Errors inherent in the use of piston activated pipettes

The accuracy and reproducibility of the volume of solution delivered from a number of piston-activated pipettes of different makes and sizes has been tested under a variety of experimental conditions. The results indicate that these devices deliver the nominal volume only when both the solution to be sampled, and the whole pipette are at the same temperature. When a temperature differential exists, the absolute accuracy is affected, and unless particular care is taken, the reproducibility is relatively poor. Thus it is only under well-controlled conditions that the behaviour of piston-activated pipettes falls within the specifications quoted by the manufacturers.     

The carpal tunnel syndrome as a probable primary deficiency of vitamin B6 rather than a deficiency of a dependency state

The mean basal specific activities and the mean % deficiencies of the activity of the glutamic oxaloacetic transaminase of the erythrocytes were identical (n.s.) for a group of eight patients with a severe carpal tunnel syndrome and for a group of eight university students. There was no significant difference in the increases in the specific activities for the patients and the students at 4 concentrations of pyridoxal 5′-phosphate. The apparent K_m for the patients and the students was 95 μM and 61 μM (n.s.) respectively. It is concluded from these data in conjunction with previous findings that the carpal tunnel syndrome is a deficiency disease of vitamin B₆, which is probably primary rather than one of a dependency state.     

Characterization of extracellular metallo- and serine-proteases of Aeromonas hydrophila strain B51

The extracellular proteases of Aeromonas hydrophila B51 were stable on heating (56 degrees C) and on storage at 4 degrees C or -20 degrees C. Inhibitor studies showed that 72% of the total activity was inhibited by EDTA (a metalloprotease inhibitor) and 26% was inhibited by phenylmethanesulphonyl fluoride (a serine protease inhibitor). Analytical isoelectric focussing revealed the presence of 33 proteins in the crude extracellular products. Using a casein overlay technique three separate zones of proteolytic activity were detected: a zone with pI 6.5-6.8, formed of two closely focussed bands (possibly isomers of the same protease) and completely inhibited by EDTA; a single band with pI 7.0, which was inhibited by EDTA; and a diffuse zone with pI 8.3-8.5, which was only partially inhibited by EDTA. It is concluded that the serine protease activity focussed in this latter zone. These results indicate the presence of at least four, and possibly five proteases. Our results differ substantially from those reported by other workers using different isolates and it is suggested that significant differences in the character of extracellular products and extracellular proteases exist between different isolates of A. hydrophila.     

Phenylalanine residues in the active site of tyrosine hydroxylase: mutagenesis of Phe300 and Phe309 to alanine and metal ion-catalyzed hydroxylation of Phe300.

Residues Phe300 and Phe309 of tyrosine hydroxylase are located in the active site in the recently described three-dimensional structure of the enzyme, where they have been proposed to play roles in substrate binding. Also based on the structure, Phe300 has been reported to be hydroxylated due to a naturally occurring posttranslational modification [Goodwill, K. E., Sabatier, C., and Stevens, R. C. (1998) Biochemistry 37, 13437-13445]. Mutants of tyrosine hydroxylase with alanine substituted for Phe300 or Phe309 have now been purified and characterized. The F309A protein possesses 40% less activity than wild-type tyrosine hydroxylase in the production of DOPA, but full activity in the production of dihydropterin. The F300A protein shows a 2.5-fold decrease in activity in the production of both DOPA and dihydropterin. The K(6-MPH4) value for F300A tyrosine hydroxylase is twice the wild-type value. These results are consistent with Phe309 having a role in maintaining the integrity of the active site, while Phe300 contributes less than 1 kcal/mol to binding tetrahydropterin. Characterization of Phe300 by MALDI-TOF mass spectrometry and amino acid sequencing showed that hydroxylation only occurs in the isolated catalytic domain after incubation with a large excess of 7, 8-dihydropterin, DTT, and Fe(2+). The modification is not observed in the untreated catalytic domain or in the full-length protein, even in the presence of excess iron. These results establish that hydroxylation of Phe300 is an artifact of the crystallography conditions and is not relevant to catalysis.     

Interactions among species with contrasting dispersal modes explain distributions for epiphytic lichens

Understanding how the biodiversity response to climate change will be modified at ecological scales, e.g. by species interactions, is a major challenge. Lichen epiphytes – the close interdependent relationship between a heterotrophic fungus and photosynthetic partner (photobiont) – are used here to explore how interaction regimes (between lichen species, and between lichens and their photobionts) explain distribution patterns along spatial climatic gradients. To do this we tested field evidence for the ‘core‐fringe hypothesis’, which proposes a facilitative interaction; sexually‐reproducing and spore‐dispersed lichens with a requirement for resynthesis with a compatible photobiont (Nostoc) are facilitated by the prior establishment of asexual lichens which disperse both the fungus and photobiont together. We used two closely related Nephroma species which differ in their reproductive mode – N. laevigatum (sexual spore‐dispersed) and N. parile (asexual) – and compared their occurrence along a bioclimatic gradient to local habitat factors, including the co‐occurrence of asexual lichens which have shared specificity for compatible Nostoc genotypes. The results showed that: 1) N. laevigatum is significantly more likely to occur on trees that have already been colonised by asexual lichens with shared specificity for Nostoc, supporting the core‐fringe hypothesis, while 2) N. parile is independent of this association (strengthening the core‐fringe hypothesis), with its response to a precipitation gradient modified by microhabitat factors. This positive test for the core‐fringe hypothesis demonstrates how interaction regimes can fundamentally alter expectations under climate change. There is an assumption that spore‐dispersed lichen species could more easily track their suitable bioclimatic space through fragmented habitat, compared to asexual species with larger and heavier propagules. However, the establishment of spore‐dispersed lichen epiphytes into new habitat may be limited by the dispersal rates of asexual species, which act as key facilitators.