Fluid Ferroelectric Filaments.

Freestanding slender fluid filaments of room-temperature ferroelectric nematic liquid crystals are described. They are stabilized either by internal electric fields of bound charges formed due to polarization splay or by external voltage applied between suspending wires. The phenomenon is similar to those observed in dielectric fluids, such as deionized water, except that in ferroelectric nematic materials the voltages required are three orders of magnitudes smaller and the aspect ratio is much higher. The observed ferroelectric fluid threads are not only unique and novel but also offer measurements of basic physical quantities, such as the ferroelectric polarization and viscosity. Ferroelectric nematic fluid threads may have practical applications in nano-fluidic micron-size logic devices, switches, and relays.

Facilitating time series classification by linear law-based feature space transformation.

The aim of this paper is to perform uni- and multivariate time series classification tasks with linear law-based feature space transformation (LLT). First, LLT is used to separate the training and test sets of instances. Then, it identifies the governing patterns (laws) of each input sequence in the training set by applying time-delay embedding and spectral decomposition. Finally, it uses the laws of the training set to transform the feature space of the test set. These calculation steps have a low computational cost and the potential to form a learning algorithm. For the empirical study of LLT, a widely used human activity recognition database called AReM is employed. Based on the results, LLT vastly increases the accuracy of traditional classifiers, outperforming state-of-the-art methods after the proposed feature space transformation is applied. The fastest error-free classification on the test set is achieved by combining LLT and the k-nearest neighbor (KNN) algorithm while performing fivefold cross-validation.

Comparison of GH, IGF-I, and testosterone with mRNA of receptors and myostatin in skeletal muscle in older men.

Growth hormone (GH), insulin-like growth factor I (IGF-I), and testosterone (T) are important mediators of muscle protein synthesis, and thus muscle mass, all of which decline with age. We hypothesized that circulating hormones would be related to the transcriptional levels of their respective receptors and that this expression would be negatively related to expression of the myostatin gene. We therefore determined content of mRNA transcripts (by RT-PCR) for GH receptor (GHR), IGF-I, androgen receptor (AR), and myostatin in skeletal muscle biopsy samples from 27 healthy men >65 yr of age. There were no significant relationships between age, lean body mass, or percent body fat and transcript levels of GHR, IGF-I, AR, or myostatin. Moreover, there were no significant correlations of serum GH, IGF-I, or T with their corresponding target mRNA levels (GHR, intramuscular IGF-I, or AR) in skeletal muscle. However, GHR was negatively correlated (r = -0.60, P = 0.001) with myostatin mRNA levels. The lack of apparent relationships of muscle transcripts with their respective ligands in healthy older adults suggests that age-related deficits in both GH and T may lead to an increase in myostatin expression and a disassociation in autocrine IGF-I effects on muscle protein synthesis, both of which could contribute to age-related sarcopenia.

A longitudinal assessment of change in VO2max and maximal heart rate in master athletes.

PURPOSE: The purpose of this study was to determine the longitudinal change in VO2max and HRmax in male and female master endurance runners and to compare these changes based upon gender, age, and change in training volume. METHODS: Eighty-six male (53.9 +/- 1.1 yr) and 49 female (49.1 +/- 1.2 yr) master endurance runners were tested an average of 8.5 yr apart. Subjects were grouped by age at first visit, change in VO2max, and change in training volume. Measurements included body composition by hydrostatic weighing, maximal exercise testing on a treadmill, and training history by questionnaire. Data were analyzed by ANOVA and multiple regression. RESULTS: VO2max and HRmax declined significantly regardless of gender or age group (P < 0.05). The rate of change in VO2max by age group ranged from -1% to -4.6% per year for men and -0.5% to 2.4% per year for women. Men with the greatest loss in VO2max had the greatest loss in LBM (-2.8 +/- 0.7 kg), whereas women with the greatest loss in VO2max demonstrated the greatest change in training volume (-24.1 +/- 3.0 km.wk-1). Additionally, women with the greatest loss in VO2max (-9.6 +/- 2.6 mL.kg-1.min-1) did not replace estrogen after menopause independent of age. HRmax change did not differ by VO2max change or training volume change in either gender. CONCLUSIONS: In conclusion, these data suggest that VO2max declines in male and female master athletes at a rate similar to or greater than that expected in sedentary older adults. Additionally, these data suggest that maintenance of LBM and VO2max were associated in men, whereas in women, estrogen replacement and maintenance of training volume were associated with maintained VO2max.

