Casein gene expression: from transfection to transgenics.
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Animals↗
Biomedical subjects
J M Rosen
Publications and source records attributed to J M Rosen.
Differential effects of the simian virus 40 early genes on mammary epithelial cell growth, morphology, and gene expression.
To study the effect of SV40 T-antigen in mammary epithelial cells, a rat beta-casein promoter-driven SV40 early-region construct was stably introduced into the clonal mouse mammary epithelial cell line HC11. With the expression of the viral T-antigens under the control of a hormone-inducible promoter, it was possible to dissociate the effects of different levels of T-antigen expression on cell growth, morphology, and gene expression. Following hormonal induction, a rapid but transient induction of T-antigen was observed, followed by a delayed induction of H4 histone mRNA. In T-antigen-positive HC11 cells cultured in the absence of EGF, the expression of basal levels of T-antigen (in the absence of hormonal induction) led to a decreased doubling time and an increased cell density. In the presence of EGF, T-antigen expression resulted additionally in an altered cell morphology. Despite the effects of T-antigen on cell growth and gene expression, the cells were unable to form colonies in soft agar and were nontumorigenic when transplanted into cleared mammary fat pads. They were, however, weakly tumorigenic in nude mice. Relatively high levels of p53 protein synthesis were observed in both the transfected HC11 cells and the parental COMMA-D cells, as compared to 3T3E fibroblasts and another mammary epithelial cell line. The HC11 and COMMA-D cells synthesized approximately equal levels of wild-type and mutated p53 proteins as defined by their reactivities with monoclonal antibodies PAb246 and PAb240, respectively. Interactions between excess p53 and T-antigen may, in part, explain the failure of these cells to display a completely transformed phenotype.
Adenovirus Early Proteins↗
Correlation of in vitro release rate and in vivo absorption characteristics of four chlorpheniramine maleate extended-release formulations.
An in vitro/in vivo correlation was established for four formulations of chlorpheniramine maleate (histamine, H1-blocker) extended-release tablets exhibiting different in vitro release rate characteristics. In vitro release rate data were obtained for 12 individual tablets of each formulation using the USP Apparatus 2, paddle stirrer at 50 rpm in 1000 ml of distilled water at 37.0 +/- 0.5 degrees C. Inspection of the individual and mean release rate data indicated that the in vitro release rate of chlorpheniramine maleate was consistent with the intended design of the four extended-release formulations. The in vivo bioavailability and pharmacokinetics of these formulations were evaluated in 24 healthy subjects under fasting conditions. Wagner Nelson analyses of the in vivo data revealed extended release absorption profiles for all four formulations. Linear regression analyses of the mean percentage of dose absorbed versus the mean in vitro release resulted in a statistically significant correlation (r2 greater than 0.98, P less than 0.001) for each formulation. Qualitative rank-order correlations were observed among all combinations of in vitro and in vivo parameters. These data support a Level A correlation between the in vitro release rate profiles and the in vivo absorption for chlorpheniramine maleate determined under fasting conditions.
Adult↗
A practical guide to international leech transportation.
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Animals↗
Diffuse pulmonary tumor microemboli from adenocarcinoma of the colon.
We have presented a rare case of pulmonary tumor microemboli from adenocarcinoma of the colon. The majority of primary tumors are adenocarcinomas, with gastric, breast, and liver carcinomas most frequently identified. Dyspnea and respiratory distress without an obvious cause is common. The history, physical examination, chest roentgenogram, ventilation-perfusion lung scan, and pulmonary angiogram are often not helpful in establishing the diagnosis. Pulmonary hypertension, present in our patient, and cor pulmonale are well described in association with diffuse pulmonary tumor microemboli. The pathologic findings are limited to the intravascular compartment exclusively, frequently in association with thrombus. The true incidence of pulmonary tumor microemboli is not known and may be underestimated. Appropriate management requires a high index of suspicion to establish the diagnosis.
Adenocarcinoma, Papillary↗
Regeneration microelectrode array for peripheral nerve recording and stimulation.
