Use of a serum gradient for separation of polykaryocytes from single cells after fusion with Sendai virus

After cell fusion, polykaryocytes and heterokaryocytes were separated from single cells by a calf serum gradient. Fractions enriched with polykaryocytes were thus obtained. Heterokaryocytes with a prevalence of nuclei from either parental cell were distributed in different fractions of the gradient. The efficiency of the separation procedure is evaluated and its possible application to problems in cell hybridization techniques is discussed.     

The error catastrophe hypothesis with reference to aging and the evolution of the protein synthesizing machinery.

Recent investigations of the thermodynamics of protein denaturation, in particular of pressure effects, have questioned the fundamental importance, hitherto assumed, of hydrophobic interactions in the native conformations of proteins. The volume changes observed on protein denaturation are incompatible with the volume changes estimated on the basis of volume effects observed in low molecular weight model systems of the aliphatic groups. In the present paper the model systems generally considered are critically discussed. It is concluded, that solutions of low molecular weight alkanes may not be any adequate models of aliphatic groups in proteins. Studies of more appropriate model systems suggest that the volume changes to be expected, when buried aliphatic groups of proteins are exposed to water, are small and positive, and mainly due to structural changes of the water. These volume changes are in accordance with the volume changes actually measured of protein denaturation, and the latter volume effects are taken as supporting evidence of the importance of hydrophobic interactions in protein confonriations.     

Molecular feature of the nerve growth factor in human serum

High molecular weight binding components which bind [¹²⁵I] mouse β nerve growth factor exist in human serum. The binding of β nerve growth factor to the serum components was inhibited at alkaline condition. After gel filtration of human serum on a Sephadex G-150 column at neutral condition, the nerve growth factor-like immunoreactivity was observed in only one peak, differing from the high molecular weight serum components. However, at alkaline condition two peaks with nerve growth factor-like immunoreactivity appeared; one was almost at the position observed at neutral pH, and the other was a new peak eluted approximately to the column volume. these results suggest that there are at least two nerve growth factor-like molecules in human serum and most of the nerve growth factor in the serum exists in a complex form associated with serum components with high molecular weight.     

Reduction of ricin and other plant toxins by thiol:protein disulfide oxidoreductases

The inhibitory effect of ricin, abrin, and modeccin on protein synthesis by a rabbit reticulocyte lysate is enhanced after preincubation of the toxins with GSH in the presence of a thiol:protein disulfide oxidoreductase purified from bovine liver. The same toxins, as well as the toxin from Viscum album, are reduced also by another thiol:protein disulfide oxidoreductase purified from rat liver cytosol.     

Enhancing effect of lithium and potassium ions on lectin-induced lymphocyte proliferation in aging and down's syndrome subjects

The effect of different concentrations of LiCl or KCl (0.6–20 meq/liter) on PHA-stimulated lymphocytes from young, old, and Down's syndrome subjects was studied. LiCl showed a dramatic enhancing effect on [³H]thymidine incorporation induced by a suboptimal dose of PHA in old subjects and Down's syndrome patients. An increase of [³H]thymidine incorporation in human lymphocytes stimulated by a suboptimal dose of PHA was also observed with KCl. This effect was higher in old subjects than that observed in young and Down's subjects. LiCl and KCl can modulate and partially restore the derangement in early events of mitogen stimulation which seems to be present in lymphocytes from both old and Down's syndrome subjects.     

Spatial learning in golden hamsters: Relationship between food-searching strategies and difficulty of the task

In an elevated maze consisting of three reconvergent radial arms, golden hamsters were tested with the same experimental rule: to choose each path without repeating any choice. However, variations of procedure concerning (a) the location of the reward in the maze, and (b) reinforcement contingencies, were introduced in order to define several problems involving variable levels of difficulty. The relationship between response strategies and difficulty of the task was then studied. The common learning criterion was the achievement of three consecutive correct daily sessions, each session corresponding to a particular sequence (pattern) of choices of paths. Response strategies were studied by analyzing the patterns obtained over the three final sessions in which an animal reached the learning criterion. Such a set of patterns (triplet) could be heterogeneous (patterns all different), mixed (two identical patterns, one different) or stereotyped (identical patterns). No relationship was found between the mean level of difficulty presented by each learning problem and the occurrence of a particular type of triplet. However, in each situation, mixed triplets were the most frequently recorded and corresponded to the medium individual speeds of learning whereas heterogeneous triplets corresponded to rapid successes and stereotyped triplets to delayed successes. These findings indicate that, whatever the problem designed to be tested in a three-arm maze, the various forms of solutions reflect different individual adaptative mechanisms.     

