Experimental Biology and Medicine

The highly conserved cardiotonic steroid (CS) binding site present on the ubiquitous membrane sodium pump, sodium, potassium-ATPase, appears to have been conserved by no force other than its capacity to bind CS: a family that includes plant-derived cardiac glycosides and putative endogenous vertebrate counterparts. Binding of ligand is inhibited by increased extracellular potassium. This implies functional coordination because inhibition of the sodium pump would be counterproductive when extracellular potassium is elevated. The interesting biology of the CS binding site continues to stimulate investigations into the identity of endogenous ligands, their role as pump regulators at the cellular level, and as mediators of body fluid balance and blood pressure regulation. In addition to inhibition of sodium and potassium transport, there is considerable recent evidence suggesting that the sodium pump may act as a cell signaling receptor activated by CS binding and responding by coordination of intracellular signaling pathways that can be dependent on and also independent of the reduction in transmembrane ion flux resulting directly from pump inhibition. This signaling may influence cell survival, growth, and differentiation. Recent insight into the biology of pump regulation by CS is reviewed.

The term spinal accessory nerve plexus may be defined as the spinal accessory nerve with all its intra- and extracranial connections to other nerves, principally cranial, cervical, and sympathetic. The term is not new. This review examines its applied anatomy in head and neck cancer and atherosclerosis. Over the centuries, general studies of neural and vascular anatomy and embryology formed a basis for the understanding upon which the plexus is described. During the past century, its anatomy and blood supply have come to be better understood. The importance of almost all of the plexus to head, neck, and upper extremity motor and sensory functions has come to be realized. Because of this understanding, surgical neck dissection has become progressively more conservative. This historical progression is traced. Even the most recent anatomic studies of the spinal accessory nerve plexus reveal configurations, new to many of us. They were probably known to classical anatomists, and not recorded in readily available literature, or not recorded at all. Human and comparative anatomic studies indicate that the composition of this plexus and its blood supply vary widely, even though within the same species their overall function is very nearly the same. Loss of any of these structures, then, may have very different consequences in different individuals. As a corollary to this statement, data are presented that the spinal accessory nerve itself need not be cut during surgical neck dissections for severe impairment to occur. In addition, data are presented supporting the theory that atherosclerosis by obstructing vessels to this plexus and its closely connected brachial plexus will very likely result in their ischemic dysfunction, often painful. Finally evidence, as well as theory, is stated concerning anatomic issues, methodology, outcome, and possible improvements in surgical procedures emphasizing conservatism.

Due to major developments in genetics over the past decade, molecular biology tests are serving promising tools in early diagnosis and follow-up of cancer patients. Recent epidemiological studies revealed that the risk for each individual to develop cancer is closely linked to his/her own genetic potentialities. Some populations that are defective in DNA repair processes, for example in Xeroderma pigmentosum or in the Lynch syndrome, are particularly prone to cancer due to the accumulation of mutations within the genome. Such populations would benefit from the development of tests aimed at identifying people who are particularly at risk. Here, we review some data suggesting that the inactivation of mismatch repair is often found in endometrial cancer and we discuss molecular-based strategies that would help to identify the affected individuals in families with cases of glandular malignancies.

Weight control is dependent on energy balance. Reduced energy expenditure (EE) associated with decreased physical activity is suggested to be a major underlying cause in the increasing prevalence of weight gain and obesity. Therefore, a better understanding of the biological determinants involved in the regulation of physical activity is essential. To facilitate interpretation in humans, it is helpful to consider the evidence from animal studies. This review focuses on animal studies examining the biological determinants influencing activity and potential implications to human. It appears that physical activity is influenced by a number of parameters. However, regardless of the parameter involved, body weight appears to play an underlying role in the regulation of activity. Furthermore, the regulation of activity associated with body weight appears to occur only after the animal achieves a critical weight. This suggests that activity levels are a consequence rather than a contributor to weight control. However, the existence of an inverse weight-activity relationship remains inconclusive. Confounding the results are the multifactorial nature of physical activity and the lack of appropriate measuring devices. Furthermore, many determinants of body weight are closely interlocked, making it difficult to determine whether a single, combination, or interaction of factors is important for the regulation of activity. For example, diet-induced obesity, aging, lesions to the ventral medial hypothalamus, and genetics all produce hypoactivity. Providing a better understanding of the biological determinants involved in the regulation of activity has important implications for the development of strategies for the prevention of weight gain leading to obesity and subsequent morbidity and mortality in the human population.

