Dihydroorotate dehydrogenase mRNA and protein expression analysis in normal and drug-resistant cells

To follow the expression of the fourth enzyme of pyrimidine de novo synthesis dihydroorotate dehydrogenase (DHODH) in cells and tissues, we studied the DHODH mRNA expression by means of RT-PCR in rat tissues. Rabbit polyclonal anti-DHODH immunoglobulins were applied for immunochemical quantification of the enzyme protein by Western blotting. In mouse B-lymphocytes, which were adapted to tolerate up to a 50-fold concentration of the DHODH inhibitor leflunomide, a 20 fold protein overexpression was measured. Southern blotting indicated DHODH gene amplification.     

Mycoplasma haemocanis infection--a kennel disease?

Mycoplasma haemocanis (formerly Haemobartonella canis) is a red blood cell parasite that causes disease mainly in immunosuppressed and splenectomized dogs. Clinical outbreak of the disease resulted in failure of a large experimental project. We aimed to identify whether M. haemocanis has increased prevalence in kennel-raised dogs. In a prospective study, we compared the prevalence of M. haemocanis in whole blood (anti-coagulated by use of EDTA) collected from pet dogs (University of Illinois, Urbana Champaign, Ill.; n = 60) with that in blood from dogs raised in three distinct kennels in western Europe (WE; n = 23), eastern Europe (EE; n = 20), and North America (NA; n = 20). Screening included antibody testing and microscopy of blood smears. The presence of M. haemocanis was identified using a polymerase chain reaction (PCR) assay for specific DNA of the organism. None of the pet dogs (0%) was test positive for M. haemocanis DNA. Mycoplasma haemocanis was found in dogs tested at all of the kennels. Infection rate in the three kennels was 30, 35, and 87%, respectively (all P < 0.001 versus control, chi2-test). Latent infection with M. haemocanis was not a single observation in kennel-raised dogs. Prevalence may be higher than that in a pet dog population. The potential exists for these latent infections to adversely affect or confound research results.     

Effects of Septal Grafts on Acetylcholine Release from Rat Hippocampus After 192 IgG-Saporin Lesion

The cholinergic inputs to the rat hippocampus were lesioned by intraseptal injections of 192 IgG-saporin. After 15 days, fetal septal cells were grafted into the hippocampus. Thirteen months later, hippocampal acetylcholine (ACh) release was studied by microdialysis. Lesioning reduced basal ACh release (100%) to 20% of normal, which was compensated for by the graft (71%). Infusion of the serotonin uptake inhibitor citalopram (100 M) enhanced ACh release to the same extent (% of basal release) in all rat groups. Systemic injection of 8-OH-DPAT (0.5 mg/kg, SC), an agonist of 5-HT_1A receptors, caused a smaller ACh release than citalopram. Acetylcholinesterase (AChE) staining and densitometric quantification revealed that the lesion-induced reduction of the AChE-staining density was compensated for by septal grafting. In conclusion, both histochemical and biochemical methods showed that cholinergic hippocampal parameters were drastically impaired by 192 IgG-saporin lesions, but were almost completely restored by septal grafting. The graft responded to intrinsic serotonergic regulation.     

The human phosphatidylinositol phosphatase SAC1 interacts with the coatomer I complex

The Saccharomyces cerevisiae SAC1 gene encodes an integral membrane protein of the endoplasmic reticulum (ER) and the Golgi apparatus. Yeast SAC1 mutants display a wide array of phenotypes including inositol auxotrophy, cold sensitivity, secretory defects, disturbed ATP transport into the ER, or suppression of actin gene mutations. At present, it is not clear how these phenotypes relate to the finding that SAC1 displays polyphosphoinositide phosphatase activity. Moreover, it is still an open question whether SAC1 functions similarly in mammalian cells, since some phenotypes are yeast-specific. Potential protein interaction partners and, connected to that, possible regulatory circuits have not been described. Therefore, we have cloned human SAC1 (hSAC1), show that it behaves similar to ySac1p in terms of substrate specificity, demonstrate that the endogenous protein localizes to the ER and Golgi, and identify for the first time members of the coatomer I (COPI) complex as interaction partners of hSAC1. Mutation of a putative COPI interaction motif (KXKXX) at its C terminus abolishes interaction with COPI and causes accumulation of hSAC1 in the Golgi. In addition, we generated a catalytically inactive mutant, demonstrate that its lipid binding capacity is unaltered, and show that it accumulates in the Golgi, incapable of interacting with the COPI complex despite the presence of the KXKXX motif. These results open the possibility that the enzymatic function of hSAC1 provides a switch for accessibility of the COPI interaction motif.     

