Bridging macroscopic and microscopic methods for the measurements of cerebral blood flow: Toward finding the determinants in maintaining the CBF homeostasis.

Methods exist to evaluate the cerebral blood flow (CBF) at both the macroscopic and microscopic spatial scales. These methods provide complementary information for understanding the mechanism in maintaining an adequate blood supply in response to neural demand. The macroscopic CBF assesses perfusion flow, which is usually measured using radioactive tracers, such as diffusible, nondiffusible, or microsphere. Each of them determines CBF based on indicator dilution principle or particle fraction principle under the assumption that CBF is steady state during the measurement. Macroscopic CBF therefore represents averaged CBF over a certain space and time domains. On the other hand, the microscopic CBF assesses bulk flow, usually measures using real-time microscopy. The method assesses hemodynamics of microvessels, ie, vascular dimensions and flow velocities of fluorescently labeled or nonlabeled RBC and plasma markers. The microscopic CBF continuously fluctuates in time and space. Smoothing out this heterogeneity may lead to underestimation in the macroscopic CBF. To link the two measurements, it is needed to introduce a common parameter which is measurable for the both methods, such as mean transit time. Additionally, applying the defined physiological and/or pharmacological perturbation may provide a good exercise to determine how the specific perturbations interfere the quantitative relationships between the macroscopic and microscopic CBF. Finally, bridging these two-scale methods potentially gives a further indication how the absolute CBF is regulated with respect to a specific type of the cerebrovascular tones or capillary flow velocities in the brain.

Repeated longitudinal in vivo imaging of neuro-glio-vascular unit at the peripheral boundary of ischemia in mouse cerebral cortex.

Understanding the cellular events evoked at the peripheral boundary of cerebral ischemia is critical for therapeutic outcome against the insult of cerebral ischemia. The present study reports a repeated longitudinal imaging for cellular-scale changes of neuro-glia-vascular unit at the boundary of cerebral ischemia in mouse cerebral cortex in vivo. Two-photon microscopy was used to trace the longitudinal changes of cortical microvasculature and astroglia following permanent middle cerebral artery occlusion (MCAO). We found that sulforhodamine 101 (SR101), a previously-known marker of astroglia, provide a bright signal in the vessels soon after the intraperitoneal injection, and that intensity was sufficient to detect the microvasculature up to a depth of 0.8 mm. After 5-8 h from the injection of SR101, cortical astroglia was also imaged up to a depth of 0.4 mm. After 1 day from MCAO, some microvessels showed a closure of the lumen space in the occluded MCA territory, leading to a restructuring of microvascular networks up to 7 days after MCAO. At the regions of the distorted microvasculature, an increase in the number of cells labeled with SR101 was detected, which was found as due to labeled neurons. Immunohistochemical results further showed that ischemia provokes neuronal uptake of SR101, which delineate a boundary between dying and surviving cells at the peripheral zone of ischemia in vivo. Finally, reproducibility of the MCAO model was evaluated with magnetic resonance imaging (MRI) in a different animal group, which showed the consistent infarct volume at the MCA territory over the subjects.

Long-term effects of hepatocyte growth factor gene therapy in rat myocardial infarct model.

We investigated the long-term effects of human hepatocyte growth factor (HGF) gene therapy in a rat myocardial infarct model. Treatment adenovirus coexpressing the HGF therapeutic gene and the human sodium/iodide symporter (NIS) reporter gene or control adenovirus expressing the NIS gene alone were injected directly into the infarct border zone immediately after permanent coronary ligation in rats (n=6 each). A uniform disease state was confirmed in the acute phase in terms of impaired left ventricular (LV) function by cine magnetic resonance imaging (MRI), large infarct extent by (99m)Tc-tetrofosmin single-photon emission computed tomography (SPECT) and successful gene transfer and expression by (99m)TcO(4)(-) SPECT. After a 10-week follow-up, repeated cine MRI demonstrated no significant difference in the LV ejection fraction between the time points or groups, but a significantly increased end-diastolic volume from the acute to the chronic phase without a significant difference between the groups. Capillary density was significantly higher in the treatment group, whereas arteriole density remained unchanged. Two-photon excitation fluorescence microscopy revealed extremely thin capillaries (2-5 µm), and their irregular networks increased in the infarct border zone of the treated myocardium. Our results indicated that single HGF gene therapy alone induced an immature and irregular microvasculature.

