Differential effects of interleukin-1α and β on the arachidonic acid cascade in human synovial cells and chondrocytes in culture

The effects of interleukin-1 and were tested on the [³H]-arachidonic acid release and the prostaglandin synthesis by human cultured synovial cells and chondrocytes. Both forms of interleukin-1 stimulated the arachidonic acid release but interleukin-1 was more potent than IL-1. Human synovial cells and chondrocytes synthesized three types of prostaglandins upon stimulation with interleukin-1 or : prostaglandin E₂, F₂ and 6-keto-prostaglandin F₁. Regarding the synthesis of these prostaglandins, IL-1 was again more potent than IL-1. A comparison between interleukin-1-stimulated synovial cells and chondrocytes revealed neither significant quantitative nor qualitative differences in both the arachidonic acid release and the prostaglandin synthesis.     

An In Vitro Study of the Hinge and Near-Field Forward Flow Dynamics of the St. Jude Medical® Regent™ Bileaflet Mechanical Heart Valve

The most widely implanted prosthetic valve is the mechanical bileaflet. Recent clinical experiences suggest that some designs are more prone to thromboembolic episodes than others. This study evaluated the hinge flow and near-field forward flow of the new St. Jude Medical^® Regent bileaflet mechanical heart valve. Laser Doppler velocimetry measurements were conducted within the hinge and near-field forward flow regions of the Regent valve. These pulsatile flow velocity measurements were animated in time to visualize the flow fields throughout the cardiac cycle. During forward flow, a recirculation region developed in the inflow pocket of the Regent hinge but was subsequently abolished by strong backflow during valve closure. Leakage velocities in the hinge region reached 0.72 m/s and Reynolds shear stresses reached 2,600 dyn/cm². Velocities in the near-field region were highest in the lateral orifice jet, reaching 2.1 m/s. Small regions of separated flow were observed adjacent to the hinge region. Leaflet motion through the Regent hinge creates a washout pattern which restricts the persistence of stagnation zones in its hinge. Based upon the results of these studies, the hematological performance of the Regent series should be at least equivalent to the performance of the Standard series. © 2000 Biomedical Engineering Society. PAC00: 8719Uv, 8780-y, 8719Hh     

Zur Wirkung eines Ovulationshemmers auf Plasma-Steroide

Zusammenfassung Unter Behandlung mit einem Ovulationshemmer (17-Äthyl-19-nortestosteron-acetat + Äthinyloestradiol, Anovlar-21) wurde eine Erhöhung freier und konjugierter 17-OH-CS im Plasma nachgewiesen. Des weiteren kam es zu einer Abnahme der 17-KS und der freien und konjugierten Oestrogene im Blut. Nach Absetzen des Medikaments trat eine weitgehende Normalisierung der Steroidkonzentrationen im Plasma ein.

---

Summary Under treatment with 17-Äthinyl-19-nortestosteron-acetat + Äthinyloestradiol (Anovlar-21) an increase of free and conjugated 17-OH-CS in plasma could be detected. Furthermore, a decrease of 17-KS and free and conjugated estrogens in blood was observed. After cessation of therapy all steroids levels tended to return to normal concentrations.

     

Attachment, invasion, chemotaxis, and proteinase expression of B16-BL6 melanoma cells exhibiting a low metastatic phenotype after exposure to dietary restriction of tyrosine and phenylalanine

