The effect of some ions on the activity of β-galactosidase and the inhibitory effect of tris (hydroxymethyl) aminomethane

The stimulating effect of phosphate and the inhibitory effect of tris-HCl on the activity of β-galactosidase inEscherichia coli was studied. The phosphate anion antagonizes the inhibitory effect of chloride. Since a similar effect is displayed by sulphate and arsenate no specific “stimulating” effect of phosphate can take place. The tris cation has also an inhibitory effect which is antagonized by univalent cations (K⁺). The resulting β-galactosidase activity reflects the antagonisms between cations and anions present in the reaction medium.     

The effect of divalent cations on the catalytic activity of the human plasma 3′-exonuclease

The 3′-exonuclease from human plasma is a soluble form of nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) (EC 3.1.4.1/EC 3.6.1.9). Here, the possibility of divalent cation influence for the 3′-exonuclease activity was investigated using the phosphorothioate congener of oligonucleotide containing all phosphorothioate internucleotide linkages of the [R_P]-configuration ([R_P-PS]-d[T₁₂]) as the substrate for this enzyme. It was found that the 3′-exonuclease is a metalloenzyme, i.e. its phosphodiesterase activity was completely abolished at 0.8 mM concentration EDTA and, in turn, it was restored in the presence of Mg²⁺ or Mn²⁺ ions. In addition, Mg²⁺ can be replaced effectively by Ca²⁺, Mn²⁺, or Co²⁺, but not by Ni²⁺ and Cd²⁺ during the hydrolysis of the phosphorothioate substrate in human plasma. In addition, the mechanism is postulated, by which a single internucleotide phosphorothioate bond of the S_P-configuration at the 3′-end of unmodified phosphodiesters (PO-oligos), or their phosporothioate analogs (PS-oligos) protects these compounds against degradation in blood.     

Induction of lymphokine-activated killer activity in mice by prothymosin α

We have recently demonstrated that prothymosin (ProT) when administered intraperitoneally (i.p.) protects DBA/2 mice against the growth of syngeneic leukemic L1210 cells through the induction of tumoricidal peritoneal cells producing high levels of tumor necrosis factor (TNF) [Papanastasiou et al. (1992) Cancer Immunol Immunother 35: 145]. In this report we tested further immunological alterations that may be caused by the administration of ProT in vivo. We demonstrate that i.p. injections of ProT enhance natural killer (NK) cell activity and induce lymphokine-activated (LAK) activity in vivo. Thus, splenocytes from ProT-treated DBA/2 animals exhibited significantly higher cytotoxic activity (up to threefold) against the NK-sensitive YAC cell line and the NK-resistant P815 and L1210 syngeneic tumor cells, as compared to splenocytes from syngeneic control mice. The enhancement of the cytotoxic profile of DBA/2 splenocytes was associated with increased percentages of CD8⁺ cells, NK cells and activated CD3⁺ cells. The ProT-induced effect persisted for 30 days after the end of the ProT treatment period and returned to normal levels 20 days later. SPlenocytes from non-treated DBA/2 animals generated high NK and LAK activities in response to ProT in vitro. The ProT-induced NK an LAK activities reached 84% and 75% respectively of what was obtained with interleukin-2 (IL-2). High concentrations of TNF and IL-2 were generated in response to ProT in LAK cultures. These findings suggest that ProT may provide an overall protective effect against tumor growth in vivo through induction of NK and LAK activities possibly indirectly via the production of IL-2 and TNF in the spleen, peritoneal cavity and probably other lymphoid organs.This work was supported by a CEC grant to M. Papamichail     

The role of possible risk factors for acute and late renal dysfunction after high-dose interleukin-2, interferon α and lymphokine-activated killer cells

