Customized media based on miniaturized screening improve growth rate and cell yield of methane-oxidizing bacteria of the genus Methylomonas

The growth of twelve methanotrophic strains within the genus Methylomonas, including the type strains of Methylomonas methanica and Methylomonas koyamae, was evaluated with 40 different variations of standard diluted nitrate mineral salts medium in 96-well microtiter plates. Unique profiles of growth preference were observed for each strain, showing a strong strain dependency for optimal growth conditions, especially with regards to the preferred concentration and nature of the nitrogen source. Based on the miniaturized screening results, a customized medium was designed for each strain, allowing the improvement of the growth of several strains in a batch setup, either by a reduction of the lag phase or by faster biomass accumulation. As such, the maintenance of fastidious strains could be facilitated while the growth of fast-growing Methylomonas strains could be further improved. Methylomonas sp. R-45378 displayed a 50 % increase in cell dry weight when grown in its customized medium and showed the lowest observed nitrogen and oxygen requirement of all tested strains. We demonstrate that the presented miniaturized approach for medium optimization is a simple tool allowing the quick generation of strain-specific growth preference data that can be applied downstream of an isolation campaign. This approach can also be applied as a first step in the search for strains with biotechnological potential, to facilitate cultivation of fastidious strains or to steer future isolation campaigns.     

Solid-phase synthesis of peptides with C-terminal asparagine or glutamine. An effective, mild procedure based on N alpha-fluorenylmethyloxycarbonyl (Fmoc) protection and side-chain anchoring to a tris(alkoxy)benzylamide (PAL) handle

Attempts to anchor Fmoc-asparagine or glutamine as p-alkoxybenzyl esters for solid-phase peptide synthesis are fraught with difficulties. A convenient and effective method to prepare peptides with C-terminal asparagine or glutamine involves quantitative attachment of N alpha-Fmoc-C alpha-tert.-butyl aspartate or glutamate via the free omega-carboxyl groups to a tris(alkoxy)benzylamino (PAL) support. Chain elongation proceeds normally by standard Fmoc chemistry, and treatment with acid, e.g., CF3COOH--CH2Cl2, 90 min at 25 degrees, releases the desired peptides in greater than 95% yields without side reactions at the C-terminus. Feasibility of the approach has been demonstrated by the syntheses of the C-terminal octapeptide from human proinsulin, H-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-OH, and the serum thymic factor pGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-OH.     

Linkage disequilibrium studies in multiple endocrine neoplasia type 1 (MEN1)

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterised by tumours of the parathyroids, pancreas and anterior pituitary. The MEN1 gene has been localised to a 2-Mb region of chromosome 11q13 by meiotic mapping studies in MEN1 families. Such studies may have a limited resolution of approximately 1 cM (i.e. 1 Mb) and we have therefore investigated 96 MEN1 families (40 British, 17 French, 12 Finnish, 7 Swedish, 7 Dutch, 7 North American, 2 Australian, 1 New Zealand, 1 German, 1 Spanish and 1 Danish) for linkage disequilibrium, in order to facilitate a finer mapping resolution. We have utilised five microsatellite DNA sequence polymorphisms from the candidate region and have accurately determined their allele sizes, which ranged from 161 bp to 272 bp. The heterozygosity and number of alleles (given in brackets), respectively, at the loci were: D11S1883 (76%, 11), D11S457 (55%, 5), PYGM (94%, 18), D11S1783 (10%, 4) and D11S449 (87%, 16). Allelic association was assessed by Chi-square 2 ×n contingency tables, by Fisher exact 2 ×n contingency tables and by a likelihood-based approach. The results of haplotype analysis revealed 91 different affected haplotypes in the 96 families, an identical affected haplotype being observed in no more than two families. These results indicate the absence of an ancestral affected haplotype. Significant linkage disequilibrium (P < 0.005) could be established amongst the microsatellite loci but not between the loci and MEN1 in either the total population or in any of the geographical sub-populations. The absence of linkage disequilibrium between MEN1 and the polymorphic loci is probably the result of the occurrence of multiple different disease-causing mutations in MEN1. Received: 1 April 1997 / Accepted: 25 June 1997     

Root growth inhibition of the holorganic layer of moder humus under spruce (Picae abies KARST.) and beech (Fagus Silvatica L.)

