Kinetic analysis of blood-brain barrier transport of amino acids

The Michaelis-Menten kinetics of blood-brain barrier transport of fourteen amino acids was investigated with a tissue-sampling, single-injection technique in the anesthetized rat. Tracer quantities of ¹⁴C-labelled amino acids and ³H₂O, used as a freely diffusible internal reference, were mixed in 0.2 ml of buffered Ringer's solution and injected rapidly into a common carotid artery. Circulation was terminated by decapitation at 15 s following injection. A brain uptake index (I_b) was determined from the ratio of ¹⁴C dpm in the brain tissue and the injection mixture divided by the same ratio for the ³H₂O reference. Brain clearance of tracer concentration of amino acid was saturable when various concentrations of unlabeled amino acid were added to the injection solution. Double reciprocal plots of the saturation data yielded K_m (mM) values that ranged from a low of 0.09 mM for arginine to a high of 0.75 mM for cycloleucine. Transport V values were determined from the relationship $\text{P =}\text{V}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ where P is the blood-brain barrier permeability constant (ml/g per min): P was calculated from the I_b for each amino acid based on a cerebral blood flow of 0.56 ml/g per min and a fractional extraction of 0.75 for the ³H₂O reference 15 s following carotid injection. The V values ranged from a low of 6.2 nmol/g per min for lysine to a high of 64 nmol/g per min for l-DOPA. Efflux of the tracer amino acid during the 15-s period after injection was assumed to be slow, since the rate constant of cycloleucine from brain to blood was low, 0.11 min^?1.     

Electrophysiological evidence for the autoregulation of β-cell secretion by insulin

Electrophysiological studies of cultured rat pancreatic β-cells using intracellular microelectrodes show that exogenous insulin over the range of 0.1–10.0 μg/ml inhibits the electrical activity due to 27.8 mM glucose in a dose-related manner. This inhibitory effect is manifested by a mean increase of the membrane potential from about ?20 to ?30 mV and inhibition of the manner of cells impaled showing spike activity from 60 to less than 10%. The inhibitory influence of insulin is rapid occuring within 5 min for the highest level used. The results provide evidence for a negative feedback role of insulin in regulating its own release.     

Decreased rate of S-adenosyl-L-homocysteine metabolism: an early event related to transformation in cells infected with Rous sarcoma virus.

An increase of methylase activity is often related to neoplastic transformation. SAH, the natural inhibitor of transmethylases, does not inhibit cell transformation induced by RSV, in contrast to one of its synthetic analogues, SIBA. This inefficiency was thought to be due to the rapid metabolism of SAH by transformed cells. We now show, that, on the contrary, 70 % of the added amount of SAH disappears in one hour in cell-free extracts of normal cell against only 14 % in extracts of transformed cells. This decreased rate of degradation occurred one day post infection. Cells infected with the non transforming RAV₁ degrade SAH at the same rate as normal cells. A decrease of SAH-hydrolase and adenosine deaminase activity was also observed in infected cells. The decrease of the first enzyme seems to be related to the transformed state, whereas that of the second enzyme seems to depend only on infection, since it is also observed in cells infected with RAV₁.     

Uncoupling of mitochondrial oxidative phosphorylation by thallium.

The mechanism of integration of λ$\text{bio}$ll, which is deleted of all the known λ recombination genes, was studied using $\text{bio}$ deleted hosts as recipients. The presence of $\text{rec}$BC DNase and $\text{exo}$I in the recipient cells affected the fate of λ$\text{bio}$ll DNA. In nine of ten $\text{imm}\text{λ}{}^{\mathrm{+}}$ transductants, insertion of the λ$\text{bio}$ll genome took place somewhere between J and N and the remaining one had abnormally permuted prophage λ. In this lysogen (#42), the sequence of prophage genes was similar to that of vegetative phage λ. The properties of lysogen #42 were compared with those of other lysogens.     

Comparison of the acid denaturation of several hemoglobins which differ in amino acid sequence

There are pronounced differences in kinetic and thermodynamic stability between human and horse hemoglobins. Since the amino acid sequences of the α, β dimers of horse and human hemoglobins differ in 61 locations, it is difficult to account for them in terms of specific direct or indirect effects of the sequence differences. Rhesus hemoglobin differs from human in only 12 locations and its stability resembles that of human more closely than does horse, although pronounced differences remain. The stabilities of rhesus ferrihemoglobin and deoxyhemoglobin (Hb⁺ and Hb °) are intermediate between those of the corresponding high-spin forms of horse and human hemoglobin; but there are only small or negligible differences between the low-spin forms (carbonylhemoglobin and oxyhemoglobin) of the two species. The equilibrium isotherm between native and acid unfolded forms of rhesus Hb⁺ resembles that of horse more than that of human, but it is slightly more stable and slightly less cooperative. The effects of octanol on the rates of unfolding of rhesus ferrihemoglobin are only slightly smaller than with human. There is no effect of octanol on the unfolding rate of any of the CO hemoglobins. Unlike the equilibria of horse and human, octanol is also without effect on the unfolding equilibrium of rhesus ferrihemoglobin, and thus qualifies as a true catalyst of the initial stage of the acid unfolding reaction of the monkey ferriprotein. Differences in stability are tentatively attributed to a limited number of the 12 differences between the two proteins.     

Studies on the rat glomerular basement membrane: Age-related changes in composition

To examine the effect of age on the glomerular basement membrane, compositional analyses were performed on membranes isolated in highly purified form from rats at different stages of their growth (35 to 200 days old). Substantial age-related changes were observed in the amino acid composition of the basement membranes. A significant correlation with age (P < 0.01) was evident in the contents of 3- and 4-hydroxyproline, threonine, serine, alanine, valine, half-cystine, hydroxylysine, and lysine. Of these amino acids, hydroxylysine and both isomers of hydroxyproline demonstrated a progressive increase with age, while the others were found to decline. The direct relationship of hydroxylysine content with age (P < 0.001) was associated with an inverse correlation of lysine with age (P < 0.001) so that the ratio of hydroxylysine to lysine increased in a highly significant manner from 0.92 at 35 days to 1.33 at 200 days. This elevation in the hydroxylysine content was accompanied by an augmentation in the number of saccharide units linked to it so that the percentage glycosylation of this amino acid was not significantly affected by age. The relative differences in the hydroxylysine and lysine levels between young and older rats were maintained in sodium dodecyl sulfateextracted membranes. These results suggest that the compositional changes observed during the aging process reflect an alteration in the subunit makeup of the basement membrane, possibly due to an increased synthesis or decreased degradation of the more collagen-like polypeptide components.