Large schools of Japanese sardines,Sardinops melanostictus, mate in single pair units at night

Synopsis We observed the behavior of the spawning group of Japanese sardine,Sardinops melanostictus, in the field at night, and also confirmed the spawning behavior in the darkness in captivity. The chasing and mating were done in a pair of 1 male and 1 female. The 1-to-1 mating was a definite finding irrespective of the group density in the field, the density in the tank in captivity, or the sex ratio. This maybe because there is little visual information during spawning at night.     

Isolation and characterization of osteoclast precursors that differentiate into osteoclasts on calvarial cells within a short period of time

Osteoclasts are formed in cocultures of mouse calvarial cells and hematopoietic cells in the presence of osteotropic factors such as 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃], parathyroid hormone (PTH) and prostaglandin E₂ (PGE₂). We isolated osteoclast precursors (OCPs) from the coculture and examined their characteristics. After coculture for 7 days of mouse calvarial cells and bone marrow cells in the absence of osteotropic factors, hematopoietic cells were recovered and applied to a Sephadex G-10 column. Cells which passed through the column were collected as OCPs. When OCPs were cultured on calvarial cell layers in the presence of 1α,25(OH)₂D₃, tartrate-resistant acid phosphatase (TRAP)–positive cells first appeared within 24 h, and their number increased thereafter. OCPs also differentiated into TRAP-positive cells within 48 h on the calvarial cell layer which had been pretreated with either 1α,25(OH)₂D₃, PTH, or PGE₂. Autoradiography using [¹²⁵I]-labeled calcitonin showed that TRAP-positive cells formed on the calvarial cell layer expressed calcitonin receptors. Direct contact between OCPs and calvarial cells was required for the differentiation of OCPs into TRAP-positive cells. Flow cytometric analysis revealed that OCPs were positive for Mac-1, Mac-2, and Gr-1 but negative for F4/80, B220 and CD3e. Calvarial cells obtained from macrophage-colony stimulating factor (M-CSF)–deficient osteopetrotic (op/op) mice did not support OCP formation. A cell preparation disaggregated from long bones of newborn mice contained OCPs that differentiated into TRAP-positive cells on calvarial cells within 48 h, but cell preparations of freshly isolated bone marrow cells and alveolar macrophages did not. These results suggest that OCPs are specific cells which are formed only in the bone microenvironment and that OCPs recognize a signal(s) expressed by stromal cells in response to osteotropic factors and differentiate into osteoclasts. J. Cell. Physiol. 177:26–35, 1998. © 1998 Wiley-Liss, Inc.     

Observation by SEM of the attachment ofAgrobacterium tumefaciens to the surface of vinca,asparagus and rice cells

Transformation of vinca cells was performed by the co-cultivation of cell-wall regenerated vinca protoplasts withAgrobacterium tumefaciens. Using thisin vitro and single cell system, attachment of the bacteria to the surface of vinca cells was observed by scanning electron microscopy (SEM). Figures of the bacteria polarly binding to the plant cell wall were often observed. AsEscherichia coli does not attach to the plant cells at all, the observed attachment ofA. tumefaciens is suggested as a characteristic feature in crown gall induction. Even though no evidence of transformation was obtained by the co-cultivation methods, a similar attachment was observed in the cell-wall regenerated protoplasts of rice. The bacteria also attached to the surface of isolated mesophyll cells of asparagus and root hairs of rice. From these observation, we concluded that the attachment is not the limiting step of crown gall induction byA. tumefaciens in monocotyledonous plants. Extracellular fibrils like pili were observed with a few strains of A.tumefaciens for the first time. These fibrils were observed regardless of their ability of attachment and infectivity.     

T cell response mediated by myeloid cell-derived IL-12 is responsible for Porphyromonas gingivalis-induced periodontitis in IL-10-deficient mice

