An amino-terminal signal sequence abrogates the intrinsic membrane-targeting information of mitochondrial uncoupling protein

Mitochondrial uncoupling protein, a polytopic integral protein of the inner membrane, is initially made in the cytoplasm as a soluble polypeptide (307 amino acids) lacking a cleavable targeting (signal) peptide. Earlier studies (Liu, X., Bell, A. W., Freeman, K. B., and Shore, G. C. (1988) J. Cell Biol. 107, 503-509) identified internal regions of the molecule that are critical for targeting and membrane insertion. Here, we demonstrate that the ability of uncoupling protein to insert into the inner membrane is abrogated when the molecule is fused behind the matrix-targeting signal of preornithine carbamyltransferase; the hybrid protein was imported across the inner membrane and deposited in the matrix where it was processed. In this context, however, the processed product remained in the matrix and was incapable of inserting into the inner membrane.     

Analysis of mononuclear cell surfaces with fluoresceinated staphylococcal protein A complexed with IgG antibody of heat-aggregated gamma-globulin.

Fluorescein-conjugated staphylococcal protein A (SPA) was complexed with either: 1) heat-aggregated IgG, 2) B cell specific antibody, or 3) T cell specific antibody and then used for an immunofluorescent analysis of mononuclear cell surfaces. Cellular Fc receptors failed to recognize the Fc region of aggregated IgG that had been blocked by SPA. Moreover, fluoresceinated SPA that had been complexed either with anti-Fab (B-cell specific) or T cell-specific antisera prevented the nonspecific binding of these reagents to the IgG-Fc receptors on mononuclear cells, thereby permitting the latter to be properly identified as B or T lymphocytes. In addition, when unconjugated SPA was added to presensitized target cells in a test for antibody-dependent cell-mediated cytotoxicity, cytolysis was abrogated.     

The site of cellulose synthesis. Hormone treatment alters the intracellular location of alkali-insoluble beta-1,4-glucan (cellulose) synthetase activities

Membrane preparations from growing regions of 8-day old Pisum sativum epicotyls contain multiple beta-1,4-glucan (cellulose) synthetase activities (UDP- or GDP-glucose: beta-1,4-glucan-glucosyl transferase), and the levels of some of these are influenced by treatments with the growth hormone, indoleacetic acid (IAA). When membranes from control epicotyl segments (zero time) are fractionated by isopycnic sedimentation in sucrose density gradients, all of the synthetase activities are associated mainly with Golgi membrane (density 1.55 g/cm3). After decapitation and treatment of epicotyls with IAA, synthetases also appear in a smooth vesicle fraction (density 1.11 g/cm3) which is rich in endoplasmic reticulum (ER) marker enzyme. Major fractions of these synthetases are not recovered in association with plasma membrane or washed cell walls. When [14-C]sucrose is supplied in vivo to segments +/- IAA, radioactive cellulose is deposited only in the wall. Cellulose or cellodextrin precursors do not accumulate in those membranes in which synthetase activities are recovered in vitro. In experiments where tissue slices containing intact cells are supplied with [14C]sugar nucleotide in vitro, alkali-insoluble beta-1,4-glucan is synthesized (presumably outside the protoplast) at rates which greatly exceeded (20-30 times) those obtained using isolated membrane preparations. Progressive disruption of cell structure results in increasing losses of this high activity. These results are consistent with the interpretation that Golgi and ER-associated synthetases are not themselves loci for cellulose synthesis in vivo, but represent enzymes in transit to sites of action at the wall:protoplast omterface. There they operate only if integrity of cellular organization is maintained.     

Bovine Heterografts for Hemodialysis

Long-term results with 52 bovine, 53 saphenous vein and 78 radial-cephalic arteriovenous fistulas (AVF) were analyzed. Side-to-end radial-cephalic AVF provided the best patency data, and remain the preferred access system for hemodialysis. Bovine AVF were next in ranking with better patency rates than for the saphenous vein AVF studied. Corrected one-year patency rates were 71 percent for bovine, 45 percent for saphenous and 91 percent for radial-cephalic AVF. The incidence of nonthrombotic complications with bovine AVF was higher than with saphenous vein AVF. Distal ischemia due to “steal” and certain bleeding and wound complications were unique to bovine AVF. Excellent dialysis blood flow rates and easy accessibility were provided by bovine grafts. When a satisfactory radial-cephalic AVF cannot be created, bovine graft AVF is an acceptable alternative for hemodialysis access.     

