The effect of rowing on endothelial function and insulin action in healthy controls and in patients with type 2 diabetes.

Patients with type 2 diabetes (T2DM) have an increased risk for cardiovascular disease. We examined the effects of 8 weeks of home-based rowing training (heart rate corresponding to 65-70% of VO(2 peak) ) on endothelial function and glucose clearance (local and systemic effects) in male subjects with T2DM (n=9) and matched controls (n=8). Before and after training (30 min every other day), all subjects underwent sequential graded brachial artery infusions of non-insulin vasodilators (acetylcholine; sodium nitroprusside; adenosine). Forearm blood flow was improved by training in controls (without and with insulin: P=0.003 and 0.05, respectively) but not in subjects with T2DM. Likewise, whole body glucose clearance increased in response to training in controls (P=0.05) but not in T2DM. However, in both groups, the capacity for local forearm glucose extraction (controls: P=0.001; T2DM: P=0.002) and clearance (controls: P<0.001; T2DM: P=0.01) were positively affected by exercise. While the subjects with T2DM did not respond to the same degree as controls to 8 weeks of home-based exercise, there are clear benefits as illustrated by improvements in local glucose disposal. Training of higher intensity or duration may be required in order to elicit a response similar to controls.

Kinetic characterization of ribonuclease-resistant 2'-modified hammerhead ribozymes.

The incorporation of 2'-fluoro- and 2'-aminonucleotides into a hammerhead ribozyme was accomplished by automated chemical synthesis. The presence of 2'-fluorouridines, 2'-fluorocytidines, or 2'-aminouridines did not appreciably decrease catalytic efficiency. Incorporation of 2'-aminocytidines decreased ribozyme activity approximately by a factor of 20. The replacement of all adenosines with 2'-fluoroadenosines abolished catalysis in the presence of MgCl2 within the limits of detection, but some activity was retained in the presence of MnCl2. This effect on catalysis was localized to a specific group of adenines within the conserved single-stranded region of the ribozyme. The decrease in catalytic efficiency was caused by a decrease in the rate constant; the Michaelis constant was unaltered. The 2'-fluoro and 2'-amino modifications conferred resistance toward ribonuclease degradation. Ribozymes containing 2'-fluoro- or 2'-aminonucleotides at all uridine and cytidine positions were stabilized against degradation in rabbit serum by a factor of at least 10(3) compared to unmodified ribozyme.

The right-to-left shunt of crocodilians serves digestion.

Abstract All amniotes except birds and mammals have the ability to shunt blood past the lungs, but the physiological function of this ability is poorly understood. We studied the role of the shunt in digestion in juvenile American alligators in the following ways. First, we characterized the shunt in fasting and postprandial animals and found that blood was shunted past the lungs during digestion. Second, we disabled the shunt by surgically sealing the left aortic orifice in one group of animals, and we performed a sham surgery in another. We then compared postprandial rates of gastric acid secretion at body temperatures of 19 degrees and 27 degrees C and rates of digestion of bone at 27 degrees C. Twelve hours after eating, maximal rates of gastric acid secretion when measured at 19 degrees and 27 degrees C were significantly less in the disabled group than in sham-operated animals. Twenty-four hours postprandial, a significant decrease was found at 27 degrees C but not at 19 degrees C. For the first half of digestion, dissolution of cortical bone was significantly slower in the disabled animals. These data suggest the right-to-left shunt serves to retain carbon dioxide in the body so that it can be used by the gastrointestinal system. We hypothesize that the foramen of Panizza functions to enrich with oxygen blood that is destined for the gastrointestinal system to power proton pumps and other energy-demanding processes of digestion and that the right-to-left shunt serves to provide carbon dioxide to gastrointestinal organs besides the stomach, such as the pancreas, spleen, upper small intestine, and liver.

Long-acting formulations for the treatment of latent tuberculous infection: opportunities and challenges.

