Coordination dynamics of heme-copper oxidases. The ligand shuttle and the control and coupling of electron transfer and proton translocation

Results are presented which, taken with evidence developed by others, suggest a general mechanism for the entry and binding of exogenous ligands (including O₂) at the binuclear site (Cu_B Fe _a3) of the heme-copper oxidases. The mechanism includes a ligand shuttle wherein the obligatory waystation for incoming ligands is Cu_B and the binding of exogenous ligands at this site triggers the exchange and displacement of endogenous ligands at Fe _a3. It is suggested that these ligand shuttle reactions might be functionally important in providing a coupling mechanism for electron transfer and proton translocation. Scenarios as to how this might happen are delineated.     

Concluding remarks

On the basis of symposium contributions onChlorella, Hibbertia, Eucalyptus, Ambrosia and on numerical approaches some fundamental problems of (bio)systematics, evolution, and taxonomic categories are discussed: Methods available for analysing affinities; conflicting evidence from phenetic, biochemical, cytogenetic and other analyses; further classification problems in cases of intermediacy, etc. While sibs of various levels and their natural hierarchy often can be objectively defined, this appears impossible for particular taxonomic levels itself (e. g. species). A single objective taxonomic system of organisms is unrealistic. Certain guiding lines for relative and practicable concepts of species and genus are proposed.Presented at the symposium Speciation and the Species Concept during the XIIth International Botanical Congress, Leningrad, July 8, 1975.     

Average phase difference theory and 1∶1 phase entrainment in interlimb coordination

The dynamics of coupled biological oscillators can be modeled by averaging the effects of coupling over each oscillatory cycle so that the coupling depends on the phase difference between the two oscillators and not on their specific states. Average phase difference theory claims that mode locking phenomena can be predicted by the average effects of the coupling influences. As a starting point for both empirical and theoretical investigations, Rand et al. (1988) have proposed d/dt= — K sin ), with phase-locked solutions =arcsin( /K), where is the difference between the uncoupled frequencies and K is the coupling strength. Phase-locking was evaluated in three experiments using an interlimb coordination paradigm in which a person oscillates hand-held pendulums. was controlled through length differences in the left and right pendulums. The coupled frequency _c was varied by a metronome, and scaled to the eigenfrequency _v of the coupled system K was assumed to vary inversely with _c. The results indicate that: (1) and K contribute multiplicatively to (2) =0 or = regardless of K when =0; (3) 0 or regardless of when K is large (relative to ); (4) results (1) to (3) hold identically for both in phase and antiphase coordination. The results also indicate that the relevant frequency is _c/_v rather than _c. Discussion high-lighted the significance of confirming =arcsin(/K) for more general treatments of phase-locking, such as circle map dynamics, and for the 11 phase-entrainment which characterizes biological movement systems.     

Identification of a cDNA encoding a plant Lewis-type alpha1,4-fucosyltransferase

Recently, it has been found that plants, including tomato (Lycopersicon esculentum), express the Lewis-a epitope, Gal1,3(Fuc1,4)GlcNAc, on some N-glycans. By searching the EST database, it was possible to identify a tomato cDNA encoding a protein, designated FucTC, of 413 amino acids with homology to plant and mammalian 1,3/4-fucosyltransferases. The cDNA was expressed in Pichia pastoris and the recombinant enzyme was found to transfer fucose from GDP-Fuc (K_m 16 M) to lacto-N-tetraose (Gal1,3GlcNAc1,3Gal1,4Glc; K_m 80 M) as well as to 1,3- and 1,4-galactosylated N-glycans. It is concluded that FucTC is responsible for the biosynthesis of Lewis-a on N-glycans in tomato.     

