The detection and isolation of protease activity associated with purified preparations of human chorionic gonadotropin.

The highly purified human CG (hCG) CR series is widely used as a reference material in immunological and biological assays. However these hormone preparations, specifically hCG (CR127), exhibit Arg-specific peptidase activity with synthetic peptide substrates. The putative serine protease-like activity associated with hCG (CR127) was almost completely inhibited by diisopropylfluorophosphate, soybean trypsin inhibitor, N-tosyl-L-phenylalanine chloromethyl ketone and to a lesser extent by N-alpha-p-tosyl-L-lysine chloromethyl ketone and was isolated after hydrophobic interaction and affinity chromatography with soybean trypsin inhibitor, which indicated the presence of exogenous protease contaminants rather than intrinsic peptidase activity. 3H-DFP labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated contaminants revealed two possible serine proteases of apparent mol wts 60,000 and 20,000. The presence of these contaminants had no apparent effect on the receptor binding capability of hCG, however the in vitro biological activity of hCG determined by maximal cAMP production, was decreased after hydrophobic interaction chromatographic purification of the hormone. These observations suggest that the serine protease-like contaminants enhance cAMP production, thereby introducing a significant source of error in biological assays that use hCG (CR127). Further purification of hCG by hydrophobic interaction and affinity chromatography is recommended before its use in bioassays or research.

Purification of human pituitary LH and thyrotrophin by hydrophobic chromatography.

A new preparative procedure is described for the efficient purification of LH and TSH from frozen human pituitary glands. LH and TSH were isolated simultaneously from crude preparations by hydrophobic-interaction chromatography followed by separation and purification by reverse-phase high-performance liquid chromatography in a single step. Highly purified hormones were prepared in good yields (45 mg LH and 20 mg TSH/1000 glands) and with high biological activities; the potencies of purified LH and TSH were 5.8 x NIH-LH-S1 equivalent in an ovarian ascorbic acid depletion assay and 7.1 U/mg against human TSH MRC Research Standard (T1 70/9) in the McKenzie assay respectively. Cross-contamination by other glycoprotein hormones was low.

Fast-atom-bombardment mass spectrometry of sulphated oligosaccharides from ovine lutropin.

The positive- and negative-ion f.a.b.-mass spectra and the fragmentation of sulphated oligosaccharides derived from ovine lutropin are described. Negative-ion f.a.b.-m.s. of methylated derivatives offers a sensitive and rapid method for screening glycans for sulphation, for defining the location of sulphated residues, and for sequencing sulphated branches. Positive-ion f.a.b.-m.s. gives complementary data on non-sulphated branches in both complex and hybrid-type sulphated structures.

Churg-Strauss syndrome associated with pregnancy.

Churg-Strauss syndrome, an uncommon condition, occurs even more rarely in association with pregnancy. One month postpartum, a 25-year-old Japanese woman was hospitalized for dyspnea, fever, diarrhea, and complaints of dysesthesia in the right lower limb and right lumbar and abdominal region. Marked eosinophilia was present. Symptoms of bronchial asthma had developed early in the pregnancy, which concluded with delivery of a healthy baby at 39 weeks of gestation. Churg-Strauss syndrome was diagnosed and prednisolone was administered with a good response leading to remission. Three years after the first pregnancy, low-dose steroid therapy was continued through another pregnancy. The patient delivered another healthy baby at 39 weeks of gestation, this time with no exacerbation of symptoms.

Acute eosinophilic pneumonia following cigarette smoking.

Two cases of acute eosinophilic pneumonia following cigarette smoking are analyzed for characteristic features. The first patient noted dyspnea 14 days after initiation of smoking. The second patient noted dyspnea 12 days after beginning to smoke. Both cases had characteristic features including occurrence at an age younger than 30 years; less than 1 month duration of cigarette smoking before onset of disease; and no identifiable cause of acute eosinophilic pneumonia apart from smoking. We believe that acute eosinophilic pneumonia following cigarette smoking, which has characteristic features as described above, should be considered as a distinct subtype of AEP.

