Protein secretion by minicells of Bacillus subtilis

Summary The minicell producing strain Bacillus subtilis IA292 was transformed with plasmids encoding the Bacillus enzymes -glucanase, -amylase and neutral protease. Purified minicells were shown to be free of detectable proteolytic activity. Minicells containing plasmids were found to synthesise all three enzymes internally, but evidence of secretion was only observed in the unique case of neutral protease secretion by minicells prepared from cultures grown in BHI medium.     

DNA degradation in minicells of Escherichia coli K-12

Summary The properties of minicell producing mutants of Escherichia coli deficient in gentic recombination were examined. Experiments were designed to test recombinant formation in conjugal crosses, survival following UV-irradiation in cells, and the state of DNA metabolism in minicells. The REC^- phenotypes are unaffected by min ⁺/^- genotypes in whole cells. In contrast to minicells produced by rec ⁺ parental cells, minicells from a recB21 strain have limited capacity to degrade linear, Hfr transferred DNA. The lack of a functional recA gene product, presumably involved in inhibiting the recBC nuclease action(s), permits unrestricted Hfr DNA breakdown in minicells produced by a recA1 strain. This results in an increase in TCA soluble products and in the formation of small DNA molecules that sediment near the top of an alkaline sucrose gradient. Unlike the linear DNA, circular duplex DNA from plasmids R64-11 or dv, segregated into the minicells, is resistant to breakdown. By using in vitro criteria, and [³²P]-labelled linear DNA from bacteriophage T₇ for substrate, we found that the ATP-dependent exonuclease of the recBC complex (exo V) is present in rec ⁺ and recA^- minicells, and is lacking in the recB21 mutant. In fact, the absence of a functional exo V in recBC^- minicells results in isolation of larger than average Hfr DNA from minicells. We suggest that recombination (REC) enzymes segregate into the polar minicells at the time of minicell biogenesis. This system should be useful for studies on DNA metabolism and functions of the recBC and recA gene products.Paper 1 in series, see Khachatourians et al., 1974.     

Lambda encodes an outer membrane protein: the lom gene.

Summary infected minicells synthesize a polypeptide (M _r=20,500) which is incorporated almost exclusively into the outer membrane of the minicell envelope. The gene (lom=lambda outer membrane) encoding this polypeptide has been mapped in the nonessential region of the genome between coordinates 39.4% and 40.7% of .     

Trehalose biosynthesis in Thermus thermophilus RQ-1: biochemical properties of the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase

The genes for trehalose synthesis in Thermus thermophilus RQ-1, namely otsA [trehalose-phosphate synthase (TPS)], otsB [trehalose-phosphate phosphatase (TPP)], and treS [trehalose synthase (maltose converting) (TreS)] genes are structurally linked. The TPS/TPP pathway plays a role in osmoadaptation, since mutants unable to synthesize trehalose via this pathway were less osmotolerant, in trehalose-deprived medium, than the wild-type strain. The otsA and otsB genes have now been individually cloned and overexpressed in Escherichia coli and the corresponding recombinant enzymes purified. The apparent molecular masses of TPS and TPP were 52 and 26 kDa, respectively. The recombinant TPS utilized UDP-glucose, TDP-glucose, ADP-glucose, or GDP-glucose, in this order as glucosyl donors, and glucose-6-phosphate as the glucosyl acceptor to produce trehalose-6-phosphate (T6P). The recombinant TPP catalyzed the dephosphorylation of T6P to trehalose. This enzyme also dephosphorylated G6P, and this activity was enhanced by NDP-glucose. TPS had an optimal activity at about 98°C and pH near 6.0; TPP had a maximal activity near 70°C and at pH 7.0. The enzymes were extremely thermostable: at 100°C, TPS had a half-life of 31 min, and TPP had a half-life of 40 min. The enzymes did not require the presence of divalent cations for activity; however, the presence of Co²⁺ and Mg²⁺ stimulates both TPS and TPP. This is the first report of the characterization of TPS and TPP from a thermophilic organism.     

