The University of Minnesota Biocatalysis/Biodegradation Database: emphasizing enzymes.

The University of Minnesota Biocatalysis/Biodegradation Database (UM-BBD, http://umbbd.ahc.umn.edu/) provides curated information on microbial catabolic enzymes and their organization into metabolic pathways. Currently, it contains information on over 400 enzymes. In the last year the enzyme page was enhanced to contain more internal and external links; it also displays the different metabolic pathways in which each enzyme participates. In collaboration with the Nomenclature Commission of the International Union of Biochemistry and Molecular Biology, 35 UM-BBD enzymes were assigned complete EC codes during 2000. Bacterial oxygenases are heavily represented in the UM-BBD; they are known to have broad substrate specificity. A compilation of known reactions of naphthalene and toluene dioxygenases were recently added to the UM-BBD; 73 and 108 were listed respectively. In 2000 the UM-BBD is mirrored by two prestigious groups: the European Bioinformatics Institute and KEGG (the Kyoto Encyclopedia of Genes and Genomes). Collaborations with other groups are being developed. The increased emphasis on UM-BBD enzymes is important for predicting novel metabolic pathways that might exist in nature or could be engineered. It also is important for current efforts in microbial genome annotation.

The proliferative and antiapoptotic effects of substance P are facilitated by formation of a beta -arrestin-dependent scaffolding complex.

A requirement for scaffolding complexes containing internalized G protein-coupled receptors and beta-arrestins in the activation and subcellular localization of extracellular signal-regulated kinases 1 and 2 (ERK1/2) has recently been proposed. However, the composition of these complexes and the importance of this requirement for function of ERK1/2 appear to differ between receptors. Here we report that substance P (SP) activation of neurokinin-1 receptor (NK1R) stimulates the formation of a scaffolding complex comprising internalized receptor, beta-arrestin, src, and ERK1/2 (detected by gel filtration, immunoprecipitation, and immunofluorescence). Inhibition of complex formation, by expression of dominant-negative beta-arrestin or a truncated NK1R that fails to interact with beta-arrestin, inhibits both SP-stimulated endocytosis of the NK1R and activation of ERK1/2, which is required for the proliferative and antiapoptotic effects of SP. Thus, formation of a beta-arrestin-containing complex facilitates the proliferative and antiapoptotic effects of SP, and these effects of SP could be diminished in cells expressing truncated NK1R corresponding to a naturally occurring variant.

Improved vectors for nisin-controlled expression in gram-positive bacteria.

A set of shuttle vectors, able to replicate in Escherichia coli and in gram-positive bacteria, containing a nisin-inducible promoter (PnisA) and genes encoding NisR and NisK, the two-component signaling mechanism for activating transcription from PnisA in the presence of nisin, was constructed. To test these vectors, Enterococcus faecalis pCF10 plasmid genes prgX, prgY, and prgZ, which respectively encode cytosolic, integral membrane, and cell surface proteins, were cloned downstream of PnisA. Increased protein expression, in the presence of nisin, was demonstrated by Western blot analysis.

Regulation of hexuronate utilization in Bacillus subtilis.

We have identified a locus essential for galacturonate utilization in Bacillus subtilis. Genes homologous to Escherichia coli and Erwinia chrysanthemi glucuronate and galacturonate metabolic genes were found in a cluster consisting of 10 open reading frames (ORFs) in the B. subtilis chromosome. A mutant of B. subtilis containing a replacement of the second and third ORFs was unable to grow with galacturonate as its primary carbon source. Galacturonate induced expression from a sigmaA-dependent promoter, exuP1, located upstream from the first ORF. The eighth ORF in this cluster (the exu locus) encodes a LacI and GalR homolog that negatively regulated expression from exuP1. A 26-bp inverted repeat sequence centered 15 bp downstream from the exuP1 start point of transcription acted in cis to negatively regulate expression from exuP1 under noninducing conditions. Expression from the exuP1 promoter was repressed by high levels of glucose, which is probably mediated by CcpA (catabolite control protein A). A sigmaE-dependent promoter, exuP2, was localized between the second and third ORFs and was active during sporulation.