Benign asbestos pleural effusion associated with pulmonary aspergilloma.

A rare case of benign asbestos pleural effusion associated with aspergilloma is reported. A chest radiograph of a 75-year-old Japanese man who was admitted with right chest pain showed a right pleural effusion and nodular shadows in the right apex and left middle lung field. Thoracocentesis revealed an exudate with atypical mesothelial cells. An open lung biopsy showed aspergilloma in the right S2 area and no evidence of malignancy. Many reactive mesothelial cells were found in the pleura. A quantitative asbestos digestion study of the lung tissue biopsy showed high-grade asbestos exposure.

Adult respiratory distress syndrome induced by a Chinese medicine, Kamisyoyo-san.

We report the first case of Kamisyoyo-san-induced adult respiratory distress syndrome (ARDS). A 59-year-old female was given Kamisyoyo-san for treatment of seborrheic dermatitis. She then presented with a respiratory illness having clinical, radiologic and functional characteristics of ARDS. Bronchoalveolar lavage fluid showed an increased number of lymphocytes, neutrophils and eosinophils. The lymphocyte stimulation test with Kamisyoyo-san was positive.

Two familial mesothelioma cases with high concentrations of soluble cytokeratin 19 fragment in pleural fluid.

We report two cases of diffuse malignant pleural mesothelioma occurring almost simultaneously in one family. Patient 1 was a 42-year-old Japanese man who had worked as an electrical engineer for 25 years. Patient 2, his mother, was 69 years old. She lived for 10 years with patient 1 after he started his work, and also worked at a shipyard herself for 6 years. The concentrations of cytokeratin subunit 19 fragment (CYFRA 21-1) in pleural fluid of the two patients were 1,500 ng/ml, and 1,200 ng/ml, respectively. Measurement of CYFRA 21-1 concentration in the pleural fluid may be a useful tool for a diagnosis of malignant mesothelioma.

Serum interleukin-5 levels in a case with allergic granulomatous angiitis.

A 50-year-old woman with allergic granulomatous angiitis (AGA) presented with an elevated serum interleukin-5 (IL-5) level upon admission to a hospital. Administration of prednisolone showed marked improvement of asthmatic symptoms, reduced eosinophil counts of peripheral blood, and normalized serum IL-5 levels. This is the first report describing an elevated serum level of IL-5 in AGA. The present findings suggest that IL-5 is involved in the pathogenesis of AGA.

Factors influencing response to treatment of pulmonary tuberculosis.

We analyzed 150 patients with pulmonary tuberculosis from 1990 to 1996 (i) to evaluate the frequency of drug resistance, (ii) to elucidate factors influencing the response to chemotherapy, and (iii) to attempt to improve the therapeutic approach. Multidrug-resistant tuberculosis strains were not found. By univariate analysis, there were 8 factors associated with an increased sputum conversion time: male gender, prior treatment, complications, progressive chest radiographic findings, a high Ziehl-Neelsen stain score, lymphocytopenia, a high erythrocyte sedimentation rate (ESR), and hypoproteinemia. Complications, prior treatment, a high Ziehl-Neelsen stain score, and a high ESR were independent predictive factors in a Cox proportional hazard model. Recursive partitioning and amalgamation (RPA) defined 3 subgroups that responded to treatment. In order to reduce the time to sputum conversion, poor responders according to the RPA should be treated with a 4-drug regimen containing pyrazinamide.

The usefulness of CYFRA 21-1 in diagnosing and monitoring malignant pleural mesothelioma.

Five patients with malignant pleural mesothelioma (MPM) were studied to determine whether CYFRA 21-1 is useful for diagnosis of this disease. In pleural effusions, the median concentration of CYFRA 21-1 from 4 patients with MPM was significantly higher than for 34 patients with benign diseases. The sensitivity of serum CYFRA 21-1 for diagnosis of MPM was 40% and its concentration changed in proportion to disease activity in all cases. Immunohistochemically, anticytokeratin 19 antibody revealed strong staining in both epithelial and sarcomatous MPM tissues. Based on these results, we conclude that measurement of CYFRA 21-1 in pleural effusions and serum may be useful for diagnosing and monitoring MPM.

