A simple and inexpensive method for cellulase and β-glucosidase production by Neurospora crassa

Summary Elevated levels of cellobiohydrolase, carboxymethylcellulase (CMCase) and -glucosidase were produced by strain STA of Neurospora crassa, grown in solid-state fermentation on untreated wheat straw supplemented with simple mineral salts. Yields as high as 6.1 units of cellobiohydrolase, 969.2 units of CMCase and 169.4 units of -glucosidase per gram of straw were obtained at optimum growth and enzyme assay conditions.     

Estimating position and velocity of a submerged moving object by the clawed frog Xenopus and by fish—A cybernetic approach

The lateral-line system is a unique facility of aquatic animals to locate predator, prey, or conspecifics. We present a detailed model of how the clawed frog Xenopus, or fish, can localize submerged moving objects in three dimensions by using their lateral-line system. In so doing we develop two models of a slightly different nature. First, we exploit the characteristic properties of the velocity field, such as zeros and maxima or minima, that a moving object generates at the lateral-line organs and that are directly accessible neuronally, in the context of a simplified geometry. In addition, we show that the associated neuronal model is robust with respect to noise. Though we focus on the superficial neuromasts of Xenopus the same arguments apply mutatis mutandis to the canal lateral-line system of fish. Second, we present a full-blown three-dimensional reconstruction of the source on the basis of a maximum likelihood argument.     

Stimulation and inhibition of anaerobic processes by heavy metals—A review

Stimulatory, inhibitory and antagonistic roles of heavy metals in anaerobic digestion are reviewed. Partitioning and speciation, variety of biotic and abiotic detoxifying mechanisms as well as acclimation are shown to hinder an accurate prediction of response based on a given total metal concentration.     

Regulation of macrophage differentiation and polarization by group IVC phospholipase A₂

Although the cellular function of group IVC phospholipase A(2) (IVC-PLA(2)) remains to be understood, the expression of IVC-PLA(2) in human monocytic THP-1 cells was increased during phorbol ester-induced macrophage differentiation. We therefore examined the role of IVC-PLA(2) in macrophage differentiation using THP-1 cells. Two THP-1 cell lines stably expressing IVC-PLA(2)-specific shRNA were established. Differentiation and maturation into macrophages on treatment with phorbol ester were facilitated by knockdown of IVC-PLA(2) without the compensatory induction of mRNA expression for other group IV and VI PLA(2)s. Furthermore, the enhancement of macrophage differentiation by IVC-PLA(2)-knockdown were abolished by treatment with lysophosphatidylcholine, a metabolite of phospholipids generated by PLA(2)-mediated hydrolysis, indicating that PLA(2) activity is necessary for the inhibition of macrophage differentiation by IVC-PLA(2). Additionally, we found that the differentiation into classically activated M1 macrophage was superior in IVC-PLA(2)-knockdown cells, whereas the differentiation into alternatively activated M2 macrophage was suppressed by IVC-PLA(2)-knockdown. These findings suggest that IVC-PLA(2) is involved in regulations of macrophage differentiation and macrophage polarization.     

Inhibition and inactivation of cytochrome P450 2A6 and cytochrome P450 2A13 by menthofuran, β-nicotyrine and menthol

Nicotine is the primary addictive agent in tobacco products and is metabolized in humans by CYP2A6. Decreased CYP2A6 activity has been associated with decreased smoking. The extrahepatic enzyme, CYP2A13 (94% identical to CYP2A6) also catalyzes the metabolism of nicotine, but is most noted for its role in the metabolic activation of the tobacco specific lung carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In this study, the inhibition and potential inactivation of CYP2A6 and CYP2A13 by two tobacco constituents, 1-methyl-4-(3-pyridinyl) pyrrole (β-nicotyrine) and (-)-menthol were characterized and compared to the potent mechanism based inactivator of CYP2A6, menthofuran. The effect of these compounds on CYP2A6 and CYP2A13 activity was significantly different. (-)-Menthol was a more efficient inhibitor of CYP2A13 than of CYP2A6 (KI, 8.2 μM and 110 μM, respectively). β-Nicotyrine was a potent inhibitor of CYP2A13 (KI, 0.17 μM). Neither menthol nor β-nicotyrine was an inactivator of CYP2A13. Whereas, β-nicotyrine was a mechanism based inactivator of CYP2A6 (KI(inact), 106 μM, kinact was 0.61 min(-1)). Similarly, menthofuran, a potent mechanism based inactivator of CYP2A6 did not inactivate CYP2A13. Menthofuran was an inhibitor of CYPA13 (KI, 1.24 μM). The inactivation of CYP2A6 by either β-nicotyrine or menthofuran was not due to modification of the heme and was likely due to modification of the apo-protein. These studies suggest that β-nicotyrine, but not menthol may influence nicotine and NNK metabolism in smokers.     

