A structural view of synthetic cofactor integration into [FeFe]-hydrogenases.

[FeFe]-hydrogenases are nature's fastest catalysts for the evolution or oxidation of hydrogen. Numerous synthetic model complexes for the [2Fe] subcluster (2FeH) of their active site are known, but so far none of these could compete with the enzymes. The complex Fe2[µ-(SCH2)2X](CN)2(CO)42- with X = NH was shown to integrate into the apo-form of [FeFe]-hydrogenases to yield a fully active enzyme. Here we report the first crystal structures of the apo-form of the bacterial [FeFe]-hydrogenase CpI from Clostridium pasteurianum at 1.60 Å and the active semisynthetic enzyme, CpIADT, at 1.63 Å. The structures illustrate the significant changes in ligand coordination upon integration and activation of the [2Fe] complex. These changes are induced by a rigid 2FeH cavity as revealed by the structure of apoCpI, which is remarkably similar to CpIADT. Additionally we present the high resolution crystal structures of the semisynthetic bacterial [FeFe]-hydrogenases CpIPDT (X = CH2), CpIODT (X = O) and CpISDT (X = S) with changes in the headgroup of the dithiolate bridge in the 2FeH cofactor. The structures of these inactive enzymes demonstrate that the 2FeH-subcluster and its protein environment remain largely unchanged when compared to the active enzyme CpIADT. As the active site shows an open coordination site in all structures, the absence of catalytic activity is probably not caused by steric obstruction. This demonstrates that the chemical properties of the dithiolate bridge are essential for enzyme activity.

Comparative ultrastructural study of cytotoxic granules in nasal natural killer cell lymphoma, intestinal T-cell lymphoma, and anaplastic large cell lymphoma.

Comparative immunohistochemical and ultrastructural studies were performed on five nasal natural killer (NK) cell lymphoma cases, two intestinal T-cell lymphoma cases, and eight anaplastic large cell lymphoma (ALCL) cases to clarify morphological differences in cytotoxic granules among these cytotoxic lymphomas. Nasal NK-cell lymphomas and intestinal T-cell lymphomas had fine azurophilic granules and displayed dot-like immunostaining of granzyme B- and T-cell intracellular antigen 1 (TIA-1), predominantly in the central area of the cytoplasm. Ultrastructurally, these NK-cell lymphomas and intestinal T-cell lymphomas had two types of cytotoxic granules, type-I granules (dense core granules) and type-II granules (multivesicular bodies), which have been demonstrated in normal large granular lymphocytes in peripheral blood. However, ALCLs did not have azurophilic granules, and only type-II cytotoxic granules were found ultrastructurally, even though they showed similar dot-like immunostained patterns of granzyme B and TIA-1, as seen in NK-cell lymphomas and intestinal T-cell lymphomas. Immunoelectron microscopy revealed that TIA-1 was primarily located at the periphery of the cytoplasmic granules in the NK-cell lymphoma and ALCL cases. These findings suggest that malignant lymphomas with a cytotoxic phenotype can be divided into two types, (azurophilic granule)+, (type-I granule)+, (type-II granule)+ lymphomas and (azurophilic granule)-, (type-I granule)-, (type-II granule)+ lymphomas.

Cloning of Corticium rolfsii glucoamylase cDNA and its expression in Saccharomyces cerevisiae.

A cDNA coding for the glucoamylase of Corticium rolfsii AHU 9627 was cloned using synthetic oligonucleotide probes that code for inner amino acid sequences of the purified enzyme. This clone (CG 15) is 1900 base pairs long and contains the entire coding region for a polypeptide of 579 residues. Comparison with amino acid sequences of other fungal glucoamylases showed homologies of 35%-56%, and most homology with that of Aspergillus niger. The expression plasmid pACG 115 was constructed by introduction of the coding region of CG 15 into a yeast expression vector pAAH 5, containing the promoter and terminator of alcohol dehydrogenase (ADHI). Saccharomyces cerevisiae AH 22, containing the recombinant plasmid pACG 115, acquired starch-saccharifying ability.

[Clinical evaluation of combined therapy of ceftazidime and tobramycin for intractable pulmonary infection mainly caused by Pseudomonas aeruginosa].

