Gamma-irradiation-induced cell cycle arrest and cell death in a human submandibular gland cell line: effect of E2F1 expression.

This study examined the effect of gamma-irradiation (5 and 10 Gy) on the human submandibular cell line (HSG). Radiation treatment (5 Gy and 10 Gy) induced a dose-dependent decrease in cell proliferation, with a G2/M arrest of the cell cycle, and an increase in cell death (cells with <2n DNA increased from 7% in control cells to 34% and 40% in 5 and 10 Gy irradiated cells, respectively). [Ca2+]i measurements demonstrated that the status of internal Ca2+ stores, and muscarinic receptor-mediated Ca2+ mobilization, in irradiated cells was comparable to that in non-irradiated cells. These data suggest that 1) irradiated HSG cells maintain normal physiology and 2) internal Ca2+ store depletion does not account for the decreased cell proliferation. To manipulate the radiation-induced cell cycle arrest, we examined the effect of the transcription factor E2F1, which has been shown to induce cell cycle progression in HSG cells (Lillibridge and O'Connell, 1997, J. Cell. Physiol., 1 72:343-350). The ability of irradiated HSG cells to express and appropriately route proteins was demonstrated by using adenovirus-mediated expression of beta-galactosidase, alpha1-antitrypsin, and aquaporin-1. Infection of HSG cells with an adenoviral vector encoding E2F1, either 12 h before or immediately following irradiation, but not post-irradiation, induced maintenance of cells in the S phase of the cell cycle, reduced the number of cells arrested at G2/M, and decreased the rate of appearance of cells with <2n DNA. While the mechanism of irradiation-induced cell death has not yet been confirmed, these data suggest that expression of the E2F1 gene product in HSG cells can be a useful strategy to manipulate cell cycle events and reduce the initial loss of cells due to radiation.

Somatic gene transfer to salivary glands.

Recent developments in gene transfer technology have expanded the range of in vivo experimentation and provided new insights that might be applicable to the treatment of human diseases. Somatic gene transfer may complement conventional transgenic animal experiments by allowing for more restricted gene expression. Salivary glands of rats are readily transduced in vivo by adenovirus vectors. This model has been used to demonstrate the effects of transferring a water channel (aquaporin) gene to glands that have been damaged by radiation. Submandibular glands that receive the aquaporin vector increase the stimulated salivary flow close to normal levels. The possible role of E2F1 in promoting cell regeneration in vivo was also explored. A vector expressing E2F1 was capable of increasing DNA synthesis in rat salivary glands, though complete mitosis was not observed. Future generations of vectors must overcome current limitations of efficiency, immunogenicity, and transient expression.

Safety of salivary gland-administered replication-deficient recombinant adenovirus in rats.

We have examined the safety of a replication-deficient recombinant adenovirus administered at a single, high dose intraductally to rat submandibular glands or systemically via the femoral vein. The virus used directed the synthesis of human aquaporin-1, a water channel protein, and is termed AdhAQP1. Comparisons were made 1 and 9 days post-infection with animals administered either a similar virus encoding no transgene or the viral suspension buffer. Animals were specifically not given anti-inflammatory drugs to impede the well-known immunopathologic response to recombinant adenoviral administration. Serum chemistries and hematological parameters were monitored. Rats were subjected to complete gross necropsy and selected tissues were evaluated by histopathology. Most clinical chemistry and hematology values were within normal ranges; however, evidence of inflammation (e.g., elevated lactic dehydrogenase, total leukocyte count) was seen. Gross pathology was normal, as was histopathology, excepting rare focal areas of necrosis. The results show that intrasalivary gland or intravenous AdhAQP1 administration leads to low levels of toxicity in rats.

Triplex-forming oligonucleotides can modulate aquaporin-5 gene expression in epithelial cells.

