IGF-I, leptin and active ghrelin levels in very low birth weight infants during the first 8 weeks of life.

AIM: We investigated the relationship between plasma insulin-like growth factor I (IGF-I), leptin, active ghrelin levels, and postnatal growth in very low birth weight (VLBW) infants. METHOD: Plasma IGF-I, leptin, and active ghrelin levels were measured at birth and at 2, 4, 6 and 8 weeks after birth in 61 VLBW infants, including 31 appropriate-for-gestational-age (AGA) and 30 small-for-gestational-age (SGA) infants. RESULTS: Insulin-like growth factor I levels were the lowest at birth, but increased gradually over the first 8 weeks of life. IGF-I was positively correlated with body weight, body length and body mass index at all time points. Leptin levels did not change over the study period. Ghrelin levels were significantly lower at birth; however, there were no significant differences between the levels after 2 weeks of age. Leptin and ghrelin levels were not correlated with anthropometrical measures. IGF-I levels at birth were significantly lower in SGA than in AGA infants, but the leptin and ghrelin levels were not significantly different between the two groups. CONCLUSION: Insulin-like growth factor I is related to length and weight gain in the prenatal and the early postnatal periods in VLBW infants, but this does not appear to be the case for leptin and ghrelin.

Does breastfeeding in the neonatal period influence the cognitive function of very-low-birth-weight infants at 5 years of age?

AIM: Breast milk is rich in docosahexaenoic acid (DHA), which is selectively concentrated in neuronal membranes and is thought to be necessary for optimal neurodevelopment. This study evaluated the relationship between breastfeeding, especially the resultant DHA level in the red blood cell (RBC) membranes of infants, and the cognitive function of very-low-birth-weight infants at 5 years of age. METHODS: Eighteen patients were classified into groups that were breastfed or formula-fed or both. We measured the DHA concentration in the RBC membranes of 18 preterm infants at 4 weeks of age. To evaluate cognitive function at the age of 5 years, we asked the children to perform five tests: the Kaufman Assessment Battery for Children, Day-Night Test, Kansas Reflection Impulsivity Scale for Preschoolers (KRISP), Motor Planning Test, and Strengths and Difficulties Questionnaire. RESULTS: The DHA level at 4 weeks after birth was significantly higher in the breastfed infants than in the formula-fed infants. The scores for the Day-Night Test, KRISP, and Motor Planning Test were significantly higher in the breastfed group. There were significant correlations between the scores for the Day-Night Test and for the KRISP and the level of DHA at 4 weeks of age. CONCLUSION: Breastfeeding in the neonatal periods increases the DHA level in preterm infants and may have an important influence on brain development not only during early infancy but also during the preschool years, especially in terms of cognitive function.

Molecular cloning and characterization of neural activity-related RING finger protein (NARF): a new member of the RBCC family is a candidate for the partner of myosin V.

Activity-dependent synaptic plasticity has been thought to be a cellular basis of memory and learning. The late phase of long-term potentiation (L-LTP), distinct from the early phase, lasts for up to 6 h and requires de novo synthesis of mRNA and protein. Many LTP-related genes are enhanced in the hippocampus during pentyrenetetrazol (PTZ)- and kainate (KA)-mediated neural activation. In this study, mice were administered intraperitoneal injections of PTZ 10 times, once every 48 h, and showed an increase in seizure indexes. Genes related to plasticity were efficiently induced in the mouse hippocampus. We used a PCR-based cDNA subtraction method to isolate genes that are expressed in the hippocampus of repeatedly PTZ-treated mice. One of these genes, neural activity-related RING finger protein (NARF), encodes a new protein containing a RING finger, B-box zinc finger, coiled-coil (RBCC domain) and beta-propeller (NHL) domain, and is predominantly expressed in the brain, especially in the hippocampus. In addition, KA up-regulated the expression of NARF mRNA in the hippocampus. This increase correlated with the activity of the NMDA receptor. By analysis using GFP-fused NARF, the protein was found to localize in the cytoplasm. Enhanced green fluorescent protein-fused NARF was also localized in the neurites and growth cones in neuronal differentiated P19 cells. The C-terminal beta-propeller domain of NARF interacts with myosin V, which is one of the most abundant myosin isoforms in neurons. The NARF protein increases in hippocampal and cerebellar neurons after PTZ-induced seizure. These observations indicated that NARF expression is enhanced by seizure-related neural activities, and NARF may contribute to the alteration of neural cellular mechanisms along with myosin V.