Hydralazine modifies Aβ fibril formation and prevents modification by lipids in vitro

Lipid oxidative damage and amyloid β (Aβ) misfolding contribute to Alzheimer's disease (AD) pathology. Thus, the prevention of oxidative damage and Aβ misfolding are attractive targets for drug discovery. At present, no AD drugs approved by the Food and Drug Administration (FDA) prevent or halt disease progression. Hydralazine, a smooth muscle relaxant, is a potential drug candidate for AD drug therapy as it reduces Aβ production and prevents oxidative damage via its antioxidant hydrazide group. We evaluated the efficacy of hydralazine, and related hydrazides, in reducing (1) Aβ misfolding and (2) Aβ protein modification by the reactive lipid 4-hydroxy-2-nonenal (HNE) using transmission electron microscopy and Western blotting. While hydralazine did not prevent Aβ aggregation as measured using the protease protection assay, there were more oligomeric species observed by electron microscopy. Hydralazine prevented lipid modification of Aβ, and Aβ was used as a proxy for classes of proteins which either misfold or are modified by HNE. All of the other hydrazides prevented lipid modification of Aβ and also did not prevent Aβ aggregation. Surprisingly, a few of the compounds, carbazochrome and niclosamide, appeared to augment Aβ formation. Thus, hydrazides reduced lipid oxidative damage, and hydralazine additionally reduced Aβ misfolding. While hydralazine would require specific chemical modifications for use as an AD therapeutic itself (to improve blood brain barrier permeability, reduce vasoactive side effects, and optimization for amyloid inhibition), this study suggests its potential merit for further AD drug development.     

Lipase immobilized by modification-coupled and adsorption–cross-linking methods: A comparative study

Candida antarctica lipase was immobilized by an adsorption and cross-linking method with NW-ZT2 and by modification-coupled method with a silica–PEG gel. The final product silica–PEG-lipase was confirmed by IR spectra. The optimum pH value, the optimum temperature, the thermo-stabilities and operational stabilities for two kinds of immobilized lipase were also determined. Results show that the silica–PEG-lipase gel was superior to the lipase immobilized by adsorption and cross-linking, however both are viable for use in transesterification reactions.     

Characterization and inhibition of aminopeptidase A by α-mercapto-β-amino acyl dipeptides

Summary In order to study the physiological role of aminopeptidase A(APA), several α-mercapto-β-amino acyl dipeptides were synthesized to obtain compounds having a high affinity for APA and a high selectivity versus aminopeptidase N (APN). Sulfornamide and carboxylate moieties which have been shown to be recognized by the S₁ subsite of the enzyme were introduced on the side chain of the α-mercapto-β-amino acyl sub-unit, the latter being coupled to dipeptides optimized to interact with the S'₁ and S'₂ subsites by means of combinatorial chemistry. Good affinities (16nM) were obtained, the selectivity factors being up to 160-fold versus APN.     

Cluster analysis and disease mapping--why,when, and how? A step by step guide.

Growing public awareness of environmental hazards has led to an increased demand for public health authorities to investigate geographical clustering of diseases. Although such cluster analysis is nearly always ineffective in identifying causes of disease, it often has to be used to address public concern about environmental hazards. Interpreting the resulting data is not straightforward, however, and this paper presents a guide for the non-specialist. The pitfalls include the fact that cluster analyses are usually done post hoc, and not as a result of a prior hypothesis. This is particularly true for investigations prompted by reported clusters, which have the inherent danger of overestimating the disease rate through "boundary shrinkage" of the population from which the cases are assumed to have arisen. In disease surveillance the problem of making multiple comparisons can be overcome by testing for clustering and autocorrelation. When rates of disease are illustrated in disease maps undue focus on areas where random fluctuation is greatest can be minimised by smoothing techniques. Despite the fact that cluster analyses rarely prove fruitful in identifying causation, they may-like single case reports-have the potential to generate new knowledge.     