Hormone release during computer-monitored sexual behavior in mature and aged male rats

Sexual behavior and the increase in plasma hormone levels of LH, prolactin, and testosterone associated with sexual behavior were examined in three age groups of sexually naive male rats. The two younger groups (5- and 11-month-old) mated normally and their behavioral latencies decreased significantly following sexual experience. Both plasma testosterone and LH concentrations increased significantly following entrance of a receptive female into the mating arena. Plasma prolactin levels rose but not significantly. However, the 27-month-old rats neither mated nor showed an increase in the three plasma hormone concentrations during exposure to a receptive female. Only basal testosterone levels were significantly lower than those of the younger animals. Low testosterone levels possibly contributed to deficiencies in both behavior and its associated hormone release. The monitoring of sexual behavior was facilitated by a computer, programmed to record, store, and analyze behavioral events.     

Strain differences in rat adrenal biosynthetic enzymes and stress-induced increases in plasma catecholamines.

We have examined in two inbred rat strains basal and stress-induced increases in plasma levels of epinephrine (EPI) and norepinephrine (NE) and compared these with activities of the adrenal enzymes involved in the synthesis of catecholamines. There were no differences in basal levels of NE and EPI in plasma of adult male rats of the Wistar-Kyoto (WKY) and Brown-Norway (B-N) strains. However, following 5 min. of intermittent footshock, plasma levels of both catecholamines were twice as high in WKY rats as in B-N rats. In the adrenals of unstressed rats, activities of tyrosine hydroxylase and dopamine-beta-hydroxylase were significantly higher in B-N rats. In addition, the adrenal weights and the contents of NE but not EPI were greater in B-N rats. Thus, in these two rat strains, the capacity of the adrenal gland to synthesize and store catecholamines appeared to be inversely related to plasma levels of NE and EPI after stress. The differences between the strains appeared to be due to differences in the rates of removal of catecholamines from the peripheral circulation as well as to differences in the rate of release of catecholamines from the sympatho-adrenal medullary system. Thus biosynthetic enzyme activities need not be related directly to the capacity to release and elevate plasma levels of catecholamines following stressful stimulation.     

Differences in the protein binding of several drugs and bilirubin in serum and heparinized plasma of rats.

The purpose of this investigation was to compare the protein binding of salicylic acid, phenytoin, warfarin and bilirubin in serum and heparinized plasma of rats. Protein binding was determined by equilibrium dialysis (drugs) or by a reaction rate method (bilirubin), using serum and plasma obtained from the same animals. The three drugs were significantly less protein bound in heparinized plasma than in serum; this difference was particularly pronounced in the case of warfarin. Addition of heparin to serum also resulted in a decrease in the protein binding of the drugs but to a lesser extent than in plasma. The protein binding of bilirubin was more extensive in plasma than in serum, irrespective of the anticoagulant used (heparin, sodium citrate, or disodium ethylenediamine-tetraacetate). It may be desirable to perform all binding studies with serum rather than plasma.     

Modified two-dimensional electrophoresis for identification and autoradiography of ribosomal proteins reacted withp-chloromercuribenzoate

Ribosomal proteins labelled with [¹⁴C]PCMB, a reversibly reacting sulfhydryl reagent, lose most of their radioactivity when separated by the conventional two-dimensional polyacrylamide gel electrophoresis. Therefore, the two-dimensional method was modified to insure stability of the PCMB-protein complex during the procedure. Proteins ofEscherichia coli ribosomes were separated by the modified method, and those which reacted with [¹⁴C]PCMB were identified by counting of stained spots and by autoradiography of dried gels.     

Two methods for the separation of human alpha-fetoprotein and albumin. (A) Affinity chromatography using Blue Sepharose CL-6B and (B) ampholyte displacement chromatography.

Two methods for the separation of α-fetoprotein (AFB) and albumin using (A) affinity chromatography on Blue Sepharose CL-6B and (B) ampholyte displacement chromatography are described. The heterogeneity of immunoreactive human AFP is demonstrated by ampholyte displacement chromatography; possibly seven variants of AFP can be distinguished by this method.     

