The role of hemolymph coagulation in innate immunity.

Invertebrate animals, which lack adaptive immune systems, have developed defense systems that respond to common antigens on the surface of potential pathogens. Hemolymph coagulation is one such defense system in innate immunity. The discovery of lipopolysaccharide-sensitive and (1-->3)-beta-D-glucan-sensitive serine protease zymogens in horseshoe crab (limulus) hemocytes, both of which trigger the coagulation cascade, has exemplified how the animals detect and respond to foreign materials.

Mitochondrial DNA replication in human T lymphocytes is regulated primarily at the H-strand termination site.

The most unique feature in the replication of mitochondrial DNA (mtDNA) is that most of the newly synthesized heavy strands (H-strands) terminate prematurely, resulting in the formation of displacement loop (D-loop) strands. Only the H-strand which proceeds past the termination site is a true nascent H-strand leading to the overall replication on a circular mtDNA molecule. The physiological significance of the D-loop formation has long been unclear. To examine the role of premature termination in mtDNA replication, we therefore developed a method for selectively measuring both the total amount of nascent H-strands and the amount of true nascent H-strands using ligation-mediated polymerase chain reaction, which, for the first time, enabled us to estimate the frequency of premature termination. The stimulation of cell proliferation with interleukin 2 and phytohemagglutinin in human peripheral T lymphocytes caused an increase in the net replication rate of mtDNA. In stimulated cells, in comparison to resting ones, the amount of true nascent H-strands increased approx. 2.6-fold while the total amount of nascent H-strands remained unchanged, indicating that premature termination decreased while the initiation of replication remained the same. Our findings thus demonstrate the first clear example that premature termination plays a primary role in the up-regulation of the net rate of mtDNA replication in human cells.

Molecular and functional organization of yeast plasmid pSR1.

The nucleotide sequence of a 6251 base-pair plasmid, pSR1, harbored in an osmophilic haploid yeast, Zygosaccharomyces rouxii (formerly Saccharomyces rouxii), was determined. No homology was detected between the sequences of pSR1 and 2-micron DNA of Saccharomyces cerevisiae. pSR1 has a pair of inverted repeats consisting of completely homologous 959 base-pair sequences, which separate two unique sequences 2654 base-pairs and 1679 base-pairs long. Each inverted repeat has an ARS sequence functional in both Z. rouxii and S. cerevisiae hosts. Short direct repeats or dyad symmetries were observed in the inverted repeats similar to those found close to the replication origin of 2-micron DNA. Three open reading frames, P, S and R, each able to encode a protein of molecular weight larger than 10,000, were found. Insertional inactivation of R gave rise to a defect in the intramolecular recombination at the inverted repeats, and that of S reduced the copy number of pSR1 in the S. cerevisiae host. The maintenance stability of the plasmid was also tested in the heterogeneous S. cerevisiae host, but the results of the insertional inactivation of P, S and R were ambiguous. pSR1 and 2-micron DNA were compatible in S. cerevisiae cells, but the protein factors encoded by these plasmids did not complement each other.

Telomerase activity in pancreatic juice differentiates ductal carcinoma from adenoma and pancreatitis.

Telomerase activity was measured in pancreatic juice obtained by endoscopic retrograde pancreatography from 34 patients (12 with ductal carcinoma, 12 with pancreatic adenoma, and 10 with pancreatitis). The activity in pancreatic juice was expressed as the number of cells of a human pancreatic cancer cell line, MIA PaCa-2, that exhibit an activity equal to that expressed in 1 microg of protein from pancreatic juice. A telomerase ladder was detected in the pancreatic juice obtained from a majority of the patients with ductal adenocarcinoma. The median value of relative telomerase activity in the carcinoma samples was 9.38 (25th percentile, 3.14; 75th percentile, 95.8), a value significantly higher than that derived from patients with either pancreatitis or pancreatic adenoma (P < 0.0001). When a threshold value of relative telomerase activity of 3.00 was used, 75% (9 of 12) of the samples obtained from patients with ductal carcinoma were positive. We conclude that telomerase activity in pancreatic juice differentiates adenocarcinoma from adenoma and pancreatitis and may serve as a useful diagnostic tool.

Telomerase elevation in pancreatic ductal carcinoma compared to nonmalignant pathological states.

