Prognostic value of tumor-infiltrating lymphocytes differs depending on histological type and smoking habit in completely resected non-small-cell lung cancer.

BACKGROUND: T-cell infiltration in tumors has been used as a prognostic tool in non-small-cell lung cancer (NSCLC). However, the influence of smoking habit and histological type on tumor-infiltrating lymphocytes (TILs) in NSCLC remains unclear. PATIENTS AND METHODS: We evaluated the prognostic significance of TILs (CD4+, CD8+, CD20+, and FOXP3+) according to histological type and smoking habit using automatic immunohistochemical staining and cell counting in 218 patients with NSCLC. RESULTS: In multivariate survival analyses of clinical, pathological, and immunological factors, a high ratio of FOXP3+ to CD4+ T cells (FOXP3/CD4) [hazard ratio (HR): 4.46, P < 0.01 for overall survival (OS); HR: 1.96, P < 0.05 for recurrence-free survival (RFS)] and a low accumulation of CD20+ B cells (HR: 2.45, P = 0.09 for OS; HR: 2.86, P < 0.01 for RFS) were identified as worse prognostic factors in patients with adenocarcinoma (AD). In non-AD, a low number of CD8+ T cells were correlated with an unfavorable outcome (HR: 7.69, P < 0.01 for OS; HR: 3.57, P < 0.02 for RFS). Regarding smoking habit in AD, a high FOXP3/CD4 ratio was poorly prognostic with a smoking history (HR: 5.21, P < 0.01 for OS; HR: 2.38, P < 0.03 for RFS), whereas a low accumulation of CD20+ B cells (HR: 4.54, P = 0.03 for OS; HR: 2.94, P < 0.01 for RFS) was confirmed as an unfavorable factor in non-smokers with AD. CONCLUSIONS: A low number of CD8+ T cells in non-AD, a high FOXP3/CD4 ratio in smokers with AD, and a low number of CD20+ B cells in non-smokers with AD were identified as independent unfavorable prognostic factors in resected NSCLC. Evaluating the influence of histological type and smoking habit on the immunological environment may lead to the establishment of immunological diagnosis and appropriate individualized immunotherapy for NSCLC.

Prolyl hydroxylase regulates axonal rewiring and motor recovery after traumatic brain injury.

Prolyl 4-hydroxylases (PHDs; PHD1, PHD2, and PHD3) are a component of cellular oxygen sensors that regulate the adaptive response depending on the oxygen concentration stabilized by hypoxia/stress-regulated genes transcription. In normoxic condition, PHD2 is required to stabilize hypoxia inducible factors. Silencing of PHD2 leads to the activation of intracellular signaling including RhoA and Rho-associated protein kinase (ROCK), which are key regulators of neurite growth. In this study, we determined that genetic or pharmacological inhibition of PHD2 in cultured cortical neurons prevents neurite elongation through a ROCK-dependent mechanism. We then explored the role of PHDs in axonal reorganization following a traumatic brain injury in adult mice. Unilateral destruction of motor cortex resulted in behavioral deficits due to disruption of the corticospinal tract (CST), a part of the descending motor pathway. In the spinal cord, sprouting of fibers from the intact side of the CST into the denervated side is thought to contribute to the recovery process following an injury. Intracortical infusion of PHD inhibitors into the intact side of the motor cortex abrogated spontaneous formation of CST collaterals and functional recovery after damage to the sensorimotor cortex. These findings suggest PHDs have an important role in the formation of compensatory axonal networks following an injury and may represent a new molecular target for the central nervous system disorders.

Prostacyclin promotes oligodendrocyte precursor recruitment and remyelination after spinal cord demyelination.

Adult oligodendrocyte precursor cells (OPCs) are located adjacent to demyelinated lesion and contribute to myelin repair. The crucial step in remyelination is the migration of OPCs to the demyelinated area; however, the mechanism of OPC migration remains to be fully elucidated. Here we show that prostacyclin (prostaglandin I2, PGI2) promotes OPC migration, thereby promoting remyelination and functional recovery in mice after demyelination induced by injecting lysophosphatidylcholine (LPC) into the spinal cord. Prostacyclin analogs enhanced OPC migration via a protein kinase A (PKA)-dependent mechanism, and prostacyclin synthase expression was increased in the spinal cord after LPC injection. Notably, pharmacological inhibition of prostacyclin receptor (IP receptor) impaired remyelination and motor recovery, whereas the administration of a prostacyclin analog promoted remyelination and motor recovery after LPC injection. Our results suggest that prostacyclin could be a key molecule for facilitating the migration of OPCs that are essential for repairing demyelinated areas, and it may be useful in treating disorders characterized by demyelination.

