Structural requirements of pyrroloquinoline quinone dependent enzymatic reactions

On the basis of crystal structures of the pyrroloquinoline quinone (PQQ) dependent enzymes methanol dehydrogenase (MDH) and soluble glucose dehydrogenase (s-GDH), different catalytic mechanisms have been proposed. However, several lines of biochemical and kinetic evidence are strikingly similar for both enzymes. To resolve this discrepancy, we have compared the structures of these enzymes in complex with their natural substrates in an attempt to bring them in line with a single reaction mechanism. In both proteins, PQQ is located in the center of the molecule near the axis of pseudo-symmetry. In spite of the absence of significant sequence homology, the overall binding of PQQ in the respective active sites is similar. Hydrogen bonding interactions are made with polar protein side chains in the plane of the cofactor, whereas hydrophobic stacking interactions are important below and above PQQ. One Arg side chain and one calcium ion are ligated to the ortho-quinone group of PQQ in an identical fashion in either active site, in agreement with their proposed catalytic function of polarizing the PQQ C5-O5 bond. The substrates are bound in a similar position above PQQ and within hydrogen bond distance of the putative general bases Asp297 (MDH) and His144 (s-GDH). On the basis of these similarities, we propose that MDH and s-GDH react with their substrates through an identical mechanism, comprising general base-catalyzed hydride transfer from the substrate to PQQ and subsequent tautomerization of the PQQ intermediate to reduced PQQ.     

Calcium requirements of Azotobacter

Summary Calcium was essential for the growth of A. vinelandii, A. chroococcum, A. beijerinckii and A. insigne in the presence and absence of combined nitrogen. A. agile and A. macrocytogenes were able to grow in the absence of calcium but growth was stimulated by the cation.Calcium could be replaced by strontium at roughly the same molar concentration.The authors present evidence suggesting that the calcium sparing effect of ammonium and sodium acetates reported in the literature may be dependent on the size of inoculum used.Professor Dr. Dr. h. c. A. Rippel zum 70. Geburtstag.     

钙依赖性突触的可塑性

突触前和突触后细胞内钙离子（[Ca^2 ]i)在短时程和长时程突触的可塑性中,发挥着重要的住处传递作用。兴奋后残留[Ca^2 ]i,可以激发短时程突触增强。突触前[Ca^2 ]i可以影响被抑制的突触前膜囊泡的更新,并准确编码突前和突触后信息,产生截然相反的长时程突触修（ＬＴＰ或ＬＴＤ）。     

Standardized, automated procedure for measurement of serum hemolytic complement activity

[This corrects the article on p. 1635 in vol. 16.].     

Bone marrow determination of complement dependent and complement independent cellular cytotoxicity

Adoptive transfer of both bone marrow and thymus cells is needed in lethally irradiated syngeneic mice to elicit CICC and CDCC responses against SRBC as assayed by the ⁵¹Cr-release cytotoxic test. The contribution of thymocytes to both CDCC and CICC was assessed by limiting dilution assays in BDF₁ and CDF₁ mice. Irradiated mice were reconstituted with a large number of bone marrow cells and graded limiting numbers of thymocytes, and were then immunized with SRBC. The limiting dilution plots conformed to the Poisson Model for both types of responses and both strains of mice. The numbers of thymocytes required for about 63% of the recipient spleens to be positive (i.e., the inoculum sizes containing one detectable antigen reactive unit) were similar for both CDCC and CICC ($\text{1}\text{1.8}\text{× 10}{}^7$ thymocytes) in both strains. Chi-square tests, at the 0.01 level of significance, were incompatible with the hypothesis that CICC and CDCC are independent of each other. Limiting dilutions of bone marrow cells with larger numbers of thymus cells, using CDF₁ mice, showed a non-Poisson curve for both CICC and CDCC. Chi-square values were compatible with the hypothesis of independent assortment of responses, as if the potential of the majority of precursors were restricted to either CICC or CDCC, but not to both. In contrast, BDF₁ mice showed a Poisson curve for CDCC and a non-Poisson curve for CICC. Chi-square values were also compatible with independent assortment of responses.     

Measurement of hemolytic complement and the third component of complement in nonhuman primates.