Telomerase activity was measured in surgically resected tissues of 20 human pancreatic ductal carcinomas, 12 adenomas, 5 pancreatitis tissues, 14 normal pancreatic ducts, and 13 normal pancreatic tissues (primarily made up of acinar cells) using a PCR-based telomerase assay. Relative telomerase activity was expressed as the equivalent telomerase intensity of the number of cells of a human pancreatic cancer cell line, MIA PaCa-2, per microgram of protein in the tissue samples. The median value (25th percentile, 75th percentile) of relative telomerase activity in pancreatic carcinomas was 13.2 (3.58, 244), which was significantly higher relative to normal tissues, normal ducts, pancreatitis tissues, and adenomas (P < 0.0001). When the cutoff value of relative telomerase activity was set at 1.00 and 3.00, the positivity rates of telomerase activity in pancreatic ductal carcinomas were 100 and 80%, respectively. Some of the adenoma samples displayed a weak telomerase ladder. However, when semiquantitatively analyzed, the relative telomerase activity of all adenoma tissues was less than 1.00 equivalent cells per microgram protein of the tissues, which was equivalent to the values encountered in normal ducts. Thus, our results indicate that reactivation of telomerase may occur at a late stage of pancreatic ductal carcinogenesis. Therefore, telomerase may be a specific marker for distinguishing pancreatic cancer from pancreatitis and adenomas.

Clotting and immune defense in Limulidae.

The blue blood of the horseshoe crab contains a sophisticated defense system very sensitive to pathogens or foreign materials. The hemocytes circulating in the hemolymph detect trace amounts of LPS molecules on the invading microorganisms and respond quickly to release the granular components into the external milieu. The coagulation system composed of three serine protease zymogens, factor C, factor B, and proclotting enzyme, and a clottable protein, coagulogen, is activated by LPS to form insoluble coagulin gel. The coagulation system also responds to beta-(1,3) glucan through the activation of unique heterodimeric serine protease zymogen, factor G. The pathogens are, thus, engulfed in the gel and subsequently killed by antimicrobial substances with various specificities, which are also released from cells. The horseshoe crab has developed two kinds of serine protease zymogens as biological sensors, factor C and factor G, which are responsive to LPS and beta-(1,3) glucan on the surface of Gram-negative bacteria and fungi, respectively. These are possible invaders for horseshoe crabs and also for most animals including humans. This novel heterodimeric serine protease zymogen, factor G, may open a new way to develop an innovative assay system to quantitate beta-(1,3) glucans. Furthermore, these LPS and beta-(1,3) glucan sensitive factors could be utilized as a unique tool to analyze other biological reactions caused by LPS or the glucan. Although the coagulation reaction in horseshoe crabs is famous, it is not the only defense mechanism of this animal. Many agglutinins are present either in hemolymph plasma or in the cell. The hemolymph plasma also has cytolytic activity against foreign cells. These cellular and humoral defense systems, in concert, defend themselves from invading foreign organisms. Such a sophisticated defense system has allowed the horseshoe crab to survive for more than 200 million years on the earth. Horseshoe crabs are often called ¿living fossils." However, they are not fossils. They are living.

A horseshoe crab receptor structurally related to Drosophila Toll.

Innate immunity against microbial pathogens relies on the pattern recognition of cell wall components on invading microbes. Recent evidence has shown that a mammalian Toll-like receptor (TLR) is activated by bacterial lipopolysaccharides (LPS). The innate immunity in invertebrates is also triggered by LPS, as seen in the hemolymph coagulation in horseshoe crab. We report the cloning of a TLR from the Japanese horseshoe crab Tachypleus tridentatus. A cDNA coding for Tachypleus Toll was isolated from a hemocyte cDNA library and the open reading frame codes for a proprotein including a signal sequence. Like Drosophila Toll, Tachypleus Toll is a type I transmembrane protein with an extracellular domain consisting of two leucine-rich repeats flanked by two cystein-rich clusters and a cytoplasmic domain exhibiting striking similarity with the cytoplasmic domain of interleukin-1 receptor. Tachypleus Toll is most similar to Drosophila Toll in the domain architecture and the overall length.

TAK1 mediates an activation signal from toll-like receptor(s) to nuclear factor-kappaB in lipopolysaccharide-stimulated macrophages.

