Essential oils for managing Pratylenchus penetrans on Easter lilies.

Easter lily bulbs for greenhouse forcing are produced in Del Norte County, California and Curry County, Oregon, USA. Pratylenchus penetrans infestation seriously affects growth of field grown bulbs. During two consecutive years of field trials containing 22 treatments, commercially prepared formulations of essential oils (EOs) were compared to an untreated control and to a standard chemical fumigant treatment (FU) (1,3-dichloropropene and metam sodium) applied preplant followed by phorate (PH) at planting to determine their value in the management of lesion nematode, and in improving plant health. The EO products Duogard, EF400, EF300, and Cinnamite were tested as preplant dips to bulblet planting stock. The treated bulblets were tested either alone, in combination with PH at-planting, at planting following FU or in combination with PH at planting following FU. The organophosphates ethoprop and fosthiazate were also tested either alone, or at a reduced rate in combination with a reduced rate of PH. With respect to bulb circumference, ten treatments consistently outperformed the control. In consecutive years, three treatments had healthier looking roots than the control. At harvest, levels of lesion nematode within roots were consistently lower in nine treatments. EOs were beneficial in mitigating nematode damage.Easter lily bulbs for greenhouse forcing are produced in Del Norte County, California and Curry County, Oregon, USA. Pratylenchus penetrans infestation seriously affects growth of field grown bulbs. During two consecutive years of field trials containing 22 treatments, commercially prepared formulations of essential oils (EOs) were compared to an untreated control and to a standard chemical fumigant treatment (FU) (1,3-dichloropropene and metam sodium) applied preplant followed by phorate (PH) at planting to determine their value in the management of lesion nematode, and in improving plant health. The EO products Duogard, EF400, EF300, and Cinnamite were tested as preplant dips to bulblet planting stock. The treated bulblets were tested either alone, in combination with PH at-planting, at planting following FU or in combination with PH at planting following FU. The organophosphates ethoprop and fosthiazate were also tested either alone, or at a reduced rate in combination with a reduced rate of PH. With respect to bulb circumference, ten treatments consistently outperformed the control. In consecutive years, three treatments had healthier looking roots than the control. At harvest, levels of lesion nematode within roots were consistently lower in nine treatments. EOs were beneficial in mitigating nematode damage.

Identification of the binding site in intercellular adhesion molecule 1 for its receptor, leukocyte function-associated antigen 1.

Intercellular adhesion molecule 1 (ICAM-1, CD54) is a member of the Ig superfamily and is a counterreceptor for the beta 2 integrins: lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18), complement receptor 1 (MAC-1, CD11b/CD18), and p150,95 (CD11c/CD18). Binding of ICAM-1 to these receptors mediates leukocyte-adhesive functions in immune and inflammatory responses. In this report, we describe a cell-free assay using purified recombinant extracellular domains of LFA-1 and a dimeric immunoadhesin of ICAM-1. The binding of recombinant secreted LFA-1 to ICAM-1 is divalent cation dependent (Mg2+ and Mn2+ promote binding) and sensitive to inhibition by antibodies that block LFA-1-mediated cell adhesion, indicating that its conformation mimics that of LFA-1 on activated lymphocytes. We describe six novel anti-ICAM-1 monoclonal antibodies, two of which are function blocking. Thirty-five point mutants of the ICAM-1 immunoadhesin were generated and residues important for binding of monoclonal antibodies and purified LFA-1 were identified. Nineteen of these mutants bind recombinant LFA-1 equivalently to wild type. Sixteen mutants show a 66-2500-fold decrease in LFA-1 binding yet, with few exceptions, retain binding to the monoclonal antibodies. These mutants, along with modeling studies, define the LFA-1 binding site on ICAM-1 as residues E34, K39, M64, Y66, N68, and Q73, that are predicted to lie on the CDFG beta-sheet of the Ig fold. The mutant G32A also abrogates binding to LFA-1 while retaining binding to all of the antibodies, possibly indicating a direct interaction of this residue with LFA-1. These data have allowed the generation of a highly refined model of the LFA-1 binding site of ICAM-1.

A comparative study of the enantiomeric separation of labeled amino acids with cyclodextrins and mixed micelles in capillary electrophoresis.