Long-acting/extended-release drug formulations have proved very successful in diverse areas of medicine, including contraception, psychiatry and, most recently, human immunodeficiency virus (HIV) disease. Though challenging, application of this technology to anti-tuberculosis treatment could have substantial impact. The duration of treatment required for all forms of tuberculosis (TB) put existing regimens at risk of failure because of early discontinuations and treatment loss to follow-up. Long-acting injections, for example, administered every month, could improve patient adherence and treatment outcomes. We review the state of the science for potential long-acting formulations of existing tuberculosis drugs, and propose a target product profile for new formulations to treat latent tuberculous infection (LTBI). The physicochemical properties of some anti-tuberculosis drugs make them unsuitable for long-acting formulation, but there are promising candidates that have been identified through modeling and simulation, as well as other novel agents and formulations in preclinical testing. An efficacious long-acting treatment for LTBI, particularly for those co-infected with HIV, and if coupled with a biomarker to target those at highest risk for disease progression, would be an important tool to accelerate progress towards TB elimination.

Comparison of an implanted abdominal aortic counterpulsation device with the intraaortic balloon pump in a heart failure model.

The abdominal aortic counterpulsation device is a round pumping chamber with a valveless opening which is implanted retroperitoneally on the abdominal aorta. The Utah driver is connected to the device through an air conduit and is synchronized on the electrocardiographic signal to provide diastolic aortic augmentation. For comparison an intraaortic balloon was also driven by the Utah driver system. The abdominal aortic counterpulsation device (stroke volume = 30, 40 and 60 ml) and the intraaortic balloon pump (balloon volume = 20 ml) were tested in dogs with acute left ventricular failure. The abdominal aortic counterpulsation device was also tested in normal animals. In acute left ventricular failure the abdominal aortic counterpulsation device at a stroke volume of 30, 40 or 60 ml decreased left ventricular end-diastolic pressure by an average of 28.56 (p less than 0.001), 39.56 (p less than 0.001) and 44.14% (p less than 0.005), respectively; aortic end-diastolic pressure by 24.11 (p less than 0.001), 26.67 (p less than 0.001) and 19.57% (p less than 0.01); and aortic systolic pressure by 18.56 (p less than 0.002), 26.0 (p less than 0.001) and 22.43% (p less than 0.005). It increased cardiac index by 27.58 (p less than 0.02), 35.59 (p less than 0.005) and 43.42% (p less than 0.001) and it provided peak aortic diastolic augmentation of 64.5 (p less than 0.001), 69.78 (p less than 0.001) and 74.43% (p less than 0.001), respectively, above the control aortic end-diastolic pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and purification of retroviral HIV-1 reverse transcriptase.

Modern molecular biology techniques have provided valuable tools which allow for the expression of large amounts of enzyme in E. coli. For potential therapeutic targets such as HIV-1 reverse transcriptase, it is desirable that the enzyme studied is pure and correlates to the active form of the enzyme found in vivo. This poses a particular challenge for those researchers studying HIV-RT since a significant degree of heterogeneity is introduced by nonspecific proteolytic cleavage of the p66 subunit by E. coli proteases. The advantage of the purification protocol presented here is that the association of monomers is facilitated by mixing an excess of p51 subunit, which is truncated at a site that is N-terminal to known bacterial cleavage sites, with p66 protein. This avoids enzymatic processing of the larger subunit since the formation of heterodimeric RT is rapid and the dimer is stable against proteolytic cleavage. Therefore, it is possible to isolate a pure homogeneous p66/p51 heterodimer. An enzyme prepared in this manner yields crystals that defract to a 3.2-A resolution. It has also been used to study both sensitivity of HIV-1 RT mutants to azidothymidine triphosphate and the kinetics of a potent nonnucleoside RT inhibitor (L-743,726). Finally, it is interesting to note the similarity of HIV-1 RT with reverse transcriptases from other lentiviruses (FIV and EIAV RT). Both of these enzymes consist of heterodimers of p66 and p51 subunits and share other biophysical characteristics. Purification of these reverse transcriptases can, in all likelihood, be optimized by using methods similar to those described in this chapter.

