Synthesis of novel fructooligosaccharides by substrate and enzyme engineering

Fructooligosaccharides (FOSs) and polyfructosides (PSs) have received particular attention due to its beneficial effects as prebiotics. Here we report the synthesis of a new class of fructooligosaccharides by substrate and enzyme engineering. Using an engineered levansucrase enzyme (SacB of Bacillus subtilis), and sucrose analogues (alpha-Xyl-1,2-beta-Fru or alpha-Gal-1,2-beta-Fru), the product profile shifted from the fructan (levan) polymer to a range of new higher oligosaccharides (xylooligofructosides), or polysaccharides (galactopolyfructosides), of varying size. Further the enzyme was tailored by random mutagenesis, for the synthesis of short-chain fructooligosaccharides to yield variant A5 (N242H), which is unable to produce polymers. It shifts its product pattern to short-chain oligosaccharides and hydrolysis and enabled in combination with the sucrose analogue Xyl-Fru for the first time the direct synthesis of a 6-kestose analogue (alpha-Xyl-1,2-beta-Fru-2,6-beta-Fru). The different glycopyranosyl-residues (i.e. galactose and xylose) that cap fructooligosaccharides may alter prebiotic and biochemical properties.     

Fructansucrase enzymes and sucrose analogues: A new approach for the synthesis of unique fructo-oligosaccharides

Fructansucrase enzymes of lactic acid bacteria use the cheap compound sucrose (Glc-Fru) to synthesize a variety of poly- and oligosaccharide products. Recently, it has been shown that a variety of sucrose analogues (e.g. Gal-Fru, Man-Fru) can be efficiently synthesized. This has exciting potential for the development of novel (fructo) oligosaccharide derivatives.     

Deep-Sea Octopus (Graneledone boreopacifica) Conducts the Longest-Known Egg-Brooding Period of Any Animal

Octopuses typically have a single reproductive period and then they die (semelparity). Once a clutch of fertilized eggs has been produced, the female protects and tends them until they hatch. In most shallow-water species this period of parental care can last from 1 to 3 months, but very little is known about the brooding of deep-living species. In the cold, dark waters of the deep ocean, metabolic processes are often slower than their counterparts at shallower depths. Extrapolations from data on shallow-water octopus species suggest that lower temperatures would prolong embryonic development periods. Likewise, laboratory studies have linked lower temperatures to longer brooding periods in cephalopods, but direct evidence has not been available. We found an opportunity to directly measure the brooding period of the deep-sea octopus Graneledone boreopacifica, in its natural habitat. At 53 months, it is by far the longest egg-brooding period ever reported for any animal species. These surprising results emphasize the selective value of prolonged embryonic development in order to produce competitive hatchlings. They also extend the known boundaries of physiological adaptations for life in the deep sea.     

Identification of point mutations by mispairing PCR as exemplified in MERRF disease

The point mutation in the tRNA(Lys) gene of mitochondrial DNA (mtDNA) from patients with myoclonic epilepsy and ragged red fibers (MERRF) was quantitatively analyzed after digestion with the restriction endonuclease Nae I of the PCR amplified DNA. Since the point mutation is not part of a restriction site for a commonly available restriction endonuclease, the Nae I restriction site was introduced by PCR using a mispairing primer. The percentage of mutated mtDNA was determined in a few hairs of five members of an affected family by counting the radioactivity of the fragments after PCR amplification with labelled dATP.     

Myoclonic epilepsy and ragged-red fiber disease (MERRF) is associated with a mitochondrial DNA tRNA(Lys) mutation

An A to G transition mutation at nucleotide pair 8344 in human mitochondrial DNA (mtDNA) has been identified as the cause of MERRF. The mutation alters the T psi C loop of the tRNA(Lys) gene and creates a CviJI restriction site, providing a simple molecular diagnostic test for the disease. This mutation was present in three independent MERRF pedigrees and absent in 75 controls, altered a conserved nucleotide, and was heteroplasmic. All MERRF patients and their less-affected maternal relatives had between 2% and 27% wild-type mtDNAs and showed an age-related association between genotype and phenotype. This suggests that a small percentage of normal mtDNAs has a large protective effect on phenotype. This mutation provides molecular confirmation that some forms of epilepsy are the result of deficiencies in mitochondrial energy production.     

