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Biomedical subjects

R D Allen

Publications and source records attributed to R D Allen.

At least 73 records · Page 4Linked to original sources
PubMed1995

Rapid bulk replacement of acceptor membrane by donor membrane during phagosome to phagoacidosome transformation in Paramecium.

The extent to which a donor membrane will be retrieved, or if it is retrieved at all after it fuses with an acceptor membrane, is usually difficult to determine. We have studied the dynamics of membrane retrieval in the phagosome system of Paramecium multimicronucleatum using six monoclonal antibody markers. Our previous freeze-fracture and transmission electron microscopic studies have indicated that extensive changes take place in the membrane of the young phagosome as it progresses through its cycle. Using immunofluorescence and immunoelectron microscopy to determine the times of entry and exit of these individual antigens into the digestive vacuole system, we showed that two hydrophilic antigens, one located on the cytosolic and one on the lumenal side of the discoidal membrane (phagosome membrane precursor), were completely retrieved from the phagosome by tubulation within the first three minutes. At the same time that this membrane was retrieved, membrane from a second population of vesicles, the acidosomes, fused with the phagosome to produce the phagoacidosome. On the basis of immunogold localization on cryosections of a total of six antigens, the two specific for phagosome/discoidal vesicle membrane as well as four specific for the acidosome/phagoacidosome membrane, this replacement is total. We also showed that in the presence of the actin-active drug cytochalasin B, this replacement was essentially prevented. However, when vacuole acidification was neutralized by ammonium chloride, this replacement process continued unaffected after a lag. Consequently, acidification, per se, is not required to trigger the replacement of the phagosome membrane. We conclude, on the basis of these studies as well as our previous freeze-fracture studies that during phagoacidosome formation most of the acceptor membrane is retrieved and is replaced by the donor membrane. This shows that at least one cell type possesses the mechanisms needed to substantially replace the membrane of a phagosomal compartment when radical and rapid changes are needed to modulate the digestive and absorptive processes.

Ammonium Chloride↗
PubMed1995

Predicting glomerular filtration rate after simultaneous pancreas and kidney transplantation.

Impairment of glomerular filtration rate (GFR) after simultaneous pancreas and kidney (SPK) transplantation is an important marker of chronic renal rejection and recurrence of diabetic glomerulopathy. The use of unmodified serum creatinine to estimate GFR, however, is limited by variations in muscle mass. In this study, predictive factors for long-term GFR were evaluated in consecutive SPK recipients (n = 33) using a Tc99m DTPA GFR reference method between 90 days and 6 years after transplantation (n = 136 measurements). Substantial variability between serum creatinine and isotopic GFR after SPK (R2 = 0.30) high-lighted the inaccuracy of an unmodified serum creatinine in the evaluation of GFR. Factors which predicted GFR apart from serum creatinine included age, sex, height and body weight. A detailed formula was derived for accurate estimation of GFR (ml/min) = [71.4 (male) or 50.4 (female)] + 5520/creatinine (mumol/l) + 0.27 x body weight (kg) - 0.50 x age (yr) - 0.29 x height (cm). This formula was more accurate in estimation of GFR in SPK recipients than six published predictive methods which were derived from chronic renal failure patients using creatinine clearance. All of these methods overestimated GFR at lower levels of renal function. Most correlated poorly with Tc99m DTPA GFR and contained a generalized systematic overestimation of GFR which ranged from 4.7 to 8.4 ml/min (p < 0.05). A simplified version for rapid calculation was also derived as GFR (ml/min) = [25 (male) or 5 (female)] + 5000/creatinine (mumol/l).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Kinetic and organ-specific patterns of cytokine expression in acute graft-versus-host disease.

Although many cytokines have been previously implicated in graft-versus-host disease (GVHD), no study to date has comprehensively evaluated their expression over time or in different tissues affected by GVHD. Using a semi-quantitative reverse transcriptase-PCR technique and a murine model of acute GVHD, we have evaluated the expression levels of mRNA for a wide range of cytokines in spleen, gut and liver tissues at weekly intervals after bone marrow transfer. The earliest cytokine responses seen were increases in IL-2, IL-10, IFN-gamma, MIP-1 alpha and TNF-alpha in the spleen, suggesting a primarily Th1 pathway. Other cytokines (IL-1 alpha, IL-10 and MIP-1 alpha) were persistently elevated in GVHD mice, but were variable depending on the tissue. These data demonstrate that a wide range of cytokines are involved in the GVHD response and that their kinetic pattern of expression is different in various affected tissues.