Identification of MK-944a: a second clinical candidate from the hydroxylaminepentanamide isostere series of HIV protease inhibitors.

Recent results from human clinical trials have established the critical role of HIV protease inhibitors in the treatment of acquired immune-deficiency syndrome (AIDS). However, the emergence of viral resistance, demanding treatment protocols, and adverse side effects have exposed the urgent need for a second generation of HIV protease inhibitors. The continued exploration of our hydroxylaminepentanamide (HAPA) transition-state isostere series of HIV protease inhibitors, which initially resulted in the identification of Crixivan (indinavir sulfate, MK-639, L-735,524), has now yielded MK-944a (L-756,423). This compound is potent, is selective, and competitively inhibits HIV-1 PR with a K(i) value of 0.049 nM. It stops the spread of the HIV(IIIb)-infected MT4 lymphoid cells at 25.0-50.0 nM, even in the presence of alpha(1) acid glycoprotein, human serum albumin, normal human serum, or fetal bovine serum. MK-944a has a longer half-life in several animal models (rats, dogs, and monkeys) than indinavir sulfate and is currently in advanced human clinical trials.

Thrombus generation within the artificial heart.

The sites of thrombus formation in the Jarvik III artificial heart were studied in 20 consecutive calf experiments. Identical design and implantation procedures were used with two different surfaces. The 10 anticoagulated calves receiving Dacron-fibrilized silicone rubber surfaces survived an average of 296 hours. In contrast, the 10 nonanticoagulated calves with smooth poyurethane hearts survived 545 hours. The polyurethane surfaces were constantly clean. However, the rough surfaces were coated with variable amounts of thrombi. The sharp angle between the diaphragm and housing contained varying amounts of thrombi deposits in 75 per cent of the cases. Thrombe deposition occurred on 41 per cent of the valves. These thrombe were the source of emboli into the kidney, brain, and lungs. Turbulences and stagnation areas generated thrombus formation. Anticoagulation did not prevent thrombus formation. It would appear that heart free of turbulence and stagnation areas must be designed to eliminate thromboembolism.

Six-month survival of a calf with an artificial heart.

A pneumatically powered artificial heart, constructed primarily from a polyurethane, was implanted in the chest of a calf and supported the calf for more than 6 months. The heart, which was designed to fit in the chest of a 90 kilogram calf, was able to suppor the animal when it weighed 180 kilograms. During the first 105 days the calf remained strong and healthy. The animal grew progressively weaker after day 106, and by day 160 right heart failure became apparent. The principal cause of the right heart failure was an obstructive growth between the right atrium and the right ventricle. An attempt to correct the problem on day 184 with an artificial heart resulted in the animal's death.

The artificial heart in human subjects.

In preparation for clinical implantation of the Utah J-7 pneumatic artificial heart as a permanent cardiac substitute, the device was implanted into five brain-dead human subjects. This report presents our results and details our two most successful trials. Three different surgical implant techniques were utilized in the five subjects. Because of the unique "no risk" situation of the subjects, the function of the artificial heart could be tested in a manner not advisable in patients, but necessary for clinical preparation. The implantable total artificial heart was able to maintain physiological hemodynamics in two subjects for 41 and 72 hours at which time the trials were electively terminated.

Survival for 18 days with a Jarvik-type artificial heart.

This is a report of an experiment wherein a calf had its natural heart replaced with an artificial heart and survived for 18 days and 20 hours. All measured physiologic parameters remained normal until the fourteenth day. Thereafter a gradual persistent rise in venous pressure and signs of a decreased cardiac output occurred. However, the animal outwardly appeared normal until the eighteenth day. During the nineteenth day it became comatose and was killed. At autopsy large thrombi were found in both atria, impairing ventricular filling, resulting in venous congestion and diminished cardiac output. This extended survival time and our ability to understand and eliminate the problems associated with artificial heart implantation give support to our hope that artificial hearts for man will be possible in the not too distant future.