Analysis of copper in brain by the mass-spectrometric integrated-ioncurrent procedure

Through use of the high-resolution double-focusing mass spectrometer, copper has been identified in various regions of the mouse, rat, guinea pig, rabbit, and human brain. The procedure depends on converting the copper (in ashed tissue) to its chloride salt, followed by derivatization with tetraphenylporphyrin (TPP) to yield a TPP chelate. After chromatographic separation, this chelate is assessed in the mass spectrometer by the integrated-ion-current procedure. Deuterated metal TPP chelates and the rare stable isotope⁶⁵Cu were used as internal standards. Whole brain values obtained were as follows: mouse, 6.67±0.16 (mean±SEM) g/g wet weight of tissue; rat, 1.06±0.05; guinea pig, 5.40±0.63; and rabbit, 7.52±0.76. In the rat, the cerebellum contained the highest concentration (1.25 g/g), and the striatum the lowest (0.70 g/g). In the human brain, the cortex (gray) and the striatum were relatively the highest copper-containing regions, with the cerebellum (white) being the lowest.     

Studies on the Mechanisms of the Monofluoroacetanilides Poisoning to Mammals

Organophosphates, phosphorothioates, phosphorodithioates, phosphites, and phosphonates were found to inhibit the fluoroacetanilide amidohydrolase of chicken liver. Above all, the inhibition by triphenyl phosphate, tri-n-butyl phosphorothioate, triphenyl phosphite, Dipterex, and DDVP were extremely remarkable. Their concentrations for 50% inhibition were about 10^?8 m or 10^?7 m, and the inhibition of triphenyl phosphate was non-competitive to the substrate. These compounds were slightly or moderately toxic to mice even when administered intraperitoneally. The specificity of the inhibition was also discussed.

The effects of the fluoroacetanilide amidohydrolase (fluoroacetanilidase) inhibitors on the fluoroaceto-p-bromoanilide (FBA) poisoning to mammals and insects were studied. Triphenyl phosphate (TPP) and tri-n-butyl phosphorothioate (TBT) were mainly used as the inhibitors. TPP was effective for reducing the oral and cutaneous toxicities of FBA to mice and rats only when applied prior to the FBA application. However, in cases of the inhaled toxicity and the combined inhaled-cutaneous toxicity, TPP or TBT were effective even when they were applied simultaneously with FBA. When TPP and TBT were administered to mice by the combined inhaled-cutaneous application, the activity of fluoroacetanilidase in liver and kidney was rapidly decreased, but it recovered within 4 to 6 days after the application. The insecticidal and miticidal activities of FBA were not affected at all both in the previous and simultaneous applications of the inhibitors. However, it was found that the fluoroacetanilides hydrolyzing enzyme of Mealy plum aphid was not inhibited by TPP even in the concentration of 10^?4 m. The mechanisms of the fluoroacetanilides poisoning to mammals and insects were discussed.     

Transport of branched-chain amino acids in Corynebacterium glutamicum

The transport of branched-chain amino acids was characterized in intact cells of Corynebacterium glutamicum ATCC 13032. Uptake and accumulation of these amino acids occur via a common specific carrier with slightly different affiniteis for each substrate (K _m[Ile]=5.4 M, K _m[Leu]=9.0 M, K _m[Val]=9.5 M). The maximal uptake rates for all three substrates were very similar (0.94–1.30 nmol/mg dw · min). The optimum of amino acid uptake was at pH 8.5 and the activation energy was determined to be 80 kJ/mol. The transport activity showed a marked dependence on the presence of Na⁺ ions and on the membrane potential, but was independent of an existing proton gradient. It is concluded, that uptake of branched-chain amino acid transport proceeds via a secondary active Na⁺-coupled symport mechanism.Abbreviations CCCP Carboxyl cyanide m-chlorophenylhydrazone - dw dry weight - MES 2[N-morpholino]ethanesulfonic acid - mon monensin - nig nigericin - TPP tetraphenylphosphonium bromide - Tris tris[hydroxymethyl]aminomethane - val valinomycin     

