Effects of colchicine on the hepatocellular transport of indocyanine green in the rat

The effects of colchicine on plasma elimination and biliary excretion of indocyanine green (ICG) and sulfobromophthalein (BSP) in rats were examined. Elimination of two different doses of ICG (6 mg and 20 mg/kg body weight) from plasma was significantly delayed when rats were treated with colchicine (3 mg/kg body weight) 3 h prior to the administration of the dye. On the other hand, disappearance of BSP (100 mg/kg) from plasma was not influenced by colchicine. The fact that the difference in the ICG elimination from plasma between colchicine-treated and saline-treated rats was minimal in the early period (i.e., 2 min after administration of the dye), but evident after its half-life (i.e., 10 min, when 6 mg/kg body weight of ICG was given), suggested that colchicine mainly affected the hepatocellular transport of ICG rather than the uptake of the dye by hepatocytes. Colchicine also significantly reduced the excretion of ICG (6 mg and 20 mg/kg) into bile but did not alter that of BSP (100 mg and 200 mg/kg). On the other hand, the same amount of lumicolchicine (3 mg/kg) did not have any effect on the biliary excretion of ICG. These results suggested that ICG is transported through hepatocytes into bile with the aid of the cytoplasmic microtubular system, whereas BSP is handled by hepatocytes in a different way.     

The permeability of capillaries among the small granule-containing cells in rat superior cervical ganglia: an ultrastructural lanthanum tracer study.

The permeability of blood capillaries associated with small granule-containing (SGC) cells in rat superior cervical ganglia was investigated at ultrastructural level by employing ionic lanthanum as an electron dense tracer. In rat superior cervical ganglia, the majority of blood capillaries were nonfenestrated. Both fenestrated and nonfenestrated capillaries were observed in the area associated with SGC cells. Lanthanum tracer was observed in the luminal surface, the interendothelial cleft and the subendothelial perivascular spaces of both fenestrated and nonfenestrated capillaries associated with SGC cells. The external lamina of the Schwann cell which surrounded the neurons, nerve fibres and SGC cells were clearly delineated by the lanthanum tracer. Furthermore, the perineuronal space, the periaxonal space, and the pericellular space of the SGC cells were readily accessible to the lanthanum ion. The results demonstrated an absence of blood-nerve barrier, blood-ganglionic and blood-SGC cell barrier to the lanthanum ion in the parenchymal area of the SGC cells in rat superior cervical ganglia. It is proposed that lanthanum may pass through the endothelial cells via 1) the fenestrae of fenestrated capillaries, 2) the intercellular junctions of both fenestrated and nonfenestrated capillaries, i.e., a paracellular pathway; and 3) the process of endocytosis/exocytosis, i.e., a transcellular pathway, to reach the subendothelial space and be distributed in the parenchyma of SGC cells in rat superior cervical ganglia.     

Measurement of plasma volume using fluorescent silica-based nanoparticles

Plasma volume (PV) is an important determinant of cardiovascular function and organ perfusion, and it is the target of infusion and diuretic therapies in daily clinical practice. Despite its fundamental importance PV is not commonly measured because available methods of tracer dilution are reliant on dye substances that suffer from numerous drawbacks including binding plasma proteins, spectral changes, and clearance kinetics that complicate analysis and interpretation. To address these issues, we have tested the utility of fluorescent nanoparticles comprised of a dye-rich silica core and polyethylene glycol-coated shell. Photophysical and visual analysis showed discrete size-gradated nanoparticle populations could be synthesized within a distribution tolerance of ±4 nm, which were optically unaffected in the presence of plasma/albumin. In normal mice, the cutoff for renal filtration of nanoparticles from blood into urine was ≤11 nm. A linear relationship between body weight and PV was readily determined in mice administered far red fluorescent nanoparticles sized either 20 or 30 nm. PV measurements using nanoparticles were correlated to values obtained with Evans blue dye. Induced expansion or contraction of PV was demonstrated with albumin or furosemide administration, respectively, in mice. Longitudinal experiments >30 min required matched untreated control mice to correct for nanoparticle loss (≈30%) putatively to the reticuloendothelial/phagocyte system. Collectively, the findings support a nanotechnology-based solution to methodological problems in measure of PV, notably in clinical settings where information on hemodynamic changes may improve treatment of injury and disease.     

