Development of new microsatellite markers from a salt-marsh sedge Carex rugulosa by compound simple sequence repeat-polymerase chain reaction.

We developed 12 polymorphic microsatellite markers from a salt-marsh sedge Carex rugulosa. The number of alleles per locus ranged from two to four, with an average of 2.75. The observed and expected heterozygosities ranged from 0.067 to 0.600 and from 0.128 to 0.620, respectively. These simple sequence repeat markers will allow the identification of genets and evaluation of the genetic diversity of C. rugulosa.

Cardiac emergency triage and therapeutic decisions using whole blood rapid troponin T test for patients with suspicious acute coronary syndrome.

Emergency triage and therapeutic decisions using the recently developed whole blood rapid troponin T test were evaluated and compared with conventional electrocardiographic (ECG) diagnosis in a total of 155 patients with chest pain who visited 16 emergency outpatient clinics in the Tokyo metropolitan area. Thirty-seven patients (23.9%) had a final diagnosis of acute myocardial infarction or high-risk unstable angina requiring emergency coronary intervention and these events were defined as acute coronary syndrome. Diagnostic values using the second-generation rapid troponin T test were evaluated according to 3 time-windows in 85 patients. The sensitivity of the test was 10% for patients assessed within 3 h after the onset, 62.5% for 3-6 h after, and 75% for more than 6h, whereas conventional ECG diagnosis had 100% sensitivity at any time-window. In contrast, the specificity of the rapid troponin T test was 100%, 100%, and 97.4%, whereas that of conventional ECG diagnosis was 25%, 57.1%, and 42.2%, respectively for the 3 time-windows. The positive predictive value of ECG diagnosis was inferior to the rapid troponin T test, which reflected the prudent attitude of physicians taking ECG decisions as positive when myocardial ischemia was suspected. The diagnostic efficacy of the rapid troponin T test was remarkable in patients with the non-ST elevation type of acute coronary syndrome. A questionnaire survey on therapeutic decisions revealed that only 10% of Tokyo outpatient institutes performed prehospital thrombolytic therapy, 30-33% administered aspirin or nitrate, and 16.7% gave heparin. The rapid troponin T test is extremely useful for cardiac emergency triage and therapeutic decision making. There is a requirement for practical guidelines for the primary therapeutic decisions for patients with suspicious acute coronary syndrome.

[Refractory non-small-cell lung cancer responding to combination chemotherapy with docetaxel, gemcitabine and cisplatin].

The combination of docetaxel (TXT), gemcitabine (GEM), and cisplatin (CDDP) produced a regression in a squamous cell carcinoma of the lung that had recurred after radiation therapy plus chemotherapy, and was resistant to the combination of carboplatin (CBDCA) with etoposide (ETP) or paclitaxel (TXL). The patient was a 62-year-old man with squamous cell lung cancer, which was first successfully treated by a combination of radiation therapy and chemotherapy, but showed local recurrence after 8 months. Although the recurrence was treated with CBDCA plus ETP and then TXL, the tumor continued to grow with symptomatic progression of airway stenosis. The tumor began to regress after the regimen of TXT, GEM and CDDP was started. This therapy achieved PR with symptomatic improvement. The combination of TXT, GEM and CDDP may be effective for recurrent non-small-cell lung carcinoma, even in patients that have failed to respond to more than one chemotherapy regimen.

Diffuse and severe left ventricular dysfunction induced by epicardial coronary artery spasm.

Endothelial dysfunction and effectiveness of treatment of calcium antagonists are suggestive of coronary artery spasm as an underlying disorder in dilated cardiomyopathy (DCM). The aim of this study is to determine whether or not the epicardial coronary artery spasm can induce severe cardiac dysfunction like DCM. Thirty-four consecutive patients with angiographically normal coronary arteries and diffuse left ventricular hypokinesis whose causes had been unknown underwent acetylcholine provocation test and left ventricular biopsy. Eight patients were excluded according to the clinical and laboratory data and biopsy findings suggesting myocarditis or other systemic diseases. According to the results of the acetylcholine provocation test, 17 patients were finally diagnosed as having DCM, and nine patients (35% of the study patients), who had acetylcholine-induced diffuse and multivessel coronary spasm, were diagnosed as having DCM-like vasospastic angina pectoris (VSA). Clinical and cardiac catheterization data including hemodynamics and biopsy findings were similar between the two groups except that left ventricular end-systolic volume was significantly greater in DCM than in DCM-like VSA. After the acetylcholine provocation test, DCM patients received both a beta blocker and an angiotensin-converting enzyme inhibitor, and DCM-like VSA patients received antianginal drugs. In echocardiographic findings at predischarge and those after 6-month drug treatment, both DCM-lke VSA and DCM showed significant reduction in end-diastolic and end-systolic diameters and significant increase in fractional shortening and ejection fraction, whereas changes in ejection fraction and fractional shortening were significantly greater in DCM-like VSA than those in DCM. Epicardial coronary artery spasm can induce diffuse and severe left ventricular dysfunction like DCM in VSA. Although antianginal drugs markedly improve left ventricular function of these patients, only the acetylcholine provocation test can identify DCM-like VSA.

