Selection of cellular genetic markers for the detection of infectious poliovirus.

AIMS: Cellular responses of an established cell line from human intestinal epithelial cells (INT-407 cells) against poliovirus (PV) infections were investigated in order to find cellular genetic markers for infectious PV detection. METHODS AND RESULTS: Gene expression profile of INT-407 cells was analysed by DNA microarray technique when cells were infected with poliovirus 1 (PV1) (sabin) at multiplicity of infection of 10-3 and incubated for 12 h. Poliovirus infection significantly altered the gene expressions of two ion channels, KCNJ4 and SCN7A. The expression profile of KCNJ4 gene was further investigated by real-time RT-qPCR, and it was found that KCNJ4 gene was significantly regulated at 24 h postinfection of PV1. CONCLUSIONS: KCNJ4 gene, coding a potassium channel protein, is proposed as a cellular genetic marker for infectious PV detection. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to show the availability of cellular responses to detect infectious PV. The selection of cellular genetic markers for infectious viruses using DNA microarray and RT-qPCR can be applicable for the other enteric viruses.

Antitumor effects of an antiangiogenic polysaccharide from an Arthrobacter species with or without a steroid.

A sulfated polysaccharide-peptidoglycan complex, DS-4152, isolated from the culture supernatant of an Arthrobacter species inhibited angiogenesis and tumor growth and enhanced the antiangiogenic activity of 11 steroid hormones by 2 to 100 times. In the presence of cortisone acetate or tetrahydro S, DS-4152 suppressed chick chorioallantoic membrane angiogenesis and murine tumor M5076 cell-induced s.c. angiogenesis. The antitumor effects of DS-4152 administered in combination with a steroid whose dose level did not affect tumor growth were examined. DS-4152 significantly inhibited the growth of s.c.-implanted B16 melanoma in combination with cortisone acetate. DS-4152 plus tetrahydro S inhibited the growth of s.c. solid tumors and prolonged the survival time of mice bearing highly metastasizing M5076. The body weight increase was not affected by any administration. On the other hand, the survival of mice with ascitic M5076 tumors was not affected by the combination of DS-4152 plus tetrahydro S. The antiangiogenic activity of DS-4152 was more potent than that of heparin. Furthermore, DS-4152 is an angiogenesis inhibitor by itself, without steroid hormones. Successive s.c. treatment with heparin caused hemorrhagic death, but with DS-4152, suppressed tumor growth without reducing body weight.

Evaluation of cerebrospinal fluid flow through a Pudenz 12 mm or a standard Rickham reservoir: phantom experiments.

We have investigated the assessment of fluid flow through a Pudenz and a Rickham shunt using radioactive tracer. Experimentally, the radioactivity clearance curve obtained, using either shunt, is defined by two exponents. The relationship between flow-rate (F) and clearance half-time (T1/2) of the first component is identical with the clearance formula (formula: see text) or log10(F) =-LOG10(T1/2) + LOG100.693V using the physical volume. The value of the second component in a Pudenz 12 mm and a standard Rickman reservoir is log10(F) = -1.3LOG10(T1/2) + 0.087 and log 10(F) =-1.8log10(T1/2) + 0.019 respectively. In clinical practice, the first component may not be recorded adequately because the interval between appearance of the first and second components is very short. To calculate the CSF flow, we recommend referring to the line of "radioactivity clearance half-time and flow-rate" of the second component instead of the calculation based on clearance formula using a physical volume.

The sulfated polysaccharide-peptidoglycan complex from an Arthrobacter species: characterization of the linkage between the two components.

