Threshold Level Of Expression Research Articles

Transgenic expression of pancreatic secretory trypsin inhibitor-1 rescues SPINK3-deficient mice and restores a normal pancreatic phenotype

Endogenous trypsin inhibitors are synthesized, stored, and secreted by pancreatic acinar cells. It is believed that they play a protective role in the pancreas by inhibiting trypsin within the cell should trypsinogen become prematurely activated. Rodent trypsin inhibitors are highly homologous to human serine protease inhibitor Kazal-type 1 (SPINK1). The mouse has one pancreatic trypsin inhibitor known as SPINK3, and the rat has two trypsin inhibitors commonly known as pancreatic secretory trypsin inhibitors I and II (PSTI-I and -II). Rat PSTI-I is a 61-amino acid protein that shares 65% sequence identity with mouse SPINK3. It was recently demonstrated that mice with genetic deletion of the Spink3 gene (Spink3(-/-)) do not survive beyond 15 days and lack normal pancreata because of pancreatic autophagy. We have shown that targeted transgenic expression of the rat Psti1 gene to acinar cells in mice [TgN(Psti1)] protects mice against caerulein-induced pancreatitis. To determine whether the autophagic phenotype and lethality in Spink3(-/-) mice were due to lack of pancreatic trypsin inhibitor, we conducted breeding studies with Spink3(+/-) heterozygous mice and TgN(Psti1) mice. We observed that, whereas Spink3(+/+), Spink3(+/-), and Spink3(-/-)/TgN(Psti1) mice had similar survival rates, no Spink3(-/-) mice survived longer than 1 wk. The level of expression of SPINK3 protein in acini was reduced in heterozygote mice compared with wild-type mice. Furthermore, endogenous trypsin inhibitor capacity was reduced in the pancreas of heterozygote mice compared with wild-type or knockout mice rescued with the rat Psti1 gene. Surprisingly, the lesser amount of SPINK3 present in the pancreata of heterozygote mice did not predispose animals to increased susceptibility to caerulein-induced acute pancreatitis. We propose that a threshold level of expression is sufficient to protect against pancreatitis.

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) functions as an oligomer and oligomerization is dependent on receptor density

Lectin-like oxidized low-density lipoprotein (LDL) receptor (LOX-1) exists as a homodimer formed by an intermolecular disulfide bond. Although the dimer is the minimum structural unit of LOX-1 on cell membranes, LOX-1 can form larger noncovalent oligomeric complexes. But, the functional unit of LOX-1 is not known. We quantitatively analyzed the correlation between cyan fluorescent protein-tagged LOX-1 expression and the fluorescence-labeled ligand (DiD-AcLDL) binding ability on each cell. The results clearly indicate that there is a threshold level of expression that enables LOX-1 to bind ligand. Above this threshold level, the ability of LOX-1 to bind ligand was proportional to its level of expression. Using the membrane impermeable crosslinker BS 3, we detected oligomers (primarily hexamers) only on the cell lines that stably expressed LOX-1 above the threshold level. In contrast, little oligomer or ligand binding was detected in cell lines expressing LOX-1 below the threshold level. Moreover, oligomerization was independent of ligand binding. These results indicate that the functional unit of LOX-1 is an oligomer and that oligomerization of LOX-1 is dependent on the receptor density on the plasma membrane.

