Two-phase stopped-flow measurement of the protonation of tetraphenylporphyrin at the liquid-liquid interface.

The formation rate of the protonated form of tetraphenylporphyrin (TPP) in a dispersed two-phase system composed of dodecane and aqueous trichloroacetic acid (TCA) was studied by means of a stopped-flow method. The protonation reaction took place at the liquid-liquid interface, and the diprotonated TPP (H(2)TPP(2+)) formed was adsorbed there. In order to determine the rate-determining process, changes in absorbance at the absorption maximum wavelengths of TPP and H(2)TPP(2+) were analyzed. The obtained rate constant for the decrease of TPP in the organic phase, 21 ± 2 s(-1), was in agreement with that for the increase of diprotonated TPP at the interface, 20 ± 3 s(-1). The observed rate constants did not show any dependence on concentrations of both TPP and the acid. The experimental results suggested the rate-determining step to be the molecular diffusion process of TPP in the stagnant layer in the organic phase side at the liquid-liquid interface, and the thickness of the stagnant layer was estimated as 1.4 × 10(-4) cm.

Maturation of major Drosophila rhodopsin, ninaE, requires chromophore 3-hydroxyretinal.

Opsin expression is extremely suppressed by carotenoid deprivation in Drosophila. Carotenoid replacement in deprived flies promotes the recovery of visual pigment with an increase in opsin, as well as the chromophore 11-cis-3-hydroxyretinal. Here, we show that opsin mRNA and opsin peptide in an intermediate step of posttranslational processing were present in carotenoid-deprived flies. By supplementing chromophore to photoreceptor cells, intermediate opsin was made mature. During this process, opsin peptide underwent multiple modifications involving glycosylation. Based on these results, we present a novel mechanism of protein regulatory expression; that is, chromophore posttranslationally controls the expression of apoprotein by promoting its maturation.

Criterion validity of the cross-cultural cognitive examination in Japan.

Criterion validity of a two-stage Cross-Cultural Cognitive Examination (CCCE) designed for epidemiologic use was evaluated in Japanese subjects by comparison with a physician's DSM-III-R diagnosis of dementia and the Hasegawa Dementia Rating Scale (the standard Japanese instrument similar to the Mini-Mental State Exam). We report on 188 subjects tested in three locations in Japan: Tokyo area, Ise, and Osaka. Subjects ranged in age (50-93 years) and education (1-22 years) and included neurology outpatients, community volunteers, and inpatients. The CCCE was 97.4% specific for dementia, with sensitivity of 88%. The correlation with the Hasegawa scale was significant (r(175) = .8230, p less than .0001). Diagnosis using the CCCE showed good validity when compared with Japanese criteria for dementia. If the instrument could be shown to be reliable and more "culture fair" than the currently available tests, it may be useful in cross-cultural epidemiologic studies of dementia.

Granuloma caused by oxidized cellulose following craniotomy.

Three cases are reported in which large granulomas developed 13 to 21 months respectively after removal of intracranial meningiomas (2 cases) and after operation for an anterior communicating artery aneurysm. In each of the cases oxidized cellulose had been used and left in place for haemostasis. All of the granulomas acted as space-occupying lesions. They could be removed operatively with good result. All of them consisted of mononuclear phagocytic and multi-nuclear giant cells and contained remnants of oxidized cellulose. No comparable cases have been reported in the literature. Diagnosis, treatment and possible aetiological factors are discussed.

Construction of a plasmid vector for the regulatable high level expression of eukaryotic genes in Escherichia coli: an application to overproduction of chicken lysozyme.

A novel expression vector pKP1500 for synthesizing unfused protein in Escherichia coli was constructed. pKP1500 perserves the tac promoter, the lacZ SD sequence, unique restriction sites (EcoRI, SmaI, BamHI, SalI, PstI and HindIII) and the rrnB terminators of pKK223-3, but the replication origin is replaced with that of pUC9. Construction of this plasmid is based upon the observation that the copy number control of pUC9 is temperature dependent. At 28 degrees C, the copy number of pKP1500 is less than 25 per chromosome, approximately the same copy number as that of pKK223-3, which contains the replication origin of pBR322, whereas at 42 degrees C, the copy number increases about 10 times and reaches up to 230 copies per chromosome. The main advantage of this system is that the temperature-dependent copy control and regulatable expression of the tac promoter make cells carrying pKP1500 derivatives stable against selective pressure by detrimental overproduction of foreign proteins at a low temperature and permits high expression of cloned DNAs at a high temperature. When chicken lysozyme cDNA carrying the initiation codon (ATG) immediately upstream from the Lys1 codon was inserted downstream from the tac promoter and the SD sequence, the pKP1500 derivative produced lysozyme at about 25% of the total cellular proteins. This value is more than 10 times higher than that obtained with the pKK223-3 derivative carrying the same lysozyme cDNA. By comparison, the expression of eukaryotic genes from the tac promoter reported by others has usually been less than a few % of the total cellular protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Point mutation of alanine (31) to valine prohibits the folding of reduced lysozyme by sulfhydryl-disulfide interchange.