Renal dysfunction is a frequently encountered adverse event following treatment with high-dose interleukin-2 (IL-2). Information about parameters predicting the severity of IL-2-associated renal function abnormalities is limited. In this study the role of possible risk factors in the development of high-dose IL-2-associated acute and long-term renal dysfunction was investigated. A total of 72 patients, who were treated with a regimen consisting of IL-2 (18 MIU m⁻² day⁻¹ by continuous infusion), interferon α (IFNα; 5 MIU m⁻² day⁻¹, intramuscularly) and lymphokine-activated killer (LAK) lymphocytes, were analysed. Serum creatinine measurements were performed daily during treatment, weekly between courses and monthly during follow-up. Pre-and posttreatment 24-h urine samples were collected for calculation of creatinine clearances. Renal dysfunction was observed in 97% of patients. Grade 1 dysfunction (according to WHO criteria) was observed in 20 patients (28%), grade 2 in 37 (51%), grade 3 in 13 (14%) and grade 4 in 0 (0%). Renal dysfunction was reversible in more than 90% of patients. Only 6 patients (8%) suffered a certain amount of permanent function loss. More severe acute renal dysfunction occurred in patients who were experiencing hypertension prior to treatment, those who suffered sepsis during treatment and in men than in women. Sepsis was also associated with irreversible function loss. Other variables such as age, performance status, diabetes mellitus, interval between nephrectomy and start of IL-2 therapy and hypotension during treatment were not important. In conclusion, with high-dose IL-2, renal dysfunction develops in almost every patient and such abnormalities are mostly reversible. Predictors for severe acute renal dysfunction are pre-existing hypertension, sepsis and sex. A septic episode also carries a risk of permanent damage. Received: 10 December 1998 / Accepted: 20 May 1999     

Role of the carboxy-terminal sequence on the biological activity of human immune interferon (IFN-γ)

The biological activity of human immune interferon depends on its carboxy-terminal structure. New genes coding for mature immune interferon molecules lacking 4, 5, 6, 8, 9, 10 and 11 amino acid residues were constructed, expressed in Escherichia coli and purified to homogeneity. Deletions with 14 and 18 carboxy-terminal amino acids were obtained by limited proteolysis of full-length immune interferon with mouse submaxillary gland ‘Arg-C’ protease and human plasmin, respectively. If a limited number of carboxy-terminal residues are removed, the antiviral and antiproliferative activities and the potential to activate macrophages is drastically enhanced. Maximal enhancement is found with the deletion of 9 or 10 residues. However, removal of 14 or more carboxy-terminal residues results in a sharp decrease of activity. We suggest that human immune interferon is synthesized as a preprotein and that its activity is modulated by proteolytic digestion.     

The inhibitory effect of Chinese herb on SARS virus infection

[Subject]Severe acute respiratory syndrome(SARS)is a contagious atypical pneumonia with a high mortality rate.SARS coronavirus(SARS-CoV)is the pathogenof SARS.We established SARS-CoVS/HIVpseudotyped(SHP)virussystemandthe cell fusion assay systemto screeninhibitors for entry of SARS-CoV.[Materials and methods]SHPor VSV-Gpseudotype(VHP)virus was made bytransfecting pCMVΔR8·2,pHR’CMV-Luc and pCMV/R-SARS-S or pMDGplasmids into293Tcells.5ng p24of SHPor VHPvirus was …     

The effect of staphylococcal δ-haemolysin on the secretory activity of the pancreatic β-cell

The secretion of insulin from isolated rat islets of Langerhans was found to be stimulated by the surface-active staphylococcal exotoxin, -haemolysin. The response was dependent on the concentration of -haemolysin, was rapid in onset, and could be maintained for at least an hour in the presence of the agent. The rate of secretion rapidly declined on removal of -haemolysin and the islets remained responsive to glucose follow!ng toxin treatment.Further characterization of the interaction of this agent with the -cell plasma membrane may provide valuable information concerning the role played by this membrane in the regulation of insulin secretion.     

Interferon-γ (IFN-γ) and interleukin-2 in the generation of lymphokine-activated killer cell cytotoxicity — IFN-γ-induced suppressive activity