In an open, multicenter trial, we investigated the clinical efficacy of a combination therapy of ceftazidime (CAZ) and tobramycin (TOB) for intractable pulmonary infections mainly caused by Pseudomonas aeruginosa. Evaluated for the utility of the combination therapy were 33 cases with pneumonia (Group I: pneumonia caused by P. aeruginosa 15, Group II: pneumonia caused by other Gram-negative bacilli 4 and pneumonia which causative organism was not determined 14) and 23 cases with chronic respiratory tract infection caused by P. aeruginosa. The results obtained are summarized as follows. 1. In Group I pneumonia, included 11 severe cases and 4 moderate cases, with a mean age of 69.3 years. Significant underlying diseases were present in 14 out of the 15 (93.3%): they included 10 cases of pulmonary diseases and 4 cerebrovascular diseases. The overall efficacy rate in these cases was 60.0%: but the efficacy rate in moderate cases was 100% and that in severe cases was 45.5%. 2. In Group II pneumonia included 16 severe cases and 2 moderate cases with a mean age of 68.2 years. Significant underlying diseases were present in 15 out of 18 (83.3%, all of the underlying diseases were pulmonary diseases) and the overall efficacy rate was 72.2% with 100% efficacy rate among moderate cases and 68.8% among severe cases. 3. In the cases with chronic respiratory tract infections caused by P. aeruginosa, the efficacy rate was 82.6% and the eradication rate was 65.2%. We consider the combination therapy of CAZ and TOB is useful for intractable pulmonary infections caused by P. aeruginosa.

[Clinical evaluation of cefteram pivoxil in respiratory tract infections].

The usefulness of cefteram pivoxil (CFTM-PI) was evaluated in 99 cases with respiratory tract infections: 32 cases with acute bronchitis, 51 cases with infectious exacerbations of chronic respiratory diseases and 16 cases with pneumonia. 1. The clinical efficacies included marked improvement in 27 cases, improvement in 51 cases, moderate improvement in 9 cases, no change in 10 cases and deterioration in 2 cases. The improvement rate was 78.8%. 2. Overall effects were excellent in 12 cases, good in 9 cases and fair in 5 cases. There was no case in which efficacy was not observed and the efficacy rate was 80.8%. 3. Bacteriological effects were classified according to the causative organisms. Eradication rate was 80.8% (21 of 26 strains), indicating an excellent antibacterial action of CFTM-PI. In particular, MICs of cefteram were below 0.05 microgram/ml against all 10 strains of Haemophilus influenzae regardless of beta-lactamase production even with an inoculum of 10(8) or 10(6) cells/ml. 4. Side effects rarely occurred and included a slight gastrointestinal irritation in 4 of 99 cases (4%). Two cases which had abnormal elevations of GOT and GPT had abnormal values prior to administration of CFTM-PI. The elevations were slight and it was possible to continue administration. The GOT and GPT values were improved after the end of administration. The above results indicate the usefulness of CFTM-PI in acute respiratory infections and infectious exacerbation of chronic respiratory diseases.

[Extraordinary examinations in tuberculous prevalence areas and family contact examinations].

A survey was made on the actual state of performance of extraordinary examinations done in high tuberculosis prevalence areas and those of family contact examinations of the index cases of tuberculosis during the period between April 1986 and November 1987 in Chiba Prefecture. The results are as follows: (1) Forty-eight extraordinary examinations were performed by 19 public health centers. (2) By these extraordinary examinations, 22 additional tuberculous patients were detected and 143 persons were indicated INH prophylaxis. There were 2 cases of mass tuberculosis infection: from which 19 patients and 140 persons for chemoprophylaxis were detected. (3) Out of 18 extraordinary examinations done for contacts of cases from danger groups, one was done actually for unnecessary case. As to tuberculin tests, they were not performed in 2 groups, and in another case, the test subjects were inappropriate. (4) The examination rate of family members of index cases was as high as 95.4%, whereas the performance of tuberculin tests was not satisfactory, especially as regards to the younger populations. The total performance rate of the tests was 29.0%, and the rate in those below 19 years of age was 56.5%, and the rate in those below 29 was 42.9%. (5) There were 6 families with tuberculosis, which included 14 patients. It is considered that in extraordinary examinations, appropriate setting of subjects and tuberculin tests for younger persons are important; in family contacts examination, tuberculin tests for younger persons are indispensable.

[Clinical experience with ofloxacin in the treatment of chronic respiratory tract disease aggravated by acute infection].

