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Objectives: To demonstrate the clinical features and prognostic impact of cyclin D1 positivity in patients with amyloid light chain amyloidosis (AL)., Methods: We consecutively included 71 patients diagnosed with AL with cyclin D1 positivity between February 2008 and January 2022. t(11;14) was examined through interphase fluorescence in situ hybridization using bone marrow cells., Results: The median age of the patients was 73 years, and 53.5% were male. The underlying diseases included symptomatic multiple myeloma, smoldering multiple myeloma, Waldenström macroglobulinemia, and monoclonal gammopathy of undetermined significance, representing 33.8%, 26.8%, 2.8%, and 36.6%, respectively. The prevalence of cyclin D1 and t(11;14) was 38.0% and 34.7%, respectively. Higher frequency of light chain paraprotein type was seen in cyclin D1-positive patients with AL than in cyclin D1-negative patients (70.4% vs 18.2%). The median overall survival (OS) of patients with AL with and without cyclin D1 expression was 18.9 months and 73.1 months, respectively (P = .019). Early death occurred in 44.4% of cyclin D1-positive patients and 31.8% of cyclin D1-negative patients. Moreover, 83.3% of cyclin D1-positive patients and 21.4% of cyclin D1-negative patients died of cardiac causes., Conclusions: Cyclin D1 immunohistochemistry accurately identified patients with t(11;14). Cyclin D1-positive patients had significantly inferior OS compared with cyclin D1-negative patients., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society for Clinical Pathology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Sucrose transporters (SUTs) are known to play critical roles in the uptake of sucrose from the apoplast in various steps of sugar translocation. Because developing pollen is symplastically isolated from anther tissues, it is hypothesized that SUTs are active in the uptake of apoplastic sucrose into pollen. To investigate this possibility, a comprehensive expression analysis was performed for members of the SUT gene family in the developing pollen of rice (Oryza sativa L.) using real-time RT-PCR combined with a laser microdissection technique. Among the five SUT genes, OsSUT1 and OsSUT3 were found to be preferentially expressed and had temporal expression patterns that were distinct from each other. Expression of OsSUT1 in pollen was confirmed by a promoter-GUS fusion assay. The physiological function of OsSUT1 in pollen was further investigated using retrotransposon insertion mutant lines. While the homozygote of disrupted OsSUT1 (SUT1-/-) could not be obtained, heterozygote plants (SUT1+/-) showed normal grain filling. Their progeny segregated into SUT1+/- and SUT1+/+ with the ratio of 1:1, suggesting that the pollen disrupted for OsSUT1 is dysfunctional. This hypothesis was reinforced in vivo by a backcross of SUT1+/- plants with wild-type plants and also by in vitro pollen germination on the artificial media. However, starch accumulation during pollen development was not affected by disruption of OsSUT1, suggesting that the sugar(s) required for starch biosynthesis is supplied by other sugar transporters.

A comprehensive analysis of the transcript levels of genes which encode starch-synthesis enzymes is fundamental for the assessment of the function of each enzyme and the regulatory mechanism for starch biosynthesis in source and sink organs. Using quantitative real-time RT-PCR, an examination was made of the expression profiles of 27 rice genes encoding six classes of enzymes, i.e. ADPglucose pyrophosphorylase (AGPase), starch synthase, starch branching enzyme, starch debranching enzyme, starch phosphorylase, and disproportionating enzyme in developing seeds and leaves. The modes of gene expression were tissue- and developmental stage-specific. Four patterns of expression in the seed were identified: group 1 genes, which are expressed very early in grain formation and are presumed to be involved in the construction of fundamental cell machineries, de novo synthesis of glucan primers, and initiation of starch granules; group 2 genes, which are highly expressed throughout endosperm development; group 3 genes, which have transcripts that are low at the onset but which rise steeply at the start of starch synthesis in the endosperm and are thought to play essential roles in endosperm starch synthesis; and group 4 genes, which are expressed scantly, mainly at the onset of grain development, and might be involved in synthesis of starch in the pericarp. The methodology also revealed that the defect in the cytosolic AGPase small subunit2b (AGPS2b) transcription from the AGPS2 gene in endosperm sharply enhanced the expressions of endosperm and leaf plastidial AGPS1, the endosperm cytosolic AGPase large subunit2 (AGPL2), and the leaf plastidial AGPL1.

Objective: Endometriosis, a common disease among women of reproductive age, is characterized by the presence of endometrial-like tissue outside the uterus. TNF-alpha induces IL-8 production in endometriotic cells through nuclear factor-kappaB (NF-kappaB) activation. Thalidomide (Thal) inhibits inflammation by down-regulating the expression of proinflammatory cytokines in tumor cells and inflammatory cells. However, the mechanism of Thal action in human endometriotic stromal cells has not yet been elucidated., Main Outcome Measures: We examined whether Thal abrogates TNF-alpha-induced up-regulation of IL-8 expression in endometriotic stromal cells., Results: Here, we show 1) that treatment of endometriotic stromal cells with TNF-alpha increased the expression of phosphorylated IkappaBalpha and degradation of total IkappaBalpha, which in turn activates NF-kappaB; 2) Thal significantly inhibits the TNF-alpha-induced expression of phosphorylated IkappaBalpha and degradation of IkappaBalpha; 3) TNF-alpha activation induced increased nuclear translocation of NF-kappaB, which was inhibited by pretreatment with either Thal or N-tosyl-L-phenylalanine chloromethyl ketone, an NF-kappaB inhibitor. Thal did not enhance the N-tosyl-L-phenylalanine chloromethyl ketone's action; and 4) Pretreatment with Thal reduced TNF-alpha-induced IL-8 protein production as well as mRNA expression., Conclusion: The current study showed for the first time that Thal treatment attenuated the expression of IL-8 by reducing TNF-alpha-induced NF-kappaB activation.

