Transcapillary escape rate of albumin in humans during exercise-induced hypervolemia

Haskell, Andrew, Ethan R. Nadel, Nina S. Stachenfeld, KeiNagashima, and Gary W. Mack. Transcapillary escape rate of albuminin humans during exercise-induced hypervolemia. J. Appl. Physiol. 83(2): 407-413, 1997.To test thehypotheses that plasma volume (PV) expansion 24 h after intenseexercise is associated with reduced transcapillary escape rate ofalbumin (TER_alb) and that localchanges in transcapillary forces in the previously active tissues favorretention of protein in the vascular space, we measured PV,TER_alb, plasma colloid osmoticpressure (COP_p), interstitialfluid hydrostatic pressure (Pi), and colloid osmotic pressure in legmuscle and skin and capillary filtration coefficient (CFC) in the armand leg in seven men and women before and 24 h after intense uprightcycle ergometer exercise. Exercise expanded PV by 6.4% at 24 h (43.9 ± 0.8 to 46.8 ± 1.2 ml/kg, P < 0.05) and decreased total protein concentration (6.5 ± 0.1 to6.3 ± 0.1 g/dl, P < 0.05) andCOP_p (26.1 ± 0.8 to 24.3 ± 0.9 mmHg, P < 0.05), although plasmaalbumin concentration was unchanged. TER_alb tended to decline (8.4 ± 0.5 to 6.5 ± 0.7%/h, P = 0.11) and was correlated with the increase in PV(r = 0.69,P < 0.05). CFC increased in the leg(3.2 ± 0.2 to 4.3 ± 0.5 µl · 100 g¹ · min¹ · mmHg¹,P < 0.05), and Pi showed a trend toincrease in the leg muscle (2.8 ± 0.7 to 3.8 ± 0.3 mmHg, P = 0.08). These datademonstrate that TER_alb isassociated with PV regulation and that local transcapillary forcesin the leg muscle may favor retention of albumin in the vascular spaceafter exercise.

     

Mutations in the SLC3A1 Transporter Gene in Cystinuria

Cystinuria is an autosomal recessive disease characterized by the development of kidney stones. Guided by the identification of the SLC3A1 amino acid–transport gene on chromosome 2, we recently established genetic linkage of cystinuria to chromosome 2p in 17 families, without evidence for locus heterogeneity. Other authors have independently identified missense mutations in SLC3A1 in cystinuria patients. In this report we describe four additional cystinuria-associated mutations in this gene: a frameshift, a deletion, a transversion inducing a critical amino acid change, and a nonsense mutation. The latter stop codon was found in all of eight Ashkenazi Jewish carrier chromosomes examined. This report brings the number of disease-associated mutations in this gene to 10. We also assess the frequency of these mutations in our 17 cystinuria families.     

Genetic and functional evidence that Type IV pili are required for social gliding motility in Myxococcus xanthus

The social gliding behaviour of Myxococcus xanthus has previously been associated with the presence of polar pili. A Tn 5 transposon insertion was isolated which introduces a defect in social gliding and is genetically linked to a known sgl locus; this insertion was found also to cause a piliation defect. A 2.7 kb section of DNA was isolated from either side of this transposon and sequenced, revealing three genes which encode amino acid sequences with substantial similarity to components of the Type IV pilus biogenesis pathway in Pseudomonas aeruginosa . The myxococcal pilA gene encodes a putative pilin precursor with a short signal sequence and processing site similar to those of other Type IV pilins. Myxococcal pilS and pilR encode amino acid sequences with similarity to PilS and PilR of P. aeruginosa , as well as to other members of the NtrB/C family of two-component regulators. Mutations within pilR and pilA that have no polar effect were demonstrated to be responsible for pilus and social motility defects. These results indicate that the pili of M. xanthus belong to the Type IV family of pili, and demonstrate that these pili are actually required for social motility.     

Increased α1 receptor density in brown adipose tissue of cafeteria-fed rats

A possible general corollary between ₁-receptor density in brown adipose tissue and the degree of activation of the tissue was investigated. For this purpose, the effect of cafeteria feeding on ₁-adrenergic receptors in brown adipose tissue of seven-week-old female rats was studied by the use of the ₁-antagonist (³H)prazosin. In cafeteria-fed rats, the K_D of the ₁-receptor for (³H)prazosin was unchanged (about 0.35 nM), but the receptor density was doubled (up to 40 fmol per mg of membrane protein). This was also observed when the results were expressed per unit of a plasma-membrane marker (5-nucleotidase). It was concluded that an increased ₁-receptor density is seen not only in cold-acclimated rats, but also in other conditions where brown fat is activated, and a possible general physiological significance of ₁-adrenergic pathways in brown adipose tissue is discussed.     