Acute Disease↗
PubMed1995

Erythrocytosis after renal transplantation: risk factors and relationship with GFR.

Post-transplant erythrocytosis (PTE) has been increasingly recognized as a complication of kidney transplantation, and several risk factors have been defined. Recent evidence suggests renal function may also play a role in hematological recovery after transplantation and risk of PTE. In this study of kidney transplant recipients (n = 123), simultaneous Tc99m DTPA GFR (n = 710) and hemoglobin levels were compared with possible clinical determinants. The frequency histogram of post-transplant hemoglobin was bell-shaped and continuously distributed above and below the arbitrary definition of PTE, suggesting that PTE is not a separate disease entity. Hemoglobin reached a plateau at 12 months after transplantation and was correlated with isotopic GFR (r = 0.46, p < 0.001). This consistent and statistically independent relationship became prominent 3 months after transplantation. Hemoglobin was independently predicted by multivariate analysis by time after transplantation, presence of polycystic renal disease, greater serum albumin and reduced serum urea (which in turn were reflected by number of infective and rejection episodes), shorter kidney anastomosis time and a higher GFR, but not by immunosuppressive therapy. Rejection or infection episodes impaired hematological recovery. The independent determinants of GFR included hematological recovery. The independent determinants of GFR included hemoglobin level, kidneys from young, male donors, fewer HLA-DR mismatches and rejection episodes, shorter time on dialysis and greater azathioprine dose. Renal function was not altered by therapeutic phlebotomy. Determination of hemoglobin level by both donor and recipient variables supports the relevance of tubular and glomerular function in control of erythrocystosis after renal transplantation. A role for renin-angiotensin mediation in the alteration of intraglomerular hemodynamics and erythropoietin secretion is postulated.

Adult↗
PubMed1994

Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase.

The success of Mycobacterium species as pathogens depends on their ability to maintain an infection inside the phagocytic vacuole of the macrophage. Although the bacteria are reported to modulate maturation of their intracellular vacuoles, the nature of such modifications is unknown. In this study, vacuoles formed around Mycobacterium avium failed to acidify below pH 6.3 to 6.5. Immunoelectron microscopy of infected macrophages and immunoblotting of isolated phagosomes showed that Mycobacterium vacuoles acquire the lysosomal membrane protein LAMP-1, but not the vesicular proton-adenosine triphosphatase (ATPase) responsible for phagosomal acidification. This suggests either a selective inhibition of fusion with proton-ATPase-containing vesicles or a rapid removal of the complex from Mycobacterium phagosomes.

Animals↗
PubMed1994

22S axonemal dynein is preassembled and functional prior to being transported to and attached on the axonemes.

In an earlier study we reported the isolation of a cytoplasmic dynein from the cytosol of Paramecium multimicronucleatum. In this study we report the isolation and characterization of two cytosolic axonemal dyneins (22S and 12S) as well as a 19S cytoplasmic dynein from the cytosol of whole or deciliated cells using preformed bovine brain microtubules. These three dynein species were characterized according to mass, morphology, vanadate photocleavage patterns, CTPase/ATPase ratios, Km and Vmax values, temperature optima and reactivity with a mAb. For comparison, 22S and 12S axonemal dyneins (ADs) were also isolated and purified from the demembranated axonemes. The 22S and 12S soluble dyneins appear to be related to ciliary ADs in that the 22S soluble dynein is three-headed while the 12S is a one-headed dynein, as determined by negative staining. Ciliary ADs and their corresponding 22S and 12S soluble dyneins isolated from the cytosol also have similar Km and Vmax values as well as vanadate photocleavage patterns and temperature optima. A mAb raised against the soluble 22S dynein reacted with the 22S ciliary dyneins but not the 12S axonemal or the 19S cytoplasmic dynein. All isolated dyneins supported similar microtubule gliding rates but had different ionic requirements for the translocation buffer. These results suggest that: (i) the two soluble 22S and 12S dyneins are precursor molecules of the ciliary dyneins, (ii) the subunits of the outer arm dynein are already assembled in the cytosol as a three-headed bouquet, and (iii) the 22S and 12S soluble dyneins are functional prior to being transported and attached to the axonemes of the cilia.