Harzkonservierte fossile Vogelfedern aus der untersten Kreide

Zusammenfassung Harzkonservierte Fossilien ermöglichen bei Anwendung adäquater Methoden die morphologische Analyse der Feinmerkmale bis zur Auflösungsgrenze des Lichtmikroskops, Beobachtung in verschiedenen Ebenen und Richtungen, und somit konkrete Rückschlüsse auf die Wirkung und Bedeutung der Einzelelemente und des Gesamtgefüges.Eine so eingehende funktionsmorphologische Analyse mit Berücksichtigung der Positionsvariation (graduell verschiedene Gestaltung in gesetzmäßiger Abhängigkeit von der Lage innerhalb der Gesamtfeder) der Einzelelemente wie Abzweigungs-, Knick-, Neigungswinkel, Krümmung, Länge, Dicke, Querschnitt, Dichte, Differenzierungsgrad der verschiedenen Abschnitte von Rhachis, Rami, Radii inklusive Häkchen und Cirren wird erstmals für fossile Vogelfedern geliefert (hier als Abriß zu einer dokumentarisch und thematisch ausführlicheren Darstellung in Stuttgarter Beiträge zur Naturkunde).Diese Federn entstammen der untersten Unterkreide und sind damit nur relativ wenig jünger alsArchaeopteryx. Sie weisen extrem differenzierten Aufbau auf, der auf hohe flugtechnische und wärmeisolierende Leistungsfähigkeit schließen läßt.Die hier vorgelegten funktionsmorphologischen Ermittlungen an fossilen Körperkonturfedern mögen auch zu einer intensiveren Analyse der bis jetzt stark vernachlässigten Untersuchung ganz normaler Körperfedern rezenter Vögel anregen. Erst dann, nach umfassender Kenntnis ihrer Ausgestaltung innerhalb der verschiedensten rezenten Vogelgruppen, läßt sich überzeugend begründen, ob und wieweit die hier vorgelegten Federn dieses Unterkreide-Vogels noch ursprüngliche Elemente (Plesiomorphien) oder ihnen eigene Sonderbildungen (Autapomorphien) aufweisen; das gilt sowohl für morphologische wie für funktionelle Elemente der Gesamtstruktur.

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Resin-preserved fossil bird's feathers from the Lowermost Cretaceous

Summary Parts of some feathers, originating from a single bird, were discovered in our collections of Lower Cretaceous amber from the Lebanon mountains — which, in general, contains the oldest terrestrial microfossils preserved with all morphological details.These contour feathers of the trunk, which are nearly as old as Archaeopteryx (Lowermost Cretaceous: Neocomian/Uppermost Jurassic: Kimmeridigian) were studied with magnifications of 500–900 in several levels by a special technique. (In normal fossils, i.e., impressions, the granulation of the sediment and the fossil's bulky carbon remainders cause a blurred image even at a magnification of merely 100).Special emphasis was laid on the study of the individual elements' gradual variation, depending on the respective position within the total feather (position variation). Where appropriate, an analysis of lengths, quantity, degree of differentiation, angle of inclination, break, and branching, cross-sectional view, curvature, etc. of the rhachis, rami, distal and proximal radii, barbicles, hooklets, etc. were undertaken. [Through measurements of the depth of details the effects caused by a sloping position (apparent variation) may be precisely separated from the real variation.]On the basis of such a detailed knowledge of structure and relative position a thorough functional analysis of the single elements as well as the total system is given.Principal features: The production of plain stability in the feather's center, and of flexibility in its apical and lateral rims; dispersion of forces in case of pressure or a pulling load; function of the hooklets (which donot serve as an interlocking mechanism while the feather is in the normal resting position, but function with increasing braking action only when a neighboring ramus diverges to a precisely defined extent from its resting position) including the mechanism of their unhooking; devices for the avoidance of harmful hooking into contacted parts of other feathers; production of maximal stability by minimal air resistance, and of minute chambers (<0,00001 mm³) with still air for optimal heat isolation.Apart from this abstract, further information, accompanied by numerous figures, will be given in a later paper in Stuttgarter Beiträge zur Naturkunde.

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Veränderte Fassung eines am 11. 10. 1971 gehaltenen Vortrages auf der 83. Jahresversammlung der Deutschen Ornithologengesellschaft in Bonn.     

Distribution and Density of Seals in Pil'tun Bay (Northeastern Sakhalin) in the Summer–Autumn Period

The results of seal counts performed during the summer–autumn period of the years 1999–2001 in Pil'tun Bay from onboard Zodiak motor boats and Mi-8 and Mi-8MTV helicopters have been analyzed. The information is presented on the density of seals in the bay. The heterogeneous distribution pattern of seals in the bay and the presence of intermittent migrations provide favorable conditions for foraging in young animals.     

