A Novel Nano-approach for Targeted Inner Ear Imaging.

During the last decade, there have been major improvements in imaging modalities and the development of molecular imaging in general. However detailed inner ear imaging still provides very limited information to physicians. This is unsatisfactory as sensorineural hearing loss is the main cause of permanent hearing loss in adults and at least 134 genetic mutations that result in congenital hearing loss have been identified. We are still unable, in most cases where gross anatomical changes are not observed, to determine the exact cause of hearing loss at a cellular or molecular level in patients using non-invasive techniques. This limitation in inner ear diagnostic modalities is a major obstacle behind the delay in discovering treatments for many of the causes of sensorineural hearing loss. This paper initially investigated the use of targeted gold nanoparticles as contrast agents for inner ear imaging. These nanoparticles have many useful characteristics such as being easy to target and possessing minimal cytotoxicity. We were able to detect the nanoparticles diffusing in the hair cells using confocal microscopy. Regrettably, despite their many admirable characteristics, the gold nanoparticles were unable to significantly enhance CT imaging of the inner ear. Consequently, we investigated liposomal iodine as a potential solution for the unsatisfactory CT contrast obtained with the gold nanoparticles. Fortunately, significant enhancement of the micro-CT image was observed with either Lugol's solution or liposomal iodine, with Lugol's solution enabling fine inner ear structures to be detected.

Misassembly of full-length Disrupted-in-Schizophrenia 1 protein is linked to altered dopamine homeostasis and behavioral deficits.

Disrupted-in-schizophrenia 1 (DISC1) is a mental illness gene first identified in a Scottish pedigree. So far, DISC1-dependent phenotypes in animal models have been confined to expressing mutant DISC1. Here we investigated how pathology of full-length DISC1 protein could be a major mechanism in sporadic mental illness. We demonstrate that a novel transgenic rat model, modestly overexpressing the full-length DISC1 transgene, showed phenotypes consistent with a significant role of DISC1 misassembly in mental illness. The tgDISC1 rat displayed mainly perinuclear DISC1 aggregates in neurons. Furthermore, the tgDISC1 rat showed a robust signature of behavioral phenotypes that includes amphetamine supersensitivity, hyperexploratory behavior and rotarod deficits, all pointing to changes in dopamine (DA) neurotransmission. To understand the etiology of the behavioral deficits, we undertook a series of molecular studies in the dorsal striatum of tgDISC1 rats. We observed an 80% increase in high-affinity DA D2 receptors, an increased translocation of the dopamine transporter to the plasma membrane and a corresponding increase in DA inflow as observed by cyclic voltammetry. A reciprocal relationship between DISC1 protein assembly and DA homeostasis was corroborated by in vitro studies. Elevated cytosolic dopamine caused an increase in DISC1 multimerization, insolubility and complexing with the dopamine transporter, suggesting a physiological mechanism linking DISC1 assembly and dopamine homeostasis. DISC1 protein pathology and its interaction with dopamine homeostasis is a novel cellular mechanism that is relevant for behavioral control and may have a role in mental illness.

Increased expression of the dopamine transporter leads to loss of dopamine neurons, oxidative stress and l-DOPA reversible motor deficits.

The dopamine transporter is a key protein responsible for regulating dopamine homeostasis. Its function is to transport dopamine from the extracellular space into the presynaptic neuron. Studies have suggested that accumulation of dopamine in the cytosol can trigger oxidative stress and neurotoxicity. Previously, ectopic expression of the dopamine transporter was shown to cause damage in non-dopaminergic neurons due to their inability to handle cytosolic dopamine. However, it is unknown whether increasing dopamine transporter activity will be detrimental to dopamine neurons that are inherently capable of storing and degrading dopamine. To address this issue, we characterized transgenic mice that over-express the dopamine transporter selectively in dopamine neurons. We report that dopamine transporter over-expressing (DAT-tg) mice display spontaneous loss of midbrain dopamine neurons that is accompanied by increases in oxidative stress markers, 5-S-cysteinyl-dopamine and 5-S-cysteinyl-DOPAC. In addition, metabolite-to-dopamine ratios are increased and VMAT2 protein expression is decreased in the striatum of these animals. Furthermore, DAT-tg mice also show fine motor deficits on challenging beam traversal that are reversed with l-DOPA treatment. Collectively, our findings demonstrate that even in neurons that routinely handle dopamine, increased uptake of this neurotransmitter through the dopamine transporter results in oxidative damage, neuronal loss and l-DOPA reversible motor deficits. In addition, DAT over-expressing animals are highly sensitive to MPTP-induced neurotoxicity. The effects of increased dopamine uptake in these transgenic mice could shed light on the unique vulnerability of dopamine neurons in Parkinson's disease.