Sentinel node biopsy in breast cancer using infrared laser system first experience with PDE camera

Background

Sentinel node biopsy (SNB) is a gold standard in staging of early breast cancer. Nowadays, routine mapping of lymphatic tract is based on two tracers: human albumin with radioactive technetium, with or without blue dye. Recent years have seen a search for new tracers to examine sentinel node as well as lymphatic network. One of them is indocyanine green (ICG) visible in infrared light.

Aim

The aim of this study is to evaluate clinical usage of ICG in comparison with standard tracer, i.e. nanocoll, in SNB of breast cancer patients.

Materials and methods

In the 1st Department of Surgical Oncology and General Surgery, Greater Poland Cancer Centre, Poznań, 13 female breast cancer patients have benn operated since September 2010. All these patients had sentinel node biopsy with nanocoll (human albumin with radioactive technetium), and with indocyanine green. The feasibility of this new method was assessed in comparison with the standard nanocoll.

Results

A lymphatic network between the place of injection of ICG and sentinel node was seen in infrared light. An area where a sentinel node was possibly located was confirmed by gamma probe. Sensitivity of this method was 100%.

Conclusion

SNB using ICG is a new, promising diagnostics technique. This procedure is not without drawbacks; nevertheless it opens new horizons in lymphatic network diagnostics.     

Interstitial exclusion of positively and negatively charged IgG in rat skin and muscle

Volume exclusion, i.e., the space not available for a specific probe, may be dependent on the probe charge. Therefore, interstitial exclusion was measured for positively and negatively charged immunoglobulin (IgG) in skin and muscle of rats by using a continuous infusion method (30). Steady-state concentration of (125)I-labeled IgG 1 (pI = 8.7) and (131)I- labeled IgG 4 (pI = 6.6) was maintained by infusion of tracer for 120-168 h with an implanted osmotic pump. At the end of the infusion period and before tissue sampling, the rat was anesthetized and nephrectomized, and (51)Cr-labeled EDTA was injected and allowed 4 h for equilibration to measure interstitial fluid volume (V(i)). Interstitial fluid was isolated from skin and muscle by using nylon wicks implanted post mortem. The relative IgG available space was measured as the ratio between labeled IgG and (51)Cr-labeled EDTA wick fluid equivalent spaces, and relative excluded volume fraction (V(e)/V(i)) was calculated as 1--V(a)/V(i). V(e)/V(i) in hindlimb skin averaged 0.37 +/- 0.05 (SE) and 0.65 +/- 0.06 (P < 0.01) for IgG 1 and 4, respectively, with corresponding figures of 0.24 +/- 0.05 and 0.51 +/- 0.04 (P < 0.01) in hindlimb muscle (n = 9 for both tissues). These experiments suggest that fixed negative charges, most likely glycosaminoglycans, influence distribution of macromolecules in the interstitium and therefore affect interstitial fluid balance.     

A strategy for efficient characterization of macromolecular heteroassociations via measurement of sedimentation equilibrium.

A method is proposed for the selection of experimental conditions for sedimentation equilibrium experiments that will provide maximal information about the values of equilibrium association constants within a given scheme for heteroassociation of two solute components. A discriminator function is proposed that indicates the sensitivity of the experimentally observed gradient or gradients to alterations in the underlying association constants. The value of this function is plotted or tabulated as a function of the concentrations of the two components, over a broad range of solution compositions. It is suggested that experiments performed with loading compositions corresponding to large absolute values of the discriminator function will yield the most information with respect to determination of the underlying association constants. This method was tested by predicting optimal conditions for three different types of sedimentation equilibrium experiments: (i) measurement of total (natural) solute absorbance; (ii) measurement of individual component gradients via measurement of tracer absorbance; and (iii) global analysis of multiple experiments. Experimental data resulting from sedimentation equilibrium experiments carried out under the specified conditions were simulated by addition of realistic levels of random error to calculated equilibrium gradients. The simulated data were then analyzed exactly as real experimental data, i.e., without prior knowledge of the underlying association constants. It was found that the highest accuracy and precision in determination of heteroassociation constants are obtained by global analysis of multiple experiments performed using significantly different loading compositions, each of which is selected from 'sensitive' regions of the discriminator map.     

