Differential patterns of expression of glycosylphosphatidylinositol-anchored carcinoembryonic antigen and alkaline phosphatase in various cancer cell lines

The expression of glycosylphosphatidylinositol (GPI-anchored) carcinoembryonic antigen (CEA) and alkaline phosphatase (ALP) on the cell surface of various cancer cell lines and a lung diploid cell line (WI38) was investigated, with exposure of the cell lines to a cell differentiation agent (sodium butyrate) to induce cell differentiation and expression of the two tumor-associated antigens. In three colon (SW1222, SW1116, and HT-29) and stomach (MKN-45) cancer cell lines, all of which are double producers of CEA and ALP, the maximum expression of GPI-anchored CEA occurred with butyrate at a lower concentration than did that of GPI-anchored ALP. GPI-anchored ALP derived from colon (SW1222 and SW1116) and stomach (MKN-45 and MKN-1) cancer cell lines was heat-stable with and without exposure to butyrate, but GPI-anchored ALP derived from lung cancer cell lines (PC-6, PC13, PC-14, WI26VA4, and WI38VA13) showed a variety of heat stabilities, depending on cell line, butyrate exposure, and SV40 transformation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Relationship between antibody productivity by activated human lymph node lymphocytes from lung cancer patients and lymphocyte subsets

Regional lymph node lymphocytes from five patients with primary lung cancer were analyzed for subset composition, and exposed in vitro to the polyclonal human B cell mitogen Staphylococcus aureus Cowan I (SACI) or the murine B cell mitogen lipopolysaccharide (LPS) and then fused with mouse myeloma cells for investigation at the clonal level of their antibody (Ab) production and its statistical relation to the original subset composition. No correlation was found between the proportion of CD19+, CD23+, or CD3+ cells in the lymphocyte sample prior to its exposure to either SACI or LPS, and the Ab production efficiency, defined as the ratio of the number of Ab producing wells to the total number of proliferating wells. For lymphocytes exposed to LPS, however, a strong correlation (r = 0.931, p = 0.02) was observed between the Ab production efficiency and the ratio of CD8+ to CD3+ cells (CD8/CD3) in the original sample at least within the ranges studied (CD8/CD3 = 0.216–0.288). For those exposed to SACI, no correlation was found between the Ab production efficiency and the CD8/CD3 ratio (r = 0.881, p = 0.12) or the proportion of CD8+ cells (r = 0.808, p = 0.19) in the original sample. These results suggest that the repertoire of B cells responsive to LPS is different at least in part from the repertoire responsive to SACI and that the ratio CD8/CD3 could serve as a practical predictor for Ab production by human lymphocytes stimulated with LPS. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nuclear Localization of the δ Subunit of Ca2+/Calmodulin-Dependent Protein Kinase II in Rat Cerebellar Granule Cells

Abstract : To examine the physiological roles of the δ subunit of Ca²⁺/calmodulin-dependent protein kinase ∥ (CaM kinase ∥δ) in brain, we examined the localization of CaM kinase ∥δ in the rat brain. A specific antibody to CaM kinase ∥δ1-δ4 isoforms was prepared by immunizing rabbits with a synthesized peptide corresponding to the unique carboxyl-terminal end of these isoforms. The prepared antibody did not recognize the α, β, and γ subunits, which were each overexpressed in NG108-15 cells. Immunoblot analysis on various regions and the nuclear fractions from rat brains suggested that some isoforms of CaM kinase ∥δ1-δ4 were abundant in the nucleus in the cerebellum. Total RNA from the cerebellum was analyzed by RT-PCR with a primer pair from variable domain 1 to variable domain 2. We detected the three PCR products δ3.1, δ3.4, and δ3 that contained the nuclear localization signal. These CaM kinase ∥δ3 isoforms were localized in the nuclei in transfected NG108-15 cells. Immunohistochemical study suggested the existence of these isoforms in the nuclei in cerebellar granule cells. These results suggest that CaM kinase ∥δ3 isoforms are involved in nuclear Ca²⁺ signaling in cerebellar granule cells.     

Involvement of distinct cellular compartments in the abnormal lymphoid organogenesis in lymphotoxin-alpha-deficient mice and alymphoplasia (aly) mice defined by the chimeric analysis.