Further structural features of the sulfated polysaccharide-peptidoglycan complex, which is produced by an Arthrobacter sp. and contains phosphorus as its minor component, were investigated. Phosphoric acid esters such as D-glucose 6-phosphate, glycerol 1-phosphate and muramic acid phosphate were isolated from the acid hydrolysate of the complex. On mild acid treatment, the complex became positive for both the Morgan-Elson reaction and acid phosphatase digestion. The mild acid hydrolysate readily formed the Morgan-Elson chromogen on heating at pH 7, indicating release of terminal reducing N-acetylglucosamine substituted on C-3 by adjacent sugars, and its release was accompanied by that of phosphomonoester. The complex released peptidoglycan fragments on the mild acid treatment, together with acid-degraded, sulfated polysaccharide chains with terminal reducing N-acetylglucosamine. A large proportion of phosphorus in the complex was shown to occur in the sulfated polysaccharide chains, and the rest as muramic acid phosphate in the peptidoglycan fragments. After mild acid treatment of the complex, 50% of total phosphorus was released as inorganic phosphate on phosphatase digestion of the hydrolysate. These results suggest that the sulfated polysaccharide chains, which are additionally phosphorylated to a low degree, are linked to the peptidoglycan fragments through acid-labile phosphodiester linkages, probably between (1 leads to 3)-linked N-acetylglucosamine 1-phosphate and muramic acid.

A sulfated polysaccharide produced by an Arthrobacter species.

A new sulfated polysaccharide was isolated from the culture supernatant of a strain of Arthrobacter sp. The polysaccharide purified with quaternary ammonium salts consists of D-galactose, D-glucose, sulfate, phosphorus, glucosamine, muramic acid, alanine, glutamic acid, glycine, and LL-diaminopimelic acid in a molar ratio of 56 : 9.0 : 68 : 6.4 : 2.0 : 1.1 : 2.1 : 1.0 : 1.2 : 1.2. The presence of the two amino sugars and four amino acids suggests that the polysaccharide, which is principally a galactan sulfate, contains small amounts of so-called peptidoglycan and that it is derived from the bacterial cell-wall polysaccharide. Gel filtration indicates the heterogeneity of the purified polysaccharide in its peptidoglycan content and molecular size. The molecular weight of its major portion was estimated to be 2.3 X 10(4) by gel filtration. The fractions GS-I and GS-II, and GS-4M and GS-5M, which were obtained by fractionation of the polysaccharide on Sephacryl S-200 and Dowex 1-X2 (Cl- form), respectively, gave almost the same chemical composition as the original polysaccharide, except in the peptidoglycan content, indicating that this polysaccharide is a series of complexes composed of essentially equal, sulfated polysaccharide chains and peptidoglycan fragments in their various ratios. The polysaccharide has [alpha]D -36 degrees and is composed predominantly of beta-glycosidic linkages, as judged from its specific optical rotation (-37 degrees) and the infrared absorption (885 cm-1) of its desulfated material. It exhibits a potent antithrombin activity (ID50, 0.82 micrograms/ml). A possible partial structure of the polysaccharide is also discussed, based on the results of periodate oxidation, Smith degradation, and alkali treatment.

Alleviation of axonal damage in acute spinal cord injury by a protease inhibitor: automated morphometric analysis of drug-effects.

The degenerating axons and axon terminals developed in Rexed's lamina VIII in the anterior horn of the L6 segment after acute spinal cord compression at Th11 level in rats were visualized by the method of Fink-Heimer and the extent of axonal damage was quantitatively assayed with the aid of an automated image analyzer. Leupeptin, a potent protease inhibitor, substantially reduced the extent of the axonal damage (17% on average).

Moyamoya disease associated with persistent primitive trigeminal artery. Report of two cases.

Two cases of moyamoya disease associated with persistent primitive trigeminal artery (PTA) are reported. The first patient was a 44-year-old man who experienced a sudden severe headache brought about by an intracerebral hematoma in the left temporoparietal lobe. Four-vessel study showed a right-sided PTA and moyamoya disease. The second patient was a 56-year-old woman with similar symptoms and a hematoma in the right temporoparietal lobe. Four-vessel study showed a left-sided PTA and moyamoya disease. Among the 212 PTA cases reported in the literature, none has been associated with moyamoya disease. Moreover, there are no cases of moyamoya disease among the 119 cases of persistent primitive hypoglossal artery (PHA), which is thought to be a vascular anomaly fundamentally similar to PTA. None of the 907 cases of moyamoya disease reported in Japan has been associated with either PTA or PHA. Nonetheless, the embryonic stage when PTA or PHA normally disappears partially overlaps that period when moyamoya-like vascular anomalies have been thought to arise. The possible developmental relationship between these two varieties of vascular abnormality is discussed.