Relationship between physiological loss, performance decrement, and age in master athletes.

BACKGROUND: The use of master athletes to describe an idealized rate of physiological loss associated with aging is quite common. The results of such studies suggest that older athletes may be able to reduce the rate of decline in functional loss. The findings of such studies have been questioned due to their limited sample size and the age range and gender of their subjects. METHODS: We examined a group of 146 male and 82 female master athletes over the age of 40 years. Physiological parameters included maximal oxygen uptake (VO2max), body composition, muscle strength, bone density, and blood chemistries. Medical histories and training records were obtained via questionnaire. RESULTS: Results demonstrated gender differences in body composition, blood chemistries, blood pressure, VO2max, muscle strength, bone density, and performance (p <.05). All metabolic parameters for men and most for women demonstrated significant losses across the age range (p <.05). In addition, strength and performance for men and women and bone density for women declined significantly with age (p <.05). The demonstrated loss rates did not differ by gender. CONCLUSIONS: Although limited by the lack of a sedentary comparison group, these data suggest that age-related losses in VO2max may not be different from data previously reported for older sedentary adults and that loss in muscle strength and performance with aging is not linear.

Recombinant human growth hormone and recombinant human insulin-like growth factor I diminish the catabolic effects of hypogonadism in man: metabolic and molecular effects.

Severe gonadal androgen deficiency can have profound catabolic effects in man. Hypogonadal men develop a loss of lean body mass, increased adiposity, and decreased muscle strength despite normal GH and insulin-like growth factor I (IGF-I) concentrations. We designed these studies to investigate whether GH or IGF-I administration to male subjects with profound hypogonadism can diminish or abolish the catabolic effects of testosterone deficiency. Moreover, we also examined the nature of the interactions among GH, IGF-I, and androgens in specific genes of the im system. A group of 13 healthy subjects (mean age, 22 +/- 1 yr) was studied at baseline (D1) and 10 weeks after being made hypogonadal using a GnRH analog (GnRHa; D2). At 6 weeks from baseline they were started on either recombinant human (rh) IGF-I (60 microg/kg, sc, twice daily) or rhGH (12.5 microg/kg, sc, daily) for 4 weeks. On each study day subjects had infusions of L-[(13)C]leucine; indirect calorimetry; isokinetic dynamometry of the knee extensors; determination of body composition (dual energy x-ray absortiometry) and hormone and growth factor concentrations, as well as percutaneous muscle biopsies. Their data were compared with those of previously studied male subjects who received only GNRHA: Administration of rhIGF-I and rhGH to the hypogonadal men had similar effects on whole body metabolism, with maintenance of protein synthesis rates, fat oxidation rates, and fat-free mass compared with the eugonadal state, preventing the decline observed with hypogonadism alone. This was further amplified by the molecular assessment of important genes in muscle function. During rhIGF-I treatment, im expression of IGF-I declined, and IGF-binding protein-4 increased, similar to the changes during GnRHa alone. However, rhGH administration was associated with a marked increase in IGF-I and androgen receptor messenger ribonucleic acid concentrations in skeletal muscle with a reciprocal decline in IGF-binding protein-4 expression in the hypogonadal men. The gene expression for myostatin did not change. These effects were accompanied by a much greater increase in plasma IGF-I concentrations after rhIGF-I (225 +/- 32 vs. 768 +/- 117 microg/L) compared with the concentrations achieved during rhGH (217 +/- 20 vs. 450 +/- 19 microg/L). We conclude that 1) rhGH and rhIGF-I both may be beneficial in preserving lean body mass and sustaining rates of protein synthesis during states of severe androgen deficiency in man; 2) GH may affect the im IGF system via an a paracrine, local production of IGF-I; 3) androgens may be necessary for the full anabolic effect of GH/IGF-I in man. These hormones, particularly GH, may play a role in the treatment of hypogonadal men rendered hypogonadal pharmacologically or those unable to take full testosterone replacement. The latter requires further study.

