CB307: A Dual Targeting Costimulatory Humabody VH Therapeutic for Treating PSMA-Positive Tumors.

Purpose: CD137 is a T- and NK-cell costimulatory receptor involved in consolidating immunologic responses. The potent CD137 agonist urelumab has shown clinical promise as a cancer immunotherapeutic but development has been hampered by on-target off-tumor toxicities. A CD137 agonist targeted to the prostate-specific membrane antigen (PSMA), frequently and highly expressed on castration-resistant metastatic prostate cancer (mCRPC) tumor cells, could bring effective immunotherapy to this immunologically challenging to address disease.

Axonal growth towards Xenopus skin in vitro is mediated by matrix metalloproteinase activity.

We have previously demonstrated that the growth of peripheral nervous system axons is strongly attracted towards limb buds and skin explants in vitro. Here, we show that directed axonal growth towards skin explants of Xenopus laevis in matrigel is associated with expression of matrix metalloproteinase (MMP)-18 and also other MMPs, and that this long-range neurotropic activity is inhibited by the broad-spectrum MMP inhibitors BB-94 and GM6001. We also show that forced expression of MMP-18 in COS-7 cell aggregates enhances axonal growth from Xenopus dorsal root ganglia explants.

Fibronectin supports neurite outgrowth and axonal regeneration of adult brain neurons in vitro.

The molecular basis of axonal regeneration of central nervous system (CNS) neurons remains to be fully elucidated. In part, this is due to the difficulty in maintaining CNS neurons in vitro. Here, we show that dissociated neurons from the cerebral cortex and hippocampus of adult mice may be maintained in culture for up to 9 days in defined medium without added growth factors.

Enhancement of axonal regeneration by in vitro conditioning and its inhibition by cyclopentenone prostaglandins.

Axonal regeneration is enhanced by the prior ;conditioning' of peripheral nerve lesions. Here we show that Xenopus dorsal root ganglia (DRG) with attached peripheral nerves (PN-DRG) can be conditioned in vitro, thereafter showing enhanced neurotrophin-induced axonal growth similar to preparations conditioned by axotomy in vivo. Actinomycin D inhibits axonal outgrowth from freshly dissected PN-DRG, but not from conditioned preparations.

A role for GATA-6 in vertebrate chondrogenesis.

The GATA family of transcription factors are known to play multiple critical roles in vertebrate developmental processes, including erythropoiesis, endoderm formation and cardiogenesis. There have been no previous demonstrations of a functional role for any GATA family member being associated with musculoskeletal development but we now identify a possible role for GATA-6 in chondrogenesis. We detect abundant levels of GATA-6 mRNA in precartilaginous condensations (PCCs) in both the axial and appendicular skeleton of mouse embryos and in committed primary chondrocyte precursors.

High-performance X-ray detectors for the new powder diffraction beamline I11 at Diamond.

The design and performance characterization of a new light-weight and compact X-ray scintillation detector is presented. The detectors are intended for use on the new I11 powder diffraction beamline at the third-generation Diamond synchrotron facility where X-ray beams of high photon brightness are generated by insertion devices. The performance characteristics of these detection units were measured first using a radioactive source (efficiency of detection and background count rate) and then synchrotron X-rays (peak stability, light yield linearity and response consistency).

Impaired axonal regeneration by isolectin B4-binding dorsal root ganglion neurons in vitro.

The subpopulation of dorsal root ganglion (DRG) neurons recognized by Griffonia simplicifolia isolectin B4 (IB4) differ from other neurons by expressing receptors for glial cell line-derived neurotrophic factor (GDNF) rather than neurotrophins. Additionally, IB4-labeled neurons do not express the laminin receptor, alpha7-integrin (Gardiner et al., 2005), necessary for optimal axonal regeneration in the peripheral nervous system.

GATA factors in vertebrate heart development and disease.

Vertebrate heart formation is dependent upon complex hierarchical gene regulatory networks, which effect both the specification and differentiation of cardiomyocytes and subsequently cardiac morphogenesis. GATA-4, -5 and -6 comprise an evolutionarily conserved subfamily of transcription factors, which are expressed within the precardiac mesoderm from early stages in its specification and continue to be expressed within the adult heart. We review here the functional roles of individual GATA transcription factors in cardiac development, normal homeostasis and disease.

Wnt2 is a direct downstream target of GATA6 during early cardiogenesis.