In rice, caryopses located at the base of the panicle have a lower growth rate than those at the tip of the panicle. The former and latter types of caryopses are called inferior and superior caryopses, respectively. Taking the different growth rate into consideration, sugar status and the expression of genes encoding carbohydrate-metabolizing enzymes in inferior caryopses were compared with those in superior caryopses. During the first 5 d after flowering, superior caryopses elongated rapidly, but inferior caryopses did not. At this phase, inferior caryopses had a low ratio of hexose to sucrose, high activity of acid invertase and the absence of the expression of the genes encoding the above enzymes except for two isoforms of cell wall invertase, OsCIN4 and INV1, in comparison with superior caryopses. At the start of caryopsis elongation in both superior and inferior caryopses, the hexose/sucrose ratio increased accompanied by gene expression of vacuolar invertase (INV3), sucrose synthase (RSus1) and ADP-glucose pyrophosphorylase (AGP-L2: D50317). Furthermore, the genes related to endospermal starch accumulation were expressed highly with the decrease in the hexose/sucrose ratio after its peak. Based on the comparison of superior and inferior caryopses, the possible mechanism of grain filling in rice is discussed.

Background: Lipopolysaccharide (LPS) is the primary mediator of gram-negative sepsis; it induces the production of macrophage-derived cytokines. It has been shown that bikunin, a Kunitz-type protease inhibitor, inhibits LPS-induced cytokine expression., Methods: To explore the role of bikunin, bikunin knockout (Bik(-/-)) mice were used for in vitro cytokine experiments and in vivo animal models., Results: We show that a higher level of LPS-mediated death was induced in Bik(-/-), compared with wild-type (wt), mice; the administration of bikunin caused a significant reduction in LPS-induced lethality; LPS significantly increased tumor necrosis factor (TNF)- alpha and interleukin-1 beta levels in Bik(-/-), relative to wt, mice after LPS challenge; concomitant administration of bikunin inhibited the LPS-induced plasma levels of these cytokines; bikunin suppressed the LPS-induced up-regulation of cytokine expression through the suppression of the phosphorylation of ERK1/2, JNK, and p38 in macrophages; and LPS-induced up-regulation of TNF- alpha expression was not enhanced in Bik(-/-) macrophages without endogenous bikunin., Conclusions: These data allow us to speculate that the increased sensitivity of Bik(-/-) mice to LPS-induced death in vivo is due to a lack of circulating bikunin in plasma. Bikunin may play a role as a potent anti-inflammatory agent.

FSH and IGF-I are both important determinants of follicle development and the process of cumulus cell-oocyte complex expansion. FSH stimulates the phosphorylation of Akt by mechanisms involving phosphatidylinositol 3-kinase (PI3-K), a pattern of response mimicking that of IGF-I. Cartilage link protein (Crtl1) is confined to the cartilaginous lineage and is assembled into a macroaggregate complex essential for hyaluronan-rich matrix stabilization. The present studies were performed to determine the actions of FSH and IGF-I on Crtl1 production in rat granulosa cells. Primary cultures of granulosa cells were prepared from 24-d-old rats. After treatments, cell extracts and media were prepared, and the Crtl1 level was determined by immunoblotting analysis using anti-Crtl1 antibodies. Here we showed that 1) treatment with FSH (> or = 25 ng/ml) or IGF-I (> or = 25 ng/ml) for 4 h increased Crtl1 production; 2) maximal stimulatory effects of FSH or IGF-I were observed at 100 or 50 ng/ml, respectively; 3) FSH caused a concentration-dependent increase in IGF-I-induced Crtl1 production and vice versa; 4) FSH and IGF-I also up-regulate the expression of Crtl1 mRNA; 5) FSH- and IGF-I-dependent Crtl1 production were abrogated by PI3-K inhibitors (LY294002 and wortmannin), and inhibition of Crtl1 production by p38 mitogen-activated protein kinase inhibitor (SB202190) was partial (approximately 30%), suggesting that PI3-K and, to a lesser extent, p38 mitogen-activated protein kinase are critical for the response. Our study represents the first report that FSH amplifies IGF-I-mediated Crtl1 production, possibly via PI3-K-Akt signaling cascades in rat granulosa cells.

To establish the significance of cell wall invertase in grain filling of rice (Oryza sativa L.), we cloned a cDNA for a cell wall invertase from developing grains of rice. The cDNA, designated OsCIN1, contains an open reading frame of 1731 bp encoding a polypeptide of 577 amino acid residues. The deduced amino acid sequence showed typical features of the cell wall invertases, including a beta-fructosidase motif and a cysteine catalytic site, and shared 78.6 and 73.7% identity with maize cell wall invertases, Incw1 and Incw2, respectively. OsCIN1 is expressed in roots, in sink- and source-leaves, and in panicles. During the course of grain filling in the caryopses, OsCIN1 transcript is detectable only in the very early stage of their development, 1-4 d after flowering, when the cell wall invertase activity is the highest and the increase in caryopsis length is rapid. In situ localization of the mRNA revealed that OsCIN1 is expressed preferentially in the vascular parenchyma of the dorsal vein, integument and its surrounding cells, and is expressed weakly in the nucellar projection and nucellar epidermis. These results suggest that, during the early stage of caryopsis development, OsCIN1 is important for supplying a carbon source to developing filial tissues by cleaving unloaded sucrose in the apoplast.

