An isoform of eIF4E is a component of germ granules and is required for spermatogenesis in C. elegans.

Control of gene expression at the translational level is crucial for many developmental processes. The mRNA cap-binding protein, eIF4E, is a key player in regulation of translation initiation; appropriate levels of eIF4E are essential for normal cell-cycle regulation and tissue differentiation. The observation that eIF4E levels are elevated during gametogenesis in several organisms suggests that eIF4E might have a specific role in gamete formation as well. We show that one of the five isoforms of C. elegans eIF4E, IFE-1, is enriched in the germline and is a component of germ granules (P granules). The association of IFE-1 with P granules requires the P-granule protein PGL-1. In vitro PGL-1 interacts directly with IFE-1, but not with the other four isoforms of eIF4E. Analysis of animals depleted of IFE-1 by RNAi shows that IFE-1 is required for spermatogenesis, specifically for efficient progression through the meiotic divisions and for the production of functional sperm, in both hermaphrodites and males. The requirement for IFE-1 is highly sensitive to temperature. IFE-1 is not required for oogenesis, as ife-1(RNAi) hermaphrodites produce viable progeny when normal sperm are supplied. Consistent with a primary role in spermatogenesis, ife-1 mRNA levels are highest in regions of the gonad undergoing spermatogenesis. Our results suggest that C. elegans spermatogenesis requires either this specific isoform of eIF4E or an elevated level of eIF4E.

The first total synthesis of (+/-)-linderol A, a tricyclic hexahydrodibenzofuran constituent of Lindera umbellata bark, with potent inhibitory activity on melanin biosynthesis of cultured B-16 melanoma cells.

[reaction in text] The first total synthesis of (+/-)-linderol A, a hexahydrodibenzofuran isolated from Lindera umbellata bark, with potent inhibitory activity on melanin biosynthesis of cultured B-16 melanoma cells was achieved via a 20-step of reaction in 7.64% overall yield starting from 4,6-dimethoxysalicylaldehyde.

The lacZ gene under the control of the 7 kb of human dystrophin muscle specific promoter is expressed in cardiac muscle but not in adult skeletal muscle in transgenic mice.

In previous transgenic studies, we reported a 0.9 kb fragment from a mouse dystrophin muscle promoter that contains the regulatory elements required for expression of dystrophin only in the right heart. In this study, to further characterize the regulation of muscle type of promoter, we analyzed promoter activity and tissue specificity using a total 14 kb fragment around the human dystrophin muscular-specific exon 1 in vitro and in vivo. In vitro analysis showed that the lacZ construct of the 7 kb promoter and 7 kb intron 1 was expressed 2.5 times as strong as the lacZ construct of only the 7 kb promoter in C2/4 myotubes. In vivo analysis revealed expression of both constructs in the whole heart, skeletal muscle and vascular smooth muscle in embryos. However, in adults, the expression in skeletal muscle disappeared. We conclude that the 7 kb upstream region and the 7 kb intronic region included responsible elements for the expression in the heart, but not in skeletal muscle in vivo. It is possible that a strong enhancer element for skeletal muscle exists in some other region.

[Intravenous pamidronate delivery to a case with multiple bone metastasis of breast cancer].

MRI and bone scintigraphy of a 64-year-old woman admitted with severe lumbago showed multiple metastatic bone cancer mainly on vertebrae, and breast cancer was found by mammography. After enucleation was performed, treatment with tegafur, tamoxifen and oral bisphosphonate/etidronate was started. Because symptoms associated with bone metastasis worsened, we began to administer 30 mg of pamidronate intravenously every 4 weeks. Since that time the extent of metastasis has been inhibited, resulting in ameliorated lumbodynia and improved quality of life.

Reductive dimerization of alkylidenemalonates using samarium(II) diiodide and 1H-NMR behavior of the dimers, 2,3-diaryl-1,1,4,4-butanetetracarboxylates.

Alkylidenemalonates were readily dimerized in the presence of SmI2 to give 2,3-disubstituted 1,1,4,4-butanetetracarboxylates as mixtures of meso and racemic isomers in moderate to good yields. The structure of the less polar isomer of tetraethyl 2,3-diphenyl-1,1,4,4-butanetetracarboxylate was determined by X-ray crystallographic analysis to be the meso form. Characteristic 1H-NMR behavior of the meso and racemic isomers is also discussed.