Summary Incubation of human lymphocytes with recombinant interleukin-2 (rIL-2) results in the generation of lymphokine-activated killer (LAK) cells capable of lysing a wide variety of tumor cells. The present study was undertaken to examine the effect of recombinant interferon (rIFN-) on LAK cell cytotoxicity generated from different peripheral blood mononuclear cell (PBMC) subpopulations. When unseparated PBMC were stimulated by rIL-2 and rIFN-, the latter induced a transient enhancement after 2 days followed by a suppression of LAK cell cytotoxicity at day 6. Enhancement of LAK cell cytotoxicity was moderate and inconstant, whereas the inhibition was strong and observed with all the donors tested. This suppression was not associated with a decrease in the [³H]thymidine uptake. PBMC depleted of adherent cells were more sensitive to the stimulation by rIL-2 and the induced cytotoxicity was not modified by rIFN-. Monocyte-enriched plastic-adherent cells, when incubated with rIL-2 and rIFN-, became cytotoxic after 2–3 days of culture and inhibited LAK cell activity after 5–6 days. Collectively, our results suggest that rIFN- affects LAK cell cytotoxicity through the activation of plastic-adherent, monocyte-rich, cells which modulate natural killer cells, first in a positive, then in a negative way.     

Phenotypic analyses of lymphokine-activated killer cells that release interferon γ and tumor necrosis factor α

Summary We recently reported that interleukin-2(IL-2)-activated peripheral blood lymphocytes and CD3⁺, lymphokine-activated killer (LAK) cell clones release tumor necrosis factor (TNF) and interferon (IFN) when stimulated with K562 erythroleukemia cells. We examined the phenotype of IL-2-activated peripheral blood leukocytes that secrete TNF and IFN when stimulated with K562 cells and demonstrated that TNF secretion is not due to the presence of contaminating mononuclear phagocytes. Further, we demonstrate that IL-2-activated natural killer (NK) cells release only IFN when stimulated with K562 cells while T lymphocytes exposed to monoclonal anti-CD3 and K562 cells secrete both TNF and IFN. However, T cells stimulated only with K562 cells did not release IFN or TNF while the admixture of these T cells with NK cells, when stimulated with K562 cells, released levels of TNF comparable to those produced by the unseparated cells. At present it is unclear whether only one or both effector cell types respond to K562 by releasing TNF or why the presence both cell types is needed.This work was supported by grants from the national Institutes of Health (CA 23074 and CA 17094) and the Arizona Disease Commission (8277-000000-1-0-YR-9301)     

The inhibitory effect of ginsan on TGF-β mediated fibrotic process

Transforming growth factor-beta (TGF-β) plays a central role in the development of fibrosis by stimulating extracellular matrix accumulation, and signals either directly or indirectly through types I, II, and III (TβRI, II, and III) TGF-β receptor complexes. Ginsan, a polysaccharide extracted from Panax ginseng, has multiple immunomodulatory effects. Here, we examine whether ginsan regulates the fibrogenic process by interfering with TGF-β signaling pathways. TGF-β treatment of murine or human normal lung fibroblasts enhanced the levels of several fibrotic markers, including smooth muscle alpha actin (α-SMA), collagen-1, and fibronectin. Interestingly, ginsan treatment either before or after TGF-β administration led to significant reductions in all of α-SMA, collagen-1, and fibronectin expression levels. Ginsan not only inhibited phosphorylation of Smad2 and Smad3, but also attenuated pERK and pAKT signaling induced by TGF-β. Moreover, ginsan restored TβRIII protein expression, which was significantly downregulated by TGF-β, but reduced TβRI and TβRII protein levels. In a murine model of bleomycin (BLM)-induced pulmonary fibrosis, ginsan significantly suppressed accumulation of collagen, α-SMA, and TGF-β. These data collectively suggest that ginsan acts as an effective anti-fibrotic agent in the treatment of pulmonary fibrosis by blocking multiple TGF-β signaling pathways.     

The effect of 17-N substituents on the activity of the opioid κ receptor in nalfurafine derivatives

We have previously reported the essential structure of the opioid κ receptor agonist nalfurafine hydrochloride (TRK-820) for binding to the κ receptor. In the course of this study, we focused on the effect of the substituent at 17-N in nalfurafine on the binding affinity for the κ receptor. The exchange of the 17-N substituent in nalfurafine from cyclopropylmethyl to fluoro-substituted alkyl groups, which are strong electron withdrawing substituents, almost completely diminished the binding affinities for the μ and δ opioid receptors, but the binding affinity for the κ receptor was still maintained. As a result, nalfurafine derivatives with 17-fluoro-substituted alkyl groups showed higher selectivities for the κ receptor than did nalfurafine itself. With regard to the κ agonistic activities, the conversion of the 17-N substituent in nalfurafine from cyclopropylmethyl to fluoro-substituted alkyl groups led to the gradual decrease of the agonistic activities in the order corresponding to their binding affinities for the κ receptor. In contrast, the derivative with the bulky 17-isobutyl group showed lower affinity and agonistic activity for the κ receptor than the derivatives with the smaller functional groups. This research suggested that both the electronic property and the steric characteristics of the 17-N substituent would have a great influence on the binding property for the κ receptor.     