Cloning and expression of a Bacillus coagulans amylase gene in Escherichia coli

Summary A partial EcoRI fragment of Bacillus coagulans DNA cloned in an Escherichia coli K12 bacteriophage host-vector system was shown to direct the synthesis of a thermostable -amylase whose activity could be detected in situ on petri plates using the iodine staining method. A 3.31 kb EcoRI fragment containing the active gene with its own promoter was subcloned in pBR322; in the new clone, called pAMY2, the amylase was shown to accumulate in the periplasmic space. The molecular weight of the enzyme, confirmed by in vivo labelling of plasmid products in minicells, was estimated to be 60000.The restriction map of the plasmid was determined for five restriction enzymes and two new plasmids with smaller DNA inserts were constructed, both directing the synthesis of amylase; one of them with a 2.2 kb PstI insert was shown to be responsible for the synthesis of a fused -lactamase--amylase protein with amylase activity.     

Salutary effect of tedisamil on post-ischemic recovery rat heart: Involvement of sarcolemmal (Na,K)-ATPase

The in vitro effect of tedisamil on the specific activity and kinetic parameters of the sarcolemmal (Na,K)-ATPase as well as its ex vivo effect on the (Na,K)-ATPase in the isolated, perfused rat hearts was determined. Five mol/l of tedisamil was added 5 min before the onset of 30 min global normothermic ischemia followed by 10 min reperfusion. At the conditions of its maximal cardioprotective effect (heart rate reduction, improved postischemic recovery of left ventricular developed pressure), the hearts were immediately used for isolation of sarcolemmal vesicles. In vitro, 1–100 mol/l of tedisamil produced a concentration-dependent stimulatory effect on (Na,K)-ATPase activity, with a peak seen at 20 mol/l (p < 0.01), while Mg-dependent ATPase was almost unchanged. Kinetic analysis revealed a significant increase in the affinity of the Na-binding sites on ATPase molecule at 20 mol/l of tedisamil. These biochemical findings were confirmed by cytochemistry. Moreover, ex vivo experiments revealed that tedisamil rendered the sarcolemmal (Na,K)-ATPase activity to be a more resistant to detrimental effects of ischemia. In conclusion, the cardioprotective action of tedisamil was accompanied with a better preservation of the specific activity of (Na,K)-ATPase.     

Substrate specificity of phenol sulfotransferase from primary cultures of bovine brain microvessel endothelium

The substrate specificity of the thermostable phenol sulfotransferase (PST) from primary cultures of brain microvessel endothelial cell monolayers was characterized. Selected catecholamines, catecholamine metabolites, and p-nitrophenol at 5, 50, and 500 M were used as substrates in PST assays of cytosol extracts. Endogenous catecholamines, epinephrine, norepinephrine, and dopamine, exhibited no detectable activity as substrates (500 M) compared to 500 M p-nitrophenol as substrate (1.8 pmol/mg/min specific activity) for the PST. In contrast, 500 M of either deaminated or 3-O-methylated metabolites of catecholamines exhibited intermediate (1.0 pmol/mg/min specific activity) to low (0.2 pmol/mg/min specific activity) activity, respectively, as substrates compared to p-nitrophenol as substrate for the PST. Additionally, 500 M of metabolites of catecholamines that were both deaminated and 3-0-methylated exhibited high activity (>3.0 pmol/mg/min specific activity) as substrates compared top-nitrophenol as substrate for the PST. Qualitatively similar results were observed at lower substrate concentrations. Therefore, results from this study suggest a potential role for PST as part of the enzymatic blood-brain barrier in regulating transendothelial passage of endogenous catecholamines between the blood and the brain.     