Synthesis of 27-Oxo, 27-Hydroxymilbemycins A3 and A4 and Novel 27-Alkoxymilbemycins A3 and A4 from Milbemycins A3 and A4 and…

27-Oxomilbemycins A₃ and A₄ and 27-hydroxymilbemycins A₃ and A₄ were identified as metabolites in soil metabolism studies of milbemycins A₃ and A₄. Chemical derivation methods were developed to synthesize 27-oxomilbemycins A₃ and A₄ and 27-hydroxymilbemycins A₃ and A₄ from milbemycins A₃ and A₄. In addition, 27-alkoxymilbemycin derivatives were also synthesized from the same precursors. Some of the synthesized compounds displayed satisfactory acaricidal activity against the organophosphorus-sensitive two-spotted spider mite (Tetranychus urticae), but did not have superior activity to corresponding milbemycins A₃ and A₄.     

Analysis of Aβ (1-40) and Aβ (1-42) monomer and fibrils by capillary electrophoresis

A method based on capillary electrophoresis (CE) with UV absorbance detection is presented to characterize synthetic amyloid beta (Aβ) peptide preparations at different aggregation states. Aggregation of Aβ (1-40) and Aβ (1-42) is closely linked to Alzheimer's disease (AD), and studying how Aβ peptides self-assemble to form aggregates is the focus of intense research. Developing methods capable of identifying, characterizing and quantifying a wide range of Aβ species from monomers to fully formed fibrils is critical for AD research and is a major analytical challenge. Monomer and fibril samples of Aβ (1-40) and Aβ (1-42) were prepared and characterized for this study. The monomer-equivalent concentration for each sample was determined by HPLC-UV, and aggregate formation was confirmed and characterized by transmission electron microscopy. The same samples were studied using CE with UV absorbance detection. Analysis by mass spectrometry of collected CE fractions was used to confirm the presence of Aβ for some CE-UV peaks. The CE-UV method reported here clearly indicates that monomeric and aggregated Aβ were electrophoretically separated, and substantial differences in the electrophoretic profiles between samples of Aβ (1-40) and Aβ (1-42) were observed. This CE-UV method can differentiate between Aβ monomer, oligomeric intermediates, and mature fibrils.     

Effect of vibrostimulation frequency upon skin mechanoreceptors innervated by Aβ-, Aδ, and C-fibers

Vibrostimulation of skin from the crus of the cat at frequencies of 10, 30, and 50/sec evokes a response of mechanoreceptors innervating group A and A fibers to the presentation of each vibrostimulus. The higher the frequency of vibrostimulation, the more clearly manifest are changes in on- and off-responses on a neurogram recorded from the n. saphenus branch. These changes are a result of a decrease in the number of impulses evoked by each vibratory beat. Mechanoreceptors innervated by fibers of group C are not excited as a result of vibrostimulation at these frequencies. After preliminary vibrostimulation, a portion of the mechanoreceptors innervated by C-fibers do not respond to presentation of a mechanical test stimulus (stretching of the skin); the higher the frequency of vibrostimulation, the smaller is the number of reacting receptors.Kirov Medical Institute, Nizhny Novgorod. Translated from Neirofiziologiya, Vol. 24, No. 1, pp. 20–27, January–February, 1992.     

Monoamine oxidases A and B are differentially regulated by glucocorticoids and “aging” in human skin fibroblasts

