Synapsoarchitectonics of the septum of the cat brain

In the medial and lateral septal nuclei, 4 types of axonal terminals are distinguished. Type I contains spherical vesicles and forms asymmetric synapses on small and middle stems and spines of the dendrites; type I terminals comprise 63% in the medial nucleus of the total number of axons, and in the lateral one--52%. Type II contains polymorphic vesicles and forms symmetrical synapses on the soma and large dendrites. In the medial nucleus they comprise 6%, and in the lateral one--3%. Type III contains either clear spherical (IIIa), or polymorphic (IIIb) vesicles, as well as 1-2 vesicles with a dense core. They form axodendritic, axospine and axosomatic synapses. In the medial nucleus they comprise 25% and 3%, respectively, in the lateral one--40% and 2%. Type IV contains a great number of vesicles with a dense core. These terminals in both septal nuclei comprise 3% and do not participate in formation of active contacts.     

Evolutionary histology and the theory of evolution (on the 100th anniversary of the birth of Academician A. A. Zavarzin

Darwin's theory did not touch upon the problem of evolution of tissues. An attempt made by Heckel to explain phylogeny of tissues, basing on principles of selection and divergence, failed. It was not at once understood that evolution of separate levels of organization possessed certain specificity. The theory of parallelism, suggested by A. A. Zavarzin, stated the notion on specific regularities in evolution of tissues. Having analysed the correlation between the theory of evolution and evolutional histology, A. A. Zavarzin demonstrated that darwinism developed predominantly at the theory of speciation. This approach is correct for the period of the new evolutionary synthesis, too. The synthetic theory of evolution does not take into consideration evolution of tissues. A. A. Zavarzin's theory contributed to reorganization of methodology of the evolutional biology. The historical method was enriched by a certain principle on specific regularities of evolution for each level of organization in the alive. Simultaneously, the genesis of the parallelism theory discovered that correct explanation of the regularities in evolution of tissues is possible only under conditions that the evolution of histostructures can be inserted into the evolution of ontogenesis and species.     

Transforming growth factor-beta inhibits endothelial cell proliferation

Transforming growth factor-beta (TGF-beta) is an inhibitor of the proliferation of bovine aortic endothelial cells in culture. Basal cell growth in serum-containing medium and cell proliferation stimulated by fibroblast growth factor (FGF) are inhibited by TGF-beta in a dose-dependent manner. Half-maximal inhibition occurs at an inhibitor concentration of 0.5-1.0 ng/ml. TGF-beta does not appear to be cytotoxic and cells treated with the inhibitor grow normally after removal of TGF-beta. High concentrations of FGF are ineffective in overcoming TGF-beta-induced inhibition of cell proliferation, suggesting that antagonism of growth factor-induced cell proliferation by TGF-beta is of a noncompetitive nature.     

Modulation of the binding characteristics of a fluorescent nucleotide derivative to the sarcoplasmic reticulum adenosinetriphosphatase

Trinitrophenyladenosine monophosphate (TNP-AMP) binding to the phosphorylated Ca2+-ATPase of sarcoplasmic reticulum results in manyfold higher fluorescence intensity and longer lifetimes of the nucleotide analogue, as compared to TNP-AMP binding to the nonphosphorylated enzyme. This is observed when the phosphoenzyme intermediate is formed either from ATP or from inorganic phosphate (Pi). An important question is whether the TNP-AMP fluorescence properties can also reflect the kinetically defined interconversions of different phosphoenzyme species during catalysis. We have approached this question by manipulating the phosphorylation conditions in a manner which is known to result in accumulation of different species of the phosphoenzyme, i.e., by variations in pH, substrates, and K+ and Ca2+ concentrations. Decreasing pH or increasing [K+] caused large decreases in fluorescence intensity at a given concentration of TNP-AMP under conditions of phosphorylation with either ATP or Pi. In contrast, low to high intravesicular Ca2+ concentrations had no effect on fluorescence during steady-state turnover. TNP-AMP titrations of the phosphorylated enzyme stabilized in different states revealed that H+ and K+ caused a shift in TNP-AMP binding affinity to the site responsible for high fluorescence enhancement, while maintaining approximately the same maximal fluorescence yield at saturation. The fluorescence lifetimes of TNP-AMP bound to phosphoenzyme did not change with variations in pH, [K+], and substrates. We conclude that the environment of that part of the TNP-AMP binding site which binds the trinitrophenyl moiety undergoes a change upon enzyme phosphorylation resulting in enhanced fluorescence yield; this change is invariant between different phosphoenzyme species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Guanosine 3',5'-cyclic monophosphate stimulates release of actively accumulated calcium in purified disks from rod outer segments of bovine retina