Liposomes for microcompartmentation of enzymes and their influence on catalytic activity

Cholinephosphotransferase (CPT), the terminal enzyme in the de novo synthesis of phosphatidylcholine (PC), has an important role in regulating the acyl group of PC in mammalian cells. A 593bp cDNA coding for the 3(')-end of the CPT gene has been cloned from guinea pig liver using degenerative oligos based on the human CPT gene. It has 85% amino acid homology with the human CPT enzyme and amino acid variations were found to cluster at few points. Restriction enzyme polymorphisms were found particularly with respect to BamHI and NcoI. Hydrophobic and helix plot analysis of the sequence shows a similar pattern to human counterpart except for amino acid residues 142-179 and 173-179. PCR analysis suggested that a predominant pseudogene may be present in guinea pig and also the intronic sequences were much shorter when compared to the human CPT gene. We are the first to report on the C-terminal 195 amino acid residues of the CPT gene from any animal species alike in many aspects of cellular metabolism. The probable differences in genomic organization and its expression in different cancer cells have been discussed here having CPT as an important target for cancer drug development.     

Altered glycosylation of acetylcholinesterase in APP (SW) Tg2576 transgenic mice occurs prior to amyloid plaque deposition

Previous studies have shown that a minor glycoform of acetylcholinesterase (AChE) is increased in Alzheimer's disease brain and cerebrospinal fluid. This glycoform can be distinguished from other AChE species by its lack of binding to concanavalin A (Con A). In this study, the temporal relationship between AChE glycosylation and Abeta deposition was examined in Tg2576 mice. There was a significant (p < 0.05) difference in AChE glycosylation in Tg2576 mice compared with age-matched background strain control mice at 4 months of age. This difference in glycosylation was also observed in 8- and 12-month-old Tg2576 mice. In contrast, Abeta plaques were only seen in the Tg2576 mice at 12 months of age, and were not detected at 4 and 8 months of age. Soluble human-sequence Abeta was detected as early as 4 months of age in the transgenic mice. The altered AChE glycosylation was due to an increase in a minor AChE isoform, which did not bind Con A, similar to that previously observed to be increased in Alzheimer's disease brain and cerebrospinal fluid. The results demonstrate that in transgenic mice altered AChE glycosylation is associated with very early events in the development of AD-like pathology. The study supports the possibility that glycosylation may also be a useful biomarker of AD.     

Procathepsin D interacts with prosaposin in cancer cells but its internalization is not mediated by LDL receptor-related protein

The cell surface binding, endocytosis, and lysosomal routing of procathepsin D (procath-D) in cancer cells are mostly independent of the mannose-6-phosphate (M6P) receptors. In an attempt to define the receptor involved, we intracellularly cross-linked procath-D with a 68-kDa protein that we identified with specific antibodies as prosaposin in human breast and ovarian cancer cell lines. In cancer cells, this protein-protein interaction was resistant to ammonium chloride or M6P treatment, indicating that it was independent of the M6P receptors. A similar interaction also occurred in the breast cancer cell culture medium between the secreted prosaposin and procath-D. Since these two precursors can be endocytosed, we then determined whether they were interacting with the same cell surface receptor. In fibroblasts, we confirmed that the endocytosis of these two proteins was different since it was generally mediated by the M6P receptors for procath-D and mostly by LRP (LDL receptor-related protein) for prosaposin. In breast cancer cells, prosaposin endocytosis was not detected, in contrast to procath-D endocytosis, suggesting that the majority of procath-D is not internalized as a complex with prosaposin. Moreover, RAP (receptor-associated protein), a ligand inhibiting LRP-mediated endocytosis, prevented internalization of prosaposin in 49-F rat fibroblasts, but did not affect procath-D M6P-independent internalization in MDA-MB231 cells. We conclude that in breast cancer cells, even though procath-D interacts intracellularly and extracellarly with prosaposin, it is endocytosed independent of prosaposin by a receptor different from the M6P receptors and the LRP.     