NMDA receptor-deficient mice display sexual dimorphism in the onset and severity of behavioural abnormalities.

N-methyl-d-aspartate (NMDA) receptor-deficient mice can be used to understand the role that NMDA receptors (NMDARs) play in the pathophysiology of neurodevelopmental disorders such as schizophrenia. Genetically modified mice with low levels of NR1 subunit (NR1 knockdown mice) have reduced receptor levels throughout development, and have robust abnormalities in behaviours that are relevant to schizophrenia. We traced the onset and severity of these behaviours at three developmental stages to understand when in development the underlying circuits depend on intact NMDAR function. We examined social behaviour, working memory, executive function, locomotor activity and stereotypy at 3, 6 and 12 weeks of age in NR1 knockdown mice and their wild-type littermates. We discovered that each of these behaviours had a unique developmental trajectory in mutant mice, and males showed an earlier onset and severity than females in several behaviours. Hyperlocomotion was most substantial in juvenile mice and plateaued in adult mice, whereas stereotypy progressively worsened with age. Impairments in working memory and sociability were sexually dimorphic, with deficits first detected in peri-adolescent males but only detected in adult females. Interestingly, executive function was most impaired in peri-adolescent mice of either sex. Furthermore, while juvenile mutant mice had some ability to problem solve in the puzzle box test, the same mice lost this ability when tested 4 weeks later. Our studies highlight key developmental periods for males and females in the expression of behaviours that are relevant to psychiatric disorders.

Diazepam improves aspects of social behaviour and neuron activation in NMDA receptor-deficient mice.

NR1 knockdown (NR1KD) mice are genetically modified to express low levels of the NR1 subunit of N-methyl-D-aspartate (NMDA) receptors, and show deficits in affiliative social behaviour. In this study, we determined which brain regions were selectively activated in response to social stimulation and asked whether differences in neuronal activation could be observed in mice with reduced sociability. Furthermore, we aimed to determine whether brain activation patterns correlated with the amelioration of social deficits through pharmacological intervention. The cingulate cortex, lateral septal nuclei, hypothalamus, thalamus and amygdala showed an increase in c-Fos immunoreactivity that was selective for exposure to social stimuli. NR1KD mice displayed a reduction in social behaviour and a reduction in c-Fos immunoreactivity in the cingulate cortex and septal nuclei. Acute clozapine did not significantly alter sociability; however, diazepam treatment did increase sociability and neuronal activation in the lateral septal region. This study has identified the lateral septal region as a neural substrate of social behaviour and the GABA system as a potential therapeutic target for social dysfunction.

N-aryl piperazine metabotropic glutamate receptor 5 positive allosteric modulators possess efficacy in preclinical models of NMDA hypofunction and cognitive enhancement.

