Participation of the nonreducing terminal beta-galactosyl residues of the neutral N-linked carbohydrate chains of porcine zona pellucida glycoproteins in sperm-egg binding.

The zona pellucida (ZP) surrounding the mammalian oocyte is composed of three glycoprotein components (ZPA, ZPB, and ZPC). Mammalian sperm bind to carbohydrate chains of a ZP glycoprotein in the initial phase of fertilization. Sperm-ligand carbohydrate chains have been characterized in mouse, cow, and pig. In pigs, triantennary/tetraantennary neutral complex-type chains from ZPB/ZPC mixture possess stronger sperm-binding activity than those of biantennary chains (Kudo et al., 1998: Eur J Biochem 252:492-499). Most of these oligosaccharides have beta-galactosyl residues at the nonreducing ends. This study used two in vitro competition assays to investigate the participation of the nonreducing terminal beta-galactosyl residues of the ligand active chains in porcine sperm binding. The removal of the nonreducing terminal beta-galactosyl residues from either the ligand active carbohydrate chains or endo-beta-galactosidase-digested glycoproteins significantly reduced their inhibition of sperm-egg binding, indicating that the beta-galactosyl residues at the nonreducing ends are involved in porcine sperm-egg binding. A correlation between the sperm-binding activity and in vitro fertilization rate is also presented.

Calmodulin binds to inv protein: implication for the regulation of inv function.

Establishment of the left-right asymmetry of internal organs is essential for the normal development of vertebrates. The inv mutant in mice shows a constant reversal of left-right asymmetry and although the inv gene has been cloned, its biochemical and cell biological functions have not been defined. Here, we show that calmodulin binds to mouse inv protein at two sites (IQ1 and IQ2). The binding of calmodulin to the IQ2 site occurs in the absence of Ca(2+) and is not observed in the presence of Ca(2+). Injection of mouse inv mRNA into the right blastomere of Xenopus embryos at the two-cell stage randomized the left-right asymmetry of the embryo and altered the patterns of Xnr-1 and Pitx2 expression. Importantly, inv mRNA that lacked the region encoding the IQ2 site was unable to randomize left-right asymmetry in Xenopus embryos, implying that the IQ2 site is essential for inv to randomize left-right asymmetry in Xenopus. These results suggest that calmodulin binding may regulate inv function. Based on our findings, we propose a model for the regulation of inv function by calcium-calmodulin and discuss its implications.

Preservation of neurotrophin expression in microglia that migrate into the gerbil's brain across the blood-brain barrier.

Microglia isolated from a mixed glial culture drawn from neonatal Mongolian gerbils were demonstrated to produce high amounts of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). The gerbil microglia retained the capability to migrate into the brain parenchyma after intra-arterial injection. We found that exogenously migrated microglia retained their BDNF and GDNF productive ability and expressed large amounts of BDNF and GDNF in damaged brain areas which suggests microglia's role as a protectant of damaged neurons. Since peripherally injected microglia exhibit specific affinity for areas of neural damage within the brain, we suggest that microglia are possible tools for cell therapy of brain damage.

A detailed deletion map of chromosome 20 in human oral squamous cell carcinoma.

Loss of heterozygosity (LOH) on the long arm of chromosome 20 (20q) has been detected in several human cancers. However, little is known about LOH on chromosome 20 in oral squamous cell carcinoma (OSCC). To determine which loci of chromosome 20 were involved in OSCC tumorigenesis, 41 cases of OSCC were examined for LOH state on chromosome 20 at 17 microsatellite loci by PCR-LOH assay. LOH occurred in 41.5% of tumors in at least one locus. Among the 17 loci, D20S48 on 20p11.2 and RPN2 on 20q12-13.1 exhibited higher frequencies of LOH, 27.6% and 31.4%, respectively. The LOH incidence was significantly higher in tumors in which the primary site was on gingiva compared with other oral sites (p=0.012). Our results indicate that allelic deletions on 20q12-13.1 and 20p11.2 may play roles in OSCC carcinogenesis, and suggest that allelic deletions on 20q might have some relation with the primary site of OSCC.

Neurosurgery at Fujita Health University, Japan.

