Characterisation of a novel homodimeric N-acetyl-beta-D-glucosaminidase from Streptococcus gordonii

An N-acetyl-beta-D-glucosaminidase (GcnA) from Streptococcus gordonii FSS2 was cloned and sequenced. GcnA had a deduced molecular mass of 72,120 Da. The molecular weight after gel-filtration chromatography was 140,000 Da and by SDS-PAGE was 70,000 Da, indicating that the native protein was a homodimer. The deduced amino acid sequence had significant homology to a glycosyl hydrolase from Streptococcus pneumoniae and the conserved catalytic domain of the Family 20 glycosyl hydrolases. GcnA catalysed the hydrolysis of the synthetic substrates, 4-methylumbelliferyl (4MU)-N-acetyl-beta-D-glucosaminide, 4MU-N-acetyl-beta-D-galactosaminide, 4-MU-beta-D-N,N'-diacetylchitobioside, and 4-MU-beta-D-N,N',N'-chitotrioside as well as the respective chito-oligosaccharides. GcnA was optimally active at pH 6.6 and 42 degrees C. The Km for 4-MU-beta-D-N,N',N'-chitotrioside, 45 microM, was the lowest for all the substrates tested. Hg2+, Cu2+, Fe2+, and Zn2+ completely inhibited while Co2+, Mn2+, and Ni2+ partially inhibited activity. S. gordonii FSS2 and a GcnA negative mutant grew equally well on chito-oligosaccharides as substrates. The S. gordonii sequencing projects indicate two further N-acetyl-beta-D-glucosaminidase activities.     

Update on estrogen signaling

Our understanding of estrogen signaling has undergone a true paradigm shift over recent years, following the discovery in 1995 of a second estrogen receptor, estrogen receptor beta (ERbeta). In many contexts ERbeta appears to antagonize the actions of ERalpha (yin/yang relationship) although there also exist genes that are specifically regulated by one of the two receptors. Studies of ERbeta knockout mice have shown that ERbeta exerts important functions in the ovary, central nervous system, mammary gland, prostate gland, hematopoiesis, immune system, vessels and bone. The use of ERbeta-specific ligands against certain forms of cancer represents one of the many pharmaceutical possibilities that have been created thanks to the discovery of ERbeta.     

Developmental switch in brain nutrient transporter expression in the rat

Normal development of both human and rat brain is associated with a switch in metabolic fuel from a combination of glucose and ketone bodies in the immature brain to a nearly total reliance on glucose in the adult. The delivery of glucose, lactate, and ketone bodies from the blood to the brain requires specific transporter proteins, glucose and monocarboxylic acid transporter proteins (GLUTs and MCTs), respectively. Developmental expression of the GLUTs in rat brain, i.e., 55-kDa GLUT1 in the blood-brain barrier (BBB), 45-kDa GLUT1 and GLUT3 in vascular-free brain, corresponds to maturational increases in cerebral glucose uptake and utilization. It has been suggested that MCT expression peaks during suckling and sharply declines thereafter, although a comparable detailed study has not been done. This study investigated the temporal and regional expression of MCT1 and MCT2 mRNA and protein in the BBB and the nonvascular brain during postnatal development in the rat. The results confirmed maximal MCT1 mRNA and protein expression in the BBB during suckling and a decline with maturation, coincident with the switch to glucose as the predominant cerebral fuel. However, nonvascular MCT1 and MCT2 levels do not reflect changes in cerebral energy metabolism, suggesting a more complex regulation. Although MCT1 assumes a predominantly glial expression in postweanling brain, the concentration remains fairly constant, as does that of MCT2 in neurons. The maintenance of nonvascular MCT levels in the adult brain implies a major role for these proteins, in concert with the GLUTs in both neurons and astrocytes, to transfer glycolytic intermediates during cerebral energy metabolism.     

Evolution of flowering in response to day length: flipping the CONSTANS switch

Day length provides an important environmental cue by signalling conditions favourable for flowering. While Arabidopsis promotes flowering in response to long days, rice promotes flowering in response to short days. Despite this difference, a recent paper reveals that the network controlling this response is highly conserved in these distantly related plants, only the activity of one component is reversed. This reveals how an important developmental process can be diversified for adaptation by using the same set of genes, but regulating them differently.     