Two forms of monoamine oxidase (MAO A and MAO B) exist which, although similar in a number of properties, can be distinguished on the basis of their substrate specificity, inhibitor sensitivity, kinetic parameters, and protein structure. These properties were used to study the molecular mechanism(s) by which glucocorticoid hormones and "aging," known to alter MAO activity in vivo, regulated the expression of MAO A and MAO B in cultured human skin fibroblasts. The addition of dexamethasone or hydrocortisone to cultures resulted in a dose- and time-dependent increase in total MAO activity, whereas the removal of hormone from cultures resulted in a time-dependent decrease in activity toward control levels. The response to dexamethasone was affected by culture conditions such as serum concentration, feeding frequency, and cellular "age." Cellular aging, in the absence of hormone, also resulted in increased levels of total MAO activity. The effects of hormones and aging on total MAO activity appeared to be selective for MAO A. The 6- to 14-fold increases in total activity were paralleled by similar increases in the activity and amount of active MAO A but less than 2- to 3-fold increases in the activity and amount of MAO B. Altered synthesis or degradation of the active enzyme appeared to account for the effects of hormones, aging, and various culture conditions on MAO activity. Inhibitor sensitivity, substrate affinity, electrophoretic mobility, and molecular turnover number of either form of the enzyme were not altered during dexamethasone treatment or during cellular aging. However, rates of active MAO synthesis were affected by hormone treatment and feeding frequency, rates of active MAO degradation by serum concentration, and rates of active MAO synthesis or degradation by aging. In summary, we have shown that glucocorticoids and cellular aging selectively affect the amount of MAO A at the level of active enzyme synthesis or degradation. Further, our finding that the expression of the two forms of MAO in human fibroblasts can be independently regulated supports the growing evidence that MAO A and MAO B are separate molecular entities.     

A rose by any other name? Rethinking the similarities and differences between male and female genital cutting

In this article, we offer a critical examination of the tendency to segregate discussion of surgical alterations to the male and female genitals into separate compartments--the first known as circumcision, the second as genital mutilation. We argue that this fundamental problem of definition underlies the considerable controversy surrounding these procedures when carried out on minors, and that it hinders objective discussion of the alleged benefits, harms, and risks. We explore the variable effects of male and female genital surgeries, and we propose a scale of damage for male circumcision to complement the World Health Organization's categorization of female genital mutilation. The origins of the double standard identified are placed in historical perspective, and in a brief conclusion we make a plea for greater gender neutrality in the approach to this contentious issue.     

Cellular DNA damage by nitrosocimetidine: A comparison with N-methyl-N′-nitroso-nitrosoguanidine and X-irradiation

Permanently proliferating lymphoblastoid cell lines (LCLs) and normal unstimulated peripheral blood leukocytes have been used to study the effects of nitrosocimetidine (NC) on cultured human lymphoid cells. The approaches that were used to assess the cells' ability to cope with NC were: (i) determination of cell survival as measured by colony formation in microtiter plates; (ii) quantitation of DNA synthesis and DNA-repair replication by isopyknic sedimentation of DNA density labeled with 5-bromo-2-deoxyuridine (BrdU); (iii) measurement of the induction of alkali labile lesions and strand breaks by NC in ³H-labeled DNA using velocity sedimentation in alkaline sucrose. In summary, treatment with NC was found to inhibit both replicative DNA synthesis and colony formation in LCLs. At the molecular level, NC treatment induced alkali labile lesions in LCL DNA and elicited DNA-repair replication in proliferating LCLs as well as unstimulated lymphocytes. Considered in total, these data indicate that NC is reactive with human DNA in the cellular environment in a manner similar to methylating nitroso compounds which have been shown to be carcinogenic. The significance of these findings will be discussed.     

Aβ Association Inhibition by Transferrin

The iron-transport glycoprotein transferrin has recently been shown to serve as a potent inhibitor of Aβ self-association. Although this novel, to our knowledge, inhibitory function of transferrin is of potential therapeutic interest for the treatment of Alzheimer’s disease, the underlying mechanism is still not fully understood. Although it has been shown that the Fe(III) sequestration by transferrin reduces oxidative damage and Aβ aggregation, it is not clear whether transferrin is also able to inhibit Aβ self-association through direct binding of Aβ. Here, using saturation transfer and off-resonance relaxation NMR spectroscopy, we show that transferrin inhibits Aβ aggregation also by preferentially binding Aβ oligomers and outcompeting Aβ monomers that would otherwise cause the growth of the Aβ oligomers into larger assemblies. This inhibitory mechanism is different from the iron-sequestration model, but it is qualitatively similar to a mechanism previously proposed for the inhibition of Aβ self-association by another plasma and cerebrospinal fluid protein, i.e., human serum albumin. These results suggest that Aβ monomer competition through direct Aβ oligomer binding might be a general strategy adopted by proteins in plasma and cerebrospinal fluid to prevent Aβ aggregation.Abbreviations used: AD, Alzheimer’s disease, CNS, central nervous system, CSF, cerebrospinal fluid, HSA, human serum albumin, ICP, inductively coupled plasma, ORR, off-resonance relaxation, SL, spin-lock, STD, saturation transfer difference, STR, saturation transfer reference, Tf, transferrin, WG, watergate water-suppression NMR technique     