Detailed biosynthetic pathway to decaprenoxanthin diglucoside in Corynebacterium glutamicum and identification of novel intermediates.

Carotenogenic mutants of Corynebacterium glutamicum were analyzed for their carotenoid content. Mutant MV10 accumulated the same carotenoids as the wild-type, decaprenoxanthin, decaprenoxanthin monoglucoside, and (2R,6R,2'R,6'R)-decaprenoxanthin di-(beta-D)-glucoside, but in three-fold higher amounts. In addition, decaprenoxanthin diglucoside fatty acid esters and the intermediates nonaprene, 2-(3-methyl-2-butenyl)-epsilon,psi-carotene, and sarcinene, 2,2'-bis(3-methyl-2-butenyl)-epsilon,epsilon-carotene were identified as minor carotenoids. The pink mutants MV40 and MV60 synthesized only lycopene. From another pink mutant, MV70, novel C(50)-carotenoids were isolated. By NMR and mass spectroscopy, nonaflavuxanthin, 2-(4-hydroxy-3-methyl-2-butenyl)-1,16-didehydro-1,2-dihydro-psi,psi-carotene, and flavuxanthin, 2,2'-bis(4-hydroxy-3-methyl-2-butenyl)-1,16,1',16'-tetradehydro-1,2,1',2'-tetrahydro-psi,psi-carotene, were identified. The identification of these intermediates revealed the detailed pathway for the formation of decaprenoxanthin derivatives in Corynebacterium glutamicum.

Myxoxanthophyll in Synechocystis sp. PCC 6803 is myxol 2'-dimethyl-fucoside, (3R,2'S)-myxol 2'-(2,4-di-O-methyl-alpha-L-fucoside), not rhamnoside.

We identified the molecular structures of the carotenoids in Synechocystis sp. PCC 6803. Myxoxanthophyll in this cyanobacterium was myxol 2'-dimethyl-fucoside, (3R,2'S)-myxol 2'-(2,4-di-O-methyl-alpha-L-fucoside). The sugar moiety of the pigment was not rhamnose but dimethylated fucose, which has not been reported in carotenoid glycosides. The other carotenoids were beta-carotene, (3R,3'R)-zeaxanthin, echinenone, (3'R)-3'-hydroxyechinenone and deoxymyxol 2'-dimethyl-fucoside, (2'S)-deoxymyxol 2'-(2,4-di-O-methyl-alpha-L-fucoside). Generally, the group of polar carotenoids in cyanobacteria is referred to as myxoxanthophyll, and the structure is considered to be myxol 2'-rhamnoside. Since the name myxoxanthophyll can not specify the sugar moiety and the identification of the sugar moiety is unfeasible in many cyanobacteria, we propose the following naming convention: when the sugar moiety is unknown, the name is myxol glycoside, when known, as in the case of rhamnose and alpha-L-fucose, they should be named myxol 2'-rhamnoside and myxol 2'-alpha-L-fucoside, respectively.

Identification of a gene required for cis-to-trans carotene isomerization in carotenogenesis of the cyanobacterium Synechocystis sp. PCC 6803.