The GATA4, 5 and 6 subfamily of transcription factors are potent transactivators of transcription expressed within the precardiac mesoderm. However, little is known of the immediate downstream targets of GATA-factor regulation during the earliest stages of cardiogenesis. Using the P19-CL6 embryonal carcinoma (EC) cell line as an in vitro model of cardiogenesis, we show that GATA6 is the most abundantly expressed of the GATA factors in presumptive cardiac cells.

GATA factors lie upstream of Nkx 2.5 in the transcriptional regulatory cascade that effects cardiogenesis.

Members of the GATA-4, -5, and -6 subfamily of transcription factors are co-expressed with the homeoprotein Nkx 2.5 in the precardiac mesoderm during the earliest stages of its specification and are known to be important determinants of cardiac gene expression. Ample evidence suggests that GATA factors and Nkx 2.

Effective gene delivery to adult neurons by a modified form of electroporation.

Non-viral methods of transfection of cDNAs into adult neurons and other post-mitotic cells are generally very inefficient. However, the recent development of Nucleofector technology developed by Amaxa Biosystems allows direct delivery of cDNAs into the nucleus, enabling transfection of non-dividing cells. In this study, we describe a reliable method for culturing large numbers of retinal cells from adult rats and using Nucleofection, we were able to transfect cDNA-encoding GFP (jellyfish green fluorescent protein) into retinal ganglion cells (RGCs) with relatively high efficiency (up to 28%).

Steroid sulfotransferase 2A1 gene transcription is regulated by steroidogenic factor 1 and GATA-6 in the human adrenal.

Sulfonation is a phase II conjugation reaction responsible for the biotransformation of many compounds including steroids, bile acids, and drugs. Humans are presently known to express at least five cytosolic sulfotransferase (SULT) enzymes, of which only two are hydroxysteroid SULT, SULT2A1, commonly known as steroid sulfotransferase, and the cholesterol sulfotransferase SULT2B1. SULT2A1 is highly expressed in the adrenal where it is responsible for the sulfation of hydroxysteroids including conversion of dehydroepiandrosterone to dehydroepiandrosterone sulfate and in the liver where it is responsible for sulfation of bile acids and circulating hydroxysteroids.

Neurotrophin-independent attraction of growing sensory and motor axons towards developing Xenopus limb buds in vitro.

The mechanisms for directing axons to their targets in developing limbs remain largely unknown though recent studies in mice have demonstrated the importance of neurotrophins in this process. We now report that in co-cultures of larval Xenopus laevis limb buds with spinal cords and dorsal root ganglia of Xenopus and axolotl (Ambystoma mexicanum) axons grow directly to the limb buds over distances of up to 800 microm and in particular to sheets of epidermal cells which migrate away from the limb buds and also tail segments in culture. This directed axonal growth persists in the presence of trk-IgG chimeras, which sequester neurotrophins, and k252a, which blocks their actions mediated via trk receptors.

Widespread expression of an extended peptide sequence of GATA-6 during murine embryogenesis and non-equivalence of RNA and protein expression domains.

The transcription factor GATA-6 is known to be a critical determinant of early vertebrate development. We have shown previously that mammalian GATA-6 genes have the potential to encode two protein isoforms, resulting from alternative, in-frame, initiator methionine codons. We have generated GATA-6 antibodies, including one specific to the longer form of GATA-6, and by immunohistochemical analysis we demonstrate here that the longer protein, which is the more potent transcriptional transactivator, is widely expressed in vivo.

Involvement of alpha7beta1 integrin in the conditioning-lesion effect on sensory axon regeneration.

Conditioning lesions of peripheral nerves improve axonal regeneration after injury and involve changes in expression of proteins required for axonal growth. Integrin alpha7beta1 expression in motor and sensory neurons increases following nerve lesions and motor axon regeneration is impaired in alpha7 integrin KO mice (J. Neurosci.

Widespread expression of an extended peptide sequence of GATA-6 during murine embryogenesis and non-equivalence of RNA and protein expression domains.

The transcription factor GATA-6 is known to be a critical determinant of early vertebrate development. We have shown previously that mammalian GATA-6 genes have the potential to encode two protein isoforms, resulting from alternative, in-frame, initiator methionine codons. We have generated GATA-6 antibodies, including one specific to the longer form of GATA-6, and by immunohistochemical analysis we demonstrate here that the longer protein, which is the more potent transcriptional transactivator, is widely expressed in vivo.