[Bronchiolo-alveolar cell carcinoma of the lung presenting as spontaneous pneumothorax].

A 30-year old man visited a local clinic complaining of dyspnea and chest pain. He had a history of hepatocystadenocarcinoma 10 years before. Pneumothorax was diagnosed. Immediately, the patient was referred to us and underwent a tube thoracostomy. Cytologic examination of pleural effusion specimens revealed malignant cells. Because pleural drainage and pleurodesis using anti-cancer agents were not effective, we performed a thoracotomy. A tumor with accompanying bulla was detected in the right S6 region and a right lower lobectomy was performed. Histologic examination of tumor specimens disclosed bronchiolo-alveolar cell carcinoma. We speculated that bullae formed by tumor invasion, had burst, resulting in pneumothorax. This was a case of asynchronous double cancer in a relatively young patient, and the first report in Japan of bronchiolo-alveolar cell carcinoma presenting with spontaneous pneumothorax as an initial symptom.

[A case of idiopathic interstitial pneumonia that responded well to intermittent intravenous administration of high doses of cyclophosphamide and a steroid].

A 78-year-old man was hospitalized on February 29, 2000 because of dyspnea. A chest radiograph showed diffuse bilateral interstitial shadows associated with pulmonary volume loss. We could not obtain histological evidence of idiopathic interstitial pneumonia (IIP) because of his advanced age and severe respiratory dysfunction. IIP was diagnosed on the basis of radiographic findings and clinical symptoms. The patient was intubated and mechanical ventilation was performed. After one course of pulsed cyclophosphamide (CPM) and methylprednisolone therapy, the hypoxemia improved and it became possible to wean the patient from the ventilator. After five courses of pulsed CPM therapy, the dose of oral corticosteroid was tapered. CPM was administered safely without any severe side effects. Pulsed CPM therapy appears to be a viable alternative method of treatment for IIP.

[Induction chemotherapy followed by adjuvant surgery (IC-AS) in patients with stage I-II small cell lung cancer (SCLC)].

Ten patients with stage I-II SCLC received IC-AS between 1984 and 1993. As induction chemotherapy, COMP-VAN alternating chemotherapy and CAV-PVP hybrid chemotherapy were administered. The former consisted of a 4-drug combination of cyclophosphamide (CPA), vincristine (VCR), methotrexate (MTX) and procarbazine alternated with a 3-drug combination of etoposide (ETP), adriamycin (ADM) and nimustine every 4 weeks. In the latter, a 3-drug combination of CPA, ADM and VCR given on day 1, and a 2-drug combination of ETP and cisplatin on day 8, were repeated every 4 weeks. All the patients had an objective response, including one complete response by induction chemotherapy. Post-operative pathology revealed SCLC in 4 patients, adenocarcinoma in 2 and no tumor (pathological CR) in 4. Four patients relapsed, and a intrathoracic relapse was experienced in only 2 patients. Six patients have died: 3 from relapsing SCLC, 2 from stomach cancer, and 1 from squamous lung cancer, who was salvaged from relapsing SCLC. The median survival time was 27.5 months, and the 3-year survival rate 37.5%. These results indicate that IC-AS is highly effective for stage I-II SCLC and warrant additional studies comparing IC-AS with chemo-radiotherapy.

Separation of human glycoprotein hormones and their subunits by reversed-phase liquid chromatography.

Separation of human pituitary follicle-stimulating hormone (FSH), luteinizing hormone (LH) and thyroid-stimulating hormone (TSH) was effected on a micro-scale by reversed-phase high-performance liquid chromatography (HPLC) on a SynChropak C1 column in series with a Vydac C4 column using a linear gradient of acetonitrile in 0.1 M triethylamine phosphate buffer at pH 6.5. Chromatography on the C4 column alone caused partial dissociation of FSH into its subunits, whereas LH and TSH remained intact. Good yields of the separated subunits were obtained after prior dissociation of each hormone, and the results show that reversed-phase HPLC is useful for the analytical and preparative separation of these structurally related hormones and their subunits.

