Metabolic functions of duplicate genes in Saccharomyces cerevisiae

The roles of duplicate genes and their contribution to the phenomenon of enzyme dispensability are a central issue in molecular and genome evolution. A comprehensive classification of the mechanisms that may have led to their preservation, however, is currently lacking. In a systems biology approach, we classify here back-up, regulatory, and gene dosage functions for the 105 duplicate gene families of Saccharomyces cerevisiae metabolism. The key tool was the reconciled genome-scale metabolic model iLL672, which was based on the older iFF708. Computational predictions of all metabolic gene knockouts were validated with the experimentally determined phenotypes of the entire singleton yeast library of 4658 mutants under five environmental conditions. iLL672 correctly identified 96%-98% and 73%-80% of the viable and lethal singleton phenotypes, respectively. Functional roles for each duplicate family were identified by integrating the iLL672-predicted in silico duplicate knockout phenotypes, genome-scale carbon-flux distributions, singleton mutant phenotypes, and network topology analysis. The results provide no evidence for a particular dominant function that maintains duplicate genes in the genome. In particular, the back-up function is not favored by evolutionary selection because duplicates do not occur more frequently in essential reactions than singleton genes. Instead of a prevailing role, multigene-encoded enzymes cover different functions. Thus, at least for metabolism, persistence of the paralog fraction in the genome can be better explained with an array of different, often overlapping functional roles.     

Genetic and cytological characterization of the RecA-homologous proteins Rad51 and Dmc1 of Schizosaccharomyces pombe

The Schizosaccharomyces pombe rad51⁺ and dmc1⁺ genes code for homologues of the Escherichia coli recombination protein RecA. Deletion of rad51⁺ causes slow growth, retardation of cell division and a decrease in viability. rad51 cells have a defect in mating-type switching. The DNA modification at the mating-type locus required for mating-type switching contributes to slow growth in the rad51 mutant. Cell mating is reduced in crosses homozygous for rad51. Ectopic expression of the dmc1⁺ gene allowed us to demonstrate that the reduction in meiotic recombination in dmc1 mutants is not caused by a disturbance of rad24 expression from the dmc1-rad24 bicistronic RNA. We describe the functional defects of terminally epitope-tagged Dmc1 and Rad51 and discuss it in terms of protein interaction. Presumptive Rad51 and Dmc1 foci were detected on spreads of meiotic chromatin.     

T cell receptor induced intracellular redistribution of type I protein kinase A

The productive activation of CD4(+) T lymphocytes, leading to proliferation and cytokine secretion, requires precise temporal regulation of intracellular cyclic AMP concentrations. The major effector molecule activated by cyclic AMP in mammalian cells is the cyclic AMP-dependent protein kinase A (PKA). The type I PKA isozyme mediates the inhibitory effects of cyclic AMP on T-cell activation. Using laser scanning confocal microscopy, we demonstrated that the regulation of PKA type I activity involves spatial redistribution of PKA type I molecules following T-cell receptor (TCR) stimulation. In resting T cells, PKA type I was located in membrane proximal regions and distributed equally across the cell. Shortly after antigen engagement, T cells and antigen-presenting cells formed an area of intense contact, known as the immunological synapse. TCR concentrated at the synapse, whereas PKA type I molecules redistributed to the opposite cell pole within 10 min after T-cell stimulation. Type I PKA redistribution was solely dependent on TCR signalling, because we observed the same temporal and spatial distribution after antibody-mediated cross-linking of the TCR-associated CD3 complex. Segregation of TCR and PKA type I molecules was maintained for at least 20 min. Thirty minutes after stimulation, PKA type I partially colocalized with the TCR. After 60 min, PKA type I distribution again approached the resting state. Considering that initial TCR signals lead to increases in intracellular cyclic AMP, PKA type I molecules may be targeted towards localized cyclic AMP accumulations or transported away from these areas, depending on the requirements of the cellular response.     