Morphometric assessment of drug effects in experimental spinal cord injury.

The effect of large doses of methylprednisolone sodium succinate (MPSS) and two protease inhibitors, leupeptin and bestatin, on experimental acute spinal cord injury was evaluated by morphometric analysis of degenerating axons with the aid of an automated image analyzer. Spinal cord injury was produced by epidural compression with a surgical clip on the T-11 segment in rats. The extent of axonal damage was assessed in Rexed's lamina VIII in the L-6 segment by measuring the amount of silver grains, representing degenerating axons and their terminals, using the Fink-Heimer method. The severity of axonal damage was expressed as the degeneration index: that is, the amount of silver grains in experimental animals/the amount of silver grains in cord-transected animals. When examined on the 7th postoperative day, axonal degeneration in MPSS-treated rats was significantly decreased, with an average degeneration index difference of 6 (p less than 0.05). Increased preservation of axons was seen in the leupeptin-treated rats sacrificed 7, 10, and 14 days after trauma. The difference in the degeneration index between the leupeptin-treated and untreated groups was 16 on Day 7 (p less than 0.001), 12 on Day 10 (p less than 0.001), and 13 on Day 14 (p less than 0.01). Bestatin had no beneficial effect. The implications for the use of calcium-activated neutral protease inhibitors in acute spinal cord injury are discussed.

Anterior osteophytectomy for cervical spondylotic myelopathy in developmentally narrow canal.

The authors present 19 cases of cervical spondylotic myelopathy in patients with developmentally narrow canal treated by microsurgical anterior osteophytectomy with interbody fusion, with follow-up periods of 1 to 8 years (mean 38 months). Postoperatively, the lower limb function, evaluated by Nurick's six-grade classification, improved two or three grades in 16 cases, one grade in two cases, and remained unchanged in one case. The upper limb function, evaluated by the authors' own four-grade classification, improved two or three grades in 11 cases, one grade in seven cases, and remained unchanged in one case. No deterioration caused by the osteophytectomy was seen. During the follow-up period, spondylolisthesis appeared 31 months postoperatively in one patient and soft disc hernia occurred 66 months postoperatively in another; these two patients were treated by a second operation and cervical traction, respectively. The authors conclude that anterior osteophytectomy with interbody fusion is applicable as a surgical treatment of cervical spondylotic myelopathy even where developmental canal stenosis is present.

Experimental spinal cord injury: quantitation of axonal damage by automated image analysis.

The severity of acute experimental spinal cord injury in rats was assessed quantitatively with the aid of an automated image analyzer by measuring the amount of degenerating axons that had developed distal to the site of mechanical insult. Spinal cord injury was produced in adult male rats by epidural compression at T-11 with a Biemer vascular clip. On the 7th postoperative day, the animals were graded according to the degree of hindlimb motor deficit, as follows: Grade 0: normal (three rats); Grade 1: crawling with difficulty (10 rats); Grade 2: some voluntary movement (nine rats); and Grade 3: no voluntary movement (nine rats). The rats were then sacrificed. The L-6 segment was chosen for selective silver impregnation of degenerating axons by the Fink-Heimer method. Silver grains, representing degenerating axons and their terminals, were accumulated in the descending tracts and in Rexed's laminae VII and VIII. The extent of axonal damage was expressed by the percentage of the area occupied by silver grains in Rexed's lamina VIII. The area occupied by silver grains was 17.0% (mean) in Grade 0 rats, 22.3% +/- 2.63% (mean +/- standard deviation) in Grade 1 rats, 28.7% +/- 3.35% in Grade 2 rats, and 35.9% +/- 2.76% in Grade 3 rats. The severity in Grade 3 rats was close to that of rats with transected cords (37.6% +/- 0.89%). The differences among the groups were statistically significant (p less than 0.001). This method may serve as a useful tool for the objective assessment of therapeutic modalities in large series of small experimental animals.