Animals↗
PubMed1994

Monoclonal antibodies with a high degree of specificity for Listeria monocytogenes serotype 4b.

Strains of Listeria monocytogenes serotype 4b account for a large fraction of sporadic listeriosis cases, as well as all major food-borne epidemics attributed to this pathogen. We have identified a set of three monoclonal antibodies which showed a high degree of specificity for strains of L. monocytogenes serotype 4b. Two of these antibodies (c74.33 and c74.180, isotypes immunoglobulin M [IgM] and IgG3, respectively) recognized all serotype 4b strains, whereas antibody c74.22 (isotype IgG1) failed to recognize certain epidemic-associated strains. The corresponding antigens were located on the surface of the bacteria and were expressed following bacterial growth in different media and over a wide range of temperatures (4, 22, and 37 degrees C). Heating L. monocytogenes cells at 80,90, or 100 degrees C abolished reactivity for c74.22 but not for c74.33 MAb. These MAbs were negative for all of the non-Listeria strains tested, including representatives of several gram-negative and gram-positive species. The surface antigen recognized by c74.22 appeared to be associated with the ability of the bacteria to enter (invade) mammalian cells in culture.

Animals↗
PubMed1994

Purification and characterization of two forms of a high-molecular-weight cysteine proteinase (porphypain) from Porphyromonas gingivalis.

Porphyromonas gingivalis, and organism implicated in the etiology and pathogenesis of human periodontal diseases, produces a variety of potent proteolytic enzymes, and it has been suggested that these enzymes play a direct role in the destruction of periodontal tissues. We now report that two cell-associated cysteine proteinases of P. gingivalis W12, with molecular masses of approximately 150 kDa (porphypain-1) and 120 kDa (porphypain-2), as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, have been separated and purified to apparent homogeneity. These proteinases appear to be SDS-stable conformational variants of a 180-kDa enzyme, and they are the largest cysteine proteinases yet purified from P. gingivalis. The purified proteinases hydrolyze fibrinogen, tosyl-Gly-L-Pro-L-Arg p-nitroanilide, and tosyl-Gly-L-Pro-L-Lys p-nitroanilide. While hydrolysis of both synthetic substrates by porphypain-1 and -2 requires activation by reducing agents, is inhibited by EDTA, and is stimulated in the presence of derivatives of glycine, the Arg-amidolytic activity is sensitive to leupeptin and H-D-tyrosyl-L-prolyl-L-arginyl chloromethyl ketone, whereas the Lys-amidolytic activity is sensitive to tosyl-L-lysyl chloromethyl ketone and insensitive to leupeptin. These data suggest that porphypains contain two types of active sites. These cell-associated P. gingivalis proteinases may contribute significantly and directly to periodontal tissue destruction.

Amino Acid Sequence↗
PubMed1993

R-(-)-beta-phenyl-GABA is a full agonist at GABAB receptors in brain slices but a partial agonist in the ileum.

R-(-)-beta-phenyl-GABA has been compared at GABAB receptors using cortical and ileal preparations. R-(-)-beta-phenyl-GABA (EC50 = 25 microM) was a less potent full agonist than R,S-(+/-)-baclofen (EC50 = 2.5 microM), in depressing CA1 population spikes of rat hippocampal slices, and 5 times less potent in attenuating the spontaneous discharges of rat neocortex. However, R-(-)-beta-phenyl-GABA (100-400 microM) was only a weak partial agonist in the ileum. All these actions were sensitive to CGP 35348 (3-aminopropyl-(P-diethoxymethyl)-phosphinic acid) and therefore mediated by GABAB receptors.

Animals↗
PubMed1993

Increased resistance to oxidative stress in transgenic plants that overexpress chloroplastic Cu/Zn superoxide dismutase.

