Biomedical subjects
Publications and source records attributed to G Lang.
Knee surgery employs both joint preserving and joint replacing techniques. Even when replacement has been decided on, the further decision as to unicompartmental vs total replacement depends on many factors. A good surgeon should be able to offer all forms of treatment.
A palindromic element (site B) located between bases -328 and -347 (relative to the start site of transcription) of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) was shown in a gel mobility shift assay to bind the human retinoic acid receptors (RARs). Greatly enhanced binding to this site was observed in the presence of both RAR and the retinoid X receptor. Retinoic acid responsiveness in F9 cells could be conferred on a thymidine kinase promoter by the presence of single or multiple copies of site B and responsiveness was abolished when this sequence was mutated to a form that could not bind RARs. However, the presence of this sequence did not render the HIV-1 LTR responsive to retinoic acid in F9 cells.
INTRODUCTION: Shock induced microcirculatory failure is proposed to be causative for the impairment of hepatic function, which contributes to the development of multiple organ failure. In order to quantify the interrelation between hepatic microcirculatory disturbances and organ dysfunction, we have analyzed hepatic microcirculation (in vivo microscopy), energy metabolism (ketone body ratio) and liver excretory function (bile flow) during hemorrhagic shock in rats. METHODS: Under chloralose anesthesia and mechanical ventilation Sprague-Dawley rats (n = 6) were laparotomized and the left liver lobe was exteriorized for in vivo fluorescence microscopy. Sinusoidal perfusion was assessed after i.v. injection of Na-fluorescein (2 mumol.kg-1), in vivo staining of white blood cells by Rhodamin-6G (0.1 mumol.kg-1 i.v.) enabled for analysis of leukocyte-endothelium interaction. Erythrocyte flux was measured by means of laser Doppler flowmetry. Hepatic function was estimated by measurement of bile flow via cannulation of the common bile duct. Ketone body ratio (acetoacetate/beta-hydroxybutyrate, Ac-Ac/beta-OHB) was measured spectrophotometrically from arterial blood and served as indicator of hepatic energy metabolism (n = 12). After baseline recordings hemorrhagic shock (MAP: 40 mmHG) was induced by blood withdrawal and maintained for a total of 2 h. Measurements were performed during baseline as well as 1 h and 2 h after induction of hemorrhage. RESULTS: After 1 h and 2 h of hemorrhagic shock sinusoidal perfusion was found markedly impaired (p < 0.05) from 100% (baseline) to 78.3 +/- 2.4% and 60.9 +/- 13.4%, accompanied by a significant fall of erythrocyte flux to 52 +/- 2% and 46 +/- 4% (p < 0.05). Leukocyte velocity was significantly (p < 0.05) reduced both in sinusoids and in postsinusoidal venules, while leukocyte adherence to the endothelial lining cells was found increased (sinusoids: 183.2 +/- 55.6% and 178.8 +/- 39.0% (p < 0.05); postsinusoidal venules: 232.4 +/- 43.1% and 297.6 +/- 65.8% (p < 0.05)). In addition, 2 h of hemorrhagic shock caused an increase of beta-OHB, while Ac-Ac levels remained unchanged. This resulted in a significant reduction of ketone body ratio. Concomitantly, bile production was significantly decreased from 20.8 +/- 2.9 microliters.min-1 during control to 6.1 +/- 1.0 microliter.min-1 after 2 h of shock (p < 0.05). The alteration of energy metabolism and the impairment of bile flow correlated significantly (p < 0.01) with the extent of microcirculatory disturbances. CONCLUSION: Liver microcirculation in hemorrhagic shock is characterized by sinusoidal perfusion failure with a reduction of erythrocyte flux, leukocyte velocity and enhancement of leukocyte adherence to the microvascular endothelial lining. Correlation of the impairment of energy metabolism and liver dysfunction with these microcirculatory disturbances may indicate their crucial role in the development of shock-induced organ failure.
We have previously identified a T lymphocyte protein which binds to a site within the LTR of the human immunodeficiency virus type 1 (HIV-1) and exerts an inhibitory effect on virus gene expression. The palindromic site (site B) recognized by this protein is related to the palindromic binding sites of members of the steroid/thyroid hormone receptor family. Here we characterize the T cell protein binding to this site as a 100 kD protein which is most abundant in T cells and which binds to site B as a 200 kD complex. This protein is distinct from other members of the steroid/thyroid hormone receptor family including the COUP protein which has a closely related DNA binding specificity.
