Modulation of Negative Work Output from a Steering Muscle of the Blowfly Calliphora Vicina

Of the 17 muscles responsible for flight control in flies, only the first basalar muscle (b1) is known to fire an action potential each and every wing beat at a precise phase of the wing-beat period. The phase of action potentials in the b1 is shifted during turns, implicating the b1 in the control of aerodynamic yaw torque. We used the work loop technique to quantify the effects of phase modulation on the mechanical output of the b1 of the blowfly Calliphora vicina. During cyclic length oscillations at 10 and 50 Hz, the magnitude of positive work output by the b1 was similar to that measured previously from other insect muscles. However, when tested at wing-beat frequency (150 Hz), the net work performed in each cycle was negative. The twitch kinetics of the b1 suggest that negative work output reflects intrinsic specializations of the b1 muscle. Our results suggest that, in addition to a possible role as a passive elastic element, the phase-sensitivity of its mechanical properties may endow the b1 with the capacity to modulate wing-beat kinematics during turning maneuvers

Reciprocal knock-in mice to investigate the functional redundancy of lamin B1 and lamin B2.

Lamins B1 and B2 (B-type lamins) have very similar sequences and are expressed ubiquitously. In addition, both Lmnb1- and Lmnb2-deficient mice die soon after birth with neuronal layering abnormalities in the cerebral cortex, a consequence of defective neuronal migration. The similarities in amino acid sequences, expression patterns, and knockout phenotypes raise the question of whether the two proteins have redundant functions. To investigate this topic, we generated "reciprocal knock-in mice"-mice that make lamin B2 from the Lmnb1 locus (Lmnb1(B2/B2)) and mice that make lamin B1 from the Lmnb2 locus (Lmnb2(B1/B1)). Lmnb1(B2/B2) mice produced increased amounts of lamin B2 but no lamin B1; they died soon after birth with neuronal layering abnormalities in the cerebral cortex. However, the defects in Lmnb1(B2/B2) mice were less severe than those in Lmnb1-knockout mice, indicating that increased amounts of lamin B2 partially ameliorate the abnormalities associated with lamin B1 deficiency. Similarly, increased amounts of lamin B1 in Lmnb2(B1/B1) mice did not prevent the neurodevelopmental defects elicited by lamin B2 deficiency. We conclude that lamins B1 and B2 have unique roles in the developing brain and that increased production of one B-type lamin does not fully complement loss of the other

Dysregulation of of phospholipid-specific phagocytosis by B1 B cells in diet-induced obese mice

B1 B cells have received increasing attention recently due to their newly discovered phagocytic and microbicidal capabilities. Several studies have demonstrated that B1 cells can phagocytize polystyrene fluorescent particles, bacteria (Staphylococcus aureus, Escherichia coli), and even apoptotic cells. Nevertheless, little is known about the biological significance of this seemingly redundant function of B1 B cells as compared to that of conventional phagocytes. Here we investigate the unique phosphotidylcholine (PtC)-specific B1 B cell phagocytosis. PtC is a major phospholipid in the biological membrane and a classical antigen recognized by B1 B cell-derived natural antibodies. These antibodies play important roles in immune defense as well as tissue homeostasis. Here we report that B1 cells preferentially phagocytose PtC-coated beads, differing from that of conventional macrophages. We further attest that these beads were truly internalized and subsequently fused with hydrolytic lysosomes indicated by increasing fluorescent intensity of a pH-sensitive dye. Despite the differences in antigen specificity, phagocytosis of both B1 cells and macrophages can be inhibited by the microtubule-inhibitor, Colchicine, in a dose-dependent manner. Most intriguingly, upon chronic high-fat diet (HFD) consumption by the host, B1 cell phagocytosis starts to lose antigen-specificity for PtC. Morphologically, some of these B1 B cells in DIO mice show enlarged cytosol and engulfed more beads, indicating a transition to macrophage-like cells. Our study suggests for the first time that B1 B cells have unique phospholipid-specific phagocytosis capacity, which is affected by diet-induced obesity

Visualisation of tissue kallikrein, kininogen and kinin receptors in human skin following trauma and in dermal diseases

During dermal injury and inflammation the serine proteases kallikreins cleave endogenous, multifunctional substrates (kininogens) to form bradykinin and kallidin. The actions of kinins are mediated by preferential binding to constitutively expressed kinin-B2 receptors or inducible kinin-B1 receptors. A feature of the kinin-B1 receptors is that they show low levels of expression, but are distinctly upregulated following tissue injury and inflammation. Because recent evidence suggested that kinin-B1 receptors may perform a protective role during inflammation, we investigated the specific occurrence of the kallikrein-kinin components in skin biopsies obtained from normal skin, patients undergoing surgery, basalioma, lichenificated atopic eczema, and psoriasis. The tissue was immunolabelled in order to determine the localisation of tissue pro-kallikrein, kallikrein, kininogen and kinin receptors. The kinin components were visualised in normal, diseased and traumatised skin, except that no labelling was observed for kininogen in normal skin. Of the five types of tissue examined, upregulation of kinin-B1 receptors was observed only in skin biopsies obtained following surgery. In essence, the expression of kinin-B1 receptors did not appear to be enhanced in the other biopsies. Within the multiple steps of the inflammatory cascade in wound healing, our results suggest an important regulatory role for kinin-B1 receptors during the first phase of inflammation following injury

Explicit excluded volume of cylindrically symmetric convex bodies

We represent explicitly the excluded volume Ve{B1,B2} of two generic cylindrically symmetric, convex rigid bodies, B1 and B2, in terms of a family of shape functionals evaluated separately on B1 and B2. We show that Ve{B1,B2} fails systematically to feature a dipolar component, thus making illusory the assignment of any shape dipole to a tapered body in this class. The method proposed here is applied to cones and validated by a shape-reconstruction algorithm. It is further applied to spheroids (ellipsoids of revolution), for which it shows how some analytic estimates already regarded as classics should indeed be emended