Transgenic tobacco plants that express a chimeric gene that encodes chloroplast-localized Cu/Zn superoxide dismutase (SOD) from pea have been developed. To investigate whether increased expression of chloroplast-targeted SOD could alter the resistance of photosynthesis to environmental stress, these plants were subjected to chilling temperatures and moderate (500 mumol of quanta per m2 per s) or high (1500 mumol of quanta per m2 per s) light intensity. During exposure to moderate stress, transgenic SOD plants retained rates of photosynthesis approximately 20% higher than untransformed tobacco plants, implicating active oxygen species in the reduction of photosynthesis during chilling. Unlike untransformed plants, transgenic SOD plants were capable of maintaining nearly 90% of their photosynthetic capacity (determined by their photosynthetic rates at 25 degrees C) following exposure to chilling at high light intensity for 4 hr. These plants also showed reduced levels of light-mediated cellular damage from the superoxide-generating herbicide methyl viologen. These results demonstrate that SOD is a critical component of the active-oxygen-scavenging system of plant chloroplasts and indicate that modification of SOD expression in transgenic plants can improve plant stress tolerance.

Base Sequence↗
PubMed1993

Differential cytokine expression in acute and chronic murine graft-versus-host-disease.

The relationship between acute and chronic graft-versus-host disease (GVHD) is not well understood. While both syndromes appear to result from recognition of host antigens by donor T cells, their pathological changes differ markedly. In light of the recent concept that helper T cells (Th) may be divided into two types based on their cytokine secretion profile and their ability to mediate cellular (Th1) or humoral (Th2) immunity, and considering the inflammatory nature of acute GVHD and the occurrence of significant B cell activation in chronic GVHD, we hypothesized that acute and chronic GVHD may be associated with differential cytokine production by activated T cells. To evaluate this hypothesis, we assessed expression of a range of cytokines in (C57BL/6 x DBA/2)F1 (B6D2F1) recipients of C57BL/6 (acute GVHD), DBA/2 (chronic GVHD) or B6D2F1 (control) spleen cells. The results reported here indicate that a wide range of cytokines, including interleukin (IL)-4, IL-10, interferon-gamma, tumor necrosis factor beta and macrophage inflammatory protein-1 alpha, are indeed differentially expressed in acute and chronic GVHD and support the concept that the pathology peculiar to acute or chronic GVHD may arise due to differential cytokine expression by activated T cells.

Acute Disease↗
PubMed1993

Upstream regulatory sequences from two beta-conglycinin genes.

Genes encoding the beta-conglycinin seed storage proteins of soybean are expressed only in seeds during specific stages of development. The different subunits of beta-conglycinin, alpha', alpha and beta, are encoded by distinct members of a gene family. Yet there are marked differences in the regulation of the genes encoding the alpha'/alpha and beta subunits. Previous work (Chen et al., EMBO J 7: 297-302, 1988) identified a seed specific transcriptional enhancer upstream of a gene encoding the alpha' subunit. Mutations were made within this region to discern its functional components. Among those identified is a 62 bp region (between -77 and -140) that contains a vicilin box consensus sequence as well as a sequence that binds the soybean nuclear factor SEF4 in vitro. A second region, which contains a sequence homologous to the core of the legumin box consensus (i.e., CATGCAT-like or RY repeat element) at -246, was also shown to affect the activity of this enhancer in transgenic plants. A series of 5' terminal deletions were used to identify regulatory elements upstream of the beta subunit gene. Two regions were identified (from -553 to -442 and from -308 to -72) that, when deleted, led to a marked reduction in gene expression. Both of these elements contain sequences that bind SEF4 in vitro. The distal element also contains an AT-rich segment that recognizes a second nuclear factor, SEF1, in vitro. Neither of these elements contains any homology to the vicilin box consensus.

Antigens, Plant↗
PubMed1993

Acidosomes: recipients of multiple sources of membrane and cargo during development and maturation.

