Hamilton Syringe Research Articles

Caveolin-1 (P132L), a Common Breast Cancer Mutation, Confers Mammary Cell Invasiveness and Defines a Novel Stem Cell/Metastasis-Associated Gene Signature

Here we used the Met-1 cell line in an orthotopic transplantation model in FVB/N mice to dissect the role of the Cav-1(P132L) mutation in human breast cancer. Identical experiments were performed in parallel with wild-type Cav-1. Cav-1(P132L) up-regulated the expression of estrogen receptor-alpha as predicted, because only estrogen receptor-alpha-positive patients have been shown to harbor Cav-1(P132L) mutations. In the context of primary tumor formation, Cav-1(P132L) behaved as a loss-of-function mutation, lacking any tumor suppressor activity. In contrast, Cav-1(P132L) caused significant increases in cell migration, invasion, and experimental metastasis, consistent with a gain-of-function mutation. To identify possible molecular mechanism(s) underlying this invasive gain-of-function activity, we performed unbiased gene expression profiling. From this analysis, we show that the Cav-1(P132L) expression signature contains numerous genes that have been previously associated with cell migration, invasion, and metastasis. These include i) secreted growth factors and extracellular matrix proteins (Cyr61, Plf, Pthlh, Serpinb5, Tnc, and Wnt10a), ii) proteases that generate EGF and HGF (Adamts1 and St14), and iii) tyrosine kinase substrates and integrin signaling/adapter proteins (Akap13, Cdcp1, Ddef1, Eps15, Foxf1a, Gab2, Hs2st1, and Itgb4). Several of the P132L-specific genes are also highly expressed in stem/progenitor cells or are associated with myoepithelial cells, suggestive of an epithelial-mesenchymal transition. These results directly support clinical data showing that patients harboring Cav-1 mutations are more likely to undergo recurrence and metastasis.

Role of Bound Zn(II) in the CadC Cd(II)/Pb(II)/Zn(II)-responsive Repressor

The Staphylococcus aureus plasmid pI258 cadCA operon encodes a P-type ATPase, CadA, that confers resistance to Cd(II)/Pb(II)/Zn(II). Expression is regulated by CadC, a homodimeric repressor that dissociates from the cad operator/promoter upon binding of Cd(II), Pb(II), or Zn(II). CadC is a member of the ArsR/SmtB family of metalloregulatory proteins. The crystal structure of CadC shows two types of metal binding sites, termed Site 1 and Site 2, and the homodimer has two of each. Site 1 is the physiological inducer binding site. The two Site 2 metal binding sites are formed at the dimerization interface. Site 2 is not regulatory in CadC but is regulatory in the homologue SmtB. Here the role of each site was investigated by mutagenesis. Both sites bind either Cd(II) or Zn(II). However, Site 1 has higher affinity for Cd(II) over Zn(II), and Site 2 prefers Zn(II) over Cd(II). Site 2 is not required for either derepression or dimerization. The crystal structure of the wild type with bound Zn(II) and of a mutant lacking Site 2 was compared with the SmtB structure with and without bound Zn(II). We propose that an arginine residue allows for Zn(II) regulation in SmtB and, conversely, a glycine results in a lack of regulation by Zn(II) in CadC. We propose that a glycine residue was ancestral whether the repressor binds Zn(II) at a Site 2 like CadC or has no Site 2 like the paralogous ArsR and implies that acquisition of regulatory ability in SmtB was a more recent evolutionary event.

Loss of Caveolin-3 Induces a Lactogenic Microenvironment that Is Protective Against Mammary Tumor Formation