Regulation of the cytotoxic effect of human ‘normal killer cells’ on tumor cell lines by neuraminidase-treated T-lymphocytes

Summary Subpopulations of peripheral blood lymhocytes (PBL) from healthy individuals were separated according to their capacity to form various rosettes and tested for their cytotoxic activity on cell lines of urinary bladder and breast carcinomas. The subpopulation exerting the highest natural cytotoxic activity was characterized by the presence of cell surface Fc-receptors and by the lack of receptors for sheep red blood cells and for C'3 on their surface. Treatment with vibrio cholera neuraminidase (VCN) increased the cytotoxicity of unseparated PBL to a level twice as high as that of untreated PBL. The attachment of T-lymphocytes to tumor monolayers was increased several fold after VCN-treatment, while the attachment of other lymphocyte subpopulations was not. Evidence is presented that the augmentation of the cytotoxicity of PBL following VCN-treatment results from the interaction of VCN-treated T-lymphocytes, attached to target cells, with normal killer cells. It is suggested that augmentation of the activity of killer cells by T-lymphocytes may play a role in antitumor defense mechanisms.Abbreviations CMC Cell-mediated cytolysis - E-rosettes Rosettes formed with sheep red blood cells - EA-rosettes Rosettes formed with red blood cells coated with antibody - EAC'-rosettes Rosettes formed with red blood cells coated with antibody and complement - FCS Heat inactivated fetal calf serum - PBL Peripheral blood lymphocytes - RBC Red blood cells - RF-TAL E-rosette forming, target-attached lymphocytes - SRBC Sheep red blood cells - VCN Vibrio cholera neuraminidase     

Interferon γ but not tumor necrosis factor α decreases susceptibility of human renal cell cancer cell lines to lymphokine-activated killer cells

Summary Human renal cell cancer (RCC) cell lines, ACHN and KRC/Y, with or without exposure to cytokines, were examined for their susceptibility to lymphokine-activated killer (LAK) cells. Flow-cytometric analysis demonstrated constitutional expression of class I antigen on both cell lines, which was enhanced by interferon (IFN), IFN and tumor necrosis factor (TNF). A 4-h⁵¹Cr-release cytotoxicity assay demonstrated that pretreatment of both cell lines with IFN or IFN, but not with TNF, decreased their susceptibility to LAK cells. IFN also decreased susceptibility to natural killer cells in a 16-h⁵¹Cr-release cytotoxicity assay. IFN treatment decreased the susceptibility of ACHN cells in a dose-dependent manner. Cold-target competition assay clearly showed that IFN- but not TNF-pretreated cells compete less effectively than do untreated target cells. Pretreatment with IFN, however, increased expression of intercellular adhesion molecule-1 (ICAM-1) to a degree comparable to that with TNF. Northern blot analyses using a 520-base-pair ICAM-1 cDNA as a probe demonstrated that more 3.3-kb mRNA is expressed in IFN- and TNF-pretreated cells. These results suggest that IFN-treated RCC cell lines may reduce their ability to be recognized by LAK cells, and that IFN-induced protection of RCC cell lines against LAK cells may depend upon a mechanism independent of the expression of class I antigens or ICAM-1 on tumor cells.     