The application of continuous monitoring microdensitometry to an analysis of NAD+ binding and 3 beta-hydroxy-delta 5-steroid dehydrogenase activity in the regressing corpus luteum of the pro-oestrous rat ovary

Summary A technique which allows for the continuous microdensitometric monitoring (at 3.3 s intervals) of an enzyme reaction has been applied to a study of 3-hydroxy-⁵-steroid dehydrogenase activity in regressing corpora lutea cells of unfixed tissue sections (5µm thick) of the pro-oestrous rat ovary. Initial reaction rates for NAD⁺ reduction by the activity of the enzyme were maintained for less than 3 min at all the concentrations (0-500µmol/l) of co-factor employed. The relationship between NAD⁺ concentration and enzyme activity under initial velocity rate conditions was hyperbolic and maximum enzyme activity was seen when the NAD⁺ concentration was greater than 250µmol/l. The apparent Michaelis—Menten constant (K _m) was 29µmol/l and V_max 0.63µmol NAD⁺ reduced/min/cm³ corpora lutea.     

Molecular cloning and characterisation of the gua regulatory region of Escherichia coli K12

Summary The regulatory region of the gua operon of Escherichia coli is contained within a 2.1 kb EcoR1 restriction fragment isolated from a pgua transducing phage. This DNA fragment was inserted into pPV33-H, a promoter-cloning vector, where it activated the gene(s) for tetracycline resistance. The level of tetracycline resistance conferred by the hybrid plasmid was reduced by the addition of guanine and increased by adenine, indicating the presence of the gua promoter. The cloned fragment codes for a polypeptide that complements in vivo the defective enzymes present in certain guaB mutants. This polypeptide was characterised using minicells and immunoprecipitation, and is presumed to correspond to the N-terminal region of IMP dehydrogenase.     

Phosphomannomutase activity in wild-type and alginate-producing strains ofPseudomonas aeruginosa

Phosphomannomutase (PMM) activity was detected in the soluble cytoplasmic fraction of crude extracts of both mucoid (alginate-producing) and nonmucoid strains ofPseudomonas aeruginosa. The enzyme activity was concentrated and partially purified from cell extracts of mucoid strain V388 by precipitation with ammonium sulfate and by molecular exclusion chromatography. These preparations catalyzed the conversion of mannose 1-phosphate to mannose 6-phosphate in a coupled assay system that contained commercial phosphomannoisomerase, phosphoglucoisomerase, and glucose 6-phosphate dehydrogenase. Catalytic activity in this system was strictly dependent on the presence of glucose 1,6-diphosphate (apparent Km, 150 M) and exhibited a pH optimum of around 9 in Bicine-NaOH buffer. PMM exhibited an apparent Km of 60 M for mannose 1-phosphate, but concentrations greater than 150 M caused significant inhibition. Specific activities of PMM were consistently higher in the soluble fractions of mucoid strains (1.2–3.6 nmol/min/mg protein) than of nonmucoid strains (0.2–0.6 nmol/min/mg protein).     

Crystal Structure and Autoactivation Pathway of the Precursor Form of Human Tripeptidyl-peptidase 1, the Enzyme Deficient in Late Infantile Ceroid Lipofuscinosis