Here, we show that functional loss of a single gene is sufficient to confer constitutive milk protein production and protection against mammary tumor formation. Caveolin-3 (Cav-3), a muscle-specific caveolin-related gene, is highly expressed in muscle cells. We demonstrate that Cav-3 is also expressed in myoepithelial cells within the mammary gland. To determine whether genetic ablation of Cav-3 expression affects adult mammary gland development, we studied the phenotype(s) of Cav-3(-/-)-null mice. Interestingly, Cav-3(-/-) virgin mammary glands developed lobulo-alveolar hyperplasia, akin to the changes normally observed during pregnancy and lactation. Genome-wide expression profiling revealed up-regulation of gene transcripts associated with pregnancy/lactation, mammary stem cells, and human breast cancers, consistent with a constitutive lactogenic phenotype. Expression levels of three key transcriptional regulators of lactation, namely Elf5, Stat5a, and c-Myc, were also significantly elevated. Experiments with pregnant mice directly showed that Cav-3(-/-) mice underwent precocious lactation. Finally, using orthotopic tumor cell implantation, we demonstrated that virgin Cav-3(-/-) mice were dramatically protected against mammary tumor formation. Thus, Cav-3(-/-) mice are a novel preclinical model to study the protective effects of a lactogenic microenvironment on mammary tumor onset and progression. Our current studies have broad implications for using the lactogenic microenvironment as a paradigm to discover new therapies for the prevention and/or treatment of human breast cancers.

Stem-cell transplantation into the frontal motor cortex in amyotrophic lateral sclerosis patients

Amyotrophic lateral sclerosis (ALS) is characterized by the selective death of motor neurons. CD133(+) stem cells are known to have the capacity to differentiate into neural lineages. Stem cells may provide an alternative treatment for ALS and other neurodegenerative diseases. Five men and five women (aged 38-62 years) with confirmed ALS were included in this study. Our institutional ethics and research committees approved the protocol. After informed consent was obtained, patients underwent Hidrogen-Magnetic Resonance Imaging (H-MRI) spectroscopy and were given scores according to an ALS functional rating scale, Medical Research Council power muscle scale and daily living activities. Bone marrow was stimulated with 300 microg filgrastim subcutaneously daily for 3 days. Peripheral blood mononuclear cells were obtained after admission by leukapheresis. The cell suspension was conjugated with anti-human CD133 superparamagnetic microbeads, and linked cells were isolated in a magnetic field. The isolated cells (2.5-7.5x10(5)) were resuspended in 300 microL of the patient's cerebrospinal fluid, and implanted in motor cortexes using a Hamilton syringe. Ten patients with confirmed ALS without transplantation were used as a control group. Patients were followed up for a period of 1 year. The autologous transplantation of CD133(+) stem cells into the frontal motor cortex is a safe and well-tolerated procedure in ALS patients. The survival of treated patients was statistically higher (P=0.01) than untreated control patients. Stem-cell transplantation in the motor cortex delays ALS progression and improves quality of life.

The Effect of Delivery Via Narrow-Bore Needles on Mesenchymal Cells

Recently, there have been numerous preclinical and human studies investigating the regenerative capacity of cell suspensions following their direct injection into a target organ: the fundamental parameters for successful (clinical) cell therapy. At present, limited data exist in the identification of factors important for the survival of these cells (i.e., morphology, viability and proliferation rates) during and following their ejection via narrow-bore needles. Primary murine mesenchymal stem cells (mMSCs) were isolated, expanded and processed into a concentrated cell suspension consisting of either HBSS or HBSS supplemented with the antioxidant n-acetyl-cysteine. This suspension was then ejected from a 10 microl Hamilton syringe, via a variety of bore-sized needles, at different ejection rates. Cell characteristics including viability, spreading and attachment, apoptosis and proliferative ability were then assessed. Following manipulation within a syringe, a decrease in the viability and cell spreading of mMSCs and a concurrent increase in the production of the caspase-3 protein, an early regulatory event in apoptosis, occurs. These detrimental effects were found to be increased when the cells were left in the syringe chamber for increased periods of time, and were similar at 5 microl/min and 1 microl/min ejection rates. However, on increasing the needle bore diameter, a significant reduction in these characteristics was observed. By comparison, mMSCs that were left to stand at room temperature (18-20 degrees C), but were not manipulated within a syringe, showed a significantly greater viability compared with manipulated cells. However, cells kept at 4 degrees C demonstrated a decreased viability compared with manipulated cells. When the mMSC were incubated with n-acetyl-cysteine, a known antioxidant, no significant change in caspase-3 production or cell spreading was observed. This study highlights potential parameters, such as minimizing the time period the cells are within the syringe and the use of wider-bore needles, involved in maintaining the high viable cell density required for the delivery of cell suspensions for cell therapy applications.