Impaired transmission through glutamatergic circuits has been postulated to play a role in the underlying pathophysiology of schizophrenia. Furthermore, inhibition of the N-methyl-d-aspartate (NMDA) subtype of ionotropic glutamate receptors (NMDAR) induces a syndrome that recapitulates many of the symptoms observed in patients with schizophrenia. Selective activation of metabotropic glutamate receptor subtype 5 (mGlu5) may provide a novel therapeutic approach for treatment of symptoms associated with schizophrenia through facilitation of transmission through central glutamatergic circuits. Here, we describe the characterization of two novel N-aryl piperazine mGlu5 positive allosteric modulators (PAMs): 2-(4-(2-(benzyloxy)acetyl)piperazin-1-yl)benzonitrile (VU0364289) and 1-(4-(2,4-difluorophenyl)piperazin-1-yl)-2-((4-fluorobenzyl)oxy)ethanone (DPFE). VU0364289 and DPFE induced robust leftward shifts in the glutamate concentration-response curves for Ca(2+) mobilization and extracellular signal-regulated kinases 1 and 2 phosphorylation. Both PAMs displayed micromolar affinity for the common mGlu5 allosteric binding site and high selectivity for mGlu5. VU0364289 and DPFE possessed suitable pharmacokinetic properties for dosing in vivo and produced robust dose-related effects in reversing amphetamine-induced hyperlocomotion, a preclinical model predictive of antipsychotic-like activity. In addition, DPFE enhanced acquisition of contextual fear conditioning in rats and reversed behavioral deficits in a mouse model of NMDAR hypofunction. In contrast, DPFE had no effect on reversing apomorphine-induced disruptions of prepulse inhibition of the acoustic startle reflex. These mGlu5 PAMs also increased monoamine levels in the prefrontal cortex, enhanced performance in a hippocampal-mediated memory task, and elicited changes in electroencephalogram dynamics commensurate with procognitive effects. Collectively, these data support and extend the role for the development of novel mGlu5 PAMs for the treatment of psychosis and cognitive deficits observed in individuals with schizophrenia.

Overlapping sites of tetratricopeptide repeat protein binding and chaperone activity in heat shock protein 90.

The sequential binding of different tetratricopeptide repeat (TPR) proteins to heat shock protein 90 (hsp90) is essential to its chaperone function in vivo. We have previously shown that three basic residues in the TPR domain of PP5 are required for binding to the acidic C-terminal domain of hsp90. We have now tested which acidic residues in this C-terminal domain are required for binding to three different TPR proteins as follows: PP5, FKBP52, and Hop. Mutation of Glu-729, Glu-730, and Asp-732 at the C terminus of hsp90 interfered with binding of all three TPR proteins. Mutation of Glu-720, Asp-722, Asp-723, and Asp-724 inhibited binding of FKBP52 and PP5 but not of Hop. Mutation of Glu-651 and Asp-653 did not affect binding of FKBP52 or PP5 but inhibited both Hop binding and hsp90 chaperone activity. We also found that a conserved Lys residue required for PP5 binding to hsp90 was critical for the binding of FKBP52 but not for the binding of Hop to hsp90. These results suggest distinct but overlapping binding sites on hsp90 for different TPR proteins and indicate that the binding site for Hop, which is associated with hsp90 in intermediate stages of protein folding, overlaps with a site of chaperone activity.

Effects of phosphorylation on binding of catecholamines to tyrosine hydroxylase: specificity and thermodynamics.

As the catalyst for the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters, the activity of tyrosine hydroxylase is tightly regulated. A principle means of posttranslational regulation is reversible phosphorylation of serine residues in an N-terminal regulatory domain. Phosphorylation of serine 40 has been shown to have a large effect on the rate constant for dissociation of dopamine and a much smaller effect on that for DOPA [Ramsey, A. J., and Fitzpatrick, P. F. (1998) Biochemistry 37, 8980-8986]. To determine the structural basis for the differences in affinity and to further test the validity of the previously proposed model for regulation, the effects of phosphorylation of serine 40 on the affinities for a series of catechols have been determined. The affinities of the unphosphorylated enzyme vary by 3 orders of magnitude due to differences in the rates of dissociation. The highest affinities are found with catecholamines which lack a carboxylate. The affinities of the phosphorylated enzyme show a much smaller range. In the case of binding of dihydroxyphenylalanine, the decrease in affinity upon phosphorylation is due primarily to a decrease in the enthalpy of the interaction. Based upon these results, a structural model for the effect of phosphorylation is proposed.

Effects of phosphorylation of serine 40 of tyrosine hydroxylase on binding of catecholamines: evidence for a novel regulatory mechanism.

