Steroid monooxygenase of Rhodococcus rhodochrous: sequencing of the genomic DNA, and hyperexpression, purification, and characterization of the recombinant enzyme.

Steroid monooxygenase of Rhodococcus rhodochrous is a Baeyer-Villigerase catalyzing the insertion of an oxygen atom between the C(17)- and C(20)-carbons of progesterone to produce testosterone acetate. The 5.1-kbp-long BamHI DNA fragment containing the steroid monooxygenase gene, smo, was cloned from the chromosomal DNA and sequenced. The smo gene is 1,650 nucleotides long, starts with a TTG codon, and ends with a TGA codon. The deduced amino acid sequence indicates that the enzyme protein consist of 549 amino acid residues with a molecular mass of 60,133. Thus, the molecular mass of the holoenzyme is 60,919. The amino acid sequence is highly homologous (41.2% identity) to that of cyclohexanone monooxygenase of Acinetobacter sp. In the upstream of the smo gene, the genes of heat shock proteins, dnaK, grpE, and dnaJ, located on the complementary strand, and the DNA-inserts of pSMO and pD1, which contains the ksdD gene, were joined at the BamHI site of the dnaJ gene. The smo gene was modified at the initiation codon to ATG and ligated with an expression vector to construct a plasmid, pSMO-EX, and introduced into Escherichia coli cells. The transformed cells hyperexpressed the steroid monooxygenase as an active and soluble protein at more than 40 times the level in R. rhodochrous cells. Purification of the recombinant monooxygenase from the E. coli cells by simplified procedures yielded about 2.3 mg of enzyme protein/g wet cells. The purified recombinant steroid monooxygenase exhibited indistinguishable molecular and catalytic properties from those of the R. rhodochrous enzyme.

Essential tyrosine residues in 3-ketosteroid-delta(1)-dehydrogenase from Rhodococcus rhodochrous.

Tetranitromethane treatment of 3-ketosteroid-Delta(1)-dehydrogenase of Rhodococcus rhodochrous caused loss of the catalytic activity in a time- and concentration-dependent manner. Peptides (P-81) and (PN-83) were isolated from tryptic digests of the native and tetranitromethane-treated enzyme proteins, respectively. PN-83 was the nitrated form of P-81. The amino acid sequence was GGAPLIDYLESDDDLEFMVYPWPDYFGK (positions 97-124 of the dehydrogenase sequence). PN-83 showed a low yield of PTH-Tyr of position 116, i.e. less than 5% of that of P-81, and instead a high yield of PTH-3-nitrotyrosine. This indicated that tetranitromethane modifies Y-116 under the experimental conditions used. Mutation of Y-104, Y-116, and Y-121 to smaller amino acid residues, Phe, Ser, or Ala, significantly changed the catalytic activity of the dehydrogenase. All of the mutants contained FAD and exhibited the same spectrophotometric properties as those of the wild type enzyme. The K(m) values for 4-androstene-3,17-dione of the Y-104, Y-116, and Y-121 mutants changed to large values. The most drastic change was observed for Y116A. The K(d) values for 1,4-androstadiene-3,17-dione of the Y116 mutants changed to 1.5-2.6-fold larger values than that of the recombinant enzyme. The Y-121 mutant enzymes exhibited catalytic activities like those of the recombinant enzyme, but the catalytic efficiencies of Y121F and Y121A drastically decreased to 0. 014-0.054% of that of the recombinant enzyme. The present results indicate that Y-121 plays an important role in the catalytic function, and that Y-116 and Y-104 act on binding of the substrate steroid.

CEA-producing mucin-negative gastric signet-ring cell carcinoma with neuroendocrine markers: a case report.

Biopsy and autopsy materials excised from a 69-year-old woman were investigated. Serum carcinoembryonic antigen (CEA) showed a high value of 955 ng/mL. A plateaulike tumor was located in the gastric cardia and fundus to the entire gastric body. It showed severe proliferation and infiltration from the mucosa to the serosa. The tumor was comprised of signet-ring cells and poorly differentiated adenocarcinoma cells, which spread into the submucosa of the pylorus, duodenum, and jejunum. Signet-ring cells had a large, eccentric vesicular nucleus and a pale cytoplasmic inclusion. Poorly differentiated adenocarcinoma cells had a pleomorphic nucleus, small eosinophilic nucleolus, and abundant eosinophilic cytoplasm. Both neoplastic cells were positive for CEA, epithelial membrane antigen, Leu-7 (CD57), and neuron-specific enolase, and were negative for cytokeratin, vimentin, and periodic acid-Schiff, Alcian blue, and mucicarmine stains. Electron microscopy showed endocrine granules with a limiting membrane measuring approximately 238 nm in diameter in the cytoplasm. The authors diagnosed this patient as having mucin-negative gastric signet-ring cell carcinoma with neuroendocrine markers, which is suggested to exist among poorly differentiated adenocarcinoma, undifferentiated carcinoma, and signet-ring cell carcinoma.