Cardiac sympathetic dysfunction contributes to left ventricular remodeling after acute myocardial infarction.

To investigate the role of the cardiac sympathetic nervous system in left ventricular remodelling, 50 patients with first-time acute myocardial infarction (AMI) and patency of the infarct-related artery after reperfusion underwent quantitative iodine-123 metaiodobenzylguanidine (MIBG) imaging at 4 days and 4 weeks (n=42), and quantitative technetium-99m tetrofosmin imaging at 2 days after AMI. They also underwent both ventriculography and coronary angiography on admission and about 4 weeks after AMI. On the basis of left ventricular end-systolic volume (LVESV), patients were divided into two groups. Patients with LVESV dilatation (n=20) had a significantly lower ejection fraction (P<0.003) and a significantly higher severity score of 99mTc-tetrofosmin (P<0.04), and total severity (P<0.01), delta extent (P<0.007) and delta severity (P<0.0008) scores of MIBG than patients without LVESV dilatation (n=30). delta severity score of MIBG was directly correlated with change in LVESV at 4 weeks (r=0.63, P<0.0001). Stepwise linear discriminant function analysis showed that delta severity score of MIBG (P<0.0002) was the only discriminator of LVESV dilatation. Patients with LVESV dilatation had higher regional washout rates in both the infarct and the non-infarct zones than patients without such dilatation. Furthermore, no MIBG parameters changed significantly between 4 days and 4 weeks after AMI. In reperfused AMI, delta severity score of MIBG was related to the degree of ventricular dilatation and was the only powerful discriminator of ventricular dilatation. These results suggest that cardiac sympathetic nervous abnormality might contribute to left ventricular remodelling in reperfused AMI. MIBG imaging may allow identification of reperfused AMI patients at high risk for left ventricular remodelling.

The mechanism of the neurotransmitter release in growth cones.

The growth cone is considered the precursor of the presynaptic terminal. To elucidate the minimal molecular machinery required for exocytosis, we examined the characteristics of alpha-latrotoxin-induced exocytosis in growth cones. In isolated growth cones (IGC), neurotransmitters were released in a SNARE-dependent manner, but rab3A cycling was blocked. By supplying rabphilin, a rab3A acceptor found in low levels in IGC, the IGC obtained as high an exocytotic efficiency as adult synaptosomes, and the complete GDP-GTP conversion of rab3A occurred on growth cone vesicles (GCV). GCVs bound SNAREs but not NSF or alpha-SNAP; whereas in the rabphilin-supplied IGC, GCVs recruited both NSF and alpha-SNAP, to form the SNARE-NSF-SNAP complex. These results suggest that rab3A cycling is dependent upon the accumulation of rabphilin and is completed later than the SNARE mechanism, and that rabphilin is involved in determining the efficiency of exocytosis by modifying the SNARE mechanism.

Cell cycle arrest and astrocytic differentiation resulting from PTEN expression in glioma cells.

OBJECT: Genetic alterations of the PTEN gene (also known as MMAC1 or TEP1) have frequently been identified in high-grade gliomas, indicating that inactivation of PTEN plays a crucial role in human glioma progression. The aim of this study was to assess the biological significance of PTEN inactivation in the development of glioma. METHODS: The authors introduced wild-type PTEN complementary DNA into four human glioma cell lines (T98G, U-251MG, U-87MG, and A172) containing endogenous aberrant PTEN alleles. The number of colonies transfected with the wild-type PTEN was reduced to 15 to 32% of those found after transfection of a control vector, suggesting growth suppression by the exogenous PTEN. To analyze phenotypic alterations produced by PTEN expression, T98G-derived clones with inducible PTEN expression were further established using a tetracycline-regulated inducible gene expression system. Induction of PTEN expression suppressed the in vitro growth of T98G cells with accumulation of G1 phase cells. Furthermore, when cells were cultured in the presence of the extracellular matrix (ECM), PTEN expression caused distinct morphological changes, with multiple and elongated cytoplasmic processes similar to those of normal astrocytes. The level of glial fibrillary acidic protein, an intermediate protein specifically expressed in differentiated astrocytes, was upregulated concomitantly. CONCLUSIONS: These findings strongly indicate that exogenous PTEN expression inhibits the proliferation of glioma cells by inducing G1 arrest and elicits astrocytic differentiation in the presence of the ECM. Inactivation of PTEN would play an important role in the enhancement of unregulated growth of undifferentiated glioma cells.