Evaluation of out-in skin transparency using a colorimeter and food dye in patients with atopic dermatitis.

BACKGROUND: Atopic dermatitis is a disease of skin barrier dysfunction and outside stimuli can cross the skin barrier. OBJECTIVES: To examine a new method for evaluating the outside to inside skin transparency with a colorimeter and yellow dyes. METHODS: In study 1, a total of 28 volunteer subjects (24 normal and four with atopic dermatitis) participated. After provocation with yellow dye, the skin colour of all the subjects was measured using a colorimeter. The skin transparency index was calculated by the changes of the skin colour to yellow. Other variables of skin function, including transepidermal water loss (TEWL) and stratum corneum hydration, were also measured. In study 2, the skin transparency index was evaluated for a cohort of 38 patients with atopic dermatitis, 27 subjects with dry skin and 29 healthy controls. RESULTS: In study 1, the measurement of skin colour (b*) using tartrazine showed good results. There was a significant relationship between the skin transparency index with tartrazine and the atopic dermatitis score (P = 0.014). No other measurements of skin function, including the TEWL, were correlated. In study 2, the skin transparency index score obtained with tartrazine in the patients with atopic dermatitis was significantly higher than that of the controls and those with dry skin (P < 0.001 and P = 0.022, respectively). However, the TEWL in patients with atopic dermatitis was not significantly higher than that of patients with dry skin and the TEWL in subjects with dry skin was not higher than that of the controls. CONCLUSIONS: This method, which used a colorimeter and food dye, is noninvasive, safe and reliable for the evaluation of out-in skin transparency and can demonstrate the characteristic dysfunction in the skin barrier in patients with atopic dermatitis.

Neonatally born granule cells numerically dominate adult mice dentate gyrus.

Hippocampal granule cells (GCs) are continuously generated in the subgranular zone of the dentate gyrus (DG) and functionally incorporated to dentate neural circuits even in adulthood. This raises a question about the fate of neonatally born GCs in adult DG. Do they exist until adulthood or are they largely superseded by adult-born GCs? To investigate this question, we examined the contributions of postnatally born GCs to the adult mouse DG. C57BL/6 mice were grouped in three different postnatal (P) ages (group 1: P0, group 2: P7, and group 3: P35) and received a daily bromodeoxyuridine (BrdU) injection for three consecutive days (P0/1/2, P7/8/9, and P35/36/37, respectively) to label dividing cells. At 6 months old, hippocampal sections were prepared from the animals and immunostained with anti-BrdU antibody and an antibody against the homeobox prospero-like protein Prox1, a marker of GCs. We defined BrdU- and Prox1-double positive cells as newborn GCs and analyzed their density and distribution in the granule cell layer (gcl), revealing that newborn GCs of each group still existed 6 months after BrdU injections and that the density of GCs born during P0-2 (group 1) was significantly higher compared with the other groups. Although the density of newborn GCs in the each group did not differ between male and female, the radial distribution of them in gcl showed some differences, that is, male newborn GCs localized toward the molecular layer compared with female ones in group 1, while to the hilus in group 2. These results suggest that GCs born in early postnatal days numerically dominate adult DG and that there exist sex differences in GC localizations which depend on the time when they were born.

Immunogenicity of human amniotic membrane in experimental xenotransplantation.