Complement, determined by hemolytic assay, and the third component of complement (C3), determined by radial immunodiffusion assay, were measured in nine nonhuman primate species. The species studied were the titi (Callicebus mollach). The sooty mangabey (Cercocebus atys), the thick-tailed galago or bushbaby (Galago crassicaudatus panganiensis), the crab-eating monkey (Macaca fascicularis), the rhesus monkey (Macaca mulatta), the bonnet monkey (Macaca radiata), the stumptailed macaque (Macaca speciosa), the yellow baboon (Papio cynocephalus), and the black-and-red tamarin (Saguinus nigricollis). Both sheep and bovine erythrocytes were used in the hemolytic complement assays. With the sheep erythrocyte system, sera from four species (yellow baboon, sooty mangabey, bonnet monkey, black-and-red tamarin) had similar titers with both antibody sensitized and non-sensitized erythrocytes. In contrast, the titers obtained using sensitized bovine erythrocytes was always higher than the values obtained using non-sensitized bovine erythrocytes. In all species, the titers for non-sensitized sheep erythrocytes was higher than the titer for non-sensitized bovine erythrocytes. When the species were compared for cross reactivity using the radial immunodiffusion assay for human C3, the rhesus monkey showed the strongest cross reaction; the thick-tailed galago, a prosimian, showed no detectable cross reactivity; and the other species examined showed intermediate degrees of reactivity.     

Structural requirements for the complement regulatory activities of C4BP

C4b-binding protein (C4BP) is a regulator of the classical complement pathway C3 convertase (C4bC2a complex). It is a disulfide-linked polymer of seven alpha-chains and a unique beta-chain; the alpha- and beta-chains are composed of eight and three complement control protein (CCP) domains, respectively. To elucidate the importance of the polymeric nature of C4BP and the structural requirements for the interaction between C4b and the alpha-chain, 19 recombinant C4BP variants were created. Six truncated monomeric variants, nine polymeric variants in which individual CCPs were deleted, and finally, four variants in which double alanine residues were introduced between CCPs were functionally characterized. The smallest truncated C4BP variant still active in regulating fluid phase C4b comprised CCP1-3. The monomeric variants were less efficient than polymeric C4BP in degrading C4b on cell surfaces. All three N-terminal CCP domains contributed to the binding of C4b and were important for full functional activity; CCP2 and CCP3 were the most important. The spatial arrangements of the first CCPs were found to be important, as introduction of alanine residues between CCPs 1 and 2, CCPs 2 and 3, and CCPs 3 and 4 resulted in functional impairment. The results presented here elucidate the structural requirements of individual CCPs of C4BP, as well as their spatial arrangements within and between subunits for expression of full functional activity.     

Calcium enhances the hemolytic action of bile salts

The lysis of human erythrocytes by bile salts in buffer containing isotonic saline was dramatically enhanced by the addition of 5-10 mM calcium chloride. All bile acids tested showed this effect, with a marked increase in lysis occurring at 0.75 mM for deoxycholate, 1 mM for chenodeoxycholate, 2.5 mM for ursodeoxycholate and 5.5 mM with cholate in the presence of 10 mM calcium chloride. The effect appeared to be specific for calcium; strontium chloride and magnesium chloride gave no stimulatory effect. The increased lysis of the erythrocytes in the presence of 1 mM deoxycholate and 1-10 mM calcium chloride was not associated with increased uptake of the bile salt by the cells (measured with [14C]deoxycholate). Using erythrocytes previously labelled with [3H]cholesterol, there was no evidence of an enhanced removal of that membrane component in the presence of calcium and deoxycholate, compared to deoxycholate alone. The sensitivity of the cells to the effect of calcium in the presence of 1 mM deoxycholate increased with the length of time of their storage at 4 degrees C. The sensitivity returned to that of fresh cells after incubation at 37 degrees C with 30 mM adenosine plus 25 mM glucose, but this treatment did not further diminish the lysis. Lysis in the presence of 10 mM calcium chloride and 1 mM deoxycholate was partially blocked by increasing the KCl concentration at the expense of NaCl. The maximum effect occurred with a buffer comprising 100 mM KCl/50 mM NaCl. A more dramatic reduction in the lysis followed the incorporation of the calcium chelator, quin2, into the cells. The lysis induced by 1 mM deoxycholate in the presence of calcium was reduced by 80% in quin-2-loaded cells compared to controls. The data suggest that bile acids can promote the influx of calcium into erythrocytes, leading to lysis as a result of the efflux of intracellular potassium and/or the uptake of sodium from the incubation medium. The data further suggest that cellular effects may occur at lower bile acid concentrations than that thought to be required for detergent damage.     

