytochemical analysis of gut morphology and major histocompatibility complex class II (MHC II) expression and the enumeration of CD3+ T cells and goblet cells. Control mice received saline only. SEB treatment of BALB/c mice caused a time- and dose-dependent enteropathy that was characterized by reduced villus height, increased crypt depth, and a significant increase in MHC II expression. An increase in the number of CD3+ T cells was observed 48 h after exposure to 100 microg of SEB. Enteric structural alterations were not apparent in SEB-treated SCID mice compared to saline-treated SCID mice. In contrast, SEB challenge of SCID mice reconstituted with a mixed lymphocyte population or purified murine CD4+ T cells resulted in enteric histopathological changes reminiscent of those observed in SEB-treated BALB/c mice. These findings implicate CD4+ T cells in this SEB-induced enteropathy. Our results show that SAg immune activation causes significant changes in jejunal villus-crypt architecture and cellularity that are likely to impact on normal physiological processes. We speculate that the elevated MHC II expression and increased number of T cells could allow for enhanced immune responsiveness to other SAgs or environmental antigens.

Animals↗

PubMed1998

The Cys-rich region of hepatitis A virus cellular receptor 1 is required for binding of hepatitis A virus and protective monoclonal antibody 190/4.

The hepatitis A virus cellular receptor 1 (HAVcr-1) cDNA codes for a class I integral membrane glycoprotein, termed havcr-1, of unknown natural function which serves as an African green monkey kidney (AGMK) cell receptor for HAV. The extracellular domain of havcr-1 has an N-terminal Cys-rich region that displays homology with sequences of members of the immunoglobulin superfamily, followed by a Thr/Ser/Pro (TSP)-rich region characteristic of mucin-like O-glycosylated proteins. The havcr-1 glycoprotein contains four putative N-glycosylation sites, two in the Cys-rich region and two in the TSP-rich region. To characterize havcr-1 and define region(s) involved in HAV receptor function, we expressed the TSP-rich region in Escherichia coli fused to glutathione S-transferase and generated antibodies (Ab) in rabbits (anti-GST2 Ab). Western blot analysis with anti-GST2 Ab detected 62- and 65-kDa bands in AGMK cells and 59-, 62-, and 65-kDa bands in dog cells transfected with the HAVcr-1 cDNA (cr5 cells) but not in dog cells transfected with the vector alone (DR2 cells). Treatment of AGMK and cr5 cell extracts with peptide-N-glycosidase F resulted in the collapse of the havcr-1-specific bands into a single band of 56 kDa, which indicated that different N-glycosylated forms of havcr-1 were expressed in these cells. Treatment of AGMK and cr5 cells with tunicamycin reduced binding of protective monoclonal Ab (MAb) 190/4, which suggested that N-glycans are required for binding of MAb 190/4 to havcr-1. To test this hypothesis, havcr-1 mutants lacking the N-glycosylation motif at the first site (mut1), second site (mut2), and both (mut3) sites were constructed and transfected into dog cells. Binding of MAb 190/4 and HAV to mut1 and mut3 cells was highly reduced, while binding to mut2 cells was not affected and binding to dog cells expressing an havcr-1 construct containing a deletion of the Cys-rich region (d1- cells) was undetectable. HAV-infected cr5 and mut2 cells but not mut1, mut3, d1-, and DR2 cells developed the characteristic cytoplasmic granular fluorescence of HAV-infected cells. These results indicate that the Cys-rich region of havcr-1 and its first N-glycosylation site are required for binding of protective MAb 190/4 and HAV receptor function.

Animals↗

PubMed1998

Comparative sequence analysis of a gene-rich cluster at human chromosome 12p13 and its syntenic region in mouse chromosome 6.

The Human Genome Project has created a formidable challenge: the extraction of biological information from extensive amounts of raw sequence. With the increasing availability of genomic sequence from other species, one approach to extracting coding and regulatory element information is through cross-species sequence comparison. To assess the strengths and weaknesses of this methodology for large-scale sequence analysis, 227 kb of mouse sequence syntenic to a gene-rich cluster on human chromosome 12p13 was obtained. Primarily through percent identity plots (PIPs) of SIM comparative sequence alignments, the sequence of coding regions, putative alternative exons, conserved noncoding regions, and correlation in repetitive element insertions were easily determined. The analysis demonstrated that the number, order, and orientation of all 17 genes are conserved between the two species, whereas two human pseudogenes are absent in mouse. In addition, apart from MIRs, no direct correlation of distribution or position of the majority of repetitive elements between the two species is seen. Finally, in examining the synonymous and nonsynonymous substitution rates in the conserved genes, a large variation in nonsynonymous rates is observed indicating that the genes in this region are diverging at different rates. This study indicates the utility and strength of large-scale cross-species sequence comparisons in the extraction of biological information from raw sequence, especially when combined with other computational tools such as GRAIL and BLAST.

Amino Acid Sequence↗

PubMed1998

Anatomic considerations of superior cluneal nerve at posterior iliac crest region.