PURPOSE: The immunogenic characterization of amniotic membrane is still unknown. This study was designed to examine the immunogenicity of human amniotic membrane, by using experimental xenotransplantation models. METHODS: Anti-human class I, class II, and Fas ligand monoclonal antibodies were used against cryopreserved amniotic membrane and cell viability tested for cryopreserved amniotic membrane. Amniotic membranes were then transplanted to the limbal area, intracorneal space, and under the kidney capsule. The scores of transparency and neovascularization after transplantation were recorded by slit lamp microscopy. Host cell infiltration was examined by hematoxylin-eosin or immunohistochemical staining. Control grafts were transplanted human cryopreserved skin grafts. RESULTS: Strong class I expression was observed in amniotic epithelium, mesenchymal cells, and fibroblasts in cryopreserved amniotic membrane. Some fibroblast cells unexpectedly expressed class II antigen. Fas ligand-positive cells were also detected in mesenchymal cells of amniotic stroma. Approximately 50% of epithelial cells of amniotic membrane cryopreserved for several months were still viable. In limbal transplantation, although some CD4(+) and CD8(+) T cells surrounded the amniotic graft, the response was mild. In intracorneal transplantation, all grafted amniotic membranes were accepted and clear, without host cell infiltration. In contrast, all skin grafts were rejected within 3 weeks after intracorneal transplantation. In amniotic membrane transplantation under the kidney capsule, extremely few host vessels and cells infiltrated the amniotic membrane; however, more host cells infiltrated the skin tissues under the kidney capsule. CONCLUSIONS: Amniotic membrane seems to be immune-privileged tissue and to contain some immunoregulatory factors, including HLA-G and Fas ligand. The amniotic membrane may be useful to supplement corneal collagen, and it may be applied not only to the ocular surface but also intracorneally.

[An immunopathological study of autoimmune keratitis in nude mice with embryonic rat thymus grafts].

PURPOSE: The purpose of the present study was to examine the keratitis detected in these mice after the xenogeneic thymus gland transplantation. METHODS: The thymus glands were collected from F 344 rat embryos and transplanted under the renal capsules of BALB/c nude mice (rat TG nude mice). Histological and immunohistological examinations and a transfer experiment of keratitis was also conducted. RESULTS: The histological image was characterized by the infiltration of mononuclear cells and neutrophils and the marked angiogenesis. The autoantibodies reacting specifically to corneal epithelial cells and corneal stroma were recognized in the blood. The deposits of immunoglobulins and complements were simultaneously observed in the corneal stroma and around the basement membrane. Corneal lesions of rat TG nude mice were therefore successfully transferred into naive nude mice by host splenic CD 4 positive cells. CONCLUSION: The present study demonstrated that autoimmunity was involved in the development of keratitis in rat TG nude mice and that these mice were the first animal models to develop autoimmune keratitis spontaneously.

GPS computer navigators to shorten EMS response and transport times.

GPS (global positioning satellite system to determine one's position on earth) units have become inexpensive and compact. The purpose of this study is to assess the effectiveness of a GPS enhanced computer street map navigator to improve the ability of EMS drivers in an urban setting to locate their destination and shorten response times. For part I, residential addresses in the city were randomly selected from a telephone directory. Two driver/navigator teams were assigned to drive to the address adhering to speed limits. One team used a standard street map, whereas the other team used a GPS computer navigator. The travel time and distance of the runs were compared. For part II, the computer GPS navigator was placed on an ambulance to supplement their normal methods of navigation to find the address requesting EMS. After the run was completed, EMS providers were interviewed to determine their opinion of whether the GPS navigator was helpful. For part I the results showed that in the 29 initial test runs, comparing the GPS team versus the standard map team, the mean distances traveled were 8.7 versus 9.0 kilometers (not significant) and the mean travel times were 13.5 versus 14.6 minutes (P=.02), respectively. The GPS team arrived faster in 72% runs. For part II the results showed that most EMS providers surveyed noted that the GPS computer navigator enhanced their ability to find the destination and all EMS providers acknowledged that it would enhance their ability to find a destination in an area in which they were unfamiliar. These results suggest that a portable GPS computer navigator system is helpful and can enhance the ability of prehospital care providers to locate their destination. Because these units are accurate and inexpensive, GPS computer navigators may be a valuable tool in reducing pre-hospital transport times.

[Preliminary report on the efficacy and safety of brief-pulse ECT in depression].

Because there is no report on the use of brief-pulse devices in Japan, we have examined the efficacy and safety of brief-pulse electroconvulsive therapy (ECT) on four treatment-resistant depressive patients (three males and one female, 55.0 +/- 17.8 years of age). The ethical committee of the National Center of Neurology and Psychiatry approved this study and written informed consent was obtained from all of the patients. ECT was administered bilaterally twice a week using atropine, propofol, and succinylcholine (or vecronium) as anesthetic medications. The Hamilton rating scale for depression scores decreased from 30.5 +/- 11.0 to 14.8 +/- 11.5 following a course of 5-12 treatments. Memory and cognitive functioning, evaluated using the Mini-Mental State Examination, Short-Memory Questionnaire and so on, had not changed a week after the last ECT. These results further support the view that brief-pulse ECT is efficacious and safe for the treatment of depression.