We previously reported that low levels of tyrosine (Tyr) and phenylalanine (Phe) alter the metastatic phenotype of B16-BL6 (BL6) murine melanoma and select for tumor cell populations with decreased lung colonizing ability. To more specifically characterize the effects of Tyr and Phe restriction on the malignant phenotype of BL6, we investigated in vitro attachment, invasion, proteinase expression, and chemotaxis of high and low metastatic BL6 variants. High metastatic variant cells were isolated from subcutaneous tumors of mice fed a nutritionally complete diet (ND cells) and low metastatic variant cells were isolated from mice fed a diet restricted in Tyr and Phe (LTP cells). Results indicate that attachment to reconstituted basement membrane (Matrigel) was significantly reduced in LTP cells as compared to ND cells. Attachment to collagen IV, laminin, and fibronectin were similar between the two variants. Invasion through Matrigel and growth factor-reduced Matrigel were significantly decreased in LTP cells as compared to ND cells. Zymography revealed the presence of M _r 92 000 and M _r 72 000 progelatinases, tissue plasminogen activator, and urokinase plasminogen activator in the conditioned medium of both variants; however, there were no differences in activity of these secreted proteinases between the two variants. Growth of the variants on growth factor-reduced Matrigel similarly induced expression of the M _r 92 000 progelatinase. The variants exhibited similar chemotactic responses toward laminin. However, the chemotactic response toward fibronectin by LTP cells was significantly increased. MFR5, a monoclonal antibody which selectively blocks function of the ₅ chain of the ₅ß₁ integrin, VLA-5, decreased the chemotactic response toward fibronectin of ND cells by 37%; the chemotactic response by LTP cells was reduced by 49%. This effect was specific for fibronectin-mediated chemotaxis since the chemotaxis toward laminin and invasion through Matrigel were not altered by the presence of MFR5. The surface expression of VLA-5 was significantly increased in LTP cells as compared to ND cells by flow cytometric analysis. These observations suggest that limitation of Tyr and Phe either directly modifies BL6 or selects for subpopulations with altered in vitro invasion, chemotaxis, and integrin expression.     

Virus-specific RNA formed in Newcastle disease virus-infected cells after suppression of protein synthesis by cycloheximide

Summary In Newcastle disease virus-infected cells the accumulation of virus-specific RNA is prevented if protein synthesis is inhibited by cycloheximide at an early stage of infection. At a later stage cycloheximide enhances the synthesis of smaller (18–35 S) virus-specific RNA while the synthesis of 49 S RNA is suppressed. This is accompanied by a corresponding change in the relative amounts of plus and minus RNA strands: the proportion of plus strand is sharply decreased. A possible significance of the phenomenon for the regulation of virus-specific RNA synthesis is discussed.     

Synoviocytes in chronic synovitis in situ and cytokine stimulated synovial cells in vitro neo-express α1, α3 and α5 chains of β1 integrins

The expression of the 1 integrins was examined immunohistochemically in synoviocytes from normal synovial membrane and from chronic synovitis of different aetiology and intensity. Normal synoviocytes were 61-positive but lacked 1 through 5. In mild inflammation type A synoviocytes neo-expressed 1, 3, and 5 chains. In severe inflammation both type A and B synoviocytes expressed 3, 4, 5, and 6 chains. The effects of inflammatory cytokines, as single agents or in combination, on the 1 integrin expression in cultured normal synoviocytes was determined by immunocytochemistry and flow cytometry. The 1 chain, while absent in unstimulated synoviocytes, was induced by interleukin-1 (IL-1), tumour necrosis factor- (TNF-), and interferon- (INF-). This effect was enhanced by combining IL-1 and TNF-. Expression of the 3 chain was up-regulated by IL-1 and, more intensely, by IFN-. Transforming growth factor (TGF-) inhibited the up-regulating effect of IL-1 and antagonized the effect of IFN- on 3 chain expression. Expression of the 5 chain was up-regulated significantly by co-stimulation through IL-1 together with TGF- or TNF-. Thus, the 1 integrin profile of cytokine activated synoviocytes in vitro resembled that of synoviocytes in synovitis in situ. These data suggest that IL-1, TNF-, IFN-, and TGF- are likely to be among the effectors regulating 1 integrin expression in synoviocytes in vivo.     

