Mechanism of the photocatalytic inactivation of Lactobacillus casei phage PL-1 by titania thin film.

The mechanism of the inactivation of Lactobacillus casei phage PL-1 suspended in a phosphate buffer by black-light (BL) -catalytic titanium dioxide (TiO2) thin film was studied. Generation of both superoxide anions (O2-) and hydroxyl radicals (*OH) was confirmed in the aqueous medium in which TiO2 film was settled with BL irradiation under gentle shaking. With BL-irradiation alone without TiO2 film, only O2- was generated to some extent. The genome DNA inside the phage particles was found to be fragmented by the treatment of PL-1 phages with BL-catalytic TiO2 film. The phage inactivation by BL-catalytic TiO2 film was inhibited by the addition of albumin in a concentration-dependent manner. BL-catalytic TiO2 film was considered to cause primarily the damage to the capsid protein through the generation of active oxygen species such as *OH, followed by damage to the genome DNA inside the phage particles.

Selective demonstration of mural nerves in ganglionic and aganglionic colon by immunohistochemistry for glucose transporter-1: prominent extrinsic nerve pattern staining in Hirschsprung disease.

OBJECTIVE: Hypertrophic nerves have long been considered a histopathologic feature of the aganglionic segment in Hirschsprung disease, but they remain incompletely explained. The purpose of this study was to define the nature and diagnostic importance of hypertrophic nerves in Hirschsprung disease and to clarify their relation to nearby smaller nerve fibers. METHODS: We used an immunoperoxidase staining technique to compare the distribution of 2 nerve markers-erythrocyte-type glucose transporter (GLUT-1), a marker of perineurium, and nerve growth factor receptor, a marker of both nerve fibers and perineurium-in aganglionic tissue (12 resected specimens and 4 rectal biopsies) and control tissue (6 autopsy specimens and 17 rectal biopsies) of children. RESULTS: In control ganglionic tissue, the myenteric and submucosal areas contained only occasional GLUT-1-positive nerves (usually less than 50 microm in diameter), but extramural extrinsic (serosal) nerves were invariably positive for GLUT-1. In aganglionic tissue, GLUT-1-positive nerves in the myenteric and submucosal areas were frequent and included both large (50-150 microm) and small (<50 microm) diameter nerves. Nerve growth factor receptor-positive fibers were frequent in all layers of all tissue studied. In aganglionic bowel, a distinct perineurium could be identified in the largest nerves, but nerve growth factor receptor had poor discrimination for small perineurium-sheathed nerves. CONCLUSION: Most nerves, of both large and small diameter, in the myenteric and submucosal plexus of aganglionic bowel are GLUT-1 positive. Serosal extrinsic nerves stain identically, supporting the interpretation that the mural nerves are of extrinsic origin. Mural GLUT-1-positive nerves, when they are multiple and especially when they are greater than 50 microm in diameter (a figure which may be used as a threshold for hypertrophic nerves), are suggestive of Hirschsprung disease.

Effect of ionic strength on the inactivation of micro-organisms by microwave irradiation.

Microwave irradiation at 2450 MHz inactivated the cells of Escherichia coli, Staphylococcus aureus and Candida albicans suspended in a phosphate buffer. The rate of cell inactivation was proportional to that of the increase in temperature accompanied by microwave irradiation. The inactivation rates of E. coli and C. albicans were affected by addition of NaCl and KCl, but not by sucrose. The maximal inactivation effect was exerted at concentrations of 0.5-1.0 mol l-1, and the end-point temperature was the highest at the same salt concentrations. Correlation of both the electroconductivity and di-electric loss of ionic solutions with the heating by microwave irradiation was discussed.

Function of 90-kDa heat shock protein in cellular differentiation of human embryonal carcinoma cells.

