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Fatigue studies of isolated, intact muscles typically utilize solutions saturated with O2. However, under in vivo fatiguing conditions, less oxygen is delivered to the muscles and they actually experience hypoxia. No studies to date have correlated the effects of acute hypoxia on the isometric contractile properties of intact muscles, skinned fibers isolated from the same muscles, and the cellular content of specific muscle proteins. Therefore, we have studied the effects of in vitro acute hypoxia on the fatigability of intact diaphragm muscle strips and on the isometric contractile properties of single Triton-skinned fibers isolated from control and hypoxic diaphragm muscles. We found that hypoxia and fatiguing stimulation per se affect the tetanic force of intact muscle strips without exhibiting any significant deleterious effects on the calcium-activated force of skinned muscle fibers dissected from the intact muscles. In contrast, fatiguing stimulation under hypoxic conditions decreased both the tetanic force of muscle strips and the calcium-activated force of skinned muscle fibers. Gel electrophoresis of muscles subjected to hypoxia and hypoxic-fatigue revealed that there is a significant reduction in three protein bands when compared to control muscles. Protein modification may be the underlying mechanism of muscle fatigue under physiologic conditions.

Insulin-like growth factors (IGFs) are important regulators of epithelial cell growth. The mitogenic activity of these factors is influenced by the levels of extracellular IGF binding proteins, including insulin-like growth factor binding protein 3 (IGFBP-3). In the present report we study the effects of epidermal growth factor (EGF) and all-trans-retinoic acid (RA) on IGFBP-3 RNA and protein levels in human papillomavirus-immortalized cervical epithelial cells. Treatment of ECE16-1 cells with 3–20 ng/ml EGF causes a marked reduction in IGFBP-3 levels. In contrast, 1 μM RA increases IGFBP-3 mRNA and protein levels in the presence or absence of 20 ng/ml EGF. The response is concentration dependent with a half-maximal increase observed at 1 nM RA. RA is able to reverse the EGF suppression when added simultaneously or 3 days after initiation of EGF treatment. Conversely, when cells are treated with RA, IGFBP-3 levels increase within 24 h and subsequent addition of EGF is without effect. Thus, the RA-dependent increase in IGFBP-3 levels is dominant over the EGF suppression. The increased IGFBP-3 levels are correlated with RA suppression of proliferation. Similar RA effects on IGFBP-3 mRNA levels were observed in other cervical epithelial cell lines (i.e., ECE16-D1, ECE16-D2, and CaSki). These results suggest that RA may act to inhibit cervical cell growth by increasing IGFBP-3 levels and reducing the extracellular concentration of free insulin-like growth factor I (IGFI) and/or, alternatively, IGFBP-3 may inhibit cell growth by direct effects on the cell, independent of IGFI. © 1994 Wiley-Liss, Inc.

Modulation of phospholipase A2 activity and sodium transport by angiotensin-(1-7). Angiotensin II (Ang II) receptors are coupled to a variety of signal transduction mechanisms. In the kidney, Ang II at nanomolar concentration binds to proximal tubular cells and stimulates phospholipase A2 (PLA2), which in turn catalyzes the hydrolysis of phosphatidylcholine into lysophosphatidylcholine (LPC) and fatty acid. This signal transduction pathway has been shown to be an important modulator of sodium transport. The kidney cortex possesses the enzyme necessary to convert angiotensin I (Ang I) directly to Ang-(1-7) bypassing Ang II as an intermediate. The present investigation was undertaken to determine whether Ang-(1-7) influences epithelial cell function by comparing this heptapeptide with Ang II as a modulator of PLA2 activity and sodium transport. Proximal tubular cells were labeled in tissue culture with 3H-choline and PLA2 activity was measured by quantitation of LPC. We found that Ang II (10-9 M to 10-6 M) significantly increased PLA2 activity (154 ± 36% to 209 ± 94%). Similar results were obtained with Ang-(1-7) (240 ± 130% to 353 ± 40%). The bioactivity of the peptides was assayed by its ability to regulate transcellular 22Na flux. Ang II (10-9 M) inhibited 22Na flux by 12 ± 2% while Ang-(1-7) (10-9 M) inhibited 22Na flux by 20 ± 5%. These results suggest that one potential role of Ang-(1-7) in the regulation of kidney epithelial electrolyte transport may involve activation of PLA2.