Neurosurgery at the Fujita Health University began in 1972 with Dr. Tetsuo Kanno. In 1973, he was joined by Dr. Kazuhiro Katada and in the year 1976, an independent neurosurgery department was established with Dr. Kanno as the Chief of Neurosurgery. Under his guidance the department continued to grow and by 1978, a neurosurgical residency program recognised by the Japanese Board of Neurosurgery was established. Integration of laboratory research and clinical experience is the hallmark of this program. The current philosophy is directed towards subspecialization and academic training. This article provides a brief overview of the rapid development of a Neurosurgical Centre to reach international acclaim under the guidance of Prof. Tetsuo Kanno.

Exogenous microglia enter the brain and migrate into ischaemic hippocampal lesions.

We compared migration of systemically injected microglia into normal brain vs. ischaemic brain using a model of ischaemic hippocampal lesion. Microglia were labeled by a fluorescent dye using our standard phagocytosis procedure of microscopic particles and then injected intra-arterially into Mongolian gerbils subjected to ischaemia reperfusion neuronal injury. Delayed death of pyramidal neurons was confirmed by conventional histological analysis and dUTP nick end labeling (TUNEL) method. Clusters of dye-tagged cells migrating into the hippocampal ischaemic lesions were confirmed histochemically to be microglia. Since peripherally injected microglia exhibit specific affinity for ischaemic brain lesions and does not exacerbate ischaemic neuronal injury in the present model, we suggest that microglia may have a potential to be used as a piggy-back ride to deliver therapeutic genes and/or drugs for CNS repair following transitory global ischaemic insult.

Localization of a novel tumor suppressor gene loci on chromosome 9p21-22 in oral cancer.

Allelic imbalance or loss of heterozygosity (LOH) studies have been used to identify regions on chromosomes that may contain putative tumor suppressor genes. Deletions of chromosome 9 regions have been observed at high frequency in many other types of sporadic tumor, whereas in oral cancer no decisive information about the allelic loss on chromosome 9 has been reported. To provide detailed understanding of the genetic alterations in oral cancer, 24 highly polymorphic markers mapped on chromosome 9 were used to examine 34 cases of oral squamous cell carcinoma (SCC). LOH was detected in 18 (53%) of 34 informative samples at one or more loci examined. On the basis of our results, three commonly deleted regions were identified and a detailed deletion map was constructed. One of the novel regions was on 9p22, where a tumor suppressor gene, interferon a cluster (IFNA) gene, was identified before. Another region was D9S157 locus at 9p22, telomeric to IFNA locus and p15/16 genes, and the third was located on 9p21 of the D9S104 locus, centromelic to methylthioadenosine phosphorylase (MTAP) gene and p15/16 genes. Thus, our data suggest that, except for p15/16 and MTAP gene, there were at least two candidate tumor suppressor genes located at chromosome 9p, and that the alteration of these genes is associated with the tumorigenesis of oral SCC.

Localization of a novel tumor suppressor gene associated with human oral cancer on chromosome 4q25.

Recent cytogenetic and molecular studies with highly polymorphic microsatellite markers have implicated allele loss involving chromosome 4 in several human cancers, which suggests the presence of multiple tumor suppressor gene (TSG) loci. However, there has been no detailed analysis of loss of heterozygosity (LOH) on chromosome 4 in oral squamous cell carcinoma (OSCC). To determine the location of a putative TSG associated with OSCC on chromosome 4, polymerase chain reaction (PCR) analysis of microsatellite polymorphisms corresponding to 17 loci was performed to screen 32 patients with OSCC. LOH was observed in the majority of the tumors (75%) in at least one of the loci. The loci on the long arm exhibited a significantly higher frequency of deletions (66%) than those of the short arm (25%). Among the loci tested, frequent LOH was centered at D4S1573 on 4q25, which represents a region of about 4 centimorgans (cM). However, no commonly deleted regions were found on the short arm of the chromosome. We detected microsatellite instability (MI) in 31% of the cases. MI was also observed more frequently on the long arm (28%) than the short arm (6%). Thus, our data indicate that alterations of chromosome 4 regions, especially the long arm, are associated with OSCC tumorigenesis and that the 4q25 region may harbor at least one putative TSG.

Evidence of multi-step oncogenesis of oral squamous cell carcinoma.