The effects of phosphorylation at Ser40 of rat tyrosine hydroxylase on the affinities of catechols have been determined with both the ferric and ferrous forms of the enzyme. Phosphorylation had no effect on the Ki value for the inhibition of the ferrous enzyme by either dopamine or DOPA when the initial rate of turnover was measured in assays. However, phosphorylation of the ferric enzyme resulted in a 17-fold decrease in affinity for DOPA and a 300-fold decrease in the affinity for dopamine, while the affinity for dihydroxynaphthalene was unchanged. The changes in binding affinity for the two catecholamines were almost exclusively due to large increases in the dissociation rate constants upon phosphorylation. These results support a novel mechanism for regulation in which phosphorylation affects binding of catecholamines to the catalytically inactive ferric form of the tyrosine hydroxylase.

Characterization of the active site iron in tyrosine hydroxylase. Redox states of the iron.

Tyrosine hydroxylase is an iron-containing monooxygenase that uses a tetrahydropterin to catalyze the hydroxylation of tyrosine to dihydroxyphenylalanine in catecholamine biosynthesis. The role of the iron in this enzyme is not understood. Purification of recombinant rat tyrosine hydroxylase containing 0.5-0.7 iron atoms/subunit and lacking bound catecholamine has permitted studies of the redox states of the resting enzyme and the enzyme during catalysis. As isolated, the iron is in the ferric form. Dithionite or 6-methyltetrahydropterin can reduce the iron to the ferrous form. Reduction by 6-methyltetrahydropterin consumes 0.5 nmol/nmol of enzyme-bound iron, producing quinonoid 6-methyldihydropterin as the only detectable product. In the presence of oxygen, reoxidation to ferric iron occurs. During turnover the enzyme is in the ferrous form. However, a fraction is oxidized during turnover; this can be trapped by added catechol or by the dihydroxyphenylalanine formed during turnover.

Identification of iron ligands in tyrosine hydroxylase by mutagenesis of conserved histidinyl residues.

Tyrosine hydroxylase catalyzes the hydroxylation of tyrosine and other aromatic amino acids using a tetrahydropterin as the reducing substrate. The enzyme is a homotetramer; each monomer contains a single nonheme iron atom. Five histidine residues are conserved in all tyrosine hydroxylases that have been sequenced to date and in the related eukaryotic enzymes phenylalanine and tryptophan hydroxylase. Because histidine has been suggested as a ligand to the iron in these enzymes, mutant tyrosine hydroxylase proteins in which each of the conserved histidines had been mutated to glutamine or alanine were expressed in Escherichia coli. The H192Q, H247Q, and H317A mutant proteins contained iron in comparable amounts to the wild-type enzyme, about 0.6 atoms/sub-unit. In contrast, the H331 and H336 mutant proteins contained no iron. The first three mutant enzymes were active, with Vmax values 39, 68, and 7% that of the wild-type enzyme, and slightly altered V/Km values for both tyrosine and 6-methyltetrahydropterin. In contrast, the H331 and H336 mutant enzymes had no detectable activity. The EPR spectra of the H192Q and H247Q enzymes are indistinguishable from that of wild-type tyrosine hydroxylase, whereas that of the H317A enzyme indicated that the ligand field of the iron had been slightly perturbed. These results are consistent with H331 and H336 being ligands to the active site iron atom.

Sequence and crystallization of Escherichia coli dethiobiotin synthetase, the penultimate enzyme of biotin biosynthesis.

The enzyme dethiobiotin synthetase (EC 6.3.3.3) has been cloned and over-expressed in Escherichia coli in such a way that milligram quantities are available. The purified enzyme has been subjected to a number of physical and chemical studies, sequenced and most notably it has been crystallized in a form that is suitable for X-ray structure determination. The cell dimensions are a = 72.8 A, b = 49.2 A, c = 61.4 A, beta = 106.2 degrees. The systematic absences are consistent with the monoclinic space group C2 with one polypeptide chain in the asymmetric unit.

