Effects of basic fibroblast growth factor and ganglioside GM1 on neuronal survival in primary cultures and on eight-arm radial maze task in adult rats following partial fimbria transections.

The effects of basic fibroblast growth factor (bFGF) and ganglioside GM1 (GM1) were evaluated alone and simultaneously in two types of experiments. First, the neuronal survival of primary culture neurons from fetal rat brain was measured. Then, performance on radial maze task in adult male rats following bilateral partial Fimbria-Fornix transections (F-F lesion) was tested. In primary culture neurons, bFGF (1-10 ng/ml) supported the neuronal survival from three regions (hippocampus, cortex and septum) of embryonic rat brain. However, GM1 (0.1-10 micrograms/ml) did not support the neuronal survival from any regions. Survival of cultured neurons was not supported by addition of 0.1 ng/ml bFGF, but when bFGF (0.1 ng/ml) and GM1 (0.1, 1 microgram/ml) were given to the cultured neurons simultaneously, the number of surviving neurons increased significantly. In the eight-arm radial maze task, where only the same four arms were baited, F-F lesion produced substantial memory impairment. In this task, administration of bFGF (10 micrograms/ml) or GM1 (1 mg/ml) alone did not produce any effects. However, when they were given simultaneously, the number of working memory errors decreased significantly, in spite of no amelioration for hippocampal choline acetyl transferase (ChAT) depletion. These findings indicate that actions of bFGF may be potentiated by the addition of GM1 in both primary neuronal cultures and radial maze task performance. These results suggest that the combination of bFGF and GM1 may synergistically improve spatial memory deficits.

Effects of acidic fibroblast growth factor on hippocampal long-term potentiation in fasted rats.

Effects of human recombinant acidic fibroblast growth factor (haFGF) on long-term potentiation (LTP) and the increase of the spike amplitude induced by weak tetanic stimulation were investigated and compared with those of CS23 (modified human basic FGF) in the dentate gyrus of fasted and nonfasted rats. haFGF didn't influence the LTP induced by the tetanus of 100 pulses at 100 Hz in both 24 hr fasted and non-fasted rats. On the other hand, the tetanus of 20 pulses at 60 Hz significantly enhanced the amplitude of population spike and facilitated the generation of LTP by the i.c.v. injection of 10 microliters of 20-40 micrograms/ml haFGF in 24 hr fasted rats but not in non-fasted rats. However, 40 micrograms/ml CS23 induced LTP when the tetanus of 20 pulses at 60 Hz was applied in both fasted and non-fasted states. These results suggest that haFGF might be one of the regulating factors of feeding and memory.

Human acidic fibroblast growth factor has trophic effects on cultured neurons from multiple regions of brain and retina.

Neurotrophic activities of human recombinant acidic fibroblast growth factor (haFGF) were evaluated on primary cultured neurons from various brain regions and compared with those of CS23, modified human basic fibroblast growth factor. Survival of cultured neurons from embryonic day 16 (E16) rat cortex and substantia nigra was significantly increased by the addition of more than 10 ng/ml haFGF and that from the hippocampus was increased by 100 and 1000 ng/ml. However, enhancement of viability by haFGF was observed only in 1000 ng/ml-treated neurons from the striatum, thalamus, colliculus and cerebellum; and it was not observed in septal neurons. Survival of cultured neurons from postnatal day 15 rat on glial feeder layer was significantly increased by the addition of 1000 ng/ml haFGF, except for neurons from the septum. CS23 (10 ng/ml) increased the survival of cultured neurons from all regions mentioned above, and its effects were stronger than those of 1000 ng/ml haFGF. Addition of more than 10 ng/ml haFGF significantly increased the survival of cultured neurons from neonatal day 2 rat retina. Addition of 1000 ng/ml haFGF increased the choline acetyltransferase activity of E16 septal neurons slightly but significantly, but didn't increase the dopamine uptake activity of embryonic day E15 ventral midbrain. These results show that haFGF is effective on limited regions and ages of brain and retina, while CS23 is effective on all regions tested.

Nucleotide sequences of immunoglobulin-epsilon pseudogenes in man and apes and their phylogenetic relationships.