Moderate hypothermia reduces hypotensive, but not hypercapnic vasodilation of pial arterioles in rats.

Two types of acid-base strategies are available for the blood gas management of patients during hypothermia: alpha-stat and pH-stat management. However, the more suitable strategy for therapeutic hypothermia is unclear. We studied the effects of hypothermia (30 degrees C) and acid-base management on reactivity to hypercapnia and hypotension in rat pial arterioles, using a closed cranial window. The baseline diameter during hypothermia decreased in the alpha-stat (PaCO2 was maintained at 35 mm Hg when measured at 37 degrees C, n = 8), but not in the pH-stat (PaCO2 was maintained at 35 mm Hg when corrected to the animal's actual temperature, n = 7). Vasodilation induced by hypotension was significantly reduced in hypothermic groups compared with the normothermic group (n = 7), whereas responses to hypercapnia were preserved. Moreover, hypotensive vasodilation was more attenuated in the pH-stat, than the alpha-stat, management. These findings show that moderate hypothermia and acid-base management alter cerebrovascular autoregulation.

3-Ketosteroid-delta1-dehydrogenase of Rhodococcus rhodochrous: sequencing of the genomic DNA and hyperexpression, purification, and characterization of the recombinant enzyme.

The gene encoding 3-ketosteroid-Delta1-dehydrogenase from Rhodococcus rhodochrous was cloned and sequenced. The gene (ksdD) consists of 1,536 nucleotides and encodes an enzyme protein of 511 amino acid residues. The amino terminal methionine residue was deleted in the mature protein. The amino acids involved in the flavin binding site are conserved in the dehydrogenase sequence. The deduced amino acid sequence is highly homologous to that from Arthrobacter simplex but less so to that from Pseudomonas testosteroni. Upstream of the gene was located a heat shock protein gene, dnaJ, and downstream, a gene of a hypothetical protein. The enzyme gene was ligated with an expression vector to construct a plasmid pDEX-3 and introduced into Escherichia coli cells. The transformed cells hyperexpressed the 3-ketosteroid-Delta1-dehydrogenase as an active and soluble protein at more than 30 times the level of R. rhodochrous cells. Purification of the recombinant 3-ketosteroid-Delta1-dehydrogenase from the E. coli cells by a simplified procedure yielded about 13 mg of enzyme protein/liter of the bacterial culture. The purified recombinant dehydrogenase exhibited identical molecular and catalytic properties to the R. rhodochrous enzyme.

Coexisting primary early gastric plasmacytoma and sarcoidosis with hypercalcaemia.

We report on a 61-year-old woman with coexisting early stage primary gastric plasmacytoma and sarcoidosis with hypercalcaemia. Laboratory data on admission showed hypercalcaemia, with 12.8 mg/dl, parathyroid hormone-related peptide (PTHrP) 1.2 pmol/l, C-PTHrP 69.5 pmol/l, and 1,25-dihydroxyvitamin D3 46.7 pg/ml. Neoplastic plasma cells proliferated in the propria mucosa of the stomach, showed a monoclonal immunoglobulin of cytoplasmic IgA (lambda light chain) and were positive for leucocyte common antigen and epithelial membrane antigen on paraffin section prepared from a stomach biopsy specimen. Russel bodies were present, as were crystals. Abundant sarcoid granulomas were observed in many of the regional lymph nodes around the stomach and in the dermis of a skin nodule. The patient underwent subtotal gastrectomy with administration of antimyeloma chemotherapy. We suggest that the hypercalcaemia in this patient was due to PTHrP production by neoplastic plasma cells.

Impaired autoregulation in an experimental model of chronic cerebral hypoperfusion in rats.