[Evaluation of whole blood rapid troponin T assay: cooperative study of general practitioners and office cardiologists in Tokyo].

The recently developed whole blood rapid assay for cardiac troponin T (TROP T) was evaluated by a cooperative study of general practitioners and office cardiologists in the Tokyo area (Tokyo TROP T Trial). Seventy patients with suspected acute myocardial infarction treated at 14 medical clinics were enrolled in this study. The diagnostic efficacy of troponin T was compared with that of electrocardiographic evaluations and clinical standards for diagnosis of acute myocardial infarction. Twelve of the 70 patients (17.1%) had final diagnoses of acute myocardial infarction or severe unstable angina necessitating emergency coronary intervention. Troponin T was judged as positive when simultaneously measured serum troponin T was more than 0.25 ng/ml. Sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 58.6%, 33.3%, and 100% for electrocardiographic evaluation, and 58.3%, 100%, 100%, and 92.1% for troponin T, respectively. Presence of bundle branch block (11/70 cases) or previous myocardial infarction (14/70 cases) caused false positive readings on electrocardiography, and resulted in the lower positive predictive value. In contrast, troponin T was false in patients admitted within 4 hours after the onset, or patients with smaller infarct. The Tokyo TROP T Trial by general practitioners and office cardiologists concluded that the combination of electrocardiographic evaluation and use of troponin T is a rapid and efficient diagnostic method for the evaluation of patients with suspected acute myocardial infarction.

[Asymptomatic cerebral infarction associated with a patent foramen ovale and atrial septal aneurysm].

We reported two young adults (a 42-year-old female and a 45-year-old male) with tension type headache who had a patent foramen ovale and atrial septal aneurysm demonstrated by transesophageal echocardiography, associated with asymptomatic cerebral infarctions. There were multiple subcortical infarctions in the frontal and parietal lobes in case 1, and cerebral infarctions in the right corona radiata, head and body of the caudate nucleus, and putamen in case 2. The two cases did not have hypertension, diabetes mellitus, hyperlipidemia, cardiac diseases detected by electrocardiography and transthoracic echocardiography, and abnormality of intracranial and extracranial arteries by ultrasound sonography and cerebral angiography. Transeosophageal echocardiography revealed atrial septal aneurysm, and showed right-to-left shunt (patent foramen ovale) by Valsalva maneuver. Two cases were diagnosed as paradoxical cerebral embolism associated with a patent foramen ovale. If asymptomatic cerebral infarctions are cryptogenic stroke, a patent foramen ovale and atrial septal aneurysm should be examined by transesophageal echocardiography with Valsalva maneuver.

Stimulation of L-type Ca2+ channel in growth cones activates two independent signaling pathways.

Although growth cones respond to various modulators of neurite outgrowth, such as neurotrophins, neurotransmitters, and cell adhesion molecules, the signal-transducing mechanisms for these modulators in growth cones are unclear. Since recent studies have suggested that the signals of these modulators are mediated by Ca2+ influx through L-type voltage-sensitive Ca2+ channels (VSCCs) in the growth cone, we examined L-type VSCC-dependent signaling pathways, using isolated growth cones (IGCs) from developing rat forebrains. Binding assays revealed that L-type VSCC is enriched in growth cone membrane and gradually decreased in amount developmentally, while N-type VSCC has the opposite tendency. In intact IGCs, Bay K 8644 (BK, an L-type agonist) induced much more rapid elevation of [Ca2+]i than that in adult synaptosomes. Ca2+-dependent phosphorylation of GAP-43 and MARCKS protein by protein kinase C (PKC) was enhanced in the IGC by BK, resulting in the release of these proteins from the membrane, which is consistent with our recent report. In addition, the Ca2+-dependent degradation of brain spectrin (fodrin) by calpain was also enhanced by BK or GABA, consequently inducing the release of alpha-actinin from the membrane skeleton of the growth cones. The activities of PKC and calpain were not inhibited by inhibitors of the other, indicating that these reactions occur independently. Our results suggest that Ca2+ influx through L-type VSCCs activates two distinct signaling branches, probably in the different domains of the growth cone, i.e., Ca2+-dependent phosphorylation of GAP-43 and MARCKS protein, and Ca2+-dependent degradation of brain spectrin and the release of alpha-actinin by calpain.