A disruptive mutant of the sll0033 gene of the cyanobacterium Synechocystis sp. PCC 6803 produced primarily cis carotenes and small amounts of all-trans carotenes, but no xanthophylls, under dark conditions. Under light conditions, however, it produced normal carotenoids, that were the same as those produced by wild-type cells grown under both light and dark conditions. When the mutant cells cultured under dark conditions were irradiated, cis-isomers of carotenes were converted to all-trans lycopene. These findings demonstrate that this gene, designated crtH, is involved in the isomerization of cis-carotenes to all-trans forms in dark conditions, and that cis-carotenes were also converted to all-trans forms under light conditions by photoisomerization.

Direct evidence for requirement of phosphatidylglycerol in photosystem II of photosynthesis.

Phosphatidylglycerol (PG) is considered to play an important role in the ordered assembly and structural maintenance of the photosynthetic apparatus in thylakoid membranes. However, its function in photosynthesis remains poorly understood. In this study we have identified a pgsA gene of Synechocystis sp. PCC6803 that encodes a PG phosphate synthase involved in the biosynthesis of PG. A disruption of the pgsA gene allowed us to manipulate the content of PG in thylakoid membranes and to investigate the function of PG in photosynthesis. The obtained pgsA mutant could grow only in the medium containing PG, and the photosynthetic activity of the pgsA mutant dramatically decreased with a concomitant decrease of PG content in thylakoid membranes when the cells grown in the presence of PG were transferred to the medium without PG. This decrease of photosynthetic activity was attributed to the decrease of photosystem (PS)II activity, but not to the decrease in PSI activity. These findings demonstrate that PG is essential for growth of Synechocystis sp. PCC6803 and provide the first direct evidence that PG plays an important role in PSII.

Beta-carotene hydroxylase gene from the cyanobacterium Synechocystis sp. PCC6803.

The ORF sll1468 of Synechocystis sp. PCC6803 was identified as a gene for beta-carotene hydroxylase by functional complementation in a beta-carotene-producing Escherichia coli. The gene product of ORF sll1468 added hydroxyl groups to the beta-ionone rings of beta-carotene (beta, beta-carotene) to form zeaxanthin (beta, beta-carotene-3,3'-diol). This newly identified beta-carotene hydroxylase does not show overall amino acid sequence similarity to the known beta-carotene hydroxylases. However, it showed significant sequence similarity to beta-carotene ketolases of marine bacteria and a green alga.

Aluminum Tolerance Acquired during Phosphate Starvation in Cultured Tobacco Cells.

Al toxicity in cultured tobacco cells (Nicotiana tabacum L. cv Samsun; nonchlorophyllic cell line SL) has been investigated in nutrient medium. In this system, Al and Fe(II) (ferrous ion) in the medium synergistically result in the accumulation of both Al and Fe, the peroxidation of lipids, and eventually death in cells at the logarithmic phase of growth (+P cells). A lipophilic antioxidant, N,N[prime]-diphenyl-p-phenylenediamine, protected +P cells from the peroxidation of lipids and from cell death, suggesting that a relationship exists between the two. Compared with +P cells, cells that had been starved of Pi (-P cells) were more tolerant to Al, accumulated 30 to 40% less Al and 70 to 90% less Fe, and did not show any evidence of the peroxidation of lipids during Al treatment. These results suggest that -P cells exhibit Al tolerance because their plasma membranes are protected from the peroxidation of lipids caused by the combination of Al and Fe(II). It seems likely that the exclusion of Fe from -P cells might suppress directly Fe-mediated peroxidation of lipids. Furthermore, since -P cells accumulated [beta]-carotene, it is proposed that this carotenoid pigment might function as a radical-trapping antioxidant in the plasma membrane of cells starved of Pi.

DNA sequence and regulation of the gene (cbpA) encoding the 42-kilodalton cytoplasmic membrane carotenoprotein of the cyanobacterium Synechococcus sp. strain PCC 7942.

