No abstract

A commonly encountered problem in orthopedics is bone protein was expressed in essentially 100% of selected cells and cartilage tissue injury which heals incompletely or within vitro and was observed in the experimental explants out full structural integrity. This necessitates development from animals after both 4 and 8 weeks in vivo, while cells of improved methods for treatment of injuries which are not transduced with a retroviral vector bearing only the neo r amenable to treatment using current therapies. An already gene in negative control explants showed no blue staining. large and growing number of growth factors which play sigWe extended our study by delivering a gene of therapeutic nificant roles in bone remodeling and repair have been relevance, human bone morphogenic protein 7 (hBMP-7), identified in the past few years. It is well established that to primary periosteal cells via retroviral vector. The hBMPbone morphogenic proteins induce the production of new 7 gene was cloned from human kidney 293 cell total RNA bone and cartilage. An efficient method of delivery of these by RT-PCR into a retroviral vector under control of the growth factors by conventional pharmacological means CMV enhancer/promoter. Hydroxyapatite secretion, prehas yet to be elucidated. We wished to evaluate the use sumably caused by overexpression of hBMP-7, was of retroviral vector-mediated gene transfer to deliver genes observed on the surface of the transduced and selected of therapeutic relevance for bone and cartilage repair. To periosteal cells, however, this level of expression was toxic determine the feasibility of using amphotropically packaged to both PA317 producer and primary periosteal cells. Subretroviral vectors to transduce primary rabbit mesenchymal sequently, the strong CMV enhancer/promoter driving the stem cells of periosteal origin, primary periosteal cells were hBMP-7 gene was replaced in the retroviral vector by a isolated from New Zealand white rabbits, transduced in weaker enhancer/promoter from the rat ␤-actin gene. Nonvitro with a retroviral vector bearing both the nuclear toxic levels of expression of hBMP-7 were confirmed at localized lacZ marker gene and the neo r gene, and selecboth the RNA and protein levels in PA317 producer and ted in G418. We used a convenient model for analysis of primary periosteal cell lines and cell supernatants. This in vivo stability of these cells which were seeded on to polywork demonstrates the feasibility of using a gene therapy mer scaffold grafts and implanted into rabbit femoral osteoapproach in attempts to promote bone and cartilage tissue chondral defects. The nuclear localized ␤-galactosidase repair using gene-modified periosteal cells on grafts.

In a companion paper (Jacquin et al., '89), the structure and function of local circuit (LC) neurons in spinal trigeminal (V) subnucleus interpolaris (Sp Vi) were described. The present report provides similar data for 44 projection neurons in Sp Vi. Of these, 25 thalamic, 16 cerebellar, 2 superior collicular, and 1 inferior olivary projecting neurons were studied. The majority responded to vibrissa(e) deflection, and all except 4 of these had multivibrissae receptive fields. The remainder were responsive to either guard hair deflection or indentation of glabrous skin. Latencies to V ganglion shocks were suggestive of monosynaptic activation from the periphery. Sp Vi projection neurons were topographically organized in a manner consistent with that of their primary afferent inputs. Nonvibrissa sensitive cells had diverse morphologies. Morphometric analyses of the more heavily sampled thalamic and cerebellar projecting, vibrissa(e)-sensitive cells indicated the following. (1) As compared to LC neurons, projection neurons had bigger receptive fields, cell bodies, dendritic trees, and axons; less circular dendritic trees; a greater preponderance of spiny dendrites and fewer axon collaterals in Sp Vi. (2) Dendritic tree extent correlated significantly with receptive field size, thus suggesting that dendritic tree size is one mechanism contributing to receptive field size in vibrissae-sensitive projection neurons. (3) V thalamic cells had significantly bigger receptive fields and dendritic trees, and also give off more local axon collaterals, than V cerebellar neurons. Collicular and inferior olivary projecting neurons shared structural and functional attributes with other Sp Vi long-range projecting cells. Structure-function relationships exist for vibrissa-sensitive projection neurons in Sp Vi. The relevant parameters correlating with projection neuron morphology are receptive field size and projection status, whereas for Sp Vi LC neurons the relevant correlative parameter is peripheral receptor association.

IL-4-induced gene-1 (Il4i1 or Fig1) initially isolated as a gene of unknown function from mouse B lymphocytes, is limited in expression to primarily immune tissues and genetically maps to a region of susceptibility to autoimmune disease. The predicted Il4i1 protein (IL4I1) sequence is most similar to apoptosis-inducing protein and Apoxin I, both l-amino acid oxidases (LAAO; Enzyme Commission 1.4.3.2). We demonstrate that IL4I1 has unique LAAO properties. IL4I1 has preference for aromatic amino acid substrates, having highest specific activity with phenylalanine. In support of this selectivity, IL4I1 is inhibited by aromatic competitors (benzoic acid and para-aminobenzoic acid), but not by nonaromatic LAAO inhibitors. Il4i1 protein and enzyme activity is found in the insoluble fraction of transient transfections, implying an association with cell membrane and possibly intracellular organelles. Indeed, IL4I1 has the unique property of being most active at acidic pH (pH 4), suggesting it may reside preferentially in lysosomes. IL4I1 is N-linked glycosylated, a requirement for lysosomal localization. Confocal microscopy of cells expressing IL4I1 translationally fused to red fluorescent protein demonstrated that IL4I1 colocalized with GFP targeted to lysosomes and with acriflavine, a green fluorescent dye that is taken up into lysosomes. Thus, IL4I1 is a unique mammalian LAAO targeted to lysosomes, an important subcellular compartment involved in Ag processing.