Simultaneous determination of RRR- and SRR-alpha-tocopherols and their quinones in rat plasma and tissues by using chiral high-performance liquid chromatography.

We established a method to simultaneously determine RRR- and SRR-alpha-tocopherol (alpha-Toc) and their quinones in biological samples by chiral-phase high-performance liquid chromatography (HPLC). Alpha-Toc had a shorter retention time than alpha-tocopherylquinones (alpha-TQ), and 2-ambo-alpha-Toc was completely separated into two peaks; the first peak was RRR-alpha-Toc and the second SRR-isomer by chiral HPLC connected Chiralcel OD-H column and Sumichiral OA4100 column. In contrast, of the two peaks of alpha-TQ, the first was the SRR-isomer. We also investigated differences in the distribution of RRR- and SRR-alpha-TQ in rat tissues after oral administration of 2-ambo-alpha-Toc by the above HPLC method. Rats deficient in vitamin E were divided into two groups, control and experimental, and tissues were collected at 3, 6, and 24 h after oral 2-ambo-alpha-Toc administration. The concentrations of RRR- and SRR-alpha-Toc and their quinones in plasma and each tissue were determined. The concentration of SRR-alpha-TQ in plasma and adrenal glands was not significantly different from RRR-alpha-TQ. However, the concentration of SRR-alpha-TQ in liver up to 6 h after oral administration was higher than that of RRR-alpha-TQ, and SRR- and RRR-alpha-TQ levels were similar at 24 h after oral administration. Therefore, we may assume that the formation of alpha-TQ in vivo was not different between RRR- and SRR-isomer and that it was not affected by the presence of alpha-Toc stereoisomers.

[Clinical results and complications of refractive surgery].

PURPOSE: To evaluate the efficacy of photorefractive keratectomy (PRK) with a scanning type excimer laser MEL-60 (AESCLUP-MEDITEC, Co). SUBJECTS AND METHOD: We performed PRK on 102 eyes of 62 myopic patients whose refraction ranged from-3.00 to -12.50 D (mean, -6.47 D) and examined the clinical results of postoperative refraction and complications. RESULTS: At 12, 18, and 24 months after the operation, the mean refraction was -1.57 +/- 1.25D, -1.63 +/- 1.51 D and -1.73 +/- 1.47 D. At 12 months after the operation, 36 eyes (46.2%) were within +/- 0.5 D of intended correction, 61 eyes (78.3%) within +/- 1.0 D, and 76 eyes (97.4%) within +/- 2.0 D. Twenty-four months after the operation, 12 eyes (37.5%) were within +/- 0.5 D, 18 eyes (56.3%) within +/- 1.0 D and 29 eyes (90.6%) within +/- 2.0 D. The complications were as follows: keratitis filamentosa was observed in 10 eyes (10.5%), decrease of contrast sensitivity in 7 eyes (7.4%), subepithelial corneal haze in 4 eyes (1.2%), steroid-induced glaucoma was 2 eyes (2.1%), increase of astigmatism in 2 eyes (2.1%), decrease of best corrected visual acuity in 2 eyes (2.1%), and corneal ulcer in 1 eyes (1.1%). CONCLUSION: PRK with a scanning type excimer laser MEL-60 was effective to reduce refractive error in low and mild myopia, but there were some complications, so that a long, careful follow-up seems necessary.

Sequence analysis of cDNA and genomic DNA, and mRNA expression of the medaka fish homolog of mammalian guanylyl cyclase C.

We isolated the cDNA and genomic DNA encoding a membrane guanylyl cyclase of medaka fish (designated as OlGC6), and determined their complete nucleotide sequences. The open reading frame for OlGC6 cDNA predicted a protein of 1,075 amino acids. Phylogenetic analysis indicated that OlGC6 is a member of the enterotoxin/guanylin receptor family. We also determined the partial genomic structure of the gene of another membrane guanylyl cyclase of medaka fish, OlGC2, which is a member of the natriuretic peptide receptor family. The intron positions relative to the protein-coding sequence are highly conserved in the intracellular domains of OlGC6, OlGC2, mammalian GC-A, and GC-E. Despite their divergent primary structures, some intron positions also seem to be conserved in the extracellular domains of different membrane guanylyl cyclase genes. Northern blot analysis demonstrated that an OlGC6 transcript of 3.9 kb is only present in the intestine, while reverse transcription (RT)-PCR analysis demonstrated that the OlGC6 transcript is present in the kidney, spleen, liver, pancreas, gallbladder, ovary, testis, brain, and eye. RT-PCR also demonstrated that OlGC6 is only expressed zygotically and that transcripts are present from 1 day after fertilization, i.e. long before the intestinal tissues begin to develop.