The gene (cbpA) coding for a carotenoid-binding protein of the cyanobacterium Synechococcus sp. strain PCC 7942 (Anacystis nidulans R2) has been cloned and sequenced. A polyclonal antibody against the protein was used to identify immunoreactive clones from a lambda gt11 expression library of Synechococcus strain PCC 7942. The initial positive clone (lambda gtAN42) contained a 0.9-kilobase (kb) chromosomal fragment, which was used to detect a larger chromosomal fragment from a lambda EMBL3 library. The lambda EMBL3 recombinant, lambda EM109, contained an 18-kb portion of the Synechococcus strain PCC 7942 chromosome. The open reading frame of cbpA encoded 450 amino acids which give rise to a protein of 49,113 daltons. The hydrophobicity plot indicates that the protein may have a 49-residue signal sequence which is cleaved to yield a mature protein of 43,709 daltons. The protein has been localized in the cytoplasmic membrane by biochemical procedures as well as by electron microscopic immunocytochemistry. Northern (RNA) blot analysis indicates that transcription of cbpA is tightly regulated by DNA topology, light intensity, and iron concentration. Transcription is greatly induced by growth under high light intensities and repressed during growth under iron-deficient conditions. The DNA gyrase inhibitor novobiocin specifically inhibited the light-induced transcription. In Northern blots, the gene-specific probe hybridized to two size classes of RNA, with lengths of 2.0 and 6.2 kb. Since cbpA appears to be a component of the 6.2-kb transcript, it is likely part of a larger operon.

Long-latency growth-promoting activity of EGF when administered to mice at the neonatal stage.

The effect of biosynthetic human epidermal growth factor (Bh-EGF) as well as mouse EGF on postnatal development of mice of ICR strain was examined. Daily administration of Bh-EGF (0.01, 0.1, and 1.0 microgram X g body wt-1 X day-1) for 30 consecutive days postpartum caused a clearly dose-dependent increase in their body weight. Furthermore, in addition to the well-known premature eyelid opening and early tooth eruption, we have also observed precocious opening of the vagina among treated females. As far as the accelerated growth rate as reflected in their body weight gain was concerned, daily administration for only five consecutive days postpartum was just as effective as the above noted 30 consecutive daily injections. As to the precocious vaginal opening, however, the susceptible 5-day-period was found to be 14-18 days after the parturition. Some of those treated females also entered the estrous cycle precociously, a few days after the precocious opening of their vagina. The microscopic examination of various organs from treated males and females revealed no apparent pathological changes. As far as the above noted effects of EGF were concerned, Bh-EGF, which is xenogenic to mice, was as potent as mouse EGF.

Isolation and Characterization of a Carotenoid-Associated Thylakoid Protein from the Cyanobacterium Anacystis nidulans R2.

A carotenoid-associated membrane protein was isolated from Anacystis nidulans R2 thylakoids. Sodium pyrophosphate and sodium bromide washed thylakoids were solubilized with the nonionic detergents dodecyl-beta-D-maltoside and octyl-beta-D-glucopyranoside, and these detergent extracts were fractionated on a sucrose density gradient. A yellow fraction from the sucrose gradient was further purified by anion-exchange and organomercuric-affinity column chromatography to yield a fraction virtually free of chlorophyll and highly enriched in both carotenoids and a 42 kilodalton polypeptide. Evidence presented in this paper suggests that the carotenoid-containing 42 kilodalton protein is thylakoid associated rather than cytoplasmic membrane associated. The purified 42 kilodalton polypeptide was used to raise polyclonal antibodies in rabbits. Immuno-chemical detection of the 42 kilodalton polypeptide on Western blots demonstrated an increased accumulation of this polypeptide in cells grown under high-light conditions relative to cells grown under low light.

Dependence on surface pH and surface substrate concentration of activity of microsome-bound arylsulfatase C and the surface charge density in the vicinity of the enzyme.