Genes for C4b-binding protein α- and β-chains (C4BPA andC4BPB) are located on chromosome 1, band 1q32, in humans and on chromosome 13 in rats

C4b-binding protein is involved in the regulation of the complement system. It is a multimeric protein composed of seven identical -chains and a single copy of a unique -chain. The latter was identified only recently and its structure determined by cDNA cloning. Both subunits in C4b-binding protein belong to the same superfamily of proteins composed predominantly of tandemly arranged short consensus repeats (SCR) approximately 60 amino acid residues in length. The gene for the human -chain is known to be located in a gene cluster on chromosome 1, band 1q32, which is called the regulators of complement activation (RCA) gene cluster. We have used cDNA probes for both - and -chains of human C4b-binding protein to localize their genes with an in situ hybridization technique. We find the genes for both chains to be located on chromosome 1, band 1q32, in the human. This suggests that the -chain gene is also a member of the RCA gene cluster and that the - and -chain genes are located close to each other. The cDNA probes for the - and -chains also were used to screen mouse-rat somatic cell hybrids using Southern blotting to localize their genes in the rat. Both the - and -chain genes were shown to be located on chromosome 13 in the rat. These are the second and third genes to be located on rat chromosome 13, and the results suggest that the genes for the - and the -chains together with the gene for coagulation factor V represent a conserved chromosomal region in rat and man.     

Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes: role of the α subunit in agonist sensitivity and desensitization

Neuronal nicotinic acetylcholine receptors (nAChRs) were expressed in Xenopus laevis oocytes after nuclear injection of complementary deoxyribonucleic acid (cDNA) expression vectors. The two receptor subtypes 4/n1 and 3/n1 were readily distinguishable from one another by ACh sensitivity and desensitization. 3/n1 receptors showed lower ACh sensitivity and stronger desensitization than 4/n1 receptors. Furthermore, although the current/voltage relationship was very similar in both receptor subtypes, the voltage dependence of desensitization was found to be strikingly different. As the n1 subunit was unchanged, the subunits must be responsible for these functional differences. Symmetric hybrid cDNAs, 43 and 34, were constructed and functional receptors were obtained by co-injection with n1. These hybrid receptors displayed an ACh sensitivity that was mainly defined by the extracellular sequence of the subunit. In contrast, no part of the subunit was found fully to determine desensitization.     

Temporal and mechanistic effects of heat shock on LPS-mediated degradation of IkappaBalpha in macrophages

Previous studies demonstrated important interactions between the heat shock response and the IB/NF-B pathway when these two pathways are induced sequentially. One such interaction involves the ability of heat shock to inhibit subsequent degradation of IB in response to a proinflammatory signal. Herein we investigated the temporal relationship between recovery from heat shock and inhibition of IB degradation, and the proximal mechanisms by which heat shock inhibits degradation of IB in macrophages. In RAW 264.7 murine macrophages, prior heat shock inhibited LPS-mediated IB degradation up to 4 h after recovery from heat shock, and this effect correlated with inhibition of LPS-mediated activation of NF-B. Beyond these recovery periods, heat shock did not inhibit IB degradation. IB kinase (IKK) assays demonstrated that heat shock inhibited LPS-mediated activation of IKK up to 1 h after recovery from heat shock. Heat shock also increased intracellular phosphatase activity, and inhibition of intracellular phosphatase activity partially reversed the ability of heat shock to inhibit both LPS-mediated degradation of IB and LPS-mediated activation of IKK. These data demonstrate that the ability of heat shock to inhibit degradation of IB is dependent on the recovery period between the heat shock stimulus and the proinflammatory stimulus. The mechanism by which heat shock inhibits degradation of IB involves dual modulation of IKK and intracellular phosphatase activity.     

The effect of treatment with recombinant γ-interferon on adjuvant-induced arthritis in rats

We have investigated the effects of recombinant rat -interferon (rIFN) on adjuvant-induced arthritis (AA). Lewis rats, inoculated in the left hind-paw with adjuvant (day 0), were given 10⁵ U/rat of rIFN daily (days 0 to 20), subcutaneously and intramuscularly on alternate days. rIFN suppressed the secondary phase of swelling of both hind-paw on and after day 18 without influencing the earlier phases, both primary and secondary, of swelling. rIFN also reduced the hind-paw bone lesions, the degree of splenomegaly, and the increase in erythrocyte sedimentation rate and plasma fibrinogen. These results indicate a new aspect of the regulatory role of IFN in chronic inflammation.     