Biochemical markers of bone turnover: part I: biochemistry and variability

With the ageing population in most countries, disorders of bone and mineral metabolism are becoming increasingly relevant to every day clinical practice. Consequently, the interest in, and the need for effective measures to be used in the screening, diagnosis and follow-up of such pathologies has markedly grown. Together with clinical and imaging techniques, biochemical tests play an important role in the assessment and differential diagnosis of metabolic bone disease. In recent years, the isolation and characterisation of cellular and extracellular components of the skeletal matrix have resulted in the development of molecular markers that are considered to reflect either bone formation or bone resorption. These biochemical indices are non-invasive, comparatively inexpensive and, when applied and interpreted correctly, helpful tools in the diagnostic and therapeutic assessment of metabolic bone disease. Part I of this article provides an overview of the basic biochemistry of bone markers, and sources of non-specific variability. Part II (to be published in a subsequent issue of this journal) will review the current evidence regarding the clinical use of biochemical markers of bone remodelling in metabolic and metastatic bone disease.     

Anti-crosslinking properties of carnosine: significance of histidine

Carnosine, a histidine-containing dipeptide, is a potential treatment for Alzheimer's disease. There is evidence that carnosine prevents oxidation and glycation, both of which contribute to the crosslinking of proteins; and protein crosslinking promotes beta-amyloid plaque formation. It was previously shown that carnosine has anti-crosslinking activity, but it is not known which of the chemical constituents are responsible. We tested the individual amino acids in carnosine (beta-alanine, histidine) as well as modified forms of histidine (alpha-acetyl-histidine, 1-methyl-histidine) and methylated carnosine (anserine) using glycation-induced crosslinking of cytosolic aspartate aminotransferase as our model. beta-Alanine showed anti-crosslinking activity but less than that of carnosine, suggesting that the beta-amino group is required in preventing protein crosslinking. Interestingly, histidine, which has both alpha-amino and imidazolium groups, was more effective than carnosine. Acetylation of histidine's alpha-amino group or methylation of its imidazolium group abolished anti-crosslinking activity. Furthermore, methylation of carnosine's imidazolium group decreased its anti-crosslinking activity. The results suggest that histidine is the representative structure for an anti-crosslinking agent, containing the necessary functional groups for optimal protection against crosslinking agents. We propose that the imidazolium group of histidine or carnosine may stabilize adducts formed at the primary amino group.     

Mitochondrial DNA mutations in multiple symmetric lipomatosis

Multiple symmetric lipomatosis (MSL) is a rare disorder of middle life characterized by large subcutaneous fat masses around the neck, shoulders and other parts of the trunk. Peripheral neuropathy is a common finding in these predominantly male patients. Employing electrophysiological measures, we found additional signs of central nervous system involvement in a majority of patients. Etiologically, there is an association with mitochondrial dysfunction. In muscle biopsy, we found ragged red fibers in 8 of 12 patients. Molecular genetic analysis revealed multiple deletions of mitochondrial DNA in one patient and the MERRF mutation at nucleotide 8344 in another. In this review, we summarize our clinical, electrophysiological morphological, biochemical and molecular genetic findings in 17 MSL patients, and give a survey of the literature. (Mol Cell Biochem 174: 271–275, 1997)     

Electron-microscopic study of the maturing rat red nucleus. I. The large-neuron population

The maturing large neurons of the rat red nucleus in animals ranging in age from 1 to 21 days of postnatal life were studied ultrastructurally. Days 1--6 were characterized by rapid morphologic maturation occurring concomitantly with the onset of synaptogenesis. Morphogenesis was confined to the soma, while the first synaptic contacts were also formed in relationship to the soma. Days 6--9 demonstrated continued somal morphogenesis exemplified by cytoplasmic expansion and by the conspicuous presence of perisomatic and growth cone processes. Proximal dendritic morphogenesis was initiated, and synaptogenesis became complex with synaptic sites occurring in relation to the neuronal soma, the perisomatic processes and proximal dendrites. Days 9--15 were characterized by the completion of somal and proximal dendritic morphogenesis and by a massive degree of synaptogenic activity. During this interval, the soma lost perisomatic and growth cone processes, while somatic spines appeared. By the end of this period the neuronal soma and the proximal dendrites appeared mature in terms of both morphology and synaptic input. Complete neuronal maturation was ultimately attained by day 21 of postnatal life.