A microsurgical anterior osteophytectomy for cervical spondylotic myelopathy.

Cervical spondylotic myelopathy was treated by a microsurgical anterior approach removing osteophytes completely. Follow-up results of 43 patients showed that 39 patients (91%) exhibited overall improvements of either 1, 2, or 3 grades on Nurick's grading. Four (9%) remained unchanged. Even severely affected myelopathic patients exhibited neurologic recovery. Cases that also had narrow cervical canals were treated by the same anterior osteophytectomy, and they showed similar responses. Six out of ten patients who had osteophytectomies without fusion developed neck and arm pain postoperatively, although the presence or absence of an interbody fusion did not affect the postoperative recovery from myelopathy.

Structural studies on an antitumor polysaccharide from Microellobosporia grisea.

The structure of the antitumor polysaccharide from the actinomycete Microellobosporia grisea has been investigated. By methylation and periodate-oxidation studies, the polysaccharide was shown to consist of (nonreducing) D-mannosyl groups, (1 leads to 4)-linked D-glucosyl residues, and 3,6-branched, (1 leads to 4)-linked D-glucosyl residues in the approximate molar ratios of 2:1:1. Periodate oxidation of the polysaccharide, followed by borohydride reduction and mild hydrolysis with acid yielded glycerol, erythritol, 2-O-beta-D-glucopyranosyl-D-erythritol, and 5-O-beta-D-glucopyranosyl-2,4-bis(hydroxymethyl)-1,3-dioxane, which were isolated in the molar ratios of 2.0:0.14:0.74:0.35. Partial hydrolysis of the polysaccharide gave alpha-D-Manp-(1 leads to 6)-D-Glcp, beta-D-Glcp-(1 leads to 4)-D-Glcp, alpha-D-Manp-(1 leads to 3)-D-Glcp, and beta-D-Glcp-(1 leads to 4)-[alpha-D-Manp-(1 leads to 3)-]-D-Glcp. From these results, it is proposed that the polysaccharide is mainly composed of tetrasaccharide repeating-units having the following structure. (formula: see text)

Structure-antitumor activity relationship of a D-manno-D-glucan from Microellobosporia grisea: effect of periodate modification on antitumor activity.

An antitumor D-manno-D-glucan from Microellobosporia grisea, an actinomycete, has a tetrasaccharide repeating-unit structure, a single alpha-D-mannosyl group being located at both O-3 and O-6 of every other beta-D-(1 leads to 4)-glucosyl residue. The D-mannosyl groups and D-glucosyl residues of the mannoglucan were polyhydroxylated to various extents by controlled periodate oxidation followed by borohydride reduction. The derivatives (PA mannoglucans) were further subjected to mild hydrolysis with acid, to give partially debranched mannoglucans (PA-H mannoglucans). These derivatives were tested for antitumor activity against Ehrlich carcinoma solid tumor in mice. The PA mannoglucans having degrees of polyhydroxylation of less than approximately 50 and 2% of the D-mannosyl groups and D-glucosyl residues, respectively, showed high antitumor activities, similar to that of the original mannoglucan, whereas further polyhydroxylation resulted in a marked decrease in, or complete loss of, the activity. The PA-H mannoglucans, lacking 5-40% of the D-mannosyl branches, still had potent antitumor activities, comparable to that of the original mannoglucan. On the basis of these results, the relationship of the structure of the mannoglucan to the antitumor activity is discussed.

The sulfated polysaccharide-peptidoglycan complex potently inhibits embryonic angiogenesis and tumor growth in the presence of cortisone acetate.

