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.     

Presence of H type 3/4 chains of ABO histo-blood group system in serous cells of human submandibular gland and regulation of their expression by the secretor gene (FUT2).

We have investigated by immunochemistry the distribution of H Type 3/4 chains of the ABO histo-blood group system in human submandibular gland using a monoclonal anti-H MBr1 antibody specific for H Type 3/4 chains, and have found the expression of H Type 3/4 chains was mainly in the serous cells. Serous cells from secretors were stained by MBr1 but not by anti-A and anti-B antibodies, whereas serous cells from nonsecretors exhibited a negative reaction with MBr1. Mucous cells were not stained by MBr1. Only a few striated duct cells showed a weak reaction with anti-H MBr1. These results suggested that the H Type 3/4 chains were distributed predominantly in the serous cells of the human submandibular gland and that secretor Type alpha(1,2)fucosyltransferase (Se enzyme) controlled the synthesis of H Type 3/4 chains in vivo. Saliva also contained H Type 3/4 chains, which were controlled by the secretor gene (FUT2). The differences in the distributions of H Type 1, H Type 2, and H Type 3/4 chains of the ABO histo blood group system in the submandibular gland are discussed.     

Tracheal motile cilia in mice require CAMSAP3 for the formation of central microtubule pair and coordinated beating

Motile cilia of multiciliated epithelial cells undergo synchronized beating to produce fluid flow along the luminal surface of various organs. Each motile cilium consists of an axoneme and a basal body (BB), which are linked by a “transition zone” (TZ). The axoneme exhibits a characteristic 9+2 microtubule arrangement important for ciliary motion, but how this microtubule system is generated is not yet fully understood. Here we show that calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), a protein that can stabilize the minus-end of a microtubule, concentrates at multiple sites of the cilium–BB complex, including the upper region of the TZ or the axonemal basal plate (BP) where the central pair of microtubules (CP) initiates. CAMSAP3 dysfunction resulted in loss of the CP and partial distortion of the BP, as well as the failure of multicilia to undergo synchronized beating. These findings suggest that CAMSAP3 plays pivotal roles in the formation or stabilization of the CP by localizing at the basal region of the axoneme and thereby supports the coordinated motion of multicilia in airway epithelial cells.     

Thy-1-Mediated phosphatidylinositol turnover in cultured rat glomerular mesangial cell

Thy-1 glycoprotein is expressed in rat glomerular mesangial cells, and anti-Thy-1 nephritis induced by anti-Thy-1 antibodies is a model of human renal diseases. In this study, we examined Thy-1-mediated biological reactions in cultured rat glomerular mesangial cells utilizing two anti-Thy-1 monoclonal antibodies (mAbs), 1-22-3 and OX-7. Incubation of the cells with these mAbs resulted in increased inositol trisphosphate (IP₃) levels. The rise in IP₃ produced by mAb 1-22-3 was greater than that produced by mAb OX-7 at the same dose. Incubation of mesangial cells with these mAbs resulted in an increase in the intracellular free calcium concentration ([Ca²⁺]i). mAb 1-22-3 induced a sustained increase in [Ca²⁺]i, while that induced by mAb OX-7 lasted 1-2 min, then decreased to the basal level. An transient increase in [Ca²⁺]i was also observed in Ca²⁺-free medium, indicating that these [Ca²⁺]i increases are due to release of Ca²⁺ from internal stores by IP₃ without calcium flux across cell membrane. When cells were pretreated with protein tyrosine kinase (PTK) inhibitors (herbimycin A or genistein), Thy-1-mediated increases in [Ca²⁺]i were inhibited. These data suggest that Thy-1 induces the production of IP₃ (including inositol 1,4,5-triphosphate, an intracellular Ca²⁺-releasing factor) and that PTKs may contribute to the Thy-1-mediated elevation of [Ca²⁺]i which presumably results from phospholipase C activation following Thy-1-mediated signaling in rat mesangial cells. © 1996 Wiley-Liss, Inc.     

Synthesis and evaluation of a novel near-infrared fluorescent probe based on succinimidyl-Cys-C(O)-Glu that targets prostate-specific membrane antigen for optical imaging

Prostate-specific membrane antigen (PSMA), which is highly expressed in both localized and metastatic prostate cancer (PCa), is an ideal target for imaging and therapy of PCa. We previously reported radiolabeled asymmetric urea derivatives as a PSMA-targeting radiotracer for single-photon emission computed tomography (SPECT) and positron emission tomography (PET) imaging. Here, based on these radiopharmaceutical probes, we designed a novel near-infrared (NIR) fluorescent imaging probe (800CW-SCE) by chemical conjugation between IRDye 800CW-Maleimide and an asymmetric urea compound, known as PSMA inhibitor, for optical imaging. In the in vitro cellular uptake study, 800CW-SCE was internalized into PSMA-positive PCa cells (LNCaP cells) but not into PSMA-negative PCa cells (PC-3 cells). Moreover, in the in vivo imaging study, the probe was highly accumulated in LNCaP tumors but not in PC-3 tumors, and remained in LNCaP tumors until 24 h after intravenous administration. These results suggest that the potent NIR conjugate may contribute to clinical intraoperative optical imaging.     

Crystal structural characterization reveals novel oligomeric interactions of human voltage‐dependent anion channel 1

Voltage‐dependent anion channel 1 (VDAC1), which is located in the outer mitochondrial membrane, plays important roles in various cellular processes. For example, oligomerization of VDAC1 is involved in the release of cytochrome c to the cytoplasm, leading to apoptosis. However, it is unknown how VDAC1 oligomerization occurs in the membrane. In the present study, we determined high‐resolution crystal structures of oligomeric human VDAC1 (hVDAC1) prepared by using an Escherichia coli cell‐free protein synthesis system, which avoided the need for denaturation and refolding of the protein. Broad‐range screening using a bicelle crystallization method produced crystals in space groups C222 and P22₁2₁, which diffracted to a resolution of 3.10 and 3.15 Å, respectively. Each crystal contained two hVDAC1 protomers in the asymmetric unit. Dimer within the asymmetrical unit of the crystal in space group C222 were oriented parallel, whereas those of the crystal in space group P22₁2₁ were oriented anti‐parallel. From a model of the crystal in space group C222, which we constructed by using crystal symmetry operators, a heptameric structure with eight patterns of interaction between protomers, including hydrophobic interactions with β‐strands, hydrophilic interactions with loop regions, and protein–lipid interactions, was observed. It is possible that by having multiple patterns of interaction, VDAC1 can form homo‐ or hetero‐oligomers not only with other VDAC1 protomers but also with other proteins such as VDAC2, VDAC3 and apoptosis‐regulating proteins in the Bcl‐2 family.