We studied the clinical usefulness of ofloxacin (OFLX) in 13 patients with chronic respiratory tract diseases aggravated by acute infections with identified causative bacteria. 1. Overall clinical efficacies were: highly effective 5, effective 6, slightly effective 2, and ineffective none, showing an efficacy rate of 84.6%. 2. In 6 patients with fever of over 37 degrees C, time lengths in days for symptoms to have been alleviated due to OFLX treatment were; 1 day: 4 cases, 3 days: 1 case and 5 days: 1 case (2.0 days on average). 3. As to bacterial transitions, in 9 of 10 patients Haemophilus influenzae was eliminated and in 1 patient it was substituted by Pseudomonas aeruginosa. In 3 patients Branhamella catarrhalis was eliminated and in 1 patient, the number of P. aeruginosa was reduced. OFLX is expected to have a potent bacteriological effect on H. influenzae and B. catarrhalis. 4. As to side effects, 1 of the 13 patients (7.7%) complained of discomfort in the epigastrium. This discomfort disappeared when a gastric mucosa protective agent was administered. There was no abnormality in laboratory test values. Judging from the above results, we consider OFLX a useful drug for the treatment of patients with chronic respiratory tract diseases aggravated by acute infections.

[Echocardiographic manifestations of postinfarction perforation with extensive dissection of the interventricular septum: report of an autopsied case].

This is a report of the echocardiographic features of postinfarction perforation associated with dissection of the interventricular septum (IVS) in a 61-year-old woman. She had acute myocardial infarction and was admitted to a nearby hospital, and later admitted to our CCU because hemodynamic deterioration. On admission, she was somnolent and hypotensive (90/64 mmHg), with sinus tachycardia, marked cyanosis, and peripheral edema. On auscultation there were a harsh holosystolic murmur over the LLSB and moist rales in both lung fields. An ECG revealed extensive anterior and inferior infarctions. Catheterization data confirmed O2 step-up in the right ventricle. Two-dimensional echocardiograms demonstrated extensive dissection of the IVS characterized by an echo-free lumen extending from the apex to the cardiac base in the long-axis and partly reaching the left ventricular free wall in the short-axis with the concomitant paradoxical movement of the IVS. Autopsy disclosed marked hemorrhagic infarction and extensive dissection of the IVS forming a lumen (5 X 5.5 X 2 cm3) corresponding exactly to the echocardiographic free space. In addition, a shunt between the right and left ventricles was confirmed by the presence of two perforations near the apex on the right and left sides of the IVS, whose diameters were 6 and 10 mm, respectively.

[Amyl nitrite test in the evaluation of left ventricular function: application to ischemic heart disease and Duchenne's progressive muscular dystrophy].

The effect of amyl nitrite (AN) inhalation on the left ventricular function was evaluated by mechanocardiography and echocardiography. The patient's group consisted of 110 cases with ischemic heart disease (IHD) and 25 cases of dystrophia musculorum progressiva (DMP) of Duchenne type. The former was a representative of impairment of blood supply and myocardial involvement, and the latter was of predominant myocardial disease. The control was age-matched 32 normals for IHD group and 17 cases for DMP group. Left ventricular function was mainly evaluated by systolic time intervals (STI) and the echocardiographic correlates. Fifty-five cases of IHD group were tested by coronary angiography and left ventriculography and these data were compared with the noninvasive measures. The results were as follows: I. IHD group: The ratio of ejection time (ET) to pre-ejection period (PEP), ET/PEP, did not change so much as in controls after AN inhalation, and this percent change was much smaller in cases with lesions of the left anterior descending artery (LAD) than in cases with lesions of the right coronary artery (RCA). On the other hand, mean posterior wall velocity (mPWV) and posterior wall excursion (PWE) changed greater in patients with LAD lesion than in those with RCA lesion. In cases with LAD stenosis, percent change in ET/PEP was smaller in cases with asynergy than in cases without it, disclosing more significant impairment of the left ventricle in the former. II. DMP group: In severe cases, ET/PEP was small even at rest, and percent change by AN inhalation was smaller than control in mild cases and smallest in severe cases. This seems to be useful in evaluating the severity of the diseased process. The mPWV and PWE showed impairment of left ventricular motion even at rest, but it was clearly showed in severe cases after AN inhalation. These results indicate that impairment of left ventricular function induces the poor response to AN inhalation and this, in turn, results in the lack of hyperactivity of the heart produced by this drug.

Aortic insufficiency caused by Takayasu's arteritis without usual clinical features.

A 30-year-old woman with rapidly progressive, fatal aortic insufficiency was studied. Pathologic studies revealed that the aortic insufficiency was caused by dilation of the aortic root due to Takayasu's arteritis that, unusually, was confined to the ascending aorta and aortic arch. When other diseases that bring about aortic insufficiency can be excluded, Takayasu's arteritis should be considered, even if most of its manifestations are absent.