Triplex-forming oligonucleotides (TFOs) may provide a useful approach to decrease gene transcription in vivo. We have identified two sequences in the rat aquaporin 5 (rAQP5) cDNA that are capable of forming a DNA triple helix. We designed four TFOs based on these sequences (a purine and a pyrimidine TFO per sequence). All four TFOs were able to bind to the rAQP5 cDNA at varying efficiencies in vitro as measured by using gel mobility shift assays. The TFOs were delivered to intact MDCK epithelial cells via adenovirus-polylysine complexes. Experiments with fluorescein-isothiocyanate-labeled oligonucleotides delivered in this way showed primarily a nuclear localization. Three of the four TFOs internalized by adenovirus-polylysine complexes were capable of decreasing rAQP5 expression in intact MDCK cells infected with a recombinant adenovirus encoding rAQP5. These data show that adenovirus-polylysine-TFO complexes can result in TFO delivery to the nucleus in intact epithelial cells and that TFOs may provide a useful way to selectively modulate rAQP5 gene expression.

In human salivary gland cells, overexpression of E2F1 overcomes an interferon-gamma- and tumor necrosis factor-alpha-induced growth arrest but does not result in complete mitosis.

Increased levels of cytokines in the salivary glands have been associated with the loss of secretory cells and reduced salivary function. It has been demonstrated that interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) treatment of a human submandibular gland (HSG) cell line causes growth arrest in the G0/G1 phase of the cell cycle, followed by apoptosis. To stimulate DNA synthesis and reverse this growth arrest, we used an adenovirus vector to overexpress the transcription factor E2F1 in HSG cells. Initially, cells were synchronized by a double thymidine block and then infected with recombinant adenovirus (AdE2F1) expressing E2F1. Cells were harvested at intervals and analyzed by flow cytometry. Greater than 50% of synchronized cells infected with AdE2F1 were in S phase by 18 hours postinfection (hpi) compared to 12% of uninfected cells. Similarly, AdE2F1 infection of HSG cells arrested by IFN-gamma and TNF-alpha treatment caused a fivefold increase in S-phase cells by 48 hpi. However, by 72 hpi, AdE2F1-infected cells showed increases in the subdiploid cell population. Forty-one percent of AdE2F1-infected cells labeled positive by TUNEL, compared to fewer than 6% for controls. Additionally, AdE2F1-infected cells (84 hpi) had low forward-angle and high side scatter light characteristics, similar to apoptotic lymphocytes. These results suggest that E2F1 accumulation in growth-arrested salivary gland cells can stimulate DNA synthesis and overcome a G0/G1 block in the cell cycle. However, E2F1 overexpression did not lead to complete mitosis in HSG cells but, rather, diverted cells into an apoptotic pathway.

Dirofilaria immitis: ultrastructural localization, molecular characterization, and analysis of the expression of p27, a small heat shock protein homolog of nematodes.

A cDNA fragment encoding the complete coding region of a 27-kDa protein (p27) of Dirofilaria immitis was cloned. Antibody to the recombinant p27 bound to hypodermal tissues of third (L3) and fourth stage larvae (L4) of D. immitis and to both the hypodermis and the cuticle of L3s of Onchocerca volvulus, as visualized by immunoelectronmicroscopy. The deduced amino acid sequence of the central and C-terminal regions of p27 (amino acids S83 to H222) is 18-36% identical to members of the sHsp/alpha-crystallin family of proteins. The homologous region is thought to be responsible for the molecular chaperone activity of members of this family. The p27 cDNA does not encode a hydrophobic signal peptide. At least two homologous yet distinct p27 genes were identified in the D. immitis genome by Southern hybridization using the p27 cDNA as a probe. The p27 transcript was 0.9 kb in length on Northern blots. The expression of p27 in L3s of D. immitis was neither upregulated by heat shock (43 degrees C) nor by incubation at the physiologic temperature of 37 degrees C. Pulse-labeling experiments of both D. immitis and Brugia malayi L3s during the L3-L4 molt in vitro showed that synthesis of p27 is also not upregulated during this developmental phase. However, p27 is expressed constitutively throughout the D. immitis L3-L4 molt and therefore by both larval stages. In addition, both female and male adult worms of this species express p27 constitutively. P27, or an allomorph thereof, was detected in each of nine species representing four nematode superfamilies, thus indicating that this molecule is ubiquitous within the phylum Nematoda. In view of the hypodermal localization of p27, its constitutive expression, and its retention among nematodes, the function of this protein in essential housekeeping roles such as that of molecular chaperone during the molting process is discussed.

Brugia malayi: the diagnostic potential of recombinant excretory/secretory antigens.