Furosemide (frusemide) is a weakly acidic diuretic drug. Its absorption is poor and variable, in part due to its restricted sites of absorption, mainly the stomach. The narrow absorption window of this drug can be explained by pH partition theory. The purpose of this study was to investigate the feasibility of widening the absorption window of furosemide by controlling the pH in distal portions of the gastrointestinal tract with officially used additives. Methacrylate copolymer (Eudragit L100-55), hydroxypropylmethylcellulose phthalate (HP-55) and hydroxypropylmethylcellulose acetate succinate (AS-MF) were selected as additives. The pH of suspensions of these additives was about 4, and the pH was adjusted to about 6-7 by the addition of NaOH. The Eudragit L100-55 suspension was found to be the most resistant to NaOH titration. When Eudragit L100-55 was used in an in-situ ileal loop experiment in rats, the pH of the intestinal contents was significantly reduced, from 7.9+/-0.1 to 5.7+/-0.1, and the plasma concentration of furosemide 15 min after administration was about 3 times higher than that in controls, 1.81+/- 0.42microg mL(-1) vs 0.63+/-0.08 microg mL(-1). However, the plasma concentration of [14C] mannitol was not changed by the co-administration of Eudragit L100-55. Furthermore, the AUC of furosemide was significantly increased by a factor of about 1.6 relative to that in controls by the co-administration of Eudragit L100-55, to 21.4+/-4.0 microg h mL(-1) from 13.3+/- 3.9 microg h mL(-1), and the gastrointestinal pH in the midgut and ileum was significantly reduced, with most of the furosemide remaining in these segments at 2 h following the oral administration of furosemide with Eudragit L100-55 to rats. These findings clearly demonstrate that the addition of Eudragit L100-55 can increase the absorption of furosemide in distal portions of the gastrointestinal tract. In conclusion, it is feasible to widen the absorption window of furosemide by controlling the pH in distal portions of the gastrointestinal tract by the co-administration of Eudragit L100-55.

The purpose of this study was to determine whether cold-stress stimulation to the soles of paws produces continuous hypertension in rats. Wistar-Kyoto rats were kept in cages with a 0 degrees C floor and 23 degrees C room temperature (cold-stressed group, n = 10) or in cages with 23 degrees C floor and 23 degrees C room temperature (control group, n = 10). BP and levels of plasma catecholamines, serum glucose, and serum insulin were measured, and the histologic characteristics of the kidney and adrenal gland were studied in all groups. After a week of localized cold-stress, BP of the experimental rats were significantly increased over those of the control rats. Significant increases were also seen in plasma epinephrine and norepinephrine, as well as serum insulin concentrations in the rats that underwent localized cold stimulation; these changes were not observed in the control rats. Fibrinoid deposition in the kidney and the intensity of neuropeptide Y-staining in the adrenal medulla were increased in the localized cold-stressed group compared with the control group. We conclude that chronic local cold stimulation to the soles is a new model of experimental hypertension.

Cumulus oocyte complex (COC) expansion is induced through hyaluronic acid production and accumulation of proteins of the inter-alpha-trypsin inhibitor family in the gonadotropin-stimulated cumulus cells. Link protein, a glycoprotein found in cartilage, interacts specifically with hyaluronic acid and stabilizes the binding of proteoglycan monomers to hyaluronic acid to form aggregates. The aim of this study was to investigate the expression of immunoreactive link protein during follicle development in rats and in cumulus cells in culture by immunohistochemistry and Western blot as well as by specific enzyme-linked immunosorbent assay. Immunohistochemical analysis revealed that the extracellular matrix of cumulus cells that were morphologically at a stage of COC expansion were markedly stained for link protein, whereas granulosa cells from immature follicles were not stained. Cumulus cells deposited link protein into the extracellular matrix in an in vitro culture system. The staining intensity was negated by the treatment with hyaluronidase, suggesting that the link protein is bound to hyaluronic acid. We have identified a 42-kDa immunoreactive link protein in rat ovary during the preovulatory period and in COC extracts. Addition of FSH to the medium of cumulus cells in culture supplemented with 10% FBS and oocyte-conditioned medium resulted in an increased rate of link protein synthesis. This work suggests that the cumulus cells synthesize the link protein that may stabilize the binding of inter-alpha-trypsin inhibitor or dermatan sulfate proteoglycan to hyaluronic acid to make up hyaluronic acid-rich matrix aggregate.

Hyaluronic acid (HA) stimulates the synthesis of interleukin (IL) 8, while dehydroepiandrosterone sulphate (DHEA-S) induces the expression of IL-8 and its receptor in the human cervical fibroblast. This has led us to investigate the effect of DHEA-S on HA-induced cervical ripening. Experiments were performed in pregnant rabbits using vaginal suppositories containing 1 mg HA, 30 mg DHEA-S, 30 mg DHEA-S + 0.1 mg HA, 30 mg DHEA-S + 1 mg HA, and 500 microl Witepsol-50 base (control). The effects were evaluated by measuring collagenase, gelatinase and elastase activities, water content, neutrophil infiltration, relative collagen concentration and histological assessment. The activities of collagenase, gelatinase and elastase were significantly increased in rabbits treated with DHEA-S + 1 mg HA compared with rabbits treated with DHEA-S + 0.1 mg HA (P < 0.009, P < 0.001, P < 0.009 respectively). Water content was markedly increased in rabbits treated with DHEA-S + 1 mg HA compared with DHEA-S + 0.1 mg HA treatment (P < 0.05). Neutrophil infiltration was markedly increased, while relative collagen concentration was significantly decreased with DHEA-S + 1 mg HA compared with the DHEA-S + 0.1 mg HA approach (P < 0.001, P < 0.002). The histology of cervices treated with DHEA-S + 1 mg HA showed the density of collagen to be markedly decreased, and collagen fibres irregularly separated. Increased vascularity with massive dilatation of blood vessels was also observed in these rabbits. We conclude that DHEA-S upregulates the HA-induced cervical ripening process.