Effects of red blood cell aggregation on myocardial hematocrit gradient using two approaches to increase aggregation

The normal transmyocardial tissue hematocrit distribution (i.e., subepicardial greater than subendocardial) is known to be affected by red blood cell (RBC) aggregation. Prior studies employing the use of infused large macromolecules to increase erythrocyte aggregation are complicated by both increased plasma viscosity and dilution of plasma. Using a new technique to specifically alter the aggregation behavior by covalent attachment of Pluronic F-98 to the surface of the RBC, we have determined the effects of only enhanced aggregation (i.e., Pluronic F-98-coated RBCs) versus enhanced aggregation with increased plasma viscosity (i.e., an addition of 500 kDa dextran) on myocardial tissue hematocrit in rapidly frozen guinea pig hearts. Although both approaches equally increased aggregation, tissue hematocrit profiles differed markedly: 1) when Pluronic F-98-coated cells were used, the normal transmyocardial gradient was abolished, and 2) when dextran was added, the hematocrit remained at subepicardial levels for about one-half the thickness of the myocardium and then rapidly decreased to the control level in the subendocardial layer. Our results indicate that myocardial hematocrit profiles are sensitive to both RBC aggregation and to changes of plasma viscosity associated with increased RBC aggregation. Furthermore, they suggest the need for additional studies to explore the mechanisms affecting RBC distribution in three-dimensional vascular beds.     

Improved molecular dynamics simulations for the determination of peptide structures

In this article a few methods or modifications proven to be useful in the conformational examination of peptides and related molecules by molecular dynamics are illustrated. The first is the explicit use of organic solvents in the simulations. For many cases such solvents are appropriate since the nmr measurements (or other experimental observations) were carried out in the same solvent. Here, the use of dimethylsulfoxide and chloroform in molecular dynamics is described, with some advantages of the use of these solvents highlighted. A constant allowing for the scaling of the nonbonded interactions of the force field, an idea previously employed in distance geometry and simulated annealing, has been implemented. The usefulness of this method is that when the nonbonded term is turned to zero, atoms can pass through each other, while the connectivity of the molecule is maintained. It will be shown that such simulations, if a sufficient driving force is present (i.e., nuclear Overhauser effects restraints), can produce the correct stereoconfiguration (i.e., chiral center) as well as configurational isomer (i.e., cis/trans isomers). Lastly, a penalty term for coupling constants directly related to the Karplus curve has been plemented into the potential energy force field. The advantages of this method over the commonly used dihedral angle restraining are discussed. In particular, it is shown that with more than one coupling constant about a dihedral angle a great reduction of the allowed conformational space is obtained. © 1993 John Wiley & Sons, Inc.     

Brain Arachidonic Acid Incorporation and Precursor Pool Specific Activity During Intravenous Infusion of Unesterified [3H]Arachidonate in the Anesthetized Rat

Abstract: Brain fatty acid incorporation into phospholipids can be measured in vivo following intravenous injection of fatty acid tracer. However, to calculate a cerebral incorporation rate, knowledge is required of tracer specific activity in the final brain precursor pool. To determine this for one tracer, unesterified [³H]arachidonate was infused intravenously in pentobarbital-anesthetized rats to maintain constant plasma specific activity for 1–10 min. At the end of infusion, animals were killed by microwave irradiation and analyzed for tracer specific activity and concentration in brain phospholipid, neutral lipid, and lipid precursor, i.e., unesterified arachidonate and arachidonoyl-CoA, pools. Tracer specific activity in brain unesterified arachidonate and arachidonoyl-CoA rose quickly ( t _1/2 < 1 min) to steady-state values that averaged <5% of plasma specific activity. Incorporation was rapid, as >85% of brain tracer was present in phospholipids at 1 min of infusion. The results demonstrate that unesterified arachidonate is rapidly taken up and incorporated in brain but that brain phospholipid precursor pools fail to equilibrate with plasma in short experiments. Low brain precursor specific activity may result from (a) dilution of label with unlabeled arachidonate from alternate sources or (b) precursor pool compartmentalization. The results suggest that arachidonate turnover in brain phospholipids is more rapid than previously assumed.     