Progress has continued to be made over the past 4 years in our understanding of the glucose-6-phosphatase (G6Pase) system. The gene for a second component of the system, the putative glucose-6-P transporter (G6PT), was cloned, and mutations in this gene were found in patients diagnosed with glycogen storage disease type 1b. The functional characterization of this putative G6PT has been initiated, and the relationship between substrate transport via the G6PT and catalysis by the system's catalytic subunit continues to be explored. A lively debate over the feasibility of various aspects of the two proposed models of the G6Pase system persists, and the functional/structural relationships of the individual components of the system remain a hot topic of interest in G6Pase research. New evidence supportive of physiologic roles for the biosynthetlc functions of the G6Pase system in vivo also has emerged over the past 4 years.

Amyloid-β peptides (Aβ) play an important role in the pathophysiology of dementia of the Alzheimer's type and in amyloid angiopathy. Aβ outside the CNS could contribute to plaque formation in the brain where its entry would involve interactions with the blood-brain barrier (BBB). Effective antibodies to Aβ have been developed in an effort to vaccinate against Alzheimer's disease. These antibodies could interact with Aβ in the peripheral blood, block the passage of Aβ across the BBB, or prevent Aβ deposition within the CNS. To determine whether the blocking antibodies act at the BBB level, we examined the influx of radiolabeled Aβ (¹²⁵I-Aβ_1-40) into the brain after ex-vivo incubation with the antibodies. Antibody mAb3D6 (élan Company) reduced the blood-to-brain influx of Aβ after iv bolus injection. It also significantly decreased the accumulation of Aβ in brain parenchyma. To confirm the in-vivo study and examine the specificity of mAb3D6, in-situ brain perfusion in serum-free buffer was performed after incubation of ¹²⁵I-Aβ_1-40 with another antibody mAbmc1 (DAKO Company). The presence of mAbmc1 also caused significant reduction of the influx of Aβ into the brain after perfusion. Therefore, effective antibodies to Aβ can reduce the influx of Aβ_1-40 into the brain.

Leptin acts within the hypothalamus to diminish food intake. During pregnancy and lactation, both circulating leptin concentrations and food intake are elevated, suggesting an ineffectiveness of leptin to reduce food intake in these mice. Thus, this study tested the ability of intracerebroventricular (ICV) leptin administration to alter food intake during pregnancy and lactation. Mice during the first, second, and third trimesters of pregnancy, lactating mice on postpartum Day 7, and age-matched female mice were used. Plasma leptin concentrations averaged 2.9 ± 0.3 ng/ml in control mice, increased steadily as pregnancy progressed (3.4 ± 0.7, 29.8 ± 4.5, and 40.5 ± 0.7 ng/ml during the first, second, and third trimesters, respectively), and remained elevated on Day 7 postpartum (26.4 ± 7.8 ng/ml). Mice were food deprived for 4 h, injected ICV with vehicle or leptin (1 μg), and food intake was subsequently measured hourly for 3 hr, and after 24 hr. Vehicle-treated pregnant mice consumed marginally more food than cycling control mice, whereas nursing dams ate two to three times as much food as controls. As expected, ICV leptin administration reduced 24-hr food intake of control mice by 2 g, or ~50%. ICV-administered leptin was as effective in reducing food Intake of pregnant and lactating mice as observed in control mice. Thus, the elevated circulating leptin concentrations observed in pregnant and nursing mice did not alter the ability of ICV-administered leptin to diminish food intake. High plasma concentrations of leptin-binding proteins observed during pregnancy, and probably during lactation, may limit the amount of endogenous leptin reaching the hypothalamus, and may consequently enable increases in food intake concomitant with elevated plasma leptin during these nutritionally demanding periods.