Frequencies of CYP2D6 mutant alleles in a normal Japanese population and metabolic activity of dextromethorphan O-demethylation in different CYP2D6 genotypes.

AIMS: To determine the frequencies of 11 CYP2D6 mutant alleles (CYP2D6*2, *3, *4, *5, *8, *10, *11, *12, *14, *17 and *18), and their relation to the metabolic capacity of CYP2D6 in Japanese subjects. METHODS: One hundred and sixty-two unrelated healthy Japanese subjects were genotyped with the polymerase chain reaction amplification method and 35 subjects were phenotyped with dextromethorphan. RESULTS: The frequencies of CYP2D6*2,*5, *10 and *14 were 12.9, 6.2, 38.6 and 2.2% in our Japanese subjects, respectively. CYP2D6*3, *4, *8, *11, *12, *17 and *18 were not detected. The mean log metabolic ratio of dextromethorphan in subjects with genotypes predicting intermediate metabolizers was significantly greater than that of heterozygotes for functional and defective alleles. CONCLUSIONS: CYP2D6*5 and CYP2D6*14 are the major defective alleles found in Japanese subjects. In addition, CYP2D6*10 may play a more important role than previously thought for the treatment of Japanese patients with drugs metabolized by CYP2D6.

Identification of the C-terminal activation domain of the NeuroD-related factor (NDRF).

NeuroD-related factor (NDRF) is a basic helix-loop-helix (bHLH) protein whose expression is restricted to the central nervous system, and is considered to be responsible for maintenance of differentiated neurons as well as neurogenesis. NDRF structurally resembles NeuroD in the bHLH region and can induce neurogenesis ectopically in ectodermal cells of the Xenopus embryo. In this study, we delineated the functional domains of NDRF. Using GAL4/NDRF fusion proteins, we identified the C-terminal activation domain (C-AD) in NDRF between amino acid positions 294 and 383. This region was highly homologous to one part of the activation domain of NeuroD. We further investigated the transactivational function of C-AD in the mouse type 1 inositol 1,4,5-trisphosphate receptor promoter, which has an NDRF site. Truncation of C-AD resulted in reduction of the activation function, whereas the DNA-binding specificity was not affected. These results suggest that C-AD has a stimulatory function in the mammalian nervous system.

Distribution of AP-2 subtypes in the adult mouse brain.

In the mammalian central nervous system (CNS), transcription factor activator protein 2 (AP-2) is one of the critical regulatory factors for neural gene expression and neural development. As AP-2 has diverged into several subtypes, i.e. AP-2alpha, -2beta, and 2.2, we investigated the distribution of the AP-2 subtypes in the adult mouse brain by in situ hybridization using subtype-specific probes. Though AP-2 was essentially expressed in most regions of the brain, the hippocampus and cerebellum Purkinje cells exhibited a relatively high concentration of transcripts of any of the AP-2 subtypes. Among AP-2alpha variants, the expression of variant 1 was considerably lower than that of variant 3. Hence, the expression pattern of AP-2alpha variant 3 is suggested to represent the major gene expression of AP-2alpha. On the other hand, the expression of AP-2beta messenger RNA (mRNA) was higher than that of AP-2alpha in many regions. Especially, the olfactory bulb, hippocampus, cerebellum, and cerebral cortex contained an abundance of these mRNAs. Different from those of AP-2alpha, AP-2beta mRNAs were detected in considerable amounts in the glanular cells as well as in Purkinje cells. AP-2.2 gene expression was weak throughout the brain. Consequently, we found that various AP-2 subtypes and variants were expressed in a similar distribution pattern with each having its own specific intensity but that their precise distribution profiles were not exactly the same. In the mature brain, AP-2 is thought to regulate neural gene expression through specific and redundant association with a target gene.