Influence of weight training exercise and modification of hormonal response on skeletal muscle growth.

To investigate the influence of carbohydrate (CHO) consumption on the acute hormonal response, and chronic adaptation to weight lifting exercise, two studies were conducted. Following a four-hour fast, seven young men (21.3 +/- 3.5 y) performed (on two occasions) a nine-station weight lifting protocol, completing 3 sets of 10 repetitions at 75% of 1RM (series 1). Randomly assigned, one session included the ingestion of a non-caloric placebo, and the other, a 6% CHO solution. For series 2, two groups of young men (21.3 +/- 1.5 y) participated in 12 weeks of progressive resistance weight training. Training for one group included the ingestion of a non-caloric placebo, and the other, a 6% CHO solution. In series 1, weight lifting exercise with CHO ingestion significantly (p < 0.05) elevated blood glucose and plasma insulin levels above baseline, as well as that occurring with the placebo. This resulted in a significant blunting of the cortisol response (7% with CHO compared to 99% with placebo). These findings indicate that CHO consumption during weight lifting exercise can modify the acute hormonal response to exercise. With series 2, CHO consumption continued to blunt the cortisol response to exercise during the twelve weeks of training. This is in contrast to significantly elevated cortisol levels observed for the placebo control group. Corresponding with the modified response patterns were differences in muscle growth. Weight training exercise with CHO ingestion resulted in significantly greater gains in both type I (19.1%) and type II (22.5%) muscle fibre area than weight training exercise alone. The difference in the cortisol response accounted for 74% of the variance (r = 0.8579, p = 0.006) of change in type I muscle fibre area, and 52.3% of the variance (r = 0.7231, p = 0.043) of change in type II muscle fibre area. These findings suggest that the modification of the cortisol response associated with CHO ingestion can positively impact the skeletal muscle hypertrophic adaptation to weigh training.

Maximal aerobic power, lactate threshold, and running performance in master athletes.

PURPOSE: This study sought to determine how lactate threshold (LT) is related to running performance in older male and female runners, if LT changes significantly with age, and if gender alters the relationship between LT and performance in older runners. METHODS: Subjects were 168 master runners (111 men, 57 women) selected from a longitudinal study, who ran at least 10 miles x wk(-1) for 5 yr or more. VO2max was measured on a treadmill and body composition by hydrostatic weighing. Blood samples taken each minute of exercise were analyzed for lactate concentration and LT determined as the breakpoint in lactate accumulation. Performance times and training histories were self-reported by questionnaire. RESULTS: Men had significantly greater body mass, fat-free mass (FFM), and VO2max (L x min(-1); mL x kg(-1) x min(-1)) than women. FFM and VO2max (L x min(-1); mL x kg(-1) x min(-1)) declined with age in both men and women. Running performance was significantly different between men and women and declined with age in both. LT (L x min(-1); mL x kg(-1) x min(-1)) was significantly different between men and women, and declined significantly with age in men, whereas LT (%VO2max) did not differ between men and women and increased significantly with age in both. VO2max (mL x kg(-1) x min(-1)) was the most significant predictor of performance in both men and women, whereas LT (L x min(-1)) added to the prediction of 5-km and 10-km performance in women. CONCLUSION: The results of this study demonstrate that VO2max (mL x kg(-1) x min(-1)) is a better predictor of performance than LT in older male and female runners. Additionally, LT as a percentage of VO2max increases significantly with age.

Increased intracellular triglyceride in C(2)C(12) muscle cells transfected with human lipoprotein lipase.