Heat shock proteins (HSPs) have been recognized as molecules that maintain cellular homeostasis during changes in the environment. Here we report that HSP90 functions not only in stress responses but also in certain aspects of cellular differentiation. We found that HSP90 showed remarkably high expression in undifferentiated human embryonal carcinoma (EC) cells, which were subsequently dramatically down-regulated during in vitro cellular differentiation, following retinoic acid (RA) treatment, at the protein level. Surprisingly, heat shock treatment also triggered the down-regulation of HSP90 within 48 h at the protein level. Furthermore, the heat treatment induced cellular differentiation into neural cells. This down-regulation of HSP90 by heat treatment was shifted to an up-regulation pattern after cellular differentiation in response to RA treatment. In order to clarify the functions of HSP90 in cellular differentiation, we conducted various experiments, including overexpression of HSP90 via gene transfer. We showed that the RA-induced differentiation of EC cells into a neural cell lineage was inhibited by overexpression of the HSP90alpha or -beta isoform via the gene transfer method. On the other hand, the overexpression of HSP90beta alone impaired cellular differentiation into trophoectoderm. These results show that down-regulation of HSP90 is a physiologically critical event in the differentiation of human EC cells and that specific HSP90 isoforms may be involved in differentiation into specific cell lineages.

Effects of microwave irradiation on bacteria attached to the hospital white coats.

A test to sterilize pieces of cloths, which are the material of hospital white coats (doctors and nurses wears), by microwave irradiation in place of autoclaving was done using a commercial 2,450 MHz microwave oven. When pieces of cloths made of cotton (35%) and polyester (65%) were contaminated experimentally with Escherichia coli or Staphylococcus aureus and irradiated by microwave, the bacteria were killed quite rapidly according to almost first-order reaction kinetics. Only after a 20-sec irradiation, when the water content of cloths was decreased from the original 48% to 35%, the relative survivals of these bacteria fell to below 1% that of the non-irradiated control. The cloths were neither browned nor crisped, even after a 10-min irradiation of microwaves.

Lack of a docking mechanism for neurotransmitter release in the aganglionic segment of bowel in patients with Hirschsprung's disease.

We examined immunohistochemically the expression and localisation of synapse-associated proteins, syntaxin (SNT) and synaptotagmin (STG) in the entire resected specimens of colon obtained at the time of pull-through operation from 15 patients with Hirschsprung's disease (HD) and 6 age-matched controls. Both antibodies showed a similar pattern of staining. In the normal colon and ganglionic colon from HD, there was strong reactivity in the submucous and myenteric plexuses in addition to staining of nerve fibres in the smooth-muscle layers. In the aganglionic colon, there was an absence or marked decrease in SNT and STG-positive nerve fibres in the smooth-muscle layers and in hypertrophic nerve trunks. Our data indicate that important proteins necessary for the docking of synaptic vesicles at the presynaptic plasma membrane are lost in fibres innervating the smooth muscle of HD and suggest that abnormal neurotransmission may have a role in the maintenance of muscle spasticity.

A temperate phage with cohesive ends induced by mitomycin C treatment of Lactobacillus casei.

A temperate phage, named PL-2, was induced from Lactobacillus casei ATCC 27092 by mitomycin C treatment of the cells at exponential growth phase. The phage had an isometric head of 45 nm in diameter and a flexible, non-contractile tail, 150 nm long and 10 nm wide, with a sharp tip. Along the tail axis, about 40 regularly spaced striae were seen. The phage DNA had complementary cohesive ends. The restriction enzyme map of the DNA was constructed by using 13 different restriction endonucleases. The size of the DNA was 35.2 kb, 83% in size of that of phage PL-1 lytic for the same Lb. casei strain.

Photocatalysis-dependent inactivation of Lactobacillus phage PL-1 by a ceramics preparation.

A ceramics preparation (Cleansand-205), which was coated with a mixture of the oxides of Si, Al, Ti, and Ag, was found to inactivate Lactobacillus phage PL-1 suspended in a buffer solution. The inactivation of phage was dependent on the amounts of Cleansand-205 added, and the reaction obeyed almost first-order reaction kinetics. The phage inactivation was considerably accelerated by the presence of light.

Effects of some calcium-related agents on the protoplast transfection of Lactobacillus casei with phage PL-1 DNA.

