Changing distribution patterns of α-glucosidase isoenzymes in extracts of antennal segments and in the haemolymph of the cotton stainer Dysdercus intermedius dist.

• 1.1. The α-glucosidase (E.C. 3.2.1.20) activities in extracts of the 2nd and 4th antennal segments (AS) and in the haemolymph of the cotton stainer were quantitatively examined by use of trehalose, p-nitrophenyl-α-glucoside, sucrose, turanose, maltose, isomaltose and palatinose as substrates.
• 2.2. Three peaks of activity were isolated by gel filtration from the extracts of both AS, but only two peaks from the haemolymph.
• 3.3. The peaks III of both AS differ in their substrate specificities from those of the peaks I and II.
• 4.4. It was evident from experiments, which used fractional extraction and variation of pH in addition to gel filtration that more than a single enzyme contributed to the formation of each peak.
• 5.5. Results are discussed with reference to the hypothesized presence of sugar reception processes in insect gustatory cells.

     

Role of the ubiquitin-binding domain of Polη in Rad18-independent translesion DNA synthesis in human cell extracts

In eukaryotic cells, the Rad6/Rad18-dependent monoubiquitination of the proliferating cell nuclear antigen (PCNA) plays an essential role in the switching between replication and translesion DNA synthesis (TLS). The DNA polymerase Polη binds to PCNA via a consensus C-terminal PCNA-interacting protein (PIP) motif. It also specifically interacts with monoubiquitinated PCNA thanks to a recently identified ubiquitin-binding domain (UBZ). To investigate whether the TLS activity of Polη is always coupled to PCNA monoubiquitination, we monitor the ability of cell-free extracts to perform DNA synthesis across different types of lesions. We observe that a cis-syn cyclobutane thymine dimer (TT-CPD), but not a N-2-acetylaminofluorene-guanine (G-AAF) adduct, is efficiently bypassed in extracts from Rad18-deficient cells, thus demonstrating the existence of a Polη-dependent and Rad18-independent TLS pathway. In addition, by complementing Polη-deficient cells with PIP and UBZ mutants, we show that each of these domains contributes to Polη activity. The finding that the bypass of a CPD lesion in vitro does not require Ub-PCNA but nevertheless depends on the UBZ domain of Polη, reveals that this domain may play a novel role in the TLS process that is not related to the monoubiquitination status of PCNA.     

CpG motifs: the active ingredient in bacterial extracts?

The use of bacteria and bacterial extracts for immunotherapy has a checkered past. Recent developments in immunology reveal that these nonspecific immune activators actually work by triggering specific receptors that are expressed by subsets of immune cells. Identification of these receptors and the molecular signaling pathways that they activate has enabled a new era of specific targeted immunotherapy using chemically synthesized mimics of pathogen molecules.     

Fimbria–fornix (FF)-transected hippocampal extracts induce the activation of astrocytes in vitro

Hippocampus is one of the neurogenesis areas in adult mammals, but the function of astrocytes in this area is still less known. In our previous study, the fimbria–fornix (FF)-transected hippocampal extracts promoted the proliferation and neuronal differentiation of radial glial cells in vitro. To explore the effects of hippocampal extracts on gliogenesis, the hippocampal astrocytes were treated by normal or ff-transected hippocampal extracts in vitro. The cells were immunostained by brain lipid-binding protein (BLBP), nestin, and SOX2 to assess their state of activation. The effects of astrocyte-conditioned medium on the neuronal differentiation of hippocampal neural stem cells (NSCs) were also investigated. After treatment of FF-transected hippocampal extracts, the number of BLBP, nestin, and Sox-positive cells were obviously more than the cells which treated by normal hippocampal extracts, these cells maintained a state of activation and the activated astrocyte-conditioned medium also promoted the differentiation of NSCs into more neurons. These findings suggest that the astrocytes can be activated by FF-transected hippocampal extracts and these activated cells also can promote the neuronal differentiation of hippocampal NSCs in vitro.     

Studies of the orotidine 5′-monophosphate decarboxylase activity of crude extracts of human cells

The specific activity of orotidine 5-monophosphate (OMP) decarboxylase in cultured human fibroblasts is an exponential function of the concentration of cell protein in the extract. Low concentrations of uridylic or cytidylic acid augment the catalytic activity of dilute solutions of cell extract but not of concentrated ones. In the presence of urea, specific activity becomes independent of protein concentration, and uridylic or cytidylic acid augments activity over all concentrations of cell extract. These results, as well as other observations, suggest that the decarboxylase may be composed of subunits which are in dynamic equilibrium with an aggregate. At least one of the subunits is likely to have catalytic activity for the reaction, though less activity than the aggregate. The effect of the mutant gene for orotic aciduria on OMP decarboxylase is easily demonstrated when cell extracts are assayed in either the presence or the absence of urea.Supported by Program Project Grants 1-PO1-GM 15419 and GM 18153-1, National Institutes of Health, United States Public Health Service.     

