Autoradiographic characterization of [3H]inositol (1,4,5) trisphosphate and [3H]inositol (1,3,4,5) tetrakisphosphate binding sites in human brain

Autoradiographic techniques were used to investigate the characteristics of tritiated inositol(1,4,5)trisphosphate ([3H]IP3) and inositol (1,3,4,5) tetrakisphosphate ([3H]IP4) binding to human brain. In brain sections [3H]IP3 exhibited a two-site binding with KD values of 87 nM and 9.3 microM respectively for the higher and lower affinity sites. [3H]IP4 also bound to two sites with KD values of 43 nM and 1.4 microM, respectively. With the conditions fixed in this study, [3H]IP3 and [3H]IP4 autoradiography in the cortex, caudate, hippocampus and cerebellum were performed. The most prominent [3H]IP3 binding among these regions was found in the cerebellum, particularly in the molecular layer. Within the hippocampus, the subiculum and the CA1 region showed much more prominent binding than the other subfields. [3H]IP4, binding was fairly homogeneous in the regions studied, with the exception of a slightly higher binding in the molecular layer of the cerebellum.     

Increased potency of Fc-receptor-targeted antigens

A major challenge for using native and modified T cell epitopes to induce or suppress immunity relates to achieving efficient uptake and processing by antigen-presenting cells (APC) in vivo. IgG Fc receptors, which are expressed constitutively by professional APC including monocytes and dendritic cells, have long been known to mediate antigen uptake in a manner leading to efficient T cell activation. We have previously demonstrated enhanced presentation of antigenic and antagonistic peptides by targeting them to the type I Fc receptor for IgG (Fc gamma RI, CD64) on human monocytes. In the present report we review the literature suggesting that CD64-targeted antigens are likely to be effective in vivo, and present data demonstrating enhanced immunogenicity in CD64 transgenic mice of a fusion protein that combines the specificities of HIV gp120 and the humanized anti-CD64 monoclonal antibody H22. Overall, these studies suggest that targeting antigens to CD64 represents an effective approach to enhancing the effectiveness of vaccines in vivo.     

Spinal arteriovenous malformations

Spinal cord arteriovenous malformations encompass a variety of distinct lesions with aberrant connections between the arterial and venous circulation. Symptomatology and clinical presentation of such lesions are directly related to their location and particular pathophysiology. In the authors' surgical experience, 90% of patients have had the same or improved neurologic function and long-term follow-up.     

Rabbit cells expressing human CD4 and human CCR5 are highly permissive for human immunodeficiency virus type 1 infection

To evaluate the feasibility of using transgenic rabbits expressing CCR5 and CD4 as a small-animal model of human immunodeficiency virus type 1 (HIV) disease, we examined whether the expression of the human chemokine receptor (CCR5) and human CD4 would render a rabbit cell line (SIRC) permissive to HIV replication. Histologically, SIRC cells expressing CD4 and CCR5 formed multinucleated cells (syncytia) upon exposure to BaL, a macrophagetropic strain of HIV that uses CCR5 for cell entry. Intracellular viral capsid p24 staining showed abundant viral gene expression in BaL-infected SIRC cells expressing CD4 and CCR5. In contrast, neither SIRC cells expressing CD4 alone nor murine 3T3 cells expressing CCR5 and CD4 exhibited significant expression of p24. These stably transfected rabbit cells were also highly permissive for the production of virions upon infection by two other CCR5-dependent strains (JR-CSF and YU-2) but not by a CXCR4-dependent strain (NL4-3). The functional integrity of these virions was demonstrated by the successful infection of human peripheral blood mononuclear cells (PBMC) with viral stocks prepared from these transfected rabbit cells. Furthermore, primary rabbit PBMC were found to be permissive for production of infectious virions after circumventing the cellular entry step. These results suggest that a transgenic rabbit model for the study of HIV disease may be feasible.     

The value of pre-operative lymphoscintigraphy in breast cancer treatment

Three patients (aged 68-75 years) with histologically confirmed relapsed or refractory high-grade non-Hodgkin's lymphoma were entered in this pilot study in which gemcitabine 800 mg/m2 was given as a 30 min i.v. infusion once a week for 3 weeks. One patient responded with complete remission and the other two with partial remission and stable disease for 2 and 3 months, respectively. Haematological toxicity was modest with grade 4 leucopenia (one cycle) and grade 4 thrombocytopenia (two cycles). The activity and mild toxicity seen with gemcitabine suggest that this agent should be further evaluated in the treatment of high-grade non-Hodgkin's lymphoma.     