Late infantile neuronal ceroid lipofuscinosis is a fatal childhood neurological disorder caused by a deficiency in the lysosomal protease tripeptidyl-peptidase 1 (TPP1). TPP1 represents the only known mammalian member of the S53 family of serine proteases, a group characterized by a subtilisin-like fold, a Ser-Glu-Asp catalytic triad, and an acidic pH optimum. TPP1 is synthesized as an inactive proenzyme (pro-TPP1) that is proteolytically processed into the active enzyme after exposure to low pH in vitro or targeting to the lysosome in vivo. In this study, we describe an endoglycosidase H-deglycosylated form of TPP1 containing four Asn-linked N-acetylglucosamines that is indistinguishable from fully glycosylated TPP1 in terms of autocatalytic processing of the proform and enzymatic properties of the mature protease. The crystal structure of deglycosylated pro-TPP1 was determined at 1.85 Å resolution. A large 151-residue C-shaped prodomain makes extensive contacts as it wraps around the surface of the catalytic domain with the two domains connected by a 24-residue flexible linker that passes through the substrate-binding groove. The proenzyme structure reveals suboptimal catalytic triad geometry with its propiece linker partially blocking the substrate-binding site, which together serve to prevent premature activation of the protease. Finally, we have identified numerous processing intermediates and propose a structural model that explains the pathway for TPP1 activation in vitro. These data provide new insights into TPP1 function and represent a valuable resource for constructing improved TPP1 variants for treatment of late infantile neuronal ceroid lipofuscinosis.Late infantile neuronal ceroid lipofuscinosis (LINCL)³ (OMIM number 204500) is a neurodegenerative lysosomal storage disease of childhood that presents typically between the ages of 2 and 4 years with the onset of seizures. Disease progression is reflected by blindness, dementia, mental retardation, and an increase in the severity of seizures. LINCL is always fatal, and the life span of patients is typically 6-15 years. LINCL is caused by mutations in TPP1 (previously named CLN2, for ceroid lipofuscinosis neuronal type 2 gene) (1), which normally encodes a lysosomal protease, tripeptidyl-peptidase 1 (TPP1, EC 3.4.14.9) (2, 3).There is currently no treatment of demonstrated efficacy for LINCL, but promising progress is being made in some directions. Proof-of-principle for virus-mediated gene therapy has been established in a mouse model of LINCL, with a significant improvement in disease phenotype achieved with the use of adeno-associated virus vectors expressing TPP1 (4-7). Affected children have also been treated with adeno-associated virus vectors, although it is too soon to determine whether significant clinical benefits have been achieved in these early trials (8). Enzyme replacement therapy, an approach that has proven successful in a number of other lysosomal storage diseases, has also been investigated in LINCL. Purified recombinant human TPP1 that contains the mannose 6-phosphate lysosomal targeting modification can be taken up by LINCL fibroblasts where it degrades storage material (9), and the protein has been introduced into the cerebrospinal fluid of the LINCL mouse model via intraventricular injection, resulting in significant uptake into the brain and some correction of neuropathology (10).For therapeutic approaches that rely upon replacing a mutant gene product with a functional protein via recombinant methods, e.g. gene and enzyme replacement therapy, a thorough understanding of the biological and biophysical properties of the protein in question are essential for success. Thus, for LINCL, considerable effort has been directed toward the investigation of TPP1, and as a result, this is a well characterized enzyme at the functional and molecular levels (reviewed in Refs. 11, 12). TPP1 encodes a 563-residue preproprotein with a cleavable N-terminal 19-residue signal sequence. The proenzyme (residues 20-563) is a soluble monomer that undergoes proteolytic cleavage in the lysosome, converting the zymogen to an active, mature protease (residues 196-563) (1). Studies on purified pro-TPP1 demonstrate that maturation is autocatalytic in vitro (13, 14) but may involve other proteases in vivo (15). TPP1 is glycosylated, and its N-linked oligosaccharides have been implicated in maturation, activity, targeting, and stability of the processed enzyme (16, 17).TPP1 is a serine protease (14) that possesses two catalytic functions as follows: a primary tripeptidyl exopeptidase activity with a pH optimum of ∼5.0 that catalyzes the sequential release of tripeptides from the unsubstituted N termini of substrates (18), and a much weaker endoproteolytic activity with a pH optimum of ∼3.0 (19). TPP1 exhibits broad substrate specificity (20) and is the only mammalian member of the S53 sedolisin family (reviewed in Ref. 21), which includes a number of unusual bacterial serine peptidases (22). High resolution crystal structures of both free and inhibitor-bound complexes have been determined for three bacterial members of this family (sedolisin (23-26), kumamolisin (27, 28), and kumamolisin-As (29, 30)), and for one (kumamolisin), the structure of a mutant, inactive precursor form has also been obtained (28). These proteins share a common subtilisin-like fold, an octahedrally coordinated calcium-binding site, and an active site that contains an unusual Ser-Glu-Asp (SED) catalytic triad, rather than the Ser-His-Asp (SHD) triad of subtilisin (31, 32). Chemical modification studies of TPP1 have revealed that Ser⁴⁷⁵ is the active site nucleophile (14). Modeling studies suggest that Glu²⁷² and Asp²⁷⁶ complete the catalytic triad and that Asp³⁶⁰ is homologous to the conserved Asn in the subtilisin family in its role in stabilization of the oxyanion of the tetrahedral intermediate during catalysis (33). Site-directed mutagenesis studies are consistent with these conclusions (14, 34).A detailed understanding of the tertiary structure of TPP1 may have implications for developing or improving therapeutic strategies. First, a high resolution model would provide the basis for targeted protein engineering efforts to design TPP1 derivatives with increased half-life prior to and/or upon delivery to the lysosome. Successful creation of a longer lived TPP1 molecule could significantly enhance gene or enzyme replacement approaches to LINCL. Second, a structural model for TPP1 could be valuable in designing derivatives tagged with protein transduction domains to facilitate crossing of the blood-brain barrier for delivery to the central nervous system from the bloodstream. In this study, we present the crystal structure of the proform of human TPP1 at 1.85 Å resolution. This model provides novel insights into the structural basis for the pH-induced auto-activation of the proform of TPP1. A structure of glycosylated pro-TPP1 has been independently determined, displaying features similar to those of deglycosylated TPP1.⁴     