Human chromosome 19 contains the neurotrophin-5 gene locus and three related genes that may encode novel acidic neurotrophins

Differentiation, survival, and function of the vertebrate neurons are controlled by multiple, target-derived neurotrophic factors. The best characterized mammalian neurotrophic factors are four structurally related 13 to 14 kDa basic proteins, collectively known as neurotrophins. Here we describe the identification of a gene cluster localized on human chromosome 19 that contains neurotrophin-5 (NT-5) and that may encode three additional acidic members of this protein family. The three novel partial open reading frames (ORFs), designated neurotrophin-6- (NT6-), NT6- and NT6-, are 95% identical to each other and 75% identical to NT5. The putative matureN-terminal portion of NT6 ORFs does not contain a typical dibasic cleavage site and lacks two out of six cysteines that are conserved among the neurotrophins. The unique structures of NT6-, -, and - suggest that if the NT6 open reading frames indeed code for functional proteins, these proteins may display novel functions and may act through a distinct class of receptors. In the human, both NTF5 and NTF6 gene loci were mapped to chromosome 19 by Southern analysis of somatic cell hybrid panels. In mouse, the NT5 gene (Ntf-5) was assigned to chromosome 7 and no sequences representing NT6 homologs were identified.     

In vitro susceptibility of“Campylobacter upsaliensis” to twenty-four antimicrobial agents

The in vitro susceptibility of 41 strains ofCampylobacter upsaliensis to 24 antimicrobial agents was determined using a broth microdilution procedure. Most isolates were susceptible to the fluoroquinolones and -lactam antibiotics tested, but all strains were resistant to trimethoprim (MBCs 128 µg/ml) and teicoplanin (MBCs 32 µg/ml). These agents may be useful in a selective isolation medium forCampylobacter upsaliensis.     

Addressing the Coming Radiology Crisis—The Society for Computer Applications in Radiology Transforming the Radiological Interpretation Process (TRIP™) Initiative

The Society for Computer Applications in Radiology (SCAR) Transforming the Radiological Interpretation Process (TRIP) Initiative aims to spearhead research, education, and discovery of innovative solutions to address the problem of information and image data overload. The initiative will foster interdisciplinary research on technological, environmental and human factors to better manage and exploit the massive amounts of data. TRIP will focus on the following basic objectives: improving the efficiency of interpretation of large data sets, improving the timeliness and effectiveness of communication, and decreasing medical errors. The ultimate goal of the initiative is to improve the quality and safety of patient care. Interdisciplinary research into several broad areas will be necessary to make progress in managing the ever-increasing volume of data. The six concepts involved are human perception, image processing and computer-aided detection (CAD), visualization, navigation and usability, databases and integration, and evaluation and validation of methods and performance. The result of this transformation will affect several key processes in radiology, including image interpretation; communication of imaging results; workflow and efficiency within the health care enterprise; diagnostic accuracy and a reduction in medical errors; and, ultimately, the overall quality of care.A position paper from the SCAR TRIP Subcommittee of the SCAR Research and Development Committee, November, 2003.A correction to the authors affiliation was made in November 2004 after online publication.     

Transient expression of myosin heavy chain MHCIα in rabbit muscle during fast-to-slow transition

The expression of an -cardiac-like myosin heavy chain, MHCI, was investigated at both the mRNA and protein levels in rabbit tibialis anterior muscle undergoing fast-to-slow transition by continuous chronic low-frequency stimulation (CLFS). According to sequence analyses of the PCR product, the MHCI isoform was found to be identical to the -cardiac MHC expressed in rabbit atrium. In muscles at different degrees of transformation, the upregulation of MHCI mRNA preceded that of the MHCI mRNA. At more advanced stages of the transformation, MHCI mRNA decayed while MHCI mRNA persisted at high levels. The expression of MHCI, therefore, was transitory. Studies at the protein level were based on immunoblotting using a monoclonal antibody (F88 12F8,1), characterized to be specific to MHCI in rabbit muscle. These studies revealed a similar relationship between initial increase and successive decline of the MHCI protein as seen at themRNA level. Immunohistochemistry of 30-day stimulated muscle revealed that up to 65% of the fibres expressed the MHCI isoform in combination with other adult MHC isoforms. The most frequent patterns of coexistence were MHCIIa+MHCI + MHCI (28%), MHCI+MHCI (18%), and MHCIIa + MHCI (11%). According to these combinations, the upregulation of MHCI may be assigned as an intermediate step in the transformation of existing fibres during theMHCIIa MHCI transition. A small fraction of fibres contained, in addition to the MHCI + MHCI and MHCIIa + MHCI combinations, developmental myosin, suggesting that MHCI was also expressed in regenerating fibres originating from satellite cell-derived myotubes.     