Increasing the rate of wound healing of acute wounds and promoting the closure of chronic ulcers is an important goal in wound therapy. Growth factors have been shown to facilitate this process; however, the systems described for growth factor delivery are not ideal. In the present report we demonstrate the feasibility of a new method of delivering growth factors to the wound site using a genetically engineered biological bandage. The bandage consysts of keratinocytes (SCC-13 cells) that are engineered by gene transfer to produce high levels of bovine growth hormone (bGH). bGH was selected for these studies because it can be easily distinguished from rat and human growth hormone in wound fluids and culture medium. The bGH-producing cells are contained and maintained in serum-free medium inside an envelope composed of a low protein binding, 0.2 μm pore size, polysulfone membrane. The genetically engineered cells cannot escape from the bandage, but the bGH is freely released into the surrounding culture medium. When placed onto a full-thickness, surgically generated wound on rats, the cells within the bandage continue to produce and release bGH into the wound for at least 3 days. This system is a safe and reliable way of providing real-time delivery of any desired biomolecule into the wound site. © 1993 John Wiley & Sons, Inc.

The synthesis of two novel hydrogel adhesive films, poly(butoxy- Entprol) and poly(butoxy/ethylene-Entprol) was accomplished by crosslinking either epoxy end-capped oligomeric-butoxy-Entprol or epoxy end-capped oligo meric-butoxy/ethylene-Entprol with 5-10 mole% diethylenetriamine. The two oligomers with Mn 1700 to 5000 were obtained in a 78-87% yield, by the reac tion of one equivalent of N,N'-bis(2-hydroxypropyl)ethylenediamine with either one equivalent of 1,4-butanediol diglycidyl ether or one equivalent of a 1:1 mole ratio mixture of 1,2,7,8-diepoxyoctane and 1,4-butanediol diglycidyl ether. Peel adhesion to glass for poly(BE) and poly(BEE) gave peel forces of 12.02 and 13.61 N, respectively, as compared to five acrylate adhesives, ranging from 4.90-11.56 N. Poly(BE) had superior adhesion to skin (3.45 N) compared to poly(BEE) (0.97 N) and the acrylate-based adhesives (0.79-2.64 N). In addition, fully hydrated poly(BE) and poly(BEE) have both a high water uptake (971% and 630%, respectively) and equilibrium water content (90% and 86%, respec tively). Both films lose adhesion with progressive hydration. These hydrogels actively support the growth of 3T3 fibroblasts and SCC-13 epithelial cells. These results strongly support the potential of these films as single-layered adhesive hydrogel dressings.

Conditions such as respiratory failure and cardiopulmonary arrest can expose the diaphragm to hypoxemia. In skeletal muscles, fatiguing stimulation renders muscles hypoxic, which has long been known to dramatically reduce muscle function. We have previously demonstrated that fatiguing stimulation under hypoxic conditions disrupts both the excitation-contraction coupling (ECC) process and the isometric contractile properties (ICP) in intact diaphragm muscle strips and the contractile properties of skinned fibers isolated from these muscles. Here we have analyzed the effects of intermittent fatiguing stimulation on specific muscle proteins in muscle strips from mouse diaphragms that have been exposed to hypoxia. We report for the first time that the effects of hypoxia-fatigue, namely to decrease maximal tetanic force, maximal calcium-activated force and calcium sensitivity of the mouse diaphragm muscle, are associated with the degradation of troponins TnI and TnC (Western blot analysis). The concentrations of TnT and actin did not change under these same conditions. Because troponins are integrally involved in regulating the interaction between actin and myosin during the cross-bridge cycle, the degradation of TnI and TnC may explain the effects of hypoxia-fatigue on the ICP. This interpretation is supported by the observations that extraction of troponins from control skinned fibers mimics the effects of hypoxia-fatigue on contractile function and that incorporation of native troponins into fibers isolated from hypoxic-fatigued muscles partially restores function.

Cervical cancer is the third most common type of cancer in women in the United States and is the leading cause of cancer deaths in women in third world countries (1,2). Although the pathogenesis of the disease is incompletely understood, human papillomavirus (HPV) is present in over 90% of all high grade cervical lesions and is strongly implicated in the process of cancer development. A common feature of these tumors is expression of the E6 and E7 viral reading frames (3,4,5). These viral genes encode factors, E6 and E7, that interact with the p53 and pRB tumor suppressor genes (6,7,8,9,10). E6 promotes the degradation of p53 via a ubiquitin-dependent pathway (10,11) and E7 interferes with pRB function (8,9,10). Expression of E6 and E7 is sufficient for immortalization of cultured epidermal keratinocytes and cultured cervical epithelial cells (12,13,14,15).