Human lactate dehydrogenase-B processed pseudogene: nucleotide sequence analysis and assignment to the X-chromosome

Two human genomic clones containing the lactate dehydrogenase-B processed pseudogene were isolated from two patients deficient in lactate dehydrogenase-B isozyme. The sequences of 3,287 nucleotides, including the pseudogenes and its flanking regions, from both clones were found to be identical except for three differences in the pseudogenes. The sequences of 1,286 nucleotides from these two pseudogenes exhibited 93% homology with the cDNA sequence of the lactate dehydrogenase-B functional gene, and the pseudogene contained 75/76 base substitutions, 11/12 single-base deletions, and 5 single-base insertions. This pseudogene was mapped to the x-chromosome by dot-blot analysis using a probe for the pseudogene or its 5' flanking sequence.     

Molecular characterization of genetic mutation in human lactate dehydrogenase-A (M) deficiency

Human lactate dehydrogenase-A mutant gene was isolated from the genomic DNA library of a patient deficient in LDH-A (Muscle) subunit. The nucleotide sequences of seven protein-coding exons were determined and a deletion of 20 base-pairs in exon 6 was found. This mutation results in a frame-shift translation and premature termination. The predicted incomplete LDH-A (M) subunit containing only 259 instead of 331 amino acids appears to be degraded rapidly, since no protein was detected immunologically (Maekawa et al., Am J Hum Genet 39:232-238, 1986). In addition, three synonymous (silent) substitutions, A to C, T to C, and G to A, were observed at codons 115, 160 and 172, respectively, in this LDH-A mutant gene.     

Granulocyte-macrophage colony-stimulating factor enhances macrophage accessory function in con A-stimulated T-cell proliferation

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been reported to augment various macrophage (M phi) functions, including antigen presentation in the antibody-producing response. We investigated the augmentative effect of GM-CSF on M phi A-cell activity in concanavalin A-stimulated T-cell proliferation. Pretreatment with GM-CSF of peritoneal M phi enhanced the T-cell proliferative response. This effect of GM-CSF was dose dependent and GM-CSF supplementation was needed at the beginning of M phi culture. We observed that GM-CSF induced M phi spreading and firm attachment accompanied with enlargement of the cytoplasm, but could not induce de novo expression of Ia antigen. GM-CSF treatment enabled M phi to produce more interleukin (IL)-1 and IL-6 upon stimulation with lipopolysaccharides or polyinosinic-polycytidylic acid, but was unable to stimulate M phi directly. This was confirmed by Northern blot analysis. These results indicate that GM-CSF augments M phi A-cell activity through the enhancement of the capacity of M phi to produce IL-1 and IL-6.     

Improved heart failure protection by FK664, a novel positive inotropic agent, in dog heart-lung preparations.

The effects of FK664, a novel positive inotropic agent, and enoximone on pentobarbital-induced heart failure were compared in dog heart-lung preparations. Both FK664 and enoximone improved the cardiac function curve in a dose-dependent manner and restored it to the control level at drug concentrations of 1 microgram/ml and 10 micrograms/ml, respectively. Therefore, the cardiotonic potency of FK664 appears to be 10 times that of enoximone. These agents were almost equal in force-rate separation of cardiac effect. Neither of the agents produced arrhythmia at any dose tested. These results suggest that FK664 may be a potent cardiotonic agent for the treatment of heart failure.     

NVL2 is a nucleolar AAA-ATPase that interacts with ribosomal protein L5 through its nucleolar localization sequence

NVL (nuclear VCP-like protein), a member of the AAA-ATPase family, is known to exist in two forms with N-terminal extensions of different lengths in mammalian cells. Here, we show that they are localized differently in the nucleus; NVL2, the major species, is mainly present in the nucleolus, whereas NVL1 is nucleoplasmic. Mutational analysis demonstrated the presence of two nuclear localization signals in NVL2, one of which is shared with NVL1. In addition, a nucleolar localization signal was found to exist in the N-terminal extra region of NVL2. The nucleolar localization signal is critical for interaction with ribosomal protein L5, which was identified as a specific interaction partner of NVL2 on yeast two-hybrid screening. The interaction of NVL2 with L5 is ATP-dependent and likely contributes to the nucleolar translocation of NVL2. The physiological implication of this interaction was suggested by the finding that a dominant negative NVL2 mutant inhibits ribosome biosynthesis, which is known to take place in the nucleolus.     

Small molecule inhibitors of the CCR2b receptor. Part 1: Discovery and optimization of homopiperazine derivatives

N,N'-Disubstituted homopiperazine derivatives have been discovered as CC-chemokine receptor 2b (CCR2b) inhibitors with submicromolar activity in the CCR2b binding assay. A 4-substituted benzyl group on one homopiperazine nitrogen was an important moiety for binding affinity to the CCR2b receptor. The SAR for CCR2b binding affinity correlated inversely with the sigma factor of the functional group on this benzyl moiety. Introduction of hydroxy groups to appropriate positions in the 3,3-diphenylpropyl group on the other homopiperazine nitrogen increased CCR2b binding activity. The synthesis of an informer library to search for alternative substructures is also described.     