To clarify the mechanism of Ca2+ involvement in the DNA transfer through cell membrane, we studied the effects of Ca2+-chelator, Ca2+-ionophore, and Ca2+-channel blocker on the protoplast transfection of Lactobacillus casei ATCC 27092 by PL-1 phage DNA in the presence of Ca2+. Ca2+-chelators, citrate, EDTA, and dipicolinic acid, inhibited the transfection probably by compensating the effect of Ca2+. Ca2+-ionophores, A23187 and N,N,N', N'-tetracyclohexyl-3-oxapentanediamide, which were expected to accelerate transfection by introducing Ca2+ into cells, inhibited the transfection. This fact indicated the absence of correlation between the entry of Ca2+ and the transport of DNA into protoplasts. Verapamil, which blocks voltage-dependent Ca2+-channel besides beta-adrenergic receptor, inhibited the transfection with little effect on the survival of the protoplasts. Both flunarizine and vinpocetine, voltage-dependent Ca2+-channel blockers, did not show the selective toxicity. D-alpha-Aminoadipic acid, a glutamate receptor-operated Ca2+-channel blocker, had no effect. Propranolol, which blocks beta-adrenergic receptor as does verapamil, inhibited the transfection without severely damaging the protoplasts. These results suggested that a kind of receptor-operated Ca2+-channel was involved in the transport of PL-1 phage DNA into the cells and that the cell membrane might have a receptor structure somewhat similar to the beta-adrenergic receptor found in mammalian cells.

Inactivation of Lactobacillus bacteriophage PL-1 by microwave irradiation.

The effect of microwave irradiation on the survival of bacteriophage PL-1, which is specific for Lactobacillus casei, was studied using a commercial 2,450 MHz microwave oven. The phages were inactivated by microwave irradiation according to almost first-order reaction kinetics. The rate of phage inactivation was not affected by the difference in the continuous or intermittent irradiation, nor by the concentrations of phages used, but was affected by the volume of phage suspensions, which prevented the loss of generated heat. Microwave irradiation of phage suspensions produced a number of ghost phages with empty heads, but fragmentation of the tail was hardly noticed. The breakage of phage genome DNA was primarily caused by the heat generated by microwave irradiation, whereas the phage DNA was not affected by the same temperature achieved by heat from outside. Thus we concluded that the phage-inactivating effect of microwave irradiation was mainly attributed to a thermal microwave effect, which was much stronger than a simple thermal exposure.

Involvement of host cell energy in the transfection of Lactobacillus casei protoplasts with phage PL-1 DNA.

Transfection of Lactobacillus casei ATCC 27092 protoplasts with phage PL-1 DNA was studied under various conditions. The process of transfection was dependent on the incubation temperature, and the apparent activation energy was calculated to be about 11 kcal/mol. Transfection was inhibited by treating the cells before protoplasting either with monoiodoacetate, N,N'-dicyclohexylcarbodiimide (DCCD), or NaN3, without affecting both the viability of uninfected cells and protoplasting. The addition of DCCD after mixing protoplasts and DNA had no influence on transfection efficiencies. The transfection of L. casei protoplasts with phage PL-1 DNA was considered to require cell energy such as proton-motive force, probably in the initial stages, although the direct involvement of cell energy in the transfer of DNA across the cell membrane is still unclear.

Restriction map of the genomic DNA of Lactobacillus casei bacteriophage PL-1 and nucleotide sequence of its cohesive single-stranded ends.

A restriction map of the genomic DNA of Lactobacillus casei phage PL-1 was constructed using the restriction endonucleases BamHI, EcoRI, HindIII, KpnI, NruI and XhoI. The PL-1 genome was 42.2 kb in size and had complementary cohesive ends forming a ring-like monomer. The cohesive ends, analysed with exonuclease III and S1 nuclease, were 3'-terminated single strands protruding from both ends. The nucleotide sequence of the cohesive ends, determined by the dideoxynucleotide method, comprised four A + T and 10 G + C pairs: 5' GAGGCCGACCGTTC 3'/3' CTCCGGCTGGCAAG 5'. Thus, the cohesive ends of PL-1 DNA were higher in G + C content than those of other known bacteriophage DNAs.

Calcium requirement for protoplast transfection mediated by polyethylene glycol of Lactobacillus casei by PL-1 Phage DNA.