Enantiomeric separations of fluorescently labeled amino acids are studied by capillary electrophoresis (CE) under a novel variety of experimental conditions. Three different labels are evaluated using two different additives: cyclodextrins (beta- and gamma-) and a dual surfactant system of sodium dodecyl sulfate and sodium taurodeoxycholate. Fluorescein-5-isothiocyanate is the best label to use in this cyclodextrin-based system, and dansyl chloride is the best label to use in this dual surfactant system. Possible limitations for separation of the enantiomers using the mixed micelle system include the fact that there is little interaction of the solute with the surfactants, the negative charge of the solute is limiting the separation window of the system, and the amount of the chiral phase available for partitioning is limited. The separations using cyclodextrins as a chiral selector show that the label affects migration order of the enantiomers, and the cyclodextrins are very effective in separating numerous enantiomers. Overall, cyclodextrins are the better buffer additive for CE use, and the dual surfactant systems, including sodium taurodeoxycholate, offer future promise.

A Ca2+-activated ATPase specifically released by Ca2+ shock from Paramecium tetraurelia.

Deciliation of Paramecium tetraurelia by a Ca2+ shock procedure releases a discrete set of proteins which represent about 1% of the total cell protein. Marker enzymes for cytoplasm (hexokinase), endoplasmic reticulum (glucose-6-phosphatase), peroxisomes (catalase), and lysosomes (acid phosphatase) were not released by this treatment. Among the proteins selectively released is a Ca2+-dependent ATPase. This enzyme has a broad substrate specificity which includes GTP, ATP, and UTP, and it can be activated by Ca2+, Sr2+, or Ba2+, but not by Mg2+ or by monovalent cations. The crude enzyme has a specific activity of 2-3 mumol/min per mg; the optimal pH for activity is 7.5. ATPase, GTPase, and UTPase all reside in the same protein, which is inhibited by ruthenium red, is irreversibly denatured at 50 degrees C, and which has a sedimentation coefficient of 8-10 S. This enzyme is compared with other surface-derived ATPases of ciliated protozoans, and its possible roles are discussed.

Relationship of human milk pH during course of lactation to concentrations of citrate and fatty acids.

Human milk pH was measured in 309 samples obtained from 52 women who had delivered at term and lactated for as long as 10 months thereafter. The mean pH decreased from 7.45 for colostrum to a nadir of 7.04 during the second week of lactation. Thereafter, the pH of milk remained between 7.0 and 7.1 until 3 months postpartum and then increased gradually to 7.4 by 10 months. The change in hydrogen ion concentration in milk was associated with corresponding changes throughout lactation in the concentration of citrate but not with the concentration of lactose. Lactose concentration increased gradually for 3 weeks; the concentration of saturated medium-chain fatty acids increased more rapidly. One interpretation of these findings is that the hydrogen ions and citrate generated by mammary secretory cell metabolism are used after the second week of lactation for de novo synthesis of fatty acids more rapidly than they are synthesized. Milk samples from ruminants were found to have concentrations of hydrogen ions and citrate that are greater than and pH that is less than the respective measurements in human milk. The significance for the recipient infant of the predictable changes in human milk pH during lactation and of the higher pH of human milk throughout lactation relative to bovine milk is unknown. However, drug excretion into milk, milk enzyme activity, milk leukocyte function, and neonatal gastrointestinal function are affected by ambient pH and may be influenced by the pH of milk.

Monoclonal antibodies to the extracellular domain of HIV-1IIIB gp160 that neutralize infectivity, block binding to CD4, and react with diverse isolates.

Ten monoclonal antibodies prepared against a soluble, recombinant form of gp160, derived from the IIIB isolate of HIV-1, were characterized. Four of the antibodies neutralized HIV-1IIIB infectivity in vitro, three blocked the binding of recombinant gp120 to CD4, three were reactive with gp41, and one preferentially reacted with an epitope on gp120 within the gp160 precursor. All three CD4 blocking antibodies bound to distinct epitopes, with one mapping to the C1 domain, one mapping to the C4 domain, and one reactive with a conformation-dependent, discontinuous epitope. Of these, the antibody reactive with the discontinuous epitope exhibited neutralizing activity against homologous and heterologous strains of HIV-1. The binding of these monoclonal antibodies to a panel of seven recombinant gp120s prepared from diverse isolates of HIV-1 was measured, and monoclonal antibodies with broad cross reactivity were identified. The epitopes recognized by 7 of the 10 monoclonal antibodies studied were localized by their reactivity with synthetic peptides and with fragments of gp120 expressed as fusion proteins in a lambda gt-11 gp160 epitope library.

