Targeted disruption of the inosine 5'-monophosphate dehydrogenase type I gene in mice

Inosine 5'-monophosphate dehydrogenase (IMPDH) is the critical, rate-limiting enzyme in the de novo biosynthesis pathway for guanine nucleotides. Two separate isoenzymes, designated IMPDH types I and II, contribute to IMPDH activity. An additional pathway salvages guanine through the activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) to supply the cell with guanine nucleotides. In order to better understand the relative contributions of IMPDH types I and II and HPRT to normal biological function, a mouse deficient in IMPDH type I was generated by standard gene-targeting techniques and bred to mice deficient in HPRT or heterozygous for IMPDH type II. T-cell activation in response to anti-CD3 plus anti-CD28 antibodies was significantly impaired in both single- and double-knockout mice, whereas a more general inhibition of proliferation in response to other T- and B-cell mitogens was observed only in mice deficient in both enzymes. In addition, IMPDH type I(-/-) HPRT(-/0) splenocytes showed reduced interleukin-4 production and impaired cytolytic activity after antibody activation, indicating an important role for guanine salvage in supplementing the de novo synthesis of guanine nucleotides. We conclude that both IMPDH and HPRT activities contribute to normal T-lymphocyte activation and function.     

Pre-analytical requirements

Correct test selection: a test must have the potential to alter patient management and have the specificity and sensitivity appropriate to the pretest probability of disease. Correct dynamic test procedure: dynamic tests may assist diagnosis and protocols must be readily available. Correct patient preparation: fasting, or other patient preparation, may reduce variability. Clear communication, to both patients and staff, of any such requirements is essential. Correct sample collection: the tube type (for blood) or container (for urine) must be appropriate for the analyte; there must be sufficient volume, avoidance of venous stasis, contaminants and haemolysis; and adequate labelling. Correct sample handling: the time and temperature before and after separation, and the centrifugation and separation procedures, must be suitable for the analyte. Accept/reject criteria must be defined. Methods require thorough evaluation of patient-related pre-analytical factors, and quantification of the effects of time, temperature, haemolysis, anticoagulant type and minimum allowable volume on sample suitability.     

The first report of <Emphasis Type="Italic">Spondias</Emphasis> native to Madagascar: <Emphasis Type="Italic">Spondias tefyi</Emphasis>, sp. nov. (Anacardiaceae)

Spondias represents a genus new to Madagascar’s native flora. Like Campnosperma, it is now known from both American and Asian tropics and Madagascar but not from continental Africa. The new species Spondias tefyi is easily distinguished from all of its Asian congeners by having the stamens shorter than the pistil and fruits brown and lenticellate at maturity (vs. greenish, yellow, orange or red, and relatively smooth). The new species is one of several Anacardiaceae whose fruits are eaten by lemurs in the Analavelona Forest, highlighting the importance of conserving this threatened subhumid forest remnant in southern Madagascar.     

CHANGES IN CELL FINE STRUCTURE DURING LENS REGENERATION IN XENOPUS LAEVIS

Changes at the level of cell fine structure have been studied during lens regeneration in the toad, Xenopus laevis, where cornea gives rise to the new lens. The transformation of these cells may be divided into three phases. (1) In the cornea, flattened cells become cuboidal and rough endoplasmic reticulum increases in amount. (2) In the new lens vesicle, cisternae of the rough ER break down into vesicles, smooth-walled vesicles and free ribosomes increase in number, and mitochondria can become enlarged and irregular, then centrally attenuated. Rudimentary cilia form. (3) As new lens fibers form, ribosomes become very numerous and low density fibrous elements and dense clumps appear in the cytoplasm. These phases are accompanied by marked nucleolar changes. The changes during the 3rd phase are similar to changes in the lens during normal development. The first two phases show an unexpected morphological complexity.     

Mannostatin A, a new glycoprotein-processing inhibitor

Mannostatin A is a metabolite produced by the microorganism Streptoverticillium verticillus and reported to be a potent competitive inhibitor of rat epididymal alpha-mannosidase. When tested against a number of other arylglycosidases, mannostatin A was inactive toward alpha- and beta-glucosidase and galactosidase as well as beta-mannosidase, but it was a potent inhibitor of jack bean, mung bean, and rat liver lysosomal alpha-mannosidases, with estimated IC50's of 70 nM, 450 nM, and 160 nM, respectively. The type of inhibition was competitive in nature. This compound also proved to be an effective competitive inhibitor of the glycoprotein-processing enzyme mannosidase II (IC50 of about 10-15 nM with p-nitrophenyl alpha-D-mannopyranoside as substrate, and about 90 nM with [3H]mannose-labeled GlcNAc-Man5GlcNAc as substrate). However, it was virtually inactive toward mannosidase I. The N-acetylated derivative of mannostatin A had no inhibitory activity. In cell culture studies, mannostatin A also proved to be a potent inhibitor of glycoprotein processing. Thus, in influenza virus infected Madin Darby canine kidney (MDCK) cells, mannostatin A blocked the normal formation of complex types of oligosaccharides on the viral glycoproteins and caused the accumulation of hybrid types of oligosaccharides. This observation is in keeping with other data which indicate that the site of action of mannostatin A is mannosidase II. Thus, mannostatin A represents the first nonalkaloidal processing inhibitor and adds to the growing list of chemical structures that can have important biological activity.     

