An immunoelectron-microscopical observation of mouse juxtaglomerular cells in the case of experimental hydronephrosis.

Changes in juxtaglomerular (JG; renin-containing) cells in experimental hydronephrosis 1 month after ureteral ligation were investigated with immunoelectron-microscopical techniques. Two types of granules, electron dense (D) and lucent (L), were observed. D type granules were labeled more intensely with gold particles than those of L type. Granules intermediate between D and L types and exocytosis of D types were observed. In the cells containing D types exclusively, gold particles were restricted to the granules, whereas in the cells containing both D and L type granules, the particles were scattered throughout the cytoplasmic cytosol. The authors discuss the mechanisms of renin release in JG cells.     

A molecular orbital study of the metabolic pathway with hydroquinone intermediate from benzene as carcinogen

A possible metabolic activation pathway of benzenein vivo is the nonenzymatic oxidation of hydroquinone producedvia the cytochrome P-450-mediate two-step oxidation of benzene. The mechanism of the further oxidation of hydroquinone and the nature of the most reactive intermediate have not yet been clarified, although it is speculated that the intermediate isp-benzoquinone and/orp-benzosemiquinone. The theoretical result of using molecular orbital calculations (ab initio and CNDO/2 methods) indicates that although the mechanism of the nonenzymatic oxidation of hydroquinone cannot yet be determined, the intermediate is thep-benzosemiquinone anion radical. It is also suggested that active-oxygen species such as hydroxyl radical, which accelerates the nonenzymatic oxidation, play an important role in the metabolic pathway in question.     

Phenylalanine Transport Across the Blood-Brain Barrier as Studied with the In Situ Brain Perfusion Technique

Unidirectional L-phenylalanine transport into six brain regions of pentobarbital-anesthetized rats was studied using the in situ brain perfusion technique. This technique allows both accurate measurements of cerebrovascular amino acid transport and complete control of perfusate amino acid composition. L-Phenylalanine influx into the brain was sodium independent and could be described by a model with a saturable and a nonsaturable component. Best-fit values for the kinetic constants in the parietal cortex equaled 6.9 X 10(-4) mumol/s/g for Vmax, 0.011 mumol/ml for Km, and 1.8 X 10(-4) ml/s/g for KD during perfusion with fluid that did not contain competing amino acids. D-Phenylalanine competitively inhibited L-phenylalanine transport with a Ki approximately 10-fold greater than the Km for L-phenylalanine. There were no significant regional differences in Km, KD, or Ki, whereas Vmax was significantly greater in the cortical lobes than in the other brain regions. L-Phenylalanine influx during plasma perfusion was only 30% of that predicted in the absence of competing amino acids. Competitive inhibition increased the apparent Km during plasma perfusion by approximately 20-fold, to 0.21 mumol/ml. These data provide accurate new estimates of the kinetic constants that describe L-phenylalanine transport across the blood-brain barrier. In addition, they indicate that the cerebrovascular transfer site affinity (1/Km) for L-phenylalanine is three- to 12-fold greater than previously estimated in either awake or anesthetized animals.     

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.     

Changes of erythrocyte deformability induced by calcium accumulation and calmodulin inhibitors

The deformability of human erythrocytes was investigated with a rheoscope to study the role of intracellular calcium in the dynamic cytoskeletal structure. Calcium was loaded to or depleted from erythrocytes with a calcium ionophore (A 23187) in a Na- or a K-HEPES buffer. (1) After calcium loading in the Na-HEPES buffer, the cell volume of erythrocytes was greatly reduced due to dehydration. On the contrary, upon calcium-loading or -depletion in the K-HEPES buffer, the intracellular calcium content could be varied in the range of 1/4 to 3 times as much as that of control cells without the reduction of mean cell volume. Further incubation without A 23187 and calcium in the K-HEPES buffer enabled the calcium-loaded erythrocytes to restore the cell shape and the ATP concentration. (2) When intracellular calcium content was increased to above 1.5 times of the normal value, the deformability was distinctly decreased. On the other hand, the deformability was unchanged when the intracellular calcium content was reduced below the normal level. (3) The deformability, once decreased due to the calcium accumulation, was recovered by the treatment with a calmodulin inhibitor, W-7 or trifluoperazine, while these drugs were not effective on the deformability of control or calcium-depleted erythrocytes. We conclude that the membrane stiffness which influence the deformability of erythrocytes, is modulated by the intracellular calcium content through the interaction between the calcium-calmodulin complex and the cytoskeletal proteins.     