The diagnostic potential of recombinant E/S antigens of the lymphatic filaria Brugia malayi was investigated by Western blot. A cDNA expression library was constructed using B. malayi male adult worm mRNA, and E/S recombinants were identified with a rabbit antiserum raised against E/S products collected in vitro from B. malayi male and female adult worms. Two of these recombinants, Bm12 and Bm14L, were studied after subcloning the cDNA inserts in an Escherichia coli plasmid expression and purification vector, obtaining the inserts' nucleotide sequence, and purifying the expressed proteins. By homology of their deduced amino acid sequence with that of previously identified proteins, Bm12 was identified as the B. malayi gp 15/400 antigen, and Bm14 as a member of the hsp90 family of heat shock proteins. The antigenic cross-reactivity of the purified recombinant proteins was assessed with 28 serum samples from patients infected with Ascaris, Trichuris, or hookworm, and also with a few samples from patients with onchocerciasis and loiasis. For Bm12, the specificity for all of the intestinal helminthiasis together was 75%. Bm14L, on the other hand, cross-reacted with all of the ascariasis serum samples with which it was tested. Presence of antibodies cross-reactive with B. malayi was confirmed in all of these serum samples by examining their antibody reactivity with Western blots of extracts of whole B. malayi adult worms. A semiquantitative (+ or -) assessment of the sensitivity of Bm12 for antibody detection was performed using 6 serum samples from patients with chronic filariasis and 24 samples from patients with microfilaremia. All of these serum samples contained anti-Bm12 antibody (sensitivity of 100%). Finally, the ability of Bm12 to detect antibody before the onset of patency was established with a longitudinal collection of serum samples obtained from 2 African green vervets (Cercopithecus aethiops) and 3 rhesus macaques (Macaca mulatta), all of which were infected with B. malayi. Anti-Bm12 antibodies were detectable in all animals between 4 and 11 weeks before patency.

Quantitative observations of hymens in prepubescent females selected for non-abuse.

The maximum hymenal opening was evaluated quantitatively in 111 prepubescent females during routine physical examinations in a pediatric subspecialty office. For comparison, an additional 53 females referred by child protective agencies were also examined. Analysis of data show "non-abused" groups may be separated statistically from "abused" groups on the basis of area of hymenal opening. The mean area for the "non-abused, non-masturbate" group was 6.4 mm2. The upper limit of area (mean + 3 S.D.) of hymenal opening in this group was 24.1 mm2. A child having a hymenal opening diameter of 6.94 mm or less has a 99% chance of being in the "non-abused" group. The area of hymenal opening for "non-abused" groups did not change with increasing age, height or weight. A skilled pediatrician knowledgeable in the area of sexual abuse may obtain clinically relevant information with ordinary office equipment and trained personnel. Regular and repeated observations of genitalia during routine health maintenance examinations are vital baseline measurements for the physical and mental health of young female patients.

Studies on human gastric mucosal zymogen granules and their zymogens.

Intact human gastric mucosal zymogen granules (ZG) were detected in specimens from surgical resections of one patient with gastric adenocarcinoma and two with benign gastric ulcer. Both large ZG with unilaminar membranes and smaller ZG with trilaminar membranes were identified by electron microscopy. The zymogens in the ZG and in mucosal extracts were separated by gel electrophoresis. Slow-moving Protease (SMP) was seen in the whole mucosal extracts but was absent from ZG. One specimen of pyloric mucosa showed a striking absence of ZG. Despite the absence of ZG, pyloric mucosa showed Pepsinogens 2-5 (constituents of PG I) as well as Pepsinogens 6 and 7 (constituents of PG II) and SMP.

A method for qualitative identification of sugars and semiquantitative determination of lactose content suitable for a variety of foods.

A semiquantitative chromatographic technique was developed and standardized using water/chloroform extracts of foods which are spotted onto heat-activated silica gel thin layer chromatographic (TLC) plates, run in butanol/acetic acid/ether/water, developed in acids anisaldehyde, and quantitated planimetrically using Purdy and Truter's formula. Clinically significant amounts of lactose were found in low-calorie sweeteners, breads, yogurt, margarine, penicillin, Gantrisin, and other commonly ingested nondairy substances.