The aim of this research was to study the effect of exogenous interleukin (IL)-8, IL-1 and IL-8 + IL-1 on uterine contractions in rabbits. Four equal groups of non-pregnant rabbits (n = 24) were investigated using either placebo or experimental drugs in the form of vaginal suppositories. The suppositories contained human recombinant IL-8 (200 ng), IL-1 (200 ng), IL-8 (200 ng) + IL-1 (200 ng) or vehicle Witepsol base, 500 microliters). Subsequently, the plasma concentration of prostaglandin (PG) E2 was estimated 3 h after the last dose of treatment. Neutrophil infiltration in the endometrial tissue was studied with anti-rabbit RT2 staining. Suppositories with IL-1 and IL-8 + IL-1 produced contractile responses with increased frequency (P < 0.003, P < 0.0005) and amplitude (P < 0.0001) in vivo, compared with vehicle. IL-1 and IL-8 + IL-1 also caused similar contractile effects with increased frequency (P < 0.01, P < 0.0007) and amplitude (P < 0.0001) in an in-vitro experiment than vehicle. The frequency and amplitude of uterine contractions were more significant with IL-8 + IL-1 than that of IL-1, both in vivo (P < 0.002, P < 0.05) and in vitro (P < 0.005, P < 0.01). IL-8 did not induce any contractions. Prostaglandin concentration was increased approximately 8-fold with IL-8 + IL-1 (P < 0.0001) and 2.5-fold with IL-1 treatment (P < 0.0001). Neutrophil numbers were significantly increased with IL-8 + IL-1 > IL-8 > IL-1 (P < 0.002, P < 0.0003 and P < 0.008) compared with vehicle. Our data suggest that IL-8 stimulates IL-1-induced uterine contractions through PGE2 production and could be an important process during labour and delivery.

We investigated the effects of dehydroepiandrosterone sulfate (DHA-S) on the production of interleukin-8 (IL-8) and expression of the interleukin-8 receptor (IL-8 R) in human cervical tissue. DHA-S increased the levels of IL-8 in cultured human cervical fibroblasts and in the supernatant in a time- and dose-dependent manner. DHA-S induced IL-8 and IL-8 R expression in human cervical fibroblasts and human pregnant cervical tissue at term in a dose-dependent manner. In addition, it induced the expression of IL-8 R in an explant culture of human cervical tissue and cultured human cervical fibroblasts in a time- and dose-dependent manner. However, dehydroepiandrosterone (DHA) only slightly induced the production of IL-8 and the expression of IL-8 R in the same cells and tissue. These results suggest that DHA-S up-regulates the autocrine system of IL-8 through the expression of IL-8 R.

Although the effects of inflammatory cytokines such as interleukin (IL)-1 and tumour necrosis factor (TNF)-alpha on ovulation have been investigated, there are few reports concerning the effect of IL-8 in the ovary. We examined the localization of IL-8 in the human ovary, and also investigated the number of IL-8 staining cells in a section of 1 mm2. The number of IL-8 staining cells was 10 +/- 4.8 in the proliferative phase, 2.0 +/- 1.9 in the secretary phase, and 2.4 +/- 2.2 in the postmenopausal phase respectively. The increased number of IL-8 staining cells could be shown in the human ovarian medulla/stroma during the proliferative phase. By serial section analysis of the ovary, the localization of IL-8 corresponded with mast cells. When immature rats were injected with pregnant mare serum gonadotrophin (PMSG) followed by an i.p. injection of human IL-8, ovarian vasodilatation and follicular growth were observed. Exogenous IL-8 induced a similar increase in follicular growth to that produced by the luteinizing hormone (LH) surge. At 6 h after IL-8 (20 microg) i.p., the follicular size was increased to almost the same size as human chorionic gonadotrophin (HCG) i.p. However, at 24 h after IL-8 i.p., the follicular size decreased. The size of follicular growth was almost the same size as control when <2 microg IL-8 was injected. At 6 h after IL-8 (10 microg) local injection, the follicular size in rats was similar to that observed with IL-8 (20 microg) i.p. We conclude that IL-8 is one of the important cytokines in the ovulatory process.

Although recent evidence suggests that certain beta-lactam antibiotics are absorbed via a specific transport mechanism, its nature is unclear. To confirm whether peptide transport in the rat can be largely ascribed to the intestinal oligopeptide transporter PepT1, the transporter has been functionally characterized and its significance in the intestinal absorption of beta-lactam antibiotics was evaluated. For evaluation of transport activity complementary RNA (cRNA) of rat PepT1 was synthesized in-vitro and expressed in Xenopus laevis oocytes. cRNA induced uptake of several beta-lactam antibiotics and the dipeptide [14C]glycylsarcosine; this was specifically inhibited by various dipeptides and tripeptides but not by their constituent amino acids or by tetra- or pentapeptides. The transport activity of PepT1 for beta-lactam antibiotics correlated well with their in-vivo intestinal transport and absorption. Furthermore, mutual inhibitory effects on uptake were observed between glyclsarcosine and beta-lactam antibiotics. Hybrid depletion of the functional expression of rat PepT1 in oocytes injected with rat intestinal epithelial total mRNA was studied using an antisense oligonucleotide corresponding to the 5'-coding region of PepT1. In oocytes injected with rat mRNA pre-hybridized with the antisense oligonucleotide against rat PepT1, the uptake of [14C]glycylsarcosine was almost completely abolished, whereas its uptake was not influenced by a sense oligonucleotide for the same region of PepT1. Similarly, the uptake of beta-lactam antibiotics was also reduced by the antisense oligonucleotide against rat PepT1. These results demonstrate that the intestinal proton-coupled oligopeptide transporter PepT1 plays a predominant role in the carrier-mediated intestinal absorption of beta-lactam antibiotics and native oligopeptides in the rat.

During pregnancy, hyaluronic acid (HA) concentration in the human cervix is very low, but increases rapidly at the onset of labour. HA has a high affinity for water molecules and hence can maintain tissue hydration. HA can stimulate collagenase production in rabbit cervix, and also stimulates migration and function of polymorphonuclear leukocytes in the tissues. It is an endogenous regulator of interleukin-1 (IL-1). We hypothesized that HA plays an essential role during cervical ripening. The effect of exogenous application of HA (20 mg) on non-pregnant and pregnant (day 23) rabbit cervices was compared with controls. HA induced cervical ripening in both pregnant and non-pregnant animals, and cervical water content was significantly increased. Tissue collagen was markedly decreased. The localization and distribution of HA and HA receptor CD44 was determined in non-pregnant and pregnant human cervical connective tissue using biotinylated HA binding protein and CD44 monoclonal antibodies. Both were widely distributed in the connective tissues, especially around the blood vessels and cervical glands. The effect of IL-8 (50, 100, 150 and 200 ng/ml) on HA production and hyaluronidase (HAase) activity was investigated in cultures of lower uterine segment collected during elective Caesarean sections. HA production was stimulated in a dose-dependent manner; there was no effect on hyaluronidase activity. HA administration (0.5, 1 and 2 mg/ml) stimulated the activities of collagenase and gelatinase together with IL-8 production in the culture supernatants. Thus HA may play an important role in cervical ripening, being involved in the regulation of cervical tissue water content, collagenolytic enzymes and cytokines.