Stimulation of monocytes/macrophages with lipopolysaccharide (LPS) results in activation of nuclear factor-kappaB (NF-kappaB), which plays crucial roles in regulating expression of many genes involved in the subsequent inflammatory responses. Here, we investigated roles of transforming growth factor-beta activated kinase 1 (TGF-TAK1), a mitogen-activated protein kinase kinase kinase (MAPKKK), in the LPS-induced signaling cascade. A kinase-negative mutant of TAK1 inhibited the LPS-induced NF-kappaB activation both in a macrophage-like cell line, RAW 264.7, and in human embryonic kidney 293 cells expressing toll-like receptor 2 or 4. Furthermore, we demonstrated that endogenous TAK1 is phosphorylated upon simulation of RAW 264.7 cells with LPS. These results indicate that TAK1 functions as a critical mediator in the LPS-induced signaling pathway.

cDNA and deduced amino acid sequence of human PK-120, a plasma kallikrein-sensitive glycoprotein.

PK-120 is a substrate for plasma kallikrein (PK), recently purified from human plasma. Here we have established the cDNA sequence for human PK-120 mRNA. The deduced amino sequence of PK-120 revealed that it consists of 902 amino acid residues with a calculated mass of 116,423 Da. The putative cleavage sites by PK have been proposed, suggesting that PK-120 may be a precursor of a bioactive peptide. Most interestingly, PK-120 showed significant sequence identities to heavy chains (HCs) of the inter-alpha-trypsin inhibitor (ITI) superfamily.

New types of clotting factors and defense molecules found in horseshoe crab hemolymph: their structures and functions.

Invertebrate animals, which lack adaptive immune systems, have developed defense systems, so-called innate immunity, that respond to common antigens on the surface of potential pathogens. One such defense system is involved in the cellular responses of horseshoe crab hemocytes to invaders. Hemocytes contain two types, large (L) and small (S), of secretory granules, and the contents of these granules are released in response to invading microbes via exocytosis. Recent biochemical and immunological studies on the granular components of L- and S-granules demonstrated that the two types of granules selectively store granule-specific proteins participating in the host defense systems. L-Granules contain all the clotting factors essential for hemolymph coagulation, protease inhibitors including serpins and cystatin, and anti-lipopolysaccharide (LPS) factor and several tachylectins with LPS binding and bacterial agglutinating activities. On the other hand, S-granules contain various new cysteine-rich basic proteins with antimicrobial or bacterial agglutinating activities, such as tachyplesins, big defensin, tachycitin, and tachystatins. The co-localization of these proteins in the granules and their release into the hemolymph suggest that they serve synergistically to construct an effective host defense system against invaders. Here, the structures and functions of these new types of defense molecules found in the Japanese horseshoe crab (Tachypleus tridentatus) are reviewed.

Tachyplesins isolated from hemocytes of Southeast Asian horseshoe crabs (Carcinoscorpius rotundicauda and Tachypleus gigas): identification of a new tachyplesin, tachyplesin III, and a processing intermediate of its precursor.

Tachyplesins and their analogs are antimicrobial peptides composed of 17 or 18 amino acid residues present abundantly in acid extracts of hemocyte debris of horseshoe crabs. We purified here tachyplesin isopeptides from hemocytes of two species of Southeast Asian horseshoe crabs, Carcinoscorpius rotundicauda and Tachypleus gigas, and determined their amino acid sequences. The major tachyplesin isolated from both species was identified, respectively, as tachyplesin I, which had previously been found in hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus). The yield from both species was very high (more than 70 mg per 100 g wet weight of hemocytes), i.e., comparable with that from T. tridentatus. In addition to tachyplesin I, a new tachyplesin isopeptide, named tachyplesin III, was also isolated from T. gigas hemocytes, in which an arginine replaced the 15th lysine of tachyplesin I. The carboxyl-terminal residue of the isolated tachyplesins I and III was confirmed, respectively, to be an arginine alpha-amide by chemical analysis. Furthermore, a tachyplesin peptide derivative with a carboxyl-terminal extension of glycine-lysine was newly found in the hemocytes of C. rotundicauda. It appeared to be an intermediate derived from a tachyplesin precursor during processing to the mature form.

Primary structure of anti-lipopolysaccharide factor from American horseshoe crab, Limulus polyphemus.