The effects of some divalent cations on protoplast transfection mediated by polyethylene glycol of Lactobacillus casei ATCC 27092 by PL-1 phage DNA in 50 mM Tris-maleate buffer (pH 6.0) were investigated. The efficiency of transfection increased about 30 times in the presence of 10 mM Ca2+. Sr2+ increased the transfection rate as well, but Ba2+, Mn2+, and Mg2+ did not. Co2+ and Zn2+ inhibited transfection. The simultaneous use of Ca2+ and Mg2+ increased the transfection efficiency. Impairment of transfection caused by lack of Ca2+ could not be reversed by the addition of Ca2+ later. A decrease in the Ca2+ concentration to an ineffective level before transfection ended immediately inhibited transfection. Protoplasts were transfected with a phage adsorption mutant resistant to PL-1, also, and these metal ions had the same effect. Multiplication of phages in the transfected protoplasts was independent of the presence or absence of calcium ions. Calcium ions seemed to be involved in the entry of PL-1 DNA into the host protoplasts.

The possible involvement of protein synthesis in the injection of PL-1 phage genome into its host, Lactobacillus casei.

The process of genome DNA injection, after adsorption, by phage PL-1 into host cells of Lactobacillus casei was monitored by using the electron microscope. Injection of DNA was inhibited by the protein-synthesis inhibitors chloramphenicol and erythromycin at concentrations where the colony-forming ability of cells not infected by phage was unaffected. The results suggest that protein synthesis may be involved in some way in the process of genome injection.

Protoplast transfection of Lactobacillus casei by phage PL-1 DNA.

Polyethylene glycol (PEG) mediated transfection of Lactobacillus casei ATCC 27092 protoplasts by phage PL-1 DNA was done. The protoplasts were obtained by treatment with purified PL-1 phage N-acetylmuramidase in the presence of citrate. Optimum conditions for transfection were 50% PEG 4,000, 15 micrograms protamine sulfate/ml, 0.15 M sucrose, and 10 mM MgSO4 in MR medium (pH 6.0). The extent of transfection was proportional to the amounts of DNA added, and the greatest efficiency of transfection after a 10-min incubation was about 3.3 x 10(5) PFU/micrograms DNA. The eclipse period of growth of progeny phages in the transfectants was 3 hr and the average burst size was 200.

Redistribution of haloperidol after electroshock: experimental evidence.

We have previously reported that plasma and red blood cell levels of haloperidol, a neuroleptic agent, significantly increased immediately after electroconvulsive shock therapy (ECT) in schizophrenic patients on long term haloperidol treatment. To elucidate the mechanism of this increase, we attempted to reproduce this phenomenon in female Wistar rats. After 4 successive days of ip administration of haloperidol (10 mg/kg body weight, once daily), rats were given ECT through corneal electrodes on the fifth day (a.c. 50 Herz, 55 mA, 2.0 sec). Haloperidol levels were determined in plasma and other major tissues using a radioreceptor assay for haloperidol distribution before and after ECT at appropriate time intervals. Plasma haloperidol level was significantly increased 1 min after ECT but tended to return to the control level (without ECT) after 5 min. A significant decrease in haloperidol concentration in tissues was not observed in any of the tissues examined including frontal cerebrum, striatum, and muscle tissues (gluteal muscles). However, the relatively high haloperidol level and the large volume of muscle tissues suggested that the muscle could be the source of the transient increase in haloperidol levels in plasma. This conclusion was also supported by the data showing no significant rise of plasma haloperidol level after ETC in rats previously given a muscle relaxant, succamethonium chloride.

Absence of age effect on plasma haloperidol neuroleptic levels in psychiatric patients.

Plasma neuroleptic levels in 41 patients (21 men, 20 women, aged 18 to 74) on haloperidol therapy were examined in relation to their age by means of radioreceptor assay. There was no significant difference among three age groups (below 45 years, 46 to 60 years, over 60 years) in the ratio of the plasma neuroleptic level to daily dose (nM/mg/kg), but a significant difference in the plasma neuroleptic level was found between the average values in parkinsonian (19.1 +/- 8.5 nM, M +/- SD) and nonparkinsonian (5.5 +/- 3.0 nM, M +/- SD) patients. There was, however, no significant difference in the incidence of parkinsonian symptoms between the young (below 60 years) and the old (over 60 years) age groups. These results suggest that in contrast to the previously reported study with chlorpromazine, the plasma neuroleptic level of haloperidol is not altered with aging and that parkinsonian symptoms induced by haloperidol occur simply in a plasma-neuroleptic-level-dependent manner.