Affinity-reversed-phase liquid chromatography assay to quantitate recombinant antibodies and antibody fragments in fermentation broth.

An automated dual-column liquid chromatography assay comprised of affinity and reversed-phase separations that quantifies the majority of antibody-related protein species found in crude cell extracts of recombinant origin is described. Although potentially applicable to any antibody preparation, we here use samples of anti-CD18 (Fab'2LZ) and a full-length antibody, anti-tissue factor (anti-TF), from various stages throughout a biopharmaceutical production process to describe the assay details. The targeted proteins were captured on an affinity column containing an anti-light-chain (kappa) Fab antibody (AME5) immobilized on controlled pore glass. The affinity column was placed in-line with a reversed-phase column and the captured components were transferred by elution with dilute acid and subsequently resolved by eluting the reversed-phase column with a shallow acetonitrile gradient. Characterization of the resolved components showed that most antibody fragment preparations contained a light-chain fragment, free light chain, light-chain dimer and multiple forms of Fab'. Analysis of full-length antibody preparations also resolved these fragments as well as a completely assembled form. Co-eluting with the full-length antibody were high-molecular-mass variants that were missing one or both light chains. Resolved components were quantified by comparison with peak areas of similarly treated standards. By comparing the two-dimensional polyacrylamide gel electrophoresis patterns of an Escherichia coli blank run, a production run and the material affinity captured (AME5) from a production run, it was determined that the AME5 antibody captured isoforms of light chain, light chain covalently attached to heavy chain, and truncated light chain isoforms. These forms comprise the bulk of the soluble product-related fragments found in E. coli cell extracts of recombinantly produced antibody fragments.

Problems Associated with Crop Rotation for Management of Pratylenchus penetrans on Easter Lily.

In Humboldt and Del Norte counties of California and Curry County, Oregon, Easter lilies (Lilium longiflotum) are grown commercially in a 3- to 6-year rotation with pasture for cattle and sheep. Bulbs are sold to greenhouse operations to produce flowering plants. The lesion nematode, Pratylenchus penetrans, is a serious detriment to Easter lily production. Both soil and planting stock are often infested; typically, a dual nematicide application is used consisting of a preplant soil fumigation followed by an at-planting application of an organophosphate or carbamate. Nematicide usage has resulted in ground-water contamination. Several factors that could lead to an improved crop rotation program were examined in five field trials in Oregon. Examining the relative nematode host status of crops for feeding cattle and sheep indicated differences in host suitability among clovers and fescues that could prove useful in development of pasture mixes. Populations of P. penetrans under continuous fallow and pasture were monitored for 4 years following harvest of Easter lilies. Populations fluctuated in both situations but generally increased on pasture plants and decreased under fallow. Nematodes were still detectable at the end of 4 years of weed-free fallow. Populations of P. penetrans on Easter lilies were followed over two successive crops. Numbers in soil peaked in July and then decreased while numbers within roots continued to increase until harvest in October.

Management Options for Pratylenchus penetrans in Easter Lily.

Alternatives to reduce or modify nematicide use for minimizing groundwater contamination in Easter lily were explored in two field trials. Alternatives to standard 1,3-dichloropropene (1,3-D) plus phorate injection in the first trial were: (i) delaying applications until after winter rains, (ii) removing roots from planting stock, (iii) 1,3-D via drip irrigation, (iv) a chitin-urea soil amendment, (v) the registered insecticide disulfoton, and (vi) several nonregistered nematicides. None of the treatments equaled the standard treatment. In the second trial, potential benefits of adding a systemic nematicide, oxamyl (OX), or a fungicide, metalaxyl (MX), to the standard treatment were explored. Preplant drip irrigation applications of metam sodium (MS), sodium tetrathiocarbonate (ST), and emulsifiable 1,3-D were evaluated alone and in combination with postplant applications of OX and MX. Several drip-applied treatments performed comparably to the standard treatment with respect to the most important criteria of crop quality, bulb circumference. Metam-sodium in combination with either or both OX and MX, 1,3-D plus OX and MX, and ST plus OX and MX provided the best results.