Two-Step Mechanism of Membrane Disruption by Aβ through Membrane Fragmentation and Pore Formation

Disruption of cell membranes by Aβ is believed to be one of the key components of Aβ toxicity. However, the mechanism by which this occurs is not fully understood. Here, we demonstrate that membrane disruption by Aβ occurs by a two-step process, with the initial formation of ion-selective pores followed by nonspecific fragmentation of the lipid membrane during amyloid fiber formation. Immediately after the addition of freshly dissolved Aβ(1-40), defects form on the membrane that share many of the properties of Aβ channels originally reported from single-channel electrical recording, such as cation selectivity and the ability to be blockaded by zinc. By contrast, subsequent amyloid fiber formation on the surface of the membrane fragments the membrane in a way that is not cation selective and cannot be stopped by zinc ions. Moreover, we observed that the presence of ganglioside enhances both the initial pore formation and the fiber-dependent membrane fragmentation process. Whereas pore formation by freshly dissolved Aβ(1-40) is weakly observed in the absence of gangliosides, fiber-dependent membrane fragmentation can only be observed in their presence. These results provide insights into the toxicity of Aβ and may aid in the design of specific compounds to alleviate the neurodegeneration of Alzheimer's disease.     

Active invasion and/or encapsulation? A reappraisal of host-cell parasitism by Cryptosporidium

Host-cell invasion by Cryptosporidium is a complex process that requires many different factors derived from both the parasite and the host cell. However, the exact natures of the processes have yet to be resolved. Here, research on different components of the invasion process is put in context, and the sequence of events and pathways associated with the establishment of Cryptosporidium in its unique niche is clarified. In addition, initial parasite-host contact, host-cell invasion and host-cell responses are described. The roles of parasite and host-cell-derived components in the invasion process are examined, as is the question of whether Cryptosporidium actively invades cells and to what extent host-cell responses are involved.     

Regulation of coenzyme A levels by degradation: the ‘Ins and Outs’

Coenzyme A (CoA) is the predominant acyl carrier in mammalian cells and a cofactor that plays a key role in energy and lipid metabolism. CoA and its thioesters (acyl-CoAs) regulate a multitude of metabolic processes at different levels: as substrates, allosteric modulators, and via post-translational modification of histones and other non-histone proteins. Evidence is emerging that synthesis and degradation of CoA are regulated in a manner that enables metabolic flexibility in different subcellular compartments. Degradation of CoA occurs through distinct intra- and extracellular pathways that rely on the activity of specific hydrolases. The pantetheinase enzymes specifically hydrolyze pantetheine to cysteamine and pantothenate, the last step in the extracellular degradation pathway for CoA. This reaction releases pantothenate in the bloodstream, making this CoA precursor available for cellular uptake and de novo CoA synthesis. Intracellular degradation of CoA depends on specific mitochondrial and peroxisomal Nudix hydrolases. These enzymes are also active against a subset of acyl-CoAs and play a key role in the regulation of subcellular (acyl-)CoA pools and CoA-dependent metabolic reactions. The evidence currently available indicates that the extracellular and intracellular (acyl-)CoA degradation pathways are regulated in a coordinated and opposite manner by the nutritional state and maximize the changes in the total intracellular CoA levels that support the metabolic switch between fed and fasted states in organs like the liver.The objective of this review is to update the contribution of these pathways to the regulation of metabolism, physiology and pathology and to highlight the many questions that remain open.     

Energy budgets do balance—A comment on a paper by Wightman and Rogers

Summary In a recently published energy budget for the larvae of the leafcutter bee (Wightman and Rogers, Oecologia (Berl.) 36 (1978) 245–257) respiration as estimated by respirometry amounted to only 67% of the respiration as estimated from the difference between assimilation and production. In this note it is shown that this discrepancy seems to result from an incorrect value of the oxycalorific equivalent and that a more reasonable value makes the two estimates of respiration agree.