Photosynthetic oxygen evolution: H/D isotope effects and the coupling between electron and proton transfer during the redox reactions at the oxidizing side of Photosystem II

The oxygen evolving complex (OEC) of Photosystem II (PS II) incorporates a Mn-cluster and probably a further redox cofactor, X. Four quanta of light drive the OEC through the increasingly oxidized states S0 S1 S2 S3 S4 to yield O2 during the transition S4 S0. It has been speculated that the oxidation of water might be kinetically facilitated by the abstraction of hydrogen. This implied that the respective electron acceptor is deprotonated upon oxidation. Whether YZ and X fulfill this expectation is under debate. We have previously inferred a 'chemical' deprotonation of X based on the kinetics of proton release (Haumann M, Drerenstedt W, Hundelt M and Junge W (1996) Biochim Biophys Acta 1273: 237–250. Here, we investigated the rates of electron transfer and proton release as function of the D2O/H2O ratio, the pH, and the temperature both in thylakoids and PS II core particles. The largest kinetic isotope effect on the rate of electron transfer (factor of 2.1–2.4) and the largest pH-dependence (factor of about 2 between pH 5 and 8) was found on S2 S3 where X is oxidized. During the other transitions both factors were much smaller ( 1.4). Electron transfer is probably kinetically steered by proton transfer only during S2 S3. These results corroborate the notion that X serves as a hydrogen acceptor for bound water during S4 S0. We propose a consistent scheme for the final reaction with water to yield dioxygen: two two-electron (hydrogen) transfers in series with a peroxide intermediate.     

Mode of uptake of sterol by Arthrobacter simplex

The mechanism of uptake of water-insoluble -sitosterol by a newly isolated strain of Arthrobacter simplex SS-7 was studied. The production of an extracellular sterol-pseudosolubilizing protein during growth of A. simplex on -sitosterol was demonstrated by isolating the factor from the cell-free supernatant and its subsequent purification by Sephadex G-150 column chromatography. The M _r of the purified sterol-pseudosolubilizing protein determined by SDS–PAGE was 19kDa. The rate of sterol pseudosolubilization (5.2×10^–3g l^–1h^–1) could not adequately account for the rate of sterol uptake (72×10^–3g l^–1h^–1) and the specific growth rate (56×10^–3 h^–1). However in the unfavourable growth condition, when the cells were treated with sodium azide at the level of 30–60% of MIC, the sterol pseudosolubilization accounted for nearly 74% of the total growth containing 96% free cells. Cellular adherence to substrate particles was found to play an active role in the normal growth of the strain on -sitosterol. Unlike sodium acetate-grown cells, whose surface activity was negligible (60mNm^–1), the sterol-grown cells had strong surface activity (40mNm^–1). The high lipid content and long chain fatty acids in the cell-wall of -sitosterol-grown cells probably contribute to the high sterol adherence activity of the cells.     

Developmental aspects of long-term callus culture ofVicia faba L.

Summary Vicia faba callus line (VFS 1), isolated from expiants of immature embryo, grew satisfactorily onMurashige andSkoog complete medium with 1.38 M 2,4-D, or with 0.92 M 2,4-D to which 1.0 M kinetin was added. It also grew well on the B 5 modified medium containing 2.3 M 2,4-D and 25.0 M kinetin. On the last of these media the cultures grew more uniformly and without necrosis. They also showed diminishing variation in polyploidy in favour of diploids and corresponding aneuploids (hypodiploids).After being cultured for nearly three years on MS containing 1.38 M 2,4-D, 8–33% of cultures of VFS 1 were able to regenerate roots when transferred to either MS half strength with 5.37 M NAA, or to a medium without 2,4-D, or else to media with the addition of kinetin only (in various concentrations).     