RBM4 interacts with an intronic element and stimulates tau exon 10 inclusion

Tau protein, which binds to and stabilizes microtubules, is critical for neuronal survival and function. In the human brain, tau pre-mRNA splicing is regulated to maintain a delicate balance of exon 10-containing and exon 10-skipping isoforms. Splicing mutations affecting tau exon 10 alternative splicing lead to tauopathies, a group of neurodegenerative disorders including dementia. Molecular mechanisms regulating tau alternative splicing remain to be elucidated. In this study, we have developed an expression cloning strategy to identify splicing factors that stimulate tau exon 10 inclusion. Using this expression cloning approach, we have identified a previously unknown tau exon 10 splicing regulator, RBM4 (RNA binding motif protein 4). In cells transfected with a tau minigene, RBM4 overexpression leads to an increased inclusion of exon 10, whereas RBM4 down-regulation decreases exon 10 inclusion. The activity of RBM4 in stimulating tau exon 10 inclusion is abolished by mutations in its RNA-binding domain. A putative intronic splicing enhancer located in intron 10 of the tau gene is required for the splicing stimulatory activity of RBM4. Immunohistological analyses reveal that RBM4 is expressed in the human brain regions affected in tauopathy, including the hippocampus and frontal cortex. Our study demonstrates that RBM4 is involved in tau exon 10 alternative splicing. Our work also suggests that down-regulating tau exon 10 splicing activators, such as RBM4, may be of therapeutic potential in tauopathies involving excessive tau exon 10 inclusion.     

The endoplasmic reticulum (ER) translocon can differentiate between hydrophobic sequences allowing signals for glycosylphosphatidylinositol anchor addition to be fully translocated into the ER lumen

The signal sequence within polypeptide chains that designates whether a protein is to be anchored to the membrane by a glycosylphosphatidylinositol (GPI) anchor is characterized by a carboxyl-terminal hydrophobic domain preceded by a short hydrophilic spacer linked to the GPI anchor attachment (omega) site. The hydrophobic domain within the GPI anchor signal sequence is very similar to a transmembrane domain within a stop transfer sequence. To investigate whether the GPI anchor signal sequence is translocated across or integrated into the endoplasmic reticulum membrane we studied the translocation, GPI anchor addition, and glycosylation of different variants of a model GPI-anchored protein. Our results unequivocally demonstrated that the hydrophobic domain within a GPI signal cannot act as a transmembrane domain and is fully translocated even when followed by an authentic charged cytosolic tail sequence. However, a single amino acid change within the hydrophobic domain of the GPI-signal converts it into a transmembrane domain that is fully integrated into the endoplasmic reticulum membrane. These results demonstrated that the translocation machinery can recognize and differentiate subtle changes in hydrophobic sequence allowing either full translocation or membrane integration.     

High-Throughput Measurement and Classification of Organic P in Environmental Samples

Many types of organic phosphorus (P) molecules exist in environmental samples¹. Traditional P measurements do not detect these organic P compounds since they do not react with colorimetric reagents^2,3. Enzymatic hydrolysis (EH) is an emerging method for accurately characterizing organic P forms in environmental samples^4,5. This method is only trumped in accuracy by Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy (³¹P-NMR) -a method that is expensive and requires specialized technical training⁶. We have adapted an enzymatic hydrolysis method capable of measuring three classes of phosphorus (monoester P, diester P and inorganic P) to a microplate reader system⁷. This method provides researchers with a fast, accurate, affordable and user-friendly means to measure P species in soils, sediments, manures and, if concentrated, aquatic samples. This is the only high-throughput method for measuring the forms and enzyme-lability of organic P that can be performed in a standard laboratory. The resulting data provides insight to scientists studying system nutrient content and eutrophication potential.     

Isolation of vascular smooth muscle cell cultures with altered responsiveness to the antiproliferative effect of heparin

Smooth muscle cell (SMC) hyperplasia in the arterial wall is an important component of both atherogenesis and post-vascular surgical restenosis. One naturally-occurring group of molecules which can suppress SMC proliferation in animal models and in cell culture systems are the complex carbohydrates of the heparan sulfate class, including heparin. In this communication, we have used retrovirus vectors to introduce several oncogenes into SMC: SV40 Large T antigen (SVLT), polyoma virus Large T antigen (PyLT), v-myc, and adenovirus E1a. We analyzed a total of 11 cultures. A combination of Western blot analysis, immunoprecipitation, and indirect immunofluorescence confirmed the expression of the infected oncogenic protein in each culture we isolated. All four oncogenes permitted the maintenance of a normal SMC phenotype, as assessed by the general morphology of cells in the light microscope and the presence of SMC-specific α-actin in an immunofluorescence assay. Doubling times in infected cells ranged from 20 to 33 hr, and final cell densities in infected cultures ranged from 4 × 10⁴ to 5 × 10⁵ cells per cm². By comparison, the parent line had a doubling time of 30 hr and reached a final cell density of 1 × 10⁵ cells per cm². Despite the differences sometimes observed in these proliferation parameters, neither one was strongly correlated with heparin responsiveness. PyLT, v-myc, and E1a all produced SMC cultures or lines which retained sensitivity to the antiproliferative activity of heparin (ED₅₀ = 50 μg/ml). In contrast, SVLT expression yielded SMC lines which were highly resistant to heparin (ED₅₀ > 300 μg/ml). These results suggest that altered responsiveness to heparin is dependent upon which oncogenic protein is being expressed in the cells. The availability of cloned, immortal SMC lines with a wide range of heparin responsiveness should aid in the understanding of the cellular and molecular mechanism of action of this potentially important growth regulator and therapeutic agent. © 1996 Wiley-Liss, Inc.