Circadian periodicity in cockroaches altered by high frequency light-dark cycles

Summary The circadian period of the freerunning activity rhythm in the cockroach is systematically altered by high frequency light-dark cycles (HF-LD) according to the ratio of light to dark within each cycle. With a standard 10 min cycle time, brief (e.g., 0.5 min) exposure to light each cycle causes the free-running period to shorten significantly in comparison to its steady-state value in constant darkness. As the ratio of light to dark in HF-LD is increased, the period of the rhythm is progressively lengthened. These findings are discussed in terms of the general proposition that light, applied throughout the circadian cycle, predictably modifies periodicity according to the asymmetrical shape of the circadian phase response curve.Abbreviations LD light-dark cycles in which cycle length is in hours - HF-LD light-dark cycles in which cycle length is in min; period of the activity rhythm; change in period of the activity rhythm - PRC phase response curve - LL constant light     

A short red light pulse during dark phase of LD-cycle perturbs the hamster's circadian clock

In this study we investigated the influence of red light, which naturally occurs during dawn and dusk, on locomotor activity and body temperature rhythms of Djungarian hamsters (Phodopus sungarus). A single weak red light pulse given 2 h before regular lights on had acute as well as long-term effects persisting for several days following exposure. The hamsters immediately stopped their locomotor activity, accompanied by a drop in body temperature. In the following undisturbed nights (LD 168) the nocturnal activity stopped earlier than usual. This lasting effect of the light pulse was more pronounced than the acute effect. The activity phase compressed gradually during 3 to 5 days after the light pulse was administered while time of activity onset was almost unaffected. It took 6 to 11 days for complete recovery of the original activity phase. The maximal activity compression and the recovery period depended on the duration of the single red light pulse and its intensity. Red light pulses of 15 min duration were about twice effective as 1 min pulses; and the effect of a red light pulse of 130 mW/m² was about 1.5 times stronger than a 30 mW/m² red light pulse. The maximal value of activity phase compression reached in this experiment was 2.5+0.2 h with a recovery period of 11.1±0.3 days following a given red light pulse of 90 mW/m² and 15 min. The morning oscillator seems to be persistently affected. This indicates a very high photosensitivity of the Djungarian hamster's circadian system to red light.Abbreviations T _b body temperature - DD constant darkness - LD light:dark cycle - LL constant light - duration of activity phase - CT circadian time - PRC phase response curve - SCN suprachiasmatic nuclei     

Overlapping of the coding regions for alpha and gamma components of penicillin-binding protein 1 b in Escherichia coli