BACKGROUND AND PURPOSE: To verify the hypothesis that impaired autoregulation may contribute to cerebral swelling or hemorrhage after a sudden recovery of perfusion pressure, we studied the chronic effects of cerebral hypoperfusion on the autoregulatory responses of the pial arterioles in situ. METHODS: Eight to 12 weeks after a carotid-jugular fistula was created in rats, experiments were performed under alpha-chloralose and urethane anesthesia. Regional cerebral blood flow (rCBF) was determined by the hydrogen clearance method, and carotid pressure was measured. Using a closed cranial window, we determined the autoregulatory responses of the arterioles (30 to 50 microns) to both hypertension induced by norepinephrine and sudden fistula closure at various mean arterial pressures (MAPs). RESULTS: rCBF on the fistula side was reduced by 27%. Carotid pressure was significantly lower than normal but was immediately increased by fistula closure. The pial arterioles showed marked elongation and enlargement. During induced hypertension, the arterioles in the fistula group started to dilate at an MAP lower than that of the control group (130 versus 180 mm Hg, respectively). The arterioles constricted when the fistula was occluded at normal MAP. However, when the fistula was occluded at an MAP higher than 130 mm Hg, the vessels dilated. CONCLUSIONS: It was demonstrated that (1) chronic hypoperfusion induced impairment of the upper limit of autoregulation and (2) sudden fistula closure under hypertensive conditions caused vasodilation of the arterioles. These findings suggest that rapid restoration of perfusion pressure is possibly followed by a pressure breakthrough phenomenon in a chronically hypoperfused cerebrovasculature.

[Follow-up study of Takayasu arteritis with aortic regurgitation].

The clinical courses of patients with Takayasu arteritis vary especially when aortic regurgitation is involved. The clinical features and outcomes of Takayasu arteritis were studied in 78 patients to clarify the influence of aortic regurgitation on the natural history, especially the earlier stages of aortic regurgitation after onset of Takayasu arteritis. During the average 12.7-year follow-up period, 7% (3/43) of patients without aortic regurgitation died, but 17% (6/35) of patients with aortic regurgitation died. Mortality was low (6%; 1/16) in patients with mild (grade II or less) regurgitation, but high (26%; 5/19) in patients with severe (greater than grade III) regurgitation. Predictors indicating patients likely to die of severe aortic regurgitation were age at onset (mean age of 30.0 years), C reactive protein and erythrocyte sedimentation rate, and presence or absence of involvement of major branches of the aortic arch. Mortality was 33% (2/6) in patients without involvement of the major branches, which was significantly higher than that of patients with such involvement (17%; 2/12). From the initial consultation, most patients with mild regurgitation remained stable, but younger patients with severe regurgitation due to acute pathological processes of the ascending aorta from the early stage and elevated erythrocyte sedimentation rate showed deterioration in their clinical courses. Younger patients with elevated erythrocyte sedimentation rate, intact major branches of the aortic arch, no signs of classical pulseless disease, and severe aortic regurgitation due to Takayasu arteritis have a poor prognosis.

Diagnostic significance of serum neuron-specific enolase and myelin basic protein assay in patients with acute head injury.

BACKGROUND: Neuron-specific enolase (NSE) and myelin basic protein (MBP) in the peripheral venous blood (PVB) have been reported to be sensitive markers for judging the prognosis of patients with head injury. However, to our knowledge, the levels of NSE and MBP in the internal jugular venous blood (IJVB) have never been studied. METHODS: In 25 patients with acute head injury, blood samples were taken from the internal jugular vein and the peripheral vein at the same time before any medical or surgical procedure was performed. The levels of NSE and MBP in the both venous blood samples were measured. The time interval between injury and sampling was 1.5-8.0 hours (mean 4.3 hours). The levels of NSE and MBP in the IJVB were compared to those in the PVB. The relationship between the clinical outcome and the serum levels of those was evaluated. RESULTS: The levels of NSE and MBP in the IJVB were almost equal to those in the PVB. The levels of NSE and MBP were significantly higher in the patients who died than in those who survived. In the survivors, the levels of NSE and MBP in the IJVB were 17.6 +/- 11.4 ng/ml and 1.4 +/- 1.5 ng/ml, whereas in the patients who died, both levels were elevated to 51.3 +/- 27.3 ng/ml (p < 0.005) and to 11.3 +/- 9.5 ng/ml (p < 0.01), respectively. CONCLUSIONS: The assay of serum NSE and MBP levels provides a reliable laboratory indicator of the degree of brain damage and allows early prediction of the prognosis in patients with acute head injury.