Human liver alcohol dehydrogenase. 2. The primary structure of the gamma 1 protein chain

The primary structure of the gamma 1 subunit of human liver alcohol dehydrogenase isoenzyme gamma 1 gamma 1 was deduced by characterization of 36 tryptic and 2 CNBr peptides. The polypeptide chain is composed of 373 amino acid residues. gamma 1 differs from the beta 1 subunit of human liver alcohol dehydrogenase at 21 positions, and from the E subunit of horse liver alcohol dehydrogenase at 43 positions including a gap at position 128 as in the beta 1 subunit. All zinc-liganding residues from the E subunit of the horse protein and the beta 1 subunit of the human enzyme are conserved, but like beta 1, gamma 1 also has an additional cysteine residue at position 286 (in the positional numbering system of the horse enzyme) due to a Tyr----Cys exchange. Most amino acid exchanges preserve the properties of the residues affected and are largely located on the surface of the molecules, away from the active site and the coenzyme binding region. However, eight positions with charge differences in relation to the E subunit of the horse enzyme are noticed. These result in a net positive charge increase of one in gamma 1 versus E, explaining the electrophoretic mobilities on starch gels. Of functional significance is the conservation of Ser-48 in gamma 1 relative to E. The residue is close to the active site but different (Thr-48) in the beta 1 subunit of the human enzyme. Thus, the closer structural relationship between human gamma 1 and horse E enzyme subunit than between beta 1 and E is also reflected in functionally important residues, explaining a greater similarity between gamma 1 gamma 1 and EE than between beta 1 beta 1 and EE.     

NMR studies of the mechanism of cyclic AMP-dependent protein kinase

NMR has been used to study the role of the divalent cation, the conformations, arrangement, and exchange rates of the enzyme-bound metal-ATP and peptide substrates, the mechanism of the phosphoryl transfer, and the structure and role of the regulatory subunit on type II cyclic AMP (cAMP)-dependent protein kinase from bovine heart. The active complex consists of an enzyme-ATP-metal bridge in which the metal is beta, gamma coordinated, with delta chirality at P beta, and a torsional angle at the adenine-ribose bond in the high-anti range (x approximately 80 degrees). The bound heptapeptide substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly is extended in conformation, forming either a coil or, less likely, a beta turn but not an alpha helix or beta sheet. The distance from the gamma-P of bound ATP analogs to the Ser-OH of the bound peptide (5.3 +/- 0.7 A) would permit a metaphosphate or an elongated phosphorane intermediate or transition state. The regulatory subunit (R2) blocks the peptide- or protein-binding site of the catalytic subunit. The 31P chemical shift of cAMP is not greatly altered on binding to R2, but the resonance is broadened to approximately 32 Hz, which indicates no chemical change but marked immobilization of bound cAMP. A narrower (approximately 7 Hz) 31P resonance at 4.44 ppm is assigned to P-serine-95 of R2 because it disappears with catalytic subunit, Mg2+, and an ADP-generating system.     

The biology ofChlamydomyxa montana: Ultrastructure of the cyst

Summary Cells ofChlamydomyxa montana Lankester photosynthesize within a cyst under bright light and ingest diatoms in an excysted amoeboid form during periods of low light intensity. Cyst cell walls are partially comprised of cellulose and vary in thickness. The cells are multinucleate and intracellular bacteria are closely associated with the nuclei. A pair of centrioles is also present adjacent to individual nuclei. Chloroplasts are numerous and thylakoids are generally organized into bands of three. No endoplasmic reticulum was found surrounding the plastids, nor was a complete girdling lamella ever observed within.C. montana chloroplasts appear to be its own but this protist does not precisely fit into an algal class.     