A microelectrode array capable of recording from and stimulating peripheral nerves at prolonged intervals after surgical implantation has been demonstrated. The microelectrode array, fabricated on a silicon substrate perforated by multiple holes (referred to as via holes), is implanted between the ends of a surgically severed nerve. Regenerating tissue fixes the device in place to provide a stable mapping between the microelectrodes and the axons in the nerve. Processes were developed for the fabrication of thin-film iridium microelectrodes, micromachined via holes, and silicon nitride passivation layers. All fabrication methods were designed to be compatible with standard CMOS/BiCMOS processes to allow for on-chip signal processing circuits in future designs. Such arrays, implanted in the peroneal nerves of rats, were used to record from and stimulate the nerves at up to 13 months postoperatively.
Animals↗
High-level expression of the rat whey acidic protein gene is mediated by elements in the promoter and 3' untranslated region.
The high-level expression of the rat whey acidic protein (WAP) gene in transgenic mice depends on the interaction of 5'-flanking promoter sequences and intragenic sequences. Constructs containing 949 bp of promoter sequences and only 70 bp of 3'-flanking DNA were expressed at uniformly high levels, comparable to or higher than that of the endogenous gene. Although this WAP transgene was developmentally regulated, it was expressed earlier during pregnancy than was the endogenous WAP gene. Replacement of 3' sequences, including the WAP poly(A) addition site, with simian virus 40 late poly(A) sequences resulted in an approximately 20-fold reduction in the expression of WAP mRNA in the mammary gland during lactation. Nevertheless, position-independent expression of the transgene was still observed. Further deletion of 91 bp of conserved WAP 3' untranslated region (UTR) led to integration site-dependent expression. Position independence was restored following reinsertion of the WAP 3' UTR into the deleted construct at the same location, but only when the insertion was in the sense orientation. The marked differences observed between the expression levels of the 3'-end deletion constructs in transgenic mice were not seen in transfected CID 9 mammary epithelial cells. In these cells, expression of the endogenous WAP gene was dependent on the interaction of these cells with a complex extracellular matrix. In contrast, the transfected WAP constructs were not dependent on extracellular matrix for expression. Thus, both the abnormal expression of WAP in cells cultured on plastic and the precocious developmental expression of WAP in transgenic mice may reflect the absence of a negative control element(s) within these recombinant constructs.
Animals↗
Artificial nerve graft using glycolide trimethylene carbonate as a nerve conduit filled with collagen compared to sutured autograft in a rat model.
A study was conducted to compare the regeneration of rat peroneal nerves across 0.5 cm gaps repaired with artificial nerve grafts (ANG) versus sutured autografts (SAG). The ANG model is composed of a synthetic biodegradable passive conduit made of glycolide trimethylene carbonate (GTMC) filled with a collagen matrix (predominantly Type I collagen, derived from calf skin, and with the telopeptide ends left intact). Axonal regeneration was studied in 11 long-term animals (two at 6 months and nine at 9 months). The nerves were studied by qualitative and quantitative histological, electrophysiological, and functional assays. Axonal regeneration with the ANG was equal to SAGs as measured by axonal diameters, physiological, and functional methods, although the SAG demonstrated statistically higher axonal counts.
Action Potentials↗
Expression of biologically active heterodimeric bovine follicle-stimulating hormone in milk of transgenic mice.
Follicle-stimulating hormone (FSH; follitropin) is a pituitary glycoprotein composed of two post-translationally modified subunits, which must properly assemble to be biologically active. FSH has been difficult to purify and to obtain in quantities sufficient for detailed biochemical studies. We have targeted FSH expression to the mammary gland of transgenic mice by using cDNAs encoding the bovine alpha and FSH beta subunits and a modified rat beta-casein gene-based expression system. Lines of bigenic mice expressing both subunits have been generated either by coinjection of the subunit transgenes or by mating mice that acquired and expressed transgenes encoding an individual subunit. Up to 60 international units (15 micrograms) of biologically active FSH per ml was detected in the milk of the bigenic mice. These lines provide a model system for studying the post-transcriptional mechanisms that effect the expression and secretion of this heterodimeric hormone.