Linker region of a halobacterial transducer protein interacts directly with its sensor retinal protein

pHtrII, a pharaonis halobacterial transducer protein, possesses two transmembrane helices and forms a signaling complex with pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, NpSRII) within the halobacterial membrane. This complex transmits a light signal to the sensory system located in the cytoplasm. It has been suggested that the linker region connecting the transmembrane region and the methylation region of pHtrII is important for binding to ppR and subsequent photosignal transduction. In this study, we present evidence to suggest that the linker region itself interacts directly with ppR in addition to the interaction in the membrane region. An in vitro pull-down assay revealed that the linker region bound to ppR, and its dissociation constant (K(D)) was estimated to be approximately 10 microM using isothermal titration calorimetry (ITC). Solution NMR analyses showed that ppR interacted with the linker region of pHtrII (pHtrII(G83)(-)(Q149)) and resulted in the broadening of many peaks, indicating structural changes within this region. These results suggest that the pHtrII linker region interacts directly with ppR. There was no demonstrable interaction between the C-terminal region of ppR (ppR(Gly224)(-)(His247)) and either the linker region (pHtrII(G83)(-)(Q149)) or the transmembrane region (pHtrII(M1)(-)(E114)) of pHtrII. On the basis of the NMR, CD, and photochemical data, we discuss the structural changes and role of the linker region of pHtrII in relation to photosignal transduction.     

Structural changes of the complex between pharaonis phoborhodopsin and its cognate transducer upon formation of the M photointermediate

pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII) is a receptor for negative phototaxis in Natronobacterium pharaonis. It forms a 2:2 complex with its transducer protein, pHtrII, in membranes, and the association is weakened by 2 orders of magnitude in the M intermediate. Such change is believed to correspond to the transfer of the light signal to pHtrII. In this paper, we applied Fourier transform infrared (FTIR) spectroscopy to the active M intermediate in the absence and presence of pHtrII. The obtained difference FTIR spectra were surprisingly similar, notwithstanding the presence of pHtrII. This result strongly suggests that the transducer activation in the ppR-pHtrII system does not induce secondary structure alterations of the pHtrII itself. On the other hand, we found that the hydrogen bond of the OH group of Thr204 is altered in the primary K intermediate, but restored in the M intermediate. The hydrogen bond of Asn74 in pHtrII is strengthened in M, presumably because of the change in interaction with Tyr199 of ppR. These facts provided a light signaling pathway from Lys205 (retinal) of the receptor to Asn74 of the transducer through Thr204 and Tyr199. Transducer activation is likely to involve a relaxation of Thr204 in the receptor and hydrogen bonding alteration of Asn74 in the transducer, during which the helices of the transducer perform rigid-body motion without changing their secondary structures.     

Serotonin 5-HT2 receptors in schizophrenic patients studied by positron emission tomography

Using positron emission tomography (PET) and [11C]N-methylspiperone (NMSP), we examined 5-HT2 receptors in the cortex of schizophrenic patients in whom we previously observed decreased prefrontal D1 receptor binding. The subjects were 10 neuroleptic-naive schizophrenic patients, 7 schizophrenic patients who were drug-free but had previously been treated with neuroleptics, and 12 normal controls. A non-significant trend towards decreased prefrontal [11C]NMSP binding was observed in the neuroleptic-treated patients, suggesting a possible effect of previous neuroleptic treatment on the alteration in cortical 5-HT2 function. However, the neuroleptic-naive patients showed no noticeable difference in cortical [11C]NMSP binding compared to controls. Our results do not rule out the role of 5-HT2 function as a crucial site of therapeutic activity of schizophrenia, but they do suggest that cortical 5-HT2 receptors might not be primarily involved in the pathophysiology of schizophrenia.     

Nuclear transfer in cattle using colostrum-derived mammary gland epithelial cells and ear-derived fibroblast cells

To assess the developmental potential of nuclear transfer embryos in cattle using mammary gland epithelial (MGE) cells derived from the colostrum, we compared the effectiveness of cloning using those cells and fibroblast cells derived from the ear. The fusion rate of the enucleated oocytes with fibroblast cells (75 +/- 4%) was significantly higher than that with MGE cells (56 +/- 7%, P<0.05). There were no significant differences in the cleavage rate (85 +/- 3% vs. 91+/- 2%) or in the developmental rate to the blastocyst stage (35 +/- 6% vs. 35 +/- 5%) using MGE cells vs. fibroblast cells as donor nuclei (P>0.05). After transfer of blastocysts derived from nuclear transfer embryos produced using MGE cells and fibroblast cells, 13% (4/31) and 16% (6/37) of recipient heifers were pregnant on Day 42 as assessed by ultrasonography, respectively. Two of the 4 and 4 of the 6 recipients of embryos with MGE cell- and fibroblast cell-derived nuclei, respectively, aborted within 150 days of pregnancy. Four live female calves were obtained from MGE cells or fibroblast cells. However, one died from internal hemorrhage of the arteria umbilicalis. The other three calves were normal and healthy. There were no differences in the pregnancy rate or calving rate when using MGE cells vs. fibroblast cells. Microsatellite DNA analyses confirmed that the cloned calves were genetically identical to the donor cows and different from the recipient heifers. We conclude that colostrum-derived MGE cells have the developmental potential to term by nuclear transfer, and the efficiency of development of those cloned embryos was the same as that of embryos obtained using fibroblast cells as donor nuclei, although there was a significant difference in the fusion rate. This method using MGE cells derived from colostrum, which is obtained easily and safely from live adult cows, is more advantageous for cloning with somatic cells.