A Bactericidal substance not dependent on complement

Сыворотка против эф фективные вещества Грам-положительных микроорганизмов (bactericidin, Б ета lysin) отличается от с истемы действитель ным по отношению к Гр ам-отрицательные ор ганизмы (антитела / properdin-д ополнение) в основно м в дополнение, не тре бующие для своей фун кции. Покушение на пр ямой дифференциации bactericidin из properdin выступил погло щения сыворотки кро лика эффективного против Грам-отрицат ельные микрооргани змы ((itSalmonella paratyphi) B) и Grampositive микроорг анизмы (ее (Bacillus anthracis)) исполь зуя zymosan (подготовка RP сыв оротке крови); это, одн ако, разрушила его ба ктерицидные в силу в отношении обоих мик роорганизмов. Погло щение бактерий, прио становления ((itSalmonella paratyphi) B), а д обыча с производств а хлороформа же резу льтат. Каолин полнос тью удаляется из bactericidin с ыворотке крови, но не которые эффективно сть противSalmonella paratyphiбыл со хранен. Bactericidin был полнос тью дифференцирова нной от антител / properdin-доп олнять Система, когд а в сыворотке была по крыты на активирова нный уголь. Даже в неб ольших количествах активированный уго ль (4 мг. / л мл. сыворотка) полностью ликвидир ованы последствия с ыворотка против (itBacillus anthracis) (т.е. bactericidin), в то время как фу нкция сыворотка про тив itSalmonella paratyphi () была полност ью сохранена. В автор ы мнению этих экспер иментов продемонст рировать полную раз ницу между сыворотк е бактерицидная сис тема против Грам-пол ожительных микроор ганизмов (bactericidin) и антите ла-дополнять Систем а активного против Г рам-отрицательная м икроорганизмов, кот орое идентично с properdin-до полнить систему опи сываемых Pillemer.     

Essential requirements for robust signaling in Hfq dependent small RNA networks

Bacteria possess networks of small RNAs (sRNAs) that are important for modulating gene expression. At the center of many of these sRNA networks is the Hfq protein. Hfq's role is to quickly match cognate sRNAs and target mRNAs from among a large number of possible combinations and anneal them to form duplexes. Here we show using a kinetic model that Hfq can efficiently and robustly achieve this difficult task by minimizing the sequestration of sRNAs and target mRNAs in Hfq complexes. This sequestration can be reduced by two non-mutually exclusive kinetic mechanisms. The first mechanism involves heterotropic cooperativity (where sRNA and target mRNA binding to Hfq is influenced by other RNAs bound to Hfq); this cooperativity can selectively decrease singly-bound Hfq complexes and ternary complexes with non-cognate sRNA-target mRNA pairs while increasing cognate ternary complexes. The second mechanism relies on frequent RNA dissociation enabling the rapid cycling of sRNAs and target mRNAs among different Hfq complexes; this increases the probability the cognate ternary complex forms before the sRNAs and target mRNAs degrade. We further demonstrate that the performance of sRNAs in isolation is not predictive of their performance within a network. These findings highlight the importance of experimentally characterizing duplex formation in physiologically relevant contexts with multiple RNAs competing for Hfq. The model will provide a valuable framework for guiding and interpreting these experiments.     

Calcium requirements for exocytosis do not delimit the releasable neuropeptide pool

Recently, it was proposed that secretory vesicles have widely varying Ca(2+) thresholds for exocytosis. This model can explain adaptation of secretory responses and predicts that incomplete release is a consequence of insufficient Ca(2+). However, membrane capacitance-based measurements have not supported varying Ca(2+) thresholds. Here, Green Fluorescent Protein (GFP) imaging is used to test whether a Ca(2+) limitation determines the size of the releasable neuropeptide pool in differentiated PC12 cells. We show that depolarization-evoked release correlates with failure to sustain fully elevated [Ca(2+)](i). However, this is coincidental because release remains incomplete when [Ca(2+)](i) is maintained at a relatively high level by application of an ionophore or by dialysis with a buffered Ca(2+) solution. Furthermore, in contradiction with the existence of high threshold vesicles, stimulating maximal release with moderate [Ca(2+)](i) prevents secretory responses to large increases in [Ca(2+)](i) induced by photolysis of the caged dimethoxynitrophenyl-EGTA-4 (DMNPE-4). Thus, optical measurements show that limited capacity for neuropeptide release in response to depolarization is not caused by an insufficient duration of [Ca(2+)](i) elevation or by variation among vesicles in Ca(2+) sensitivity for exocytosis.     

Calcium dependent modulation of fast axonal transport

The highly differentiated structure of the neuron poses special problems for the intracellular movement of molecules throughout the cell. Molecular transport distances from the synthesizing neuron cell body along the axon (which has no substantial synthetic capabilities) to the axon terminal are very great. The transported substances, transport support structures, translocator motors, and control elements are currently the focus of intense research. Interruption of this flow of molecules could have disastrous effects upon the cell and ultimately the organism resulting in neuropathological conditions. Calcium plays a critical role in modulating fast-axonal transport (FAT) speeds. Before discussing the effect of calcium on FAT, we summarize our broad perspective on the role of axonal transport in neurologic disease.