Acidosomes are organelles that in Paramecium are responsible for the acidification of phagosomes before phagosomes fuse with lysosomes. Using a combination of (a) the quick-freeze deep-etch (QF-DE) technique, (b) monoclonal antibodies (mAbs) that label specific membrane pools including those of the acidosomes, and (c) horseradish peroxidase (HRP)-uptake studies, we followed the development of acidosomes from the Golgi complex as well as the rapid transfer of HRP into the acidosomes. We also studied some of the characteristics of the involved membrane pools. Morphologically, acidosomes were first detected in the cytosol near the ER and Golgi stacks as clumps of tubules and vesicles, which apparently coalesced to form larger spherical or elongated preacidosomes. These clumped vesicles and preacidosomes had a QF-DE morphology resembling that of the mature acidosomes and were specifically labeled with mAbs that also labeled mature acidosomes. Within 10 s HRP cargo could be internalized by acidosomes while they were docked at the nascent vacuole membrane. This rapid uptake of HRP along with membrane occurs by vesicle fusion, a conclusion supported by QF-DE images. Thus the acidosome obtains its membrane from at least two sources, from the trans-Golgi network, and from the small HRP-containing vesicles. Cargo can also be acquired from two sources, the Golgi apparatus and the transport vesicles. Since it acquires non-particulate exogenous marker we conclude that the acidosome is linked to the endocytic pathway.

Animals↗
PubMed1993

Osmoregulation in Paramecium: the locus of fluid segregation in the contractile vacuole complex.

In a previous study, monoclonal antibody DS-1 was found to specifically label the decorated spongiome along the radial arms of the contractile vacuole complexes in Paramecium multimicronucleatum. Fluorescein isothiocyanate-conjugated DS-1, when injected into cells, labels the radial arms initially, but with increasing postinjection time both the intensity of fluorescence and the number of fluorescently labeled radial arms were reduced. When these cells were fixed after 45 minutes and probed indirectly using a second fluorochrome, little new label was seen on the already fluorescein-labeled radial arms. Thin sections showed that the amount of decorated tubules along some collecting canals decreased from the proximal to the distal end and vesicles, which were never seen in control cells, appeared next to the decorated spongiome. These results suggested that the decorated spongiome was undergoing disassembly and sequestration into one region of the cell. The injected DS-1 also reduced the expulsion frequency of the contractile vacuoles in a dose-, time- and site-dependent manner. The contractile vacuole complexes near the injection site were affected more than those farther from the site, but the sizes of both contractile vacuoles were only transiently affected so that fluid output per cell was reduced by approximately 60%. Beyond 45 minutes postinjection, both the expulsion frequency and total fluid output began to recover as the DS-1 was sequestered into one part of the cell. This region persisted in cells up to 18 hours but disappeared by 24 hours, which coincided with the full recovery of the expulsion frequency and of decorated spongiome along the radial arms. The contractile vacuole, the collecting canals and the smooth spongiome were morphologically unaffected. These results indicate that when the decorated spongiome is dissociated from the contractile vacuole complex, the complex's function is strongly inhibited, showing the decorated spongiome to be the site of fluid segregation.

Animals↗
PubMed1993

Vacuolar H(+)-ATPase of Dictyostelium discoideum. A monoclonal antibody study.

A Dictyostelium membrane fraction rich in vacuolar proton pumps, previously described by Nolta et al. (J. Biol. Chem. 266, 18,318-18,323, 1991), was used as the immunogen for production of monoclonal antibodies. We obtained antibodies that recognized polypeptides of 100 kDa and 68 kDa, corresponding to the two largest subunits of the vacuolar proton pump. In indirect immunofluorescence experiments, these two subunits were located on an interconnected collection of tubules and vacuoles. On frozen thin sections they were found principally on membranes of vacuoles and collections of small vesicles typically located just internal to the plasma membrane. These vesicles and vacuoles had electron-translucent lumens. No other structures in axenically grown Dictyostelium cells were labeled to a significant extent by these two antibodies. Using an affinity-purified antibody to calmodulin and a monospecific antibody to the B subunit of the chromaffin granule vacuolar ATPase, markers known to label the membranes of the contractile vacuole complex in Dictyostelium (Zhu and Clarke, J. Cell Biol. 118, 347-358, 1992; Heuser et al., J. Cell Biol. 121, 1311-1327, 1993), we showed that the 100 kDa and 68 kDa subunits had the same distribution as these two markers. Co-localization was seen in both interphase and mitotic cells. Thus, our results support the conclusion that vacuolar proton pumps are located principally on the membranes of the contractile vacuole complex in Dictyostelium. In addition, in indirect immunofluorescence experiments, these monoclonal antibodies provided improved images of the organization of the contractile vacuole system.

Animals↗