Much of the knowledge about the cell biology of lipoprotein lipase (LPL) in vitro has been gained from adipose tissue model systems. However, the importance of skeletal muscle lipoprotein lipase (SMLPL) to both lipoprotein and muscle metabolism remains unclear. Although the production of LPL in cultured myocytes has been documented, the amount of enzyme activity produced is small. To develop a more suitable tissue culture model for SMLPL, mouse C(2)C(12) myoblasts were stably transduced with a retroviral vector encoding the full-length human LPL (hLPL) cDNA. Control cells were transduced with a vector encoding beta-galactosidase. LPL expression was assayed as a function of cell growth by measuring LPL activity on days 3, 7, 9, 11, and 14 after subculture. The hLPL-transduced myoblasts increasingly overexpressed both heparin-releasable (HR) and intracellular (IN) LPL activity compared to nontransduced myoblasts (P < 0.001 at Day 11) and myoblasts transduced with the control vector (P < 0.001 at Day 11). This increase occurred while LPL mRNA levels remained stable between days 3 and 14. As expected, IN LPL activity was also increased in the transduced cells. High levels of LPL activity were also obtained after differentiating the C(2)C(12) cells into myotubes by serum deprivation. Additionally, throughout the time course, C(2)/LPL cells had greater amounts of intracellular triglyceride than both the C(2)C(12) and the C(2)/beta-GEO cells (P = 0.005 and P < 0.001, respectively) with the largest differences seen on day 14 of the time course (P = 0.001, C(2)/LPL vs C(2)C(12) (r) or C(2)/beta-GEO cells). Thus, C(2)C(12) myoblasts stably transduced with hLPL markedly overexpressed both HR and IN LPL activity compared to control cells which, in turn, was associated with increases in intracellular triglyceride content. Because LPL regulation in tissues is mostly posttranslational, this new in vitro model will permit the in-depth study of the posttranslational regulation of SMLPL and provide new insights into the fate of lipoprotein-derived fatty acids in muscle.

The inability of hormone replacement therapy or chronic running to maintain bone mass in master athletes.

BACKGROUND: Previous studies have demonstrated equivocal findings on the effect of chronic running on bone mass in post-menopausal women. The purpose of this study was to determine the effect of chronic running alone and in conjunction with hormone replacement therapy (HRT) on bone mineral density (BMD) in postmenopausal women. METHODS: Forty-three women [15 premenopausal 48.1 +/- .4 yrs (Pre); 13 postmenopausal 57.3 +/- 2.3 yrs (Post); and 15 HRT-treated postmenopausal 56.8 +/- 1.5 yrs (PostE)] served as subjects. All were chronic runners (duration > 5 yrs, > 10 miles per week). BMD was determined by dual energy x-ray absorptiometry, VO2 max on a treadmill, body composition by hydrostatic weighing, knee strength by KinCom dynamometer, and training and menstrual history by questionnaire. Analysis of covariance with Tukey post hoc tests was utilized to compare the groups. RESULTS: The groups were similar in body weight, VO2 max, years training, and miles run per week. Pre and PostE did not differ in total or spine BMD. However, Pre had greater hip BMD than PostE (.973 +/- .03 vs .876 +/- .03 g/cm2; p < .05). As well, Pre had greater BMD of the hip (.973 +/- .03 vs .805 +/- .03 g/cm2; p < .05), spine (1.047 +/- .04 vs .870 +/- .04 g/cm2; p < .05), and total body (1.115 +/- .02 vs .996 +/- .03 g/cm2; p < .05) than Post. CONCLUSIONS: These results suggest that (a) chronic running + HRT is insufficient to protect hip BMD and (b) chronic running alone provides no protection for bone mass in postmenopausal women.

Oral arginine does not stimulate basal or augment exercise-induced GH secretion in either young or old adults.

