The iron transport protein, transferrin, and the iron storage protein ferritin were examined immunohistochemically along with iron in a number of brain regions from normal and aged humans. Two age groups were examined: a middle-aged group (28-49 years), and an older group (60-90 years). Transferrin, ferritin, and iron are found throughout all brain regions examined, predominantly in the perikaryal cytoplasm of cells that are small and round, fitting the description of oligodendrocytes. These cells are present in the optic nerve and in both the gray and white matter of the cerebral cortex, cerebellum, and olfactory bulb in both age groups. Ferritin is also found in microglial cells in the gray matter of most of these brain regions. In the subcortical regions examined (corpus striatum, hippocampus, amygdala), in addition to oligodendrocytes, astrocytes can frequently be observed that contain transferrin, ferritin, and iron. There is an age-related alteration in cell labeling: astrocytes in both gray and white matter contained transferrin in the oldest age group, whereas in the younger group the subcortical transferrin immunoreactivity was confined mostly to oligodendrocytes. Ferritin in the subcortical brain regions is also present in astrocytes but is primarily confined to those in the gray matter, even in the oldest age group. Iron is found predominantly in oligodendrocytes, although a few iron-positive astrocytes and microglia can be identified. These results indicate that (1) normally oligodendrocytes contain much of the iron and iron-binding proteins found in the brain; and (2) an increase in age is associated with altered cellular distribution of iron-binding proteins, but the altered distribution is specific to glial cells. These results suggest glial cells may have previously undescribed functions related to metal regulation and sequestration.

In this study, we show that Salmonella produces an O-antigen capsule coregulated with the fimbria-and cellulose-associated extracellular matrix. Structural analysis of purified Salmonella extracellular polysaccharides yielded predominantly a repeating oligosaccharide unit similar to that of Salmonella enterica serovar Enteritidis lipopolysaccharide O antigen with some modifications. Putative carbohydrate transport and regulatory operons important for capsule assembly and translocation, designated yihU-yshA and yihVW, were identified by screening a random transposon library with immune serum generated to the capsule. The absence of capsule was confirmed by generating various isogenic ⌬yih mutants, where yihQ and yihO were shown to be important in capsule assembly and translocation. Luciferase-based expression studies showed that AgfD regulates the yih operons in coordination with extracellular matrix genes coding for thin aggregative fimbriae and cellulose. Although the capsule did not appear to be important for multicellular behavior, we demonstrate that it was important for survival during desiccation stress. Since the yih genes are conserved in salmonellae and the O-antigen capsule was important for environmental persistence, the formation of this surface structure may represent a conserved survival strategy.

Background: Certain types of potassium channels (known as Eag1, KCNH1, Kv10.1) are associated with the production of tumours in patients and in animals. We have now studied the expression pattern of the Eag1 channel in a large range of normal and tumour tissues from different collections utilising molecular biological and immunohistochemical techniques.

In the budding yeast Saccharomyces cerevisiae, entry into meiosis and its successful completion depend on two positive regulators, Ime1 and Ime2. Ime1 is a transcriptional activator that is required for transcription of IME2, a serine/threonine protein kinase. We show that in vivo Ime2 associates with Ime1, that in vitro Ime2 phosphorylates Ime1, and that in living cells the stability of Ime1 depends on Ime2. Diploid cells with IME2 deleted show an increase in the level of Ime1, whereas haploid cells overexpressing IME2 show a decrease in the stability of Ime1. Furthermore, the level of Ime1 depends on the kinase activity of Ime2. Using a mutation in one of the ATPase subunits of the proteasome, RPT2, we demonstrate that Ime1, amino acids 270 to 360, is degraded by the 26S proteasome. We also show that Ime2 itself is an extremely unstable protein whose expression in vegetative cultures is toxic. We propose that a negative-feedback loop ensures that the activity of Ime1 will be restricted to a narrow window.Successful progression and completion of the mitotic cell cycle depends on transcriptional and proteolytic regulation. These two processes determine the availability of cyclins and cyclin-dependent kinase (CDK) inhibitors that govern the sequential activation of CDKs (18,28,31,35). Initiation and progression through the meiotic cycle should also be subjected to transcriptional and proteolytic regulation. Indeed, in budding yeast a transcriptional cascade governs initiation and progression through the meiotic cell cycle (4). Yet there is no direct evidence concerning proteolysis of either positive or negative meiotic regulators. This report focuses on the regulated degradation of one of the two positive regulators of meiosis in Saccharomyces cerevisiae, Ime1, by the other, Ime2.IME1 encodes a transcriptional activator (30, 47) that is necessary for the transcription of meiosis-specific genes (48). Ime1 is tethered to promoters of early meiosis-specific genes, such as IME2, by a specific DNA-binding protein, Ume6 (39). Diploid cells with deletions of IME1 arrest at G 1 prior to the initiation of premeiotic DNA replication (22). The transcription of IME1 is regulated by nutrients. In vegetative cultures with glucose as the sole carbon source, IME1 is silent, but in the presence of acetate, low levels of IME1 mRNA are observed (22). Under meiotic conditions, i.e., nitrogen depletion and the presence of a nonfermentable carbon source such as acetate, transcription of IME1 is induced transiently in MATa/ MAT␣ diploids (22). It is not known whether this transient transcription reflects transient availability of the Ime1 protein.In addition, the IME1 promoter is subject to positive autoregulation (40,43,44), as well as negative-feedback regulation by both Ime1 and Ime2 (43,48,49).Another important regulator of meiosis and sporulation is the serine/threonine protein kinase Ime2 (12,24,34,48,49). Diploid cells with deletions of IME2 show a 5-to 12-h delay in the transcription of early meiosis-specific genes, a reduction...