Synapsis and chiasma formation in Caenorhabditis elegans require HIM-3, a meiotic chromosome core component that functions in chromosome segregation.

Meiotic chromosomes are organized about a proteinaceous core that forms between replicated sister chromatids. We have isolated a Caenorhabditis elegans gene, him-3, which encodes a meiosis-specific component of chromosome cores with some similarity to the yeast lateral element protein Hop1p. Antibodies raised against HIM-3 localize the protein to condensing chromosomes in early prophase I and to the cores of both synapsed and desynapsed chromosomes. In RNA interference experiments, chromosomes appear to condense normally in the absence of detectable protein but fail to synapse and form chiasmata, indicating that HIM-3 is essential for these processes. Hypomorphs of him-3, although being synapsis proficient, show severe reductions in the frequency of crossing-over, demonstrating that HIM-3 has a role in establishing normal levels of interhomolog exchange. Him-3 mutants also show defects in meiotic chromosome segregation and the persistence of the protein at the chromosome core until the metaphase I-anaphase I transition suggests that HIM-3 may play a role in sister chromatid cohesion. The analysis of him-3 provides the first functional description of a chromosome core component in a multicellular organism and suggests that a mechanistic link exists between the early meiotic events of synapsis and recombination, and later events such as segregation.

[Three cases with low levels of serum copper due to long-term enteral nutrition].

Three bed-ridden patients who had had only one kind of enteral nutrition without sufficient copper element during a few years showed very low levels of serum copper. Two of them also had leukopenia. The abnormal findings disappeared after the feeding nutrients rich in copper element. The leukopenia may have been due to copper deficiency rather than zinc deficiency. We confirmed that long-term parenteral nutrition must contain trace elements, for example copper.

Rapid increase in cytosolic calcium ion concentration mediated by acetylcholine receptors in cultured retinal neurons and Müller cells.

PURPOSE: This study was conducted to detect the presence of muscarinic or nicotinic receptors in cultured retinal neurons and Müller cells. METHODS: Pure Müller cell cultures and cocultures of retinal neurons and Müller cells were used; the former, obtained from adult rabbit retinas, and the latter, retinal neurons from neonatal rats, were cocultured with Müller cells. Intracellular calcium ion concentration ([Ca2+]i) following the administration of acetylcholine, a cholinesterase inhibitor (trichlorfon), nicotine or muscarinic agonist with or without a receptor antagonist was monitored using the calcium ion indicator, fura-2. RESULTS: Acetylcholine and trichlorfon induced rapid increase in [Ca2+]i in half of either cell type. Trichlorfon induced positive response in coculture but not in the pure Müller cell cultures. This positive response was blocked only partially in the presence of atropine. Approximately 30-40% of neurons responded to nicotine at 5 microM, which was significantly blocked by alpha-bungarotoxin at 50 nM. No response to nicotine could be detected in Müller cells. Approximately 50% of neurons responded to muscarine at 50 microM, but 500 microM was required for the formation of calcium transients in 50% of Müller cells. The muscarine inducement of rapid increase in [Ca2+]i was blocked by atropine. The agonist of M1 (a muscarinic receptor subtype), McN-A-343, at 0.5 microM induced the most significant and rapid increase in [Ca2+]i both in neurons and Müller cells. McN-A-343 administration at 0.05 microM induced positive response in half the neurons, but only in approximately 10% of Müller cells. Such positive response was not observed following preincubation with the M1 antagonist, pirenzepine, at 50 microM. CONCLUSIONS: Cocultured retinal neurons enhance the release of acetylcholine following anticholinesterase administration, and approximately half the neurons were found to possess muscarinic and nicotinic receptors. However, Müller cells appeared to possess only the less sensitive muscarinic receptor. Muscarinic receptor subtypes on either type of cell contained at least M1.

PGL-1, a predicted RNA-binding component of germ granules, is essential for fertility in C. elegans.

Germ cells are distinct from somatic cells in their immortality, totipotency, and ability to undergo meiosis. Candidates for components that guide the unique germline program are the distinctive granules observed in germ cells of many species. We show that a component of germ granules is essential for fertility in C. elegans and that its primary function is in germline proliferation. This role has been revealed by molecular and genetic analyses of pgl-1. PGL-1 is a predicted RNA-binding protein that is present on germ granules at all stages of development. Elimination of PGL-1 results in defective germ granules and sterility. Interestingly, PGL-1 function is required for fertility only at elevated temperatures, suggesting that germline development is inherently sensitive to temperature.