BACKGROUND: Growth hormone (GH) helps maintain body composition and metabolism in adults. However, basal and peak GH decline with age. Exercise produces a physiologic GH response that is subnormal in elderly people. Arginine (Arg) infusion can augment GH secretion, but the efficacy of oral Arg to improve GH response to exercise has not been explored. We investigated whether oral Arg increases GH secretion in young and old people at rest and during exercise. METHODS: Twenty young (Y: 22.1 +/- 0.9 y; SEM) and 8 old (O: 68.5 +/- 2.1 y) male and female subjects underwent three different trials following determination of their one-repetition maximum strength (1-RM); exercise only (EO; 3 sets, 8-10 reps at 85% of 1-RM; on 12 separate resistive lifts), Arg only (5.0 g), or Arg + exercise. Blood samples were collected between successive lifts, and GH (ng x ml(-1)) was determined via RIA. RESULTS: In Y vs O: Basal GH secreted (area under the curve) was 543.6 +/- 84.0 vs 211.5 +/- 63.0. During EO, values were 986.6 +/- 156.6 and 517.8 +/- 85.5. Both were significantly lower in the older individuals (p < .05). Oral Arg alone did not result in any increase in GH secretion at rest (310.8 +/- 73.2 vs 262.9 +/- 141.2). When Arg was coadministered during exercise, GH release was not affected in either the young or old and appeared to be blunted in the young compared to the exercise only trial in the young. CONCLUSION: Based upon these findings, we concluded that oral Arg does not stimulate GH secretion and may impair GH release during resistive exercise.

Eccentric muscle action increases site-specific osteogenic response.

PURPOSE: Strain magnitude is known to be a primary determinant of the osteogenic response to loading. However, whether bone adaptation to muscle loading is determined primarily by load magnitude is unclear. The purpose of this study was to determine the contribution of load magnitude from muscle action on the site-specific osteogenic response. METHODS: Twenty young women (12 exercise, 8 control) served as subjects. Bone mineral density (BMD) of the whole body and mid-femur segment and body composition were determined by dual-energy x-ray absorptiometry. Knee extension and flexion strengths were determined on a KinCom dynamometer, with surface electromyography of the vastus lateralis muscle. Exercise subjects trained three times weekly for 18 wk on a KinCom. One leg trained using eccentric knee extension and flexion, and the opposite leg trained using concentric knee extension and flexion. RESULTS: Eccentric exercise demonstrated greater force production with lower integrated electromyographic signal (IEMG) compared with concentric exercise. Significant increases in muscle strength occurred in both exercised legs (P < 0.05), which were of similar relative change. However, only the eccentric trained leg significantly increased mid-femur segment BMD (+3.9%, P < 0.05) and mid-thigh segment lean mass (+5.2%, P < 0.05). CONCLUSIONS: These results suggest that eccentric muscle training is more osteogenic than concentric muscle training and that eccentric training is more efficient by attaining higher force production with lower IEMG.

Age-related blunting of growth hormone secretion during exercise may not be soley due to increased somatostatin tone.

Age-related declines in growth hormone (GH) secretion may result from augmented somatostatin (SRIH) tone and/or diminished GH-releasing hormone (GHRH) secretion. We assessed GH release during exercise without and with pyridostigmine (PYR), which indirectly suppresses SRIH. GH levels were measured throughout exercise and recovery in 12 young men (mean +/- SEM, 20.8 +/- 0.4 years) and seven old men (66.1 +/- 1.9). The area under the GH curve (GH-AUC) was greater in young versus old men during a short-term maximal exercise test (12.9 +/- 2.8 v 1.5 +/- 0.2 ng x min(-1) x mL(-1), P = .002) and a 1-hour 60% maximal (submaximal, 10.0 +/- 1.5 v 3.0 +/- 1.0 ng x min(-1) x mL(-1), P = .001) cycle exercise bout. PYR increased the GH-AUC in young and old men during maximal (20.9 +/- 5.2 v 4.9 +/- 1.8) and submaximal (12.3 +/- 1.6 v 4.7 +/- 1.5) exercise (P < .05). The greater GH response to maximal versus submaximal exercise suggests a role for adrenergic modulation of GHRH during exercise. However, the failure of PYR to restore the responses of the old to those of the young suggests that increased SRIH tone does not completely explain the age difference in GH secretion during exercise.