Animals↗
A comparison of suture and tubulization nerve repair techniques in a primate.
This study compared standard methods of nerve repair, epineurial or perineurial sutures with a technique termed fascicular tubulization using a biodegradable polyglycolic acid tube in a nonhuman primate model. Electrophysiologic analysis demonstrated that the percentage of proximal axons that conducted across the repair site did not significantly differ among the three techniques while epineurial suture repairs were associated with significantly longer conduction delays across the repair site compared with the other two techniques. Even though fascicular tubulization using the current polyglycolic acid tube resulted in regeneration equal to the currently perceived best suture repair technique, associated technical problems with the current tube design indicate that this fascicular tubulization technique cannot, at present, be considered as an alternative to present clinically used nerve suture techniques.
Animals↗
Comparison of nerve repair techniques: suture vs. avitene-polyglycolic acid tube.
A study was designed to determine whether a completely sutureless technique of nerve repair using avitene and polyglycolic acid (PGA) tube could provide a better repair than the standard suture repair technique. Randomized peroneal nerves of 18 male Sprague-Dawley rats were used. The study was divided into two parts. The first part was designed to test the adhesive and tensile strength of avitene at the second postoperative day in seven animals. The tensile strength of the suture repair at 498 mN +/- 130 was found to be statistically equivalent (p = 0.77) to the repair using avitene and PGA tube at 474 mN +/- 192. The second part of the study evaluated axonal regeneration in 11 animals. Evaluation by electrophysiology revealed a significant difference (p = 0.05) between the mean percentage of Integrated Mean Compound Action Potential for the suture repaired nerve (53.1 +/- 17.6 percent) and that of the avitene and PGA tube repaired nerve (72.0 +/- 17.9). The mean axonal count and mean fiber diameter for the suture repair technique (1,879 +/- 225 and 4.3 +/- 0.4 microns, respectively) were not significantly different (p = 0.61 and 0.67, respectively) from those of the avitene-PGA tube repair technique (1,938 +/- 398 and 4.2 +/- 0.4 microns, respectively).
Animals↗
The artificial nerve graft: a comparison of blended elastomer-hydrogel with polyglycolic acid conduits.
A study was undertaken to compare the regeneration of rat peroneal nerves across a 0.5-cm gap repaired with either a permanent, porous or a resorbable, non-porous artificial nerve graft. The resorbable, impermeable artificial nerve graft was a synthetic passive conduit made from polyglycolic acid (PGA). The permanent, porous artificial nerve graft conduit was manufactured from a hydrophilic elastomeric biopolymer (HEB), and four variations were tested. Qualitative histology on short-term animals revealed similar inflammatory reactions to HEB and PGA. Axonal regeneration was evaluated in longer-term animals after three, four, and six months by qualitative and quantitative histology. Qualitative histology on longer-term animals demonstrated both artificial nerve grafts to be anti-immunogenic. All PGA-artificial nerve graft repairs among three-, four-, and six-month rats contained myelinated axons, as did all HEB-1 repairs. However, three other HEB-graft varieties accounted for a 25 percent failed regeneration rate. Quantitative histologic comparison of repair-site cross-sections in viable PGA and HEB matched pairs demonstrated statistically equivalent myelinated axon counts but larger average myelinated fiber diameters in HEB repairs, with p = .001.
Animals↗
Monitoring in microvascular surgery.
The importance of monitoring in microvascular surgery is underscored by the high reported salvage rates of failing free flaps and replants. In this overview, we begin by defining the physiology of ischemic tissue with emphasis given to the no-reflow phenomenon and the secondary critical ischemia times. Based on the physiological changes accompanying ischemia, several variables are defined that can be monitored to reflect the vascular state of a free flap or replant. Multifarious monitoring systems are then reviewed, including clinical observation, temperature, isotope clearance, ultrasonic Doppler, laser Doppler, transcutaneous oxygen tension, reflection plethysmography, dermofluorometry, pH, electromagnetic flowmetry, serial hematocrits, interstitial fluid pressure, and magnetic resonance imaging.