Sildenafil is a potent and selective inhibitor of the cyclic GMP-specific phosphodiesterase (PDE5) that is very effective in the treatment of male impotence. It inhibits breakdown of cyclic guanosine monophosphate (cGMP) formed in penile smooth muscle cells in response to stimulation by nitric oxide resulting in muscle relaxation. PDES is widely distributed in the body, being present in the vasculature, platelets, and kidneys. In the kidney, PDE5 is involved in the regulation of sodium excretion and renin secretion. The aim of the present investigation was to investigate the effect of sildenafil, in doses used clinically, on renin secretion in human subjects. The studies were performed in two groups of healthy normotensive subjects: one in which sodium intake was unrestricted, and one in which sodium intake was restricted to 600 mg/day. Blood pressure and heart rate were monitored throughout the study, and blood samples for the measurement of plasma cGMP and cAMP concentrations and plasma renin activity (PRA) were collected. After control measurements, the subjects ingested a capsule containing sildenafil or placebo. Cardiovascular measurements and blood sampling continued for the next 120 min. Sildenafil had only minor cardiovascular effects. Diastolic pressure tended to be lower and heart rate was generally higher after sildenafil than after placebo, but the differences were small. Sildenafil caused a prompt and sustained increase in plasma cGMP concentration and a more gradual increase in plasma cAMP concentration. After the subjects received placebo, there was a progressive decrease in PRA during the 2-hr observation period, presumably reflecting the circadian rhythm in renin secretion. In contrast, PRA failed to decrease after the subjects received sildenafil, thus indicating that sildenafil exerts a stimulatory action on renin secretion. This action on renin secretion may help explain why sildenafil only has minor effect on blood pressure despite the widespread distribution of PDE5 in vascular tissues.

It is generally assumed that phosphate (Pi) effluxes from proximal tubule cells by passive diffusion across the basolateral (BL) membrane. We explored the mechanism of BL PI efflux in proximal tubule-like OK cells grown on permeable filters and then loaded with ³²P. BL efflux of ³²P was significantly stimulated (P < 0.05) by exposing the BL side of the monolayer to 12.5 mM Pi, to 10 mM citrate, or by acid-loading the cells, and was inhibited by exposure to 0.05 mM Pi or 25 mM HCO₃; by contrast, BL exposure to high (8.4) pH, 40 mM K⁺, 140 mM Na gluconate (replacing NaCl), 10 mM lactate, 10 mM succinate, or 10 mM glutamate did not affect BL ³²P efflux. These data are consistent with BL PI efflux from proximal tubule-like cells occurring, in part, via an electro-neutral sodium-sensitive anion transporter capable of exchanging two moles of intracellular acidic H₂PO4^– for each mole of extracellular basic HP04^– or for citrate.

The present study investigated whether genistein, a broad-spectrum tyrosine kinase inhibitor, could increase the myofilament Ca²⁺ sensitivity and partially reverse postischemic depressed myocardial function. Left ventricular papillary muscles were isolated from adult Wistar rats and loaded with the Ca²⁺ indicator, aequorin. The use of fluorocarbon immersion with hypoxia simulated a model of ischemia. Myofilament responsiveness to Ca²⁺ was evaluated from force-[Ca²⁺], relationship recorded during tetani in papillary muscles. Protein levels of troponin I (Tnl) were measured in postischemic papillary muscles with the Western blot technique. Isometric contraction was depressed during the period of ischemia and remained low after 60 min of reoxygenation without a corresponding significant change of peak [Ca²⁺], in the control group (n = 7). In contrast, the depression of isometric contraction was ameliorated during ischemia in muscle preparations in the presence of genistein (2 μM; n = 8), and postischemic depressed myocardial contractility partially recovered after a 60-min reperfusion. The myofilament Ca²⁺ responsiveness was significantly increased in papillary muscles in the presence of genistein. Protein levels of Tnl were reduced in postischemic papillary muscles, whereas genistein partially restored decreased protein levels of Tnl. Our results reveal that genistein produces an effective attenuation of postischemic depressed myocardial function and improves myofibrillar Ca²⁺ responsiveness in rat myocardium.