Periodontal disease is a chronic inflammatory disease in the oral cavity, which culminates in alveolar bone loss. Porphyromonas gingivalis is a consensus periodontal pathogen that has been implicated in adult forms of periodontitis. We previously demonstrated that IL-10-deficient mice exhibit a hyperinflammatory phenotype and are highly susceptible to P. gingivalis-induced periodontitis, indicating an important anti-inflammatory effect of IL-10 in suppressing bone loss. In this study, we analyzed the pathway(s) by which IL-10 deficiency leads to severe P. gingivalis-induced periodontitis. Because Stat3 is essential in IL-10 signaling, immune cell-specific Stat3-deficient mice were subjected to P. gingivalis infection to identify the key IL-10-responsive cells in preventing periodontitis. Myeloid cell-specific Stat3-deficient mice exhibited increased periodontal bone loss (p < 0.001), whereas T cell- and B cell-specific Stat3 mice were resistant, suggesting that macrophages (MP) and/or polymorphonuclear leukocytes are the key target cells normally suppressed by IL-10. Myeloid cell-specific Stat3-deficient mice exhibited elevated gingival CD40L gene expression in vivo compared with wild-type controls (p < 0.01), and Stat3-deficient MPs exhibited vigorous P. gingivalis-stimulated IL-12 production in vitro and induced elevated Ag-specific T cell proliferation compared with wild-type MPs (p < 0.01). Of importance, both IL-12p40/IL-10 and T cell/IL-10 double-deficient mice were resistant to P. gingivalis-induced periodontitis, demonstrating roles for both IL-12p40 and T cells in pathogenesis in a hyperinflammatory model of disease. These data demonstrate that P. gingivalis-induced periodontitis in IL-10-deficient mice is dependent upon IL-12p40-mediated proinflammatory T cell responses.     

Tissue specificity in fasting glucose utilization in slightly obese diabetic patients submitted to bariatric surgery

Objective:

The present study was planned to investigate, by means of quantitative FDG‐PET, how bariatric surgery (BS) modifies the metabolic pattern of the whole body and different tissues in slightly obese patients with type 2 diabetes mellitus (T2DM).

Design and Methods:

Before, 1 and 4 months after BS, 21 consecutive slightly obese T2DM patients underwent blood sampling to estimate plasma levels of glucose, insulin, glycosylated hemoglobin. At the same time points, these patients underwent a dynamic ¹⁸F‐FDG PET study of thorax and upper abdomen in fasting state and after washout of T2DM therapy. Gjedde‐Patlak analysis was applied to estimate glucose uptake in the whole body and in different tissues (myocardium, skeletal back muscle, adipose tissue, and liver).

Results:

Surgical intervention quickly lowered levels of both insulin and glucose documenting an amelioration of glucose tolerance. Similarly, skeletal muscle and myocardial glucose uptake significantly increased soon after surgery (P < 0.001 and P < 0.01 at 1 month versus baseline, respectively) and remained substantially stable thereafter. By contrast, glucose uptake slightly decreased from its baseline values in the liver (P < 0.01 at 4 months) while no response could be documented over time in the adipose tissue.

Conclusions:

These findings document that BS‐induced modification of glucose homeostasis in slightly obese T2DM patients is mostly due to an increase in muscle glucose consumption. The surgically modified metabolic pattern of these patients might be of interest as a new model to investigate mechanism underlying insulin resistance.     

SLAM (CD150)-independent measles virus entry as revealed by recombinant virus expressing green fluorescent protein

Wild-type measles virus (MV) strains use human signaling lymphocyte activation molecule (SLAM) as a cellular receptor, while vaccine strains such as the Edmonston strain can use both SLAM and CD46 as receptors. Although the expression of SLAM is restricted to cells of the immune system (lymphocytes, dendritic cells, and monocytes), histopathological studies with humans and experimentally infected monkeys have shown that MV also infects SLAM-negative cells, including epithelial, endothelial, and neuronal cells. In an attempt to explain these findings, we produced the enhanced green fluorescent protein (EGFP)-expressing recombinant MV (IC323-EGFP) based on the wild-type IC-B strain. IC323-EGFP showed almost the same growth kinetics as the parental recombinant MV and produced large syncytia exhibiting green autofluorescence in SLAM-positive cells. Interestingly, all SLAM-negative cell lines examined also showed green autofluorescence after infection with IC323-EGFP, although the virus hardly spread from the originally infected individual cells and thus did not induce syncytia. When the number of EGFP-expressing cells after infection was taken as an indicator, the infectivities of IC323-EGFP for SLAM-negative cells were 2 to 3 logs lower than those for SLAM-positive cells. Anti-MV hemagglutinin antibody or fusion block peptide, but not anti-CD46 antibody, blocked IC323-EGFP infection of SLAM-negative cells. This infection occurred under conditions in which entry via endocytosis was inhibited. These results indicate that MV can infect a variety of cells, albeit with a low efficiency, by using an as yet unidentified receptor(s) other than SLAM or CD46, in part explaining the observed MV infection of SLAM-negative cells in vivo.     