To understand the phylogenetic relationships between hominoids, the nucleotide sequences of immunoglobulin-epsilon processed pseudogenes from chimpanzee, gorilla and orangutan were determined. The basic structures of these processed pseudogenes agreed with their human counterpart. Although the degrees of nucleotide differences between man and the African apes had no statistical significance, all the analytical data examined supported the theory that chimpanzee is the closest relative of man. This result was consistent with that deduced by our recent qualitative study. Studies on the nucleotide sequences of globin genes have suggested that the molecular clock runs more slowly in hominoids than in non-hominoid primates. According to the present data, however, further retardation of the evolutionary rate was not observed in the human lineage. Assuming that orangutan diverged 14 million years ago and that the evolutionary rate between the orangutan lineage and the lineage leading to the other three species is constant, the divergence dates of chimpanzee and gorilla were estimated to be 4.9(+/- 0.9) and 5.9(+/- 0.9) million years ago, respectively.

Nucleotide sequences of immunoglobulin epsilon genes of chimpanzee and orangutan: DNA molecular clock and hominoid evolution.

To determine the phylogenetic relationships among hominoids and the dates of their divergence, the complete nucleotide sequences of the constant region of the immunoglobulin epsilon-chain (C epsilon 1) genes from chimpanzee and orangutan have been determined. These sequences were compared with the human epsilon-chain constant-region sequence. A molecular clock (silent molecular clock), measured by the degree of sequence divergence at the synonymous (silent) positions of protein-encoding regions, was introduced for the present study. From the comparison of nucleotide sequences of alpha1-antitrypsin and beta- and delta-globin genes between humans and Old World monkeys, the silent molecular clock was calibrated: the mean evolutionary rate of silent substitution was determined to be 1.56 X 10(-9) substitutions per site per year. Using the silent molecular clock, the mean divergence dates of chimpanzee and orangutan from the human lineage were estimated as 6.4 +/- 2.6 million years and 17.3 +/- 4.5 million years, respectively. It was also shown that the evolutionary rate of primate genes is considerably slower than those of other mammalian genes.

Vasodilation produced by forskolin compared with that produced by adenosine in rabbit coronary artery.

We compared the relaxing action of forskolin with that of adenosine in rabbit coronary artery and analyzed the pharmacological properties of this effect of forskolin. In preparations of this artery precontracted by 10-16 mM KCl, the addition of forskolin (10(-9)-10(-5) M) and adenosine (10(-9)-10(-4) M) to the organ bath produced dose-dependent relaxations. The mean EC50 for the relaxing action of forskolin was 4.5 X 10(-8) M and that of adenosine was 3.6 X 10(-7) M, forskolin being 10 times more potent than adenosine. Relaxation produced by forskolin was not affected by treatment with propranolol (10(-5) M), atropine (10(-6) M), or 8-phenyltheophylline (10(-6) M), but was competitively inhibited by ouabain (10(-6) M). The relaxation produced by adenosine was inhibited by 8-phenyltheophylline (10(-6) M) competitively and by ouabain (10(-6) M) noncompetitively. In preparations precontracted by a higher concentration of KCl, 40 mM, forskolin produced full relaxation with a shift of the dose-response curve to the right; adenosine did not produce full relaxation. Both forskolin and adenosine attenuated the maximum contraction produced by Ca2+. These findings indicate that the vasorelaxing effect of forskolin was not due to activation of beta-adrenoceptors, muscarinic receptors, or adenosine receptors, whereas that of adenosine was due to activation of adenosine receptors. Increased availability of intracellular Ca2+ competitively inhibited the relaxation induced by forskolin and noncompetitively inhibited the relaxation induced by adenosine. Both forskolin and adenosine noncompetitively inhibited contraction induced by Ca2+.

The complete nucleotide sequence of the influenza virus neuraminidase gene of A/NJ/8/76 strain and its evolution by segmental duplication and deletion.

The neuraminidase (NA) gene from A/New Jersey (NJ)/8/76 (H1N1, formerly Hsw1 N1) strain isolated in 1976 was cloned into pBR322 and its complete nucleotide sequence was determined. The NJ8 NA gene is 1458 nucleotides long and the sequence predicted the primary structure of the NA molecule comprising of 469 amino acids with a molecular weight of 51,628. Comparison with other NA sequences of the N1 subtype strains which were isolated in 1933-1934 identified the highly variable regions at the amino-terminal stalk region and the carboxy-terminal regions. Potential glycosylation sites encoded by a 15 base-pair unit sequence are arrayed tandemly at the stalk regions. The lengths of stalk regions are highly variable because of segmental deletions in old NA genes. Possible mechanisms for such deletions are discussed.