In combination with cortisone acteate, the sulfated polysaccharide (SP)-peptidoglycan (PG) complex produced by an Arthrobacter species was found to inhibit both embryonic angiogenesis of chick chorioallantoic membrane (CAM) and growth of solid Sarcoma 180 tumor in mice. Three fractions obtained from the SP-PG complex by gel filtration showed a great difference in the inhibitory effect on angiogenesis of the CAM, whereas in ex vivo study such a difference was reduced. Of the three fractions, SP-PG-L having the lowest molecular weight and peptidoglycan content exhibited the highest antiangiogenic and antitumor activities, even higher than those of heparin. Neither desulfation nor sulfation of the SP-PG complex greatly affected the antiangiogenic activity, and dextran sulfates and mannoglucan sulfates were lacking the activity. This suggested that glycosyl sequences of sulfated polysaccharides are more important for the activity, rather than content or intramolecular distribution of sulfate groups.

Roentgenological study of the sagittal diameter of the cervical spinal canal in normal adult Japanese.

The sagittal diameter of the cervical spinal canal on roentgenograms in normal adult Japanese aged 15 years or over, 505 males and 492 females, was investigated to define the normal distribution and lower limit. Lateral roentgenograms of cervical spinal canals were taken at a constant focus-film distance of 1.5 m. The mean +/- SD magnification coefficient was 1.17 +/- 0.02. The mean +/- SD sagittal diameters of the cervical spinal canals at each vertebral level were: C-1, 21.0 +/- 2.2 mm; C-2, 18.0 +/- 1.7 mm; C-3, 15.8 +/- 1.5 mm; C-4, 15.2 +/- 1.5 mm; C-5, 15.3 +/- 1.5 mm; C-6, 15.7 +/- 1.5 mm; and C-7, 15.9 +/- 1.4 mm. The lowest mean -2 SD values were: C-1, 16.6 mm; C-2, 14.6 mm; C-3: 12.8 mm; C-4, 12.2 mm; C-5, 12.3 mm; C-6, 12.7 mm; and C-7, 13.1 mm. The smallest diameter was at the C-4 level, but there was no significant difference between values at the C-4 and C-5 levels. Males had significantly larger diameters than females (mean difference 0.8 mm) (p < 0.01). Younger subjects had greater diameters than older subjects. The incidence of spondylotic changes was 40.1% in subjects aged 50-59 years, 57.7% in those aged 60-69 years, and 76.6% in those aged 70-79 years. Males had a higher incidence than females. Ossification of the posterior longitudinal ligament was observed in 2.1% of all subjects. This study suggests that patients with a sagittal diameter in the cervical spinal canal of less than 12 mm have a high risk of cervical myelopathy.

Extraneural metastasis of intracranial germinoma with syncytiotrophoblastic giant cells--case report.

A 14-year-old boy presented with an extraneural metastasis of germinoma with syncytiotrophoblastic giant cells (STGC). The tumor was located in the pineal area, suprasellar cistern, and frontal base, and associated with the slight elevation of the beta-human chorionic gonadotropin (HCG) serum level. Following radiation therapy, the tumor completely disappeared, and the beta-HCG level decreased to normal. Two years after the initial treatment, metastasis of the tumor to the left femur was observed without recurrence of the primary lesion. The biopsy specimen revealed germinoma, the diagnosis was germinoma with STGC because of the associated elevation of HCG level. The metastatic tumor was treated by chemotherapy with cisplatin and etoposide, and the tumor completely disappeared. With adjuvant chemotherapy, he has been recurrence-free for 3 years. Extraneural metastasis occurs more frequently from germinoma with STGC than germinoma. Aggressive chemotherapy combined with radiation therapy in the initial treatment might have to be tried to avoid metastasis from germinoma with STGC.

Primitive neuroectodermal tumor in the spinal epidural space--case report.