The Fate of Implanted Syngenic Muscle Precursor Cells in Injured Striated Urethral Sphincter of Female Rats

We studied the outcome of syngenic skeletal muscle precursor cells (MPCs) implanted in the striated urethral sphincter of the female rat. These cells were injected at the site of a longitudinal sphincterotomy performed 21 days before implantation. MPCs were isolated from the striated hindlimb muscles of syngenic adult rats and were infected with a retrovirus carrying the gene for either the green fluorescent protein (GFP) or the beta-galactosidase enzyme (beta-gal). MPCs (2 x 10(5)) were injected longitudinally at the site of the lesion in 48 animals using a 10-microl Hamilton syringe. Then the whole urethras were excised from 2 h up to 90 days for cross section immunocytochemistry analysis. All the urethras exhibited connective tissue in place of the injury of the striated fibers. Two hours after injection a cluster of small round basophilic cells was observable at the site of injection and some of them expressed GFP or beta-gal. A few GFP- and beta-gal-positive cells were already detectable 7 days after injection. A large amount of injected cells probably died after injection. Many striated fibers of the urethra became GFP positive from day 7 until day 21, suggesting that few MPCs were allowed to incorporate the divided extremities of the striated fibers from day 7. Unfortunately, we did not observe centronucleated regenerated fibers in this experiment.

Formation of eicosanoids, E2/D2 isoprostanes, and docosanoids following decapitation-induced ischemia, measured in high-energy-microwaved rat brain

Inflammatory lipid mediators derived from arachidonic acid (AA) and docosahexaenoic acid (DHA) modify the pathophysiology of brain ischemia. The goal of this work was to investigate the formation of eicosanoids and docosanoids generated from AA and DHA, respectively, during no-flow cerebral ischemia. Rats were subjected to head-focused microwave irradiation 5 min following decapitation (complete ischemia) or prior to decapitation (controls). Brain lipids were extracted and analyzed by reverse-phase liquid chromatography-tandem mass spectrometry. After complete ischemia, brain AA, DHA, and docosapentaenoic acid concentrations increased 18-, 5- and 4-fold compared with controls, respectively. Prostaglandin E(2) (PGE(2)) and PGD(2) could not be detected in control microwaved rat brain, suggesting little endogenous PGE(2)/D(2) production in the brain in the absence of experimental manipulation. Concentrations of thromboxane B(2), E(2)/D(2)-isoprostanes, 5-hydroxyeicosatetraenoic acid (5-HETE), 5-oxo-eicosatetraenoic acid, and 12-HETE were significantly elevated in ischemic brains. In addition, DHA products such as mono-, di- and trihydroxy-DHA were detected in control and ischemic brains. Monohydroxy-DHA, identified as 17-hydroxy-DHA and thought to be the immediate precursor of neuroprotectin D(1), was 6.5-fold higher in ischemic than in control brain. The present study demonstrated increased formation of eicosanoids, E(2)/D(2)-IsoPs, and docosanoids following cerebral ischemia. A balance of these lipid mediators may mediate immediate events of ischemic injury and recovery.

Activation of 5-HT2A/C Receptors Counteracts 5-HT1A Regulation of N-Methyl-D-aspartate Receptor Channels in Pyramidal Neurons of Prefrontal Cortex

Abnormal serotonin-glutamate interaction in prefrontal cortex (PFC) is implicated in the pathophysiology of many mental disorders, including schizophrenia and depression. However, the mechanisms by which this interaction occurs remain unclear. Our previous study has shown that activation of 5-HT1A receptors inhibits N-methyl-d-aspartate (NMDA) receptor (NMDAR) currents in PFC pyramidal neurons by disrupting microtubule-based transport of NMDARs. Here we found that activation of 5-HT2A/C receptors significantly attenuated the effect of 5-HT1A on NMDAR currents and microtubule depolymerization. The counteractive effect of 5-HT2A/C on 5-HT1A regulation of synaptic NMDAR response was also observed in PFC pyramidal neurons from intact animals treated with various 5-HT-related drugs. Moreover, 5-HT2A/C stimulation triggered the activation of extracellular signal-regulated kinase (ERK) in dendritic processes. Inhibition of the β-arrestin/Src/dynamin signaling blocked 5-HT2A/C activation of ERK and the counteractive effect of 5-HT2A/C on 5-HT1A regulation of NMDAR currents. Immunocytochemical studies showed that 5-HT2A/C treatment blocked the inhibitory effect of 5-HT1A on surface NR2B clusters on dendrites, which was prevented by cellular knockdown of β-arrestins. Taken together, our study suggests that serotonin, via 5-HT1A and 5-HT2A/C receptor activation, regulates NMDAR functions in PFC neurons in a counteractive manner. 5-HT2A/C, by activating ERK via the β-arrestin-dependent pathway, opposes the 5-HT1A disruption of microtubule stability and NMDAR transport. These findings provide a framework for understanding the complex interactions between serotonin and NMDARs in PFC, which could be important for cognitive and emotional control in which both systems are highly involved.