Characterization of 16α-hydroxyprogesterone dehydroxylase in cell extracts of the intestinal anaerobic bacterium,Eubacterium sp. 144

Eubacterium sp. strain 144 is an intestinal anaerobic bacterium which catalyzes a two-step biotransformation of 16 α-hydroxy progesterone to 17-isoprogesterone. 16α-Hydroxyprogesterone dehydroxylase (16α-dehydroxylase) catalyzes the first reaction, yielding Δ¹⁶-progesterone. The present study examined properties of the 16α-dehydroxylase in cell extracts of strain 144. The enzyme exhibited a broad pH range (5.8–8.0) without a distinct pH optimum. 16α-Dehydroxylase was active at high (15–30% v/v) methanol concentrations and a distinct optimum occurred at 25% (v/v). The enzyme was less active with ethanol or short-chain glycols and was inhibited by propanol and butanol. Substrates included 16α-hydroxyprogesterone, 16α-hydroxypregnenolone and Δ¹⁶-progesterone; however, the specific activity with the latter steroid was only 15% of that obtained with the 16α-hydroxysteroids. The substrate saturation curve for 16α-hydroxyprogesterone was hyperbolic and a Lineweaver-Burk plot of the data was linear with an apparent K_m of 0.56 ± 0.08 mM. 16α-Dehydroxylase was stable at 60°C for 10 min but was inactivated (70%) at 65°C. An M_r of 285 000 was estimated for 16α-dehydroxylase by gel permeation HPLC of strain 144 cell extract.     

A procedure for the Cherenkov counting of high energy β and γ emitting radionuclides in coloured plant extracts

Summary Elimination of count rate variations caused by the absorption of Cherenkov radiation by pigments in coloured solutions was achieved by placing these solutions in opaque walled cylinders immersed in colourless liquids contained in standard counting vials. Good counting efficiencies for a range of and emitting radionuclides were obtained by selecting colourless liquids with high refractive index.     

Algae extracts and methyl jasmonate anti-cancer activities in prostate cancer: choreographers of ‘the dance macabre’

Background

GSAO (4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid) and PENAO (4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid) are tumour metabolism inhibitors that target adenine nucleotide translocase (ANT) of the inner-mitochondrial membrane. Both compounds are currently being trialled in patients with solid tumours. The trivalent arsenical moiety of GSAO and PENAO reacts with two matrix facing cysteine residues of ANT, inactivating the transporter. This leads to proliferation arrest and death of tumour and tumour-supporting cells.

Results

The two reactive ANT cysteine residues have been identified in this study by expressing cysteine mutants of human ANT1 in Saccharomyces cerevisiae and measuring interaction with the arsenical moiety of GSAO and PENAO. The arsenic atom of both compounds cross-links cysteine residues 57 and 257 of human ANT1.

Conclusions

The sulphur atoms of these two cysteines are 20 Å apart in the crystal structures of ANT and the optimal spacing of cysteine thiolates for reaction with As (III) is 3-4 Å. This implies that a significant conformational change in ANT is required for the organoarsenicals to react with cysteines 57 and 257. This conformational change may relate to the selectivity of the compounds for proliferating cells.     

About paired correlation of π-electrons in tissue lipid extracts of animals of different evolutionary levels

With aid of optical methods, the presence of the paired correlations of π-electrons has been revealed in phospholipids as well as in triacylglyceride molecules. Used for analysis were lipid extracts of individual representatives of animals of various evolutionary levels—cartilaginous and bony fish and mammals differing by the content of unsaturated fatty acids in lipids. It has been established that the necessary condition for formation of electron pairs is interaction of lipid molecules with each other. An opinion is put forward that in the liquid crystal structure of the membrane monolayer there are two zones able to form electron pairs—the zone of location of ester bonds and the zone in the region of double bonds. Besides, the paired correlation in the phospholipid molecule electron system is accompanied by the absence of electric resistance of the membrane monolayer, which provides the monolayer superconductivity at low rates of movements of the “electron fluid.” It is to be noted that the very fact of the presence of the electron pair implies transfer of energy by small portions, which does not allow excitation of individual phospholipid molecules in the monolayer and promotes stability of the native membrane. Our data agree with the known statement of A. Pulman and B. Pulman that the life dynamicity is determined by dynamicity of the electron cloud in coupled or partially coupled systems.     

β-Carotene biosynthesis by extracts of the C115 mutant of Phycomyces blakesleeanus

A cell extract of the yellow C115 car-42 mad-107(?) mutant of Phycomyces blakesleeanus, capable of converting MVA-[2-¹⁴C] into isoprenoids, was used to investigate the formation of β-carotene. The incorporation of radioactivity into β-carotene was reduced by the addition of unlabelled carotenes, solubilised using detergent, to the incubation mixtures. On reisolation of these carotenes after anaerobic incubations, they were found to carry radioactivity. The relative efficiencies of these carotenes as trapping agents are discussed in relation to the pathways of carotene cyclisation and to the apparent operation of a system for the negative feedback control of carotene biosynthesis.     