Preparation and properties of recombinant corynebacterial sarcosine oxidase: evidence for posttranslational modification during turnover with sarcosine.

The genes encoding the four subunits of sarcosine oxidase from Corynebacterium sp. P-1 were isolated and overexpressed in a single step by using indicator plates to screen a genomic library for colonies that generated hydrogen peroxide in a sarcosine-dependent reaction. The genomic library was constructed by inserting size-fractionated genomic DNA, previously subjected to partial digestion by Sau3AI, into pBluescript II SK (+). At least 1.0 kb, but less than 4.0 kb, can be deleted from the 3' end of the original cornyebacterial insert (7.3 kb) without affecting sarcosine oxidase expression, consistent with the estimated 5.0-kb operon size. Recombinant sarcosine oxidase is isolated as a heterotetramer containing equimolar amounts of covalent and noncovalent flavin, identical to that observed for enzyme isolated from Corynebacterium sp. P-1. Despite its similar flavin content, recombinant enzyme exhibits significantly different spectral properties than enzyme from Corynebacterium sp. P-1 (values shown in parentheses) [epsilon 450 = 9.7 (12.7) mM-1 cm-1; A368/A450 = 1.0 (0.83); A280/A450 = 16.9 (12.2)]. This difference is due to the fact that about half of the covalent flavin in recombinant enzyme forms a reversible covalent 4a-adduct with a cysteine residue (lambda max = 383 nm; epsilon 383 = 7.3 mM-1 cm-1). The equilibrium is shifted in favor of adduct dissociation by oxidizing the cysteine residue with hydrogen peroxide or by alkylation with methyl methanethiosulfonate in a reaction that is fully reversible upon addition of excess dithiothreitol. The cysteine residue is also oxidized during aerobic turnover with sarcosine. Reaction of the cysteine residue with hydrogen peroxide (or a precursor) formed during turnover partially competes with the release of hydrogen peroxide into solution, as judged by the effect of catalase on this reaction. Although the same specific activity is observed for recombinant enzyme and enzyme from Corynebacterium sp. P-1, the recombinant enzyme exhibits a pronounced lag in an NADH peroxidase-coupled assay. The lag is eliminated by prior disruption of the 4a-thiolate adduct via reaction with hydrogen peroxide or methyl methanethiosulfonate. The results show that the 4a-thiolate adduct is an inactive form of sarcosine oxidase that can be activated by reaction with sarcosine in what appears to be the first example of a posttranslational modification associated with turnover. Complete activation occurs in vivo when sarcosine oxidase is produced in Corynebacterium sp. P-1, where enzyme synthesis is induced by growth of the organism with sarcosine as the source of carbon and energy.(ABSTRACT TRUNCATED AT 400 WORDS)

Properties of a high-potential flavin analogue and its use as an active site probe with clostridial flavodoxin.