Relationship between the cytoplasm and the vacuole phosphate pool in Acer pseudoplatanus cells

The Pi concentration of Acer pseudoplatanus cells in the two major intracellular compartments, the cytoplasm and the vacuole, has been studied using 31P NMR. For sycamore cells containing approximately 2 mM of total Pi, the cytoplasmic Pi and the vacuolar Pi concentrations were approximately 6 and 1.5 mM, respectively. When the cells were transferred to a phosphate-deficient medium, the vacuolar Pi decreased rapidly while the cytoplasmic Pi decreased slowly during the first 48 h, indicating that Pi in the cytoplasm was maintained at the expense of the vacuolar Pi. When the Pi-starved cells (i.e., those containing less than 0.5 mumol of total Pi/g wet wt) were transferred to a medium containing 300 microM Pi, Pi entered the cells rapidly and accumulated in the cytoplasm. Once the cytoplasmic Pi pool was filled, Pi was taken up in the vacuole until the vacuole Pi pool was filled. On the contrary when the non-Pi-starved cells were transferred to a phosphate-rich medium (i.e., containing 45 mM Pi), Pi entered the cells slowly by diffusion and accumulated in the vacuole but not in the cytoplasm. These results demonstrate that the Pi content of the cytoplasm is maintained at the expense of the vacuolar Pi pool when sycamore cells are transferred to either a phosphate-deficient or a phosphate-rich medium.     

Release of lutropin (LH) and follitropin (FSH) in cattle after administration of a new gonadoliberin (GnRH) analogue in comparison with the gonadoliberin decapeptide.

A gonadoliberin (GnRH) analogue nonapeptide (Hoe 766) was administered intramuscularly in concentrations between 2.5 and 50 μg to m?ture cows in order to study the response of lutropin (LH) and follitropin (FSH). Results were compared with those from experiments of the GnRH decapeptide (Hoe 471). Plasma LH and FSH were radio-immunologically determined. Increasing doses of GnRH analogue up to 15–20 μg caused an approximately linear increase in total plasma LH and FSH until the response reached a plateau. With these amounts peak values were about 60 fold higher for LH and 3.5 fold higher for FSH than basal levels about 135 minutes after injection. Higher values lasted for more than 6 h for LH and about 5 h for FSH. The LH response was much greater and more prolonged than for FSH.Doses of the nonapeptide analogue 50 to 70 times lower than the GnRH decapeptide provoked about the same height and duration of LH and FSH response.     

Interactions between an alpha-helix and a beta-sheet. Energetics of alpha/beta packing in proteins

Conformational energy computations have been carried out to determine the favorable ways of packing a right-handed alpha-helix on a right-twisted antiparallel or parallel beta-sheet. Co-ordinate transformations have been developed to relate the position and orientation of the alpha-helix to the beta-sheet. The packing was investigated for a CH3CO-(L-Ala)16-NHCH3 alpha-helix interacting with five-stranded beta-sheets composed of CH3CO-(L-Val)6-NHCH3 chains. All internal and external variables for both the alpha-helix and the beta-sheet were allowed to change during energy minimization. Four distinct classes of low-energy packing arrangements were found for the alpha-helix interacting with both the parallel and the anti-parallel beta-sheet. The classes differ in the orientation of the axis of the alpha-helix relative to the direction of the strands of the right-twisted beta-sheet. In the class with the most favorable arrangement, the alpha-helix is oriented along the strands of the beta-sheet, as a result of attractive non-bonded side-chain-side-chain interactions along the entire length of the alpha-helix. A class with nearly perpendicular orientation of the helix axis to the strands is also of low energy, because it allows similarly extensive attractive interactions. In the other two classes, the helix is oriented diagonally relative to the strands of the beta-sheet. In one of them, it interacts with the convex surface near the middle of the saddle-shaped twisted beta-sheet. In the other, it is oriented along the concave diagonal of the beta-sheet and, therefore, it interacts only with the corner regions of the sheet, so that this packing is energetically less favorable. The packing arrangements involving an antiparallel and a parallel beta-sheet are generally similar, although the antiparallel beta-sheet has been found to be more flexible. The major features of 163 observed alpha/beta packing arrangements in 37 proteins are accounted for in terms of the computed structural preferences. The energetically most favored packing arrangement is similar to the right-handed beta alpha beta crossover structure that is observed in proteins; thus, the preference for this connectivity arises in large measure from this energetically favorable interaction.     