The type-2 depleted form of ascorbate oxidase from zucchini has been prepared in crystals and characterised by X-ray crystallography and EPR spectroscopy. The X-ray structure analysis by difference-Fourier techniques and refinement shows that, on average, about 1.3 Cu atoms/ascorbate oxidase monomer are removed. The copper is lost from the trinuclear site whereby the EPR-active type-2 copper is depleted most; type-1 copper is not affected. This observation indicates preferential formation of a 1 Cu-depleted form with the hole equally distributed over all three copper sites. Each of these 1 Cu-depleted species may represent an anti-ferromagnetically coupled copper pair which is EPR-silent and could explain the disappearance of the type-2 EPR signal.
Herpesvirus saimiri induces T-cell lymphomas in various species of New World monkeys and in rabbits, and it is able to immortalize monkey T lymphocytes in vitro. Sequences responsible for these effects have been localized to a region of the genome that varies significantly among the virus subgroups A, B, and C. We now report that infection of human blood lymphocytes and thymocytes with strains of subgroup C, in contrast to viruses of the other subgroups, yields continuously proliferating T-cell lines with the phenotype of mature CD4- or CD8-positive cells. Infection with strains of Herpes-virus saimiri subgroup C can thus be used to generate human T-cell lines for a variety of immunological and developmental studies.
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In diabetes prone BB rats a relative increase of serine protease (SP)-positive lymphocytes (39.8 +/- 10%) was observed in peripheral blood at the time of diabetes manifestation (DM) compared with non-diabetic healthy Sprague Dawley control rats (Co: 10.3 +/- 4%), with BB rats at age of premanifestation (PM: 14.7 +/- 4%) and beyond age of expected manifestation (non-diabetic animals, ND: 25.2 +/- 4%). Similar absolute numbers were found in diabetic BB rats in comparison with Sprague Dawley rats. In PM, absolute numbers were lower in comparison with diabetic BB rats. SP granular positivity was found restricted to OX8+ lymphocytes. SP granule-bearing OX8+ lymphocytes were more frequently seen in the BB rat strain (PM: 74.3 +/- 8%; DM: 79.4 +/- 8%; ND: 78 +/- 10%) compared with normal rats (Co: 32.5 +/- 8%). Absolute numbers were lower in PM animals in comparison with DM rats. OX8+ cells were found in a higher relative number in DM animals (49.1 +/- 7%) compared with controls (28.2 +/- 3%), PM (26.3 +/- 5%) and ND (34 +/- 2%) animals. T lymphocytes expressing the W3/25+ marker, invariably negative for SP granules, were present in a higher relative number in ND (49.8 +/- 7%) and the control group (52.3 +/- 10%) compared with PM (31 +/- 8%) and DM (38 +/- 11%) animals. Absolute numbers of the OX39+ lymphocyte subpopulation were decreased in PM and DM-BB rats in comparison with the control group.
Genomic sequences located at the 3' flanking region of the human CD2 gene confer high level tissue-specific, position-independent expression of the gene when introduced in the germ line of mice. In order to further characterize these sequences a range of deletions, from the 3' end were produced and transgenic mice were generated with the human CD2 (hCD2) gene linked to these deleted fragments. This allowed us to establish the minimum sequences necessary for the copy-dependent transgene expression. 2.1 kb or 1.5 kb of 3' flanking sequences linked to a hCD2 mini-gene is sufficient to allow T-cell specific, copy-dependent, integration-independent expression in transgenic mice. 1.1 kb of 3' sequences results in the gene being expressed in a T-cell specific manner, but copy-dependent, integration-independent expression was not observed in a small number of transgenic animals. 0.2 or 0.5 kb of 3' flanking sequences were insufficient to allow expression above the level previously found with a human CD2 gene which lacked 3' flanking sequences. We conclude that the Locus Control Region (LCR) effect is caused by 1.5 kb of flanking sequences immediately 3' to the polyadenylation signal of the gene.