The reduction potential of flavin bearing a methylsulfonyl moiety (MeSO2) in place of a methyl group at position 8 is increased by more than 150 mV as compared with normal flavin. This substitution is accompanied by a substantial increase in reactivity with various reductants, including NADH, and greatly (10(3)-fold) enhanced susceptibility toward nucleophilic attack by sulfite at N(5). 1,5-Dihydro-8-(methylsulfonyl)riboflavin exhibits two intense, well-resolved absorption bands (lambda max = 310, 362 nm) in a region where most other reduced flavins exhibit weak, characterless absorption. This unusual spectrum is attributable to a shift of pi-electron density from the N(5) atom into the benzene ring. It is observed only with reduced flavins bearing a strongly electronegative substituent (MeSO2, CN) at the 8-position. The effect is abolished by replacing the hydrogen at N(5) with a bulky group, like sulfite, which interferes with sp2 hybridization at N(5). Reaction of 8-MeSO2-substituted flavins with thiols results in nucleophilic displacement of MeSO2- in a reaction that is about 10(3)-fold faster than an analogous nucleophilic displacement reaction observed with 8-halo-substituted flavins. The flavin ring acts as a redox switch in controlling electrophilicity at the 8-position, as judged by the fact that the displacement reactions are observed only with the oxidized flavins. Initial studies to evaluate 8-MeSO2-substituted flavins as active site probes were conducted with flavodoxin from Clostridium beijerinckii MP. 8-MeSO2FMN is rapidly bound to apoflavodoxin, accompanied by absorbance and fluorescence changes similar to those observed for FMN binding. 1,5-Dihydro-8-MeSO2FMN flavodoxin exhibits spectral properties (lambda max = 323, 382 nm) similar to those of the corresponding free flavin, except for a bathochromic shift due to a change in the polarity of the flavin environment. As judged by peak resolution and intensity, the spectral properties of 1,5-dihydro-FMN flavodoxin (lambda max = 311, 362 nm) appear to lie about midway between those observed for the free 1,5-dihydro forms of FMN versus 8-MeSO2FMN. This suggests that the protein environment may favor enhanced resonance delocalization of pi-electron density into the benzene ring of bound 1,5-dihydro-FMN, as compared with the free flavin. This hypothesis is consistent with previous NMR studies and with a proposal that electron transfer from reduced flavodoxin to other redox proteins occurs through this region of the ring. 8-MeSO2FMN bound to flavodoxin reacts readily with exogenous thiols but does not react with sulfite.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of 5-deazaflavin on energy transduction during catalysis by Escherichia coli DNA photolyase.

DNA photolyase from Escherichia coli contains 1,5-dihydroFAD (FADH2) plus 5,10-methenyltetrahydropteroylpolyglutamate. The action spectrum observed for apoenzyme reconstituted with 5-deazaFADH2 (EdFADH2) matched its absorption spectrum after correction for the presence of a small amount of inactive 5-deazaFADox. The quantum yield for dimer repair with EdFADH2 (phi EdFADH2 = 0.110) was 6-fold lower than that observed with apoenzyme reconstituted with FADH2. Excited-state redox potential calculations indicate that 5-deazaFADH2 singlet is a better one-electron donor (E = -3.5 V) than FADH2 singlet (E = -2.7 V). Other studies indicate that the quantum yield for electron transfer from reduced flavin singlet to pyrimidine dimer (0.88) is unaffected when FADH2 is replaced by 5-deazaFADH2. Enhanced back electron transfer from pyrimidine dimer radical to flavin radical may account for the decreased quantum yield observed with EdFADH2 since, in the ground state, 5-deazaFADH. is a better oxidant than FADH.. The action spectrum observed for apoenzyme reconstituted with 5-deazaFADH2 plus 5,10-CH(+)-H4folate (EPtedFADH2) matched the absorption spectrum determined for enzyme-bound 5-deazaFADH2, indicating that the pterin chromophore was inactive as a sensitizer. This differs from results obtained with native enzyme, where pterin acts as a sensitizer via efficient singlet-singlet energy transfer to FADH2. The quantum yield for dimer repair by 5-deazaFADH2 bound to EPtedFADH2 (phi EPtedFADH2 = 0.0318) was 28.9% of that observed for EdFADH2. Spectroscopic studies indicate that singlet-singlet energy transfer in EPtedFADH2 is very efficient but only occurs in the "wrong" direction, i.e., from excited 5-deazaFADH2 to pterin.(ABSTRACT TRUNCATED AT 250 WORDS)

Energy transduction during catalysis by Escherichia coli DNA photolyase.