We examined biopsy samples from one oral cancer and three precancerous lesions of the tongue of an 81-year old woman by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and sequence analyses using 18 oligonucleotide primer pairs of adenomatous polyposis coli (APC) gene and 5 primers of p53 gene. Normal tongue epithelium adjacent to lesions was used as a control. The four lesions harbored the common mutation of APC gene that was not detected in the control. At codon 1621 in exon 15 of the APC gene there was a C to G substitution resulting in serine (TCA) to stop codon (TGA). No mutation of p53 gene was detected in any samples of the control and three precancerous lesions of the tongue. On the other hand, an A to G substitution at codon 170 in exon 5 of p53 gene resulting in glutamic acid (ACG) to glycine (GCG) was detected in the DNA of her tongue cancer. These results may suggest that the four lesions have the same origin, and that multi-step oncogenesis had occurred, the APC gene alteration being one of the early events in the process of tumorigenesis and p53 gene alteration involved in the late events.

Brain-specific gene expression by immortalized microglial cell-mediated gene transfer in the mammalian brain.

The intra-arterial injection of immortalized microglia transfected with the lacZ gene, resulted in the expression of beta-galactosidase in the rat brain at 48 h and the activity of -galactosidase was detected for up to 3 weeks post-injection. More than 30-fold higher activity of beta-galactosidase was detected in the brain than in the liver, lung or spleen at 48 h post-injection. This method allows us to easily deliver the gene of interest to the brain without influencing other organs. Our brain-targeting gene delivery system can facilitate gene therapy of several brain disorders, including brain tumor, metabolic disorders, and degenerative disorders, as well as investigation into the roles of particular genes in brain function and development.

Allelic loss on chromosome 22 in oral cancer: possibility of the existence of a tumor suppressor gene on 22q13.

In order to understand the detail of genetic alternation on chromosome 22, we performed polymerase chain reaction analysis of microsatellite polymorphisms corresponding to 13 loci on chromosome 22. We examined 33 primary carcinoma tissues, 5 metastatic tissues and corresponding normal tissues. We detected microsatellite instability (MI) in 14 (42.4%) of 33 cases in this study. Loss of heterozygosity (LOH) was observed in at least one locus in 24 (72. 7%) of the 33 cases. Among the loci examined, LOH was restricted to D22S274 on chromosome 22q13 in 11 (40.7%) of 27 informative cases. No significant correlation between histological differentiation and LOH was observed. These observations suggest that the incidence of LOH at chromosome 22q is high and is associated with the carcinogenesis of oral squamous cell carcinoma (SCC). The D22S274 locus may play an important role in the development of oral SCC and be the site harboring a putative tumor suppressor gene.

Successful resection of arteriovenous malformations in eloquent areas diagnosed by surface anatomy scanning and motor evoked potential.

Successful resection of cerebral arteriovenous malformations (AVMs) involving the sensorimotor cortex was achieved in 17 cases. The theoretical basis for performing resection of AVMs in eloquent areas is the fact that the brain in and around the nidus about 1 mm in thickness is considered not to be functioning. It is also considered that any center of important function, when an AVM is involved, shifts to the near-by cortex from the original site. Nevertheless, it is critically important to recognize the cortex functioning as sensorimotor centers before and during operation. For this purpose, we have used surface anatomy scanning (SAS) in combination with magnetic resonance angiography. SAS is found to be very useful for the recognition of the topographical relationship between the surface anatomy and AVM. During operation, the motor cortex is identified with motor evoked potential. We have found that, in some cases, the motor center has shifted to the accessory motor cortex. With these information, it is possible to start resection of the lesion from dissection of the main feeders and dissection of the nidus from a silent cortex toward the critical area. Apparent neurological improvements were achieved in 15 of 17 patients treated surgically (88%). With this result, we think that AVMs in eloquent areas can be treated successfully when the surgery is well-designed and well-oriented with the combined use of diagnostic imaging and monitoring. As for control of intraoperative bleeding, careful attention to small but important surgical techniques avoids troublesome bleeding during AVM surgery.

Clinical efficacy of lactulose in cirrhotic patients with and without subclinical hepatic encephalopathy.