The effect of zinc on the 5α-reduction of testosterone by the hyperplastic human prostate gland

The present studies were performed to evaluate the role of zinc in the regulation of testosterone 5α-reduction by the 800 g supematants prepared from human benign prostate hyperplasia specimens. The results show that when zinc is added at low concentrations the 5α-reduction of testosterone is increased but at higher cation concentrations the metabolism is significantly inhibited. This decrease was mediated by both a non-competitive inhibition of the binding of testosterone to the 5α-reductase enzyme and by a reduction in the formation of the NADPH cofactor. We have also demonstrated that the decreased synthesis of NADPH was produced by a competitive inhibition of both G6P and NADP binding to the G6PD enzyme. The data also suggests that the increase in testosterone metabolism observed at low zinc concentrations does not produce any changes in the binding of testosterone to the 5α-reductase enzyme. In spite of the above observations we were unable to establish any correlation between the endogenous zinc content of the tissue and the the in vitro capacity of the BPH samples to 5α-reduce testosterone. The present study suggests a possible physiological role for the regulation of testosterone metabolism by zinc in the human prostate gland.     

The PucC protein of Rhodobacter capsulatus mitigates an inhibitory effect of light-harvesting 2 α and β proteins on light-harvesting complex 1

Rhodobacter capsulatus contains lhaA and pucC genes that have been implicated in light-harvesting complex 1 and 2 (LH1 and LH2) assembly. The proteins encoded by these genes, and homologues in other photosynthetic organisms, have been classified as the bacteriochlorophyll delivery (BCD) family of the major facilitator superfamily. A new BCD family phylogenetic tree reveals that several PucC, LhaA and Orf428-related sequences each form separate clusters, while plant and cyanobacterial homologues cluster more distantly. The PucC protein is encoded in the pucBACDE superoperon which also codes for LH2 α (PucA) and β (PucB) proteins. PucC was previously shown to be necessary for formation of LH2. This article gives evidence indicating that PucC has a shepherding activity that keeps the homologous α and β proteins of LH1 and LH2 apart, allowing LH1 to assemble properly. This shepherding function was indicated by a 62% reduction in LH1 levels in ΔLHII strains carrying plasmids encoding pucBA along with a C-terminally truncated pucC gene. More severe reductions in LH1 were seen when the truncated pucC gene was co-expressed in the presence of C-terminal PucC::PhoA fusion proteins. It appears that interaction between truncated PucC::PhoA fusion proteins and the truncated PucC protein disrupts LH1 assembly, pointing towards a PucC dimeric or multimeric functional unit.     

Effect of environmental conditions on the α-glucosidase inhibitory activity of mulberry leaves

Mulberry leaves have been used as the sole food for silkworms in sericulture, and also as a traditional medicine for diabetes prevention. Mulberry leaf components, for example 1-deoxynojirimycin (1-DNJ), inhibit the activity of α-glucosidase and prevent increased blood glucose levels, and they are highly toxic to caterpillars other than silkworms. The α-glucosidase inhibitory activity of mulberry leaves changes with the season, but it is unknown which environmental conditions influence the α-glucosidase inhibitory activity. We investigated in this study the relationship between the α-glucosidase inhibitory activity and environmental conditions of temperature and photoperiod. The results demonstrate that low temperatures induced decreasing α-glucosidase inhibitory activity, while the induction of newly grown shoots by the scission of branches induced increasing α-glucosidase inhibitory activity. These results suggest that the α-glucosidase inhibitory activity was related to the defense mechanism of mulberry plants against insect herbivores.     

The impact of the C-terminal domain on the interaction of human DNA topoisomerase II α and β with DNA

Background

Type II DNA topoisomerases are essential, ubiquitous enzymes that act to relieve topological problems arising in DNA from normal cellular activity. Their mechanism of action involves the ATP-dependent transport of one DNA duplex through a transient break in a second DNA duplex; metal ions are essential for strand passage. Humans have two isoforms, topoisomerase IIα and topoisomerase IIβ, that have distinct roles in the cell. The C-terminal domain has been linked to isoform specific differences in activity and DNA interaction.

Methodology/Principal Findings

We have investigated the role of the C-terminal domain in the binding of human topoisomerase IIα and topoisomerase IIβ to DNA in fluorescence anisotropy assays using full length and C-terminally truncated enzymes. We find that the C-terminal domain of topoisomerase IIβ but not topoisomerase IIα affects the binding of the enzyme to the DNA. The presence of metal ions has no effect on DNA binding. Additionally, we have examined strand passage of the full length and truncated enzymes in the presence of a number of supporting metal ions and find that there is no difference in relative decatenation between isoforms. We find that calcium and manganese, in addition to magnesium, can support strand passage by the human topoisomerase II enzymes.