No previous studies describe the anatomic relationship of the superior cluneal nerve to the posterior iliac crest and thoracolumbar fascia. In the current study, 15 cadavers were dissected to determine the relationship of the superior cluneal nerve to the posterior iliac crest and thoracolumbar fascia. The distances from the medial branch of the superior cluneal nerve to the posterior superior iliac crest and the midline were 64.7 +/- 5.3 mm and 81.0 +/- 9.2 mm, respectively. The distances between the level of the iliac crest and perforating points of the superior cluneal nerve on the thoracolumbar fascia were 5.8 +/- 1.8 mm inferiorly for the medial branch, 2.2 +/- 1.8 mm superiorly for the intermediate branch, and 12.0 +/- 4.4 mm superiorly for the lateral branch, respectively. The proximal dissection above the perforating point of the nerve showed that the medial branch of the superior cluneal nerve is confined within a tunnel consisting of the thoracolumbar fascia and the superior rim of the iliac crest as it passes over the iliac crest. The intermediate and lateral branches of the superior cluneal nerve either pierce the thoracolumbar fascia or pass through an orifice or fissure in the thoracolumbar fascia. In two specimens, the medial branches of the superior cluneal nerve were constricted within the osteofibrous tunnel. The nerve was entrapped between the rigid fibers of the thoracolumbar fascia and the iliac crest.

Aged↗

PubMed1997

Specific interaction of the PDZ domain protein PICK1 with the COOH terminus of protein kinase C-alpha.

PICK1 is a protein kinase C (PKC) alpha-binding protein initially identified using the yeast two-hybrid system. Here we report that PICK1 contains a PDZ domain that interacts specifically with a previously unidentified PDZ-binding domain (QSAV) at the extreme COOH terminus of PKCalpha and that mutation of a putative carboxylate-binding loop within the PICK1 PDZ domain abolishes this interaction. The PDZ-binding domain in PKCalpha is absent from other PKC isoforms that do not interact with PICK1. We also demonstrate that PICK1 can homooligomerize through sequences that are distinct from the carboxylate-binding loop, suggesting that self-association and PKCalpha binding are not mutually exclusive. A Caenorhabditis elegans PICK1-like protein is also able to bind to PKCalpha, suggesting a conservation of function through evolution. Association of PKCalpha with PICK1 provides a potential mechanism for the selective targeting of PKCalpha to unique subcellular sites.

Amino Acid Sequence↗

PubMed1997

Vulnerability of the recurrent laryngeal nerve in the anterior approach to the lower cervical spine.

STUDY DESIGN: To perform anatomic dissections and measurements of the recurrent laryngeal nerve between the inferior thyroid artery and superior border of the clavicle (mid-portion) on both sides. OBJECTIVES: To determine quantitatively the differences in course and location between the recurrent laryngeal nerves on both sides and to relate this to the vulnerability of the recurrent laryngeal nerve during an anterior approach to the lower cervical spine. SUMMARY OF BACKGROUND DATA: The midportion of the recurrent laryngeal nerve is usually encountered in the anterior approach to the lower cervical spine, especially on the right side. No quantitative regional anatomy describing the course and location of the mid-portion of the recurrent laryngeal nerve is available in the literature. METHODS: Fifteen adult cadavers were used for dissections of the recurrent laryngeal nerve. The length of the recurrent laryngeal nerve between the superior border of the clavicle and the inferior thyroid artery, and the angle of the recurrent laryngeal nerve with respect to sagittal plane, were measured bilaterally. In addition, six cross-sections at C7 were obtained to determine the linear distances between esophagotracheal groove and the recurrent laryngeal nerve. RESULTS: The recurrent laryngeal nerve on the right runs in a superior and medial direction, with an angle of 25.0 degrees +/- 4.7 degrees relative to sagittal plane, compared with 4.7 degrees +/- 3.7 degrees on the left. The length of the recurrent laryngeal nerve between the superior border of the clavicle and the inferior thyroid artery is 23.0 +/- 4.4 mm on the left, and 22.8 +/- 4.3 mm on the right. The recurrent laryngeal nerve lies deep within the esophagotracheal groove on the left, but 6.5 +/- 1.2 mm anterior and 7.3 +/- 0.8 mm lateral to the esophagotracheal groove on the right. CONCLUSIONS: The recurrent laryngeal nerve on the right side is highly vulnerable to injury if ligature of the inferior thyroid vessels is not performed as laterally as possible or if retraction of the midline structures along with the recurrent laryngeal nerve is not performed intermittently. Avoiding injury to the recurrent laryngeal nerve, especially on the right side, is a major consideration during an anterior approach to lower cervical spine.

Aged↗

PubMed1997

Determination of capsaicin and zucapsaicin in human serum by high-performance liquid chromatography with fluorescence detection.