The role of leptin in neonatal growth and bone metabolism has been investigated, but not simultaneously. The objectives of this study were to determine if leptin relates to bone mass during rapid growth; if consumption of maternal milk is related to elevated circulating concentrations of leptin resulting in higher fat mass; and if glucocorticoids result in higher fat mass and reduced bone mass due to elevated leptin. Thirty-two piglets were randomized to either a suckling or milk substitute plus either dexamethasone (DEX) or placebo injection for 15 days beginning at 5 days of age. Milk and blood samples were obtained at baseline, and after 15 days, blood was sampled again for measurement of leptin and bone biochemistry. Weight at baseline plus weight and length after 15 days were recorded, followed by measurement of whole body bone mineral content, bone area, and fat mass using dual energy x-ray absorptiometry. At baseline, plasma leptin was elevated in suckled piglets. Piglets that suckled had elevated fat mass as did those who received DEX. However, DEX resulted in suppressed weight and length, bone mass, and bone metabolism. Leptin was similar among groups after the 15 days. After accounting for body size and treatment effects, piglet plasma leptin was predictive of bone and fat mass. Leptin circulating early postnatally is linked to body composition, specifically fat and bone mass. Elevations in fat mass and reductions in bone mass observed after 15 days of DEX treatment are not related to leptin metabolism. Both human and porcine neonates share similar characteristics with respect to relationships of leptin with fat and bone mass.

Eosinophilic leukocytes have been implicated as primary effector cells in inflammatory and allergic diseases. When activated by cytokines, human eosinophils secrete and produce a variety of proinflammatory or tissue damaging substances. Although well known for their chemoattractant effects, little is known about the precise contribution of the eosinophil-selective chemokines, eotaxin, eotaxin-2, and eotaxin-3 to the effector functions of eosinophils. This forms the central focus of these investigations for which clone 15-HL-60 human eosinophilic cells were used as the in vitro model. Investigation results suggest that all three subtypes of eotaxin directly stimulate eosinophil superoxide anion generation that is inhibited by neutralizing eotaxin antibody or pretreatment of cells with the receptor antibody anti-CCR3. Pretreatment or co-treatment with each of the eotaxins augmented phorbol myristate-induced superoxide generation. Concentration-dependent degranulation of eosinophil peroxidase was noted for all three chemokines, and potentiation of calcium lonophore-induced degranulation was observed with eotaxin pretreatments. Results of interleukin-5 pretreatment studies suggest that the eotaxin chemokines may act cooperatively to enhance effector functions of eosinophils. Collectively, the present studies have advanced knowledge of the eotaxin family of chemokines to include eosinophil priming and modulation of eosinophil activation and secretion effector functions.

Many studies have shown that metallothionein (MT) can be increased significantly by different oxidative insults in multiple organ systems. However, the increase in MT production often falls to protect against oxidative tissue injury. On the other hand, recent studies using a cardiac-specific, MT-overexpressing, transgenic mouse model have shown that MT protects against oxidative heart injury. Thus, the present study was undertaken to test the hypothesis that prior increase in MT levels is required to prevent oxidative injury. Oxidative heart injury was induced by doxorubicin (DOX), an important anticancer drug that causes severe cardiotoxicity through oxidative stress. Cardiac-specific, MT-overexpressing, transgenic mice and wild-type (WT) FVB mice were treated with OOX at 20 mg/kg. Four days after the treatment, MT concentrations were markedly elevated in the WT mouse heart. The elevated MT concentrations were comparable with those found in the transgenic mouse heart, which did not show further MT elevation in response to DOX challenge. Severe oxidative injury occurred in the heart of WT mice, including myocardial lipid peroxidation, morphological changes as examined by electron microscopy, high levels of serum creatine kinase activity, and decreased total glutathione concentrations in the heart. However, all of these pathological changes were significantly inhibited in the MT-transgenic mice. Therefore, this study demonstrates that there is a correlation between MT induction and oxidative stress in the DOX-treated mouse heart. However, MT can protect the heart from oxidative injury only if it is present prior to induction of oxidative stress.