Ten cDNAs of genes that were induced by dehydration stress were cloned by differential screening from the highly drought-tolerant legume, cowpea (Vigna unguiculata), a major crop in West Africa. The clones were collectively named CPRD (cowpea clones responsive to dehydration). Northern blot analysis revealed that nine of the CPRD genes were induced by dehydration stress, but the timing of induction of mRNA synthesis varied among the CPRD genes. We analyzed the effects of other environmental stresses on the expression of the CPRD8, CPRD14 and CPRD22 genes, and we found that these genes were strongly induced by high-salinity stress but not by cold or heat stress. Drought-stressed cowpea plants accumulated abscisic acid (ABA) to a level that was 160 times higher than that in unstressed plants. The CPRD8 and CPRD22 genes were induced to a significant extent by the application of exogenous ABA but the CPRD14 gene was not. These results indicate the existence of at least two signal-transduction pathways between the detection of water stress and the expression of CPRD genes in cowpea. Sequence analysis of CPRD8 and CPRD22 cDNAs revealed that they encoded putative proteins that were related to old yellow enzyme and group 2 LEA proteins, respectively. The protein encoded by CPRD14 exhibited sequence homology to dihydroflavonol-4-reductase (DFR) and vestitone reductase (VR). Old yellow enzyme, DFR and VR have not been identified as drought-inducible proteins in other plants, whereas LEA genes have been well characterized as drought-inducible genes. The various gene products might function to protect cells from environmental stress.

Because the significance of P-glycoprotein in the in-vivo secretion of beta-blockers in intestinal epithelial cells is unclear, the secretory mechanism for beta-blockers and other drugs has been evaluated. Uptake of the beta-blockers acebutolol, celiprolol, nadolol and timolol, and the antiarrhythmic agent, quinidine by the multidrug-resistant leukaemic cell line variant K562/ADM was significantly lower than that by drug-sensitive K562 cells, suggesting that these beta-blockers are transported by P-glycoprotein out of cells. The reduced uptake of acebutolol by the drug-resistant K562/ADM cells was reversed by treating the cells with anti-P-glycoprotein monoclonal antibody, MRK16, whereas no such alteration in uptake was observed for drug-sensitive K562 cells. Acebutolol uptake by K562/ADM cells was, moreover, markedly enhanced, in a concentration-dependent manner, in the presence of the specific P-glycoprotein inhibitors, MS-209 and cyclosporin. Caco-2 cells were used for evaluation of the role of P-glycoprotein in intestinal permeability to drugs in-vitro. Basolateral-to-apical transport of acebutolol was twice that in the reverse direction. A similar polarized flux was also observed in the transport of vinblastine, but not in that of acetamide or mannitol. When in-vivo intestinal absorption was evaluated by the rat jejunal loop method, with simultaneous intravenous administration of a P-glycoprotein inhibitor, cyclosporin, intestinal absorption of both acebutolol and vinblastine increased 2.6- and 2.2-fold, respectively, but no such enhancement was observed in the absorption of acetamide. The effect of cyclosporin on the intestinal absorption of several drugs was further examined, and the extent of the contribution of P-glycoprotein as an absorption barrier to those drugs was evaluated. ATP depletion by occlusion of the superior mesenteric artery resulted in a clear increase in epithelial permeability to vinblastine, but not to 3-O-methylglucose or acetamide, indicating that vinblastine is secreted by ATP-dependent P-glycoprotein into the lumen. These findings demonstrate that P-glycoprotein plays a role as an absorption barrier by transporting several drugs from intestinal cells into the lumen.

The aim of this research was to study and compare the mechanism of action of interleukin (IL)-8, IL-1beta dehydroepiandrosterone sulphate (DHEA-S) and prostaglandin (PG)E2 on the cervix. Five equal groups of pregnant rabbits (n = 45) were tested by either placebo or tested drugs in the form of vaginal suppositories once daily for 3 days. The suppositories contained human recombinant IL-8 (100 ng), IL-1beta (200 ng), DHEA-S (10 mg) or PGE2 (1 mg). All rabbits were tested by one dose, two doses or three doses. Consistency, dilatation and water contents were estimated 24 h after the last dose of treatment. Leukocyte infiltration of the cervices was studied after staining the cervical tissue sections with antirabbit RT2 monoclonal antibodies. Relative collagen concentration was assessed after staining with Picrosirius Red. Collagenase, gelatinase and elastase activities were measured in 100 mg of homogenized cervical connective tissue. Water contents were significantly increased in all tested cervices. Neutrophil numbers were increased in IL-8 and IL-1beta groups after the second dose of treatment (P < 0.0005 and 0.001 respectively). In the PGE2 group, neutrophils were increased after the third dose of treatment, whereas in DHEA-S group no significant changes were observed. Collagen content was significantly decreased in IL-8, IL-1beta and PGE2 groups after the first dose of treatment (P < 0.004, and 0.005 and 0.03 respectively). In the DHEA-S group, the decrease in collagen content occurred after the third dose (P < 0.05). Collagenase activity was markedly increased in IL-8, IL-1, and DHEA-S groups after the second dose of treatment (P < 0.001, 0.003 and 0.007 respectively). No significant increase in collagenase activity was found in PGE2 group. Gelatinase activity was significantly increased in IL-8, IL-1beta, PGE2 and DHEA-S groups after the second dose of treatment (P < 0.008, 0.01, 0.003 and 0.05 respectively). Also, elastase activity was increased after the second dose of treatment in all groups (P < 0.001, 0.001, 0.001 and 0.006 respectively). Our data suggest that ripening of the cervix in rabbit can be initiated by different mechanisms. Cytokines play a vital role in cervical ripening, especially IL-8 and IL-1. IL-8 is one of the factors which could ripen the cervix in a manner similar to the physiological process at term.