An α-mating-type-specific mutation causing specific defect in sexual agglutinability in the yeast Saccharomyces cerevisiae

We have identified a recessive -mating-type-specific gene agl causing agglutinability defect without significant effects on other sexual activities. a cells carrying agl showed sexual agglutination with cells but cells carrying agl showed sexual agglutination with neither cells nor a cells. cells carrying agl produced pheromone and responded to a pheromone just like wild cells. cells carrying agl showed a little decreased but significant mating ability when tested on solid media or membrane filter. The agl mutant is different from any -specific ste mutants found so far in many sexual activities. The agl gene is recessive, and unlinked to the mating type locus. Biological significance of the mating type agglutinability is discussed based on the results obtained with the mutant.     

Expression of calcium channel subunits in the normal and diseased human myocardium

We investigated the expression of ₁ and subunits of the L-type Ca²⁺ channel on the protein level in cardiac preparations from normal human heart ventricles and from the hypertrophied septum of patients with hypertrophic obstructive cardiomyopathy (HOCM). 1,4-Dihydropyridine (DHP) binding and immunorecognition by polyclonal antibodies directed against the C-terminal amino acid sequences of the ₂ and ₃ subunits were used for detection and quantification of ₁, ₂, and ₃ subunits. B_max of high-affinity DHP binding was 35±2 fmol/mg protein in HOCM and 20±2 fmol/mg protein in normal human hearts (P<0.05). In rabbit hearts the anti-₂ subunit antibody immunoprecipitated 80% of the total amount of DHP-labeled Ca²⁺ channels present in the assay. Under identical experimental conditions 25% of labeled Ca²⁺ channels were recovered in the immunoprecipitates of both normal and HOCM ventricles. A similar partial immunoprecipitation was observed in pig hearts. Immunoblot analysis demonstrated that the ₂ subunit was associated with the DHP receptor/Ca²⁺ channel in cardiac muscle of rabbit, pig, and human heart. In neither of these purified cardiac Ca²⁺ channels was the ₃ subunit isoform detected. Our results suggest that both ₁ and ₂ subunit expression is upregulated in HOCM in a coordinate manner.Abbreviations B _max Maximal number of binding sites - DHP 1,4-Dihydropyridine - HOCM Hypertrophic obstructive cardiomyopathy - NH Normal human heart     

Activated charcoal is as effective as fuller's earth or bentonite in paraquat poisoning

Summary In vitro investigations have shown that the adsorption capacity of activated charcoal (Kohle-Compretten, Ultracarbon, E. Merck, Darmstadt, FRG) is just as high as that of Fuller's earth (Surrey powder, Laporte Industries Ltd., Luton, GB) or Bentonite BP W.B. (Steetley Minerals Ltd., Milton Keynes, GB). Fuller's earth (Fullererde) from another manufacturer has had very poor adsorption properties and is thus not suitable for the treatment of paraquat poisoning. Animal experiments have shown that the curative effect of activated charcoal given 0.5, 1, 2, and 3 h after ingestion of 200 and 300 mg paraquat/kg body weight is equally as good or even better than that of Fuller's earth or Bentonite BP W.B.. Activated charcoal is a substitute of equal value to these mineral soils.     