Transient Receptor Potential Vanilloid-4 Has a Major Role in Visceral Hypersensitivity Symptoms

The transient receptor potential vanilloid-4 (TRPV4) is an osmosensitive channel that responds to mechanical stimulation. We hypothesized that TRPV4 could be important in visceral nociception and in the development of hypersensitivity. TRPV4 expression was investigated by immunohistochemistry and reverse transcription-polymerase chain reaction. Calcium signaling and patch-clamp studies were performed in dorsal root ganglia (DRG) neurons validating the use of 4alphaPDD as a selective TRPV4 agonist. The effects of TRPV4 activation on visceral nociception were evaluated in mice that received intracolonically TRPV4 agonist (4 alpha-phorbol 12,13-didecanoate [4alphaPDD]) and in TRPV4-deficient mice in which abdominal muscle contractions in response to colorectal distention (CRD) were recorded. Intervertebral injections of TRPV4 or mismatch small interfering RNA (siRNA) were used to specifically down-regulate TRPV4 expression in sensory neurons and to investigate the role of TRPV4 in basal visceral nociception or in protease-activated receptor 2 (PAR(2)) activation-induced visceral hypersensitivity. TRPV4 agonist 4alphaPDD specifically activated a cationic current and calcium influx in colonic projections of DRG neurons and caused dose-dependent visceral hypersensitivity. TRPV4-targeted but not mismatched siRNA intervertebral treatments were effective at reducing basal visceral nociception, as well as 4alphaPDD or PAR(2) agonist-induced hypersensitivity. Effects of the TRPV4 ligand were lost in TRPV4-deficient mice. 4alphaPDD selectively activates TRPV4 in sensory neurons projecting from the colon, and TRPV4 activation causes visceral hypersensitivity. TRPV4 activation is implicated in the nociceptive response to CRD in basal conditions and in PAR(2) agonist-induced hypersensitivity. These results suggest a pivotal role for TRPV4 in visceral nociception and hypersensitivity.

Mechanism and Regulation of the Two-component FMN-dependent Monooxygenase ActVA-ActVB from Streptomyces coelicolor

The ActVA-ActVB system from Streptomyces coelicolor is a two-component flavin-dependent monooxygenase involved in the antibiotic actinorhodin biosynthesis. ActVB is a NADH:flavin oxidoreductase that provides a reduced FMN to ActVA, the monooxygenase that catalyzes the hydroxylation of dihydrokalafungin, the precursor of actinorhodin. In this work, using stopped-flow spectrophotometry, we investigated the mechanism of hydroxylation of dihydrokalafungin catalyzed by ActVA and that of the reduced FMN transfer from ActVB to ActVA. Our results show that the hydroxylation mechanism proceeds with the participation of two different reaction intermediates in ActVA active site. First, a C(4a)-FMN-hydroperoxide species is formed after binding of reduced FMN to the monooxygenase and reaction with O(2). This intermediate hydroxylates the substrate and is transformed to a second reaction intermediate, a C(4a)-FMN-hydroxy species. In addition, we demonstrate that reduced FMN can be transferred efficiently from the reductase to the monooxygenase without involving any protein.protein complexes. The rate of transfer of reduced FMN from ActVB to ActVA was found to be controlled by the release of NAD(+) from ActVB and was strongly affected by NAD(+) concentration, with an IC(50) of 40 microm. This control of reduced FMN transfer by NAD(+) was associated with the formation of a strong charge.transfer complex between NAD(+) and reduced FMN in the active site of ActVB. These results suggest that, in Streptomyces coelicolor, the reductase component ActVB can act as a regulatory component of the monooxygenase activity by controlling the transfer of reduced FMN to the monooxygenase.