Lipoprotein lipase is expressed in cultured Schwann cells and functions in lipid synthesis and utilization.

We have previously demonstrated that lipoprotein lipase (LPL; triacylglycero-protein acylhydrolase, EC 3.1.1.34) is most likely expressed in the non-neuronal cells of the spinal cord, and glial cells may thus be the site of expression in the peripheral nervous system as well. We investigated the expression of LPL in cultured 1. 17 cells, an immortalized rat sciatic nerve Schwann cell line. The 1. 17 cells were shown to express LPL mRNA by reverse transcriptase-polymerase chain reaction analysis. The 1.17 Schwann cells demonstrated heparin-releasable lipolytic activity that was inhibited by the lipase inhibitor tetrahydrolipstatin in a dose-dependent manner. Preincubation of 1.17 cells with an anti-rat LPL antiserum reduced the heparin-releasable lipolytic activity to <10% of that measured in untreated cells. To investigate the role of LPL in Schwann cell lipid metabolism, 1.17 cells were incubated for up to 24 h with an emulsified [14C]triolein substrate and the incorporation of [14C]triolein radioactivity into various cellular lipids was examined in the presence of either anti-rat LPL antiserum or preimmune serum. Inhibiting LPL activity reduced the incorporation of 14C into cellular polar lipids, diacylglycerol, and cholesteryl esters by >80% at 2 and 6 h after addition of the radiolabeled substrate. At 24 h, radioactivity in diacylglycerol and cholesteryl esters was similar in cells treated with anti-LPL antiserum or preimmune serum, whereas 14C incorporation into polar lipids was still reduced by >60%. Separation of the polar lipids into individual lipid species revealed no specific changes in triolein-derived radioactivity incorporation across the phospholipid species examined. These results suggest that LPL-mediated hydrolysis of exogenous triacylglycerol is an important source of free fatty acids for the Schwann cell and thus may play a critical role in myelin biosynthesis in the peripheral nervous system.

Prevention of diet-induced obesity in transgenic mice overexpressing skeletal muscle lipoprotein lipase.

Transgenic (Tg) FVB/N mice were produced that overexpress human lipoprotein lipase (LPL) in skeletal muscle using the muscle creatine kinase promoter and enhancers. It was hypothesized that, by overexpressing LPL in muscle, high fat feeding-induced obesity would be prevented by diverting lipoprotein-derived triglyceride fatty acids away from storage in adipose tissue to oxidation in muscle. Mice were examined both at 6 wk of age before high fat (HF) feeding and at 19 wk of age after 13 wk of HF (46.1% fat) or high carbohydrate (HC) feeding (11.5% fat). At 6 wk in heterozygous Tg mice, LPL was increased 11-fold in white muscle and 2.5-fold in red muscle, but not in cardiac muscle or spleen, brain, lung, kidney, or adipose tissue. Plasma triglycerides (mg/dl) were lower in Tg mice (87 +/- 7 vs. 117 +/- 7, P < 0.0001), and glucose increased (201 +/- 9 vs. 167 +/- 8 mg/dl, P = 0.029). There were no differences in body weight between Tg and nontransgenic (nTg) mice; however, carcass lipid content (% body wt) was significantly decreased in male Tg mice at 6 wk (7.5 +/- 1.0 vs. 9.0 +/- 1.0%, P = 0.035). Body composition was not different in female Tg mice at 6 wk. Overall, when Tg mice were fed either a HC or HF diet for 13 wk, plasma triglycerides (P < 0.001) and free fatty acids (P < 0.001) were decreased, whereas plasma glucose (P = 0.01) and insulin (P = 0.05) were increased compared with nTg mice. HF feeding increased carcass lipid content twofold in both male (10.3 +/- 1.1 vs. 21.4 +/- 2.6%, HC vs. HF, P < 0.001) and female nTg mice (6.7 +/- 0.9 vs. 12.9 +/- 1.8%, P = 0.01). However, the targeted overexpression of LPL in skeletal muscle prevented HF diet-induced lipid accumulation in both Tg male (10.2 +/- 0.7 vs. 13.5 +/- 2.2%, HC vs. HF, P = NS) and female Tg mice (6.8 +/- 0.6 vs. 10.1 +/- 1.4%, P = NS). The potential to increase LPL activity in muscle by gene or drug delivery may prove to be an effective tool in preventing and/or treating obesity in humans.