Summary The mode of biosynthesis of penicillin-binding protein(PBP)-1 b in Escherichia coli was investigated by use of the plasmid carrying the ponB(PBP-1 b) gene region. Analyses of the products synthesized in minicells and in vitro showed that PBP-1 b was synthesized as two molecular species corresponding to the and components of PBP-1 b. The coding regions for the and components were located within the ca. 3.7 kb MluI-HincII fragment and transcribed in the direction from the HincII to the MluI site. The capacity for producing the component was abolished by a deletion extending to the MluI site ca. 0.7 kb inward from the HincII end of the ca. 3.7 kb fragment; the remaining 3.0 kb region with the MluI site at both ends directed the production of the component alone. The production of the component was enough to correct all the known defects caused by a ponB mutation. In addition to these results, the analyses for cross-reacting materials produced in correspondence to the various deletions indicated that the coding regions for the and components overlapped and that the N-terminal portion was responsible for the difference between the two components. The distal region about 0.7 kb long inward from the MluI end of the MluI-HincII fragment was dispensable for producing the functional PBP-1 b, although the PBP-1 b produced was curtailed. By a larger distal deletion reaching almost to the middle of the MluI-HincII fragment, the polypeptide produced for PBP-1 b lost the ability to bind penicillin and still retained a low but significant activity for glycan synthesis. We suggest, therefore, that the polypeptide portion required for transglycosylase activity resides on the N-terminal half of PBP-1 b, followed by the middle portion necessary for penicillin-binding and the C-terminal part dispensable for the function of PBP-1 b.     

Antimicrobial activity and biofilm formation inhibition of green tea polyphenols on human teeth

The antimicrobial effects and biofilm formation inhibition of tea polyphenols (TPP) extracted from Korean green tea (Camellia sinensis L) were evaluated against 12 oral microorganisms. Effective antimicrobial activity against all microorganisms tested, including Lactobacillus spp. (Lactobacillus acidophilus and Lactobacillus plantarum), Streptococcus spp. (Streptococcus mutans, Streptococcus sanguis, Streptococcus sobrinus, Streptococcus mitis, and Streptococcus salivarius), Staphylococcus aureus, Neisseria meningitidis, Escherichia coli, Enterobacter cloacae, Enterococcus faecalis, and Candida albicans, was shown at 2,000 μg/mL TPP within 5 min of incubation. Scanning electron microscopy (SEM) analysis revealed various morphological changes, such as the presence of perforations, the formation of cell aggregates, and the leakage of cytoplasmic materials from cells treated with TPP, depending on the bacteria. The potential role of TPP in biofilm formation inhibition on human teeth was evaluated in BHI broth with 2 mixed strains of S. mutans and S. sanguis. SEM analysis showed biofilm formation on the surface of a tooth shaken only in saline solution, whereas almost no biofilm was observed on a tooth incubated in TPP solution. This result suggests that TPP is effective against adherent cells of S. mutans and S. sanguis. Thus, TPP would be useful for development as an antimicrobial agent against oral microorganisms, and has great potential for use in mouthwash solutions for the prevention and treatment of dental caries.     

Subcellular distribution of polysaccharide depolymerase and glycoside hydrolase enzymes in rumen ciliate protozoa

The distribution of polysaccharide depolymerase and glycoside (acid) hydrolase activity in nine genera of rumen entodiniomorphid and holotrich ciliate protozoa was examined by differential centrifugation. Sedimentable activity was detected in all of the protozoa examined and occurred principally in fractions that were prepared by centrifugation at 1000g for 10 min, 10,000g for 10 min, and 20,000g for 20 min (fractions F1, F2, and F3). Acid phosphatase was present in these subcellular fractions which contained membrane-bound vesicles 0.1–0.8 m in size. The enzyme location profile of the subcellular fractions differed within the genera examined. The distribution of the enzyme activity in the subcellular fractions indicated the presence of distinct populations of hydrolase-containing organelles and other functional vesicles in the rumen ciliates.