In vivo gene transfer into the testis by electroporation and viral infection--a novel way to study testis and sperm function.

The study of gene function in testis and sperm has been greatly assisted by creation of transgenic mice by injection of a transgene into the fertilised egg. However this approach is costly and laborious and is not applicable to other species of importance for the study of sperm function, such as the hamster. We have investigated alternative ways of expressing transgenes in mouse and hamster testis and sperm by in vivo gene transfer. DNA expression constructs were introduced into the testis by injection of DNA followed by electroporation, or by injection of a lentiviral vector. Expression of fluorescent proteins was assessed by fluorescence microscopy. In vivo gene transfer by electroporation led to expression of a fluorescent reporter protein and a fluorescently tagged version of sperm protein phospholipase C zeta in hamster and mouse testis and epididymal sperm. In vivo gene transfer by lentiviral infection led to high level expression of a fluorescent reporter protein in male germ cells. In conclusion, in vivo gene transfer offers a novel way to study gene function in testis and sperm and may also have potential as a way of creating transgenic versions of important model organisms such as the hamster.

Proteasomal interactors control activities as diverse as the cell cycle and glutaminergic neurotransmission.

The six regulatory non-redundant ATPases in the base of the 19 S regulator of the 26 S proteasome belong to the AAA superfamily of ATPases. Yeast two-hybrid genetic screens, biochemical analyses and cell biological studies have identified and characterized new interactors of the human S6 (rpt3) and S8 (rpt6) ATPases of the 19 S regulator of the 26 S proteasome. The S6 ATPase interacts with gankyrin. This protein is found in purified human 26 S proteasomes and in a smaller complex(es) containing CDK4 and free S6 ATPase. Gankyrin overexpression causes the phosphorylation of the retinoblastoma protein (pRb) and the release of E2F transcription factor to trigger the expression of DNA synthesis genes. Gankyrin is oncogenic in nude mice and is overexpressed in hepatocellular carcinoma cells (HCCs). The S8 ATPase interacts with members of the large Homer-3 protein family. There are three Homer genes; the Homer 1 and 2 gene products control trafficking and calcium-store-related functions of metabotropic glutamate receptors (e.g. mGluR1alpha). Homer-3A11 by binding to the S8 ATPase brings mGluR1alpha to the 26 S proteasome for degradation. The degradation of mGluR1alpha is blocked by proteasomal inhibitors and by overexpression of the N-terminus of Homer which binds to the receptor. The S8 ATPase and mGluR1alpha are co-localized in Purkinje dendrites in rat cerebellum. The data are discussed in terms of the regulation of the cell cycle and glutaminergic receptor functions by the 26 S proteasome.

Evidence for an excitatory amino acid pathway in the brainstem and for its involvement in cardiovascular control.

The source and possible role of excitatory amino acid projections to areas of the ventrolateral medulla (VLM) involved in cardiovascular control were studied. Following the injection of [3H]D-aspartate ([3H]D-Asp), a selective tracer for excitatory amino acid pathways, into vasopressor or vasodepressor areas of the VLM in rats, more than 90% of retrogradely labelled neurones were found in the nucleus of the solitary tract (NTS). Very few of the [3H]D-Asp-labelled cells were immunoreactive for tyrosine hydroxylase, none for phenylethanolamine-N-methyltransferase or gamma-aminobutyric acid. The density of labelled cells in the NTS was similar to that obtained with the non-selective tracers wheat germ agglutinin-horseradish peroxidase (WGA-HRP) and WGA-colloidal gold, but these tracers also labelled other cell groups in the medulla. Furthermore, the decrease in blood pressure, caused by pharmacological activation of neurones in the NTS of rats, or by electrical stimulation of the aortic depressor nerve in rabbits could be blocked by the selective N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovalerate injected into the caudal vasodepressor area of the VLM. This area corresponds to the termination of [3H]D-Asp transporting NTS neurones. These results provide evidence that a population of NTS neurones projecting to the VLM use excitatory amino acids as transmitters. Among other possible functions, this pathway may mediate tonic and reflex control of blood pressure via NMDA receptors in the VLM.

Evidence for increased epidermal growth factor receptors in human sarcomas.

The results of an immunocytochemical study of the epidermal growth factor receptor (EGFR) in 35 human soft-tissue sarcomas, using a murine monoclonal antibody (MAb) EGF-R1, are reported. In many of the tumours staining was stronger than in the adjacent stroma, suggesting increased levels of receptor. Particularly strong staining was seen in one epithelioid sarcoma and in the spindle-cell component of a synovial sarcoma. Binding studies carried out on an epithelioid sarcoma cell line established from one of the specimens, using radiolabelled EGF, showed that approximately 8% of the receptors were of high affinity with a dissociation constant (KD) of approximately 10(-10)M, while the remainder were of lower affinity with a KD of 10(-9)M. The cells expressed a total of 1.7 X 10(6) receptors/cell which is equivalent to that found in some epidermoid tumours where gene amplification has been demonstrated. These data suggest that, as with other tumours recently reported, increased levels of epidermal growth factor receptor may be related to transformation.

Glycoprotein PAS-0 from the milk fat globule membrane carries antigenic determinants for epithelial membrane antigen.

Epithelial membrane antigen (EMA) can be found in human tissues using antisera raised against defatted human cream. PAS-0 is a glycoprotein which has been extracted from human milk fat globule membranes. Using a polyclonal antiserum and a series of monoclonal antibodies, we have shown that the major antigenic determinant for EMA is carried by PAS-0. A more detailed comparison of the two glycoproteins has been made by establishing a set of radioimmunoassays using the different antibodies.

Definition of three species-specific monoclonal antibodies recognizing antigenic structures present on human embryonal carcinoma cells which undergo modulation during in vitro differentiation.

Three novel species-specific monoclonal antibodies 5.1.H, 8.7.D and 13.7.A raised against semi-purified detergent solubilized fractions of the Tera 1 embryonal carcinoma (EC) cell line are restricted in their in vitro distribution to undifferentiated human EC cell lines. Competition experiments have established that distinct antigenic specificities are seen by the 3 different monoclonal antibodies. All 3 antigens 5.1.H, 8.7.D and 13.7.A, defined by these monoclonal antibodies, undergo developmental regulation and cease to be expressed on Tera 2 clones 5 and 12 after retinoic-acid-induced differentiation and on LICR LON HT 39/7 cells after phorbol-ester-induced differentiation. These results taken together with the extremely limited in vivo tissue distribution of the defined antigens suggest that the 5.1.H, 8.7.D and 13.7.A monoclonal antibodies define distinct onco-foetal antigens.