Time-related effects of enzymatic disaggregation on model human lung carcinomas.

Enzyme cocktails used to prepare tumor cell suspensions may influence yield, viability, and cytology, thus time-related cocktail effects on model human lung carcinomas were examined. A549, NCI-H125, and NCI-H460 carcinomas were completely disaggregated at 25 degrees C over 2 h with either (mg/ml) collagenase/DNAase (C/D, 1/0.1), collagenase/hyaluronidase/DNAse (C/H/D, 1/0, 1/0.1), or polymyxa protease/DNAse (PP/D, 3/0.1). Trypan blue viabilities, total yields, viable yields, and flow cytometric percent tumor cells (TC) were measured every 20-30 min (n = 4-7 per tumor type). The final percentages of TC, mononuclear cells (MN), polymorphonuclear cells (PMN), lymphocytes, and necrotic cells were determined by cytology (n = 4-5 per tumor type). The time-dependent measurements showed that 1) disaggregation was progressive and complete with all cocktails; 2) viability was stable or increasing with all cocktails; 3) percent TC was stable for all cocktails, but lower for PP/D than C/D in final suspensions; and 4) PP/D gave lower final total yields, higher final viabilities, but the same final viable yields as the C cocktails, suggesting selective elimination of dead cells by PP/D. Final cytology measurements showed that PP/D gave a lower percent MN and a higher percent PMN than C cocktails. Cocktail effects may importantly influence cell suspension properties.

Dose-related doxorubicin effect in an orthotopic secondary lung cancer screen.

Lung cancer is the leading cause of cancer-related death of both sexes in the United States and promises to be a major problem in the world community for decades. We are developing an orthotopic (organ specific) secondary screening system to measure the uptake and efficacy of new lung cancer agents. The elements of the system are: (1) orthotopic growth of a model human lung cancer (NCI-H460 large cell carcinoma) in the right caudal lobe of the nude rat; (2) 1-hr ex vivo pulmonary perfusion treatment of the tumor-bearing lungs; and (3) soft agar clonogenic assay of the enzymatically disaggregated tumor cells. This study characterizes dose-response aspects of the system. Perfusion of tumor-bearing lungs with 0, 1, 10, and 100 micrograms/ml doxorubicin resulted in a dose-related reduction in surviving fraction from 1.01 +/- 0.41 to 0.019 +/- 0.006 (P < 0.05) without significant treatment-related increases in lung weight or perfusion pressure. Tumor and lung drug levels were also dose-related, with lung levels exceeding tumor levels at all doses. The tumor drug level at the 100 micrograms/ml dose was 62 +/- 16 ng/mg. There was a strong negative correlation between the measured tumor drug level and surviving fraction in the clonogenic assay (R2 = 0.47, P = 0.0005). This new screening system is capable of demonstrating dose-related uptake and tumoricidal activity of doxorubicin on an orthotopic, model human large cell lung carcinoma. It may be useful for the secondary screening of agents active against human lung cancer.

Transforming growth factor-beta 1 regulation of signal transduction in two renal epithelial cell lines.