Sucrose transport into vacuoles isolated from barley mesophyll protoplasts

Sucrose transport has been investigated in vacuoles isolated from barley mesophyll protoplasts. Rates of sucrose transfer across the tonoplast were even higher in vitro than in vivo indicating that the sucrose transport system had not suffered damage during isolation of the vacuoles. Sucrose transport is carrier-mediated as shown by substrate saturation of transport and sensitivity to a metabolic inhibitor and to competitive substrates. A number of sugars, in particular maltose and raffinose, decreased uptake of sucrose. Sorbitol was slowly taken up but had no effect on sucrose transport. The SH-reagent p-chloromercuribenzene sulfonate inhibited sucrose uptake completely. The apparent K_m of the carrier for sucrose uptake was 21 mM. Transport was neither influenced by ATP and pyrophosphate, with or without Mg²⁺ present, nor by protonophores and valinomycin (with K⁺ present). Apparently uptake was not energy dependent. Efflux experiments with preloaded vacuoles indicated that sucrose unloading from the isolated vavuoles is mediated by the same carrier which catalyses uptake. The vacuole of mesophyll cells appears to represent an intermediary storage compartment. Uptake of photosynthetic products into the vacuole during the light apparently minimizes osmotic swelling of the small cytosolic compartment of vacuolated leaf cells when photosynthetic productivity exceeds the capacity of the phloem for translocation of sugars.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazincethane-sulfonic acid - pCMBS p-chloromercuribenzene sulfonate Dedicated to Professor Dr. W. Simonis on the occasion of his 75th birthday     

Effect of Carbon Source and the Role of Cyclic Adenosine 3′,5′-Monophosphate on the Caulobacter Cell Cycle

The expression of cell cycle events in Caulobacter crescentus CB13 has been shown to be associated with regulation of carbohydrate utilization. Growth on lactose and galactose depends on induction of specific enzymes. Prior growth on glucose results in a delay in enzyme expression and cell cycle arrest at the nonmotile, predivisional stage. Dibutyryl cyclic adenosine 3',5'-monophosphate (AMP) was shown to stimulate expression of the inducible enzymes and, thus, the initiation of the cell cycle. beta-Galactosidase-constitutive mutants did not exhibit a cell cycle arrest upon transfer of cultures from glucose to lactose. Furthermore, carbon source starvation results in accumulation of the cells at the predivisional stage. The cell cycle arrest therefore results from nutritional deprivation and is analogous to the general control system exhibited by yeast (Hartwell, Bacteriol. Rev. 38:164-198, 1974; Wolfner et al., J. Mol. Biol. 96:273-290, 1975), which coordinates cell cycle initiation with metabolic state. Transfer of C. crescentus CB13 from glucose to mannose did not result in a cell cycle arrest, and it was demonstrated that this carbon source is metabolized by constitutive enzymes. Growth on mannose, however, is stimulated by exogenous dibutyryl cyclic AMP without a concomitant increase in the specific activity of the mannose catabolic enzymes. The effect of cyclic AMP on growth on sugars metabolized by inducible enzymes, as well as on sugars metabolized by constitutive enzymes, may represent a regulatory system common to both types of sugar utilization, since they share features that differ from glucose utilization, namely, temperature-sensitive growth and low intracellular concentrations of cyclic guanosine 3',5'-monophosphate.     

Ionic inhibition of catalytic phosphorylation of histone by bovine brain protein kinase.

The effects of various ions commonly found in protein kinase assays upon the rate of histone phosphorylation catalyzed by the highly purified bovine brain enzyme, protein kinase I, have been investigated. Sodium, potassium, and magnesium were found to inhibit histone phosphorylation by protein kinase I in a similar manner. The degree of inhibition by any of these cations was demonstrated to be directly proportional to the square root of the ionic strength of the assay medium. The relationship between the ionic strength of the assay medium and the rate of histone phosphorylation catalyzed by protein kinase I was employed to correct the rate of histone phosphorylation at various magnesium acetate concentrations to a standard ionic strength. When this was done an analysis of the previously postulated rate law for histone phosphorylation c atalyzed by protein kinase I gave a binding constant for the magnesium-ATP complex which was in agreement with that expected for this complex on the basis of various binding constants available in the literature. These results demonstrate that it is unnecessary to postulate a specific ion inhibition process for protein kinase I by the ions employed in this study. They also support the reasonable assumption that magnesium ion binds to ATP at or prior to the rate-determining step in histone phosphorylation catalyzed by protein kinase I. The expression developed in this paper for the effect of ionic strength upon protein kinase I activity can now be used to correct activity measurements made under various assay conditions to a standard assay state, allowing facile comparisons of kinetic data. It should be possible to develop similar expressions for other protein kinases and substrates to permit useful interpretation of kinetic data.