Conclusions/Significance

The C-terminal domain of topoisomerase IIβ, but not that of topoisomerase IIα, alters the enzyme''s K_D for DNA binding. This is consistent with previous data and may be related to the differential modes of action of the two isoforms in vivo. We also show strand passage with different supporting metal ions for human topoisomerase IIα or topoisomerase IIβ, either full length or C-terminally truncated. They all show the same preferences, whereby Mg > Ca > Mn.     

Observations on the effect of hurricane “Carla” on insect activity

Observations in the laboratory under conditions of controlled temperature and relative humidity on the effects of low barometric pressures on insect activity resulting fron hurricane Carla, one of the largest and most destructive hurricanes to affect the middle coast of Texas, revealed that stable flies (STOMOXYS CALCITRANS (L.)), house flies (MUSCA DOMESTICA L.), and Mexican fruit flies (ANASTREPHA LUDENS (Loew)), cockroaches (BLATELLA GERMANICA (L.)), PERIPLANETA AMERICANA (L.), and BLABERUS GIGANTEUS (L.) and mosquitoes (AEDES AEGYPTI (L.), exhibited nervous activity concomitant with the reduction in barometric pressure. Field studies throughout the critical storm period also indicated unusual arthropod activity. Following the hurricane, certain insect species emerged in abnormally large numbers, particularly fall armyworms (LAPHYGMA FRUGIPERDA (J.E. Smith)), asps (MEGALOPYGE OPERCULARIS (J. E. Smith)), and southern green stink bugs (NEZARA VIRIDULA (L.)).

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Zusammenfassung WÄhrend des Hurrikans Carla, einer der grössten und verheerendsten Orkane, die je auf die mittlere Küste von Texas eingewirkt haben, wurden im Laboratorium mit kontrollierter Temperatur und Feuchte, die Wirkungen von niedrigem Barometerdruck auf die InsektenaktivitÄt untersucht. Stallfliegen (STOMOXYS CALCITRANS (L.)), Hausfliegen (MUSCA DOMESTICA L.), Mexikanische Fruchtfliegen (ANASTREPHA LUDENS (Loew)), Schaben (BLATELLA GERMANICA L.), PERIPLANETA AMERICANA (L.) und BLABERUS GIGANTEUS (L.) und Mücken (AEDES AEGYPTI (L.)) zeigten wÄhrend der Verminderung des Barometerdruckes eine verstÄrkte AktivitÄt. Bei Studien im Freien wÄhrend der kritischen Orkanperiode wurde ebenfalls eine ungewöhnlich grosse AktivitÄt der Arthopoden beobachtet. Nach dem Orkan tauchten bestimmte Insektenarten in abnorm grosser Zahl auf. besonders (LAPHYGMA FRUGIPERDA (J.E.Schmith)), (MEGALOPYGE OPERCULARIS(J.E.Schmith)) und. (NEZARA VIRIDULA (L.)).

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Résumé L'effet, sur l'activité des insectes, des basses pressions barométriques résultant de l'ouragan Carla, un des plus vastes et des plus destructeurs qui aient affecté la cÔte moyenne du Texas, a été. observé au laboratoire dans des conditions de température et d'humidité relative constantes. Les mouches des étables (STOMOXYS CALCITRANS (L.)), les mouches domestiques (MUSCA DOMESTICA L.), les mouches à fruits mexicaines (ANASTREPHA LUDENS (Loew)), les cafards (BLATELLA GERMANICA (L.)), PERIPLANETA AMERICANA (L.) et BLABERUS GIGANTEUS (L.) et les moustiques (AEDES AEGYPTI (L.)) ont manifesté une activité accrue correspondant à la réduction de la pression barométrique. Des études faites en plein air pendant la période d'ouragan ont également permis d'observer chez les arthropodes une activité inaccoutumée. A la suite de l'ouragan sont apparus en quantités anormales certains insectes tels que le(LAPHYGMA FRUGIPERDA (J.E. Smith)), le (MEGALOPYGE OPERCULARIS (J.E.Smith)) et le (NEZARA VIRIDULA (L.)).