Chromatic Regulation of Photosystem Composition in the Cyanobacterial Photosynthetic System: Kinetic Relationship between Change of Photosystem Composition and Cell Proliferation

Kinetics of the change of photosystem (PS) composition in cyanobacteriainduced by chromatic light were studied in relation to cellproliferation. The study was made for two unicellular strains,Synechococcus NIBB 1059 and Synechocystis (Aphanocapsa) PCC6714. We found that (1) the change to a higher or lower PS I/IIratio was due to acceleration or suppression of apparent PSI formation, and (2) it progressed on a similar time scale tothat of the cell proliferation. The apparent rate constant ofthe change in the PS I/II ratio was proportional to that ofcell proliferation, µ, when this was low, but at highvalues of µ the increase in the rate constant of the changein the PS I/II ratio became smaller, causing a deviation fromthe linear relationship. Results indicate that under autotrophicconditions, the photoregulated composition change occurs asa result of thylakoid development, which accompanies cell proliferation. (Received June 23, 1986; Accepted December 5, 1986)     

Characterization of revertants derived from a mouse DNA temperature-sensitive mutant strain, tsFT20, which contains heat-labile DNA polymerase alpha activity

One spontaneous and four N-methyl-N'-nitro-N-nitrosoguanidine-induced revertants of a mouse FM3A mutant, tsTF20, which has heat-labile DNA polymerase alpha activity and cannot grow at 39 degrees C, were isolated and characterized with respect to the thermolability of their DNA polymerase alpha activity, the intracellular level of enzyme activity, growth rate, cell cycle progression, and frequency of initiation of DNA replication at the origin of replicons. DNA polymerase alpha activity in the extracts from the revertant cells showed partial recovery of heat stability. The intracellular level of enzyme activity of the revertant cells was lower than that of wild-type cells even at 33 degrees C. The level of enzyme activity in the revertant cells decreased considerably after a temperature upshift to 39 degrees C, but the DNA synthesizing ability of these cells did not decrease as much as the level of enzyme activity. The growth rates of the wild-type and revertant lines were almost the same at 33 degrees C. At 39 degrees C, the rate for the wild-type increased considerably compared to that at 33 degrees C, while little difference in the growth rates of the revertant lines was observed at the two temperatures. Therefore, the doubling times of the revertant cells were relatively increased compared to those of wild-type cells cultured at the restrictive temperature. Flow microfluorometric analysis and cell cycle analysis to measure labeled mitosis revealed that the increase in the doubling time was due mainly to the increase in the duration of the S phase. Analysis of the center-to-center distance between replicons by DNA fiber autoradiography indicated that the frequency of replicon initiation per unit length DNA at a given time was reduced in the revertant cells growing at 39 degrees C.     

Requirement of the Escherichia coli dnaA gene function for ori-2-dependent mini-F plasmid replication.

The mini-F plasmids pSC138, pKP1013, and pKV513 were unable to transform Escherichia coli cells with a dnaA-defective mutation under nonpermissive conditions. The dnaA defect was suppressed for host chromosome replication either by the simultaneous presence of the rnh-199 (amber) mutation or by prophage P2 sig5 integrated at the attP2II locus on the chromosome, both providing new origins for replication independent of dnaA function. The dnaA mutations tested were dnaA17, dnaA5, and dnaA46. dnaA5 and dnaA46 are missense mutations. dnaA17 is an amber mutation whose activity is controlled by the temperature-sensitive amber suppressor supF6. Under permissive conditions in which active DnaA protein was available, the mini-F plasmids efficiently transformed the cells. However, the transformants lost the plasmid as the cells multiplied under conditions in which DnaA protein was inactivated or its synthesis was arrested. As controls, plasmids pSC101 and pBR322 were examined along with mini-F; pSC101 behaved in the same manner as mini-F, showing complete dependence on dnaA for stable maintenance, whereas pBR322 was indifferent to the dnaA defect. Thus, ori-2-dependent mini-F plasmid replication seems to require active dnaA gene function. This notion was strengthened by the results of deletion analysis which revealed that integrity of at least one of the two DnaA boxes present as a tandem repeat in ori-2 was required for the origin activity of mini-F replication.