Comparison of the effects of cereal and legume proteinaceous seed extracts on α-amylase activity and development of the Sunn pest

The Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), is a significant limiting factor in the production of wheat and barley in many areas of the world. In the current study, the effect of semi-purified proteinaceous extracts of seeds on digestive enzymes, and the growth and development of the Sunn pest were studied. The results showed that the purified α-amylase inhibitor from Triticum aestivum (type І) and rice semi-purified seed extract did not significantly affect the Sunn pest α-amylase activity. However, bean and cowpea seed extracts significantly affected α-amylase activity in vitro. For example, the bean seed extract at concentrations of 0.125 and 2.0 mg · mL^− 1 inhibited α-amylase activity of the pest by 15% and 45%, respectively, while the cowpea seed extract, at the same concentrations, inhibited α-amylase activity of the pest by 9% and 40%, respectively. Further, incorporation of the seed extracts into the insect diet showed that the rice seed extract did not affect insect development time, while bean and cowpea seed extracts at high concentrations (e.g., 3.0%) significantly affected nymphal development time and survivability (P > 0.05). These results show that semi-purified seed extracts affect α-amylase activity, developmental time, and survivability but not the adult weight of the Sunn pest.     

Biosynthesis of peptides containing α-aminoadipic acid and cysteine in extracts of a Cephalosporium sp

1. Three intracellular peptides found in small amount in a Cephalosporium sp. were rapidly labelled when dl-[(14)C]valine was added to a shaken suspension of the organism. More (14)C was incorporated into peptide P3, delta-(l-alpha-aminoadipyl)-l-cysteinyl-d-valine, than into peptide P2 (containing alpha-aminoadipic acid, cysteine, valine and glycine) or peptide P1 (containing beta-hydroxyvaline in place of the valine in peptide P2). 2. Peptides P3 and P2, but not peptide P1 were formed in a broken-cell system from the Cephalosporium sp. in the presence of delta-(l-alpha-aminoadipyl)-l-cysteine and dl-[(14)C]valine. No synthesis was observed in the presence of delta-(d-alpha-aminoadipyl)-l-cysteine or of dl-alpha-amino[(14)C]adipic acid and l-cysteinyl-l-valine or l-cysteinyl-d-valine. 3. The biosynthesis of these peptides was catalysed by the particulate fraction of the broken-cell system, whereas that of glutathione was catalysed by the supernatant fraction. 4. These results are discussed in relation to penicillin N and cephalosporin C biosynthesis.     

Antifeedant effects of in vitro culture extracts of the neem tree,Azadirachta indica against the desert locust (Schistocerca gregaria (Forskål))

Callus and micropropagated shoots were initiated from leaf explants of the neem tree, Azadirachta indica A. Juss. A variety of whole plant and in vitro cell cultures from neem seedlings of Ghanian origin were tested for insect antifeedant compounds using the desert locust (Schistocerca gregaria (Forskål)). Feeding suppression occurred when whole extracts of seed, leaf, callus, suspension and shoot cultures were tested in no-choice feeding bioassays. Controls of sucrose, carrot callus and the plant growth medium showed no feeding deterrence. Azadirachtin, the main known antifeedant in neem seed kernels, was quantified from a seed extract by HPLC but was not detected in any of the other extracts. Antifeedancy was determined during batch growth of a suspension culture which had been in culture for 5 months; results indicated that antifeedants were still being formed and that levels increased after maximum biomass was attained.     

Specific detection of α-amylase activity in crude plant extracts after isoelectric focusing

A new method which utilizes Procion Red MX 2B amylopectin for the detection of α-amylase in crude plant extracts is described. The substrate is specific only against α-amylase hydrolysis and β-amylase does not attack it. Paper containing Procion Red MX 2B amylopectin applied to gels after isoelectric focusing reveals α-amylase isoenzymes as white bands. When this technique is used, heat-inactivation of β-amylase is not required.     

Nuclear assembly of purified Crythecodinium cohnii chromosomes in cell—free extracts of Xenopus laevis eggs

Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation,nuclear envelope assembly,and nuclear reconstitution.Dinoflagellate Crythecodinium cohnii is a kind of primitive eukaryote which possesses numerous permanently condensed chromosomes and discontinuous double-layered nuclear membrane throughout the cell cycle.The assembled nuclei,being surrounded by a continuous double membrane containing nuclear pores and the uniformly dispersed chromatin fibers are morphologically distinguishable from that of Dinoflagellate Crythecodinium cohnii.However,incubation of dinoflagellate Cyrthecodinium cohnii chromosomes in the extracts from dinoflagellate Crythecodinium cohnii cells does not induce nuclear reconstitution.     

Induction of apoptosis in purified animal and plant nuclei by Xenopus egg extracts

We have developed a cell-free system that can trigger the nuclei purified from mouse liver and suspensioncultured carrot cells to undergo apoptosis as defined by the formation of apoptotic bodies and nucleosomal DNA fragments.The effects of different divalent cations and cycloheximide on DNA cleavage in this system were assessed.The fact that nuclei of plant cells can be induced to undergo apoptosis in a cell-free animal system suggests that animals and plants share a common signal transduction pathway triggering in the initiation stage of apoptosis.