The activation of neutrophils was studied in preeclampsia (n = 10) and eclampsia (n = 20) compared to normotensive controls (n = 10) and nonpregnant essential hypertensives (n = 10). Plasma elastase levels were raised in preeclampsia (0.53 +/- 0.32 microgram/mL, P < .002) and eclampsia (1.26 +/- 0.8 microgram/mL, P < .001) respectively compared to normal pregnancies (0.032 +/- 0.009 microgram/mL). The plasma elastases were more elevated in eclamptic cases compared to essential hypertensive (0.53 +/- 0.27 microgram/mL; P = .01) patients. We analyzed the correlation among elastase values, systolic (SBP), mean blood pressures (MBP), endothelin-1 (ET-1) levels and sera cytotoxicity (as measured by fura-2 release from human umbilical venous endothelial cell culture) in eclamptic cases. SBP and MBP were significantly correlated with plasma elastase levels in preeclampsia (r = 0.67, 0.63, respectively; P < .03) and eclampsia (r = 0.49, 0.49, respectively; P < .02). ET-1 levels were correlated with SBP (P = .003) and MBP (P = .001) and corresponding elastase levels (r = 0.606, P < .003) in eclamptic patients. Doses of 10, 25, and 50 pmol/mL of ET-1 increased elastase release in human neutrophil cultures dose and time dependently. Cytotoxicity of eclamptic sera correlated (P < .001) to the corresponding plasma elastase values. Therefore, this study suggests that neutrophil activation and ET-1 induced neutrophil activation occurs in this disease.

Bestatin is an immunomodulatory peptide that stimulates the humoral and cell-mediated immune system. It also has an inhibitory effect on multiple aminopeptidases. Recently we found that aminopeptidase N inactivates interleukin-8 in vitro. Bestatin successfully suppresses the effect of aminopeptidase N on interleukin-8. During cervical maturation many biochemical changes occur including decrease in collagen concentration and increase in collagenase and elastase activities. Interleukin-8, which has a potent neutrophil chemotactic effect, was found to induce cervical ripening in rabbits. The combination of interleukin-8 with bestatin also induced cervical ripening by providing approximately regular levels of neutrophil numbers, collagenase, and elastase activities. We therefore suggest that this regulatory mechanism also takes place in vivo through the inhibitory effect of bestatin on aminopeptidase N.

Aminopeptidase (APN) was found to degrade interleukin-8 (IL-8) and inactivate its chemotactic activity. The chemotactic activity of IL-8 was decreased by APN or neutrophil plasma membranes dose- and time-dependently. The chemotactic activity was not inactivated in the presence of bestatin or WM15 monoclonal antibody. The expression of IL-8 was measured by flow cytometry. On lipopolysaccharide (LPS) stimulation, IL-8 expression increased for 60 min and then decreased markedly. In contrast, on treatment with LPS and bestatin, the expression of IL-8 increased continuously for at least 120 min. These results suggest that the expression and release of IL-8 from phagocytic cells are regulated by the proteolytic effect of APN on IL-8.

The aggregation of bovine serum albumin (BSA) in the solid state and in solution was studied to elucidate the effect of water mobility on the aggregation rate and mechanism. The results suggest that the freeze-dried BSA forms covalently-bonded aggregates via disulphide bonding during storage in the moistened solid state and in solution. The aggregation rate largely depended on the water content. The freeze-dried BSA to which a small amount of water was added (in the moistened state) was found to be more liable to aggregation than that in solution. The aggregation rate in in the moistened solid state exhibited a maximum at a very low level of moisture. In contrast, the aggregation rate in solution increased with increasing ratio of water to protein (with decreasing protein concentration). The results suggest that the increased rate is related to the increase in water mobility, as measured by the spin-lattice relaxation time, T1, of water using 17O NMR, with increasing ratio of water to protein.

A sensitive and reliable method for liquid chromatographic (LC) determination of zearalenone and alpha-zearalenol in barley and Job's-tears was investigated. The method by which these toxins were determined involves addition of an internal standard (zearalenone 6'-oxime) to barley and Job's-tears samples. Extracts from grain samples were cleaned up by passage through chromatography on piperidinohydroxypropyl Sephadex LH-20 as a lipophilic gel. Individual toxins were resolved by LC on a reversed-phase (ODS) column with fluorescence detection. The detection limit is estimated to be 0.2 ng for zearalenone and alpha-zearalenol standards. Known amounts of zearalenone and alpha-zearalenol (25-1250 ng) were added to a barley sample (5 g). Average recoveries for alpha-zearalenol and zearalenone, respectively, ranged from 96 to 102% (mean CV, 3.6%) and from 96 to 103% (mean CV, 3.3%). This method is applicable to determination of alpha-zearalenol and zearalenone in barley and Job's tears with satisfactory sensitivity and accuracy.

We measured the concentration of zinc coproporphyrin I (ZnCP-I), a characteristic component of meconium, in maternal plasma by fluorometry after HPLC. We obtained plasma samples from 89 women: 35 at weeks 10-40 of normal pregnancy, 41 shortly after normal delivery, 4 from patients with amniotic fluid embolism (AFE), and 9 from non-AFE patients with intra- or postpartum shock caused by genital bleeding. The plasma ZnCP-I concentration was 97 (SD 83, range 38-240) nmol/L in the AFE patients, 11 (SD 9.2) nmol/L in the non-AFE patients, 12 (SD 7.9) nmol/L during normal pregnancy, and 26 (SD 10) nmol/L shortly after normal delivery. We suggest that measuring ZnCP-I in maternal plasma by fluorometry on HPLC is a rapid, noninvasive, and sensitive method for diagnosing AFE and propose 35 nmol/L as the cutoff value for the ZnCP-I concentration in maternal plasma for the diagnosis of AFE.