Improved lipid visualization with a modified osmium tetroxide method using ultrasonic treatment and intensification with imidazole or triazole.

Formalin fixed autopsy tissue containing lipids were cut into 1-5 mm thick blocks, washed well, then postfixed in 2% OsO4 in 0.03 M veronal acetate buffer for 30, 60, 90, 120, or 180 min with or without ultrasonic treatment. Tissues exposed to ultrasound for 90 min showed superior penetration of OsO4 and well preserved histological architecture. Tissues also were immersed for 1 hr in veronal acetate buffer (pH 7.4) containing 0.5% imidazole or triazole and compared with untreated controls. Paraffin sections, 4 microns thick, were examined under a light microscope with an image analyzer. Both intensity and percentage area of osmium blackening were significantly higher in samples immersed in imidazole or triazole than in untreated controls. No difference was observed between imidazole- and triazole-immersed samples. The OsO4 method, modified by ultrasound treatment and imidazole- or triazole-immersion, can be applied to routine formalin fixed autopsy materials for improved lipid visualization.

[Completely thrombosed large aneurysm of the distal middle cerebral artery: a case report].

A 19 year old male was admitted for evaluation after a seizure. Physical and neurological examination was normal. CT demonstrated an enlarged, high density mass in the right parietal lobe. MRI showed a homogeneous high intensity T1 weighted mass, surrounded by a low intensity T2 weighted rim in the right parietal lobe. Angiography did not show any abnormal findings. A diagnosis of cavernous angioma with primary bleeding in the subcortical region of the right parietal lobe was made after radiological examination. Histological examination showed a completely thrombosed aneurysm. The mechanism of the complete thrombosis and the growth of this large aneurysm and the shortcomings of radiological examination are discussed.

The effects of a carotid-jugular fistula on cerebral blood flow in the cat: an experimental study in the acute period.

The purpose of the present study is to investigate the effects of feline carotid-jugular (CJ) fistula (Spetzler's model) on cerebral blood flow (CBF) in the acute period after creation of the fistula. Using laser-Doppler flowmetry, cortical CBF was measured on the fistula side of 11 cats. Temporary occlusion and opening of the fistula led to an immediate increase and decrease, respectively, in cortical CBF. However, CBF returned to baseline within 1 minute, on average. CO2 reactivity in the closed fistula was preserved. It is suggested that cerebral hemodynamic changes due to Spetzler's CJ fistula model are minimal in the acute period after fistulization.

Pharmacokinetics and pharmacodynamics of serum glycerol in patients with brain edema: comparison of oral and intravenous administration.

To clarify the pharmacokinetics and effect of ICP reduction of glycerol administered orally, the serum concentration of glycerol was measured by the enzymatic method and ICP was measured by the subdural balloon method in severe head injured patients with brain edema and increased ICP. Sequential change of serum glycerol concentration and its relationship to the reduction of ICP were analyzed. The results showed that the pharmacokinetics of glycerol through oral administration were similar to that of intravenous glycerol administration and the changes of ICP were also similar to that of intravenous glycerol administration. We determined that glycerol can be administered either per oral or per venous to obtain the same results for treatment of brain edema with raised ICP.

[Intracranial extracerebral glioma].

A case of solitary leptomeningeal extracerebral glioma is reported. A 75-year-old man was admitted to our hospital because of headache and right hemiparesis. CT scan and carotid angiography revealed a tumor in the left convexity. At operation, the tumor was located between the dura mater and the arachnoid membrane and adhered to the brain surface only in a limited area. Histological study including immunostain and electron microscopy showed the tumor as anaplastic oligo-astrocytoma. We speculate that our case may originate from a heterotopic glial nest in the dural border cell layer of dura mater. This explanation seems likely because the tumor was located mostly in the subdural space.

Growth of the mammary gland in rhino mouse.