Dependence on the salt concentration of the activity of microsome-bound arylsulfatase C [EC 3.1.6.1] from rat liver was examined. The activity increased with increasing salt concentration in the reaction medium in the whole pH range tested. This effect can be explained by the dependence of the reaction rate on the surface pH and the surface concentration of the ionic substrate. The dependence on salt concentration of the activity of the microsome-bound arylsulfatase C and the pH-dependences of Vmax and Km of the enzyme were used for the estimation of pH at the microsomal surface. The two values of the surface pH (surface potential) and the salt concentration were applied to the Gouy-Chapman equation. The value of -0.39 +/- 0.08 X 10(-3) elementary charge/A2 was obtained as the surface charge density in the vicinity of the microsome-bound arylsulfatase C. This was smaller than the over-all value for microsomes (-1.08 +/- 0.04 X 10(-3) elementary charge/A2; Masamoto, K. (1982) J. Biochem. 92, 365-371). This suggests that the anion concentration in the vicinity of the enzyme on microsomes is lower than that in the bulk aqueous phase and is higher than the average value at the microsomal surface when the salt concentration is low.

[Acute toxicity test of garlic extract].

The acute toxicity toxicity test of garlic extract was studied in Wistar rats and ddY mice. The LD50 values of garlic extract by P.O., I.P. and S.C. administration were estimated over 30 ml/kg respectively in male and female of both rodents. In 30 ml/kg of I.P. group, five of ten in male rats and one of ten in female rats were died within a day after administration, however no specific signs due to garlic extract were observed in survivals for 7 days.

[Chronic toxicity test of garlic extract in rats].

The influence of garlic extract on the chronic toxicity test were examined orally in Wistar rats for 6 months. There were no toxic symptoms due to garlic extract even at dose level of 2000 mg/kg for 5 times a week during 6 months. High dose of garlic extract did not inhibit the body weight gain, while the food consumption decreased slightly for the nutritional effects of it in both male and female rats. There were no significant differences in urinary, hematological and serological examinations compared each groups. In the histopathological findings, no toxic signs were observed on any of the tissues and organs examined.

Surface charge densities and ionic substrate concentrations at the membrane surface in smooth microsomes isolated from rat liver.

Salt-induced pH changes of smooth microsomes from rat liver were examined. At pHs higher than 4.9 addition of salt to a microsomal suspension induced a decrease in pH, while at pHs lower than 4.9 it induced a pH increase. The salt-induced pH changes were explained by the change in the degree of dissociation of ionizable groups of membranes due to the change of surface potential and surface pH. On comparison of the experimental data with those of calculations with the Gouy-Chapman equation, a value of 3.1+/-0.1 X 10(-3) carboxyl groups/A2 was obtained, which gives a maximal surface charge density of -1.08+/-0.04 X 10(-3) elementary charge/A2 at neutral pH and -19.2 mV surface potential at 0.15 M monovalent salt. Due to the surface charges of smooth microsomal membranes the surface pH and surface concentrations of ionic substrates become different from those in the bulk aqueous phase depending on the salt concentration. This explains part of the salt-concentration dependence of the activities of membrane enzymes in vitro. The importance of the surface concentrations of ionic substrates of enzymes of smooth microsomal membranes in vivo is also suggested.

Surface potential dependence of the distribution of charged dye molecules onto photosynthetic membranes.

Partition of merocyanine dyes, which have a negative charge, onto photosynthetic membranes of chloroplasts and bacteria was analyzed by measuring the fluorescence intensity change, absorbance change, and amount of dye in the supernatant after centrifugation. The partition depended on the surface potential, which is a function of valence and concentration of ions in the medium. The distribution of dyes between the membrane and aqueous phase was determined after centrifugation. The logarithm of the ratio of distribution was linearly related to the logarithm of salt concentration as predicted from the Gouy-Chapman theory and the Boltzmann distribution. Plots of the logarithm of fluorescence intensity against the logarithm of KCl and MgSO4 concentrations gave two straight lines with a slope ratio of about two. The absorbance change upon salt addition was also explained by the Gouy-Chapman theory. The use of these dyes as probes of the surface potential of membranes is discussed.