Humans↗
The uterine blush on In-111 labeled leukocyte scintigraphy.
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Adult↗
A heterologous hormone response element enhances expression of rat beta-casein promoter-driven chloramphenicol acetyltransferase fusion genes in the mammary gland of transgenic mice.
Previous studies have demonstrated that the entire rat beta-casein (R beta C) gene and a -524/+490 R beta C fragment-chloramphenicol acetyltransferase (CAT) fusion gene are expressed preferentially in the mammary gland of transgenic mice in a developmentally regulated fashion. However, transgene expression was infrequent, less than 1% of that observed for the endogenous gene, and varied as much as 500-fold, presumably due to the site of chromosomal integration. To determine whether a heterologous hormone-responsive enhancer could be used to increase both the level and frequency of expression in the mammary gland, a fragment derived from the mouse mammary tumor virus long terminal repeat containing four hormone response elements (HREs) was inserted into the R beta C promoter at a site not known to contain transcriptional regulatory elements. Transgenic mice generated which carried HRE-enhanced R beta C-CAT fusion genes expressed CAT activity in the mammary glands of all founder lines examined at levels that were on average 13-fold greater than for lines generated with similar constructs not carrying HREs. In the highest expressing line, the level of HRE-enhanced transgene expression was found to be developmentally regulated, increasing 14-fold in the mammary gland from virgin to day 10 of lactation. In this line, expression was also observed in the thymus and spleen; however, the level of CAT activity was 4-fold lower than in the mammary gland and was not developmentally regulated. In adrenalectomized mice, the administration of dexamethasone stimulated CAT expression in the mammary gland but not in the thymus and spleen. These studies demonstrate that in the context of the R beta C promoter, the HRE functions in the mammary gland to increase both the frequency and level of transgene expression.
Animals↗
Tissue-specific, high level expression of the rat whey acidic protein gene in transgenic mice.
The importance of intragenic and 3' flanking sequences in the control of the temporal, hormonal and tissue-specific expression of milk whey acidic protein (WAP) has been demonstrated in transgenic mice. Mouse lines carrying a 4.3 kb genomic clone containing the entire rat WAP gene minus 200 bp of the first intron with 0.949 kb of 5' and 1.4 kb of 3' flanking DNA were generated. In eight of nine independent lines of mice analyzed, WAP transgene expression was detected at levels ranging from 1% to 95% (average, 27%) of the endogenous gene. The transgene was expressed preferentially in the mammary gland. Although developmentally regulated during pregnancy and lactation, the temporal pattern of WAP transgene expression differed from the endogenous gene. A precocious increase in expression of the transgene was detected at 7 days of pregnancy, several days earlier in pregnancy than the major increase observed in endogenous mouse WAP mRNA. The rat WAP transgene was translated and secreted into the milk of transgenic mice at levels comparable to the endogenous mouse WAP. This is the first report of a gene that is negatively regulated in dissociated cell cultures as well as in transfected cells, yet is expressed efficiently in the correct multicellular environment of the transgenic mouse.
Animals↗
Atypical presentation of pulmonary embolism.
Today we have discussed an interesting patient with an atypical presentation of pulmonary embolism. We have outlined a suggested algorithm to aid in the diagnosis and management of this disease. References 8 through 24 in the reference section are suggested readings that offer further insight into the diagnosis and management of this entity.
Aged↗
Angiosarcoma of the face and scalp.
Angiosarcomas of the face and scalp are aggressive, malignant neoplasms that are associated with a poor prognosis. Radiotherapy and chemotherapy alone have failed to give satisfactory results in the treatment of this disease, so surgery continues to play the major role in its management. In this article we report on 3 patients with angiosarcoma of the face and scalp recently seen in our institution. The clinical presentation, pathophysiology, and prognosis are extensively reviewed from the recent literature. We conclude with a discussion of treatment options in the management of this disease, with emphasis on surgical management.
Aged↗