The rhino mouse, so called because of its characteristic rhinocerous-like skin cannot raise its young. Although the animal has rudimentary mammary glands, these are thought to be defective due to their impossibility to raise their young. Morphological observations of the female mammary glands of rhino mice (hrrh/hrrh) were carried out and the results were compared with those of their normal litter mates (hrrh/+) at various ages from 1 month to 1 year. No morphological differences in mammary gland development was seen between the rhino mice and their normal litter mates, and normal secretory changes were observed in the rhino mice after delivery. Mouse mammary tumor virus (MMTV) expression and the occurrence of hyperplastic alveolar nodule (HAN) were analogous to these features in phenotypically normal hrrh/+ mice. Further, the sweat glands in the rhino mouse were normal. Thus, anomalies in the rhino mouse appeared to be limited to the skin and pilosebaceous apparatus.

Mammary tumour induction by pituitary grafting in male mice: an animal model for male breast cancer.

Isologous anterior pituitary grafting, 4 each, to 3-4-month-old SHN and SLN male mice resulted in an appearance of mammary tumours from 8 months of age and the incidence at 12 months reached 53.8% in each strain. All tumours were diagnosed as type B adenocarcinomas. In association with the results, normal mammary gland growth and mouse mammary tumour virus (MMTV)-gp52 antigen levels in the submaxillary glands were stimulated by the treatment in these strains. The effect of pituitary grafting was much less in GR/A male mice in which no mammary tumours appeared.

Tenascin staining positivity and the survival of patients with invasive breast carcinoma.

Tenascin immunohistochemical staining was investigated in 82 patients with primary invasive breast carcinoma followed for at least 5 years after surgery; the staining was analyzed in relation to clinicopathological factors. An intense tenascin staining pattern was noted in 59 patients (72%), but no definite correlation was found between tenascin positivity and clinical stage, lymph node metastasis, or estrogen receptor status. However, tenascin-positive patients had a significantly better prognosis than tenascin-negative patients. Tenascin staining may therefore be a useful marker for predicting the survival of breast cancer patients.

Antithrombotic effect of argatroban on the pial vessels of the rat: a study with He-Ne laser-induced thrombus formation.

The antithrombotic effect of the synthetic thrombin inhibitor (2R,4R)-4-methyl-1-[N2-(3-methyl-1,2,3,4-tetrahydro-8-quinolinesulfon yl)-L-arginyl]-2-piperidinecarboxylic acid monohydrate (argatroban) was investigated in cerebral vessels of the rat. An occlusive thrombus was formed in pial vessels using a He-Ne laser in a closed cranial window technique. Argatroban retarded the formation of thrombi in a dose-dependent manner. The antithrombotic effect of a single intravenous dose of argatroban at 0.5 mg/kg was diminished after 30 min in arterioles and after 50 min in venules, respectively. The antithrombotic activity was maintained, however, by continuous intravenous infusion (2 mg/kg/h).

Cellular proliferation in the anterior pituitary gland of normal adult rats: influences of sex, estrous cycle, and circadian change.

Proliferative activity of the anterior pituitary gland in 10 week-old male and female rats under normal conditions was investigated by counting mitotic figures and using single and double immunostaining of 5-bromo-2'-deoxyuridine (BrdU), proliferating cell nuclear antigen (PCNA), and six pituitary hormones. To determine which proliferative changes depend on the estrous cycle and circadian changes, respectively, six groups of female and two groups of male rats were studied at various times of day. Additionally, BrdU-incorporated cells were further classified by the six types of hormones they contained, or as immunonegative cells. Cell proliferative activity in the females fluctuated drastically with the highest activity in estrus and the lowest in diestrus. In the males, proliferative activity was at a relatively low level, and was similar to that in females in proestrus or early estrus, with the greater activity at night. Identified by their pituitary hormones, the distribution of the proliferating cells was almost the same in each sex, with prolactin (PRL) cells accounting for the highest proportion, followed by growth hormone (GH) cells, and adrenocorticotropic hormone (ACTH), luteinizing hormone (LH), follicle stimulating hormone (FSH), and thyroid stimulating hormone (TSH) cells. These percentages agreed well with previously reported levels of cell types among all pituitary cells of the rat. It is therefore suggested that the life span and cycle of rat pituitary cells does not differ among cell types. In another test, male and female rats were given BrdU continuously via an osmotic pump for 8 days to compare cell proliferative activity between sexes, exclusive of the influence of estrous cycle and circadian changes. In this way, we were able to demonstrate that the cumulative incorporation of BrdU in females was consistently twice as high as in males over a constant period of time, and to conclude that cell renewal occurs at a doubled rate in the pituitary of female rat.