Enzymic synthesis of the trisaccharide core region of the carbohydrate chain ofN-glycoprotein

Transmannosylation from mannotriose (Man1-4Man1-4Man) to the 4-position at the nonreducing end N-acetylglucosaminyl residue ofN,N-diacetylchitobiose was regioselectively induced through the use of -d-mannanase fromAspergillus niger. The enzyme formed the trisaccharide Man1-4GlcNAc1-4GlcNAc (3.7% of the enzyme-catalysed net decrease ofN,N-diacetylchitobiose) from mannotriose as a donor andN,N-diacetylchitobiose as an acceptor. Mannobiose (Man1-4Man) was also shown to be useful as a donor substrate for the desired trisaccharide synthesis.Abbreviations Man d-mannose - (M _n) (n=1–5) -linkedn-mer of mannose - GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1–4)-2-acetamido-2-deoxy-d-glucose     

Organization of α-chain genes among Hb G-Philadelphia heterozygotes in association with Hb S, β-thalassemia,and α-thalassemia-2

The percentages of the -chain variant Hb G-Philadelphia (Hb G) or ₂ 68 AsnLys₂ were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another with lower values (33.9±3.4%, N=63). Among the newborn heterozygotes, two babies fell in the category with the higher proportion of Hb G while 16 babies gave values between 25 and 34%. Studies of -chain gene organization on the parents of one neonate with a Hb G level of 27% at birth and 37% at 8 months excluded the presence of chromosomes with triplicated -chain genes which could lead to the ^0G/ genotype. Rather, these studies on five Hb G heterozygotes from three families confirmed the linkage between Hb G and a specific type of -thalassemia-2 associated with the presence of a 16-kbp Bgl II fragment which most probably carries the ^G locus since it has been found in 19 Hb G heterozygotes studied to date. The presence of an -thal-2 heterozygosity and three -chain genes (^0G/) was confirmed among Hb G heterozygotes with lower proportions of this variant. It is likely that the even lower values found in some newborn could arise through defective assembly of ^G- dimers. The presence of an -thal-2 homozygosity and two active -chain genes, one on each chromosome (^0G/⁰), was confirmed among heterozygotes with the higher proportion of Hb G. One of each of these categories was present in each of the three families investigated. This type of variability in the number of active -chain genes due to a heterozygosity or a homozygosity for -thalassemia-2 explains the trimodality of Hb S percentages among heterozygotes and the atypical hematological or biosynthetic features among patients with -thalassemia and sickle-cell syndromes.This research was supported by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution No. 0693 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.     

Inhibitory Effects of Bombusae concretio Salicea on Neuronal Secretion of Alzheimer's β-Amyloid Peptides, a Neurodegenerative Peptide

Alzheimer's disease (AD) is characterized by the age-related deposition of -amyloid (A) 40/42 peptide aggregates in vulnerable brain regions. Multiple levels of evidence implicate a central role for A in the pathophysiology of AD. A is generated by the regulated cleavage of a = 700 amino acid A precursor protein (APP). Full-length APP can undergo proteolytic cleavage either within the A domain to generate secreted sAPP or at the N-terminal and C-terminal domain(s) of A to generate amyloidogenic A peptides. Several epidemiological studies have reported that estrogen replacement therapy protects against the development of AD in postmenopausal women. The aim of this study was to elucidate the antioxidant neuroprotective mechanism of Bombusae concretio Salicea (BC). BC was effective protectants against oxidative glutamate toxicity in the murine neuroblastoma cells (N2a) and human neuroblastoma cells (SK-N-MC). BC exhibited similar protective properties against oxidative glutamate toxicity and H₂O₂ toxicity. BC exhibited an antioxidant activity at approximately 20 g/ml. BC of 5 g/ml was ineffective in preventing the oxidative modification of LDL. The half-maximal effective concentration for BC was 16 g/ml. These results suggested that BC supplementation in elderly men may be protective in the treatment of Alzheimer's disease (AD). We report here that treatment with BC increases the secretion of the nonamyloidogenic APP fragment, sAPP and decreases the secretion of A peptides from N2a cells and rat primary cerebrocortical neurons. These results raise the possibility that BC supplementation in elderly men may be protective in the treatment of AD.     