We isolated a substance from an organic extract of meconium (in neutral pH) that exhibited a porphyrin-like fluorescence peak at 580 nm on excitation at 405 nm and deduced its structure. Spectrometric data suggest that the substance is not free coproporphyrin I or free coproporphyrin III, but is a chelate of coproporphyrin I with Zn2+. We also detected a chelate of coproporphyrin III with Zn2+ by HPLC. These substances may be useful as new indicators of the presence of meconium.

In order to determine the epitope of metallothionein (MT) to a murine monoclonal antibody (MT 189-14-7) which had been produced by immunization with rat MT 2 (Kikuchi et al. (1988) Mol. Immunol. 25, 1033-1036), various lengths of synthetic oligopeptides were tested for their inhibitory activities in competitive radioimmunoassay (RIA). The amino-terminal acetylated pentapeptide, AcMDPNC, exhibited an inhibitory activity comparable to that of native MTs, whereas the acetylated tetrapeptide, AcMDPN, and the deacetylated heptapeptide, MDPNCSC, were much less inhibitory. The results suggest that the major part of the epitope structure of MT to the MT 189-14-7 monoclonal antibody is located within the amino-terminal acetylated pentapeptide, AcMDPNC. The specificities of polyclonal rabbit anti-MT antisera raised against the same immunogen were also determined by using various animal MTs and synthetic peptides as inhibitors in the RIA. Among three antisera tested, two reacted with several amino-terminal oligopeptides similarly to the MT 189-14-7 antibody. The major epitope structures to these polyclonal antibodies were shown to be located within the acetylated tetrapeptide, AcMDPN. Another antiserum contained at least two different populations of antibodies: one consisted of antibodies reactive with the amino-terminal synthetic peptides, while the other was not reactive with them. These results suggest that, in the rabbit also, the amino-terminal region common to various animal MTs can be an epitope to antibodies raised against rat MT, as shown in the mouse. Moreover, the results indicate that the synthetic amino-terminal peptides are useful for determination of the specificity of polyclonal rabbit anti-MT antibodies, which have been widely used for the quantification of MTs.

Protein kinase [EC 2.7.1.37] of human erythrocyte membranes was solubilized with 0.5 M NaCl in 5 mM phosphate buffer, pH 6.7 at 4 degrees C and purified on a CM-Sephadex C-50 column, followed by affinity chromatography on a histone-Sepharose 4B column. The purified protein kinase gave a single band (molecular weight; 41,000) on examination by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH of the enzyme was 8.0 and a millimolar range of concentration of Mg2+ was required for its maximum activity. Histone and protamine were well phosphorylated by the protein kinase but casein and phosvitin were poor phosphate acceptors for the enzyme. The enzymic activity was not stimulated by cyclic AMP (cAMP). A cAMP-finding protein from human erythrocyte membranes inhibited the activity of the protein kinase, but the activity was restored with cAMP. A heat stable protein inhibitor from rabbit skeletal muscle also inhibited this enzyme. From these observations, this protein kinase seemed to be a catalytic subunit of the membrane bound cAMP-dependent protein kinase. This enzyme was strongly inhibited with Ca2+ in the presence of 1 mM MgCl2. Various sulfhydryl reagents and polyamines also had inhibitory activity on the protein kinase. Natural substrates of the enzyme were investigated using heat treated membranes and 0.5 M NaCl extracted membrane residues. Band 4.1, 4.2, and 4.5 proteins were phosphorylated but band 2 (spectrin) and band 3 proteins were poor substrates for this protein kinase.

A cyclic AMP-binding protein of human erythrocyte membranes was solubilized with 0.3% Triton X-100 in 10 mM Tris-HCl (pH 7.4) at 4 degrees C, and purified by DEAE-cellulose column chromatography and affinity chromatography on a cyclic AMP derivative-fixed Sepharose 4B column. The purified cyclic AMP-binding protein showed a single band (molecular weight: 49,000) on examination by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the band was specifically labeled with a photoaffinity analogue of cyclic AMP, 8-N3-cyclic [2-3H]AMP. This protein bound 1.6 mol of cyclic AMP per molecule with an association constant of 3.8 x 10(9) M-1 and the optimum pH for binding was 7.4. The protein inhibited the activity of a purified protein kinase from human erythrocyte membranes [Suzuki, K., Terao, T., & Osawa, T. (1981) J. Biochem. 89, 1--11], while cyclic AMP restored the enzymic activity. The amino acid composition of this protein was different from those of cytoplasmic cyclic AMP-binding proteins. These observations indicate that the cyclic AMP-binding protein purified in this work is the regulatory subunit of a membrane bound cyclic AMP-dependent protein kinase.

The existence of specific binding proteins for alpha-casein, a well-known potent chemoattractant, on guinea pig peritoneal macrophages was demonstrated. Binding of 3H-alpha-casein to macrophages was found to be specific and reversible. Its association constant and the number of binding sites were 5.0 X 10(-6) M and 7.5 X 10(5) per cell, respectively. The presence of formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe), another chemoattractant, at a chemotactically active concentration (10(-11) to 10(-6) M) reduced this binding significantly. Pretreatment of macrophages with proteases such as trypsin and chymotrypsin decreased the number of binding sites for alpha-casein and the chemotactic response to alpha-casein. alpha-Casein binding proteins were isolated from radiolabeled macrophage surface membranes by affinity chromatography on a column of alpha-casein Sepharose 4B. SDS-polyacrylamide gel electrophoresis of the isolated proteins revealed three radioactive bands; two (MW 130,000 and 65,000) were glycoproteins, and the other (MW 100,000) was a protein which contained little carbohydrate. These three proteins were also isolated from the same source by affinity chromatography using a column of fMet-Leu-Phe-AH-Sepharose. This result, together with the observation that fMet-Leu-Phe inhibited the binding of alpha-casein to macrophages, indicated that alpha-casein and the tripeptide have at least partly common binding sites on guinea pig macrophages. Some of the proteins obtained in this work may constitute chemotactic receptors on the macrophage membrane.