A spare or an individual? Cloning and the implications of monozygotic twinning.

The creation of Dolly, the cloned sheep, raises the scenario of cloning in humans. Neither the case for, nor against, the ethics of cloning in humans is discussed in this paper. Instead, it considers the neglected issue of the likely happiness or otherwise of the resulting children if they are born as monozygotic twins or triplets. The advantages and disadvantages of twinship are discussed in detail, and it is concluded that recognized medical risks, and incompletely understood psychological effects, should be given serious consideration.

cse15, cse60, and csk22 are new members of mother-cell-specific sporulation regulons in Bacillus subtilis.

We report on the characterization of three new transcription units expressed during sporulation in Bacillus subtilis. Two of the units, cse15 and cse60, were mapped at about 123 degrees and 62 degrees on the genetic map, respectively. Their transcription commenced around h 2 of sporulation and showed an absolute requirement for sigmaE. Maximal expression of both cse15 and cse60 further depended on the DNA-binding protein SpoIIID. Primer extension results revealed -10 and -35 sequences upstream of the cse15 and cse60 coding sequences very similar to those utilized by sigmaE-containing RNA polymerase. Alignment of these and other regulatory regions led to a revised consensus sequence for sigmaE-dependent promoters. A third transcriptional unit, designated csk22, was localized at approximately 173 degrees on the chromosome. Transcription of csk22 was activated at h 4 of sporulation, required the late mother-cell regulator sigmaK, and was repressed by the GerE protein. Sequences in the csk22 promoter region were similar to those of other sigmaK-dependent promoters. The cse60 locus was deduced to encode an acidic product of only 60 residues. A 37.6-kDa protein apparently encoded by cse15 was weakly related to the heavy chain of myosins, as well as to other myosin-like proteins, and is predicted to contain a central, 100 residue-long coiled-coil domain. Finally, csk22 is inferred to encode a 18.2-kDa hydrophobic product with five possible membrane-spanning helices, which could function as a transporter.

A sigma E dependent operon subject to catabolite repression during sporulation in Bacillus subtilis.

To identify genes expressed at intermediate stages of Bacillus subtilis sporulation, we screened for sigma E-dependent promoters. One promoter that we found drives expression of an operon consisting of at least five open reading frames (ORFs). The predicted products of the first three ORFs are very homologous to enzymes involved in fatty acid metabolism, including acetyl coenzyme A (acetyl-CoA) acetyltransferase (thiolase), 3-hydroxybutyryl-CoA dehydrogenase, and acyl-CoA dehydrogenase, respectively. We showed that the fourth ORF encoded a third isozyme of citrate synthase in B. subtilis. Genetic evidence and primer extension results showed that transcription of this operon is directed by the mother cell compartment-specific sigma factor, sigma E, and so the operon was named mmg (for mother cell metabolic genes). Furthermore, we found that a sequence (mmgO) with homology to a catabolite-responsive element mediates glucose repression of mmg promoter activity during sporulation and that this repression was lost in a ccpA mutant.

The death of a twin.

Death or disability is much more common in multiple births than single children, especially in the perinatal period. Parents face particular problems in that their loss may be underestimated; their grieving may be impeded by the confusion between the live and the dead baby, and the constant reminder in the survivor may be painful. The surviving twin often suffers profoundly from the loss and may need lifelong support. The value of the twinship should always be respected.

Paediatric care of twins.

For many parents, the joy of having twins and triplets is reduced by the unexpected emotional as well as physical and financial stresses which they experience with children who may suffer from prematurity and low birthweight, and who have a higher risk of language delay and disability. Paediatric care should start from the time of ultrasound diagnosis. The Multiple Births Foundation holds regular meetings for expectant parents. Twins Clinics are held in three centres in the UK as well as three special clinics (Growth, Supertwins and Bereavement), where families may discuss problems relating to the twinship as well as sharing their experiences with parent volunteers.

Is there a role for a twins clinic? An evaluation of parents' responses.