Transbilayer movement of NBD-labeled phospholipids in red blood cell membranes: outward-directed transport by the multidrug resistance protein 1 (MRP1)

The outward movement (flop) of fluorescently labeled analogues of phosphatidylserine (PS) and phosphatidylcholine (PC) in human and murine red blood cells (RBC) was examined. 1-Oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl (C6-NBD) analogues of PS and PC were incorporated in the inner leaflet of the plasma membrane through the action of aminophospholipid translocase or through equilibration upon prolonged incubation, respectively. After removal of noninternalized probe, externalization of C6-NBD-PS or C6-NBD-PC from the inner to outer leaflet was monitored by continuous incubation of the cells in the presence of bovine serum albumin. Flop rates for both probes in intact human RBC were virtually identical (t1/2 approximately 1.5 h), confirming earlier findings by Bitbol et al. [Bitbol, M., et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6783-6787] and Connor et al. [Connor, J., et al. (1992) J. Biol. Chem. 267, 19412-19417]. Flop activity in resealed RBC ghosts could only be found upon coinclusion of both ATP and oxidized glutathione (GSSG). Furthermore, flop in intact cells was sensitive to verapamil (IC50 = 5-7 microM), vincristine (IC50 = 20 microM), and indomethacin (IC50 = 50 microM), suggesting the involvement of proteins conferring multidrug resistance (MDR). Experiments with RBC from knock-out mice for multidrug resistance P-glycoproteins (Mdr1a/1b-/- and Mdr2-/-) and multidrug resistance protein 1 (Mrp1-/-) revealed that Mrp1 is responsible for the observed flop of the fluorescent lipid analogues. We found no indications for outward transport of endogenous PS by any of these drug-transporting proteins as measured by a sensitive prothrombinase assay. Neither aminophospholipid translocase nor Ca2+-induced lipid scramblase activities were affected in RBC of these knock-out mice. We conclude that lipid floppase activity, as detected with lipid probes, reflects the activity of MRP1 recognizing the modified lipid analogues as xenobiotics to be expelled from the cell.     

A dual-luciferase reporter system for studying recoding signals

OBJECTIVE: Growth hormone status is an important determinant of serum IGF-I but it is well known that hypopituitary adults with pronounced GH-deficiency (GHDA) may exhibit normal IGF-I levels. To elucidate possible causes of this apparent paradox we compared the significance of putative IGF-I predictors in GHDA and normal subjects. DESIGN: A cross-sectional study. SUBJECTS: Twenty-seven GHDA (9 females, 18 males, mean +/- SE age 44 +/- 1 years) and 27 healthy control subjects (9 females, 18 males, mean +/- SE age 43 +/- 2 years). RESULTS: Serum IGF-I and IGFBP-3 were significantly lower in GHDAs, but a considerable overlap existed (IGF-I (microgram/l) 87 +/- 12 (GHDA) vs 177 +/- 10 (Control) (P < 0.001)). In both Controls and GHDA, IGF-I was higher in males than females (Control: 196 +/- 12 vs 138 +/- (P = 0.004); GHDA: 97 +/- 16 vs 56 +/- 11 (P = 0.05)). In GHDA, males on testosterone substitution had the highest IGF-I concentrations. The molar IGF-I:IGFBP-3 ratio was significantly lower in GHDAs (0.18 +/- 0.01 vs 0.23 +/- 0.02 (P = 0.002)). IGFBP-1 (microgram/l) was significantly elevated in GHDAs (6.28 +/- 1.11 vs 3.07 +/- 0.32 (P < 0.001)) despite comparable fasting insulin levels. Percentage total body fat (TBF, DEXA, waist/hip ratio, and intra-abdominal fat (CT) were all elevated in GHDAs. IGF-I correlated positively with lean body mass (DEXA) and negatively with TBF and IGFBP-1 in both groups. IGF-I correlated negatively with age in CON but not in GHDAs, whereas IGF-I correlated positively with IGFBP-3 only in GHDAs. Multiple regression analysis revealed that age and IGFBP-1 were the only significant predictors of IGF-I in CON, whereas IGFBP-3 and, to a lesser extent TBF, were the only independent predictors of IGF-I in GHDAs. Neither peak stimulated GH, nor physical fitness contributed in any equations in the two groups. CONCLUSIONS: 1) IGF-I levels are regulated by several variables in addition to GH status 2) age per se is an independent negative determinant in healthy subjects but not in GHDA 3) it is probable that some cases of paradoxically high IGF-I levels in GHDA are secondary to inappropriately elevated IGFBP-3 levels. 4) in mid-adulthood males have higher IGF-I levels than females and it is likely that testosterone directly stimulates IGF-I. The influence of gender and sex steroids must therefore be accounted for when comparing IGF-I levels between hypopituitary and healthy subjects.     

Depth perception during diplopia is direct

Although depth is experienced with targets at large disparities when they are seen as double or diplopic, whether that depth is as direct as with fused targets has been a matter of considerable uncertainty. Researchers have often claimed that judgments of the depth of diplopic targets during simple near/far tasks rely upon indirect associations with eye-muscle proprioception or a copy of the vergence drive signal. We designed a four-alternative task that could not be performed without a direct appreciation of depth. Observers judged the depths of each of two Gabor stereo pairs presented simultaneously. Disparities were always above each observer's measured diplopia threshold. The signs of the disparities were varied independently and observers reported the perceived depth near and far for each target. Our results demonstrate conclusively that depth during diplopia requires neither proprioception nor an efferent copy but is direct.