Evolution of mirror images by sexually asymmetric mating behavior in hermaphroditic snails.

Directionally asymmetric animals generally exhibit no variation in handedness of whole-body architecture. In contrast, reversed chirality in both coil and entire anatomy has frequently evolved in snails. We demonstrate a nonrandom pattern and deterministic process of chiral evolution, as predicted by the following hypothesis. Bimodal shell shapes are associated with discrete mating behaviors in hermaphroditic pulmonates. Flat-shelled species mate reciprocally, face-to-face. This sexual symmetry prevents interchiral mating because genitalia exposed by a sinistral on its left side cannot be joined with those exposed by a dextral on its right. Thus, selection against the chiral minority, resulting from mating disadvantage, stabilizes chiral monomorphism. Tall-shelled species mate nonreciprocally: the 'male' copulates by mounting the 'female's' shell, mutually aligned in the same direction. This sexual asymmetry permits interchiral copulation with small behavioral adjustments. Therefore, the positive frequency-dependent selection is relaxed, and reversal alleles persist longer in populations of tall-shelled species. We verified both the assumption and the prediction of this hypothesis: significantly lower interchiral mating success in a low-spired species and higher chiral evolution rate in high-spired taxa. Sexual asymmetry is the key to understanding the accelerated chiral evolution in high-spired pulmonates.

Regeneration of hemisectioned spinal cords with and without supporting materials.

Nerve regeneration in hemisectioned spinal cords with and without supporting materials was examined in rats. Cell migration and newly formed capillaries surrounded by laminin-positive materials appeared at the lesioned site 7 days postoperatively. Reactive astrocytes increased at the lesioned site and extended into the spinal cord within 3 weeks. However, cell reactions decreased by 4 weeks and a cavity formed at the lesioned site in all animals. Regenerated axons were found only proximal to the lesioned site at 4 to 8 weeks. In the presence of supporting material (sciatic nerve containing proliferated Schwann cells or membrane filter), no cavity formed in the spinal cord and glial reactions were modest around the supporting material. Regenerated axons with growth cones were observed in the supporting material. However, no regenerated axons penetrated into the distal part of the spinal cord. Spinal cord axons can regenerate in the presence of supporting materials, but appropriate microenvironments, such as those found in the injured sciatic nerve, are required for spinal axons to traverse a lesion and penetrate into the spinal cord.

Purification and characterization of perlecan fragment in urine of end-stage renal failure patients.

We found a new spot on the two-dimensional electrophoresis pattern of the urine protein from hemodialysis patients. In order to identify the protein forming this new spot, the protein was purified by five steps of chromatography. It was shown that the amino acid sequence of this new protein from the N-terminal to the 20th amino acid was identical with the sequence from the 4197th to 4216th amino acid of perlecan, which is the core protein of the proteoglycan localizing in the systemic capillary basement membranes. It was also found that the molecular weight (25,000 daltons) of this new protein was comparable to the calculated molecular weight of the molecular region of the perlecan from the 4197th amino acid to the C-terminal. Lastly, it was shown that the antibodies against this new protein reacted with the perlecan produced by human fibroblasts. All these findings indicated that the new protein is a perlecan fragment.

Effective method for purification of lysozyme from human urine.

Lysozyme in the urine of a hemodialysis patient was purified in two steps: DEAE Sephadex chromatography followed by Sephacryl chromatography. The Sephacryl S-100 column chromatographed fraction showing lytic activity was proven to give one band on SDS-PAGE and to have a molecular mass of 14500, in agreement with that of lysozyme. The N-terminal amino acid sequence of this purified protein was identical to that of lysozyme. These results indicate that the protein purified was indeed lysozyme. The specific affinity of lysozyme for Sephacryl S-100 may explain the greater purity of the same protein isolated by this method.

Peripheral nerve regeneration in a silicone tube: effect of collagen sponge prosthesis, laminin, and pyrimidine compound administration.