PIKfyve Negatively Regulates Exocytosis in Neurosecretory Cells

Regulated secretion depends upon a highly coordinated series of protein-protein and protein-lipid interactions. Two phosphoinositides, phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3-phosphate, are important for the ATP-dependent priming of the secretory apparatus prior to Ca(2+)-dependent exocytosis. Mechanisms that control phosphoinositide levels are likely to play an important role in priming fine tuning. Here we have investigated the involvement of PIKfyve, a phosphoinositide 5-kinase that can phosphorylate phosphatidylinositol 3-phosphate to produce phosphatidylinositol 3,5-bisphosphate on large dense core vesicle exocytosis from neuroendocrine cells. PIKfyve localizes to a subpopulation of secretory granules in chromaffin and PC12 cells. Nicotine stimulation promoted recruitment of PIKfyve-EGFP onto secretory vesicles in PC12 cells. YM-201636, a selective inhibitor of PIKfyve activity, and PIKfyve knockdown by small interfering RNA potentiated secretory granule exocytosis. Overexpression of PIKfyve or its yeast orthologue Fab1p inhibited regulated secretion in PC12 cells, whereas a catalytically inactive PIKfyve mutant had no effect. These results demonstrate a novel inhibitory role for PIKfyve catalytic activity in regulated secretion and provide further evidence for a fine tuning of exocytosis by 3-phosphorylated phosphoinositides.

Transparent Adult Zebrafish as a Tool for In Vivo Transplantation Analysis

The zebrafish is a useful model for understanding normal and cancer stem cells, but analysis has been limited to embryogenesis due to the opacity of the adult fish. To address this, we have created a transparent adult zebrafish in which we transplanted either hematopoietic stem/progenitor cells or tumor cells. In a hematopoiesis radiation recovery assay, transplantation of GFP-labeled marrow cells allowed for striking in vivo visual assessment of engraftment from 2 hr-5 weeks posttransplant. Using FACS analysis, both transparent and wild-type fish had equal engraftment, but this could only be visualized in the transparent recipient. In a tumor engraftment model, transplantation of RAS-melanoma cells allowed for visualization of tumor engraftment, proliferation, and distant metastases in as little as 5 days, which is not seen in wild-type recipients until 3 to 4 weeks. This transparent adult zebrafish serves as the ideal combination of both sensitivity and resolution for in vivo stem cell analyses.

Inflammatory responses in the rat brain in response to different methods of intra-cerebral administration

Direct intra-cerebral administration of substances into the brain parenchyma is a common technique used by researchers in neuroscience. However, inflammatory responses to the needle may confound the results obtained following injection of these substances. In this paper we show that the use of a glass micro-needle for intra-cerebral injection reduces mechanical injury, blood–brain barrier breakdown and neutrophil recruitment in response to the injection of vehicle or interleukin-1, compared to using a 26-gauge Hamilton syringe. Therefore, the use of a glass micro-needle to inject substances intra-cerebrally appears to cause minimal injection artefact and should be the method of choice.

Intravitreous Injection for Establishing Ocular Diseases Model

Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as direct application of local treatment. This video introduces how to use simple and inexpensive tools to finish the intravitreous injection procedure. Use of a 1 ml syringe, instead of a Hamilton syringe, is used. Practical tips for how to make appropriate injection needles using glass pipettes with perfect tips, and how to easily connect the syringe needle with the glass pipette tightly together, are given. To conduct a good intravitreous injection, there are three aspects to be observed: 1) injection site should not disrupt retina structure; 2) bleeding should be avoided to reduce the risk of infection; 3) lens should be untouched to avoid traumatic cataract. In brief, the most important point is to reduce the interruption of normal ocular structure. To avoid interruption of retina, the superior nasal region of rat eye was chosen. Also, the puncture point of the needle was at the par planar, which was about 1.5 mm from the limbal region of the rat eye. A small amount of vitreous is gently pushed out through the puncture hole to reduce the intraocular pressure before injection. With the 45 degrees injection angle, it is less likely to cause traumatic cataract in the rat eye, thus avoiding related complications and influence from lenticular factors. In this operation, there was no cutting of the conjunctiva and ocular muscle, no bleeding. With quick and minor injury, a successful intravitreous injection can be done in minutes. The injection set outlined in this particular protocol is specific for intravitreous injection. However, the methods and materials presented here can also be used for other injection procedures in drug delivery to the brain, spinal cord or other organs in small mammals.