Avoiding pitfalls in bone marrow engraftment analysis: a case study highlighting the weakness of using buccal cells for determining a patient's constitutional genotype after hematopoietic stem cell transplantation

Background aimsAn accurate and reliable assessment of bone marrow engraftment (BME) after hematopoietic stem cell transplantation (HSCT) is based on the ability to distinguish between recipient and donor cells at selected polymorphic short tandem repeat (STR) DNA loci. Buccal cells are an important source of DNA for determining the recipient's constitutional genotype, particularly in patients transplanted before the STR evaluation.MethodsGenomic DNA was extracted from the recipient buccal cells and from isolated CD3⁺ (T-cell lymphocyte) and CD33⁺ (myelocyte) cells after HSCT. BME analysis was performed using a STR-based polymerase chain reaction amplification method followed by fragment-size analysis for assessing the recipient-derived or donor-derived composition of cell lineage-specific peripheral blood DNA.ResultsWe identified three cases of complete buccal epithelial cell engraftment after HSCT detected by BME analysis, potentially leading to misinterpretation of testing results if these cells were used as the sole source for determining the recipient's genotype.ConclusionsThese cases suggest that complete engraftment of buccal epithelial cells may be a common finding in patients receiving HSCT, drawing attention to important issues such as the type of samples used for determining a patient's constitutional genotype that may confound testing results. This study also highlights the need for careful interpretation of the BME testing results in the context of the clinical findings.     

Extrafloral nectar increases seed removal by ants in Turnera ulmifolia

To investigate whether extrafloral nectar (EFN) increases seed dispersal in Turnera ulmifolia, we measured seed removal on plants with and without EFN. Plants producing EFN had more seeds removed than control plants, suggesting that EFN does play a role in seed dispersal. This is a novel function of EFN.     

Cellular acidosis in rodents exposed to cadmium is caused by adaptation of the tissue rather than an early effect of toxicity

Proton (¹H) Nuclear Magnetic Resonance (NMR) spectroscopy was used to investigate the biochemical response of bank voles and wood mice (two wild rodent species frequently found on metal-contaminated sites) to chronic cadmium (Cd) insult. Similar effects, in terms of both metabolic changes (consistent with cellular acidosis) and induced metallothionin (MT) production were observed in all animals. These changes appeared to be an adaptation of the liver to toxic insult rather than onset of a toxic effect, and, in common with previous studies, were more marked in bank voles than wood mice. This may have reflected the greater Cd intake and assimilation of the former but was not explained by differences in concentrations of free (non MT-bound) Cd; concentrations of which were negligible in both voles and mice. Responses to Cd insult were detected in both species even though their bodies contained cadmium concentrations well below the World Health Organisation critical renal concentration of 200 μg/g dry mass.     

Control of bovine placental progesterone synthesis: roles of cholesterol availability and calcium-activated systems

It was previously reported that dispersed bovine placentome secretes progesterone and that the steroidogenic activity of these cells is stimulated by a calcium-mediated, cyclic nucleotide independent mechanism. In the present study, the influence of substrate availability was explored and the roles of calmodulin and protein kinase C in progestin production examined. Incubation of dispersed fetal cotyledon cells with 25-hydroxycholesterol (25-OH-C), a soluble sterol which readily enters cells and is metabolized to steroid hormones, increased progesterone secretion in a dose-dependent manner. The response to 25-OH-C was dependent on the extracellular calcium concentration. Methyl isobutyl xanthine (MIX) alone also increased pregnenolone as well as progesterone secretion, and the combination of 25-OH-C and MIX stimulated progesterone secretion was inhibited by trifluoperazine. The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), caused no major effects on steroidogenesis but the stimulatory effects of MIX or the ionophore A23187 were enhanced in its presence. These findings suggest that (1) basal progesterone secretion by fetal cotyledon cells is limited by cholesterol availability; (2) MIX increases steroidogenesis in part by increasing the synthesis of pregnenolone, but its actions are expressed independently of cholesterol availability; (3) both calmodulin and protein kinase C may participate in the modulation of bovine placental steroidogenesis.