Possible roles of AMPA/KA receptor in cultured Müller cells.

In order to identify the function of the AMPA/KA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate) receptor in cultured Müller cells, we studied the difference in the affinity of this receptor for cultured Müller cells and retinal neurons (neurons), comparing these to the characteristics of cultured Müller cells exposed to kainate (KA), a neurotoxic amino acid. The difference in the cellular responses of Müller cells and neurons to AMPA was evaluated by measuring the rapid change in intracellular calcium ions. Results showed that neurons were more sensitive to AMPA than Müller cells; the percentage of cells responding to AMPA was always higher for neurons. When Müller cells were exposed to 0.5 mM KA, the morphology of the cells was not affected and the concentration of lactate dehydrogenase in the culture solution did not increase. However, the percentage of cells responding to a high concentration of AMPA was higher in the Müller cells exposed to KA than in those not exposed. These findings indicated that the affinity of the AMPA receptor was lower in Müller cells than in neurons, and that Müller cells were resistant to neurotoxic amino acids. It was also suggested that when an extremely large amount of neurotoxic amino acid was released into the retina as a result of ischemia, Müller cells actively protected the neurons.

[Possible role of the AMPA/KA receptors in cultured Müller cells].

AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate)/KA (kainate) receptors have been demonstrated in cultured Müller cells in a study on cytosolic free calcium ions ([Ca2+]i). The action of these receptors may be expressed under pathological rather than physiological conditions. 1. Critical concentration in response to AMPA was determined in retinal neurons and Müller cells. At 0.05mM AMPA, in all neurons and in only a limited number of Müller cells (20%) cytosolic calcium transients occurred. 2. The level of lactate dehydrogenase (LDH) and the change in [Ca2+]i following AMPA administration were measured before and after exposure to 0.5mM KA. Neither morphological change nor leakage of LDH could be detected after 24 hours. When AMPA was administered, the responsive cell number was higher for KA-exposed cells than for control cells. Müller cells may be concluded to resist neurotoxic agents and may possibly be involved in the survival mechanism of retinal neurons.

Complete atrioventricular block during left heart catheterization.

A 75-year-old male underwent cardiac catheterization for frequent ventricular premature contractions and reduced left ventricular function. ECG on admission showed complete right bundle branch block (RBBB) and left anterior hemiblock. Right heart catheterization was performed uneventfully, but complete atrioventricular block (CAVB) occurred suddenly when a pig-tail catheter was inserted into the left ventricle. Electrophysiological study identified this CAVB as HV block, and demonstrated HV prolongation in sinus rhythm. The coronary angiogram revealed no obstructive lesion or spasm with ergonovine. The reproducibility of the CAVB was demonstrated. The final CAVB developed and persisted for more than one week, requiring the implantation of a permanent pacemaker (DDD). Complete atrioventricular block induced during left heart catheterization is very rare. Pre-existing RBBB with left anterior or posterior hemiblock and reduced left ventricular function may be common factors in patients with this condition. Emergency pacing equipment should always be on-line when left heart catheterization is conducted in such patients.

Synthetic approaches to 3-amino-1,4-naphthoquinone-2-carboxylic acid derivatives and photochemical synthesis of novel 1,4,5,10-tetrahydro-5,10-dioxo-2H-naphth[2,3-d][1,3]oxazine derivatives.

Synthesis of 3-alkylamino-1,4-naphthoquinone-2-carboxylic acid (2) was attempted. Although 3-(1-piperidinyl)-1,4-naphthoquinone-2-carboxylic acid (2a) was not obtained, probably because of its instability, the esters (18a, b) of 2a could be prepared. 2-Alkylamino-3-hydroxymethyl-1,4-naphthoquinones (9a-g) were photochemically cyclized to the 1,4,5,10-tetrahydro-5,10-2H-naphth[2,3-d][1,3]oxazines (19a-g), containing a novel heterocyclic ring system in moderate yields. Anti-bacterial activity of the prepared compounds was weak or insignificant.

Homologous recombination of monkey alpha-satellite repeats in an in vitro simian virus 40 replication system: possible association of recombination with DNA replication.