A test of root growth inhibition of spruce and beech roots, according to Lynch's procedure (1977), shows the inhibitory effects of soil solution extracted from the holorganic layers (Of₂-Oh) under beech and spruce. Molecular gel filtration of soil solutions shows that the molecular weights vary over a wide range, from less than 100 to over 40,000 daltons. Chemical analysis, using CGC, HPLC and sometimes MS shows only negligible concentrations of simple aliphatic (C₁-C₅) and aromatic acids in the free state. Using the fraction scheme of Forsyth (1977) and the carbazole procedure, it is shown that uronic acids represent only a small percentage of the carboxylic acids, and have no inhibitory effects on root growth. By analogy with results of other authors, the presence of polycarboxylic acids in the soil solution are considered to be the main cause of root growth inhibition.     

Supramaximal cycle tests do not detect seasonal progression in performance in groups of elite speed skaters.

Seven female and eight male elite junior skaters performed cycle ergometer tests at four different times during the 1987/1988 season. The tests consisted of a Wingate-type 30-s sprint test and a 2.5-min supramaximal test. The subjects were tested in February, May and September 1987 and in January 1988. Maximal oxygen consumption was measured during the 2.5-min test. With the exception of the maximal oxygen consumption of the women in May which was about 6% lower than in the other three tests, no seasonal changes in the test results could be observed--this, in spite of a distinct increase in training volume (from 10 to more than 20 h.week-1) and training intensity in the course of the season. When the test data were compared to those of elite senior skaters, it appeared that the junior skaters showed the same values for mean power output during the sprint test [14.2 (SD 0.4) W.kg-1 for the men and 12.6 (SD 0.5) W.kg-1 for the women] and maximal oxygen consumption [63.1 (SD 2.8) ml.kg-1.min-1 for the men and 55.3 (SD 3.5) ml.kg-1.min-1 for the women, respectively] as found for senior skaters. It seemed, therefore, that the effects of training in these skaters had already levelled off in the period before they participated in this investigation. In contrast to previous studies, no relationship could be shown between the test results and skating performance. This was most likely due to the homogenous character of the groups (mean standard deviations in power and oxygen consumption were only 5%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Complement components and terminal complement complex in oesophageal smooth muscle of patients with achalasia.

This study investigates whether patients with achalasia exhibit autoimmune reactions with subsequent complement activation within oesophageal smooth muscle, vessels and neurones. Oesophageal muscular biopsies from 8 patients undergoing surgery for achalasia and from 6 patients operated for oesophageal cancer were investigated by immunofluorescence for the presence of the complement components C1q, C4, C3c, C3d, C9 and the C9 neoantigen of the terminal C5b-C9 complement complex. Tissues were also investigated for the expression of immunoglobulins (G,A,M) and of the antigens of rubella and varicella zoster viruses. In addition, sera of both patient groups were tested for the presence of autoantibodies against Auerbach's plexus. The terminal complement complex C5b-C9 was found within muscle cells from all patients with achalasia but in only one specimen from a patient with cancer. Two patients with achalasia also exhibited the terminal complement complex as well as IgM within ganglion cells. Muscle cells stained positive for the complement component C9 in all five patients with achalasia in whom this test was performed but in none of the control tissues. In addition, sera from four patients with achalasia contained antibodies against Auerbach's plexus. Studies for the complement components C1q, C4, C3c and for antigens of rubella and varicella zoster viruses revealed negative results in all patients and controls. The results of this study suggest that a complement activation is involved in the autoimmune pathogenesis of achalasia. However, the triggering mechanism of this phenomenon remains to be determined.