Casein is an essential cofactor in autoantibody reactivity directed against the C-terminal SmD1 peptide AA 83-119 in systemic lupus erythematosus

The C-terminal peptide SmD1(83-119) has been identified as an important autoantigen in systemic lupus erythematosus (SLE). ELISA studies have shown that roughly 70% of all sera from patients with SLE react with this peptide. Previous findings revealed that the addition of blocking agents and sample dilution buffers influences the discrimination between positive and negative anti-SmD1(83-119) sera in SLE. The aim of the present study was to identify possible cofactors in the anti-SmD1(83-119) reactivity. We therefore tested SLE sera (n=6) for anti-SmD1(83-119) reactivity by ELISA and analysed the effects of different blocking agents (1% skim milk, 1% gelatin, and 1% BSA). In our investigation, lipids were extracted from skim milk using dichlomethane, and the putative fraction was tested to assess the assay's ability to discriminate between positive and negative sera. The effects of enzymatic digestion by casein were analyzed, and different concentrations of casein were used to determine the role of this protein in the detection of anti-SmD1(83-119) antibodies by ELISA. Furthermore, rabbits were immunized with SmD1(83-119) adsorbed to casein and control proteins. One percent skim milk was the most effective blocking agent and sample dilution buffer for the discrimination between positive and negative sera. As demonstrated by SDS electrophoresis, the discriminative capacity was influenced by enzymatic digestion of skim milk proteins, but not by lipid extraction. Differences in anti-SmD1(83-119) reactivity upon variation of the casein concentration suggest that the protein plays a significant role in the detection of anti-SmD1(83-119) antibodies. However, our immunisation studies did not show any effect of casein on anti-SmD1(83-119) reactivity, suggesting that it has no immunogenic effect on the anti-SmD1(83-119) response. In conclusion, casein seems to be an important cofactor in autoantibody reactivity directed against the C-terminal SmD1(83-119) peptide and probably functions by changing the conformation of the peptide's critical epitope.     

Uncertainties − discrepancies in immunology

Summary: The many immunological observations and results from in-vitro or in-vivo experiments vary, and their interpretations differ enormously. A major problem is that within a normal distribution of biological phenomena, which are measurable with many methods, virtually anything is possible. Within a coevolutionary context, the definition of biologically relevant thresholds is an important key to improve our understanding of weaknesses and strengths of the immune system. This review is a personal view, comparing textbook rules and experiments using model antigens with observations on immunity against infections or tumors to critically evaluate our perception and understanding of specificity, affinity maturation, antigen presentation, selection of the class of the immune response, immunological memory and protective immunity, positive selection of T cells and self/nonself discrimination.     

Responsiveness of C-fiber nociceptors to punctate force-controlled stimuli in isolated rat skin: lack of modulation by inflammatory mediators and flurbiprofen

Although cutaneous C-fiber nociceptors show dramatic inflammatory sensitization to heat, they do not appear to get sensitized to the mechanical stimulation by von Frey hairs. We employed force-controlled punctate electromechanical stimulation to receptive fields of 61 characterized C-fibers in the isolated rat skin-saphenous nerve preparation. In general: low-in contrast to higher-threshold units showed greater dynamic sensitivity and response magnitude, an earlier onset and a stronger degree of adaptation, the latter due to the linear rise of the force stimulus. On this methodological basis three groups of units were subject to a mix of inflammatory mediators, to flurbiprofen or to control solution. Subsequent mechanostimulation revealed a good reproducibility of the control response and no significant changes in the treatment groups. In conclusion, even refined mechanostimulation was unable to demonstrate sensitization of the predominant nociceptor classes in the rat skin.     

