Monoclonal antibodies Ki-S3 and Ki-S5 yield new data on the 'Ki-67'proteins

Abstract. The monoclonal antibody (mab) Ki-67 has been used for about 10 years, mainly in tissue sections, to monitor proliferating cells, but so far only very little is known about the proteins it recognizes. The new mabs Ki-S3 and Ki-S5 detect proliferating cells in frozen and paraffin-embedded tissues. They recognize proteins with the same molecular mass as Ki-67 in Western blot and for the first time also in immunoprecipitation experiments. With these mabs we were able to enrich and purify the Ki-67 proteins. Protein sequencing of four peptides of the digested proteins corresponded to the cDNA-deduced amino acid sequence already published for the Ki-67 proteins.
Since we were able to immunoprecipitate the Ki-67 proteins, we performed various immunoprecipitation experiments to obtain more information about the nature of these proteins. After radiolabelling L428 cells with [³⁵S]-methionine we were able to immunoprecipitate the Ki-67 proteins after only 5 min of labelling time. In turnover experiments the Ki-67 proteins could not be detected 3 h after the end of labelling. These data indicate a halt-life of the Ki-67 proteins of about 90 min.
Labelling experiments with [³²P]-orthophosphate revealed that the Ki-67 proteins are phosphorylated. After dephosphorylation was blocked with okadaic acid or cell growth was arrested by means of Colcemid, the phosphorylation of the Ki-67 proteins was greatly increased, indicating that the Ki-67 proteins are phosphorylated via serine and threonine, and that the phosphorylation of the Ki-67 proteins increases in cycling cells. Labelling experiments with [³H]-mannose and [³H]-glucose revealed that the protein is weakly N -glycosylated.     

Native Enzyme Mobility Shift Assay (NEMSA): a new method for monitoring carboxyl group modification of carboxymethylcellulase from Aspergillus niger

A simple, sensitive, accurate and more informative assay for determining the number of modified groups during the course of carboxyl group modification is described. Monomeric carboxymethylcellulase (CMCase) from Aspergillus niger was modified by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of glycinamide. The different time-course aliquots were subjected to non-denaturing PAGE and the gel stained for CMCase activity. The number of carboxyl groups modified are directly read from the ladder of the enzyme bands developed at given time. This method showed that after 75 min of modification reaction there were five major species of modified CMCases in which 6 to 10 carboxyls were modified.     

Myelin basic protein purified on an ion-exchange continuous polymer bed in the presence of ethylene glycol and salt possesses activity againstp-nitrophenyl acetate

In this paper we describe a fast and mild method based on the use of a unique cation exchanger and buffers containing ethylene glycol and salt for the purification of the myelin basic protein (MBP; MW 18.5 kDa). MBP thus purified hydrolyses catalytically p-nitrophenyl acetate. This esterase activity facilitates not only the purification of MBP but also indicates that probably it is in its native state, i.e. there is a good chance that the purified molecules are structurally and chemically identical. This is a prerequisite to obtain crystals appropriate for x-ray diffraction and other studies.Abbreviations used MBP myelin basic protein - MW molecular weight - kDa kilo Dalton - octyl-POE n-octylpolydisperse oligooxyethylene - CHAPS 3-3-cholamidopropyl dimethylammonio-1-propane-sulfonate - CTAB cetyltrimethylammonium bromide - SDS sodium dodecyl sulfate - SDS-PAGE polyacrylamide gel electrophoresis in the presence of SDS - G 3707 heptaoxyethylene lauryl ether - TWEEN-20 polyoxyethylenesorbitan-monolaurat - EDTA ethylenediaminetetraacetic acid - HEPES N-(2-hydroxyethyl)-piperazine-N-(2-ethanesulfonic acid)     

Effects of decreased glutathione peroxidase activity on the pentose phosphate cycle in mouse lung

The effects of reducing glutathione peroxidase activity in the lung by changing dietary selenium intake has been investigated. In animals that were exposed to room air, selenium effects were confined to glutathione peroxidase activity, whereas under conditions of oxidant stress (ozone) the decrease in glutathione peroxidase activity prevented the stimulation of the pentose phosphate cycle (assayed by measuring glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities) which has been reported to increase in response to oxidant stress. The suppression of glutathione peroxidase activity was found to depend on dietary selenium concentration. The physiological significance of this observation may be related to the process of injury and repair in the lung.     

