Appearance of purine-catabolizing enzymes in fix and fix root nodules on soybean and effect of oxygen on the expression of the enzymes in callus tissue

The appearance of enzymes involved in the formation of ureides, allantoin, and allantoic acid, from inosine 5′-monophosphate was analyzed in developing root nodules of soybean (Glycine max). Concomitant with development of effective nodules, a substantial increase in specific activities of the enzymes 5′-nucleotidase (35-fold), purine nucleosidase (10-fold), xanthine dehydrogenase (25-fold), and uricase (200-fold), over root levels was observed. The specific activity of allantoinase remained constant during nodule development. With ineffective nodules the activities were generally lower than in effective nodules; however, the activities of 5′-nucleotidase and allantoinase were 2-fold higher in ineffective nodules unable to synthesize leghemoglobin than in effective nodules. Since the expression of uricase has been shown to be regulated by oxygen (K Larsen, BU Jochimsen 1986 EMBO J 5: 15-19), the expression of the remaining enzymes in the purine catabolic pathway were tested in response to variations in O₂ concentration in sterile soybean callus tissue. Purine nucleosidase responded to this treatment, exhibiting a 4-fold increase in activity around 2% O₂. 5′-Nucleotidase, xanthine dehydrogenase, and allantoinase remained unaffected by variations in the O₂ concentration. Hence, the expression of two enzymes involved in ureide formation, purine nucleosidase and uricase, has been demonstrated to be influenced by O₂ concentration.     

Expression of nodule-specific uricase in soybean callus tissue is regulated by oxygen

In soybean root nodules the enzyme uricase is expressed concomitantly with nodule development. The initial expression of this protein does not depend on active nitrogen fixation, as demonstrated by analysis of uricase activity in effective and ineffective root nodules. However, the maximal level of uricase activity is determined by the infecting Rhizobium japonicum strain. Sterile root cultures and callus tissue, devoid of the microsymbiont, were incubated at varying oxygen concentrations and analyzed for uricase activity. The specific activity of uricase was increased by lowering the oxygen concentration, with the highest activity obtained around 4−5% oxygen. The increase in uricase activity was due to increased uricase synthesis, as demonstrated by in vivo labelling of callus culture followed by immunoprecipitation with antibodies raised against highly purified nodule uricase.     

岷江百合类萌发素蛋白基因LrGLP2的克隆及表达特性分析

类萌发素蛋白是植物中普遍存在的一类可溶性糖蛋白,在植物抗逆胁迫中起着重要作用。依据岷江百合编码GLP的EST序列设计引物,采用快速扩增cDNA末端技术,从岷江百合犯ilium regale Wilson）克隆得到一个新的GLＰt基因的全长eDNA序列,命名为LrGLP2。LrGLP2全长cDNA为921bp,含有654bp的开放阅读框,49bp5’非编码区以及218bp3’UTR,编码217个氨基酸的蛋白质。LrGLP2编码蛋白质与已知植物GLPs家族成员间的同源性和聚类分析表明LrGLP2与来源于水稻（Oryza sativa）、节节麦似egilopstauschii）、葡萄（Vitis vinifera）中的GLPs具有较高的相似性。qRT-PCR分析显示,LrGLP2在岷江百合正常生长发育的根中有一定量的表达,而在茎和叶中几乎检测不到表达量。水杨酸、茉莉酸以及H202处理均不同程度抑制LrGLP2的转录水平,但乙烯处理能明显诱导LrGLP2的表达。此外,岷江百合接种尖孢镰刀菌（Fusari—umoxysporumfsp．tiliO后,LrGLP2在接种后2h表达迅速上调,12h表达量急剧上升,至24h表达量达到最大值,之后表达量下降,可见￡rGLP2参与岷江百合对尖孢镰刀菌的防卫反应。     

Interaction of Schiff base with bovine serum albumin: site-specific photocleavage

A Schiff-base ligand with donor/acceptor substituents viz. 2, 3-bis?[(2-hydroxy-4-diethylamino) (phenyl) (methylene)]amino?-2-butenedinitrile was synthesized, its binding properties with bovine serum albumin (BSA) and its site-specific photocleavage in the presence of cobaltous chloride have been evaluated. The Schiff-base ligand showed increase in absorption with a 5-nm red shift in the absorption maximum consistent with the binding of Schiff-base ligand to hydrophobic sites on the protein. The binding plot obtained from the absorption titration gives a binding constant of 6.4 +/- 0.3 x 10(4) M(-1). The CD spectrum of BSA in presence of the ligand shows that binding of the ligand leads to a change in the helicity of the protein. This ligand has been found to induce site-specific photocleavage of the protein in the presence of cobaltous chloride. The gel electrophoresis pattern of a photolyzed sample of BSA/Schiff-base ligand/cobaltous chloride shows that protein is cleaved into two polypeptide fragments, indicating site-specific binding for the ligand to the protein.     