[5-S-cysteinyldopa as a tumor marker for primary uveal malignant melanoma].

The clinical significance of 5-S-cysteinyldopa (5-S-CD), a major intermediate in melanin synthesis, was evaluated as a potential diagnostic tumor marker for uveal melanoma. Serum concentrations of 5-S-CD in 6 out of 7 patients with uveal melanoma in the absence of extraocular metastases were close to those of controls. In contrast, serum concentrations of 5-S-CD were found to be elevated in 3 patients with systemic metastases of melanoma. In addition, 5-S-CD in intraocular fluids, including both aqueous and vitreous humor, was elevated in patients with uveal melanoma regardless of the presence or absence of systemic metastases. These results suggest that 5-S-CD in the intraocular fluid may serve as a useful biochemical marker for the diagnosis of uveal melanoma.

Antithrombotic effects of NE-6505, a novel anion-binding exosite inhibitor.

NF-6505, a bi-O-Tyr-sulfated decapeptide, which specifically interacts with the anion-binding exosite of the thrombin molecule, was chemically synthesized and assessed for its antithrombotic effects in vitro and in vivo. The IC50 value of this peptide on fibrin-clot formation in vitro was about 0.05 microgram/ml, which indicated a potency similar to that of a recombinant hirudin. NF-6505 caused a 2-fold prolongation of activated partial thromboplastin time when intravenously administered at 1 mg/kg in rats. In a rat venous thrombosis model, a bolus intravenous administration of this peptide dose-dependently inhibited the thrombus formation with an ED50 value of 0.03 mg/kg, a value smaller than that of recombinant hirudin (ED50 = 0.1 mg/kg) or of argatroban (ED50 = 0.2 mg/kg). These results suggest that NF-6505 is a highly potent and safe agent for the clinical treatment of venous thrombosis diseases.

[A patient with epilepsy manifesting reversible memory dysfunction--a neuropsychological, electroencephalographical and radiological study].

A 49-year-old man was admitted to our hospital because of epileptic seizures and memory dysfunction. He had been experiencing seizures several times a day since the age of 43 years. Despite antiepileptic drug therapy (sodium valproate [VPA] and clonazepam [CZP], he suffered from frequent complex partial seizures originating in the temporal lobe, and he had a memory disturbance since age 47. When carbamazepine (CBZ) was substituted for VPA and CZP, the epileptic seizures stopped and the memory disturbance improved. Noninvasive regional cerebral blood flow (rCBF) measurements using 99mTc-HMPAO-SPECT imaging were performed twice. The initial measurements on admission showed overall decreased rCBP that was more prominent in the cerebral cortex than in the subcortical nuclei. A follow-up SPECT examination after clinical improvement revealed a marked overall increase in rCBF, especially in the cerebral cortex. The SPECT findings suggest that the memory disturbance in this patient may have been associated with the overall cerebral blood hypoperfusion. This overall hypoperfusion can be attributed to the frequent complex partial seizures and/or the adverse effect of VPA and CZP. SPECT can provide important information suggesting the pathogenesis of memory disturbance associated with epilepsy.

Biodiscrimination of alpha-tocopherol stereoisomers in humans after oral administration.

We investigated changes in the concentrations of the stereoisomers of alpha-tocopherol in serum and lipoproteins in seven normal, healthy women aged 21-37 y who had received oral administration of natural and synthetic alpha-tocopheryl acetate. This study was conducted in three separate periods of 28 d each; there was a 3-mo washout period between each experimental period. During the first period the subjects were administered a daily dose of 100 mg RRR-alpha-tocopherol/d, whereas in the second and third periods 100 mg all-rac-alpha-tocopheryl acetate/d and 300 mg all-rac-alpha-tocopheryl acetate/d were given, respectively. Blood samples were collected 3 d before each treatment and 1, 3, 7, 14, and 28 d after treatment. alpha-Tocopherol stereoisomer concentrations in serum and lipoproteins (very-low-, low-, and high-density lipoproteins) were determined by the chiral HPLC method. The bioavailability of RRR-alpha-tocopherol was greater than that of all-rac-alpha-tocopheryl acetate. When bioavailability was estimated from the increase in the concentration of RRR- or all-rac-alpha-tocopherol in serum, bioavailability of RRR-alpha-tocopherol administered at 100 mg/d was not different from that of all-rac-alpha-tocopheryl acetate administered at 300 mg/d. 2R-Isomers and small amounts of 2S-isomers were detected in the serum lipoproteins of subjects administered all-rac-alpha-tocopheryl acetate.