Regeneration of transected peripheral nerve with a 10-mm gap encased in a silicone tube was evaluated in the presence of collagen sponge with or without laminin, or with systemic administration of a pyrimidine compound, MS-818. The sciatic nerve of 20 adult rats was transected and the proximal and distal nerve stumps were fixed in a silicone tube. The lumen of the silicone tube was empty, or filled with a collagen sponge alone or with a laminin-soaked collagen sponge. Also, a pyrimidine compound was injected intraperitoneally after implantation of the empty silicone tube. Three weeks later, the contents of the silicone tubes were processed for histological examination of regenerated nerve fibers. Other animals were observed 6, 12, and 18 months after surgery to examine the long-term effects of the collagen sponge on nerve regeneration. All animals had regenerated tissue within the tube 3 weeks after nerve transection. The diameter of the tissue decreased toward the distal stump in the empty tube, but was the same throughout the full length in the collagen sponge-containing tube. Immunohistochemical studies revealed that the nerve fibers extended beyond the midline of the regenerated tissue in animals treated with a laminin-containing collagen sponge or receiving a pyrimidine compound. Long-term observation showed the regenerated nerve was thick as the proximal stump and many neurofilament- and peripheral myelin-positive fibers were observed around the collagen sponge. Collagen sponge assists the progress of regenerated tissues in silicone tubes, and laminin-containing prostheses and administration of a pyrimidine compound enhance peripheral nerve regeneration.

[A case of the localized fibrous mesothelioma which size decreased temporarily].

A 46-year-old woman complained of a back pain and was pointed the abnormal shadow on chest X-ray by the physician, so she consulted our hospital in January 1987. After a few weeks, the size of the shadow decreased with no treatment, then we followed up her with roentgenograms. Since '91, its size had increased again. Needle biopsy did not give us the diagnosis, but we performed the operation because we suspected some malignant disease. We resected the tumor with the enough surgical margin. Its size was 4 x 3 x 3 cm, its surface was white and smooth. Its was elastic hard and had the pedicle which jointed the visceral pleura. Histological diagnosis was the localized fibrous mesothelioma.

[A case of UIP with autoimmune hemolytic anemia].

A 70-year-old male was admitted because of cough and sputum. Chest X-ray showed bilateral interstitial shadows, and usual interstitial pneumonitis was diagnosed by TBLB and BAL. He was followed without medication. The dyspnea increased and the abnormal shadow spread on chest X-ray. Autoimmune hemolytic anemia was newly diagnosed because the RBC count was 195 x 10(4)/mm3, Hb was 8.6 g/dl, and direct Coombs test and warm antibody were positive. In this case, there appears to be a relationship between deterioration of interstitial pneumonia and the on-set of autoimmune hemolytic anemia.

Treatment and prevention of tethered and retethered spinal cord using a Gore-Tex surgical membrane.

The incidence of the tethered cord syndrome after repair of spinal dysraphism is not insignificant. A retethered spinal cord may also develop after an untethering operation. In order to treat and/or prevent the tethered and retethered spinal cord, the authors developed and successfully used a new method in 12 cases. After complete release and reconstruction of the spinal cord, a Gore-Tex surgical membrane was placed over the cord and fixed to the lateral dural surface with stay sutures. During a postoperative follow-up period ranging from 23 months to 7 years, no further neurological deterioration was observed in the 12 patients and magnetic resonance imaging studies showed no adhesion of the spinal cord to the operative site. It is concluded that this simple new method is effective for the treatment and prevention of tethering and/or retethering of the spinal cord, although a longer follow-up study is required.

Regulation by glucagon of serine: pyruvate/alanine: glyoxylate aminotransferase gene expression in cultured rat hepatocytes.

The effects of glucagon on serine: pyruvate/alanine: glyoxylate aminotransferase (SPT/AGT) gene expression were studied in primary cultured rat hepatocytes. When hepatocytes had been precultured for 16-18 h under serum- and hormone-free conditions, the addition of glucagon caused (after a lag period of about 2 h) a remarkable increase in the cellular level of SPT/AGT mRNA by 4 h in a time- and dose-dependent manner. The induced mRNA was that for mitochondrial SPT/AGT, as judged by ribonuclease protection analysis. A nuclear run-on assay revealed that activation of transcription is responsible for the increase in mitochondrial SPT/AGT mRNA and that the maximal rate of transcription occurs 1.5 h after glucagon addition. The effect of glucagon was mimicked by 8-bromo-cAMP and suppressed by N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide, an inhibitor of cAMP-dependent protein kinase (protein kinase A), while both 12-O-tetradecanoylphorbol-13-acetate and A23187 were without effect in elevating the SPT/AGT mRNA level, suggesting that the cAMP/protein kinase A system is involved in the regulation of SPT/AGT gene expression. In hepatocytes precultured for 16-18 h under serum- and hormone-free conditions, the glucagon-induced transcription was severely inhibited by cycloheximide. When the preculture was for 2 h, on the other hand, the activation of transcription by glucagon was more rapid, and the inhibition by cycloheximide was less than that observed with cells precultured for 16-18 h, suggesting that a short-lived protein factor is involved in the hormonal regulation. The glucagon-induced expression of the SPT/AGT gene was also turned off by dexamethasone.