Subcellular localization of Aleutian mink disease parvovirus proteins and DNA during permissive infection of Crandell feline kidney cells.

Confocal microscopy allowed us to localize viral nonstructural (NS) and capsid (VP) proteins and DNA simultaneously in cells permissively infected with Aleutian mink disease parvovirus (ADV). Early after infection, NS proteins colocalized with viral DNA to form intranuclear inclusions, whereas VP proteins formed hollow intranuclear shells around the inclusions. Later, nuclei had irregular outlines and were virtually free of ADV products. In these cells, inclusions of viral DNA with or without associated NS protein were embedded in cytoplasmic VP protein. These findings implied that ADV replication within an infected cell is regulated spatially as well as temporally.     

Chlorate as a Transport Analog for Nitrate Absorption by Roots of Tomato

Several studies have indicated that chlorate (ClO3-) and nitrate (NO3-) may share a common transport system in higher plants. Here, we compared the interactions between ClO3- and NO3-uptake by roots of intact tomato (Lycopersicon esculentum cv T5) plants. Exposure to ClO3- for more than 2 h inhibited both net ClO3- and K+ uptake, presumably because of ClO3- toxicity; consequently, subsequent measurements were conducted after short exposures to ClO3-. The apparent affinity and apparent maximum rate of absorption for net ClO3- and NO3- uptake were very similar. Interactions between ClO3- and NO3- transport were complex; 50 [mu]M NO3- acted as a mixed inhibitor of net ClO3- uptake, but 50 [mu]M ClO3- had no significant effect on net NO3- uptake, and 500 [mu]M ClO3- had no significant effect on 15NO3- influx. If the two ions share a single common high-affinity transport system, it is much more selective for NO3- than would be suggested by the similarity of net NO3- and ClO3- uptake kinetics. Our results indicate that, although NO3- may interfere with root ClO3- uptake, ClO3- is not a useful analog for the root high-affinity NO3- transport system.     

Correlation between lipid plane curvature and lipid chain order.

The 1-palmitoyl-2-oleoyl-phosphatidylethanolamine: 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPE:POPC) system has been investigated by measuring, in the inverted hexagonal (HII) phase, the intercylinder spacings (using x-ray diffraction) and orientational order of the acyl chains (using 2H nuclear magnetic resonance). The presence of 20 wt% dodecane leads to the formation of a HII phase for the composition range from 0 to 39 mol% of POPC in POPE, as ascertained by x-ray diffraction and 2H nuclear magnetic resonance. The addition of the alkane induces a small decrease in chain order, consistent with less stretched chains. An increase in temperature or in POPE proportion leads to a reduction in the intercylinder spacing, primarily due to a decrease in the water core radius. A temperature increase also leads to a reduction in the orientational order of the lipid acyl chains, whereas the POPE proportion has little effect on chain order. A correlation is proposed to relate the radius of curvature of the cylinders in the inverted hexagonal phase to the chain order of the lipids adopting the HII phase. A simple geometrical model is proposed, taking into account the area occupied by the polar headgroup at the interface and the orientational order of the acyl chains reflecting the contribution of the apolar core. From these parameters, intercylinder spacings are calculated that agree well with the values determined experimentally by x-ray diffraction, for the variations of both temperature and POPE:POPC proportion. This model suggests that temperature increases the curvature of lipid layers, mainly by increasing the area subtended by the hydrophobic core through chain conformation disorder, whereas POPC content affects primarily the headgroup interface contribution. The frustration of lipid layer curvature is also shown to be reflected in the acyl chain order measured in the L alpha phase, in the absence of dodecane; for a given temperature, increased order is observed when the curling tendencies of the lipid plane are more pronounced.     

Methylammonium as a Transport Analog for Ammonium in Tomato (Lycopersicon esculentum L.)

Methylammonium (CH3NH3+) has been widely used as an analog of ammonium (NH4+) for examining transport in bacteria and fungi. We compared the kinetics of root CH3NH3+ and NH4+ uptake from solution culture in intact tomato (Lycopersicon esculentum cv T5) plants. Efflux of NH4+ and CH3NH3+ was negligible. The apparent maximum rate of absorption (apparent Vmax) was similar for NH4+ and CH3NH3+, but the apparent affinity (apparent Km) was about 10-fold greater for NH4+ than for CH3NH3+. In characterizing the interaction between NH4+ and CH3NH3+ transport, we used [15N]NH4+ and [14C]CH3NH3+ as well as improved methods for analysis of nonisotopic CH3NH3+ and NH4+. CH3NH3+ acted as an inhibitor of NH4+ influx. Relatively low concentrations of NH4+ strongly inhibited CH3NH3+ influx. Treatments with 1 mM methionine sulfoximine that blocked NH4+ assimilation had little influence on NH4+ inhibition of CH3NH3+ influx. These results suggest that the two ions share a common transport system in tomato, but because this transport system has a much greater affinity for NH4+, CH3NH3+ may be used as a transport analog only when ambient concentrations of NH4+ are very low.     

The sight test fee: effect on ophthalmology referrals and rate of glaucoma detection.