Inhibition of in vivo tumorigenicity and invasiveness of a human glioblastoma cell line transfected with antisense uPAR vectors

Our previous studies showed that glioblastomas express increased urokinase-type plasminogen activator receptors (uPARs) in comparison to low-grade gliomas (Yamamoto et al., Cancer Res., 54, 5016-5020, 1994). To explore whether downregulation of uPAR inhibits tumor formation and invasiveness, a human glioblastoma cell line was transfected with a cDNA construct corresponding to 300 bp of the human uPAR's 5¢ end in an antisense orientation, resulting in a reduced number of uPA receptors. Co-culture studies with tumor spheroids and fetal rat brain aggregates showed that antisense SNB19-AS1 cells expressing reduced uPAR failed to invade fetal rat brain aggregates. Intracerebral injection of SNB19-AS1 stable transfectants failed to form tumors and were negative for uPAR expression in nude mice. Thus uPAR appears in this model to be essential for tumorigenicity and invasion of glioblastomas in vivo.     

Pituitary corticotroph adenoma with crooke’s hyalinization

The diagnosis of pituitary corticotroph adenoma relies on the demonstration of a loss of the normal feedback control of adrenocorticotropic hormone (ACTH) biosynthesis by cortisol. The marked variability in the degree of ACTH suppression by glucocorticoids in these tumors, however, greatly enhances the difficulty in distinguishing Cushing’s disease from other syndromes of glucocorticoid excess. To illustrate this variability, we describe the clinical, biochemical, and morphological characteristics of a pituitary corticotroph adenoma in a 63-year-old woman, who presented with symptoms of a sellar mass but did not initially have florid Cushing’s disease. Light and electron microscopy of the pituitary tumor showed a corticotroph adenoma with Crooke’s hyalinization of the tumor cells, characterized by the accumulation of keratin immunoreactive microfilaments similar to those observed in normal corticotrophs in the presence of excess glucocorticoids. This case illustrates an unusual clinical presentation that may be associated with pituitary corticotroph adenoma showing Crooke’s hyalinization.     

Efficacy of oocytes donated by older women in an oocyte donation programme

Population and insemination studies indicate that women experiencedeclining fertility with ageing. The question therefore ariseswhether older women are suitable oocyte donors. This study addressesthis issue by examining the relationship between oocyte donorage and clinical outcome in a large oocyte donation programme.We retrospectively reviewed data from 458 consecutive oocytedonation cycles completed by 164 different designated oocytedonors. Data were divided into two groups: group A, cycles withdonors aged 21–30 years at the time of follicular aspiration(193 cycles, 88 donors); and group B, cycles with donors aged31–40 years at the time of follicular aspiration (265cycles, 86 donors). Five donors, because of ageing during repetitivedonations, contributed data to groups A and B. In a given cycle,all oocytes for a recipient came from only one designated donor.Comparing the two donor groups, there was no difference in theamount of gonadotrophin used to achieve optimal stimulation;however, more oocytes were obtained from group A than groupB donors (16.8 ± 6.9 and 15.1 ± 8.1 respectively,P < 0.05). Similar percentages of oocytes were fertilizedin each group, resulting in the transfer of comparable numbersof embryos (4.5 ± 1.1 and 4.4 ± 13 respectively).Comparable clinical pregnancy rates were achieved (group A,36%; group B, 37%). The spontaneous abortion rates were alsosimilar (group A, 20%; group B, 12%), resulting in comparableongoing and delivered pregnancy rates per cycle (group A, 29%;group B, 32%) and per embryo transferred (group A, 6.4%; groupB, 7.3%). In conclusion, women of proven fertility should notbe excluded from donating oocytes simply because of their age.There exists a cohort of fertile women who resist the decreasingfecundity and increasing spontaneous abortion rates associatedwith ageing. With careful screening, many women of proven fertilitycan donate oocytes until the age of 40 years with an efficacyequal to that of younger women. Given the relative shortageof suitable oocyte donors, and increasing requests from recipientswith previous donor oocyte babies to obtain oocytes from thesame, now older, donor, the findings of this study are of practicalclinical importance.