Application of monoclonal antibody, specific for intracellular Orientia tsutsugamushi, to immunofluorescent antibody test for determining antibiotic susceptibility

The simple quantification of viable intracellular bacteria is important for the study of an obligate intracellular bacterium, Orientia tsutsugamushi. We applied a novel monoclonal antibody (M686-13)--specific for intracellular Orientia--to an immunofluorescent antibody (IFA) test for determining antibiotic susceptibility of O. tsutsugamushi. M686-13 did not react with Orientia that was inhibited by doxycycline, although bacterial particles still remained in the cells. This preferential staining of proliferating bacteria made the IFA test rapid and precise. Using this method, we could successfully measure the minimal inhibitory concentration (MIC) of a Korean strain of O. tsutsugamushi to doxycycline and clindamycin. This method may be used in other procedures to evaluate the growth of Orientia.     

Interaction of aldehydes with collagen: effect on thermal, enzymatic and conformational stability

Stabilization of type I rat tail tendon (RTT) collagen by various aldehydes, viz. formaldehyde, gluteraldehyde, glyoxal and crotanaldehyde was studied to understand the effect of each on the thermal, enzymatic and conformational stability of collagen. The aldehydes have been found to increase the heat stability of rat tail tendon collagen fibres from 62 to 77-86 degrees C. The increase in thermal stability was found to be in a species dependent manner. The variation in the thermal stability of collagen brought about by aldehydes was in the order of formaldehyde > gluteraldehyde > glyoxal > crotanaldehdye. The aldehydes also impart a high degree of stability to collagen against the activity of the degrading enzyme, collagenase. The order of enzymatic stability brought about by aldehydes follows the same trend as the thermal stability brought about by them. This shows that the number of cross-links formed influence both the thermal and enzymatic stability in the similar manner. The effect of various aldehydes on the secondary structure of collagen was studied using circular dichroism and it was found that the aldehydes lead to changes in the amplitude of the circular dichroic (CD) spectrum but did not alter the triple helical conformation of collagen. The secondary structure of collagen is not significantly altered on interaction with different aldehydes.     

Fluorescence resonance energy transfer from tryptophan to a chromium(III) complex accompanied by non-specific cleavage of albumin: a step forward towards the development of a novel photoprotease

A chromium(III) complex, transdiaqua [N, N'-propylenebis(salicylideneimino)chromium(III)]perchlorate ([Cr(salprn)(H2O)(2)]ClO(4)) in the presence of sodium azide and upon photoexcitation was found to bring about non-selective cleavage of bovine serum albumin (BSA). Electron paramagnetic resonance (EPR) evidence has been obtained for the formation of a Cr(V) species upon photolysis of a solution containing the chromium(III) complex and sodium azide. This Cr(V) species non-selectively cleaves BSA. The fluorescence excitation spectrum of BSA-[Cr(salprn)(H2O)(2)]+ adduct showed a band at lambda(max)(ex) nm due to charge transfer transition of the chromium(III) complex as well as a prominent band at 290 nm attributable to tryptophan absorption. This indicated an efficient Forster type fluorescence energy transfer (FRET) from the tryptophan residues to the chromium(III) complex indicating that the Cr(III) complex binds in the vicinity of the tryptophan residue.     

A Schiff base complex of chromium(III): an efficient inhibitor for the pathogenic and invasive potential of Shigella dysenteriae

A Schiff base complex of chromium(III), transdiaqua[N,N'ethylenebis (salicylideneimine)chromium(III)]perchlorate, [Cr(salen)(OH(2))(2)](+), was found to have an inhibitory effect on the growth of Shigella dysenteriae. The chromium(III) complex was found to cure (remove) the invasive plasmid and thereby render the microbe more sensitive to the tested antibiotics. The loss in the catalytic activity of the isolated endo-alpha-N-acetyl galactosaminidase on mucin as a substrate was also observed in the presence of [Cr(salen)(OH(2))(2)](+). This suggests that [Cr(salen)(OH(2))(2)](+) is toxic to the microbe and could make the microbe non-pathogenic and non-invasive, thus establishing its role in microbiological applications to reduce the toxic potentials of a microbe.     

Progress and recent trends in biotechnological methods for leather processing

Global environmental regulations are changing the leather-processing industry. Pre-tanning and tanning processes contribute 80-90% of the total pollution in the industry and generate noxious gases, such as hydrogen sulfide, as well as solid wastes, such as lime and chrome sludge. The use of enzyme-based products is currently being explored for many areas of leather making. Furthermore, enzymes are gaining increasing importance in the de-hairing process, eliminating the need for sodium sulfide. This review discusses emerging novel biotechnological methods used in leather processing. One significant achievement is the development of a bioprocess-based de-hairing and fiber-opening methodology to reduce toxic waste.