Crystallization and preliminary X-ray crystallographic analysis of spruce budworm antifreeze protein.

Antifreeze proteins have the ability to bind to ice with high affinity and inhibit further crystal growth. The insect antifreeze protein from spruce budworm exhibits very high thermal hysteresis activity and is implicated in the protection of overwintering larvae from freezing. This protein has been crystallized in 20-25% polyethylene glycol (Mr 6000), 0.4 M NaCl, 0.1 M Tris-HCl, pH 8.5, by vapor diffusion using the hanging drop method. The resulting crystals are very thin (typically <0.01 mm in the shortest dimension), and only after repeated seeding could crystals be grown large enough for data collection using synchrotron radiation. The crystals belong to the monoclinic space group C2, with cell dimensions a = 82.28 A, b = 62.29 A, c = 63.63 A, and beta = 113.7 degrees. Molecules in the asymmetric unit are related by a twofold axis of symmetry with two molecules present. Native data to a resolution of 2.6 A have been collected with 90.3% completeness and a Rsym of 6.9%.     

Intra- and extracellular forms of alpha-glucosidase from Aspergillus niger.

An intracellular α-glucosidase (α-glu₁) of Aspergillus niger was purified and its properties were compared to those of a secreted α-glucosidase (α-glu_E). The estimated molecular weight of α-glu_I was 95,000 by gel filtration (α-glu_E = 63,000); it is a glycoprotein possessing 29 mol of mannose, 6 mol of glucosamine, and 14 mol of glucose (α-glu_E has 5–6 and 2 mol of mannose and glucosamine, respectively). The K_m′s of α-glu₁ for p-nitrophenyl-α-d-glucopyranoside and maltose were 1.49 and 1.04, respectively, slightly lower than those of α-glu_E. In addition, at 65 °C α-glu_I enzymatic activity decayed fivefold faster than that of α-glu_E, and anti-α-glu_E antibody did not recognize α-glu_I. While some of these distinctions between the enzymes could be ascribed to conformational differences, the great dissimilarity in molecular weight (approximately 32,000) and lack of reactivity with anti-α-glu_E argue against α-glu_I being related to α-glu_E. The antibody covalently coupled to horseradish peroxidase (Ab-Px) was used as a probe to determine the cellular location of α-glu_E by electron microscopic immunocytology. It was found on both sides of the plasma membrane (pm) and in the outer of the two layers of the cell wall. This may mean that α-glu_E is synthesized at the inner surface of the pm, is extruded through the pm, becomes associated with the outer layer of the cell wall (perhaps as enzyme—substrate complex), and is eventually released into the growth medium.     

The vascular endothelium as a target tissue in acute cadmium toxicity

Sensitivity of tissues to the acute toxicity of cadmium is reviewed. It is concluded that the initial effect of acute cadmium administration is on the integrity and permeability of the vascular endothelium; other necrotic changes occur secondarily to this effect. In a sensitive tissue, not all of the endothelial cells are susceptible to cadmium. Furthermore, after necrosis of the sensitive cells, the resistant cells proliferate and result in regeneration of the vasculature and subsequent acquired resistance to the metal. It is found that sex hormones are probably important in determining susceptibility and response of tissues to cadmium. The role of metallothionein in these phenomena remains to be elucidated.     

Partial purification of two forms of Choline kinase and separation of Choline kinase from sphingosine kinase of rat brain

Choline kinase of rat brain was purified approximately 200,000 fold using acid precipitation, ammonium sulphate fractionation, Q-Sepharose, Octyl-Sepharose and AH-Sepharose chromatography. The ability of this enzyme to catalyze the phosphorylation of choline, ethanolamine (Etn), monomethylethanolamine (MeEtn), dimethylethanolamine (Me₂Etn) and sphingosine was investigated. Choline kinase was separated from sphingosine kinase. The fraction with highly purified choline kinase had four major polypeptides with different molecular masses and possessed activities towards choline, Etn, MeEtn and Me₂Etn. Two forms of choline kinase were obtained when the enzymatically active fractions eluted from the Q-Sepharose column were subjected to a horizontal isoelectrofocusing electrophoresis. One form focused around pH 4.7 and is able to phosphorylate choline, Etn, MeEtn and Me₂Etn. The other form focused around pH 10 and possessed only choline kinase activity. The latter form of choline kinase did not display classical Michaelis-Menten's mechanism but revealed a positive co-operative pattern for two choline binding sites. This form was purified to apparent homogeneity with a approximate molecular mass of 14.4 kDa.Abbreviations Etn ethanolamine - MeEtn N-monomethylethanolamine - Me₂Etn N, N-dimethylethanolamine     

Investigation of Sexual Process in Interspecific Crosses Between Gossipium hirsutum and G. arboreum

The present investigation is the first of a series of cytological researches on inter-specific hybridization of cottons. The present report deals with the cytological behavior of sex and related cells revealed in crossing Gossipium hirsutum (female parent) with G. arboreum. Self-pollenated G. hirsutum served as control. The germination of pollen grains on the stigma and the growth of pollen tubes in the style and the entry of pollen tubes into the micropyles in crosses are in a fairly normal' manner when compared with controls. The percentages of pollen tubes entering ovules of the crossed and controls have no significant difference, being 48.2% in crosses and 54.7% in controls. Fertilization proceeds also normal in cross pollinated ovules. About 92.7% of pollinated ovules are found to have completed the fusion of sexual elements. It is thus: shown that the growth of foreign pollen tubes and fusion of female elements with foreign sperms are not the factors which cause failure in hybridization. The development of endosperm in crosses is abnormal. The number of free nuclei of hybrid endosperm is much smaller than that of controls; the formation of hybrid cellular endosperm is 6 or 7 days earlier than that of controls. The hybrid endosperm cells start to abort soon after their formation. About 14 days after pollination they become completely disintegrated. The hybrid embryo which thus far developed normally becomes arrested in its development and aborts also after that time. It may be concluded that the sterility of crosses between G. hirsutum and G. arboreum is primarily attributed to the abortion of hybrid endosperm. The sound development of the hybrids should be sought in the improvement nutrition of the hybrid embryo from resource other than their own endosperm.