Characterization ofCardiobacterium hominis: Antibiotic susceptibility,morphological variations,plasmid and membrane analysis

Trypticase soy broth and Mueller Hinton broth lacking yeast extract resulted in morphological alterations (pleomorphism) ofCardiobacterium hominis, as demonstrated by light and transmission electron microscopy. Outer membrane protein profiles were influenced by the composition of growth media, especially in the region of porin proteins. In contrast, the protein composition of inner membranes was not influenced by media composition.C. hominis isolates tested were susceptible to 16 -lactam antibiotics. All isolates exhibited minimal inhibitory concentrations of 1 µg/ml to 13 of the 16 -lactams tested. The changes in outer membrane profiles induced by media composition did not influence susceptibility to -lactam antibiotics. None of the isolates examined contained detectable plasmids, -lactamase, or resistant mutants. Characteristic -lactam-induced morphological defects were observed.     

The β-1,3-glucan-binding protein from the crayfish Pacifastacus leniusculus,when reacted with a β-1,3-glucan,induces spreading and degranulation of crayfish granular cells

Summary A -1,3-glucan-binding protein (GBP) was purified from crayfish plasma, and incubated with laminarin (L), a -1,3-glucan. The GBP reacted with laminarin (GBP-L) induced strong spreading and partial degranulation of isolated and separated crayfish granular haemocytes. However, neither the GBP nor laminarin alone induced any changes in the crayfish granular cells. When monolayers of granular haemocytes were incubated with 20 g of GBP-L, more than 82% of the haemocytes were affected. The activity of GBP-L on granular cells was dose-dependent and a plateau was reached at 10 g of GBP-L. The degranulation of crayfish haemocytes induced by GBP-L seemed to occur by a regulated exocytosis, since it was strongly inhibited by specific blockers of this process such as SITS or calmidazolium. Monospecific anti-GBP antibodies also totally blocked the effect of GBP-L on crayfish granular cells. Indirect immunofluoresence staining demonstrated that the GBP-L could bind to the surface of granular cells, whereas GBP did not bind or bound very weakly to the haemocyte surface.     

Conformation of the circular dumbbell d〈pCGC-TT-GCG-TT〉: Structure determination and molecular dynamics

Summary The circular DNA decamer 5-dpCGC-TT-GCG-TT-3 was studied in solution by means of NMR spectroscopy and molecular dynamics in H₂O. At a temperature of 269 K, a 50/50 mixture of two dumbbell structures (denoted L2L2 and L2L4) is present. The L2L2 form contains three Watson-Crick C-G base pairs and two two-residue loops in opposite parts of the molecule. On raising the temperature from 269 K to 314 K, the L2L4 conformer becomes increasingly dominant (95% at 314 K). This conformer has a partially disrupted G(anti)-C(syn) closing base pair in the 5-GTTC-3 loop with only one remaining (solvent-accessible) hydrogen bond between NH of the cytosine dC(1) and O6 of the guanine dG(8). The opposite 5-CTTG-3 loop remains stable. The two conformers occur in slow equilibrium (rate constant 2–20 s^–1). Structure determination of the L2L2 and L2L4 forms was performed with the aid of a full relaxation matrix approach (IRMA) in combination with restrained MD. Torsional information was obtained from coupling constants. Coupling constant analysis (³J_HH, ³J_HP, ³J_CP) gave detailed information about the local geometry around backbone torsion angles , , and , revealing a relatively high flexibility of the 5-GTTC-3 loop. The values of the coupling constants are virtually temperature-independent. Weakly constrained molecular dynamics in solvent was used to sample the conformational space of the dumbbell. The relaxation matrices from the MD simulation were averaged over r^–3 to predict dynamic NOE volumes. In order to account for the 1:1 conformational mixture of L2L2 and L2L4 present at 271 K, we also included S² factors and r^–6 averaging of the r^–3-averaged relaxation matrices. On matrix averaging, the agreement of NOE volumes with experiment improved significantly for protons located in the thermodynamically less stable 5-GTTC-3 loop. The difference in stability of the 5-CTTG-3 and 5-GTTC-3 loops is mainly caused by differences in the number of potential hydrogen bonds in the minor groove and differences in stacking overlap of the base pairs closing the minihairpin loops. The syn conformation for dC(1), favored at high temperature, is stabilized by solvation in the major groove. However, the conformational properties of the dC(1) base, as deduced from R-factor analysis and MD simulations, include a large flexibility about torsion angle .     