Ventilatory control during exercise in calves with artificial hearts.

To determine the role of cardiac reflexes in mediating exercise hyperpnea, we investigated ventilatory responses to treadmill exercise in seven calves with artificial hearts and seven controls. In both groups, the ventilatory responses were adequate for the metabolic demands of the exercise; this resulted in regulation of arterial PCO2 and pH despite the absence of cardiac output increase in the implanted group. In this group, there was a small but significant reduction of arterial PO2 by 4 +/- 3 Torr and a rise of blood lactate by 1.1 +/- 1 mmol/l. When cardiac output was experimentally increased in the implanted calves to a level commensurate with that spontaneously occurring in the control calves, ventilation was not affected. However, experimental reductions of cardiac output led to an immediate augmentation of exercise hyperpnea by 4.56 +/- 4.3 l/min and a further significant lactate increase of 1.2 +/- 1.22 mmol/l that was associated with a significant decrease in the exercise O2 consumption (0.32 +/- 0.13 l/min). These observations indicate that neither cardiac nor hemodynamic effects of increased cardiac output constitute an obligatory cause of exercise hyperpnea in the calf.

Pseudononapeptide bombesin antagonists containing C-terminal Trp or Tpi.

Seven new antagonists of bombesin (Bn)/gastrin-releasing peptide (GRP) containing C-terminal Trp or Tpi (2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-3-carboxylic acid) in a reduced peptide bond were synthesized by solid phase methods and evaluated biologically. The reduced bond in four [Leu13 psi(CH2NH)Trp14]Bn(6-14) analogs was formed by reductive alkylation at the dipeptide stage. In the case of three [Leu13 psi(CH2N)Tpi14]Bn(6-14) analogs, the Trp dipeptide with reduced bond was reacted with formaldehyde to form the corresponding Tpi derivative. These Tpi-containing analogs have a new reduced bond which is structurally more constrained. Leu13 psi(CH2N)Tpi14 analogs inhibit [125I][Tyr4]bombesin binding to Swiss 3T3 cells with IC50 values of 2-4 nM, compared to 5-10 nM for Leu13 psi(CH2NH)Trp14 analogs. Leu13 psi(CH2N)Tpi14 analogs are also more potent than Leu13 psi(CH2NH)Trp14 analogs in growth inhibition studies using Swiss 3T3 cells. The two best bombesin antagonists of this series, [D-Trp6,Leu13 psi(CH2N)Tpi14]Bn(6-14) (RC-3415) and [Tpi6,Leu13 psi(CH2N)Tpi14]Bn(6-14) (RC-3440), inhibited GRP-stimulated growth of Swiss 3T3 cells with IC50 values less than 1 nM. RC-3440 was also active in vivo, suppressing GRP(14-27)-stimulated serum gastrin secretion in rats. Bombesin/GRP antagonists, such as RC-3440, containing the new reduced bond (CH2N) reported herein are very potent.

A ventricular assist device powered by conditioned skeletal muscle.

BACKGROUND: We are developing and testing a new ventricular assist device (VAD) to be powered by conditioned skeletal muscle. METHODS: To evaluate the VAD hardware and to develop a muscle training regimen, 8 calves have been used in studies in which the right latissimus dorsi muscle was employed. The experiments were carried out to an approximately 4-month duration. RESULTS: There was significant conversion of type II (fast twitch) to type I (slow twitch) muscle fibers. This did not correlate well, however, with device performance. The device stroke volumes ranged from approximately 17 to 90 cc. This variability of outcome occurred despite the fact that identical hardware, surgical procedures, and training regimens were employed. CONCLUSIONS: The results from the first eight studies lead us to speculate that perfusion may be important even when the muscle is working at pressures much lower than systemic blood pressure levels. In an attempt to augment tissue perfusion, we plan to investigate thermally induced angiogenesis as a possible mechanism for increasing blood flow to the tissue.

Developing an artificial fallopian tube: successful in vitro trials in mice.