Activation of the mouse inositol 1,4,5-trisphosphate receptor type 1 promoter by AP-2.

Inositol 1,4,5-trisphosphate receptor (IP3R) functions as a Ca2+ channel that increases the intracellular Ca2+ upon binding to inositol trisphosphates. IP3R is expressed ubiquitously and consists of a multigene family. Since the type 1 IP3R (IP3R1) is highly expressed in the cerebellar Purkinje cells and moderately in hippocampus in the mammalian central nervous system (CNS), it is regarded as a neural member of this gene family. In this work, we investigated transcriptional regulation of the mouse ip3r1 gene. A DNaseI footprinting assay demonstrated that a sequence from -95 to -75, designated as box-II, was a binding site for a cerebellum-enriched factor. A consensus sequence for AP-2 was located in box-II. An electrophoretic mobility shift assay with anti-AP-2 antibody revealed that AP-2 is capable of binding to box-II. Deletion analysis of box-II showed that flanking sequences beside the box-II motif were required for the stable binding. We demonstrated by transient luciferase assay that exogenously expressed AP-2 activated box-II-dependent transcription. Moreover, we showed that endogenous AP-2 induced by retinoic acid also activated transcription via box-II in P19 cells. In-situ hybridization of the mouse brain revealed that AP-2 was predominantly expressed in the cerebellar Purkinje cells and hippocampal CA1 region, where IP3R1 is also highly expressed. From these observations, AP-2 binding to box-II is thought to be responsible for IP3R1 gene regulation in the CNS.

Transcriptional regulation of mouse type 1 inositol 1,4,5-trisphosphate receptor gene by NeuroD-related factor.

The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) is a Ca2+ channel protein that is expressed abundantly in the CNS, such as in the cerebellar Purkinje cells and hippocampus. We previously demonstrated that the box-I element, which is located -334 relative to the transcription initiation site of the mouse IP3R1 gene and includes an E-box consensus sequence, is involved in the up-regulation of such IP3R1 gene expression. Furthermore, the previous study also indicated that some CNS-related basic helix-loop-helix (bHLH) factors bind to the box-I and activate IP3R1 gene expression. In this study, we demonstrated that one of the CNS-related bHLH factors, neuronal differentiation factor (NeuroD)-related factor (NDRF), specifically bound to the box-I sequence with a ubiquitously expressed bHLH protein, E47, and activated IP3R1 gene expression. In situ hybridization of adult mouse brain revealed that IP3R1 and NDRF mRNA were co-expressed in many subsets of neurons, highly in Purkinje cells and hippocampus and moderately in cerebral cortex, olfactory bulb, and caudate putamen. Furthermore, the spatiotemporal expression patterns of these two genes resembled one another throughout postnatal development of the mouse CNS. From these results, we suggest that NDRF is involved in the tissue-specific regulation of IP3R1 gene expression in the CNS.

Enhanced expression of a new class of liver-enriched b-Zip transcription factors, hepatocarcinogenesis-related transcription factor, in hepatocellular carcinomas of rats and humans.