A simple method for measurement of inulin in nanoliter samples using glass capillaries

Summary A simple method using glass capillaries instead of microcuvettes for measurement of inulin in nanoliter samples is given. Inulin was determined with anthron reagent (5 or 10 nl samples +3 l anthron reagent). Glass capillary tubes (o.d.=1 mm, i.d.=0.68 mm, length=150 mm) in which the chemical reaction took place during incubation at 56°C were directly introduced into the optical system of a Zeiss spectrophotometer PMQ II with sphere attachment and objective.Extinction was measured vertically to the axis of the capillary. The changes of extinction of 20 different capillaries with the blank at different positions was only 1.13×10^–3. The exactness of measurement in the concentration range of 100 200 400 750 1500 3000 mg-% inulin was for 5nl/3 l: 19.8 11.0 6.7 4.7 3.0 2.2%. 10nl/3 l: 13.0 8.4 5.1 3.9%.This method of measurement may also be applicable for other colorimetric reactions with nanoliter samples.This work was supported by Fonds zur Förderung der wissenschaftlichen Forschung.     

In situ expression of β1, β3 and β4 integrin subunits in non-neoplastic endothelium and vascular tumours

Endothelial cells play an important role in adhesive interactions between circulating cells and extracellular matrix proteins. In vitro studies have shown that many of these processes are mediated by a superfamily of heterodimeric transmembrane glycoproteins called integrins. The distribution patterns of 1, 3 and 4 integrin subunits in endothelial cells (EC) in situ were examined immunohistochemically on serial forzen sections of a wide range of non-neoplastic tissues and of vascular tumours, both benign and malignant. Expression of the 1 subunit was a constitutive feature of EC. Among the 1-associated subunits, 5 and 6 were broadly distributed in EC, irrespective of vessel size and microenvironment. The 3 subunit displayed intermediate levels of expression with a slight preference for small vessel EC. Presence of 1 was confined to EC of capillaries and venules/small veins. Expression of 2 in EC was inconsistent. With rare exceptions, the 4 chain was absent in EC. The 3 and v subunits were expressed in most EC, though not always concomitantly. In contrast to the 1 chain, however, these integrin subunits were absent in EC of glomerular capillaries and were expressed variably in sinusoidal EC. The 4 chain was evenly present in the great majority of EC, except for those of large vessels. In vascular tumours, the patterns of 1 and 1 to 6 subunit expression generally corresponded to those found in their non-neoplastic counterparts. Expression of 3, v and 4 chains, however, decreased in neoplasia, especially in angiosarcomas. These data show that EC dispose of broad and at the same time differential repertoires of integrin subunits that presumably reflect vessel-type associated functional differences among these cells. In vascular tumours, the orthologous distribution patterns of 1 and 1 to 6 chains are conserved in most instances while the amounts of 3, v and 4 subunits expressed in EC tend to decrease in the course of malignant transformation.Dedicated to Prof. Dr. med. Dres. h.c. Wilhelm Doerr on the occasion of his 80th birthday     

The in vitro motility activity of β-cardiac myosin depends on the nature of the β-myosin heavy chain gene mutation in hypertrophic cardiomyopathy

Several mutations in the -myosin heavy chain gene cause hypertrophic cardiomyopathy. This study investigates (1) the in vitro velocities of translocation of fluorescently-labelled actin by -myosin purified from soleus muscle of 30 hypertrophic cardiomyopathy patients with seven distinct -myosin heavy chain gene mutations: Thr124Ile, Tyr162Cys, Gly256Glu, Arg403Gln, Val606Met, Arg870His, and Leu908Val mutations; and (2) motility activity of -myosin purified from cardiac and soleus muscle biopsies in the same patients. The velocity of translocation of actin by -myosin purified from soleus or cardiac muscle of 22 normal controls was 0.48 ± 0.09 m s–1. By comparison, the motility activity was reduced in all 30 patients with -myosin heavy chain gene mutations (range, 0.112 ± 0.041 to 0.292 ± 0.066 m s–1). Notably, the Tyr162Cys and Arg403Gln mutations demonstrated significantly lower actin sliding velocities: 0.123 ± 0.044, and 0.112 ± 0.041 m s–1, respectively. -myosin purified from soleus muscle from four patients with the Arg403Gln mutation had a similar actomyosin motility activity compared to -myosin purified from their cardiac biopsies (0.127 ± 0.045 m s–1 versus 0.119 ± 0.068 m s–1, respectively). Since these seven mutations lie in several distinct functional domains, it is likely that the mechanisms of their inhibitions of motility are different