OBJECTIVE--To assess changes, if any, in the numbers of referrals and outcome of glaucoma referrals to the hospital eye service since the introduction of the sight test fee on 1 April 1989. DESIGN--Review of referral records and clinical notes. SETTING--Referrals to the Bristol Eye Hospital. SUBJECTS--51,919 patients referred to the Bristol Eye Hospital between 1984 and 1992. 9438 case notes of patients referred between 1987 and 1991 were examined in detail. MAIN OUTCOME MEASURES--Numbers of referrals; rate of adult true positive glaucoma referrals. RESULTS--Referrals to the Bristol Eye Hospital were between 13.7% and 19.0% fewer than expected after the introduction of the sight test fee. True positive glaucoma referrals were reduced by the same proportion. CONCLUSIONS--The numbers of patients being identified as requiring treatment or follow up for potentially blinding glaucoma have declined by nearly one fifth since the introduction of the sight test fee. An increased prevalence of preventable blindness may result.     

Intrachain disulfide bond in the core hinge region of human IgG4.

IgG is a tetrameric protein composed of two copies each of the light and heavy chains. The four-chain structure is maintained by strong noncovalent interactions between the amino-terminal half of pairs of heavy-light chains and between the carboxyl-terminal regions of the two heavy chains. In addition, interchain disulfide bonds link each heavy-light chain and also link the paired heavy chains. An engineered human IgG4 specific for human tumor necrosis factor-alpha (CDP571) is similar to human myeloma IgG4 in that it is secreted as both disulfide bonded tetramers (approximately 75% of the total amount of IgG) and as tetramers composed of nondisulfide bonded half-IgG4 (heavy chain disulfide bonded to light chain) molecules. However, when CDP571 was genetically engineered with a proline at residue 229 of the core hinge region rather than serine, CDP571 (S229P), or with an IgG1 rather than IgG4 hinge region, CDP571(gamma 1), only trace amounts of nondisulfide bonded half-IgG tetramers were observed. Trypsin digest reversephase HPLC peptide mapping studies of CDP571 and CDP571(gamma 1) with on-line electrospray ionization mass spectroscopy supplemented with Edman sequencing identified the chemical factor preventing inter-heavy chain disulfide bond formation between half-IgG molecules: the two cysteines in the IgG4 and IgG1 core hinge region (CPSCP and CPPCP, respectively) are capable of forming an intrachain disulfide bond. Conformational modeling studies on cyclic disulfide bonded CPSCP and CPPCP peptides yielded energy ranges for the low-energy conformations of 31-33 kcal/mol and 40-42 kcal/mol, respectively. In addition, higher torsion and angle bending energies were observed for the CPPCP peptide due to backbone constraints caused by the extra proline. These modeling results suggest a reason why a larger fraction of intrachain bonds are observed in IgG4 rather than IgG1 molecules: the serine in the core hinge region of IgG4 allows more hinge region flexibility than the proline of IgG1 and thus may permit formation of a stable intrachain disulfide bond more readily.     

Coloboma Hyperactive Mutant Mice Exhibit Regional and Transmitter-Specific Deficits in Neurotransmission

Abstract: The mouse mutant coloboma ( Cm /+), which exhibits profound spontaneous hyperactivity and bears a deletion mutation on chromosome 2, including the gene encoding synaptosomal protein SNAP-25, has been proposed to model aspects of attention-deficit hyperactivity disorder. Increasing evidence suggests a crucial role for SNAP-25 in the release of both classical neurotransmitters and neuropeptides. In the present study, we compared the release of specific neurotransmitters in vitro from synaptosomes and slices of selected brain regions from Cm /+ mice with that of +/+ mice. The release of dopamine (DA) and serotonin (5-HT) from striatum, and of arginine vasopressin and corticotropin-releasing factor from hypothalamus and amygdala is calcium-dependent. Glutamate release from and content in cortical synaptosomes of Cm /+ mice are greatly reduced, which might contribute to the learning deficits in these mutants. In dorsal striatum of Cm /+ mutants, but not ventral striatum, KCI-induced release of DA is completely blocked and that of 5-HT is significantly attenuated, suggesting that striatal DA and 5-HT deficiencies may be involved in hyperactivity. Further, although acetylcholine failed to induce hypothalamic corticotropin-releasing factor release from Cm /+ slices, restraint stress increased plasma corticosterone levels in Cm /+ mice to a significantly higher level than in +/+ mice, suggesting an important role for arginine vasopressin in hypothalamic-pituitary-adrenal axis activation. These results suggest that reduced SNAP-25 expression may contribute to a region-specific and neurotransmitter-specific deficiency in neurotransmitter release.     

Localisation and measurement of VIP in the genitourinary system of man and animals

VIP is present in the genitourinary system of man and animals. In man the highest concentrations are found in the penis, the uterus and vagina and in the urinary bladder. VIP nerves heavily innervate the erectile tissue of the male external genitalia, the uterine smooth muscle and blood vessels, the seromucous glands of the cervix, and the lamina propria and vaginal epithelium. In the urinary bladder, VIP nerves are located beneath the transitional epithelium, in the lamina propria and in the smooth muscle. Other areas well innervated by VIP nerves include the prostate, seminal vesicles and vasa deferentia. Chemical (phenol- and 6-OHDA) or surgical (hypogastric or pelvic nerve section) extrinsic denervation fail to deplete the genitourinary system of its VIP content, supporting the view that VIP-containing nerves originate from local ganglion cells. Indeed, neuronal cell bodies containing VIP are seen in the paracervical ganglia of the female genitalia, the para- or intramural bladder ganglia and scattered through the base of the cavernosum body, the neck of the bladder and the prostate. The finding of elevated levels of VIP in the local circulation after induced penile erection in man and mammals and the ability of VIP to relax the detrusor muscle of the bladder suggests that the peptide may be involved in penile erection and bladder relaxation, as does the marked VIP depletion in the penis or bladder in patients suffering from diabetic impotence or bladder instability.