Rat hepatocarcinogenesis-related transcription factor (HTF) was earlier identified as a b-Zip transcription factor in chemically induced rat hepatocellular carcinoma (HCC) by cDNA subtraction, and its structure was found to be different from that of the conventional b-Zip proteins. We investigated htf gene expression in rat tissues by Northern analysis and found that HTF expression was ubiquitous but was enriched in the liver. HTF expression increased concomitantly with HCC development in rat liver, and the HTF-containing DNA-binding factor also increased. Stimulated HTF gene expression also was observed in rat regenerating livers. From the results of various assays, X-box-binding protein 1/Tax-response element binding factor 5 was suggested to be a human homologue of rat HTF. In humans, HTF gene expression was also abundant in the liver and was revealed to be specifically stimulated in HCCs, but not in other types of cancers. To our knowledge, HTF is the first example of a liver-enriched transcription factor that exhibits HCC-associated gene expression. Injection of anti-HTF antibody decreased the growth rate of cultured HCC cells. Consequently, HTF is thought to participate in hepatocyte growth as well as in hepatocarcinogenesis.

Cloning of a novel rat gene, DB83, that encodes a putative membrane protein.

Using a partial cDNA sequence and a 5'-RACE technique, we isolated a novel cDNA from rat liver referred to as DB83. DB83 had four hydrophobic trans-membrane domains and one N-myristoylation site as well as multiple possible phosphorylation sites. The db83 gene was highly expressed in the liver and significantly in brain, lungs and kidneys. We suggest that DB83 is a tissue-specific putative membrane protein. p6

Aryl C-glycosides: physiologically stable glycomimetics of sialyl Lewis X.

In the course of the search for physiologically stable, structurally simple, and low molecular weight sLeX mimetics, aryl C-glycosides with carboxylic acid functionality 2 were found to be extremely potent inhibitors against L- and P-selectins with IC50 in the low microM range.

Electronmicroscopic study of human herpesvirus 6-infected human T cell lines superinfected with human immunodeficiency virus type 1.

Human herpesvirus 6 (HHV-6) has been proposed as one of the co-factors responsible for the development of acquired immunodeficiency syndrome (AIDS) in human immunodeficiency virus (HIV) carriers. We analyzed the interaction between HHV-6 and HIV-1 in superinfected cells. Cell-free HIV-1 could superinfect human T cell lines, MT-4 and Molt-4, which had been previously infected with HHV-6. Both HHV-1 and HHV-6 replicated in the same cells. We observed two types of morphologically distinguished cells as early as 4 days after superinfection. One type (D) was degenerate cells with intracellular and extracellular HHV-6 and with less HIV-1 virions. The other type (I) was relatively intact cells with both HIV-1 and HHV-6 virions. Replication of HIV-1 was more active in the type I as compared with type D cells. The level of HIV-1 reverse transcriptase (RT) activity in the culture supernatants of cells superinfected on day 0 declined after day 7, while that in the supernatants of cell cultures infected with HIV-1 alone remained high between days 12 and 40. These results suggest that the superinfection of the HHV-6-infected cells with HIV-1 may induce a degenerative process in these cells.

Electron microscopic study of human immunodeficiency virus type 1 (HIV-1) core structure: two RNA strands in the core of mature and budding particles.

The basic structure of human immunodeficiency virus type 1 (HIV-1) has been investigated morphologically; however, the internal structure of HIV-1 core is not well understood. We studied the internal structures by transmission electron microscopy. We modified the method for electron staining of ultrathin sections and processed electron microscopic photographs using a computer. We confirmed that a mature HIV-1 particle had two copies of RNA strands in a cone-shaped core. These two RNA strands formed a coiling structure and interwound each other, and were already present in the late budding stage.

[Correlation between MR imaging and histopathological findings of cystic metastatic brain tumors].

To clarify the correlation between the histopathological findings and MR signal intensity of the cyst wall, fifteen cystic metastatic brain tumors of eleven patients were imaged using a 0.5T MR unit just before surgery, and the MRI findings were correlated with the histopathological findings of resected lesions. On T2-weighted images, all cyst walls showed hypointensity. On T1-weighted images, the intensity of the cyst wall could be classified into three groups, compared with the cerebral cortex. Walls with hyperintensity on T1WI(group H; n = 6) consisted of ample tumor cells, blood vessels and connective tissues, suggesting viable tumor cells. Iso-intense walls on T1W1(group I; n = 3)had abundant reactive glial tissues. Hypointense walls on T1W1 (group L; n = 5)revealed hemorrhage and/or hemosicerin in the wall, suggesting hemorrhagic necrosis. Thus a good correlation was demonstrated between the MR signal intensities and histopathological findings of cyst walls of cystic metastatic brain tumors. This may contribute not only to more precise diagnosis on MRI but also to more planning for treatment of cystic brain metastases.