Tracer measurements of non-equilibrium volumes of distribution

According to conventional theory the product of the transport flowrate and the mean transit time of a tracer through a system yields the equilibrium volume of distribution for the tracer, regardless of tracer kinetics or space geometry. Experimental results do not support this notion. The influence of measurement time on the volume measured with a bolus technique is addressed using systems theory to analyze a tissue-impedance form of the Sangren-Sheppard model. Assymptotic solutions show that the volume estimates are governed by a time constant, tau, related to diffusion in the tissue, to tissue capacity, and to wall permeability, and by a dimensionless ratio, f, describing a relation of tau to vascular transport time. A third parameter, g, describing the relative contributions to overall resistance to diffusion of effective permeability and of limited diffusivity in the tissue, is shown to be of less importance. The derived tau is similar to but not equivalent to the often cited "characteristic time". The "equilibrium" volume of distribution is defined as that which would be measured if equilibrium were allowed to establish. The "non-equilibrium" volume of distribution is defined as that which would be measured given finite times and is shown to approach the "equilibrium" volume as such times increase. Tracer equilibration is not required to accurately measure the "equilibrium" volume. When there is no flow limitation (f much less than 1) a measurement time of tau (plus vascular transit time) would yield a "non-equilibrium" volume only 33% of the "equilibrium" volume; a time of 2 tau would yield 55%; a time of 10 tau would yield effectively the total equilibrium volume. Finite diffusivity in tissue and permeability restrictions can have significant effects on the proportion of the volume measured.     

Exercise stroke volume relative to plasma-volume expansion

The effects of plasma-volume (PV) expansion on stroke volume (SV) (CO2 rebreathing) during submaximal exercise were determined. Intravenous infusion of 403 +/- 21 ml of a 6% dextran solution before exercise in the upright position increased SV 11% (i.e., 130 +/- 6 to 144 +/- 5 ml; P less than 0.05) in untrained males (n = 7). Further PV expansion (i.e., 706 +/- 43 ml) did not result in a further increase in SV (i.e., 145 +/- 4 ml). SV was somewhat higher during supine compared with upright exercise when blood volume (BV) was normal (i.e., 138 +/- 8 vs. 130 +/- 6 ml; P = 0.08). PV expansion also increased SV during exercise in the supine position (i.e., 138 +/- 8 to 150 +/- 8 ml; P less than 0.05). In contrast to these observations in untrained men, PV expansion of endurance-trained men (n = 10), who were naturally PV expanded, did not increase SV during exercise in the upright or supine positions. When BV in the untrained men was increased to match that of the endurance-trained subjects, SV was observed to be 15% higher (165 +/- 7 vs. 144 +/- 5 ml; P less than 0.05), whereas mean blood pressure and total peripheral resistance were significantly lower (P less than 0.05) in the trained compared with untrained subjects during upright exercise at a similar heart rate. The present findings indicate that exercise SV in untrained men is preload dependent and that increases in exercise SV occur in response to the first 400 ml of PV expansion. It appears that approximately one-half of the difference in SV normally observed between untrained and highly endurance-trained men during upright exercise is due to a suboptimal BV in the untrained men.     

Determination of circulating blood volume by measurement of indocyanine green dye in hemolysate: a preliminary study

In 8 emergency care patients blood volume was determined using Cr5I labelled erythrocytes and indocyanine green (ICG). Prior to measurement of ICG in blood with a spectrophotometer, the blood was hemolyzed with Triton-X. A close correlation of r = 0.97 between the Cr51 and the ICGTR-X estimates was found; the ICGTR-X volume was about 3% lower than the Cr51 volume. In five additional in vitro experiments the ICGTR-X method was found to reflect real volumes with an insignificant error of less than 1%. Blood volume determination with ICGTR-X cannot be applied in cases of circulatory failure. ICG should be administered in a dose of 0.5 mg/kg of body weight. For calibration purposes, a two point calibration curve (point 1: point of intersection of x and y axis; point 2: 5 mg ICG/1 of blood) is sufficient. From these preliminary experiments it is concluded that the ICGTR-X method is a rapid and simple technique of blood volume determination with multiple reproducibility which can be carried out in any clinical laboratory.     

A multienzyme bioluminescent time-resolved pyrophosphate assay

We have developed a high-sensitivity assay for measurement of inorganic pyrophosphate (PPi) in adenosine 5'-triphosphate (ATP)-contaminated samples. The assay is based on time-resolved measurements of the luminescence kinetics and implements multiple enzymes to convert PPi to ATP that is, in turn, utilized to produce light and to hydrolyze PPi for measurement of the steady state background luminescence. A theoretical model for describing luminescence kinetics and optimizing composition of the assay detection mixture is presented. We found that the model is in excellent agreement with the experimental results. We have developed and evaluated two algorithms for PPi measurement from luminescence kinetics acquired from ATP-contaminated samples. The first algorithm is considered to be the method of choice for analysis of long, i.e., 3-5 min, kinetics. The activity of enzymes is controlled during the experiment; the sensitivity of PPi detection is about 7 pg/ml or 15 pM of PPi in ATP-contaminated samples. The second algorithm is designed for analysis of short, i.e., less than 1-min, luminescence kinetics. It has about 20 pM PPi detection sensitivity and may be the better choice for assays in microplate format, where a short measurement time is required. The PPi assay is primarily developed for RNA expression analysis, but it also can be used in various applications that require high-sensitivity PPi detection in ATP-contaminated samples.     