Structure-activity studies on C-terminal hirudin peptides containing sulfated tyrosine residues.

To clarify the role of the negative charge of the C-terminal region of hirudin, we chemically synthesized the C-terminal peptide of hirudin variant-1 (HV-1), HV-1-(54-65), and its analogs, [E61Y,E62Y]HV-1-(54-65) and [E62Y]HV-1-(54-65), and then sulfated the Tyr residue(s) in these peptides by both enzymic and chemical methods. Enzymic O-sulfation of Tyr residues in the peptides by use of sulfotransferase isolated from Eubacterium A-44 allowed us to produce four kinds of the sulfated peptide, whose C-terminal sequences were -PEY(SO3H)YLQ, -PYY(SO3H)YLQ, -PYYY(SO3H)LQ and -PYY(SO3H)Y(SO3H)LQ. On the other hand, all Tyr residues in the peptides were successfully sulfated by chemical reaction with N,N'-dicyclohexylcarbodiimide in the presence of sulfuric acid. Based on the analysis of structure-activity relationships of these sulfated peptides for thrombin inhibition, the Tyr62 and Tyr63 bisulfated peptide GDFEEIPEY(SO3H)Y(SO3H)LQ was found to be the most potent inhibitor of thrombin among the products tested. No increase in potency was observed by further substitution of Glu61 with Tyr(SO3H). The inhibitory activity by substitution with Tyr(SO3H) at position 63 was greater than that obtained by the substitution at position 62.

Anticoagulant peptides; synthesis, stability and antithrombin activity of hirudin C-terminal-related peptides and their disulfated analog.

We designed a unique anticoagulant decapeptide, which possesses two O-sulfated tyrosine residues, based on the structure of hirudin's C-terminal functional domain. We first prepared a series of octa-, nona- and decapeptides with no sulfation, Suc-Phe-Glu-Pro-Ile-Pro-Glu-Tyr-Tyr-X-OH [X = bond, Leu or Leu-Gln], by a solution phase method and measured their thrombin times (TT) using human thrombin and rabbit plasma. The shortest octapeptide (3a) showed full antithrombin activity comparable to that of the lead compound hirudin (54-65), and the longest decapeptide (3c) prolonged TT most potently with an IC50 value of 5.8 microM. We consequently converted 3c to a disulfated decapeptide (NF-22) with SO3.pyridine complex and compared its antithrombin activity with that of known hirudin-related peptides: hirugen, MDL28050 and hirulog-1. NF-22 showed potent antithrombin activity with an IC50 value of 0.3 microM, being more potent than hirugen and MDL28050 (IC50 values of 4.0 microM and 1.1 microM, respectively). NF-22 was as potent as hirulog-1. NF-22 showed no change in activity in aqueous solution for 10 d at 60 degrees C, and remained about 90% unchanged in rat plasma on incubation for 24h at 37 degrees C, whereas the corresponding unsulfated peptide (3c) was completely digested under the same condition. NF-22 appears to be one of the most potent and stable peptide anticoagulants among the hirudin analogs.

Improved production of phenomycin by a genetically engineered Escherichia coli.

We established an improved production of an antitumor polypeptide anti biotic, phenomycin (PHM), by using a genetically engineered Escherichia coli. Phenomycin consists of 89 natural amino acids without intramolecular disulfide bridge. PHM gene was synthesized as a fusion gene in which PHM at the C-terminus and the residues 1 approximately 20 of Hirudin variant 1 (HV1) at the N-terminus connected by a factor Xa recognition sequence (Ile-Glu-Gly-Arg). E coli JM 109 transformed with a plasmid containing the synthesized gene expressed a fusion protein and the trypsinization of the fusion protein purified by ultrafiltration and ion-exchange chromatography gave efficiently recombinant PHM at a final yield of 50 mg/liter of culture. This PHM yield was six times higher than that obtained by a natural PHM producing strain of Streptoverticillium baldacci. Recombinant PHM was not distinguishable from natural PHM in all aspects observed.