In the preceding paper in this issue, we described the overproduction of one mutant chicken lysozyme in Escherichia coli. Since this lysozyme contained two amino acid substitutions (Ala31----Val and Asn106----Ser) in addition to an extra methionine residue at the NH2-terminus, the substituted amino acid residues were converted back to the original ones by means of oligonucleotide-directed site-specific mutagenesis and in vitro recombination. Thus, four kinds of chicken lysozyme [Met-1Val31Ser106-, Met-1Ser106-, Met-1Val31- and Met-1 (wild type)] were expressed in E. coli. From the results of folding experiments of the reduced lysozymes by sulfhydryl-disulfide interchange at pH 8.0 and 38 degrees C, followed by the specific activity measurements of the folded enzymes, the following conclusions can be drawn: (i) an extra methionine residue at the NH2-terminus reduces the folding rate but does not affect the lysozyme activity of the folded enzyme; (ii) the substitution of Asn106 by Ser decreases the activity to 58% of that of intact native lysozyme without changing the folding rate; and (iii) the substitution of Ala31 Val prohibits the correct folding of lysozyme. Since the wild type enzyme (Met-1-lysozyme) was activated in vitro without loss of specific activity, the systems described in this study (mutagenesis, overproduction, purification and folding of inactive mutant lysozymes) may be useful in the study of folding pathways, expression of biological activity and stability of lysozyme.

Immunohistochemical demonstration of substance P-containing nerve fibres in glomus tumours.

Substance P (SP), S-100 protein, methionine-enkephalin, serotonin and myelin basic protein were studied in two solitary glomus tumours of the skin by peroxidase-antiperoxidase immunohistochemistry. Multiple SP-containing nerve fibres were distributed in the parenchyma of the tumour among proliferating glomus cells, and in the oedematous stroma of the tumour. Positive staining for myelin basic protein was detected in nerve fascicles in the capsule of the tumour, but not within the glomus tumour. S-100 protein immunoreactivity was found in nerve fascicles in the capsule of the tumour, and in addition, a few cells positive for S-100 protein were scattered throughout the stroma of the tumour. No positive staining for methionine-enkephalin and serotonin was found. The present finding may explain the clinical experience that the tumour is tender and can cause severe paroxysmal pain, because SP is known to be a primary sensory afferent neurotransmitter for mediating nociception. A possible role of SP for vasodilation in the glomus tumour is also discussed.

[Concentration of antibiotics (cefoperazone) in human brain, brain tumor tissue and cerebrospinal fluid].

The transfer of cefoperazone (CPZ) into cerebrospinal fluid (CSF), brain or brain tumor tissue was studied in 13 cases with brain tumor, chronic subdural hematoma and benign intracranial hypertension in 1982. The peak values of CPZ in serum came up immediately after its rapid intravenous administration and then decreased exponentially. The concentration of CPZ in CSF started to increase with a long delay of about 60 min. The average peak level in CSF remained 21.6 micrograms/ml and corresponded to 10.3% of the peak level in serum. The best transfer of chloramphenicol into CSF has been reported, while that of CPZ would be one of the next. The CPZ levels in CSF showed a slower decay than in serum. The concentration of CPZ in brain reached the peak level in less than 30 min and the average peak level was 36.5 micrograms/g cerebral tissue. The brain to blood rate of the CPZ concentration was 11.1%. The CPZ levels in the brain showed a rapid decrease like the transition of antibiotic levels in serum. The antibiotic levels in brain tumors were divided into two groups. The one showed sharp peak about one tenth of the values in serum. The other was of a slowly increasing type.

Molybdenum independence of nitrogenase component synthesis in the non-heterocystous cyanobacterium Plectonema.

The cyanobacterium Plectonema boryanum (IU 594-UTEX 594) fixes N2 only in the absence of combined N and of O2. We induced nitrogenase by transfer to anaerobic N-free medium and studied the effect of Mo starvation on nitrogenase activity and synthesis. Activity was first detected within 3 h after transfer by the acetylene reduction assay in controls, increasing for at least 25 h. Cells grown on nitrate and Mo and then transferred to N-free, Mo-free medium produced 8% of the control nitrogenase activity. Addition of W to the Mo-free medium reduced the activity to 0.5%. Under both Mo starvation conditions, nitrogenase protein components were synthesized. Component II of the cyanobacterial enzyme was detected by in vitro complementation with Mo-containing component I from Klebsiella pneumoniae or Azotobacter vinelandii but not Clostridium pasteurianum. Component I activity was restored by addition of Mo to cultures in which new enzyme synthesis was blocked by chloramphenicol. Acidified extracts of Plectonema induced in Mo-containing medium contained the Fe-Mo cofactor required to activate extracts of the Azotobacter mutant UW45 in vitro, but they did not activate extracts of Mo-starved Plectonema. Analysis of 35SO4(2-)-labeled proteins by polyacrylamide gel electrophoresis suggested that Mo is required for the conversion of a high-molecular-weight precursor to component I in Plectonema.

Activation of inactive nitrogenase by acid-treated component I.

When Azotobacter vinelandii was derepressed for nitrogenase synthesis in a N-free medium containing tungstate instead of molybdate, an inactive component I was synthesized. Although this inactive component I could be activated in vivo upon addition of molybdate to the medium, it could not be activated in vitro when molybdate was added to the extracts. Activation occurred, however, when an acid-treated component I was added to extracts of cells derepressed in medium containing tungstate. Acid treatment completely abolished component I activity. Mutant strains UW45 and UW10 were unable to fix N(2). Both strains synthesized normal levels of component II but produced inactive component I. Acid-treated component I activated inactive component I in extracts of mutant strain UW45 but not mutant strain UW10. This activating factor could be obtained from N(2)-fixing Klebsiella pneumoniae, Clostridium pasteurianum, and Rhodospirillum rubrum.