The effects of injecting [D-Ser(But)6] Des Gly-NH2(10) luteinizing hormone releasing hormone ethylamide in early, mid and late seasonal anoestrus on gonadotrophin secretion and luteal function in the ewe

The ability of the luteinizing hormone releasing hormone (LH-RH) analogue [D-Ser(Bu(t))(6)] Des-Gly-NH(2)(10) LH-RH ethylamide to stimulate the secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) and to induce ovulation and luteal function in seasonally anoestrous ewes was investigated by injecting the analogue at three stages of the anoestrus (day 118, day 182 and day 235 of the year). After injection on day 118, eight of nine ewes ovulated and all of the former secreted progesterone during the subsequent 20 days. After injection on day 182, six of the nine ewes ovulated, of which none showed luteal function. Only two of the nine ewes were not already secreting progesterone on day 235. Both of these responded to the analogue by secreting normal luteal levels of progesterone. The mean LH peak heights in response to injection at the three stages showed no significant differences from one another. The mean FSH peak heightafter injection on day 182 was significantly lower than the mean FSH peak height associated with the other two challenges (P < 0.05). On day 116 of the following year, 20 ewes were treated with the analogue as before. The high progesterone levels confirmed the results of the day 118 challenge in the previous year. However, none of the ewes conceived when inseminated artificially 24 and 36 hours after analogue treatment.     

Ribosomal subunit orientations determined in the monomeric ribosome by single and by double-labeling immune electron microscopy

The energies of two and three-chain antiparallel and parallel β-sheets have been minimized. The chains were considered to be equivalent. In each case, chains consisting of four and of eight l-alanine residues, respectively, with CH₃CO- and -NHCH₃ end groups were examined. Computations were carried out both for chains constrained to have a regular structure (i.e. the same φ and ψ dihedral angles for each residue) and for chains in which the regularity constraint was relaxed. All computed minimum-energy β-sheets were found to have a right-handed twist, as observed in proteins. As in the case of right-handed α-helices, it is the intrastrand non-bonded interaction energy that plays the key role in forcing β-sheets of l-amino acid residues to adopt a right-handed twist. The non-bonded energy contribution favoring the right-handed twist is the result of many small pairwise interatomic interactions involving the C^βH₃ groups. Polyglycine β-sheets, lacking the C^βH₃ side-chains, are not twisted. The twist of the poly-l-alanine sheet diminishes as the number of residues per chain increases, in agreement with observations. The twist of the four-residue chain increases somewhat (because of interstrand non-bonded interactions, also involving the C^βH₃ groups) in going from a single chain to a two-chain antiparallel structure, but then decreases slightly in going from a two-chain to a three-chain structure. β-Sheets in observed protein structures sometimes have a larger twist than those in the structures computed here. This may be due to irregularities in amino acid sequence and in hydrogenbonding patterns in the observed sheets, or to long-range interactions in proteins. The minimized energies of parallel β-sheets are considerably higher than those of the corresponding antiparallel β-sheets, indicating that parallel β-sheets are intrinsically less stable. This finding about the two kinds of β-sheets agrees with suggestions based on analyses of β-sheets observed in proteins. The energy difference between antiparallel and parallel β-sheets is due to closer packing of the chains and a more favorable alignment of the peptide dipoles in the antiparallel structures. The hydrogen-bond geometry in the computed antiparallel structures is very close to that proposed by Arnott et al. (1967) for the β-form of poly-l-alanine.     

Regulation of stable RNA synthesis and ppGpp levels in growing cells of Escherichia coli

Under the balanced condition of growth of E. coli cells, no distinct difference is observed in stable RNA and protein synthesis between CP78 (rel⁺) and CP79 (rel⁻), whereas a considerable difference is present in RNA accumulation between NF161 (rel⁺) and NF162 (rel⁻), where NF161 < NF162. The RNA content of NF161 is lower than that of NF162 in four different cultures with different growth rates. These two sets of isogenic pairs of rel⁺ and rel⁻ strains are commonly used in the study of rel gene function; however, NF161 is a mutant in the spoT gene whose product may be responsible for the degradation of ppGpp. The basal levels of ppGpp in these four strains growing with three different growth rates were examined: NF161 (rel⁺spoT⁻) has a much higher content of ppGpp than do other strains. Furthermore, the contents of ppGpp tend to be lower when the above four strains are growing at a faster rate. Thus a close correlation seems to exist between the content of RNA and the basal level of ppGpp under the condition of balanced growth.