Parallel lines of evidence have suggested that light initiates changes in both cGMP metabolism and calcium levels in rod outer segments (ROS). We report that cGMP stimulates release of a pool of Ca2+ actively accumulated within purified ROS disks. Disks were purified and actively loaded with 45Ca2+ by an associated ATP-dependent calcium uptake activity as previously described [Puckett, K.L., Aronson, E.T., & Goldin, S.M. (1985) Biochemistry 24, 390-400]. Spikes of 45Ca2+ released from disks were observed in a rapid superfusion system. The Ca2+ release was specifically stimulated by physiological levels of cGMP (Kapp approximately 20 microM; Hill coefficient = 1.7). 8-Bromo-cGMP could also activate the release mechanism, but cAMP was ineffective. At cGMP levels of greater than or equal to 100 microM, approximately 20% of the loaded Ca2+ was released. The Ca2+ release rate at saturating cGMP levels reached a maximum within the 10-s time resolution of the assay system. In contrast to other recent reports of cGMP activation of ROS ion conductances, the majority of the release activity terminated in a spontaneous manner, suggestive of an intrinsic inactivation process. The amount of Ca2+ released and the release kinetics were similar to the presence or absence of an unbleached pool of rhodopsin. Cyclic nucleotides did not stimulate release from disks passively equilibrated with 45Ca2+, i.e., in the absence of ATP but otherwise under identical conditions. Preincubation of the disks with cGMP also reduced the level of ATP-dependent Ca2+ uptake (approximately 30%); this apparent inhibition may be due to activation of the release mechanism, rather than direct modulation of the uptake activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic studies on the activation of pyrophosphate-dependent phosphofructokinase from mung bean by fructose 2,6-bisphosphate and related compounds

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) was purified from the mung bean Phaseolus aureus. The enzyme is activated by fructose 2,6-bisphosphate at nanomolar concentrations. The enzyme exhibits Michaelis-Menten kinetics, and the reaction mechanism, deduced from initial velocity studies in the absence of inhibitors as well as product and dead-end inhibition studies, is rapid equilibrium random in the presence and absence of fructose 2,6-bisphosphate. In the direction of fructose 6-phosphate phosphorylation, saturating fructose 2,6-bisphosphate (1 microM) increases V congruent to 9-fold and increases V/KMgPPi and V/KF6P about 30-fold. In the reverse direction (phosphate phosphorylation), the same concentration of activator has little if any effect on V or the Km for inorganic phosphate (Pi) and Mg2+ but does increase V/KFBP about 42-fold. No changes were observed in any of the other rate constants. The binding affinity of fructose 2,6-bisphosphate to all enzyme forms is identical. The activator site of the mung bean PPi-PFK binds fructose 2,6-bisphosphate with a Kact of 30 nM with the 2,5-anhydro-D-glucitol 1,6-bisphosphate (the most effective analogue) 33-fold less tightly. Of the alkanediol bisphosphate series, 1,4-butanediol bisphosphate exhibited the tightest binding (Kact congruent to 3 microM). These and a series of other activating analogues are discussed in relation to the activator site.     

A protein kinase copurified with chick oviduct progesterone receptor

A magnesium-dependent protein kinase activity was copurified with both the molybdate-stabilized 8S form of the chick oviduct progesterone receptor (PR) and its B subunit. In each case, purification was performed by hormonal affinity chromatography followed by ion-exchange chromatography. The Km(app) values of the phosphorylation reaction for [gamma-32P]ATP and calf thymus histones were approximately 1.3 X 10(-5) M and approximately 1.6 X 10(-5) M, respectively, and only phosphorylated serine residues were found in protein substrates, including PR B subunit. Physicochemical parameters of the enzyme [pI approximately 5.3, Stokes radius approximately 7.2 nm, sedimentation coefficient (S20,w) approximately 5.6 S, and Mr approximately 200,000] were compared to those of purified forms of PR (B subunit, pI approximately 5.3, Stokes radius approximately 6.1 nm, and Mr approximately 110,000; 8S form, Stokes radius approximately 7.7 nm and Mr approximately 240,000). The results suggest that most of the protein kinase activity copurified with both oligomeric and monomeric forms of PR belongs to an enzyme distinct from currently known receptor components. Its physiological significance remains unknown.     

Site-directed labeling of a monoclonal antibody: targeting to a disulfide bond

We have designed and synthesized crabescein, the first member of a class of fluorescent labels that add across disulfide bonds. Crabescein is a fluorescein derivative that reports the rotational correlation time of the immunoglobulin G (IgG) segment to which it is covalently bound. Chemical analysis of the IgG labeled with crabescein indicates that the fluorophore is inserted into the third disulfide bond (cysteine-229 of mouse IgG2a) in the hinge region. The rotational correlation time of this labeled macromolecule was measured as a single exponential with a decay constant of 26.8 ns. This is in contrast to the double exponential with decay constants of 14.3 and 0.2 ns for the same IgG when labeled with fluorescein via a conventional labeling reagent in which the probe is bound to the macromolecule by one-point attachments. Thus, crabescein is the prototype of a class of fluorescent and phosphorescent probes that, by virtue of their two-point attachments to proteins, faithfully report on the dynamics of the segment of macromolecule to which they are covalently bound.     

In nondividing cells, histone H1(0) is synthesized and deposited onto chromatin without accompanying phosphorylation

The phosphorylation of H1 histone subfractions was measured in mouse neuroblastoma cells stopped from dividing by three treatments that block cell division: 5 mM butyrate, 2% dimethyl sulfoxide, and serum withdrawal. H1 histone phosphorylation decreased in response to all three treatments, but the response differed in its timing and its extent for the different H 1 subfractions. The different decreases in phosphorylation correlated well with the differential decreases in biosynthesis of the individual H1 subfractions; however, an exception to this parallel decrease in synthesis and phosphorylation was observed in the case of histone H1(0). Phosphorylation of H1(0) was absent in each of the three treatments after 2 days, despite the continued synthesis and deposit of H1(0) on the chromatin. Thus, despite the fact that H1(0) was being synthesized and that the other newly synthesized H1 subfractions were phosphorylated at this time, the phosphorylation of H1(0) became uncoupled from its synthesis after prolonged treatments blocking cell division.