Kinetics of Neutral Amino Acid Transport Across the Blood-Brain Barrier

Neutral amino acid (NAA) transport across the blood-brain barrier was examined in pentobarbital-anesthetized rats with an in situ brain perfusion technique. Fourteen of 16 plasma NAAs showed measurable affinity for the cerebrovascular NAA transport system. Values of the transport constants (Vmax, Km, KD) were determined for seven large NAAs from saturation studies, whereas Km values for five small NAAs were estimated from inhibition studies. These data, together with our previous work, provide a complete set of constants for prediction of NAA influx from plasma. Among the NAAs, Vmax varied at least fivefold and Km varied approximately 700 fold. The apparent affinity (1/Km) of each NAA was related linearly (r = 0.910) to the octanol/water partition coefficient, a measure of NAA side-chain hydrophobicity. Predicted influx values from transport constants and average plasma concentrations agree well with values measured using plasma perfusate. These results provide accurate new estimates of the kinetic constants that determine NAA transport across the blood-brain barrier. Furthermore, they suggest that affinity of a L-alpha-amino acid for the transport system is determined primarily by side-chain hydrophobicity.     

Low serum reverse T3 levels in patients with primary hyperparathyroidism

Although patients with primary hyperparathyroidism (1 degree HPT) were euthyroid, we measured serum thyroid hormone levels in 16 patients with 1 degree HPT together with 17 patients with hypercalcemia due to malignant diseases (HCM). In patients with 1 degree HPT, serum levels of T3, T4 and T3U were within normal range, but serum rT3 (reverse T3) levels (205 +/- 37 pg/ml, mean +/- SD) were significantly decreased as compared with those in normal controls (276 +/- 44 pg/ml, P less than 0.01). A significant inverse correlation was observed between the serum levels of rT3 and parathyroid hormone (PTH) (r = 0.54, P less than 0.05). After parathyroidectomy, serum rT3 levels were significantly elevated (240 +/- 56 pg/ml) compared to preoperative levels (P less than 0.01). Low levels of serum rT3 seemed to be attributed to the high levels of serum PTH. On the other hand, serum levels of T3 and T4 were low and serum rT3 levels were high in patients with HCM. Low serum rT3 allows for the differentiation of patients with 1 degree HPT from those with HCM.     

Structure and function relationship of staphylocoagulase

The bacterial protein staphylocoagulase binds stoichiometrically to human prothrombin, resulting in a coagulant complex, staphylothrombin. The enzymatic properties of staphylothrombin differ from those of -thrombin in their substrate specificities toward natural and synthetic substrates, in addition to their interaction with protease inhibitors. In order to obtain information about the region of staphylocoagulase that interacts with human prothrombin, staphylocoagulase was cleaved by -chymotrypsin. Limited -chymotryptic cleavage of staphylocoagulase yielded three large fragments, of 43, 30, and 20 kD. The 43-kD fragment exhibited a high affinity for human prothrombin (K_d=1.7 nM), which is comparable to the affinity observed using intact staphylocoagulase (K_d=0.46 nM). A complex of the 43-kD fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. The 30-kD fragment exhibited weaker affinity for prothrombin (K_d=120 nM.) While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. The 20-kD fragment was found only to bind to prothrombin. The NH₂-terminal sequence analyses of these fragments revealed that the 43-kD fragment constitutes the NH₂-terminal portion of staphylocoagulase, and contains the 30-kD and 20-kD fragments. It is therefore concluded that the functional region of staphylocoagulase for binding and activation of prothrombin is localized in the NH₂-terminal region of the intact protein. The 43-kD fragment contained 324 amino acids with a molecular weight of 38,098. The 43-kD fragment had an unusual amino acid composition based on a sequence in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounted for more than 45% of the total. A comparison of the amino acid sequence of the 43-kD fragment with that of streptokinase did not reveal any obvious sequence homology. There was also no sequence homology with that of trypsin, -chymotrypsin, and elastase.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Effect of calcium overload on the phosphoinositide breakdown in the rat left ventricular papillary muscle

We investigated the effect of Ca²⁺ overload on the phospholipase C-catalyzed hydrolysis of phosphoinositides in the rat left ventricular papillary muscle. Ca²⁺ overload on the papillary muscle was induced by treatment with 0.3 mM ouabain in Ca²⁺-containing medium following either Ca²⁺-containing or Ca²⁺-free superfusion. The phosphoinositide breakdown was evaluated by determining accumulations of [³H]inositol phosphates ([³H]IPs) in the tissues prelabeled with [³H]inositol. Ca²⁺ repletion following Ca²⁺-free superfusion resulted in a rapid but small increase in resting tension that was not followed by contracture, nor was it associated with a significant increase in [³H]IPs accumulations. Treatment with ouabain following Ca²⁺-containing superfusion increased resting tension after a lag period of several minutes and produced contracture associated with an increase in [³H]IPs accumulations. The ouabain induced increases in resting tension, and accumulations of [³H]IPs were significantly potentiated by prior Ca²⁺-free superfusion instead of Ca²⁺-containing superfusion. There was a significant positive correlation between increases in resting tension and the phosphoinositide breakdown. The increased resting tension and the accumulations of [³H]IPs were not antagonized by treatments with prazosin plus atropine or indomethacin, but were abolished by superfusion with Ca²⁺-free buffer solution. Although the enhanced phospholipase C-catalyzed hydrolysis of phosphoinositides appears to be a consequence rather than a cause of increased intracellular Ca²⁺, such a biochemical change may provoke a positive feedback mechanism to develop the muscle contracture through the putative intracellular messenger action of inositol triphosphate and diacylglycerol.Abbreviations [³H]IPs [³H]Inositol Phosphates - IP Inositol Phosphate - IP₂ Inositol Bisphosphate - IP₃ Inositol Trisphosphate - PI Phosphatidylinositol - PI-4-P Phosphatidylinositol-4-phosphate - PI-4,5-P₂ Phosphatidylinositol 4,5-bisphosphate - PRZ Prazosin - ATR Atropine - INDO Indomethacin - min Minutes     