Overcoming the blockade at the upstream of caspase cascade in Fas-resistant HTLV-I-infected T cells by cycloheximide

In spite of carrying large amount of Fas death receptor on the cell surface, Human T cell lymphotropic virus type-I (HTLV-I)-infected T cell lines are resistant to Fas-mediated cytotoxicity. We investigated the mechanism(s) of HTLV-I-induced Fas resistance. Western blotting and enzymatic activity analyses revealed that the Fas-elicited apoptotic signal in HTLV-I-infected T cells was intervened at the level(s) prior to the activation of caspase-8. Upon stimulation, the clustering of Fas receptors scarcely occurred in HTLV-I-infected cells. Cycloheximide treatment converted the resistant cells to sensitive cells; the presence of short-lived anti-apoptotic molecule(s) that can block the caspase-8 activation within HTLV-I-infected T cells is suggested.     

Fibin, a novel secreted lateral plate mesoderm signal, is essential for pectoral fin bud initiation in zebrafish

We identified a novel secreted protein, fibin, in zebrafish, mice and humans. We inhibited its function in zebrafish embryos by injecting antisense fibin morpholino oligonucleotides. A knockdown of fibin function in zebrafish resulted in no pectoral fin bud initiation and abolished the expression of tbx5, which is involved in the specification of pectoral fin identification. The lack of pectoral fins in fibin-knockdown embryos was partially rescued by injection of fibin RNA. fibin was expressed in the lateral plate mesoderm of the presumptive pectoral fin bud regions. Its expression region was adjacent to that of tbx5. fibin expression temporally preceded tbx5 expression in presumptive pectoral fin bud regions, and not abolished in tbx5-knockdown presumptive fin bud regions. In contrast, fibin expression was abolished in retinoic acid signaling-inhibited or wnt2b-knockdown presumptive fin bud regions. These results indicate that fibin is a secreted signal essential for pectoral fin bud initiation in that it potentially acts downstream of retinoic acid and wnt signaling and is essential for tbx5 expression. The present findings have revealed a novel secreted lateral plate mesoderm signal essential for fin initiation in the lateral plate mesoderm.     

SAP regulation of follicular helper CD4 T cell development and humoral immunity is independent of SLAM and Fyn kinase

Mutations in SH2D1A resulting in lack of SLAM-associated protein (SAP) expression cause the human genetic immunodeficiency X-linked lymphoproliferative disease. A severe block in germinal center development and lack of long-term humoral immunity is one of the most prominent phenotypes of SAP(-) mice. We show, in this study, that the germinal center block is due to an essential requirement for SAP expression in Ag-specific CD4 T cells to develop appropriate follicular helper T cell functions. It is unknown what signaling molecules are involved in regulation of SAP-dependent CD4 T cell help functions. SAP binds to the cytoplasmic tail of SLAM, and we show that SLAM is expressed on resting and activated CD4 T cells, as well as germinal center B cells. In addition, SAP can recruit Fyn kinase to SLAM. We have now examined the role(s) of the SLAM-SAP-Fyn signaling axis in in vivo CD4 T cell function and germinal center development. We observed normal germinal center development, long-lived plasma cell development, and Ab responses in SLAM(-/-) mice after a viral infection (lymphocytic choriomeningitis virus). In a separate series of experiments, we show that SAP is absolutely required in CD4 T cells to drive germinal center development, and that requirement does not depend on SAP-Fyn interactions, because CD4 T cells expressing SAP R78A are capable of supporting normal germinal center development. Therefore, a distinct SAP signaling pathway regulates follicular helper CD4 T cell differentiation, separate from the SLAM-SAP-Fyn signaling pathway regulating Th1/Th2 differentiation.     

Increased expression of p62 in expanded polyglutamine-expressing cells and its association with polyglutamine inclusions

Huntington's disease is a progressive neurodegenerative disorder that is associated with a CAG repeat expansion in the gene encoding huntingtin. We found that a 60-kDa protein was increased in Neuro2a cells expressing the N-terminal portion of huntingtin with expanded polyglutamine. We purified this protein, and, using mass spectrometry, identified it as p62, an ubiquitin-associated domain-containing protein. A specific p62 antibody stained the ubiquitylated polyQ inclusions in expanded polyglutamine-expressing cells, as well as in the brain of the huntingtin exon 1 transgenic mice. Furthermore, the level of p62 protein and mRNA was increased in expanded polyglutamine-expressing cells. We also found that p62 formed aggresome-like inclusions when p62 was increased in normal Neuro2a cells by a proteasome inhibitor. Knock-down of p62 does not affect the formation of aggresomes or polyglutamine inclusions, suggesting that p62 is recruited to the aggresome or inclusions secondary to their formation. These results suggest that p62 may play important roles as a responsive protein to a polyglutamine-induced stress rather than as a cross-linker between ubiquitylated proteins.