TRYPTOPHAN TRANSPORT ACROSS THE BLOOD-BRAIN BARRIER DURING ACUTE HEPATIC FAILURE

Abstract— Tryptophan transport across the blood-brain barrier was studied using a single injection dual isotope label technique, in the following three conditions: normal rats, rats with portacaval shunts, and rats with portacaval shunts followed 65 h later by hepatic artery ligation. In both normal rats and those with acute hepatic failure the tryptophan transport system was found to be comprised of two kinetically distinct components. One component was saturable and obeyed Michaelis-Menten kinetics (normal: V_max= 19.5 nmol.min^?1.g^?1. K_m= 113 μM; hepatic failure: V_max, = 33.8 nmol.min^?1.g^?1, K_m= 108 μM), and the second was a high capacity system which transported tryptophan in direct proportion to concentration over the range tested (normal: K= 0.026 ml.min^?1.g^?1; hepatic failure: K= 0.067 ml.min^?1.g^?1). Since the saturable low capacity component transports several neutral amino acids, and their collective plasma concentration is high in relation to the individual K_ms, tryptophan transport by this component is reduced by competitive inhibition under physiological conditions. Thus it was calculated that in normal rats approx 40% of tryptophan influx occurs via the high capacity system. During acute hepatic failure transport via both components was increased substantially, approximately doubling the rate of tryptophan penetration of the blood-brain barrier at all concentrations tested. The contribution by the high capacity component became even more significant than in normal rats, accounting for about 75% of all tryptophan passage from plasma to brain. Brain tryptophan content was 29.9 nmol/g in normal rats and rose to 45.2 nmol/g in rats with portacaval shunts and 50.5 nmol/g in those with acute hepatic failure, correlating with the increased rate of tryptophan transport. In a previous study we found that plasma competing amino acids were greatly increased during acute hepatic failure. Calculations predict that these increased concentrations would cause a reduction in tryptophan transport by the low capacity system. However, because of the increase in the rate of transport by the high capacity component, net tryptophan entry across the blood-brain barrier was actually increased. This increased rate of transport clearly contributes to the increased content of brain tryptophan found during hepatic failure.     

Survival and transfer in the human gut of poorly mobilizable (pBR322) and of transferable plasmids from the same carrier E. coli

We examined the survival of a host Escherichia coli K-12 bacterium containing two transferable plasmids (pLM2, pSL222-4) and one poorly mobilizable plasmid (pBR322), and the transfer of these three plasmids to endogenous bacteria in the human intestinal tract. The survival of this plasmid-carrying host organism in four human volunteers was 3.5 to 6 days at recovery rates of 10^?1 to 10^?4. This finding was similar to our previous survival data on the same organism bearing a single plasmid. The K-12 strain appeared to be under a strong selective disadvantage in the human gut, since, even when bearing a tetracycline-resistant plasmid, its titer did not increase despite the administration of tetracycline. Studies of transferability showed that, while the transfer-depressed incFII plasmid pSL222-4 transferred at a frequency of 10^?1 in culture, its transfer in the human gut was much less frequent. The number of new recipients per donor cell ingested was about 10^?5, which included new recipients arising by multiplication. The recovery of pSL222-4 transcipients was enhanced by the administration of tetracycline on day 6. Neither the transfer-repressed, broad host range incP plasmid pLM2, nor the plasmid pBR322, could be detected in any endogenous host bacteria. Using the transfer and mobilization frequencies obtained in culture and the number of new recipients of pSL222-4 in the intestinal tract, we estimated that any in vivo mobilization of pBR322 to a new recipient could not occur at a frequency higher than 10^?12.     

Genetic analysis of antibiotic resistance in Streptococcus pyogenes.

The genetics of antibiotic resistance in mutant strains of Streptococcus pyrogenes was studied. Utilizing a type 6 strain (9440) primarily resistant to strepttomycin (Strr), classes of mutant strains were isolated that were resistant to one of the following antibiotics: rifampin (Rifr), erythromycin (Eryr), thiostrepton (Tstr), spiramycin (Sprr), fusidic acid (Fusr), gramicidin (Grcr), ethidium bromide (Ebrr), kanamycin (Kanr), neomycin (Neor), oleandomycin (Oler), gentamicin (Genr), and novobiocin (Novr). Transduction experiments separated antibiotic resistance markers into two distinct groups: transducible markers, including Fusr, Bacr, Ksg+, Spcr, Eryr, Sprr, Rifr, Stlr, and Tstr (Bacr, Ksgr, Spcr, and Stlr refer to resistance to bacitracin, kasugamycin, spectinomycin, and streptolydigan, respectively), and nontransducible markers, including Grcr, Ebrr, Kanr, Neor, Oler, Genr, and Novr. By means of two- and three-point crosses, transducible markers (excluding tst) were located in three separate linkage groups. spr was found to be linked with ery and spc in the order spc-ery-spr, whereas in a separate linkage group the order was determined to be str-fus-bac-ksg. The third linkage group contained the rif and stl markers.     