Augmentation mammaplasty.

In the early 1900s, the best method for enlarging small breasts was to inject them with paraffin. Within 50 years, researchers turned to the free grafting of autogenous material to achieve breast enlargement. Then came the fast and easy silicone injections of the 1950s and 1960s. For the last 20 years, the surgical implantation of alloplastic materials has been used for augmentation mammaplasty. This article examines the development of augmentation mammaplasty, identifies the current procedures and looks at the possibility for overcoming the greatest complication, capsular contracture. The management of capsular contracture is difficult because it is the natural, enhanced scarring response of tissue that has been subjected to hosting a foreign body, the prosthesis. There is no simple solution to this problem, only the possibility that through manual manipulation of the augmented breast tissue, the intensity of the contracture can be minimized.

Expansion exercises: modifying contracture of the augmented breast.

Capsular contracture, the condition wherein fibrous tissue encapsulates the implanted silastic gel prosthesis used in breast augmentation, remains the most frequent complication in surgical enlargement of the small breast. Since the condition is a natural, wound-healing response to an implanted foreign body, management of the problem must be geared to the modification of this response. Expansion exercises, manual manipulation of the affected breast tissue for controlling capsule formation, was evaluated. Results showed that subjects who did not use expansion exercises had a higher incidence of capsular contracture than the subjects who did.

Forced migration and destination choice: Armenian forced settlers and refugees in the Russian Federation.

PIP: The Soviet Republic has experienced major population shifts between its 15 states since 1991. Large numbers of refugees and forced migrants are seeking asylum from successor states in Russia. The Federal Migration Service (FMS) of Russia keeps official registers of refugees and forced settlers, facilitates resettlement, and integrates migrants into society. This study examined the role of the FMS in resettling dislocated persons from Armenia to Russia. Data were obtained from official sources. 49.8% of immigrants to Russia come from the Central Republics. However, Armenia's refugee and forced migrant population is a larger share of its total population (about 5%, compared to 4.4% of Central Asian republics). A 1993 survey revealed that about 70% of the urban populations in Erevan, Gyumri, and Ashtarak would leave Armenia if the opportunity arose. 50% wanted their children to emigrate. In 1989 and 1993, the top receiving areas in Russia were Krasnodarskii Krai, Rostovskaia, and Stavropolskii Krai. In 1989, about 60% of the Armenian population in Russia lived in these territories. Ordinary least squares models indicate that 30% of the variance in Armenian resettlement in Russia, was predicted by high concentrations of Armenian residents and cost of living. Other structural factors, such as unemployment, urbanization, or new construction, were unrelated. Findings suggest that individual choice may be more important in determining residential location than migration policy.^ieng

Medium for the accumulation of extracellular hemolysin and protease by Aeromonas hydrophila.

A medium for the accumulation of extracellular hemolysin (300 to 1,600 hemolytic units per ml) and protease (2 to 3 proteolytic units per ml) was developed for an anaerogenic strain of Aeromonas hydrophila. In this medium, growth yields were less but levels of accumulated toxin were greater or equivalent when compared with the same responses in brain heart infusion and nutrient broths. The medium was considered to be partially defined since the conditions for maximum observed hemolysin accumulation (1,600 hemolysin units per ml) were not identified. The results showed that iron and zinc contributed to the control of the extracellular accumulation of both toxins. Whereas iron exerted an inhibitory effect, zinc stimulated the accumulation of both toxins.

Fetal cartilage xylosyltransferase activity and skeletal growth in sheep.