Seventy-five cirrhotic patients with hyperammonemia in the past or at the time of the study were randomly divided into two groups (treated with lactulose or nontreated) in 14 hospitals in Japan. Thirty-six cirrhotic patients were diagnosed as having subclinical hepatic encephalopathy (SHE), and 39 were diagnosed as non-SHE. SHE was diagnosed when the results of all three of the quantitative psychometric tests used (number connection test, and symbol digit and block design tests of the Wechsler adult intelligence scale [revised]) were abnormal as compared with age-matched normal values. The mean number of abnormal psychometric test results and the prevalence of SHE were used for a quantitative evaluation of the efficacy of the lactulose treatment. Twenty-two of the SHE patients were treated with lactulose (45 mL/d) for 8 weeks, and the other 14 SHE patients did not receive lactulose. In the SHE patients administered lactulose, the results of the quantitative psychometric evaluation were significantly improved at 4 and 8 weeks after the beginning of the lactulose administration. The SHE had disappeared in 10 (50%) of the 20 treated patients at week 8, but it persisted in 11 (85%) of the untreated 13 patients. We concluded that lactulose treatment in cirrhotic patients with SHE is effective with respect to psychometric tests.

Migration activity of microglia and macrophages into rat brain.

We examined the entry of intra-arterially injected microglia and macrophages into the brain using a rat muscle graft model to compare their respective abilities to invade the brain parenchyma. Isolated microglia without any activation treatment entered into the brain with or without the muscle graft, while macrophages activated by phorbol 12-myristate-13-acetate (PMA) entered the brain only in the presence of the muscle graft. These results suggest that microglia have a higher affinity for the brain than macrophages.

[Primary biliary cirrhosis (PBC)-CREST overlap syndrome complicated with Sjögren's syndrome].

We reported two sisters of primary biliary cirrhosis (PBC)-CREST overlap syndrome complicated with Sjögren's syndrome (SS). Both patients had Raynaud's phenomenon, sclerodactylia, telangiectasia, chronic sialoadenitis, chronic nonsuppurative destructive cholangitis by liver biopsy and were positive for anti-centromere antibodies. This is the first report of two sisters of PBC-CREST overlap syndrome complicated with SS.

Stereoscopic synthesized brain-surface imaging with MR angiography for localization of a peripheral mycotic aneurysm: case report.

A patient with subacute bacterial endocarditis and a peripheral mycotic aneurysm is presented. We used a combined multi-slice surface anatomy scanning (SAS) and contrast-enhanced MR angiography image to determine the exact location of the small lesion by applying a skin marker on the scalp and visualizing the relationship of the marker to the brain surface structures and to the lesion. This technique was useful for the removal of a small peripheral aneurysm using only a limited craniotomy.

Surgical treatment of internal carotid siphon aneurysms.

Surgical treatment of internal carotid artery aneurysms around the carotid siphon is discussed. The surgical approach to the aneurysms in this region, is as follows: 1. A fronto-temporal approach with the patient in a 45 degrees semi-sitting position to decrease venous pressure. 2. A Dolenc approach cutting a part of the dura mater of the superior orbital fissure to facilitate removal of the anterior clinoid process and unroofing of the optic canal. 3. Opening the medial triangle followed by transection of the optic canal dural sheath. Carotid siphon aneurysms can be divided into three groups anatomically; aneurysms of the ophthalmic segment (C2), those of the clinoid segment (C3), and those of the horizontal segment (C4). We present 29 cases of aneurysms arising from the C2 or C2/3 segment, 14 cases arising from the C3 or C3/4 segment, and 11 cases arising from the C4 segment. Anatomic localization of the aneurysms was established preoperatively by angiography and three-dimensional CT imaging. Small aneurysms of the ophthalmic segment projecting infero-medially can be clipped using a contralateral approach via the prechiasmatic root. Aneurysms of the ophthalmic segment projecting superiorly can be clipped following resection of the anterior clinoid process. The clinoid process should be resected intradurally with direct visualization of the aneurysms. Straight side-angled clips are suitable for these aneurysms. Carotid cave aneurysms, which include aneurysms of the ophthalmic segment oriented infero-medially and of the clinoid segment projecting postero-medially, can be clipped using curved fenestrated clips via Dolenc's extradural approach. For accurate clipping, opening of the medial triangle and full mobilization of the IC at the clinoid segment and optic nerve by unroofing the optic canal are required. Aneurysms of the horizontal portion are clipped after full exposure of the artery in the cavernous sinus only when the aneurysms are large and symptomatic. We used the fronto-temporal and Dolenc approaches and applied fenestrated clips to aneurysms oriented or postero-medially and straight or oblique clips to aneurysms projecting antero-laterally. Out of 40 aneurysms which underwent surgical clipping, 37 resulted in good post-operative recovery. There were three deaths secondary to complications of vasospasm and three cases with post-operative visual loss. The classification of these aneurysms and the surgical techniques we employed are discussed in detail.