The present studies examine the effect of transforming growth factor-beta 1 (TGF-beta 1) on signal transduction pathways in two cultured renal epithelial cell lines. TGF-beta 1 promotes basal and agonist-stimulated adenylate cyclase activity in LLC-PK1 but not MDCK cell membranes. TGF-beta 1 stimulation of LLC-PK1 membrane adenylate cyclase activity occurs quickly and can be attenuated by pertussis toxin pretreatment. Both TGF-beta 1 and adenosine 3',5'-cyclic monophosphate (cAMP) exert comparable effects on [3H]thymidine uptake in LLC-PK1 cells, suggesting that TGF-beta 1 regulation of adenylate cyclase activity potentially plays a role in mediating biological responses to TGF-beta 1. The activities of protein kinase C and phospholipase A are not affected by TGF-beta 1 in either LLC-PK1 or MDCK cells. Both TGF-beta 1 and epidermal growth factor (EGF) increase expression and induce the appearance of new forms of the cAMP response element binding protein (CREB) in LLC-PK1 cells. These effects of TGF-beta 1 and EGF on CREB appear to be specific since neither TGF-beta 1 nor EGF alters expression of an activating transcription factor in LLC-PK1 cells. The effect of TGF-beta 1 and EGF to alter expression of CREB does not affect CREB binding to its regulatory element in LLC-PK1 cell lysates. These results suggest that some of the biological effects of TGF-beta 1 may be attributed to stimulation of adenylate cyclase activity and cAMP formation as well as to enhanced expression and/or modification of the CREB transcription factor in LLC-PK1 cells.

A multifunctional prokaryotic protein expression system: overproduction, affinity purification, and selective detection.

A series of plasmid vectors, pRSET A, B, and C, have been developed for high-level protein expression in prokaryotes and have been characterized. Based upon the T7 RNA polymerase-driven pET system, the pRSET vectors encode recombinant proteins as fusions with a multifunctional leader peptide containing a hexahistidyl sequence for purification on Ni(2+)-affinity resins, a tyrosine residue for radioiodination, and an enterokinase proteolytic cleavage site for leader peptide removal. Monoclonal antibodies (MAbs) to two epitopes on the leader peptide, which also contains amino acids 1-12 of the T7 gene 10 major capsid protein, were developed and provide for universal immunological detection of pRSET-expressed fusion proteins. Subcloning of protein-encoding DNA is facilitated by an 11-site polylinker which is offset for all three ribosomal reading frames, and an f1(+) origin of DNA replication permits single-stranded DNA synthesis for site-directed mutagenesis protocols. Representative fusion proteins overexpressed in Escherichia coli were successfully purified under both denaturing and nondenaturing conditions by single-step Ni2+ affinity chromatography. Purification was independent of recombinant protein solubility in sonicated or freeze-thawed E. coli lysates. Isolation of MAbs for selective recognition of either of two leader peptide epitopes was demonstrated by immunoprecipitation, but this selectivity was less evident under conditions for Western blotting. In combining the utility of T7 RNA polymerase-directed expression with several recent advances in protein purification and detection, the pRSET vectors will serve as a powerful resource for a variety of studies in protein biochemistry.

Structural determinants outside of the leucine zipper influence the interactions of CREB and ATF-2: interaction of CREB with ATF-2 blocks E1a-ATF-2 complex formation.

Dimerization of leucine zipper-containing proteins has been associated characteristically with the formation of a coiled-coil structure between two compatible leucine zipper motifs. In the present study we demonstrate the association of the leucine zipper of cAMP response element-binding protein (CREB) with a zinc finger motif of ATF-2. The association of the CREB leucine zipper with the ATF-2 zinc finger is stabilized if the ATF-2 leucine zipper is intact, implying that the preferred interactive structure of ATF-2 juxtaposes the amino-terminal zinc finger motif of this protein with the carboxy-terminal leucine zipper of this same protein. Furthermore, we demonstrate that the association of the CREB leucine zipper with the ATF-2 zinc finger in vitro blocks the association of the adenoviral E1a protein with ATF-2. Similarly, overexpression of full-length CREB, or a truncated version of this protein corresponding to the carboxy-terminal 74 amino acids that make up the DNA-binding and dimerization domains, can block the ATF-2-mediated transcriptional stimulation by E1a in vivo. Mutation of the ATF-2 zinc finger motif stimulates DNA binding of this protein, and abolishes interactions with E1a and CREB proteins. These results demonstrate that the structural conformation of ATF-2 is critical for DNA binding and protein-protein interactions and, further, that leucine zippers can mediate protein-protein interactions with structural motifs other than leucine zippers.