Effectiveness of out-of-hours biochemistry investigations.

A survey was carried out of doctors who used their out-of-hours biochemistry service to find out why requests for investigations were made, how often the results altered patient management, and whether they could define areas where investigations were unproductive. Of 107 questionnaires distributed, 147 (88%) were completed. In 86% the requests were for diagnosis or immediate patient management and in 35% the results actually altered management. Senior clinical staff were more efficient than their juniors in instigating biochemical investigations that proved to be effective. In no instance where the clinical staff predicted that it was unlikely that the results would alter management was management altered. It is suggested that joint reviews of case notes by junior and senior clinical staff would prove to be the most appropriate way to increase the effectiveness and efficiency of clinical investigation.     

Stereospecificity of peptide transport by germinating barley embryos

The stereospecific requirements for peptide transport in the scutellum of germinating barley (Hordeum vulgare) embryos are described. Replacement of an L-amino acid residue in a peptide by its D-stereoisomer decreases the affinity of the peptide for the transport site, leading to a reduction in transport. Substitution of a second D-residue reduces affinity still further. The extent to which transport is inhibited depends upon the position of the D-residue in the primary sequence, with D-residues at the C-terminus of the peptide having the greatest effect. Competition between D- and L-peptides indicates that they both enter via the same transport system. Although D-amino acids can be accumulated when presented as a peptide, these same D-residues are not transported when supplied as the free amino acids. L-Leu-D-leu is accumulated intact against a concentration gradient, indicating the operation of an active transport mechanism that can function without the involvement of peptidase activity.     

Physiological and developmental implications of motor unit anatomy

There is increasing evidence that the architectural design and arrangement of the fibers within a motor unit have important physiological and developmental ramifications. Limited data, however, are available to directly address this issue. In the present study the physiological properties of one motor unit in each of seven cat tibialis anterior (TA) muscles were determined. Each of these units then was repetitively stimulated to deplete the glycogen in all muscle fibers within the unit. Subsequently, the length, type of ending, and spatial distribution of fibers sampled from these physiologically and histochemically typed motor units were determined. Four fast fatigable (FF), one fast, fatigue resistant (FR), and two slow (S) motor units (MU) were studied. The samples consisted of all those glycogen-depleted fibers (9-27) contained within a single fascicle or a circumscribed area of each of the motor unit territories. The mean fiber lengths for the two slow motor units were 35.9 and 45.5 mm. The mean fiber lengths for the fast motor unit samples ranged from 8.8 to 48.5 mm. Some fibers of both the fast and slow units reached lengths of 58 mm. Most of the fibers in the slow units extended the entire distance between the proximal and distal musculotendinous planes, had relatively constant cross-sectional areas, and terminated at the tendon as blunt endings. In contrast, the majority of the fibers in the fast units terminated intrafascicularly at one end, and the cross-sectional area decreased progressively along their lengths, that is, showed a tapering pattern for a significant proportion of their lengths. Therefore, the force generated by units that end midfascicularly would appear to be transmitted to connective tissue elements and/or adjacent fibers. All fibers of a fast unit within a fascicle were located at approximately the same proximo-distal location. Thus, developmentally the selection of muscle fibers by a motoneuron would seem to be influenced by their spatial distribution. The architectural complexities of motor units also have clear implications for the mechanical interactions of active and inactive motor units. For example, the tension capabilities of a motor unit may be influenced not only by the spatial arrangement of its own fibers, but also by the level of activation of neighboring motor units.     

Bioprocess development to improve foreign protein production from recombinant Streptomyces.

Bioprocessing strategies to improve production of the heterologous protein parathion hydrolase from recombinant Streptomyces lividans were investigated. Initial limitations to increased production were overcome by using large amounts of nutrients and feeding these nutrients throughout the fermentation. Batch addition of such large amounts of nutrients resulted in byproduct acid accumulation. Our data suggest that byproducts resulted from incomplete utilization of peptide medium ingredients and not from an overflow of glucose catabolism. Over extended fed-batch operation, oxygen transfer became limiting and these limitations were overcome by sparging oxygen-enriched gas. When cultivation was continued past about 90 h, we observed that despite nutrient feeding and oxygen enrichment enzyme activities no longer increased. Our results show that during such late cultivation periods the rates of enzyme synthesis and deactivation became balanced. If synthesis is prevented, either by a nutritional limitation or by the addition of the protein synthesis inhibitor chloramphenicol, enzyme activities were observed to decrease. Since deactivation rate constants in these experiments were similar to those observed in cell-free studies, and because extracellular protease activities were not detected in our fermentation, it appears that deactivation results from the inherent instability of the parathion hydrolase enzyme.     

Recovery of parasitic nematodes from fish by digestion or elution.

Two methods, digestion and elution, were used to recover parasitic nematodes from 470 flatfish belonging to species in the family Pleuronectidae. Samples of similar fish were collected from market lots; half of each sample was subjected to digestion, and half was subjected to elution (sedimentation). The edible (flesh) and the inedible (viscera) portions of each fish were analyzed separately. The total number of nematodes recovered by digestion was 1,110, which was not significantly greater than the 922 nematodes recovered by elution. However, digestion recovered 1,062 nematodes of the anisakine genera Anisakis and Phocanema, which are potentially pathogenic for human consumers of raw of semiraw fish. This number is significantly greater than the 608 pathogenic nematodes recovered by elution. Digestion also recovered 242 more nematodes from the edible flesh than did elution. Conversely, more nonpathogenic nematodes were recovered by elution. Approximately half the fish (240) had been collected in Boston markets, and the other half (230) had been collected in San Francisco markets. Fish from San Francisco each contained an average of eight nematodes, and those from Boston contained an average of less than one nematode per fish.     