Evaluating pulmonary vascular permeability with radiolabeled proteins: an error analysis

Using techniques of mathematical simulation, we compared two methods of evaluating pulmonary vascular permeability, i.e., transvascular protein flux. Both methods calculate a transport rate constant [pulmonary transcapillary escape rate (PTCER)] after making external radiation detection measurements of an intravenously administered radiolabeled protein. With one method, lung tissue time-activity data are acquired by positron emission tomography (PET) and are interpreted with a two-compartment model. With the other method, the time-activity data are acquired with simple detector probes and are interpreted by linear regression after normalizing for various physical factors (slope-intercept or SI method). The results show that significant errors in calculating PTCER can result from using the SI method, because it ignores the effects of back-flux on the tissue time-activity measurements. Both methods produce errors if the data analysis includes activity from vascular volumes not involved in tracer exchange with the extravascular compartment. Significant errors can also occur with the PET method, particularly when permeability is nearly normal, if pulmonary vascular volume changes significantly during the period of data collection. On balance, the PET method appears to be the method of choice for accurately evaluating pulmonary vascular permeability by protein flux measurements, although both methods may be useful in clinical applications.     

Microtechnique for Most-Probable-Number Analysis

A microtechnique based on the most-probable-number (MPN) method has been developed for the enumeration of the ammonium-oxidizing population in soil samples. An MPN table for a research design ([8 by 12] i.e., 12 dilutions, 8 replicates per dilution) is presented. A correlation of 0.68 was found between MPNs determined by the microtechnique and the standard tube technique. Higher MPNs were obtained with the microtechnique with increased accuracy in endpoint determinations being a possible cause. Considerable savings of time, space, equipment, and reagents are observed using this method. The microtechnique described may be adapted to other microbial populations using various types of media and endpoint determinations.     

Glucose isotope, carbon recycling, and gluconeogenesis using [U-13C]glucose and mass isotopomer analysis

Experimental determinations of glucose carbon recycling using 14C or 13C glucose tracer often underestimate true Cori cycle activity because of dilution and exchange of isotope tracer through the tricarboxylic acid (TCA) cycle. The term glucose isotope recycling therefore is used to distinguish recycling of isotope from recycling of glucose carbon, the actual quantity of circulating glucose recycled. Recently, per-labeled glucose ([U-13C6]glucose) has been used to estimate glucose appearance rate and glucose isotope recycling. Chemical structural information determined by mass isotopomer analysis has been used to correct for dilution of isotope through the TCA cycle. In this report, we present experiments in the study of glucose turnover and recycling using [U-13C6]glucose. Methods of single injection and continuous infusion of [U-13C6]glucose are compared. A formula for the calculation of a dilution factor using TCA cycle parameters is derived. In this study of six rabbits, glucose turnover rate ranged from 3.4 to 8.8 mg/kg/min, and glucose m + 3 mass isotopomer recycling from 7 to 12%. The rate of pyruvate carboxylation (Y) was comparable to that of citrate synthetase, having an average relative flux of 0.89. Applying the correction factor for tracer dilution to the observed mass isotopomer recycling, we determined glucose carbon recycling (or Cori cycle activity) to be 22-35% of hepatic glucose output.     