Nuclear translocation of Cyclin B1 marks the restriction point for terminal cell cycle exit in G2 phase

Upon DNA damage, cell cycle progression is temporally blocked to avoid propagation of mutations. While transformed cells largely maintain the competence to recover from a cell cycle arrest, untransformed cells past the G1/S transition lose mitotic inducers, and thus the ability to resume cell division. This permanent cell cycle exit depends on p21, p53, and APC/CCdh1. However, when and how permanent cell cycle exit occurs remains unclear. Here, we have investigated the cell cycle response to DNA damage in single cells that express Cyclin B1 fused to eYFP at the endogenous locus. We find that upon DNA damage Cyclin B1-eYFP continues to accumulate up to a threshold level, which is reached only in G2 phase. Above this threshold, a p21 and p53-dependent nuclear translocation required for APC/CCdh1-mediated Cyclin B1-eYFP degradation is initiated. Thus, cell cycle exit is decoupled from activation of the DNA damage response in a manner that correlates to Cyclin B1 levels, suggesting that G2 activities directly feed into the decision for cell cycle exit. Once Cyclin B1-eYFP nuclear translocation occurs, checkpoint inhibition can no longer promote mitotic entry or re-expression of mitotic inducers, suggesting that nuclear translocation of Cyclin B1 marks the restriction point for permanent cell cycle exit in G2 phase

The role of hypoxia and complement receptor 2 or toll-like receptor 2 on B1 B cell effector function

Master of ScienceDivision of BiologySherry D. FlemingProfessional phagocytes play a critical role in maintaining homeostasis within a host through phagocytic, microbicidal, and inflammatory activity. Complement receptors (CR) and toll-like receptors (TLRs) aid in phagocytosis and stimulate these cells to enhance the immune response. Environmental factors such as hypoxia, prevalent at sites of tissue damage or infection, induce a similar effect. Systemic components such as opsonins may further enhance phagocyte activity. Similar to professional phagocytes, B1 B cells exhibit a broad range of immunological activity as well as expression of CRs and TLRs. Despite extensive studies with other phagocytes, the effects of CRs and TLRs expression, hypoxic stimulation, or opsonization on B1 B cell function remain unclear. We tested the hypothesis that TLR2 stimulation, hypoxia, CR2 expression, or opsonins would enhance B1 B cell phagocytic and inflammatory activity. Negatively selected peritoneal cavity B1 B cells from the (PerC) of wild type, Tlr2[superscript]-[superscript]/[superscript]-, and Cr2[superscript]-[superscript]/[superscript]- mice, or a B1 B-like cell line, Wehi 231, were subjected to normoxia or hypoxia with or without particles for phagocytosis, TLR2 agonists, or CR2 ligands. The PerC of Tlr2[superscript]-[superscript]/[superscript]- mice contained an altered B1 B cell subset distribution while Cr2[superscript]-[superscript]/[superscript]- mice exhibited a normal repertoire. We demonstrated that hypoxia significantly downregulated inflammatory cytokine production by B1 B cells, while upregulating phagocytic activity in a TLR2 or CR2 dependent manner. TLR2 or CR2 deficiency altered constitutive production of B1 B cell associated cytokines. The CR2 ligand C3d, an opsonin, significantly enhanced the phagocytic activity of B1 B cells but failed to stimulate cytokine production. However, Cr2[superscript]-[superscript]/[superscript]- B1 B cells phagocytosed C3d-coated particles suggesting multiple CR may play a role in B1 B cell phagocytosis. Overall, the data suggest TLRs, CRs, hypoxia, and opsonization all contribute to B1 B cell effector function similar to professional phagocytes

Cyclin B1/Cdk1 phosphorylation of mitochondrial p53 induces anti-apoptotic response.

The pro-apoptotic function of p53 has been well defined in preventing genomic instability and cell transformation. However, the intriguing fact that p53 contributes to a pro-survival advantage of tumor cells under DNA damage conditions raises a critical question in radiation therapy for the 50% human cancers with intact p53 function. Herein, we reveal an anti-apoptotic role of mitochondrial p53 regulated by the cell cycle complex cyclin B1/Cdk1 in irradiated human colon cancer HCT116 cells with p53(+/+) status. Steady-state levels of p53 and cyclin B1/Cdk1 were identified in the mitochondria of many human and mouse cells, and their mitochondrial influx was significantly enhanced by radiation. The mitochondrial kinase activity of cyclin B1/Cdk1 was found to specifically phosphorylate p53 at Ser-315 residue, leading to enhanced mitochondrial ATP production and reduced mitochondrial apoptosis. The improved mitochondrial function can be blocked by transfection of mutant p53 Ser-315-Ala, or by siRNA knockdown of cyclin B1 and Cdk1 genes. Enforced translocation of cyclin B1 and Cdk1 into mitochondria with a mitochondrial-targeting-peptide increased levels of Ser-315 phosphorylation on mitochondrial p53, improved ATP production and decreased apoptosis by sequestering p53 from binding to Bcl-2 and Bcl-xL. Furthermore, reconstitution of wild-type p53 in p53-deficient HCT116 p53(-/-) cells resulted in an increased mitochondrial ATP production and suppression of apoptosis. Such phenomena were absent in the p53-deficient HCT116 p53(-/-) cells reconstituted with the mutant p53. These results demonstrate a unique anti-apoptotic function of mitochondrial p53 regulated by cyclin B1/Cdk1-mediated Ser-315 phosphorylation in p53-wild-type tumor cells, which may provide insights for improving the efficacy of anti-cancer therapy, especially for tumors that retain p53