To study homologous recombination between repeated sequences in an in vitro simian virus 40 (SV40) replication system, we constructed a series of substrate DNAs that contain two identical fragments of monkey alpha-satellite repeats. Together with the SV40-pBR322 composite vector encoding Apr and Kmr, the DNAs also contain the Escherichia coli galactokinase gene (galK) positioned between two alpha-satellite fragments. The alpha-satellite sequence used consists of multiple units of tandem 172-bp sequences which differ by microheterogeneity. The substrate DNAs were incubated in an in vitro SV40 DNA replication system and used to transform the E. coli galK strain DH10B after digestion with DpnI. The number of E. coli galK Apr Kmr colonies which contain recombinant DNAs were determined, and their structures were analyzed. Products of equal and unequal crossovers between identical 172-bp sequences and between similar but not identical (homeologous) 172-bp sequences, respectively, were detected, although those of the equal crossover were predominant among all of the galK mutant recombinants. Similar products were also observed in the in vivo experiments with COS1 cells. The in vitro experiments showed that these recombinations were dependent on the presence of both the SV40 origin of DNA replication and SV40 large T antigen. Most of the recombinant DNAs were generated from newly synthesized DpnI-resistant DNAs. These results suggest that the homologous recombination observed in this SV40 system is associated with DNA replication and is suppressed by mismatches in heteroduplexes formed between similar but not identical sequences.

Calf thymus histone H1 is a recombinase that catalyzes ATP-independent DNA strand transfer.

An activity that catalyzes the strand transfer from linear double-stranded tetracycline-resistance gene (tetr) DNA to circular M13mp8-tetr viral DNA was detected in a crude extract from calf thymus. This activity was purified to near, if not complete, homogeneity as judged by NaDodSO4/polyacrylamide gel electrophoresis. We have tentatively named this protein calf thymus strand-transfer protein 1 (CTST1). The apparent molecular mass of the protein was 35 kDa by gel electrophoresis. Its sedimentation coefficient was approximately 1.5 S in glycerol gradient centrifugation. These values led us to examine the possibility that CTST1 is histone H1. Western blot analysis of CTST1 with anti-rat liver histone H1 antiserum showed that CTST1 crossreacts with the serum, indicating that CTST1 is histone H1. The mobility of CTST1 was identical to one of the subtypes of calf thymus histone H1 by NaDodSO4/polyacrylamide gel and acetic acid/urea/polyacrylamide gel electrophoreses. We have also confirmed the above conclusion by showing that calf thymus histone H1 has a strand-transfer activity with a specific activity comparable to that of CTST1. The reaction required homologous substrates, but neither Mg2+ nor ATP. The reaction also required stoichiometric amounts of protein. The purified CTST1 fraction lacked detectable exo- and endonuclease activities and also lacked a DNA helicase activity.

Illegitimate recombination mediated by calf thymus DNA topoisomerase II in vitro.

We have found that purified calf thymus DNA topoisomerase II mediates recombination between two phage lambda DNA molecules in an in vitro system. The enzyme mainly produced a linear monomer recombinant DNA that can be packaged in vitro. Novobiocin and anti-calf thymus DNA topoisomerase II antibody inhibit this ATP-dependent recombination. The recombinant molecules contain duplications or deletions, and most crossovers take place between nonhomologous sequences of lambda DNA, as judged by the sequences of recombination junctions. Therefore, the recombination mediated by the calf thymus DNA topoisomerase II is an illegitimate recombination that is similar to recombination mediated by Escherichia coli DNA gyrase or phage T4 DNA topoisomerase. The subunit exchange model, which has been suggested for the DNA gyrase-mediated recombination, is now generalized as follows: DNA topoisomerase II molecules bind to DNAs, associate with each other, and lead to the exchange of DNA strands through the exchange of topoisomerase II subunits. Illegitimate recombination might be carried out by a general mechanism in organisms ranging from prokaryotes to higher eukaryotes.

Squamous cell carcinoma found in the cardiac region of the stomach of an aged orangoutang.

A spontaneous tumor was found in the cardiac region of the stomach of an aged orangoutang (Pongo pygmeans) in the Ueno Zoological Garden. This tumor, which was 10 cm in diameter and had invaded the serosa, was diagnosed as a squamous cell carcinoma. Metastases were found in the lymph nodes at various sites and in the liver, spleen and bronchus.