Gut-derived norepinephrine plays a critical role in producing hepatocellular dysfunction during early sepsis

Although plasma norepinephrine (NE) increases and hepatocellular function is depressed during early sepsis, it is unknown whether gut is a significant source of NE and, if so, whether gut-derived NE helps produce hepatocellular dysfunction. We subjected rats to sepsis by cecal ligation and puncture (CLP), and 2 h later (i.e., early sepsis) portal and systemic blood samples were collected and plasma levels of NE were assayed. Other rats were enterectomized before CLP. Hepatocellular function was assessed with an in vivo indocyanine green (ICG) clearance technique, systemic levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6 were determined, and the effect of NE on hepatic ICG clearance capacity was assessed in an isolated, perfused liver preparation. Portal levels of NE were significantly higher than systemic levels at 2 h after CLP. Prior enterectomy reduced NE levels in septic animals. Thus gut appears to be the major source of NE release during sepsis. Enterectomy before sepsis also attenuated hepatocellular dysfunction and downregulated TNF-alpha, IL-1beta, and IL-6. Perfusion of the isolated livers with 20 nM NE (similar to that observed in sepsis) significantly reduced ICG clearance capacity. These results suggest that gut-derived NE plays a significant role in hepatocellular dysfunction and upregulating inflammatory cytokines. Modulation of NE release and/or hepatic responsiveness to NE should provide a novel approach for maintaining hepatocellular function in sepsis.     

Lipoteichoic acid is a major component of the Bacillus subtilis periplasm

Cryo-electron microscopy (cryo-EM) of frozen-hydrated specimens allows high-resolution observation of structures in optimally preserved samples. In gram-positive bacteria, this method reveals the presence of a periplasmic space between the plasma membrane and an often differentiated cell wall matrix. Since virtually nothing is known about the composition of its constituent matter (i.e., the periplasm), it is still unclear what structures (or mechanism) sustain a gram-positive periplasmic space. Here we have used cryo-EM of frozen-hydrated sections in combination with various labels to probe the model gram-positive organism Bacillus subtilis for major periplasmic components. Incubation of cells with positively charged gold nanoparticles showed almost similar levels of gold binding to the periplasm and the cell wall. On cells whose cell walls were enzymatically hydrolyzed (i.e., on protoplasts), a surface diffuse layer extending ~30 nm from the membrane was revealed. The thickness and density of this layer were not significantly altered after treatment with a nonspecific protease, whereas it was labeled with anti-lipoteichoic acid (LTA) antibodies conjugated to nanogold. Further, the LTA layer spans most of the thickness of the periplasmic space, which strongly suggests that LTA is a major component of the B. subtilis periplasm.     

FUNCTIONAL CELL COMPARTMENTS IN A RAT MODEL FOR HUMAN ACUTE MYELOCYTIC LEUKAEMIA

Functional cell compartments were studied in a rat model for human acute myelocytic leukaemia (AML). This was done by tracing the distribution of injected ⁵¹Chromium-labelled leukaemic cells in the body. It was concluded that two functional compartments can be distinguished in acute leukaemia, i.e., a rapidly exchangeable pool of cells (including the circulating blood pool, the marginal noncirculating blood pool and the rapidly exchangeable tissue pool; RETP) and a slowly exchangeable tissue pool (SETP). The sizes of these various compartments were roughly quantified at various stages of the disease by calculations based on the principle of isotope dilution and organ weight measurements. As the leukaemia progresses, the size of the SETP increases significantly relative to the size of the RETP. Simultaneously, the exchange rates of leukaemic cells between the organs and the blood decrease. The blood transit time of leukaemic cells was also significantly prolonged, as is the case in human AML.     

Artificial gravity in space flight

Clearly, physiologic adaptation to terrestrial life for all animals is assured only by frequent encounters with gravity. Indeed, upon exposure to weightlessness in space flight, losses of physiologic functions quickly begin. Some physiologic parameters change more rapidly than others, but the deconditioning process starts rapidly. The rates of functional losses for all affected parameters are interesting in that they appear to approach a limit; i.e., losses of these functions may not continue until indefinitely. The regulation of this functional asymptotic response to space is not known, but probably based on functional requirements of the body to life itself and perhaps genetic expression. The latter controlling mechanism (DNA) functions only on aquatic (weightless) animals on Earth--land animals must stimulate these physiologic functions as they relate to gravity on a regular frequent basis. This loss of regulation upon entering the weightless environment is fascinating since land-based animals including the humans have evolved from millions (perhaps billions) of years of terrestrially adapted ancestors. One would expect some DNA involvement in the regulation of its physiology, but it appears to be absent. Therefore, if the functional debilitation of space is to be denied, we must begin to understand the adaptation process of the sole basis for the control of our physiologic processes on land; i.e., how gravity regulates our biologic functions. To learn about this regulatory mechanism, some inquiry into how aquatic animals first adapted to living on land might be helpful.