Tobacco plants were genetically transformed with the gene encoding hepatitis B surface antigen (HBsAg) linked to a nominally constitutive promoter. Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg revealed the presence of HBsAg in extracts of transformed leaves at levels that correlated with mRNA abundance. This suggests that there were no major inherent limitations of transcription or translation of this foreign gene in plants. Recombinant HBsAg was purified from transgenic plants by immunoafflnity chromatography and examined by electron microscopy. Spherical particles with an average diameter of 22 nm were observed in negatively stained preparations. Sedimentation of transgenic plant extracts in sucrose and cesium chloride density gradients showed that the recombinant HBsAg and human serum-derived HBsAg had similar physical properties. Because the HBsAg produced in transgenic plants is antigenically and physically similar to the HBsAg particles derived from human serum and recombinant yeast, which are used as vaccines, we conclude that transgenic plants hold promise as low-cost vaccine production systems.Hepatitis B virus infection is one ofthe most widespread viral infections of humans and causes acute and chronic hepatitis and hepatocellular carcinoma (1). The infectious viral particle (Dane particle) is a 43-nm double-shelled sphere that consists of a core containing the 3.2-kilobase (kb) DNA genome bound to the core protein, surrounded by the viral envelope containing phospholipids and the major surface antigen [hepatitis B surface antigen (HBsAg)] (2). In addition to Dane particles, the serum of infected individuals also contains 22-nm subviral particles in great excess over virions. These noninfectious particles contain the elements of the viral envelope, including the major 24-kDa peptide that occurs in glycosylated and unglycosylated forms (2).Because the host range of hepatitis B virus is limited to humans and chimpanzees, and since the virus cannot be propagated in cell culture, HBsAg for use in vaccines was purified from the serum of infected individuals until a recombinant form (rHBsAg) was produced in yeast (3). The immunogenic yeast-derived rHBsAg occurs in the form of spherical particles with an average diameter of 17 nm. Integration of the peptides into the phospholipid-containing particles greatly enhances their immunogenic properties (4). Subsequent work showed that the peptides present in the yeastderived particles were much less extensively disulfide-linked than in the human material but that such linking could be induced in vitro (5).Intramuscular injection of serum-derived HBsAg or yeastderived rHBsAg in healthy individuals results in effective immunization and protection from viral infection (6, 7). In many areas ofthe developing world, however, the expense of immunization programs for large segments of the population is prohibitive. This has led us to attempt the expression of rHBsAg in plants with the hope of developing a less ex...

A high-affinity uptake mechanism for [3H]-gamma-aminobutyric acid (GABA) has been localized to type H1 cone horizontal cells and type Ab pyriform amacrine cells in the retina of the goldfish by light and electron microscopy autoradiography. By stimulating isolated retinas with colored lights during incubation we have been able to use [3H]-GABA uptake as a probe of light-evoked changes in membrane potential. All colors of lights increase and darkness decreases [3H]-GABA uptake by H1 cone horizontal cells. Our model of voltage dependence of GABA uptake predicts that all colors of light should hyperpolarize H1 cone horizontal cells and other investigators have shown by intracellular recording and dye-marking that type H1 cone horizontal cells hyperpolarize to all wavelengths of light. We have also obtained evidence that dark-induced depolarization of cone horizontal cells leads to release of GABA. Type Ab pyriform amacrine cells show maximal [3H]-GABA uptake in darkness and when exposed to green or blue lights, but red lights dramatically suppress uptake. We predict these neurons to be red-depolarizing, and recent intracellular recordings and dye-marking by Famiglietti et al. ('77) support our conclusions. Synaptic relations of apparently GABA-ergic neurons were investigated in the electron microscope. We propose type H1 cone horizontal cells to be both pre- and post-synaptic to red-sensitive cones and type Ab pyriform amacrine cells to be both pre- and post-synaptic to red-sensitive center-depolarizing bipolar cells.

Gendicine (recombinant human p53 adenovirus), developed by Shenzhen SiBiono GeneTech Co. Ltd., was approved in 2003 by the China Food and Drug Administration (CFDA) as a first-in-class gene therapy product to treat head and neck cancer, and entered the commercial market in 2004. Gendicine is a biological therapy that is delivered via minimally invasive intratumoral injection, as well as by intracavity or intravascular infusion. The wild-type (wt) p53 protein expressed by Gendicine-transduced cells is a tumor suppressor that is activated by cellular stress, and mediates cell-cycle arrest and DNA repair, or induces apoptosis, senescence, and/or autophagy, depending upon cellular stress conditions. Based on 12 years of commercial use in >30,000 patients, and >30 published clinical studies, Gendicine has exhibited an exemplary safety record, and when combined with chemotherapy and radiotherapy has demonstrated significantly higher response rates than for standard therapies alone. In addition to head and neck cancer, Gendicine has been successfully applied to treat various other cancer types and different stages of disease. Thirteen published studies that include long-term survival data showed that Gendicine combination regimens yield progression-free survival times that are significantly longer than standard therapies alone. Although the p53 gene is mutated in >50% of all human cancers, p53 mutation status did not significantly influence efficacy outcomes and long-term survival rate for Ad-p53-treated patients. To date, Shenzhen SiBiono GeneTech has manufactured 41 batches of Gendicine in compliance with CFDA QC/QA requirements, and 169,571 vials (1.0 × 10 vector particles per vial) have been used to treat patients. No serious adverse events have been reported, except for vector-associated transient fever, which occurred in 50-60% of patients and persisted for only a few hours. The manufacturing accomplishments and clinical experience with Gendicine, as well as the understanding of its cellular mechanisms of action and implications, could provide valuable insights for the international gene therapy community and add valuable data to promote further developments and advancements in the gene therapy field.