Native DNA photolyase from Escherichia coli contains 1,5-dihydroFAD (FADH2) plus 5,10-methenyltetrahydropteroylpolyglutamate. Quantum yield and action spectral data for thymine dimer repair were obtained by using a novel multiple turnover approach under aerobic conditions. This method assumes that catalysis proceeds via a (rapid-equilibrium) ordered mechanism with light as the second substrate, as verified in steady state kinetic studies. The action spectrum observed with native enzyme matched its absorption spectrum and an action spectrum simulated based on an energy transfer mechanism where dimer repair is initiated either by direct excitation of FADH2 or by pterin excitation followed by singlet-singlet energy transfer to FADH2. The quantum yield observed for dimer repair with native enzyme (phi Native = 0.722 +/- 0.0414) is similar to that observed with enzyme containing only FADH2 (phi EFADH2 = 0.655 +/- 0.0256), as expected owing to the high efficiency of energy transfer from the natural pterin to FADH2 [EET = 0.92]. The quantum yield observed for dimer repair decreased (2.1-fold) when the natural pterin was partially (68.8%) replaced with 5,10-CH(+)-H4folate (phi obs = 0.342 +/- 0.0149). This is consistent with the energy transfer mechanism (phi calc = 0.411 +/- 0.0118) since a 2-fold lower energy transfer efficiency is observed when the natural pterin is replaced with 5,10-CH(+)-H4folate (EET = 0.46) (Lipman & Jorns, 1992). The action spectrum observed for 5,10-CH(+)-H4folate-supplemented enzyme matched a simulated action spectrum which exhibited a small (5 nm) hypsochromic shift as compared with the absorption spectrum (lambda max = 385 nm).(ABSTRACT TRUNCATED AT 250 WORDS)

Growth inhibitory and biocidal activity of some isothiazolone biocides.

Similar patterns of growth inhibition were observed for the three biocides, benzisothiazol-3-one (BIT), 5-chloro-N-methylisothiazol-3-one (CMIT) and N-methylisothiazol-3-one (MIT) against Escherichia coli ATCC 8739 and Schizosaccharomyces pombe NCYC 1354. After periods of induced stasis, proportional to biocide concentration, growth proceeded at an inhibited rate. Extrapolation of the static periods and inhibited growth rates against biocide concentration gave minimum growth inhibitory concentration estimates of 0.1-0.5 micrograms/ml for CMIT, 15-20 micrograms/ml for BIT and 40-250 micrograms/ml for MIT. Patterns of growth inhibition by CMIT and induced morphological changes in inhibited cultures suggested this compound to also inhibit initiation of DNA replication. Growth inhibitory activity was rapidly quenched by the addition of thiol-containing materials such as glutathione and cysteine. The activity of CMIT was additionally quenched by the presence of the non-thiol amino acids valine and/or histidine. These results suggest that the chlorinated isothiazolones can react with amines as well as with essential thiol groups.

Some comparisons of pig and sheep liver cytosolic aldehyde dehydrogenases.

1. The pig enzyme was purified to homogeneity and was found to be a tetramer of apparently identical subunits. 2. The pig enzyme was found to contain 1 mol NADH/mol enzyme which is tightly bound, which is not directly involved in catalysis and which so far has not been removed from the enzyme so as to produce an active apoenzyme. 3. The pig enzyme seems to contain only one functioning active site/tetramer. 4. The pig and sheep enzymes are compared in respect of NADH binding, substrate specificity, immunological response and surface charge.

Short-term dietary regulation of lipogenesis in the lactating mammary gland of the rat.

Short-term (6 hr) withdrawal of chow diet from lactating rats decreases the rate of lipogenesis in mammary gland by 87%. This inhibition is in part explained by a 60% decrease in the extraction of glucose (the major lipogenic precursor) by the mammary tissue. These changes are not accompanied by any significant alteration in the arterial concentrations of glucose, lactate or insulin; the concentration of acetoacetate did increase by about 30%. Removal of food for 6 hr did not alter the activation state of acetyl-CoA carboxylase or the total activity of the enzyme. Glucose utilization by mammary gland acini from short-term starved rats was not depressed although a higher proportion of the glucose appeared as lactate in the medium and consequently less glucose was converted to lipid. Insulin was able to reverse these changes. Glucagon, adrenaline or cAMP did not inhibit glucose utilization or lipogenesis in isolated acini. It is concluded that the inhibition of lipogenesis in mammary gland after short-term withdrawal of food is mainly due to decreased extraction of glucose. The signal for this change does not appear to be an alteration in plasma insulin and it is postulated that there may be an intestinal factor(s) which acts synergistically with insulin.