Immunosuppression has been related to the incidence of tumor apparition, including endocrine tumors. The intrasplenic ovarian tumor (luteoma) is a typical benign endocrine tumor that develops under high gonadotropin stimulation and, from the immunological perspective, is located in a critical organ involved in immune response. To establish if immunosuppression could alter the development of this experimental tumor, the effects of cyclosporin A (CsA) and dexamethasone (Dex) were evaluated. After surgery, tumor-bearing and sham animals were kept without treatment for 4 weeks; thereafter, they were distributed into CsA (25 mg/kg), Dex (0.1 mg/kg), or vehicle (75:25 castor oll:ethanol) groups and were injected on alternate days for 50 days. Body weight was evaluated weekly. Animals were sacrificed after a jugular vein blood sample was obtained. Thymi were weighed. Tumors were measured and placed in formaline for histological studies. Serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL), and estradiol were measured by radioimmunoassay. Hematological parameters were determined. CsA induced a significant decrease in survival rates both in tumor-bearing and sham animals (P < 0.01). Dex significantly impaired weight increase in both groups of animals. CsA induced a significant weight loss in sham animals, not observed in tumor-bearing animals. Dex induced thymus weight loss in both groups, whereas CsA induced thymus weight loss only in sham animals. Only Dex induced a decrease in lymphocyte number in both groups. CsA induced an increase in monocyte number only in sham animals. Treatments did not alter LH, FSH, or estradiol, whereas PRL was increased by CsA only in sham rats. Neither Dex nor CsA induced any significant variations in tumor volume, nor did they alter tumor histology. In addition, no visible metastases or alterations in other organs were observed. We conclude that, though immunological parameters were altered by the treatments, immunosuppressor drugs did not condition tumor development. In addition, tumors secrete one or more factor/s that counteract CsA effect.

The ability of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) to influence epithelial interleukin (IL)-8 responses to the intestinal bacterium Lactobacillus plantarum 299v was analyzed in the human HT-29 colonic epithelial cell line. In the absence of TNF-α, IL-8 mRNA expression was not detectable by Northern blot analysis in HT-29 cells alone or in HT-29 cells co-cultured with L. plantarum 299v. However, TNF-α induced IL-8 mRNA expression, and co-culture of TNF-α-treated HT-29 cells with L. plantarum 299v significantly increased IL-8 mRNA expression above levels induced by TNF-α alone in an adhesion-dependent manner. The increase in IL-8 mRNA expression was not observed in TNF-α-treated HT-29/L. plantarum 299v co-cultures using heat-killed lactobacllli or when L. plantarum adhesion was prevented using mannoside or a trans-well membrane. Paradoxically, IL-8 secretion was decreased in TNF-α-treated HT-29 cells with L. plantarum 299v relative to cells treated with TNF-α alone. TNF-α-mediated responsiveness to L. plantarum 299v was further investigated by analyzing expression of a coreceptor for bacterial cell wall products CD14. HT-29 cells expressed CD14 mRNA and cell-surface CD14; however, TNF-α did not alter CD14 mRNA or cell-surface expression, and blockade of CD14 with monoclonal antibody MY4 did not alter the IL-8 response to L. plantarum 299v in TNF-α-treated HT-29 cells. These results indicate that although TNF-α sensitizes HT-29 epithelial cells to intestinal lactobacilli, the bacteria exert a protective effect by downregulating IL-8 secretion.