Discovery of the True Peroxy Intermediate in the Catalytic Cycle of Terminal Oxidases by Real-time Measurement

The sequence of the catalytic intermediates in the reaction of cytochrome bd terminal oxidases from Escherichia coli and Azotobacter vinelandii with oxygen was monitored in real time by absorption spectroscopy and electrometry. The initial binding of O(2) to the fully reduced enzyme is followed by the fast (5 micros) conversion of the oxy complex to a novel, previously unresolved intermediate. In this transition, low spin heme b(558) remains reduced while high spin heme b(595) is oxidized with formation of a new heme d-oxygen species with an absorption maximum at 635 nm. Reduction of O(2) by two electrons is sufficient to produce (hydro)peroxide bound to ferric heme d. In this case, the O-O bond is left intact and the newly detected intermediate must be a peroxy complex of heme d (Fe (3+)(d)-O-O-(H)) corresponding to compound 0 in peroxidases. The alternative scenario where the O-O bond is broken as in the P(M) intermediate of heme-copper oxidases and compound I of peroxidases is not very likely, because it would require oxidation of a nearby amino acid residue or the porphyrin ring that is energetically unfavorable in the presence of the reduced heme b(558) in the proximity of the catalytic center. The formation of the peroxy intermediate is not coupled to membrane potential generation, indicating that hemes d and b(595) are located at the same depth of the membrane dielectric. The lifetime of the new intermediate is 47 micros; it decays into oxoferryl species due to oxidation of low spin heme b(558) that is linked to significant charge translocation across the membrane.

Development of a microfluidic device for determination of cell osmotic behavior and membrane transport properties

An understanding of cell osmotic behavior and membrane transport properties is indispensable for cryobiology research and development of cell-type-specific, optimal cryopreservation conditions. A microfluidic perfusion system is developed here to measure the kinetic changes of cell volume under various extracellular conditions, in order to determine cell osmotic behavior and membrane transport properties. The system is fabricated using soft lithography and is comprised of microfluidic channels and a perfusion chamber for trapping cells. During experiments, rat basophilic leukemia (RBL-1 line) cells were injected into the inlet of the device, allowed to flow downstream, and were trapped within a perfusion chamber. The fluid continues to flow to the outlet due to suction produced by a Hamilton Syringe. Two sets of experiments have been performed: the cells were perfused by (1) hypertonic solutions with different concentrations of non-permeating solutes and (2) solutions containing a permeating cryoprotective agent (CPA), dimethylsulfoxide (Me 2SO), plus non-permeating solute (sodium chloride (NaCl)), respectively. From experiment (1), cell osmotically inactive volume ( V b) and the permeability coefficient of water ( L p) for RBL cells are determined to be 41% [ n = 18, correlation coefficient ( r 2) of 0.903] of original/isotonic volume, and 0.32 ± 0.05 μm/min/atm ( n = 8, r 2 > 0.963), respectively, for room temperature (22 °C). From experiment (2), the permeability coefficient of water ( L p) and of Me 2SO ( P s) for RBL cells are 0.38 ± 0.09 μm/min/atm and (0.49 ± 0.13) × 10 −3 cm/min ( n = 5, r 2 > 0.86), respectively. We conclude that this device enables us to: (1) readily monitor the changes of extracellular conditions by perfusing single or a group of cells with prepared media; (2) confine cells (or a cell) within a monolayer chamber, which prevents imaging ambiguity, such as cells overlapping or moving out of the focus plane; (3) study individual cell osmotic response and determine cell membrane transport properties; and (4) reduce labor requirements for its disposability and ensure low manufacturing costs.

Immobilization of Mesenchymal Stem Cells and Monocytes in Biocompatible Microcapsules to Cell Therapy

The aim of cell therapy is to replace, repair, or enhance the function of damaged tissues or organs. Several factors complicate the development of cellular therapies. Of primary importance is protection of the implanted cells from the host's immune system. Cells are encapsulated in selectively semipermeable and biocompatible membranes that block entry of immune mediators but allow outward diffusion of active molecules produced by the cells. The immobilization of mesenchymal stem cells and monocytes, in micrometric (30-60 microm) alginate-barium microcapsules based on atomization processes, has been achieved successfully. This size is necessary to the administration of microcapsules via injection (Hamilton syringe with a needle size of 100 microm) and aerosol. Microencapsulated cells survive at least 2 weeks after preparation in vitro.