[Surgical decompression for the C-1 and C-2 sensory roots and upper cervical cord in a case with cervical myelopathy and occipital neuralgia due to bilateral fenestration of vertebral artery: a case report].

A case of bilateral fenestration of the vertebral artery at the level of the atlas in a patient who had occipital neuralgia and cervical myelopathy is presented. MRI and vertebral angiogram demonstrated the fenestrated vertebral artery compressing the upper cervical cord. Surgical decompression for the C-1 and C-2 sensory roots and the upper cervical cord was performed. Fenestration of the vertebral artery is mostly of no clinical significance. However, considering the pathway of the fenestrated vertebral artery, it is quite possible that the fenestrated vertebral artery might compress the neural structures, resulting in some clinical problems. Although occipital neuralgia may result from a variety of causes, this case was caused by the fenestrated vertebral artery compressing the C-1 and C-2 sensory roots. The authors wish to emphasize that microsurgical vascular decompression may be the only effective treatment in such cases as well as in facial spasm and trigeminal neuralgia.

[A case of brain metastasis from hepatocellular carcinoma].

Reported is the case of a patient who underwent surgical resection of a brain metastasis from a hepatocellular carcinoma. The 62-year-old male was admitted to hospital because of headaches and a left hemiparesis. Six years earlier he had undergone transcatheter arterial embolization for a hepatocellular carcinoma. Further, one year ago the lower lobe of his right lung had been resected because of a pulmonary metastasis from the same tumor. A neurological examination on admission revealed disorientation, dressing apraxia, and a left hemiparesis. A CT scan revealed two highly dense masses with peripheral low dense areas in the right temporoparietal region, which were heterogenously enhanced with a contrast medium. Right carotid angiogram showed tumor stains in the same region. Also, a magnetic resonance T1 weighted image showed highly intense masses, and a T2 weighted image showed low intensity masses with prominent brain edema. Thus, a right fronto-temporo-parietal craniotomy was performed, and the two masses were removed. Histological examination revealed hepatocellular carcinoma. The postoperative course was uneventful, and the left hemiparesis improved gradually, enabling the patient to walk without assistance. A brain metastasis from a hepatocellular carcinoma has been rarely reported in the literature since the survival period is very short due to rapid disease progression at the primary site, so that most reports have been based on postmortem examination. The MRI, CT, and the angiographic findings are included in this report.

Lactone and cyclic ether analogues of platelet-activating factor. Synthesis and biological activities.

Six-membered lactone and tetrahydropyran analogues of platelet-activating factor (PAF), 4-11, and related antagonistic derivatives 41-46 were synthesized. None of the delta-lactones 4-7 showed PAF-like activities, while the corresponding cyclic ethers 8, 9 and 11 were slightly active. Some of the cyclic antagonists showed more potent inhibitory activities than the open chain antagonist CV-3988 against platelet aggregation (rabbit platelet-rich plasma, IC50) and hypotension (rat, ID50) induced by C16-PAF: e.g. dl-3-[6-[O-(trans-3-heptadecylcarbamoyloxytetrahydropyran-2- yl)methyl]phosphonoxy]hexylthiazolium (inner salt) (41d) (IC50 5.5 x 10(-7) M, ID50 0.046 mg/kg, i.v.); dl-3-[5-[O-(cis-3-heptadecylcarbamoylthiotetrahydropyran-2- yl)methyl]phosphonoxy]pentylthiazolium (inner salt) (43c) (IC50 5.7 x 10(-7) M, ID50 0.076 mg/kg, i.v.).