1104. Imaging of sec-R Directed and PEG-Stabilized Gene Transfer Nanoparticles in CF Mice

Compacted DNA targeted to the serpin enzyme complex receptor (sec-R) can deliver amounts of human CFTR DNA sufficient for partial correction of the chloride transport defect and NOS-2 downregulation in the noses of CF knock out mice. To examine the distribution of these compacted DNA particles in vivo, we used radioscintigraphy imaging. We modified our DNA compacting agent (a polymer of poly-L-lysine, chemically conjugated to the C105Y sec-R targeting ligand) with radiolabeled I-125. Prior to dosing, 12 S489X/FABP-hCFTR were imaged by X-ray for proper anatomical alignment with radiograph images. Following complex formulation, 9 mice were dosed intranasally (IN) with 25 |[mu]|g of sec-R targeted radiolabeled hCFTR DNA (specific activity 50 |[mu]|Ci each), while 3 control mice received administration of free I-125 (specific activity 50 |[mu]|Ci each). We used a small animal gamma scanner to analyze radio images at 2, 4, 24, and 48 hrs following nasal administration of the dose. At each time 3 animals were sacrificed, frozen and stored for subsequent sectioning. For each mouse, I-125 scintigraphic images, x-ray images, and autoradiographs were aligned and analyzed. Targeted delivery resulted in higher total retained activity than the carrier control. Both test and control groups begin to exhibit thyroid uptake by 2 hrs, while significantly higher activities were observed in the nasal and lower airways for animals that received the targeted complexes vs. controls. The highest observed activity was in thyroid, liver and bladder. Autoradiographic images correlated well with their corresponding in vivo scintigraphic images. For mice that received targeted complexes, strong signals were measured in both abdominal (stomach and liver) and bladder regions indicating possible ingestion during or aspiration following administration, as well as secretion into the bladder. This rapid clearance by 2 hrs was observed for all mice. Regional analysis showed significantly (p<0.05) higher activities for the targeted delivery in nose, lung and liver vs. controls, 2 and 4 hrs post administration. By 24 hrs, activities in the upper airways and lungs were back to the background level, while activity in the thyroid remained significant at 24 and 48 hrs. The data indicate a rapid clearance of sec-R targeted compacted DNA (by 2 hrs), followed by the appearance of at least the labeled component of the complex (the ligand) in the thyroid and bladder by 4 hrs. We also examined expression conferred by second generation PEG-stabilized compacted nanoparticles containing the Firefly luciferase gene using bioluminescent imaging. Expression was detected in lung 2 and 4 days following intra-tracheal (IT) but not IN administration. Lung homogenates from both methods of administration exhibited luciferase activity although levels in the IT group were significantly higher. These data suggest that a threshold level of expression, achieved only with IT administration, is necessary for successful bioluminescent detection . Development of these real-time imaging techniques has allowed us to better assess the localization and site of activity of our gene transfer complex formulations.

Transcription-independent triggering of the extrinsic pathway of apoptosis by human papillomavirus 18 E2 protein.

Cervical carcinomas are most frequently associated with human papillomaviruses (HPV), whose E6 and E7 oncogenes products induce cellular immortalization. The papillomavirus E2 protein is a transcription factor, which represses the expression of the viral oncogenes, and activates viral DNA replication during the vegetative viral cycle. This protein is specifically inactivated in HPV18-associated carcinoma cells, suggesting that E2 functions prevent carcinogenic progression. Indeed, ectopic expression of E2 in cervical carcinoma cells strongly inhibits cell proliferation. Here we show that above a threshold level of expression, the E2 protein induces apoptosis, independently of other viral functions. The amino-terminal domain is responsible for this apoptotic activity, but surprisingly with no involvement of its transcriptional functions. The death pathway triggered by E2 relies on activation of the initiator caspase 8, specific of the extrinsic pathway of apoptosis. E2 itself is cleaved by caspases during cell death, providing an example of an apoptotic inducer that is itself a target for caspase processing. The autonomous proapoptotic activity of HPV18 E2 described here may counteract the proliferative functions of viral oncogenes, and renders the inactivation of E2 crucial for carcinogenic progression.

Genetics of Time of Internode Elongation and Duration of Rosette Habit in Carthamus tinctorius L. and C. flavescens Spreng1

Rosette habit is an important component in the length of the life cycle of safflower (Carthamus spp.). Rosette habit results from nonelongation of internodes. Segregating populations indicated that time of internode elongation, a measure of duration of rosette habit, was influenced by a major gene and modifier genes in C. tinctorius L. (cultivated safflower) and C. flavescens Spreng (an annual herbaceous weed, indigenous to the Middle East). All crosses showed that earliness of internode elongation had dominance, ranging from partial to over dominance. At threshold levels of expression, the frequency of rosette habit in some C. tinctorius genotypes changed with environments; however, the mean length of the first internode of these nonrosette plants was from 1/5 to 1/2 that of ‘Gila,’ which is noted for its nonrosette habit in a wide range of environments.