To evaluate parental responses to the Twins Clinic, postal questionnaires were sent to the parents of 141 pairs of twins, born outside the maternity hospital who were referred to the clinic in the first three years. The main outcome measures were: the sources of, and reasons for, referral; previous attempts to find help; experiences at the clinic and outcome of the visit. One hundred and thirteen families (80%) replied, of whom 101 (89%) were self-referred. The most common problems for which twins were referred related to behaviour (34) and development (28). In 99 (88%) cases, parents had already sought assistance from other sources (39 from health professionals) but only 29 had found this helpful. In 95 (84%) cases, parents felt there had been a positive outcome from attending the clinic, in the form of reassurance (87), change of management (39), successful support for applications (19) or information (11). Parents' responses indicate that the Twins Clinic meets the needs of families with multiples who are inadequately served by existing sources of help. These will only be improved through education of health professionals (and of the community as a whole) so as to obviate the need for Twins Clinics.

Gestational age correction for height in preterm children to seven years of age.

Correction for gestational age continues to make a difference to the height SD score (SDS) to the age of seven years in very preterm babies. The height SDS for children born at 28 weeks' gestation increased by 0.25 SDS when postconceptual age was used instead of real age. Extrapolating from these results, the effect of correction would be an increase of approximately 0.32 SDS for a seven-year-old of 24 weeks' gestation. Unsatisfactory growth may be masked by a steady or increasing real age SDS in a few children. The risks of stopping using postconceptual age at two or three years include both false confidence in genuine cases of growth retardation and misinterpretation of a decrease in height SDS as evidence of growth retardation. As the number of very preterm babies who survive increases so does the importance of these observations.

Coordination of low birthweight seven-year-olds.

The coordination and laterality of a group of 171 seven-year-old children, free from major disability, with a birthweight of 2,000 g or less, were examined and compared with those of normal birthweight peers. More low birthweight children were left-handed and of mixed or undetermined hand, foot and eye dominance. Left-handedness may adversely affect some areas of performance of normal birthweight but not of low birthweight children. Low birthweight children performed significantly less well in tests of both fine and gross motor coordination. Girls tended to perform better than boys in fine motor tests. In the low birthweight group there was a correlation between IQ and coordination.

Enamel defects in the primary dentition of children born weighing less than 2000 g.

The findings for enamel defects in the primary dentition of a group of low birthweight (LBW) children were related to their perinatal medical histories. Examination of 110 LBW children and 93 control children aged 5 years showed that significantly more of the LBW children (71%) had hypoplasia than controls (15%), but there was no difference in opacities alone (LBW 25%, control 26%). In the LBW group, defects were seen more often in children classified as ill during the perinatal period, who received ventilator support or intravenous alimentation and in those children born at less than 32 weeks gestation, compared to the LBW children without these perinatal problems. It is speculated that the high incidence and the cause of enamel defects in sick preterm infants may be due to oxygen deprivation and mineral substitute depletion.

Lung function in children of low birth weight.

Lung function was recorded in a cohort of 130 age specific children of low birth weight (under 2000 g) and a reference population of 120 unselected local schoolchildren at 7 years of age. Children of the cohort were similar in height and forced vital capacity to the reference group, but had significantly reduced forced expiratory volume in 0.75 second and expiratory flow indices. Although neonatal respiratory illness was associated with reduced airway function, we were unable to confirm that this was a consequence of oxygen treatment or mechanical ventilation. Low birth weight, however, was closely associated with poor airway function independent of neonatal respiratory illness. Other factors of importance included the male gender and maternal smoking. The reduction in airway function observed in the low birthweight children was associated with cough but not wheeze. The disparity between the relatively well preserved vital capacity and reduced airway function suggests that very low birth weight, and hence prematurity, has its greatest effect on the subsequent growth of airway function. The absence of an association between neonatal oxygen score or mechanical ventilation and childhood lung function suggests that the long term effect of neonatal respiratory treatment is small compared with that of birth weight, maternal smoking, and male sex.