Chromatographic methods for amylases

This review surveys recent developments in chromatographic methods for the separation of amylases from complex extracts, including the separation of isozymes. It contains two tables with the properties and molecular characteristics of α- and β-amylases from different sources as well as an updated review of methods for the determination of amylase activity. The main subject of this review is a detailed evaluation of the application of newly developed chromatographic methods for the purification of amylases.     

Combined genetic and physiological analysis of a locus contributing to quantitative variation

The natural variation of many traits is controlled by multiple genes, individually referred to as quantitative trait loci (QTL), that interact with the environment to determine the ultimate phenotype of any individual. A QTL has yet to be described molecularly, in part because strategies to systematically identify them are underdeveloped and because the subtle nature of QTLs prevents the application of standard methods of gene identification. Therefore, it will be necessary to develop a systematic approach(es) for the identification of QTLs based upon the numerous positional data now being accumulated through molecular marker analyses. We have characterized a QTL by the following three-step approach: (1) identification of a QTL in complex populations, (2) isolation and genetic mapping of this QTL in near-isogenic lines, and (3) identification of a candidate gene using map position and physiological criteria. Using this approach we have characterized a plant height QTL in maize that maps to chromosome 9 near the centromere. Both map position and physiological criteria suggest the gibberillin biosynthesis gene dwarf3 as a candidate gene for this QTL.     

Purification and properties of DNA topoisomerase II from Daucus carota cells

Topoisomerase II was partially purified from Daucus carota cellsby a procedure including ammonium sulphate fractionation, ion-exchange,and affinity chromatography steps. The type II enzyme, identifiedfor its ability to unknot knotted P4 DNA and decatenate Trypanosomacruzi kDNA, requires ATP and Mg²⁺ for activity. The unknottingactivity was sensitive to an inhibitor of the mammalian typeII enzyme, the drug VP16 (IC₅₀ 32 mmol m^–3), whereas inhibitorsof DNA gyrase showed a limited effect on activity. The SDS-PAGEanalysis of the dsDNA cellulose fraction revealed the presenceof four polypeptides of apparent molecular masses of 72, 71,34, and 33 kDa among which only a polypeptide of about 70 kDacrossreacted with antibodies against yeast topoisomerase II.Immunoprecipitation experiments with monoclonal antibodies tothe and ß isoforms of the human enzyme confirmedthe recognition of a polypeptide of 70 kDa. The sedimentationcoefficient (S) of the topoisomerase II in the phosphocellulosefraction, calculated by analytical glycerol gradient, was 6.1corresponding to a molecular mass of about 123 kDa. Resultssuggest the presence in carrot of a protein of molecular massof 70 kDa having the typical properties of an eukaryotic topoisomeraseII and carrying epitopes recognized by MoAbs to both human and ß enzymes. The 70 kDa polypeptide might then representthe monomer of a homodimer enzyme of 123 kDa. Key words: Daucus carota, topoisomerase II, immunoprecipitation     

Modulated red blood cell survival by membrane protein clustering

Human and murine blood cells treated with ZnCl₂ and bis(sulfosuccinimidyl)suberate (BS³) (a cross linking agent) undergo band 3 clustering and binding of hemoglobin to red blood cell membrane proteins. These clusters induce autologous IgG binding and complement fixation, thus favouring the phagocytosis of ZnCl₂/BS³ treated cells by macrophages. The extension of red blood cell opsonization can be easily modulated by changing the ZnCl₂ concentration in the 0.1–1.0 mM range thus providing an effective way to affect blood cell recognition by macrophages. In fact, murine erythrocytes treated with increasing ZnCl₂ concentrations have proportionally reduced survivals when reinjected into the animal. Furthermore, the organ sequestration of ZnCl₂/BS³ treated cells strongly resembles the typical distribution of the senescent cells. Since the ZnCl₂/BS³ treatment can also be performed on red blood cells loaded with drugs or other substances, this procedure is an effective drug-targeting system to be used for the delivery of molecules to peritoneal, liver and spleen macrophages.     

Modulation of cisPlatin cytotoxicity by interleukin-1α and resident tumor macrophages

The modulation of cisPlatin cytotoxicity by interleukin-1 (IL-1α) was studied in cultures of SCC-7 tumor cells with and without tumor macrophages to examine potential mechanisms for the synergistic antitumor activity of cisPlatin and IL-1α in SCC-7 solid tumors. Neither IL-1α nor tumor macrophages affected the survival of clonogenic tumor cells and IL-1α had no direct effect on tumor cell growthin vitro. Macrophages had no direct effect on cisPlatin sensitivity (IC₉₀=6.0 μM), but, the addition of IL-1α (500–2000U/ml) to co-cultures of cisPlatin pretreated tumor cells and resident tumor macrophages increased cell killing (IC₉₀=3.1 μM). Similar responses were seen in primary cultures treated with cisPlatin before IL-1α. The modulation of cisPlatin cytotoxicity by IL-1α exhibited a biphasic dose response that paralleled the IL-1α dose dependent release of H₂O₂by resident tumor macrophages. Further, IL-1α modification of cisPlatin cytotoxicity was prompt and inhibited by catalase. CisPlatin and exogenous H₂O₂ (50 μM) produced more than additive SCC-7 clonogenic cell kill and hydroxyl radicals played an important role in the response. Interleukin-1 modulation of cisPlatin cytotoxicity was schedule dependent. IL-1α treatment for 24 hrs, before cisPlatin, produced drug resistance (IC₉₀=11.1 μM). Our study shows that IL-1α can stimulate tumor macrophages to release pro-oxidants that modify cellular chemosensitivity in a schedule and dose dependent fashion. Our findings may also provide a mechanistic explanation for the synergistic antitumor activity of cisPlatin and IL-1αin vivo.     

Ecology, nutrition, and timing of reproductive events in an Asian primate, the Hanuman langur (Presbytis entellus)

Ecological conditions, feeding and ranging behaviour as well as physical condition of female Hanuman langurs ( Presbytis entellus ) were studied in order to analyse the major influences on the timing of reproductive events. The langurs inhabited a semi-evergreen forest dominated by Sal trees ( Skorea robusta ). The climate was dominated by the summer monsoon lasting from May to September. Availability of plant phytophases was markedly seasonal, with fruiting as well as flushing of the six main food plants during the monsoon. Feeding and ranging behaviour of the females varied with food availability and its distribution. High dietary quality was only obtained during a short period from May to July. Physical condition of the females varied in accordance with dietary quality and energy expenditure, with a good physical condition during the second half of the year. Births occurred from January until June, with most births around the energetically worst period of the year. The results indicate that, in contrast to several other non-human primates, most females of this population gave birth too early to meet the optimum lactation requirements. Complete weaning mainly took place during an energetically bad time of the year (October to December). However, it can be inferred that the reduction of suckling bouts prior to the next conception took place when high quality food was available. Conceptions were confined to the months of July to November, coinciding with the time of best physical condition. Moreover, females who conceived were in a better physical condition than those who failed to conceive, suggesting a primary influence of nutrition on the probability of conception. Seasonality and timing of births may be the result of a regular seasonal shortfall of resources influencing the ability of the females to ovulate and to conceive.