Structural characterization of the closed conformation of mouse guanylate kinase

Guanylate kinase (GMPK) is a nucleoside monophosphate kinase that catalyzes the reversible phosphoryl transfer from ATP to GMP to yield ADP and GDP. In addition to phosphorylating GMP, antiviral prodrugs such as acyclovir, ganciclovir, and carbovir and anticancer prodrugs such as the thiopurines are dependent on GMPK for their activation. Hence, structural information on mammalian GMPK could play a role in the design of improved antiviral and antineoplastic agents. Here we present the structure of the mouse enzyme in an abortive complex with the nucleotides ADP and GMP, refined at 2.1 A resolution with a final crystallographic R factor of 0.19 (R(free) = 0.23). Guanylate kinase is a member of the nucleoside monophosphate (NMP) kinase family, a family of enzymes that despite having a low primary structure identity share a similar fold, which consists of three structurally distinct regions termed the CORE, LID, and NMP-binding regions. Previous studies on the yeast enzyme have shown that these parts move as rigid bodies upon substrate binding. It has been proposed that consecutive binding of substrates leads to "closing" of the active site bringing the NMP-binding and LID regions closer to each other and to the CORE region. Our structure, which is the first of any guanylate kinase with both substrates bound, supports this hypothesis. It also reveals the binding site of ATP and implicates arginines 44, 137, and 148 (in addition to the invariant P-loop lysine) as candidates for catalyzing the chemical step of the phosphoryl transfer.     

UNEP/SETAC life cycle initiative: background,aims and scope

The conclusions about the development of the content of the LC Initiative are the following:

In vitro production of ovarian steroids in yellow perch (Perca flavescens): effects of photothermal manipulation, gonadotropin and phorbol ester

We investigated the capacity of ovaries of yellow perch to produce steroid hormones in vitro and the ovarian response to gonadotropin and phorbol ester during the annual reproductive cycle. The effects of photothermal manipulation on perch gonadal steroidogenesis and its regulation have also been examined. Initially, all females kept indoors were exposed to the same water temperature and photoperiod. By the end of August, following the first sampling, fish were submitted to different photothermal regimes. Group A was maintained under photothermal conditions characteristic for southern Ohio. Group B was submitted to a condensed light/temperature regime designed to accelerate physiological changes that depend on photothermal stimuli. In group A, basal in vitro production of ovarian estradiol (E2) was the highest in October and November, and hCG significantly stimulated E2 secretion during the entire period of vitellogenesis (October-January). In this group, the highest production of basal testosterone (T) was observed before spawning. hCG-stimulated production of T was highest at the beginning of vitellogenesis. Gonadotropin stimulated T production before spawning, a time when gonadotropin was unable to stimulate E2 production. Phorbol ester (PDBu) stimulated E2 and T production during vitellogenesis at the same time points as hCG did (E2: December, January; T: December). hCG-stimulated T production was not mimicked by PDBu in April. Condensing of the photothermal cycle resulted in diminished ovarian production of E2 during vitellogenesis. Moreover, the fish submitted to a condensed photothermal cycle demonstrated augmented T production during the postvitellogenic stage of ovarian development. Ovaries of group B did not respond to PDBu. Generally, the seasonal fluctuations in ovarian capacity to produce E2 and T as well as in gonadal responsiveness to gonadotropin observed in female yellow perch illustrate the dynamic nature of ovarian endocrine function. The lack of response to gonadotropin with regard to E2 production prior to spawning is not due to insensitivity to gonadotropin, but rather due to some deficiency in steroidogenesis (e.g. reduced aromatase activity). It appears also that ovarian steroidogenesis and its regulation are dependent on annual changes of photothermal conditions.