The biological response to retinoic acid (RA) and synthetic derivatives (retinoids) is mediated by three nuclear retinoic acid receptors, RAR alpha, beta and gamma. To explore the potential of retinoids as receptor subtype selective activators, we employed a transcriptional activation assay. Hybrid receptors that recognize an estrogen response element were used to avoid measuring activities of endogenous retinoic acid receptors. In response to retinoic acid, the three hybrid receptors ER-RAR alpha, ER-RAR beta and ER-RAR gamma exhibited the same induction profile as the corresponding wild type receptors RAR alpha, RAR beta, and RAR gamma. Three different retinoids, analogs of 6'-substituted naphthalene-2-carboxylic acid, elicited strong transcriptional activation of gamma receptor while no activation of alpha receptor was observed. Conversely, two retinobenzoic acid analogs showed a limited alpha selectivity. We conclude that retinoids with unique profiles of retinoic acid receptor subtype selectivity can be defined and tested for their impact on cellular differentiation and for therapeutical applications.
To investigate the requirements for CD2 expression in the activation of T lymphocytes via the CD3-TCR complex, we produced and characterized a series of CD2-variants of the IL-2 producing Jurkat leukemia cell line, J32 (surface phenotype, CD2+, CD3+, CD28+). These mutants were derived by radiation and immunoselection, and were cloned under limiting dilution conditions. A total of 3 out of 30 of these mutants selectively lost the expression of both CD2 surface molecules and CD2 mRNA, and retained the expression of the CD3-TCR complex and the CD28 molecule. A mitogenic combination of anti-CD2 antibodies (9.6 + 9-1) failed to stimulate activation of these variants as measured by mobilization of intracellular Ca2+ and by IL-2 production. The CD2- mutants stimulated with anti-CD3 or anti-TCR mAb revealed an 8- to 32-fold decrease in IL-2 production and IL-2 mRNA accumulation as compared with the parental cells. No alteration of CD3-TCR-induced mobilization of intracellular Ca2+ was observed in the CD2- mutants. Reconstitution of CD2 expression by gene transfer in two J32 CD2- mutants restored IL-2 production and IL-2 mRNA accumulation in responses to both anti-CD2 and anti-CD3-TCR mAb. These results are the first direct demonstration of the requirement for CD2 molecules in optimizing IL-2 response in human T cells stimulated via CD3-TCR complex.
Mössbauer spectra from frozen solutions of ovotransferrin were recorded in a variety of applied external magnetic fields and at various temperatures in a small applied field. The results were fitted to a simple model for the electronic structure at the iron site. This model requires admixtures of the free ion 6S and 4P states, indicating a weak cubic crystal field. Possible implications of this model regarding the binding site are discussed.
40 invasive carcinomas and 80 preinvasive lesions of the uterine cervix were studied immunohistochemically; 40 benign lesions served as controls. On histological and immunohistochemical examination, invasive and preinvasive carcinomas were subdivided in the squamous (large cell, ectocervical) type and the reserve cell (small, large or clear cell, endocervical) type. Immunohistochemically, 100% of the invasive and preinvasive squamous cell carcinomas were positive with anticytokeratins 13, 14, 16 and negative with anticytokeratin 8 and anti-CEA. Most of the invasive and preinvasive reserve cell carcinomas showed a coexpression of cytokeratins 13, 14, 16, 8 and CEA. The subdivision of invasive carcinomas of the ecto- and endocervix into squamous cell and reserve cell types made by means of their structural differences is substantiated and re-evaluated by their immunohistochemical reactions. Both types of carcinomas retain the complex pattern of cytokeratins shown by their cells of origin. The reserve cell carcinomas, in addition, acquire a coexpression for CEA that indicates malignant transformation. The subdivision is of clinical importance because both types of carcinomas vary in their mode and speed of invasion and spread and in their association with HPV infection.
Morphological characters in red deer (Cervus elaphus), which serve as criteria for selective hunting, were examined in relation to electrophoretic variation in three populations from the Vosges in eastern France. From the polymorphic loci examined, certain alleles at Idh-2, Me-1 and Acp-1 showed significant associations with a special development of body and antler characters selected for by hunters. Idh-2(125) was associated with larger hind foot length in females and a higher number of antler points in males. Me-1(90) and Acp-1(100) were associated with small spikes. The populations studied differed from one another in the duration and intensity of selective hunting and the increase or decrease in the respective allele frequencies could be explained by selection for large body size, a high number of antler points and against small spikes in yearlings, rather than by genetic drift. Among other morphological characters examined, the length of the main beam was significantly associated with the allele Acp-2(100). In contrast, no associations could be detected between overall heterozygosity and the development or the degree of asymmetry (in paired structures) of any of the morphological traits in question. Although no obvious differences in the overall values of polymorphism or heterozygosity were found between the populations, selective hunting leads towards a change in allele frequencies and eventually to the loss of one or the other rare allele.