A reversed-phase high-performance liquid chromatographic (HPLC) assay was developed to analyze capsaicin and zucapsaicin (civamide) in human serum at concentrations from 1 to 100 ng/ml. Human serum specimens were extracted twice with hexane-methyl tert.-butyl ether (1:1). The chromatographic separation was carried out on a C18 column at 40 degrees C using a mobile phase consisting of 40% acetonitrile in water with 5% tetrahydrofuran and 1% acetic acid. The concentration of the eluting compounds was monitored by a fluorescence detector with excitation at 270 nm and an emission cutoff of 300 nm. No interferences were observed from the extract of blank serum. The standard curves were linear in the detection range. The relative standard deviation of the assay was better than 8.4%. The limit of detection was 0.5 ng/ml.

Analgesics, Non-Narcotic↗

PubMed1997

Regulation of C-C chemokine production by murine T cells by CD28/B7 costimulation.

C-C chemokines play an important role in recruitment of T lymphocytes to inflammatory sites. T lymphocytes secrete chemokines, but the activation requirements for chemokine production by T cells are uncertain. We studied the regulation of C-C chemokine production by CD28 costimulatory signals by murine T lymphocytes. Splenocytes from BALB/c mice cultured with anti-CD3 mAb expressed macrophage-inflammatory protein (MIP)-1alpha mRNA and secreted MIP-1alpha, which was inhibited by anti-B7-1 plus anti-B7-2 mAbs. MIP-1alpha production by Ag-stimulated T cells from DO.11.10 TCR transgenic mice was augmented by anti-CD28 mAb and increased compared with DO.11.10/CD28(-/-) cells. When T cell costimulation was provided by IL-2, MIP-1alpha was not enhanced. Studies with IL-2, IL-4, STAT4, and STAT6 knock-out mice suggested that chemokine production is controlled by pathways different from those regulating T cell differentiation. Thus, CD28 costimulation may amplify an immune response by stimulating T cell survival, proliferation, and production of chemokines that recruit T cells to inflammatory sites.

Animals↗

PubMed1997

Identification of positively charged residues contributing to the stability of plasminogen activator inhibitor 1.

Plasminogen activator inhibitor 1 (PAI-1), a member of the serpins, has a unique conformational flexibility. A typical characteristic is its intrinsic lability resulting in the conversion of the active conformation to a latent conformation. In the present study, we have evaluated the effect of substitution of positively charged residues located at the turn connecting strand s4C with strand s3C, either with negatively charged or with neutral residues, on the functional stability of PAI-1. The following mutants were constructed, purified and characterized in comparison to wild-type (wt) PAI-1: PAI-1-R186E,R187E (Arg186--> Glu and Arg187--> Glu), PAI-1-H190E,K191E (His190--> Glu and Lys191--> Glu) and PAI-1-H190L,K191L (His190--> Leu and Lys191--> Leu). In contrast to wtPAI-1 the mutants exhibited no inhibitory activity. Whereas latent wtPAI-1 can be reactivated (up to a specific activity of 78+/-19%) by treatment with guanidinium chloride, a similar treatment applied to these mutants resulted in a significant but relatively small increase of specific activity (i.e. to 14%). Evaluation of the functional stability (at 37 degrees C, pH 5.5, 1 M NaCl revealed a strongly decreased functional stability compared to wtPAI-1 (i.e. 3-9 h for the mutants vs. > 24 h for wtPAI-1). Further characterization by heat denaturation studies and plasmin susceptibility confirmed that removal or reversal of the positive charge on the turn connecting s4C with s3C results in PAI-1 mutants with a highly accelerated conversion of active to latent forms. We can therefore conclude that the pronounced positive charge in the turn connecting s4C with s3C is of the highest importance for the functional stability of PAI-1.

Electrophoresis, Polyacrylamide Gel↗

PubMed1997

Nucleotide sequences and gene organization of TaqI endonuclease isoschizomers from Thermus sp. SM32 and Thermus filiformis Tok6A1.

Eight TaqI isoschizomer genes, two from Yellowstone National Park, one from Japan, two from New Zealand, two from Portugal, and one from the Azores (1000 miles west of Portugal), were PCR-amplified and sequenced. Sequence alignment of isoschizomers isolated from close geographical locations shows identical or almost identical protein sequences, while isoschizomers from distant sites demonstrate considerable diversity, ranging from 54 to 75% in amino acid identity. Accordingly, these isoschizomers were arranged into four geographical groups, i.e., USA as represented by Thermus aquaticus YT1, Japan by Thermus thermophilus HB8, New Zealand by Thermus filiformis Tok6A1, Portugal by Thermus sp. SM32. The complete ORFs of two new representative genes, tfiTok6A1I and tsp32IR, were obtained by bubble PCR. Unlike M . TaqI-R.TaqI and M . TthHB8I-R . TthHB8I which exhibit an unusual 13-codon overlap, the methylase and endonuclease genes are each separated by 15 nucleotides in the TfiTok6A1I and Tsp32IR restriction-modification systems. Phylogenetic analysis suggests that initially TfiTok6A1I diverged from a common ancestor, then Tsp32IR branched out, and finally TaqI and TthHB8I diverged from each other during evolution.

Amino Acid Sequence↗