Physiological and biochemical effects of the mycotoxin patulin on Chang liver cell cultures.

Patulin exhibits both cytotoxic and cytopathic effects on cultured Chang liver cells. The LD50 found was 1.85 mug per ml of patulin. Effects on growth were observed with as little as 0.1 mug per ml of patulin; a 50% reduction in growth was observed at 0.38 mug per ml of patulin. Using a challenge dose of 2.5 mug per ml of patulin, the cytotoxic effect was reversible after an exposure of 10 min, but was not reversible after 20 min. Protein synthesis was depressed after 60 min and RNA synthesis after 20 min of contact with patulin. Neither protein nor RNA synthesis was completely inhibited after 260 min.     

Biofabrication with chitosan

The traditional motivation for integrating biological components into microfabricated devices has been to create biosensors that meld the molecular recognition capabilities of biology with the signal processing capabilities of electronic devices. However, a different motivation is emerging; biological components are being explored to radically change how fabrication is achieved at the micro- and nanoscales. Here we review biofabrication, the use of biological materials for fabrication, and focus on three specific biofabrication approaches: directed assembly, where localized external stimuli are employed to guide assembly; enzymatic assembly, where selective biocatalysts are enlisted to build macromolecular structure; and self-assembly, where information internal to the biological material guides its own assembly. Also reviewed are recent results with the aminopolysaccharide chitosan, a material that offers a combination of properties uniquely suited for biofabrication. In particular, chitosan can be directed to assemble in response to locally applied electrical signals, and the chitosan backbone provides sites that can be employed for the assembly of proteins, nucleic acids, and virus particles.     

Tyrosinase Reaction and Subsequent Chitosan Adsorption for Selective Removal of a Contaminant from a Fermentation Recycle Stream

In the industrial production of penicillin V, the phenoxyacetate precursor is added to the fermentor to direct biosynthesis. When used for producing semisynthetic penicillins, the penicillin V is often hydrolyzed to 6-aminopenicillanic acid with the regeneration of the phenoxyacetate precursor. To reduce raw-material as well as waste-disposal costs, it is desirable to recycle the phenoxyacetate precursor. Unfortunately, the recycle stream is generally contaminated by the p-hydroxylated derivative of this precursor. We examined a two-step approach to eliminate this contaminant. In the first step the tyrosinase enzyme was used to selectively convert the p-hydroxyphenoxyacetate contaminant to a reactive intermediate—presumably its quinone. In the second step, the tyrosinase-generated reactive intermediate was allowed to react with and strongly bind to chitosan. In contrast, the phenoxyacetate precursor was neither oxidized by tyrosinase nor bound to chitosan. When concentrated phenoxyacetate solutions were tested, the combination of tyrosinase and chitosan effectively converted low levels of the p-hydroxyphenoxyacetate contaminant and removed its products from solution, while the concentration of the phenoxyacetate precursor was unaffected.     

Spread of chloroquine resistance in Plasmodium falciparum

Malaria resistant to chloroquine has now been confirmed in more than 40 countries. The drug was introduced in 1934, but was not in large-scale use until the early 1950s. Anecdotal reports suggest that resistance emerged as early as 1957 both in Colombia and along the then Cambodia-Thailand border area. But by 1960, resistance in these areas was confirmed - and may represent two separate events. Resistance spread rapidly, with a new focus of resistance confirmed in East Africa by 1977. Chloroquine resistance represents a severe problem both for prophylaxis and treatment of malaria. In this aricle, David Payne traces the spread of resistance and discusses some of its implications.     

Ca2+/Calmodulin-dependent Protein Kinase IIα (αCaMKII) Controls the Activity of the Dopamine Transporter: IMPLICATIONS FOR ANGELMAN SYNDROME

The dopamine transporter (DAT) is a crucial regulator of dopaminergic neurotransmission, controlling the length and brevity of dopaminergic signaling. DAT is also the primary target of psychostimulant drugs such as cocaine and amphetamines. Conversely, methylphenidate and amphetamine are both used clinically in the treatment of attention-deficit hyperactivity disorder and narcolepsy. The action of amphetamines, which induce transport reversal, relies primarily on the ionic composition of the intra- and extracellular milieus. Recent findings suggest that DAT interacting proteins may also play a significant role in the modulation of reverse dopamine transport. The pharmacological inhibition of the serine/threonine kinase αCaMKII attenuates amphetamine-triggered DAT-mediated 1-methyl-4-phenylpyridinium (MPP(+)) efflux. More importantly, αCaMKII has also been shown to bind DAT in vitro and is therefore believed to be an important player within the DAT interactome. Herein, we show that αCaMKII co-immunoprecipitates with DAT in mouse striatal synaptosomes. Mice, which lack αCaMKII or which express a permanently self-inhibited αCaMKII (αCaMKII(T305D)), exhibit significantly reduced amphetamine-triggered DAT-mediated MPP(+) efflux. Additionally, we investigated mice that mimic a neurogenetic disease known as Angelman syndrome. These mice possess reduced αCaMKII activity. Angelman syndrome mice demonstrated an impaired DAT efflux function, which was comparable with that of the αCaMKII mutant mice, indicating that DAT-mediated dopaminergic signaling is affected in Angelman syndrome.