We aimed to study the biochemical consequences of T-cell activation via the CD2 antigen in mouse T cells. The lack of stimulatory monoclonal antibodies against the mouse CD2 antigen led us to analyse this problem in transgenic mice carrying and expressing the human CD2 gene. Monoclonal antibodies to the human CD2 antigen that were mitogenic for human T cells induced proliferation of mouse T cells from the CD2 transgenic mice. Stimulation was accompanied by rapid phosphorylation of the murine CD3 gamma chain and T-cell receptor zeta chain. These results demonstrate that the human CD2 antigen is functional in the CD2 transgenic mice and indicate a considerable conservation of the signal transducing processes and also the activation mechanisms between mouse and man.
A serological survey for Coxiella burnetii was undertaken on a randomly selected population of 103 Ontario sheep flocks. Twenty-two flocks had at least one positive ewe; seven flocks had two or more reactors. The positive flocks were geographically clustered northwest of Guelph. Crutch-clipping of the ewe's wool prior to lambing, and total confinement housing at lambing in winter and spring seemed to lower the probability of seroreactivity of the flock (p less than 0.05). The study suggests that sheep are not a major reservoir for Coxiella burnetii in Ontario.
Low temperature photolysis of nitric oxide from the nitrosyl complexes of ferric myoglobin (NO-Fe(III)Mb) and manganese(II)-porphyrin-substituted myoglobin (NO-Mn(II)Mb) was examined by electron paramagnetic resonance (EPR) spectroscopy in order to elucidate the electronic and structural natures of the photoinduced intermediates of these hemoprotein-ligand complexes trapped at low temperature. The photoproduct of NO-Fe(III)Mb at 5 K exhibited entirely new X-band EPR absorptions in the magnetic field strength from 0 to 0.4 tesla. The widespread absorption together with distinct, sharp zero-field absorption was consistently observed in the photoproduct of the isoelectronic NO-Mn(II)Mb. These novel ERP signals indicate a spin-coupled pair with an effective spin of S = 2 between the high spin metal center (S = 5/2) and the photodissociated NO (S = 1/2) trapped adjacent to the metal center. On the other hand, the photolyzed form of nitrosyl complexes of Fe(III)- and Mn(II)-Glycera hemoglobins, in which the distal histidine of Mb is replaced by a leucyl residue, exhibited somewhat broader EPR absorptions similar to those of the corresponding native Fe(III)- or unliganded Mn(II)-Glycera hemoglobins, respectively, indicating that the photodissociated NO molecule moved farther away from the metal center in the heme pocket. These observations show the importance of the interaction of the distal residue with the ligand in determining the nature of the photolyzed states.
The ability to measure ATP synthesis rates using 31P-NMR spectroscopy is demonstrated in the normal, ischemic, and postischemic myocardium in vivo. Cardiopulmonary bypass (CBP) was employed to induce 20 min of global myocardial ischemia, and to conduct magnetization transfer measurements during the ischemic episode and following reperfusion and return to normal circulation. For the first few minutes of ischemia, transfer of magnetization from ATP gamma to Pi was extensive and the resultant fractional reduction (delta M/M0) in the Pi resonance intensity reached approximately 100%. Subsequent to reperfusion and stabilization off CPB and on normal circulation, both the fractional reduction and the spin-lattice relaxation time, T1*, of the Pi resonance were determined when ATP gamma spins were saturated. Under these conditions, the unidirectional ATP synthesis rate was 0.41 +/- 0.09 (SEM, N = 4) mumol/s/g wet wt. The data suggest that in the canine myocardium in vivo, glycolytic enzymes mediate a very rapid exchange between Pi and ATP gamma-phosphates during early phases of ischemia; in the postischemic reperfused myocardium, however, the glycolytic contribution to the unidirectional Pi----ATP rate measured by NMR in vivo is relatively small compared to that observed in glucose-perfused, postischemic rat hearts.