Activated Platelets and Monocytes Generate Four Hydroxyphosphatidylethanolamines via Lipoxygenase

12/15-Lipoxygenase (LOX) mediates immune-regulatory activities not accounted for by its known free acid eicosanoids, suggesting that additional lipids may be generated by activated cells. To characterize novel LOX-derived lipids, a lipidomic approach was utilized. Ionophore-activated interleukin-4-treated human peripheral monocytes generated up to 10-fold more esterified 15-hydroxyeicosatetraenoic acid (15-HETE) than free in a phosphatidylinositol 3-kinase- and protein kinase C-sensitive manner. Precursor scanning electrospray ionization/tandem spectroscopy for m/z 319 (HETE, [M-H](-)) showed 4 ions at m/z 738, 764, 766, and 782 that were identified using tandem spectroscopy and MS3 as specific diacyl and plasmalogen 15-HETE phosphatidylethanolamines. Using H (18)(2)O water, the compounds were shown to form by direct oxidation of endogenous phosphatidylethanolamine (PE) by 15-LOX, with PE being the preferred phospholipid pool containing 15-HETE. Similarly, human platelets generated 4 analogous PE lipids that contained 12-HETE and increased significantly in response to ionophore, collagen, or convulxin. These products were retained in the cells, in contrast to free acids, which are primarily secreted. Precursor scanning of platelet extracts for the major platelet-derived prostanoid, thromboxane B2 (m/z 369.2), did not reveal PE esters, indicating that this modification is restricted to the LOX pathway. In summary, we show formation of PE-esterified HETEs in immune cells that may contribute to LOX signaling in inflammation.

Empty Class II Major Histocompatibility Complex Created by Peptide Photolysis Establishes the Role of DM in Peptide Association

DM catalyzes the exchange of peptides bound to Class II major histocompatibility complex (MHC) molecules. Because the dissociation and association components of the overall reaction are difficult to separate, a detailed mechanism of DM catalysis has long resisted elucidation. UV irradiation of DR molecules loaded with a photocleavable peptide (caged Class II MHC molecules) enabled synchronous and verifiable evacuation of the peptide-binding groove and tracking of early binding events in real time by fluorescence polarization. Empty DR molecules generated by photocleavage rapidly bound peptide but quickly resolved into species with substantially slower binding kinetics. DM formed a complex with empty DR molecules that bound peptide with even faster kinetics than empty DR molecules just having lost their peptide cargo. Mathematical models demonstrate that the peptide association rate of DR molecules is substantially higher in the presence of DM. We therefore unequivocally establish that DM contributes directly to peptide association through formation of a peptide-loading complex between DM and empty Class II MHC. This complex rapidly acquires a peptide analogous to the MHC class I peptide-loading complex.

Novel Role of Y1 Receptors in the Coordinated Regulation of Bone and Energy Homeostasis

The importance of neuropeptide Y (NPY) and Y2 receptors in the regulation of bone and energy homeostasis has recently been demonstrated. However, the contributions of the other Y receptors are less clear. Here we show that Y1 receptors are expressed on osteoblastic cells. Moreover, bone and adipose tissue mass are elevated in Y1(-/-) mice with a generalized increase in bone formation on cortical and cancellous surfaces. Importantly, the inhibitory effects of NPY on bone marrow stromal cells in vitro are absent in cells derived from Y1(-/-) mice, indicating a direct action of NPY on bone cells via this Y receptor. Interestingly, in contrast to Y2 receptor or germ line Y1 receptor deletion, conditional deletion of hypothalamic Y1 receptors in adult mice did not alter bone homeostasis, food intake, or adiposity. Furthermore, deletion of both Y1 and Y2 receptors did not produce additive effects in bone or adiposity. Thus Y1 receptor pathways act powerfully to inhibit bone production and adiposity by nonhypothalamic pathways, with potentially direct effects on bone tissue through a single pathway with Y2 receptors.