Structure of the human immunoglobulin C epsilon 2 gene, a truncated pseudogene: implications for its evolutionary origin.

Cloning of the overlapping DNA fragments together with Southern hybridization experiments showed the organization of the human C epsilon and C alpha gene cluster as 5'-C epsilon 2-14 kilobases-C alpha 1----C epsilon 1-13 kilobases-C alpha 2-3'. Comparison of the nucleotide sequences of the C epsilon 1 and C epsilon 2 genes revealed that four deletions have taken place in the C epsilon 2 gene and its flanking regions. The three deleted regions in the 5' side of the C epsilon 2 gene are partially filled with shorter inserted sequences. One of them has removed the CH1 and CH2 exons and a portion of the epsilon switch (S epsilon) region. The S epsilon region and the CH4 exon still retain the functional structures, whereas the CH3 exon has been inactivated by deleting its 5' intervening sequence necessary for splicing. The tetranucleotide T-G-G-G (or T-G-G-C), which is usually found in close proximity of the class-switch recombination sites in mouse myelomas, is located 5' to the three deletion sites. The results imply that the mechanism responsible for the heavy chain class-switch recombination might be relevant to the evolutionary mechanism of creation of the truncated C epsilon 2 gene. The other deletion in the 3' flanking region of the C epsilon 2 gene may be due to slipped mispairing of the short direct repeat (C-C-C-C-C) at both ends.

Cloning of human immunoglobulin epsilon chain genes: evidence for multiple C epsilon genes.

An active human epsilon chain gene was cloned from a phage library containing partial EcoRI digests of IgE-producing myeloma DNA, using the human JH (joining) gene fragment as a probe. The epsilon chain gene clone was identified by partial nucleotide sequence determination. The germ-line constant region gene of the epsilon chain (C epsilon gene) was cloned from a human fetal liver DNA library, using the cloned epsilon chain gene as a probe. Comparative studies on the human and mouse germ-line epsilon chain genes revealed that the switch (S) sequence is more conserved than the coding sequence. Restriction endonuclease BamHI digestion of human DNA produced three C epsilon fragments of 3.0, 6.5, and 9.2 kilobases, which were named C epsilon 1, C epsilon 2, and C epsilon 3 genes, respectively. We found the three C epsilon gene fragments in all of the human DNA preparations from eleven individuals. The C epsilon gene expressed in the myeloma was identified as the C epsilon 1 gene. Because the C epsilon 2 gene is deleted from the myeloma DNA, the order of the C epsilon genes is likely to be 5'-C epsilon 2-C epsilon 1-C epsilon 3-3', assuming that all the C epsilon genes are on chromosome 14. The germ-line C epsilon 3 gene was also cloned from the myeloma DNA. Characterization of the C epsilon 3 gene revealed that it does not have the S region, suggesting that it might be a pseudogene.

Long terminal repeat-like elements flank a human immunoglobulin epsilon pseudogene that lacks introns.

There are at least three immunoglobulin epsilon genes (C epsilon 1, C epsilon 2, and C epsilon 3) in the human genome. The nucleotide sequences of the expressed epsilon gene (C epsilon 1) and one (C epsilon 3) of the two epsilon pseudogenes were compared. The results show that the C epsilon 3 gene lacks the three intervening sequences entirely and has a 31-base A-rich sequence 16 bases 3' to the putative poly(A) addition signal, indicating that the C epsilon 3 gene is a processed gene. The C epsilon 3 gene sequence is homologous to the five separate DNA segments of the C epsilon 1 gene; namely, a segment in the 5'-flanking region (100 bases) and four exons, which are interrupted by a spacer region or intervening sequences. Long terminal repeat (LTR)-like sequences which contain TATAAA and AATAAA sequences as well as terminal inverted repeats are present in both 5'- and 3'-flanking regions. The 5' and 3' LTR-like sequences do not, however, constitute a direct repeat, unlike transposable elements of eukaryotes and retroviruses. The 3' LTR-like sequence is repetitive in the human genome, but is not homologous to the Alu family DNA. Models for the evolutionary origin of the processed gene flanked by the LTR-like sequences are discussed. The C epsilon 3 gene has a new open frame which codes potentially for an unknown protein of 292 amino acid residues.