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Abstract. By means of the velocity sedimentation technique for cell separation, single cell suspensions from the testes of the mouse could be separated into at least seven peaks each with a different sedimentation velocity. These were named and characterized as follows: a (12 mm/hr); # (10 mm/hr); y (6.5 mm/ hr); a (4.3 mm/hr); 0 (broad peak with median 2.5 mm/hr); K (1.1 mm/hr); X (0.75 mm/hr). Tritiated thymidine was injected into thirty groups of mice. The spermatogonial cells of each group were separated at one hour and then at daily intervals, and the acid insoluble activity of each fraction was measured. This method enabled us to determine the differentiation patterns of mouse spermatogenesis by following the thymidine label with time. It was found that the spermatogonia and primary spermatocytes formed the 0 peak with S-phase spermatogonia and primary spermatocytes having a similar sedimentation velocity of 2.1 mm/hr. The ,3 cells were identified as late pachytenes and diplotene cells, the y cells as secondary spermatocytes, the 6 cells as earliest spermatids, the K cells as spermatids, and the X cells as mature spermatozoa.

After goldfish retinas had been incubated for 1 hr with Fy-3H1aminobutyric acid, we found by autoradiography that the label was localized to a few restricted types of retinal cells. In particular, external and internal horizontal cells from light-stimulated retinas were more heavily labeled than corresponding cells from retinas kept in darkness. Some other cells and tissues in the retina also incorporated the labeled acid. Light stimulation, however, did not cause a pronounced change in the amount of label associated with these cells. Among these were some heavily labeled cells on the vitreal side of the inner nuclear layer, and scattered grains associated with the ganglion cell and optic nerve layers. Electrophoresis of retinal extracts after incubation with the labeled acid also showed that light-stimulated retinas contained about 40-100% more radioactivity than retinas kept in darkness, and that 90% of this activity remained as acid.

For determination of possible neurotransmitters synthesized by photoreceptor cells, turtle retinas were dissociated into single cells with proteolytic enzymes. These cells were partially separated by velocity sedimentation to yield a fraction rich in photoreceptors. Individual photoreceptor cells were then sucked into a micropipette and incubated with labeled precursors of known or suspected neurotransmitters. After incubation, the radioactive products were analyzed by high-voltage electrophoresis. Of all the chemicals tested, turtle photoreceptor cells synthesized only acetylcholine, suggesting that these cells may be cholinergic.Identities of neurotransmitters in the central nervous system of vertebrates are known for only a few cell types (1). Thus, although we now have a fair understanding of the neuronal connections and physiological behavior of various retinal cell types (2-7), the chemistry of synapses in the retina is still unknown. A direct way of obtaining this information is by examination of the ability of single, intact, and identified cells to synthesize known or suspected neurotransmitters. The vertebrate retina is a convenient part of the central nervous system for such an analysis, because most retinal cell types can be readily identified even when they are isolated.The presence of several known or suspected neurotransmitters, such as' acetylcholine, 'y-aminobutyric acid, and dopamine,'has been suggested or demonstrated in some vertebrate retinas (8-11). There is, however, no direct evidence that any of these compounds are synthesized or used in a particular cell type. In order to assign possible chemical transmitters to individual cell types, single cells were dissociated by proteolytic enzymes, separated by velocity sedimentation, and analyzed by incubation of selected cells with labeled precursors of known or suspected neurotransmitters. Photoreceptors were chosen for this initial analysis because they are first-order sensory neurons of the visual pathway. Turtle photoreceptors were used because they are unusually large and contain brightly colored oil droplets that permit easy identification. In addition, their structure permits analysis of transmitter synthesis by the cell bodies in the absence' of the pedicles, which may contain postsynaptic fragments from horizontal and bipolar cells. Furthermore, the anatomy (12) and physiology (13) of turtle photoreceptors have been studied in detail. MATERIALS AND METHODSCell Dissociation. All media and Ringer's solutions were supplemented with 1000 units/ml of penicillin G and 0.5 mg/ ml of streptomycin sulphate (Microbiological Associates, 1987 Inc., Bethesda, Md.), and sterilized (18). For cell dissociation and separation, isotonic calcium-free Ringer's solution supplemented with ethyleneglycol bis(aminoethyl) tetraacetic acid (EGTA) was used (in g/liter: 7.3. NaCl-0.5 NaHCO3-0.07 NaH2PO4-0.25 KCl-0.1 MgCl2-2 glucose; 5 mM EGTA; pH 7.2).Fresh-water turtles (Pseudemy8 scripta elegans, with shells about 20 cm long) were adapted to darknes...

Goldfish retinas incubated with L-glutamate-IdC (UL) were found to synthesize -~-aminobutyric acid-IdC (GABA-14C) The accumulation of newly synthesized GABA was enhanced by physiological stimulation of the retina with flashing light; and this increase was directly proportional to the logarithm of the light intensity. The total GABA content was also higher in light-stimulated than in dark-adapted retinas, although the glutamate content remained unchanged No &fferences were found in the cell-free activities of glutamate decarboxylase (EC 4 1.t 15) and GABA-glutamate transaminase (EC 2.6