The hormone receptor-like protein Gpr1p physically interacts with phosphatidylinositol-specific phospholipase C (Plc1p) and with the G␣ protein Gpa2p, as shown by two-hybrid assays and co-immune precipitation of epitope-tagged proteins. Plc1p binds to Gpr1p in either the presence or absence of Gpa2, whereas the Gpr1p/Gpa2p association depends on the presence of Plc1p. Genetic interactions between the null mutations plc1⌬, gpr1⌬, gpa2⌬, and ras2⌬ suggest that Plc1p acts together with Gpr1p and Gpa2p in a growth control pathway operating in parallel to the Ras2p function. Diploid cells lacking Gpr1p, Plc1p, or Gpa2p fail to form pseudohyphae upon nitrogen depletion, and the filamentation defect of gpr1⌬ and plc1⌬ strains is rescued by activating a mitogen-activated protein kinase pathway via STE11-4 or by activating a cAMP pathway via overexpressed Tpk2p. Plc1p is also required for efficient expression of the FG(TyA)::lacZ reporter gene under nitrogen depletion.In conclusion, we have identified two physically interacting proteins, Gpr1p and Plc1p, as novel components of a nitrogen signaling pathway controlling the developmental switch from yeast-like to pseudohyphal growth. Our data suggest that phospholipase C modulates the interaction of the putative nutrient sensor Gpr1p with the G␣ protein Gpa2p as a downstream effector of filamentation control.

Neuron-glia interactions play a key role in maintaining and regulating the central nervous system. Glial cells are implicated in the function of dopamine neurons and regulate their survival and resistance to injury. Parkinson's disease is characterized by the loss of dopamine neurons in the substantia nigra pars compacta, decreased striatal dopamine levels and consequent onset of extrapyramidal motor dysfunction. Parkinson's disease is a common chronic, neurodegenerative disorder with no effective protective treatment. In the 6-OHDA mouse model of Parkinson's disease, doxycycline administered at a dose that both induces/represses conditional transgene expression in the tetracycline system, mitigates the loss of dopaminergic neurons in the substantia nigra compacta and nerve terminals in the striatum. This protective effect was associated with: (1) a reduction of microglia in normal mice as a result of doxycycline administration per se; (2) a decrease in the astrocyte and microglia response to the neurotoxin 6-OHDA in the globus pallidus and substantia nigra compacta, and (3) the astrocyte reaction in the striatum. Our results suggest that doxycycline blocks 6-OHDA neurotoxicity in vivo by inhibiting microglial and astrocyte expression. This action of doxycycline in nigrostriatal dopaminergic neuron protection is consistent with a role of glial cells in Parkinson's disease neurodegeneration. The neuroprotective effect of doxycycline may be useful in preventing or slowing the progression of Parkinson's disease and other neurodegenerative diseases linked to glia function.

No abstract

KCNN3, encoding the small conductance calcium-activated potassium channel SK3, harbours a polymorphic CAG repeat in the amino-terminal coding region with yet unproven function. Hypothesizing that KCNN3 genotypes do not influence susceptibility to schizophrenia but modify its phenotype, we explored their contribution to specific schizophrenic symptoms. Using the Göttingen Research Association for Schizophrenia (GRAS) data collection of schizophrenic patients (n = 1074), we performed a phenotype-based genetic association study (PGAS) of KCNN3. We show that long CAG repeats in the schizophrenic sample are specifically associated with better performance in higher cognitive tasks, comprising the capacity to discriminate, select and execute (p < 0.0001). Long repeats reduce SK3 channel function, as we demonstrate by patch-clamping of transfected HEK293 cells. In contrast, modelling the opposite in mice, i.e. KCNN3 overexpression/channel hyperfunction, leads to selective deficits in higher brain functions comparable to those influenced by SK3 conductance in humans. To conclude, KCNN3 genotypes modify cognitive performance, shown here in a large sample of schizophrenic patients. Reduction of SK3 function may constitute a pharmacological target to improve cognition in schizophrenia and other conditions with cognitive impairment.