The activity of UDP-D-xylose:proteoglycan core protein beta-D-xylosyltransferase (EC 2.4.2.26), the enzyme that catalyzes the initiation of the polysaccharide chain linkage to the core protein of proteoglycans, was measured in costal cartilage from 20 fetal sheep of 65-138 days gestation. Activity of the enzyme was estimated from the transfer of [14C]xylose from UDP-[14C]xylose to silk as the acceptor protein. The specific activity decreased approximately 10-fold and was found to be highly correlated with the decremental rate of growth in length of the fetal vertebral column. These observations, together with the known gestational decrease in the in vitro rate of uptake of radiolabeled sulfate by ovine fetal cartilage, a subsequent step in proteoglycan synthesis, support the hypothesis that normal fetal skeletal growth is dependent during the last one-half of gestation on the activity of xylosyltransferase in cartilage.

Decreases in ovine fetal cartilage sulfate uptake and serum sulfate during gestation.

In vitro assays for [35S]sulfate uptake by ovine fetal costal cartilage were used to assess gestational changes in cartilage metabolism. Addition of 20% normal human serum to the incubation medium increased fetal cartilage [35S]sulfate incorporation into glycosaminoglycans. Both basal and human serum-stimulated uptakes of [35S]sulfate by fetal sheep cartilage decreased from midgestation to full term. The incremental response in [35S]sulfate uptake that was stimulated by human serum decreased as gestation proceeded to full-term. Fetal serum sulfate concentration decreased logarithmically during gestation, raising the possibility that cartilage sulfate uptake might become substrate limited as full term is approached. Perfusion of seven late gestation sheep fetuses for 7 days with Na2SO4 to achieve serum sulfate concentrations similar to those observed earlier in gestation resulted in a 33% increase in mean cartilage [35S]sulfate uptake compared with that of control twin fetuses, but uptake was not increased to values that occurred spontaneously earlier in gestation. These results suggest that the decreasing rate of [35S]sulfate uptake by fetal cartilage during the last half of gestation is associated only minimally with decreasing serum sulfate levels and is most consistent with intrinsic change in resting chondrocyte metabolism during gestation.

Epitope mapping of the human immunodeficiency virus type 1 gp120 with monoclonal antibodies.

A soluble form of recombinant gp120 of human immunodeficiency virus type 1 was used as an immunogen for production of murine monoclonal antibodies. These monoclonal antibodies were characterized for their ability to block the interaction between gp120 and the acquired immunodeficiency syndrome virus receptor, CD4. Three of the monoclonal antibodies were found to inhibit this interaction, whereas the other antibodies were found to be ineffective at blocking binding. The gp120 epitopes which are recognized by these monoclonal antibodies were mapped by using a combination of Western blot (immunoblot) analysis of gp120 proteolytic fragments, immunoaffinity purification of fragments of gp120, and antibody screening of a random gp120 gene fragment expression library produced in the lambda gt11 expression system. Two monoclonal antibodies which blocked gp120-CD4 interaction were found to map to adjacent sites in the carboxy-terminal region of the glycoprotein, suggesting that this area is important in the interaction between gp120 and CD4. One nonblocking antibody was found to map to a position that was C terminal to this CD4 blocking region. Interestingly, the other nonblocking monoclonal antibodies were found to map either to a highly conserved region in the central part of the gp120 polypeptide or to a highly conserved region near the N terminus of the glycoprotein. N-terminal deletion mutants of the soluble envelope glycoprotein which lack these highly conserved domains but maintain the C-terminal CD4 interaction sites were unable to bind tightly to the CD4 receptor. These results suggest that although the N-terminal and central conserved domains of intact gp120 do not appear to be directly required for CD4 binding, they may contain information that allows other parts of the molecule to form the appropriate structure for CD4 interaction.

Assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type 1 recombinant human immunodeficiency virus envelope glycoprotein (gp120) expressed in Chinese hamster ovary cells.