Low density lipoprotein and apoprotein B induce increases in inositol trisphosphate and cytosolic free Ca2+ via pertussis toxin-sensitive GTP-binding protein in vascular smooth muscle cells

Low density lipoprotein (LDL), a major cholesterol-carrying lipoprotein in the plasma, binds to its receptor through apoprotein B (Apo-B). The addition of LDL and Apo-B induced rapid (5 s), but transient increase in the inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) level with K0.5 values of 1.1 and 0.07 microgram/ml, accompanied by increases of cytosolic free Ca2+ concentration [( Ca2+]i), in vascular smooth muscle cells (VSMC). The increases by LDL and Apo-B were both reduced by pretreatment of the VSMC with pertussis toxin. The early change in Ins-1,4,5-P3 involving a GTP-binding protein may function as an initial signal for the action of LDL in VSMC.     

Primary structure of an N-linked sugar chain derived from glucoamylase of Rhizopus niveus

The primary structure of the N-linked sugar chain of Rhizopus niveus glucoamylase (major component) was investigated. The carbohydrate moiety was released from the polypeptide backbone by Flavobacterium sp. endo-beta-N-acetylglucosaminidase digestion. Studies using the method of exoglycosidase digestion of the fluorescent pyridylamino derivative, gel-permeation chromatography on Bio-Gel P-4 and 400-MHz 1H-NMR spectroscopy revealed that the most abundant structure is (Man)8-GlcNac-ol.     

A complete deletion mutant of the Escherichia coli dnaKdnaJ operon

Southern hydridization analyses of genomic DNAs from various dnaJ mutants of Escherichia coli showed that mutant K7052, which has well characterized dnaK706 and dnaJ705 double mutantions, is a deletion mutant. The deletion is about 8.0 kb long and encompasses the whole of the dnaKdnaJ operon.     

Plasmid expression and maintenance during long-term starvation-survival of bacteria in well water.

Strains of enteric bacteria and pseudomonads containing plasmid R388::Tnl721 (Tpr, Tcr) or pRO101 (Hgr, Tcr) were starved for over 250 days in sterile well water to evaluate effects of starvation-survival on plasmid expression and maintenance. Viable populations dropped to between approximately 0.1 and 1% of the initial populations. Escherichia coli(pRO101) and Pseudomonas cepacia(pRO101) lost both viability and plasmid expression at a lower rate than strains containing R388::Tnl721. Three patterns of host-plasmid interaction were detected: (i) no apparent loss of plasmid expression, (ii) loss of plasmid expression on initial recovery with subsequent expression upon resuscitation, and (iii) loss of capability to produce functional plasmid resistance.     

Effect of Growth Rate and Starvation-Survival on the Viability and Stability of a Psychrophilic Marine Bacterium

Cell populations of the marine bacterium ANT-300, from either batch or continuous culture with dilution rates ranging from D = 0.015 h⁻¹ to D = 0.200 h⁻¹, were monitored for viability, direct counts, and optical density for 98 days under starvation conditions. Three stages of starvation survival were observed for each of the cell populations. Although direct counts remained at 2 × 10⁷ to 3 × 10⁷ cells ml⁻¹ throughout the starvation period, large fluctuations occurred in cell viability during stage 1 (0 to 14 days) of starvation survival. Stage 2 (14 to 70 days) involved an overall decrease in viability for each of the cell populations; the rate of viability loss was dependent upon the growth rate. Cell viability stabilized at approximately 0.3% of the direct count in stage 3 (70 to 98 days). Long-term starvation corresponded to the prolongation of stage 3 starvation survival. Cell volumes for each of the cell populations decreased with the length of the starvation period. However, the cell volume of starved cells was also dependent more on growth rate than on the length of the time starved. We hypothesize that the cell population with the slowest growth rate is most closely representative of cells found in the oligotrophic marine environment.     

Transient expression of the β-glucuronidase gene introduced into barley coleoptile cells by microinjection

A -glucuronidase gene was introduced directly into barley (Hordeum vulgare L. cv. Kobinkatagi) coleoptile cells by microinjection and transient expression of the gene was examined. Inner epidermis tissue of coleoptiles was excised and injected with plasmid DNA, pBI221, carrying cauliflower mosaic virus 35S promoter, -glucuronidase gene, and a nopaline synthase polyadenylation region. Histochemical assay for -glucuronidase production showed positive enzyme activity only in coleoptile cells injected with plasmid DNA. Expression of the -glucuronidase gene was examined chronologically using honogenates of injected coleoptile tissues. Glucuronidase activity first appeared after 6 hr, reached the maximum level 24 hr after injection, and decreased afterwards. These results suggest that microinjection of coleoptile tissues may be a useful approach for the genetic engineering of Gramineae plants in which protoplast regeneration is difficult.