This report describes the design and testing of an artificial fallopian tube for the treatment of tubal infertility. Within the device, mouse eggs incubated with sperm were fertilized and a microinfusion pump was used to transport the fertilized ova through the tube. Normal offspring resulted from transfer of the developing embryos into pseudopregnant recipients. These results provide encouraging evidence that an artificial fallopian tube warrants further investigation as a potential alternative to in vitro fertilization.

Synthesis, purification and biological evaluation of porcine corticotropin-releasing factor.

The 41-residue sequences of recently identified porcine corticotropin-releasing factors [Ile40]pCRF and [Asn40]pCRF were assembled on a benzhydrylamine resin support. Deprotection and cleavage from the resin were accomplished by HF treatment. The crude peptides were purified by HPLC. The homogeneity of the final materials, obtained in 0.2% and 0.4% overall yield for [Ile40]pCRF and [Asn40]pCRF respectively, was assessed after the isolation by HPLC and amino acid analysis. Both sequences of the synthetic 41-residue pCRF stimulated the release of corticotropin (ACTH) from superfused rat pituitary cells on a column, the responses being related to a log-dose of CRF in the range of 1-20 ng/ml. [Ile40]pCRF and [Asn40]pCRF also augmented the in vivo release of ACTH in rats pretreated with chlorpromazine, morphine and Nembutal. [Ile40]pCRF appeared to be equipotent to ovine CRF and about twice as active as [Asn40]pCRF. The data indicate that synthetic porcine [Ile40]pCRF and [Asn40]pCRF have high biological activity.

Development of a microcontroller-based automatic control system for the electrohydraulic total artificial heart.

An automatic physiological control system for the actively filled, alternately pumped ventricles of the volumetrically coupled, electrohydraulic total artificial heart (EHTAH) was developed for long-term use. The automatic control system must ensure that the device: 1) maintains a physiological response of cardiac output, 2) compensates for an nonphysiological condition, and 3) is stable, reliable, and operates at a high power efficiency. The developed automatic control system met these requirements both in vitro, in week-long continuous mock circulation tests, and in vivo, in acute open-chested animals (calves). Satisfactory results were also obtained in a series of chronic animal experiments, including 21 days of continuous operation of the fully automatic control mode, and 138 days of operation in a manual mode, in a 159-day calf implant.

A moving-actuator type electromechanical total artificial heart--Part I: Linear type and mock circulation experiments.

A new type of motor-driven total artificial heart system with a moving-actuator mechanism has been developed. The prototype system consists of a brushless dc motor inside of a rolling-cylinder, two arc-shaped pusher-plates and two polyurethane sacs. The moving-actuator type electromechanical pump has structural advantages of small size and light weight, as compared to other reported motor-driven pumps with fixed-actuator mechanisms. The results of the mock circulation tests are reported in this paper with a cardiac output of 9 L/min at an aortic pressure of 120 mmHg and a heart rate of 120 bpm. The fulfillment of the basic control requirements of the artificial heart was also confirmed, i.e., preload sensitive and afterload insensitive cardiac output response and balanced right and left ventricular outputs.

A moving-actuator type electromechanical total artificial heart--Part II: Circular type and animal experiment.

A new type of electromechanical total artificial heart (TAH) based on circular rolling-cylinder mechanism was developed to overcome critical problems in motor-driven artificial hearts such as large size and difficulties in fitting the heart to atrial remnants and arterial vessels. Its performance and reliability were evaluated in mock circulation and in an animal implant experiment. The total weight and volume of the pump is 650 g and 600 mL, respectively. This new pump was implanted in a calf for total heart replacement and 96 h of survival was achieved. The whole system, including pump, controller, and control algorithm performed well enough to improve the prospect of eventual clinical application of our TAH system.

State-of-the-art and clinically applied pneumatic artificial hearts.

This article is intended to inform the reader of the current state of the art of pneumatically powered artificial hearts, including animal experiments and information available on 11 recipients of orthotopically placed devices.