Neuraminidase assay utilizing sialyl-oligosaccharide substrates with tritium-labeled aglycone.

A new method for the radioisotopic assay of neuraminidase activity has been developed. The substrate utilized, α-d-N-acetylneuraminosyl-(2 → 3′)-lactit[³H]ol, was prepared by reduction of α-d-N-acetylneuraminosyl-(2 → 3′)-lactose with tritiated borohydride and purified by ion-exchange chromatography. After incubation with neuraminidase, the reaction mixtures were applied to small columns of AG 1-X2 (formate) in order to remove free sialic acid and unhydrolyzed substrate. The lactit[³H]ol released by neuraminidase action was then recovered by washing the columns with distilled water and quantitated by utilizing a liquid scintillation spectrometer. Studies with bacterial, avian, and mammalian neuraminidases are described.     

A mathematical model for the continuous culture of microorganisms utilizing inhibitory substrates

A mathematical model is presented for both batch and continuous cultures of microorganisms utilizing inhibitory substrates. The key feature of the model is the use of a inhibition function to relate substrate concentration and specific growth rate. Simulation studies show that the primary result of inhibition by substrate in a batch culture is an increase in the lag time whereas in continuous culture inhibition by substrate may result in process instability. The model should be of value in investigations of the stability of biological processes used for the treatment of certain industrial wastes such as those containing phenols, thiocyanates, nitrates, ammonia, volatile acids, etc., which are known to be inhibitory to many of the organisms metabolizing them.     

Aminooxyacetic acid inhibits the malate-aspartate shuttle in isolated nerve terminals and prevents the mitochondria from utilizing glycolytic substrates

Aminooxyacetate, an inhibitor of pyridoxal-dependent enzymes, is routinely used to inhibit gamma-aminobutyrate metabolism. The bioenergetic effects of the inhibitor on guinea-pig cerebral cortical synaptosomes are investigated. It prevents the reoxidation of cytosolic NADH by the mitochondria by inhibiting the malate-aspartate shuttle, causing a 26 mV negative shift in the cytosolic NAD+/NADH redox potential, an increase in the lactate/pyruvate ratio and an inhibition of the ability of the mitochondria to utilize glycolytic pyruvate. The 3-hydroxybutyrate/acetoacetate ratio decreased significantly, indicating oxidation of the mitochondrial NAD+/NADH couple. The results are consistent with a predominant role of the malate-aspartate shuttle in the reoxidation of cytosolic NADH in isolated nerve terminals. Aminooxyacetate limits respiratory capacity and lowers mitochondrial membrane potential and synaptosomal ATP/ADP ratios to an extent similar to glucose deprivation. Thus, the inhibitor induces a functional 'hypoglycaemia' in nerve terminals and should be used with caution.     

Fungi from koala (Phascolarctos cinereus) faeces exhibit a broad range of enzyme activities against recalcitrant substrates

Aims:  Identification of fungi isolated from koala faeces and screening for their enzyme activities of biotechnological interest.
Methods and Results:  Thirty-seven fungal strains were isolated from koala faeces and identified by the amplification and direct sequencing of the internal transcribed spacer (ITS) region of the ribosomal DNA. The fungi were screened for selected enzyme activities using agar plates containing a single substrate for each target class of enzyme. For xylanase, endoglucanase, ligninase (ligninolytic phenoloxidase) and protease over two-thirds of the isolates produced a clearing halo at 25°C, indicating the secretion of active enzyme by the fungus, and one-third produced a halo indicating amylase, mannanase and tannase activity. Some isolates were also able to degrade crystalline cellulose and others displayed lipase activity. Many of the fungal isolates also produced active enzymes at 15°C and some at 39°C.
Conclusions:  Koala faeces, consisting of highly lignified fibre, undigested cellulose and phenolics, are a novel source of fungi with high and diverse enzyme activities capable of breaking down recalcitrant substrates.
Significance and Impact of the Study:  To our knowledge, this is the first time fungi from koala faeces have been identified using ITS sequencing and screened for their enzyme activities.     

Dynamic secondary ion mass spectrometry imaging of microbial populations utilizing C-labelled substrates in pure culture and in soil

We demonstrate that dynamic secondary ion mass spectrometry (SIMS)-based ion microscopy can provide a means of measuring (13)C assimilation into individual bacterial cells grown on (13)C-labelled organic compounds in the laboratory and in field soil. We grew pure cultures of Pseudomonas putida NCIB 9816-4 in minimal media with known mixtures of (12)C- and (13)C-glucose and analysed individual cells via SIMS imaging. Individual cells yielded signals of masses 12, 13, 24, 25, 26 and 27 as negative secondary ions indicating the presence of (12)C(-), (13)C(-), (24)((12)C(2))(-), (25)((12)C(13)C)(-), (26)((12)C(14)N)(-) and (27)((13)C(14)N)(-) ions respectively. We verified that ratios of signals taken from the same cells only changed minimally during a approximately 4.5 min period of primary O(2)(+) beam sputtering by the dynamic SIMS instrument in microscope detection mode. There was a clear relationship between mass 27 and mass 26 signals in Pseudomonas putida cells grown in media containing varying proportions of (12)C- to (13)C-glucose: a standard curve was generated to predict (13)C-enrichment in unknown samples. We then used two strains of Pseudomonas putida able to grow on either all or only a part of a mixture of (13)C-labelled and unlabelled carbon sources to verify that differential (13)C signals measured by SIMS were due to (13)C assimilation into cell biomass. Finally, we made three key observations after applying SIMS ion microscopy to soil samples from a field experiment receiving (12)C- or (13)C-phenol: (i) cells enriched in (13)C were heterogeneously distributed among soil populations; (ii) (13)C-labelled cells were detected in soil that was dosed a single time with (13)C-phenol; and (iii) in soil that received 12 doses of (13)C-phenol, 27% of the cells in the total community were more than 90% (13)C-labelled.     

Taphonomic experiments resolve controls on the preservation of melanosomes and keratinous tissues in feathers

Fossils are a key source of data on the evolution of feather structure and function through deep time, but their ability to resolve macroevolutionary questions is compromised by an incomplete understanding of their taphonomy. Critically, the relative preservation potential of two key feather components, melanosomes and keratinous tissue, is not fully resolved. Recent studies suggesting that melanosomes are preferentially preserved conflict with observations that melanosomes preserve in fossil feathers as external moulds in an organic matrix. To date, there is no model to explain the latter mode of melanosome preservation. We addressed these issues by degrading feathers in systematic taphonomic experiments incorporating decay, maturation and oxidation in isolation and combination. Our results reveal that the production of mouldic melanosomes requires interactions with an oxidant and is most likely to occur prior to substantial maturation. This constrains the taphonomic conditions under which melanosomes are likely to be fossilized. Critically, our experiments also confirm that keratinous feather structures have a higher preservation potential than melanosomes under a range of diagenetic conditions, supporting hitherto controversial hypotheses that fossil feathers can retain degraded keratinous structures.