PCR-assisted contig extension: stepwise strategy for bacterial genome closure

Finishing is rate limiting for genome projects, and improvements in the efficiency of complete genome-sequence compilation will require improved protocols for gap closure. Here we report a novel approach for extending shotgun contigs and closing gaps that we termed PCR-assisted contig extension (PACE). PACE depends on the capture of rare mismatched interactions that occur between arbitrary primers and template DNA of unknown sequence, even under highly stringent conditions, by means of elevated PCR-cycle repetition and the use of specific anchoring primers corresponding to adjacent regions of known sequence. Using PACE, we have generated extensions with an average of 1 kb from all contigs generated from the shotgun sequencing of a 5-Mb genome, which closed the majority of gaps with a single round of experimentation. This included the generation of multiple extensions for contigs that terminated in one of the eight copies of the rRNA operon. We calculate that the switch from shotgun sequencing to PACE should occur between 5- and 8-fold genome coverage for maximum benefit and minimum overall cost. PACE is a robust and straightforward strategy that should simplify the finishing phase of bacterial genome projects.     

Retroposon compensatory mechanism hypothesis not supported: Zfa knockout mice are fertile

It is hypothesized that autosomal retroposons compensate for the loss of their inactivated essential X-chromosome progenitors during spermatogenesis. Here we test this Retroposon Compensatory Mechanism (RCM) hypothesis using the Zfy gene family. The mouse autosomal retroposon Zfa is expressed in testes at the same developmental time points at which Zfx levels decline, which correspond to the time of male sex chromosome inactivation, suggesting that Zfa may compensate for the loss of Zfx during spermatogenesis. We examined the effect of Zfa-targeted mutagenesis on spermatogenesis in three genetically distinct mouse strains. Surprisingly, Zfa knockout mice showed no detectable fertility, sperm count, or testes morphology defects. We therefore conclude that Zfa is not an essential gene for spermatogenesis and fertility. This surprising finding now challenges the RCM hypothesis at least for the Zfy gene family. It also forces us to reevaluate the original data underpinning the RCM hypothesis for this family and to propose alternative hypotheses.     

Validation of new aromatase monoclonal antibodies for immunohistochemistry: progress report

Intratumoral aromatase is a potential therapeutic target for the treatment of postmenopausal estrogen-dependent breast cancers. Therefore, reliable methods should be developed for routine application for the detection of intratumoral aromatase. A multi-center collaborative group has been established to generate and validate new aromatase monoclonal antibodies (MAbs). A recombinant GST–aromatase fusion protein was expressed in baculovirus and the purified protein was used for immunization of mice either as a native or formalin-fixed antigen. Hybridomas were generated using standard techniques and screened biochemically prior to immunohistochemistry (IHC) evaluation in human placenta, ovary and breast cancer tissues. Twenty-three MAbs selected by biochemical assays were further evaluated by IHC of paraffin-embedded tissue sections including normal ovary, and placenta, and a small series of 10 breast carcinomas. Of the 23 MAbs, 2 (clones 677 and F2) were determined to specifically stain cell types known to express aromatase in normal tissues. In breast carcinomas staining of malignant epithelium, adipose tissue, normal/benign and stromal compartments was detected. IHC was performed and independently evaluated by three pathologists (HS, TJA and SGS), each using the same evaluation criteria for staining intensity and proportion of immunopositive cells. With these two MAbs, interpathologist and intralaboratory variations were minimal in comparison with differences which could be detected between tissue specimens and antibodies.     

LETM1, a gene deleted in Wolf-Hirschhorn syndrome, encodes an evolutionarily conserved mitochondrial protein

The leucine zipper-, EF-hand-containing transmembrane protein 1 (LETM1) has recently been cloned in an attempt to identify genes deleted in Wolf-Hirschhorn syndrome (WHS), a microdeletion syndrome characterized by severe growth and mental retardation, hypotonia, seizures, and typical facial dysmorphic features. LETM1 is deleted in almost all patients with the full phenotype and has recently been suggested as an excellent candidate gene for the seizures in WHS patients. We have shown that LETM1 is evolutionarily conserved throughout the eukaryotic kingdom and exhibits homology to MDM38, a putative yeast protein involved in mitochondrial morphology. Using LETM1-EGFP fusion constructs and an anti-rat LetM1 polyclonal antibody we have demonstrated that LETM1 is located in the mitochondria. The present study presents information about a possible function for LETM1 and suggests that at least some (neuromuscular) features of WHS may be caused by mitochondrial dysfunction.