A 4-year-old boy presented with a rare case of an epidural tumor causing compression of the thoracic spinal cord manifesting as rapid worsening of gait disturbance. The tumor was grossly totally resected, and radiotherapy and chemotherapy were subsequently administered. The histological diagnosis was primitive neuroectodermal tumor. He has been free of recurrence for more than 6 years. Early tumor resection followed by chemotherapy and irradiation is recommended for patients with primitive neuroectodermal tumor and unstable symptoms.

[Magnetic resonance imaging of diseased cervical and lumbar intervertebral discs].

Magnetic resonance images (MRI) of diseased cervical and lumbar intervertebral discs involving both intrinsic and extrinsic cord lesions were examined using either a 0.15 T resistive or a 0.5 T superconductive magnetic imaging system. High resolution images were obtained by means of a surface coil in most cases. The vertebrae, intervertebral discs, and spinal cord were delineated in greatest detail on spin-echo (SE) images with a long repetition time (TR) and a short echo time (proton density-weighted image), on which the spinal cord was appreciated without overshadowing by the cerebrospinal fluid-filled subarachnoid space. Protrusion of degenerated intervertebral discs into the spinal canal was clearly demonstrated not only on sagittal but also on parasagittal and transverse views. The location of protruded discs and compression of the spinal cord, caudal sac, and nerve roots were well visualized three-dimensionally. Pathological features of intervertebral discs were better appreciated on T2-weighted images with long TR and SE pulse sequences. Degeneration of intervertebral discs resulted in decreased signal intensity in cases involving lumbar disc lesions but not those involving cervical disc lesions. In a case of suspected myelomalacia, the intrinsic cord lesion resulting from traumatic disc protrusion appeared as focal low signal intensity on T1-weighted images and as somewhat high intensity on T2-weighted images. The inversion recovery sequence with median inversion time displayed an inferior image of low contrast and was judged uninformative in comparison to SE images. The authors' observations demonstrate that MRI is an essential diagnostic technique for spinal cord disorders.(ABSTRACT TRUNCATED AT 250 WORDS)

[An operative procedure of anterior osteophytectomy with interbody fusion].

Surgical anatomy of an anterior approach to the cervical spine, together with osteophytectomy for cervical spondylotic radiculomyelopathy, was described. Recognition of fascial compartments is important to avoid unnecessary injuries to the vital structures such as oesophagus, trachea, carotid artery and recurrent nerve. Detailed operative procedures of microsurgical osteophytectomy removing all the compressing components against the cervical cord and radicula was also described. Biomechanics of the cervical spine must be borne in mind beforehand when cervical spine operation is considered.

[EC-IC bypass surgery using a long vein graft--reconstructive procedures for the occluded long vein grafts].

EC-IC bypass using a long vein graft has an advantage creating a large amount of blood flow immediately after the anastomosis, but on the other hand, disadvantage of relatively frequent incidence of the vein graft occlusion. In this report, we present three kinds of reconstructive operative procedures for the stenotic or occluded long vein grafts. Type A: A long vein graft bypass between external carotid and posterior cerebral artery was found occluded intraoperatively by the thrombosis occurred where the vein graft was injured during harvesting. Reconstruction was made simply by resecting the occluded segment of the graft and end-to-end suturing. Type B: A long vein graft used in subclavian artery-middle cerebral artery bypass was occluded three days postoperatively at the supraclavicular fossa by bleeding from the anastomosis site. The vein graft was found compressed and thrombosed. We reconstructed the occluded bypass by resecting the occluded supraclavicular segment and interposing a short vein graft with end-to-end anastomosis. Then, thrombectomy of the remaining vein graft was followed. Type C: A long vein graft used in external carotid-middle cerebral artery bypass stenosed at the anastomosis site with the external carotid artery a day after the operation. The stenotic bypass was successfully reconstructed by bridging a new short vein graft from another portion of the external carotid artery on the long vein graft distal to the stenotic site. All the long vein grafts we have done reconstructive surgery have been working well one to four years follow-up periods. So, we conclude that whenever a long vein graft occludes it should be reconstructed promptly before the vein graft becomes necrotic.