Sensitivity of NGF-responsive dorsal root ganglion neurons to semaphorin D is maintained in both neonatal and adult mice.

Using a coculture assay of DRG neurons and aggregates of cells transfected with individual semaphorins, we have investigated the ability of semaphorins A, D, and E to inhibit axonal growth from DRG neurons. We show that axons of these neurons that grow in response to NGF remain responsive to semaphorin D in neonatal and in adult mice, although sensitivity may decline in the latter. Consistent with these findings, expression of the semaphorin receptor, neuropilin-1, is maintained in the DRGs of adult mice.

The role of retinoic acid receptors in neurite outgrowth from different populations of embryonic mouse dorsal root ganglia.

Dorsal root ganglion (DRG) neurons can be categorised into at least three types, based upon their neurotrophin requirement for survival. We have analysed the expression of the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs) in NGF, NT-3 and BDNF dependent neurons isolated from embryonic day (E)13.5 mouse DRG.

The human and mouse GATA-6 genes utilize two promoters and two initiation codons.

GATA-6 has been implicated in the regulation of myocardial differentiation during cardiogenesis. To determine how its expression is controlled, we have characterized the human and mouse genes. We have mapped their transcriptional start sites and demonstrate that two alternative promoters and 5' noncoding exons are utilized.

GATA-2 is a maternal transcription factor present in Xenopus oocytes as a nuclear complex which is maintained throughout early development.

We show that Xenopus oocytes and embryos contain GATA-2, stored in nuclei as a non-chromatin-bound complex. Its binding site specificity is different from that of GATA-1, having a much higher affinity for the motif with a core GATC sequence. This binding site preference was markedly reduced by either release of the factor with deoxycholate or purification on a DNA affinity column, suggesting a role for a cofactor(s).

GATA factors and the origins of adult and embryonic blood in Xenopus: responses to retinoic acid.

The transcription factors, GATA-1, -2 and -3 play essential roles in the differentiation of haematopoietic cells. To study the process of blood formation during vertebrate development we have used the expression of these GATA factors to locate haematopoietic cells in Xenopus embryos and to act as sensors for the effects of all-trans retinoic acid (RA), a signalling molecule which influences both anteroposterior patterning and haematopoietic differentiation. GATA factor expression was detected in the leading edge of the gastrulating mesoderm, in the ventral blood island (VBI) and dorsolateral plate (DLP) mesoderms and in a population of cells between the VBI and DLP.

Anteriorization of CRABP-I expression by retinoic acid in the developing mouse central nervous system and its relationship to teratogenesis.

We have investigated the role that cellular retinoic acid binding protein I (CRABP-I) may play in the development of the murine hindbrain. Since the central nervous system (CNS) represents a major site of the teratogenic action of retinoic acid (RA), we have also determined the effects of exposure of high levels of RA on CRABP-I expression within the CNS. Expression of CRABP-I can first be detected within the presumptive hindbrain of presomitic mouse embryos and later also appears in neural crest cells and neural crest derivatives; it is thus tissue specific at these early stages.

Restricted expression of a zinc finger protein in male germ cells.

Zfp-37 is a zinc finger protein gene expressed in male germ cells. The cDNA detected two transcripts on Northern blots of testis RNA, with expression first detected at around day 19. To establish the pattern of expression of the protein we have raised antibodies to ZFP-37 and used them on thin sections of testis and on Western blots.

Negative control of Xenopus GATA-2 by activin and noggin with eventual expression in precursors of the ventral blood islands.

To increase our understanding of haematopoiesis during early vertebrate development, we have studied the expression pattern of the transcription factor GATA-2 in Xenopus embryos, and asked how this is regulated. We show that the blood island precursors of the ventral mesoderm express GATA-2 RNA at neural tube stages, some 5 hours before globin RNA is detected in their derivatives. Prior to this however, GATA-2 is expressed much more widely within the embryo.

The mouse t-complex gene, Tcp-11, is under translational control.

The mouse t-complex is known to harbour genes which affect male fertility. Tcp-11 is a t-complex gene which is only expressed in male germ cells and from its position is a candidate for a distorter, one of the two types of genetic element involved in transmission ratio distortion. Antibodies raised to TCP-11 protein made in E.