The complete amino acid sequence of anti-lipopolysaccharide (LPS) factor purified from the hemocytes lysate of the American horseshoe crab, Limulus polyphemus, was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, clostripain, and Staphylococcus aureus V8 protease. Upon sequencing the peptides by the automated Edman method, the following primary structure was obtained: (Sequence: in text). During the sequence analysis, two species of the protein, which differed from each other at one locus, were found and characterized. L. polyphemus anti-LPS factor was a basic protein consisting of a single polypeptide chain of 101 residues and a calculated molecular weight of 11,786 or 11,800. The hydrophobic NH2-terminal sequence and the clustering of positive charges found in the disulfide loop yielded a typical amphipathic character of this protein. Moreover, L. polyphemus anti-LPS factor showed 83% sequence identity with the Tachypleus tridentatus protein, and the sequence similar to that observed in the EF-hand structure was found to contain in the COOH terminal portions of these proteins, although its function is unknown.

Separation of large and small granules from horseshoe crab (Tachypleus tridentatus) hemocytes and characterization of their components.

We designed a method for separating two types of granules, a smaller (S) but dense and a larger (L) but less dense granule from hemocytes of the horseshoe crab (Tachypleus tridentatus), using continuous sucrose density gradient centrifugation. The isolated L-granules contained at least three clotting factors plus a clottable protein, coagulogen, as the major component. The known anti-lipopolysaccharide factor and 7 additional unknown protein components were also present in the L-granules. Two known natural substrates, Pro-rich protein and 8.6 kDa protein, for limulus transglutaminase [Tokunaga, F., Yamada, M., Miyata, T., Ding, Y.-L., Hiranaga-Kawabata, M., Muta, T., Iwanaga, S., Ichinose, A., & Davie, E.W. (1993) J. Biol. Chem. 268, 252-261] were present in the L-granules. On the other hand, the isolated S-granules contained antimicrobial tachyplesins I and II (17 amino acids in length) as the major component, in addition to 6 unidentified proteins with molecular masses of less than 30 kDa. The structural analyses of tachyplesin analogs indicated that all these peptides of mature form are stored in the S-granules, together with a processing intermediate containing the COOH-terminal Gly-Lys sequence. We also found an Arg-rich protein of 22 kDa and a Leu-rich protein of 30 kDa in S-granules. Based on these observations, we speculate that protein components in L-granules, which probably contain all the factors essential for the limulus clotting system, participate in immobilization of invading microbes and that factors in the S-granules containing tachyplesins contribute to a self-defense system against invaders.

Role of hemocyte-derived granular components in invertebrate defense.

Figure 2 illustrates an outline of the cellular and humoral defense systems in limulus. On the basis of the knowledge described above, it is suggested that granular components present in L and S granules in the hemocytes play a decisive role in the biological defense for this animal. The isolated L granules contain at least three clotting factors plus coagulogen as the major component. The known anti-LPS factor and a number of additional unknown protein components are also present in the L granules. On the other hand, the isolated S granules contain antimicrobial tachyplesins as the major component, in addition to six unidentified proteins. We speculate that the L-granule-derived protein components, which probably contain all the factors essential for the Limulus clotting system participate, in immobilizing invading microbes, and that the S-granule-derived tachyplesins contribute to a self-defense system against invaders. Although we have not mentioned hemolymph plasma components, there are many humoral factors, such as proteinase inhibitors, alpha 2-macroglobulin, various lectins, C-reactive protein, and polyphemin, all of which are important for antimicrobial defense. Furthermore, Liu and colleagues have reported several endotoxin-binding proteins and a cell-adhesion protein found in the Limulus hemocytes. Although the exact functions of these substances are unknown, they may act in concert with other components to provide biological defense for the animal. Nevertheless, compared to our knowledge of mammalian blood cells, much less remains to be learned of biological/physiological events in horseshoe crab hemocytes.

Erythroblastic transformation of Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia associated with marked chromosomal rearrangements.

In a 22-year-old female with Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) a tumor consisting of megaloblastic proerythroblasts appeared in the right ilio-femoral region 2 years and 8 months after the diagnosis of the disease and was treated effectively with irradiation. She developed erythroblastic transformation 3 months after the tumor appeared. Cytogenetic study of the bone marrow cells in the acute phase revealed marked chromosomal rearrangements such as ring, dicentric, or tricentric chromosomes.

Chlorpropamide alters AVP-receptor binding of rat renal tubular membranes.