Site-specific N-glycosylation of human chorionic gonadotrophin--structural analysis of glycopeptides by one- and two-dimensional 1H NMR spectroscopy.

Glycopeptides representing individual N-glycosylation sites of the heterodimeric glycoprotein hormone human chorionic gonadotrophin (hCG) were obtained from subunits hCG alpha (N-glycosylated at Asn-52 and Asn-78) and hCG beta (N-glycosylated at Asn-13 and Asn-30) by digestion with trypsin and chymotrypsin, respectively. Following purification by reverse-phase HPLC and identification by amino acid sequencing, the glycopeptides were analysed by one- and two-dimensional 1H NMR spectroscopy. The results are summarized as follows: (i) oligosaccharides attached to Asn-52 of hCG alpha comprised monosialylated 'monoantenary' NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3[Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N1-4'), disialylated diantennary NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3[NeuAc alpha 2-3-Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N2), and the monosialylated hybrid-type structures NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3[Man alpha 1-3Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N1-A) and NeuAc alpha 2-3Gal-beta 1-4GlcNAc beta 1-2Man alpha 1-3[Man alpha 1-3(Man alpha 1-6)Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N1-AB) in a ratio approaching 5:2:2:1; (ii) Asn-78 of hCG alpha carried N2 and N1-4' almost exclusively (ratio approximately 3:2); (iii) both N-glycosylation sites of hCG beta contained predominantly component N2, partially (approximately 25%) and completely alpha 1-6-fucosylated at the N-acetylglucosamine linked to Asn-13 and Asn-30, respectively. The distinct site-specific distribution of the oligosaccharide structures among individual N-glycosylation sites of hCG appears to reflect primarily the influence of the surrounding protein structure on the substrate accessibility of the Golgi processing enzymes alpha-mannosidase II, GlcNAc transferase II and alpha 1,6-fucosyltransferase.

NMR investigations of the N-linked oligosaccharides at individual glycosylation sites of human lutropin.

Human lutropin or luteinizing hormone (hLH) is a heterodimeric glycoprotein, composed of two subunits. hLH alpha (N-glycosylated at Asn52 and Asn78) and hLH beta (N-glycosylated at Asn30). The sugar chains were liberated by hydrazinolysis from intact hLH beta and from glycopeptides obtained after tryptic digestion of hLH alpha, subsequently reduced and fractionated as alditols by anion-exchange and ion-suppression amine-adsorption HPLC and identified mainly by one-dimensional (1D) and two-dimensional (2D) 1H-NMR spectroscopy. The results indicate predominantly diantennary. N-acetyllactosamine-type structures at all three glycosylation sites. The oligosaccharides attached to Asn52 (hLH alpha) and Asn30 (hLH beta) show a remarkably similar pattern, with mainly chain-terminating 4-sulphated 2-deoxy-2-N-acetylamino-D-galactose (GalNAc) and a sulphated/sialylated structure as the major single component. However, virtually all N-glycans on the beta subunit bear a fucose residue alpha 1-6-linked to the proximal GlcNAc, whereas those at Asn52 (and Asn78) of the alpha subunit are predominantly non-fucosylated. The oligosaccharides at Asn78 (hLH alpha) are sialylated rather than sulphated and contain the unique sequence NeuAc alpha 2-6 GalNAc beta 1-4GlcNAc beta 1-2 Man alpha 1-3 as part of the majority of mono- and disialylated compounds. The major single constituent at Asn78 has the following structure: [formula, see text]

Site-specific N-glycosylation of ovine lutropin. Structural analysis by one- and two-dimensional 1H-NMR spectroscopy.