Role of Cation and Anion Uptake in Salt-stimulated Elongation of Lettuce Hypocotyl Sections

The role of cation and anion uptake in salt-stimulated growth of light-grown, GA₃-treated lettuce (Lactuca sativa L.) hypocotyl sections was investigated. Potassium chloride (10 mm) causes a 2-fold increase in the growth rate of GA₃-treated hypocotyl sections without affecting the growth rate of sections incubated in the absence of GA₃. Salt uptake is the same in both treatments, and furthermore the uptake of cation and anion is stoichiometric during the first 24 hours under all incubation conditions. The importance of the anion for cation uptake is demonstrated in experiments with benzenesulfonate⁻ and iminodiacetate²⁻. When K⁺ and Na⁺ are supplied only as the benzenesulfonate and iminodiacetate salts, growth and cation uptake are markedly reduced compared to KCl and NaCl. Calculation of the osmotic potential of salt-treated sections based on measurement of K⁺ and Cl⁻ uptake suggests that the observed increase in tissue osmolality is a result of salt uptake. Similarly, uptake of ions can account for the shift in water potential when sections are incubated in 10 mm KCl. We conclude that the change in growth rate of light-grown, GA₃-treated sections caused by the addition of KCl or NaCl to the incubation medium results solely from decreased water potential of the tissue due to ion uptake.     

The effect of coenzyme A and structurally related thiols on the mammalian fatty acid synthetase

A comparison was made of the structural features of thiol compounds which can interact with the mammalian fatty acid synthetase. Three functional characteristics were examined: (i) the ability of the free thiols, at low concentrations, to satisfy the essential thiol requirement of the enzyme, (ii) the ability of the free thiols, at higher concentrations, to inhibit enzyme activity, and (iii) the ability of the malonyl esters of these thiol compounds to act as substrates for fatty acid synthesis. The relative effectiveness of the various thiols studied was identical in all three roles. Coenzyme A and N-hexanoylcysteamine were the most effective, pantetheine and N-butyrylcysteamine were less effective, and N-acetylcysteamine was totally ineffective. These results lend strong support to our hypothesis (A. Stern, B. Sedgwick, and S. Smith, 1982, J. Biol. Chem.257, 799–803) that the various effects of CoA and structurally related thiols are localized at one and the same site, namely, the site of transfer of substrates between coenzyme A ester form and enzyme-bound form.     

Gelation of liposome interior. A novel method for drug encapsulation.

Liposomes can be loaded with weak acids and bases, which exist in solutions in equilibrium with membrane permeable uncharged form, using various gradients across their membranes. Because in some cases the estimated drug concentration in the loaded liposomes exceeds their aqueous solubility we investigated the physical state of the liposome encapsulated anticancer drug Doxorubicin. X-Ray diffraction, electron microscopy, and test tube solubility experiments have shown that upon encapsulation the drug molecules form a gel-like phase.     

Fecundity of Euphausia lucens (Hansen) - laboratory evidence for multiple broods

There has been much controversy in the past as to how many broods euphausiids are capable of producing per season. Several authors have suggested that multiple broods are possible, although this has not been demonstrated in the laboratory. In the present study, two Euphausia lucens females were shown to spawn continuously over a period of at least 2.5 months. The euphausiids produced ≈ 33–40 broods during this period, representing 134–192% of their body carbon. Daily egg production rates varied from 0 to 86 eggs, representing a mean of 3–4% of their body carbon d⁻¹. Egg production rates were often severely depressed during moulting, and were also somewhat lower during experimental manipulation (changing food conditions). The results suggest that egg production by E. lucens, and perhaps by many other euphausiid species, are considerably higher than was previously thought.     

Clustering of lipid-bound annexin V may explain its anticoagulant effect.

In 1985 we isolated a new vascular anticoagulant protein VAC alpha, now called annexin V, with a high binding affinity (Kd less than 10(-10) M) for phospholipids. Its anticoagulant effect was attributed to displacement of coagulation factors from the phospholipid membrane. The present study demonstrates that the inhibition of prothrombinase activity by annexin V strongly depends on the curvature of the membrane surface and on the calcium concentration. Half-maximal inhibition of prothrombinase on and binding of annexin V to small vesicles, composed of 20% phosphatidylserine and 80% phosphatidylcholine, requires 2-3 mM calcium. With large vesicles and planar bilayers considerably less calcium is required for inhibition of prothrombinase and for lipid binding. Half-maximal binding of annexin V to large vesicles and to planar bilayers occurs at 0.7 and 0.2 mM calcium, respectively. This seemingly confirms the displacement model. The displacement of coagulation factors, however, proved to be incomplete, with residual surface concentrations of factors Xa, Va, and prothrombin sufficient for effective production of thrombin. Cryoelectron microscopy revealed that annexin V binding to large vesicles caused planar facets, indicating the formation of large sheets of clustered annexin V. Apparently, the formation of these two-dimensional arrays is promoted by calcium and hampered by high surface curvature. It is speculated that the complete inhibition (greater than 99%) of prothrombinase activity by annexin V is caused by the reduced lateral mobility of prothrombin and factor Xa in rigid sheets of annexin V covering the membrane.