This report describes the structural characterization of the recombinant envelope glycoprotein (rgp120) of human immunodeficiency virus type 1 produced by expression in Chinese hamster ovary cells. Enzymatic cleavage of rgp120 and reversed-phase high performance liquid chromatography were used to confirm the primary structure of the protein, to assign intrachain disulfide bonds, and to characterize potential sites for N-glycosylation. All of the tryptic peptides identified were consistent with the primary structure predicted from the cDNA sequence. Tryptic mapping studies combined with treatment of isolated peptides with Staphylococcus aureus V8 protease or with peptide:N-glycosidase F followed by endoproteinase Asp-N permitted the assignment of all nine intrachain disulfide bonds of rgp120. The 24 potential sites for N-glycosylation were characterized by determining the susceptibilities of the attached carbohydrate structures to peptide:N-glycosidase F and to endo-beta-N-acetylglucosaminidase H. Tryptic mapping of enzymatically deglycosylated rgp120 was used in conjunction with Edman degradation and fast atom bombardment-mass spectrometry of individually treated peptides to determine which of these sites are glycosylated and what types of structures are present. The results indicate that all 24 sites of gp120 are utilized, including 13 that contain complex-type oligosaccharides as the predominant structures, and 11 that contain primarily high mannose-type and/or hybrid-type oligosaccharide structures.

Characterization of recombinant human factor VIII.

Recently, complete human factor VIII DNA clones have been obtained and subsequently expressed in baby hamster kidney cells (Wood, W. I., Capon, D. J., Simonsen, C. C., Eaton, D. L., Gitschier, J., Keyt, B., Seeburg, P. H., Smith, D. H., Hollingshead, P., Wion, K. L., Delwart, E., Tuddenham, E. G. D., Vehar, G. A., and Lawn, R. M. (1984) Nature 312, 330-337). The recombinant factor VIII (rVIII) protein secreted from these cells has now been purified allowing its structural analysis and comparison to plasma-derived factor VIII (pdVIII). Analysis of purified rVIII by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it consists of multiple polypeptides with relative mobilities (Mr) ranging from 80,000-210,000. The same pattern of polypeptides is also observed for pdVIII resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins associated with rVIII are recognized by pdVIII antibodies in a Western blot. When rVIII and pdVIII are subjected to isoelectric focusing they are resolved into a similar pattern of protein bands. Thrombin, factor Xa, and activated protein C, which modulate factor VIII activity by proteolysis, process rVIII in the same manner they do pdVIII. As is the case for pdVIII, thrombin activation of rVIII coagulant activity correlates with the generation of subunits with Mr of 73,000, 50,000 and 43,000. These subunits appear to form a metal-(perhaps Ca2+) linked complex. EDTA inactivates thrombin-activated rVIII and pdVIII, with the activity being regenerated after the addition of a molar excess of MnCl2. The results suggest that rVIII is structurally and functionally very similar to pdVIII.

Ontogeny of ovine fetal liver and kidney plasma membrane insulin receptors and fetal growth.

This investigation was performed to define certain characteristics of insulin-receptor interaction during the last 2 months of gestation in fetal sheep liver and kidney. Twenty-one sheep carrying a total of 46 fetuses were sacrificed at various gestational ages from 94 days to term; fetal and maternal livers and kidneys were analyzed by a radioreceptor assay for insulin binding characteristics. Specific binding of insulin to partially purified ovine fetal liver and kidney plasma membranes increased as gestation approached term, at which time specific binding was two- to fourfold greater to fetal than to maternal tissues. Associated with increased specific binding were late gestational increases in affinity of insulin for receptors in both fetal liver and kidney and an earlier increase in insulin receptor concentration in fetal kidney. These observations in fetal sheep liver and kidney are similar to reported observations in other species. However, the increase in specific binding of insulin to male fetal liver membranes was exponential; in contrast, there was no apparent increase in specific binding to female fetal liver membranes during the gestational interval surveyed. Both the weights and the vertebral column lengths of these fetuses were shown by multivariate analysis to be significantly affected by the interaction between specific binding of insulin and fetal sex. However, in 30 additional sheep fetuses we observed no difference between male and female fetuses in the increase with time in liver glycogen content. The lack of sex difference in this postreceptor event is consonant with the demonstrated dissociation between liver insulin receptors and glycogen synthesis in the late fetal rat. Our observations suggest that late gestational differences between male and female sheep fetuses in insulin specific binding to liver and, possibly, to other tissues such as cartilage, muscle, and/or fat, that are coupled to postreceptor events may account for differences in fetal growth between the sexes.