The Asn-linked carbohydrate structures of the heterodimeric glycoprotein hormone lutropin from ovine pituitary glands have been investigated at each of its three glycosylation sites using one- and two-dimensional 400-MHz 1H-NMR spectroscopy. Highly purified, biologically active ovine lutropin (oLH) was dissociated and separated into its alpha and beta subunits (oLH alpha, glycosylated at Asn56 and Asn82; oLH beta glycosylated at Asn13). Oligosaccharides from intact oLH beta and from glycopeptides obtained after tryptic digestion of oLH alpha were released by hydrazinolysis and subsequently fractionated according to charge and size by anion-exchange and ion-suppression amine-adsorption HPLC, respectively. 1H-NMR analysis revealed, that monosulphated, mostly hybrid-type, oligosaccharides predominate at both glycosylation sites of oLH alpha, whereas a disulphated, diantennary N-acetyllactosamine-type structure accounts for more than 60% of total oligosaccharides in the beta subunit. Furthermore, the saccharides attached to the beta subunit are almost completely fucosylated (Fuc alpha 1-6) at the reducing terminal GlcNAc, whereas the sugar chains in oLH alpha are either approximately 50% fucosylated (Asn82) or contain fucose only to a minor extent (Asn56). The results clearly indicate a distinct subunit- and site-specific synthesis of oligosaccharides in ovine lutropin and suggest that biosynthesis is effectively influenced by the surrounding polypeptide chain(s) at a given site.

The asparagine-linked oligosaccharides at individual glycosylation sites in human thyrotrophin.

The asparagine-linked carbohydrate structures at each of the three glycosylation sites of human thyrotrophin were investigated by 400 MHz 1H-NMR spectroscopy. Highly purified, biologically active human thyrotrophin (hTSH) was dissociated into its subunits hTSH alpha (glycosylated at Asn 52 and Asn 78) and hTSH beta (glycosylated at Asn 23). The alpha-subunit was further treated with trypsin which gave two glycopeptides that were subsequently separated by reverse-phase HPLC and identified by amino acid sequence analysis. The oligosaccharides were liberated from hTSH alpha glycopeptides and from intact hTSH beta by hydrazinolysis, and were fractionated as alditols by anion-exchange and ion-suppression amine-adsorption HPLC preparatory to structural analysis. The N-glycans present on hTSH were mainly diantennary complex-type structures with a common Man alpha 1-3 branch that terminated with 4-O-sulphated GalNAc. The Man alpha 1-6 branch displayed structural heterogeneity in the terminal sequence, with chiefly alpha 2-3-sialylated Gal and/or 4-O-sulphated GalNAc. The relative amounts of the two major complete diantennary oligosaccharides and their core fucosylation differed according to glycosylation site; the sulphated/sialylated diantennary oligosaccharide was most abundant at the two sites on the alpha-subunit, whereas the disulphated, core-fucosylated oligosaccharide was more plentiful on the beta-subunit. Some interesting structural features, not previously reported for the N-glycans of hTSH, included 3-O-sulphated galactose (SO4-3Gal) and peripheral fucose (Fuc alpha 1-3GlcNAc) in the Man alpha 1-6 branch of some diantennary structures; the former suggests the presence of a hitherto uncharacterized galactose-3-O-sulphotransferase in thyrotroph cells of the human anterior pituitary gland.

[Gingival metastasis of large-cell lung cancer that produced G-CSF].

A 68-year-old man was referred to our hospital for further examination of a gingival mass. Chest radiographs and magnetic resonance imaging disclosed a bulky mass originating in the upper portion of the left lung, in contact with a chronic empyema lesion that first occurred after resection for pulmonary tuberculosis. Examination of a specimen obtained by percutaneous needle biopsy of the mass led to the diagnosis of large-cell carcinoma. Laboratory findings on admission showed marked leukocytosis (48,100/microliter) without evidence of severe a bacterial infection. The level of G-CSF in serum was abnormally high (246 pg/ml, normal value: < 30 pg/ml). Chemotherapy with vindesine, ifosfamide, and cisplatin resulted in shrinkage of the gingival mass, and a decrease in the G-CSF level to 66 pg/ml. Immunohistochemical staining with an anti-G-CSF monoclonal antibody to the primary lung tumor and the gingival mass obtained at autopsy was positive for cytoplasmic G-CSF.