Influence of protein hydrolysate baits on oviposition in the apple maggot, Rhagoletis pomonella

Ovipositional responses of apple maggot (AM), Rhagoletis pomonella (Walsh), females were studied in the laboratory on apples (var: Golden Delicious) treated with different rates of four protein hydrolysate baits in choice and no-choice tests. Protein hydrolysate baits at rates of 0.5 and 1% had no significant effect, but oviposition was greatly reduced at higher rates of 5 and 10%. Apple maggot females exposed to apples treated with protein hydrolysate baits at a rate of 10% made 41–71% fewer punctures and laid 41–73% fewer eggs than in untreated control. No oviposition activity was shown on apples treated with 25 and 100% Nulure®. In no-choice tests the AM females laid 75–96% fewer eggs in apples treated with 10 and 25% Nulure compared to controls and no oviposition occurred in apples treated with 100% Nulure. Apple maggot females arrived in similar numbers on apples treated with 10% Nulure and untreated apples, but only 5% of those arriving on Nulure-treated apples showed ovipositor boring with no egg deposition while 60% of females arriving on untreated apples showed ovipositor boring activity and laid an average of 2.5 eggs per apple. In another experiment, individual AM females displayed similar behavioral responses to 10% Nulure-treated apples; none of the 56 females tested on treated apples displayed ovipositor boring activity, but 59% of the females (N=56) tested on untreated apples displayed ovipositor boring within 5 min of their arrival. Ninetyeight percent of AM females stayed and fed on fruit surfaces for 5 min on Nulure-treated apples without ovipositor boring compared to only 2% on untreated apples. Of the females that arrived on untreated apples, 39% flew away within 5 min without ovipositor boring compared to only 2% of those that arrived on Nulure-treated apples. Results of these two behavioral experiments suggest that upon arrival on a protein bait-treated apple, an apparent change of behavior occurs in AM females and instead of attempting to oviposit, they attempt to feed on fruit surfaces resulting in reduced oviposition activity. These results indicate that the feeding and oviposition-related activities of AM females are probably mutually exclusive and that the feeding behavior preempts oviposition activities on host fruits treated with higher rates of protein hydrolysate baits.     

Characterisation and Regional Localisation of Pre- and Postsynaptic Glycoproteins of the Chick Forebrain Showing Changed Fucose Incorporation Following Passive Avoidance Training

To identify those glycoproteins whose synthesis or modification is necessary for memory formation, we have studied the uptake of radiolabelled fucose into synaptic plasma membranes (SPMs) and postsynaptic densities (PSDs) derived from two specific left and right forebrain loci, at two different times after training of 1-day-old chicks on a one-trial passive avoidance learning task. To increase the reliability of the comparison, a double-labelling method was used. Tissue samples from intermediate medial hyperstriatum ventrale (IMHV) and lobus parolfactorius (LPO) were isolated at 6 and 24 h after training. At both times, training resulted in region-specific changes, both increases and decreases, in incorporated radioactivity into pre- and postsynaptic glycoproteins. After 6 h, there was a relative decline in incorporation into both SPMs and PSDs of the right IMHV of trained chicks, a decline that persisted in the PSDs until 24 h. A small decline in incorporation in SPMs from the right LPO of trained chicks at 6 h was reversed by 24 h, by which time there was a 64% increase in incorporation into SPMs and a 24% increase into PSDs of the left LPO. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of left and right hemisphere samples containing LPO revealed that 6 h after training the main effect was presynaptic, including a reduction of incorporation into high molecular mass glycoproteins, of 150-180 kDa, and an increase in a lower molecular mass (41 kDa) fraction. By 24 h after training, a left hemisphere presynaptic glycoprotein of molecular mass approximately 50 kDa showed the biggest increase in fucosylation. In addition, a wide group of postsynaptic glycoproteins of both hemispheres, in the ranges 150-180, 100-120, and 33 kDa now showed increases in incorporation. Some other fractions showed decreases. These results are in accord with previous data on incorporation obtained using the amnesic agent 2-deoxygalactose. They also support the hypothesis that memory formation involves the strengthening of connections between pre- and postsynaptic neurons of the LPO by growth or modulation of pre- and postsynaptic structures.     

On the computation of the tertiary structure of globular proteins. III. Inter-residue distances and computed structures

An analysis of geometrical models for computing the tertiary structure of globular proteins from the primary structure is presented. The roles of initial configuration, input information on inter-residue distances and the errors in this information are delineated. It is shown that for local information like that on secondary structure, the calculated structure is very sensitive to errors and to the initial configuration. Thus, such information is far from adequate for predicting the tertiary structure. On the other hand, global information on all the inter-residue distances is quite insensitive to errors. A semi-empirical method is presented to estimate these distances and the calculated structures are given for two proteins—pancreatic trypsin inhibitor and parvalbumin. These structures have good resemblances to those determined by X-ray diffraction. A strategy for further refinement of the method is indicated.