Both lymphotoxin-alpha (LTalpha)-deficient mice and alymphoplasia (aly) mice, a natural mutant strain, manifest a quite similar phenotype: lack of lymph nodes (LN) and Peyer's patches (PP), with disturbed spleen architecture. The mechanisms underlying the defective lymphoid organogenesis in these mice were investigated by generating aggregation chimeras; ex vivo fused morulae were implanted into pseudo-pregnant host females and allowed to develop to term. Chimeric mice between LTalpha-deficient mice and wild-type mice restored LN and PP almost completely, suggesting that LTalpha expressed by circulating bone marrow-derived cells is essential for lymphoid organogenesis as well as for organization of spleen architecture. By contrast, chimeric mice between aly mice and wild-type mice showed only limited restoration of LN and PP. This suggests that the putative aly gene product does not act as a circulating ligand for lymphoid organogenesis, like LTalpha. Rather, abnormal development of lymphoid organs in aly mice seems most likely due to the defective development of the incipient stromal cells of the LN and PP. Supporting this hypothesis, up-regulation of VCAM-1 on aly mouse embryonic fibroblasts by signals through LTbetaR, which is exclusively expressed by nonlymphoid cells, was disturbed. These studies demonstrate that LTalpha and the putative aly gene product together control lymphoid organogenesis with a close mechanistic relationship in their biochemical pathways through governing the distinct cellular compartments, the former acting as a circulating ligand and the latter as a LTbetaR-signaling molecule expressed by the stroma of the lymphoid organs.     

UNC-16 alters DLK-1 localization and negatively regulates actin and microtubule dynamics in Caenorhabditis elegans regenerating neurons

Neuronal regeneration after injury depends on the intrinsic growth potential of neurons. Our study shows that UNC-16, a Caenorhabditis elegans JIP3 homolog, inhibits axonal regeneration by regulating initiation and rate of regrowth. This occurs through the inhibition of the regeneration-promoting activity of the long isoform of DLK-1 and independently of the inhibitory short isoform of DLK-1. We show that UNC-16 promotes DLK-1 punctate localization in a concentration-dependent manner limiting the availability of the long isoform of DLK-1 at the cut site, minutes after injury. UNC-16 negatively regulates actin dynamics through DLK-1 and microtubule dynamics partially via DLK-1. We show that post-injury cytoskeletal dynamics in unc-16 mutants are also partially dependent on CEBP-1. The faster regeneration seen in unc-16 mutants does not lead to functional recovery. Our data suggest that the inhibitory control by UNC-16 and the short isoform of DLK-1 balances the intrinsic growth-promoting function of the long isoform of DLK-1 in vivo. We propose a model where UNC-16’s inhibitory role in regeneration occurs through both a tight temporal and spatial control of DLK-1 and cytoskeletal dynamics.     

Sequence analysis of cDNAs encoding precursors of starfish asterosaps

To understand how starfish sperm activating peptides (asterosaps) are synthesized in the ovary, we cloned cDNAs encoding asterosaps and elucidated their nucleotide sequences. The mRNA encoding asterosaps was synthesized only in the oocytes, but not in the follicle cells, and the length was 3.7 kb. The cDNA clones contained multiple isoforms of asterosaps. We assume that asterosap precursors are large prepolypeptide chains with an unusual “rosary‐type” structure made of 10 successive similar stretches of 51–55 residues. Each stretch finishes with a “spacer” of 17–21 residues immediately followed by the sequence of one asterosap isoform. The N‐terminal of this precursor has 19–21 successive glutamine‐rich repeating units. Maturation of the precursor may require endopeptidases that cleave both C‐ and N‐sites of lysine‐arginine. Dev. Genet. 25:130–136, 1999. © 1999 Wiley‐Liss, Inc.     

Benzylideneoxymorphone: A new lead for development of bifunctional mu/delta opioid receptor ligands

Opioid analgesic tolerance remains a considerable drawback to chronic pain management. The finding that concomitant administration of delta opioid receptor (DOR) antagonists attenuates the development of tolerance to mu opioid receptor (MOR) agonists has led to interest in producing bifunctional MOR agonist/DOR antagonist ligands. Herein, we present 7-benzylideneoxymorphone (6, UMB 246) displaying MOR partial agonist/DOR antagonist activity, representing a new lead for designing bifunctional MOR/DOR ligands.     

Discovery of SMP-304, a novel benzylpiperidine derivative with serotonin transporter inhibitory activity and 5-HT1A weak partial agonistic activity showing the antidepressant-like effect

We report the discovery of a novel benzylpiperidine derivative with serotonin transporter (SERT) inhibitory activity and 5-HT_1A receptor weak partial agonistic activity showing the antidepressant-like effect. The 3-methoxyphenyl group and the phenethyl group of compound 1, which has weak SERT binding activity, but potent 5-HT_1A binding activity, were optimized, leading to compound 35 with potent and balanced dual SERT and 5-HT_1A binding activity, but also potent CYP2D6 inhibitory activity. Replacement of the methoxy group in the left part of compound 35 with a larger alkoxy group, such as ethoxy, isopropoxy or methoxy-ethoxy group ameliorated CYP2D6 inhibition, giving SMP-304 as a candidate. SMP-304 with serotonin uptake inhibitory activity and 5-HT_1A weak partial agonistic activity, which could work as a 5-HT_1A antagonist, displayed faster onset of antidepressant-like effect than a representative SSRI paroxetine in an animal model.