Dimeric succinyl-concanavalin A was cross-linked with ethylenediamine using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide a condensing agent. Thus, a divalent dimer and a tetravalent tetramer composed mostly of covalently cross-linked subunits bearing altered net charges were obtained. Photoaffinity labeling of the cross-linked dimer with p-azidophenyl alpha-D-mannopyranoside resulted in a specific label for its saccharide-binding site and yielded a nonvalent dimer and a monovalent dimer (showing no subunit exchange). However, hemagglutination and glycogen precipitation data suggested that the labeled binding site is shielded but not destroyed by the label and can still bind weakly an external saccharide ligand possibly due to unsteadiness of the shielding label. Although nonvalent and monovalent derivatives were mitogenic as well as divalent and tetravalent derivatives for mouse splenic lymphocytes, binding and stimulation experiments indicated that their stimulating efficiencies after binding to the cells were far lower than those of the multivalent counterparts. Their activities were inhibited by methyl alpha-D-mannopyranoside, suggesting that the weak activities of nonvalent and monovalent derivatives were due to the labeled sites entirely and partly, respectively. We suggest that the triggering of lymphocyte mitogenesis by concanavalin A may depend on cross-linkage of cell surface receptors.

Three beta-N-acetylhexosaminidases [EC 3.2.1.52] and one beta-galactosidase [EC 3.2.1.23] were purified from the culture filtrate of streptococcus 6646 group K by a combination of column chromatographies on p-aminophenyl beta-D-thiogalactopyranoside-substituted Sepharose and N-(paminophenyl)oxamic acid-substituted Sepharose. These beta-N-acetylhexosaminidases showed optimal activities between pH 5.0 and 5.5 and could hydrolyze synthetic and glycopeptidic substrates. Glycolipids such as GM2, asialo-GM2, and globoside I were no susceptible to these beta-hexosaminidases. beta-Galactosidase, which was purified more than 11,000-fold, had a substrate specificity rather similar to that of beta-galactosidase from E. coli. This enzyme was inhibited by EDTA and activated by Mn2+, Ca2+, and Mg2+. Problems pertinent to the application of affinity chromatography to the purification of glycosidases are also discussed.

Concanavalin A (Con A) was labeled with p-azidophenyl alpha-D-mannopyranoside under ultraviolet irradiation and the reaction products were separated by affinity chromatography on Sephadex G-100 at pH 5. One of the Con A derivatives thus obtained was characterized as a monovalent dimer at pH 5 and a divalent tetramer at pH 7 by sedimentation equilibrium and equilibrium dialysis, indicating that this photoaffinity labeling did not alter the quaternary structure of Con A. In agreement with these results, the labeled Con A did not show the capacity to precipitate glycogen at pH 5, but it formed precipitates with glycogen at pH 7. Although its hemagglutinating activity was found to be weaker than that of the native Con A, the dose-response cure of the labeled Con A in the mitogenic stimulation of human peripheral lymphocytes was almost identical to that of the native con A.

The guanosine 3',5'-cyclic monophosphate (cGMP) level in the mouse splenic lymphocytes was increased about 2- to 3-fold by concanavalin A. This increase was completely dependent on the presence of Ca2+ in the medium. Homogenates of mouse splenic lymphocytes contained significant guanylate cyclase [EC 4.6.1.2] activity in both the 105,000 X g (60 min) particulate and supernatant fractions and both fractions required Mn2+ for full activity. Calcium ion (3mM) activated soluble guanylate cyclase 3-fold at a relatively low concentration of Mn2+ (less than 1mM) but inhibited the particulate enzyme slightly at all Mn2+ concentrations tested. Concanavalin A itself did not stimulate either fraction of guanylate cyclase. Thus these results suggest that elevation of the cGMP level in lymphocytes by concanavalin A might be brought about by stimulation of Ca2+ uptake and activation of soluble guanylate cyclase by the latter.

Monovalent succinyl-concanavalin A was prepared by photoaffinity labeling of succinyl-concanavalin A with p-azidophenyl a-D-mannopyranoside. This concanavalin A derivate showed a weak but definite mitogenic acitivity against mouse splenic lymphocytes and human peripheral lymphocytes, suggesting that direct receptor cross-linkage on the cell surface is not an obligatory prerequisite for the activation of lymphocytes.

For the development of therapeutic agents that possess tissue-specific carriers, a method was devised to synthesize an artificial protein hybrid conjugate containing a moiety which binds to a cell membrane receptor and an active fragment of a toxic protein. By the introduction of an activated sulfhydryl group into concanavalin A (Con A), a conjugate of Con A and the ricin A-chain cross-linked with a disulfide linkage was synthesized. The purified conjugate was studied with regard to its inhibitory activity against protein synthesis in cell-free and cultured cell systems. The Con A-rich A-chain conjugate retained about one-third the inhibitory activity of ricin in a cell-free protein synthesis system. It also was highly toxic to cultured normal cells. These results indicate that the conjugate is a structural and functional analog of ricin and that the original membrane-binding chain (B-chain of ricin) could be replaced by Con A. Transformed cells were insensitive to this conjugate and required a longer preincubation time. The sensitivity of the normal cells was reduced in the presence of local anesthetics.

Using a digital imaging fluorescence microscope, we have detected a rapid transient increase in the free cytosolic calcium concentration in a single rat basophilic leukemia cell (RBL-2H3) after antigen stimulation. Calcium ions were transported very rapidly (within 1 s) after a lag time (about 10 s at 37 degrees C) from the external environment into the cytoplasm. On the basis of the present experimental results we conclude that the gradual changes in the overall fluorescence intensity observed for a cell suspension are due to the distribution of different lag times shown by different cells as to the calcium influx through membrane calcium channels.