The effects of chlorpropamide on AVP-receptor binding in rat renal tubular basolateral membranes were investigated utilizing [3H][Arg8]vasopressin (AVP). Our data indicate that chlorpropamide alters AVP-receptor binding in a competitive manner.

Molecular mechanism of hemolymph clotting system in Limulus.

Limulus (horseshoe crab) hemolymph is known to be very sensitive to bacterial endotoxin (LPS), which causes a rapid coagulation response. Hemolymph contains a single type of hemocyte that undergoes aggregation, adhesion, and degranulation in response to LPS. The granule contents are released into the hemolymph, where they form an insoluble gel. We have characterized four components involved in this coagulation response that comprise a cascade of three serine protease zymogens (factor C, factor B, and proclotting enzyme) and one clottable protein (coagulogen). Of these components, factor C sensitive to LPS is a protein composed of five complement-related domains ("Sushi" or SCR), an EGF-like domain, and a C-type lectinlike domain as well as a putative amino-terminal LPS-binding domain. This domain structure is very similar to that of selectin family of cell adhesion molecules, suggesting that it might also function as a cell adhesion molecule after the release into the hemolymph. Factor B and the proclotting enzyme share a common Cys-rich motif ("cliplike" domain) in the amino-terminal portions. This domain is also found in a putative serine protease zymogen ("easter") in Drosophila, which is essential for normal embryonic development. All four of the components of the cascade and an antibacterial protein (anti-LPS factor) are localized to a specific type of the hemocyte granule. Another antibacterial peptide (tachyplesins I and II) is localized in a distinct granule population. The contents of both granule populations are released into the hemolymph in response to LPS, where they cooperate in immobilization and killing of Gram-negative bacteria.

Semi-quantitative analysis of telomerase in pancreatic ductal adenocarcinoma.

Using a polymerase chain reaction-based amplification assay, we measured telomerase activity in surgically resected pancreatic ductal carcinomas (n = 16 cases) and normal ducts (n = 6), comparing findings with the telomerase activity of a human pancreatic cancer cell line, MIA PaCa-2, as a standard, i.e., relative telomerase activity was determined. Telomerase activity was expressed as the equivalent telomerase intensity of the number of cells of MIA PaCa-2 per microgram protein of tissue samples. The median value for telomerase activity in normal pancreatic ducts was 0.13 and the 25th and 75th percentile were 0.01 and 0.76. The median value for telomerase activity in pancreatic ductal adenocarcinoma was 34.7 (25th percentile, 4.98; and 75th percentile, 296), significantly higher than that of normal ducts (P < 0.001). When the cut-off value was set at 1.0 and 3.0, the telomerase positivity rate of pancreatic ductal adenocarcinomas was 100% and 81.3%, respectively. Telomerase may be specific marker for pancreatic ductal carcinomas.

Xanthogranulomatous pyelonephritis.

A 76-year-old woman presented with spiking fever and right back pain. Ultrasonography (US) revealed that her right kidney was enlarged. Computed tomography (CT) showed the parenchyma was replaced by non-enhancing masses but the overall kidney shape was maintained. These findings were compatible with those of xanthogranulomatous pyelonephritis (XP). The nonenhancing masses on CT coincided with the multiple butter yellow nodules of the resected kidney and microscopically these lesions were proved to be abscesses with xanthoma cells. In this case, the US and CT findings reflected the pathological feature of XP and thus these techniques are thought to be useful for the diagnosis of XP.

[Effects of analogues and drugs on arginine vasopressin receptor binding in rat renal tubular basolateral membrane].

The methods of basolateral membrane isolation from rat kidney and 3H-AVP receptor assay using this basolateral membrane preparation were established. Then, the effects of analogues and drugs on AVP-receptor binding were studied. Specific 3H-AVP binding was inhibited by LVP, dDAVP and oxytocin in that order. Among the various agonistic or antagonistic drugs to AVP (fluoride, cyclophosphamide, mechlorethamine, chlorpropamide), only chlorpropamide inhibited 3H-AVP binding to the membrane. The Kd value calculated by Scatchard analysis was 1.30 +/- 0.28 nM (n = 4, M +/- SD), and it was increased to 2.69 +/- 0.32 nM (n = 5, M +/- SD) by adding 1 mM of chlorpropamide, while Bmax was unchanged. Our data show that 1 mM chlorpropamide decreases receptor affinity for AVP, and alters AVP-receptor binding in a competitive manner.