Uptake of sewage derived nitrogen by<Emphasis Type="Italic"> Ulva rigida</Emphasis> (Chlorophyceae) in Bahía Nueva (Golfo Nuevo,Patagonia, Argentine)

Uptake kinetics of nitrogen derived from sewage–seawater mixtures (2.5–20% v/v effluent) were determined in the laboratory for Ulva rigida (Chlorophyceae) native from Bahía Nueva (Golfo Nuevo, Patagonia, Argentine). In terms of nitrogen concentration, experimental enrichment levels varied between 53.7 and 362.3M of ammonium and between 0.77 and 6.21M of nitrate+nitrite. Uptake rates were fitted to the Michaelis–Menten equation, with the following kinetic parameters: ammonium: V_max = 591.2molg^–1h^–1, K _s=262.3M, nitrate+nitrite: V _max=12.9molg^–1h^–1, K _s=3.5M). Both nutrients were taken up simultaneously, but ammonium incorporation was faster in all cases. The results show a high capability of Ulva rigida to remove sewage-derived nitrogen from culture media. In the field, most of the nitrogen provided by the effluent would be tied up in algal biomass, supporting low nitrogen levels found at a short distance away from the source.     

Expression,purification, characterization,and deletion mutations of phosphorylase kinase γ subunit: identification of an inhibitory domain in the γ subunit

A catalytic fragment, _1-298, derived from limited chymotryptic digestion of phosphorylaseb kinase (Harris, W.R.et al., J. Biol. Chem., 265: 11740–11745, 1990), is reported to have about six-fold greater specific activity than does the subunit-calmodulin complex. To test whether there is an inhibitory domain located outside the catalytic core of the subunit, full-length wild-type and seven truncated forms of were expressed inE. coli. Recombinant proteins accumulate in the inclusion bodies and can be isolated, solubilized, renatured, and purified further by ammonium sulfate precipitation and Q-Sepharose column. Four out of seven truncated mutants show similar ( _1-353 and _1-341) or less ( _1-331 and _1-276) specific activity than does the full-length wild-type , _1-386. Three truncated forms, _1-316, _1-300, and _1-290 have molar specific activities approximately twice as great as those of the full-length wild-type and the nonactivated holoenzyme. All recombinant s exhibit similarK _m values for both substrates, i.e., about 18M for phosphorylaseb and about 75 M for MgATP. Three truncated s, _1-316, _1-300, and _1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (V _max/K _m) than that of the full-length wild-type and a 3.5- to 4.5-fold greater efficiency than that of the truncated _1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of , which is located at _301-331· _1-290, but not _1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the subunit to exhibit phosphotransferase activity. Both _1-290 and _1-300 have several properties similar to full-length wild-type , including metal ion responses (activation by free Mg²⁺ and inhibition by free Mn²⁺) pH dependency, and substrate specificities.     

The frequency of the γ chain variant AγT in different populations,and its use in evaluating γ gene expression in association with thalassemia

Summary The occurrence of the ^AT chain (i.e. ^A75 Ile Thr) in different populations was evaluated through a study of 4250 cord blood samples and blood samples from more than 350 SS¹ patients. High frequencies were observed in Italy, Yugoslavia, Turkey, Holland, but also in Japan, Vietnam, and India. The chain is (nearly) absent in the Black population of Ghana and Kenya, and low frequencies were observed in China and Australian aborigines. Only a few adult SS patients (18 out of 357) were ^AT heterozygotes. The chromosomes with the ^AT globin gene were mapped through an evaluation of the presence of 10 different restriction sites. The ^AT chromosomes from different populations were closely related and had the same subhaplotypes of [--++-+] (Hinc II 5 to ; Xmn I 5 to ^G; Hind III in ^G and ^A; Hinc II in and 3 to ), quite different from the subhaplotypes seen for ^AT negative chromosomes.² This suggests a common ancestor which may have originated in Southern Europe. An evaluation of the chain production by both chromosomes in SS patients and -thalassemia heterozygotes was possible for subjects with an ^AT heterozygosity. It was concluded that in -thalassemia trait, the chain synthesis is directed for about two-thirds by the thalassemic chromosome and for about onethird by the normal chromosome; the contribution by the normal chromosome decreases with a decrease in total chain production.This is contribution #0890 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA