Geitler's “Plattenband” in the diatomSynedra cf.ulna in the light of TEM investigations

The ultrastructure of the diatomSynedra cf.ulna was examined paying special attention to the Plattenband (platelet band). This structure was first described byGeitler in 1948 on the basis of LM observations and denotes a linear array of dictyosomes along the apical axis of the cell. The present investigation confirmsGeitler's observations in all essential details and demonstrates that the dictyosomes are arranged along polarized nuclear extensions running towards the cell poles. Laterally the extensions are accompanied by a number of microtubules. In large cells the total length of the nucleus thus may reach 400 µm and more. Since only the central part of the nucleus is DNA-positive with DAPI and acridine orange, the nuclear nature of the backbone of the Plattenband cannot be recognized by LM techniques. TEM investigation of serial apical and transapical sections, however, prove unambiguously the identity with extended parts of the nucleus.Dedicated to Prof. DrLothar Geitler on the occasion of his 90th birthday.     

Effects of hypoxia and intracellular iron chelation on hypoxia-inducible factor-1α and -1β in the rat carotid body and glomus cells

The present investigation provides for the first time, unambiguous information on the occurrence of hypoxia-inducible factors (HIF-1 and HIF-1 proteins) in normoxia (Nx) and their interaction with hypoxia (Hx) and intracellular Fe²⁺ chelation in the rat carotid body (CB) glomus cells. HIF-1 bound to HIF-1 translocated into the nucleus is identified on the basis of immunohistochemistry and immunofluorescence. In Nx, a weak expression of HIF-1 was observed in CB glomus cells. However, exposure of CB and glomus cells to Hx (Po₂7 Torr) and Nx with ciclopirox olamine (CPX, 5 M) for 1 h showed a significant (P<0.001) increase in HIF-1 protein. The CBs and glomus cells exposed to Nx, Hx, and Nx with CPX showed a constant level of HIF-1 protein expression. HIF-1 subunit is continuously synthesized and degraded under normoxic conditions, while it accumulates rapidly following exposure to low oxygen tensions. Hydroxylation of HIF-1 by prolyl hydroxylase for proteasomal degradation was dependent on iron, 2-oxoglutarate, and oxygen concentration. The intracellular iron that acts as a cofactor for prolyl hydroxylase activity belongs to the labile iron pool and can be easily chelated. Thus, chelation of intracellular labile iron by CPX in Nx significantly increased HIF-1 in CB glomus cells. Thus, the results are consistent with the hypothesis that HIF-1 which is present in the glomus cells translocates to the nucleus during exposure to Hx and to CPX in Nx.     

Localization of sulphated polysaccharides by X-ray microanalysis in Laminaria saccharina

Summary Analysis of thick sections of Laminaria thallus by X-ray electron microscope microanalysis using EMMA 4 shows that sulphated polysaccharide is located in secretory canals, the middle lamella of cell walls and on the thallus surface. Sulphation occurs in the Golgi bodies of specialised secretory cells. These results confirm previous reports using histochemical and autoradiographic techniques.     

T lymphocytes can mediate lysis of autologous melanoma cells by multiple mechanisms: Evidence with a single T cell clone

Summary The specificity analysis of a CD3⁺, WT31⁺, CD8⁺ cytotoxic T lymphocyte (CTL) clone (CTL 49), isolated from peripheral blood lymphocytes of a melanoma patient (no. 665) after mixed lymphocyte culture with an HLA-A2⁺ allogeneic lymphoblastoid cell line (VSKB-LCL), revealed that CTL 49 could lyse, in addition to HLA-A2⁺ lines, autologous HLA-A2^– melanoma (Me665/2) and K562 targets. Killing of VSKB-LCL, but not of Me665/2, could be inhibited by anti-CD3 and by anti-HLA-A2 antibodies or by modulation of the CD3 complex. Cold-target competition studies showed that K562, but not VSKB-LCL, could compete with Me665/2 for lysis by CTL 49. However, unlike K562, Me665/2 could be lysed by CTL 49 in a Ca²⁺-independent fashion in 4 h and 18 h assays. CTL 49 expressed mRNA specific for tumor necrosis factor (TNF) and, to a lesser extent, for lymphotoxin (TNF). Exposure of the clone to anti-CD3 antibodies induced the expression of interferon(IFN)--specific and the up-regulation of TNF- and TNF-specific mRNA. Antibodies to TNF, TNF and IFN reduced the lysis of Me665/2, but not of K562, by CTL 49 in 18-h cytotoxic assays. Antibodies to TNF and to IFN almost completely inhibited the lysis seen on Me665/2 (but not on K562), in 96-h assays, by supernatants isolated from VSKB-LCL- or anti-CD3-stimulated CTL 49 cells. Taken together, these data indicate that major-histocompatibility-complex-independent lysis of autologous tumor cells and of natural killer reference targets by the same alloreactive T cell clone are activities related at the level of target recognition but distinct at the level of the lytic hit. Thus, efficient lysis of autologous tumor cells results from a complex mechanism based upon direct effector-target interaction as well as on cytokine-mediated cytolytic effects.     

Brevibacterium divaricatum: Influence of nutritional conditions on the wall composition and release of uncross-linked peptidoglycan chains into medium

Purified cell walls, originating from penicillin-treated (3 g/ml, 1 h) and-untreated Brevibacterium divaricatum cells grown on complex (CM) and glucose minimal medium with (MM) or without (Ca-free MM) calcium carbonate, were isolated by two procedures. Electron micrographs and chemical analysis revealed no differences between identically isolated walls with respect to the presence or absence of either penicillin or calcium carbonate in the glucose growth medium. On the contrary, the appearance and peptidoglycan content of the walls was greatly dependent on the procedure used for their isolation and the walls isolated from the cells grown on complex medium contained more materials other than peptidoglycan. It was shown that the presence of calcium carbonate in the glucose minimal medium was essential for accumulation of large amounts of peptidoglycan chains into the medium. Penicillin-induced interruption of cell wall synthesis was prerequisite for manifestation of the calcium carbonate stimulating effect.Abbreviations CM complex medium - MM chemically defined minimal medium based on glucose and containing calcium carbonate - Ca-free MM MM modified only by the omission of calcium carbonate - ET-walls Enzyme treated walls - FPR-walls French press-ruptured walls     

Activities ofβ-glucanases andβ-glucosidases during blastospore formation inSaccharomycopsis fibuligera

Summary The activities of three glycosidases, -glucosidase and (1,3)- and (1,6)-glucanases have been monitored during growth and blastospore formation inSaccharomycopsis fibuligera. The assays were carried out on the cell-free culture and in a cell-free extract and a wall autolysate preparation from the growing cells. In complex medium containing 1% glucose an increase in the level of all three enzymes was associated with the transition from mycelium to blastospores. When the level of glucose was increased to 5% blastospore formation was repressed and the level of -glucanases only increased at the end of the fermentation. The -glucosidase activity increased during the growth phase. In a defined medium in which slow growth in a wholly yeast-like form was observed, growth was not associated with a high level of -glucanase activity.     

Correlated and uncorrelated fitness landscapes and how to tell the difference

The properties of multi-peaked fitness landscapes have attracted attention in a wide variety of fields, including evolutionary biology. However, relaively little attention has been paid to the properties of the landscapes themselves. Herein, we suggest a framework for the mathematical treatment of such landscapes, including an explicit mathematical model. A central role in this discussion is played by the autocorrelation of fitnesses obtained from a random walk on the landscape. Our ideas about average autocorrelations allow us to formulate a condition (satisfied by a wide class of landscapes we call AR(1) landscapes) under which the average autocorrelation approximates a decaying exponential. We then show how our mathematical model can be used to estimate both the globally optimal fitnesses of AR(1) landscapes and their local structure. We illustrate some aspects of our method with computer experiments based on a single family of landscapes (Kauffman's N-k model), that is shown to be a generic AR(1) landscape. We close by discussing how these ideas might be useful in the tuning of combinatorial optimization algorithms, and in modelling in the experimental sciences.     

Isolation and culture of cotton ovule epidermal protoplasts (prefiber cells) and analysis of the regenerated wall

Culture protocols were developed and characterization of the regenerated cell walls was performed for protoplasts of cotton (Gossypium hirsutum L., L., var. Acala SJ-2) ovule epidermal cells. This work was undertaken in order to extend studies concerning nutritional effects and regulation of nucleotide sugar incorporation into -1,3- and -1,4-glucan components of cotton fiber cell walls. Protein and carbohydrate polymers and recovered from the culture medium. Analysis of a cellular fraction indicated that the majority of ¹⁴C incorporated from [¹⁴C] glucose was present in the hot-water-soluble fraction of the cells. The majority of label incorporated into cell wall material could be solubilized with acetic-nitric reagent, indicative of noncellulosic material, and characterized as -1,3-linked glucans. Only 5 to 15% of the regenerated cell wall could be characterized as -1,4-linked glucose indicative of cellulose.     

Cytochemistry and biochemistry of acid phosphatases

Summary Acid phosphatases of the rat ventral prostate were studied cytochemically using different substrates. The results were compared to findings on isoelectric focussing gels stained for acid phosphatase activity. This is a highly specific and reproducible method which allows the distinction between secretory androgen-dependent and lysosomal acid phosphatases. Activity of lysosomal acid phosphatase was increased after castration, while the activity of the secretory enzyme gradually decreased after androgen deprivation. None of the substrates tested was selectively hydrolyzed by either secretory or lysosomal acid phosphatase. Phenylphosphate, creatine phosphate and choline phosphate were found to be inappropriate substrates for histochemical purposes, however, reproducible results were obtained with -naphthylphosphate, -glycerophosphate and p-nitrophenylphosphate. The method of isoelectric focussing (pH range 4.0–8.0) of enzymes with subsequent histochemical staining demonstrated lysosomal enzymes at pH 7.9 and 8.2 respectively. Small amounts of identical enzymes were found in liver, kidney, blood or epididymis. Secretory acid phosphatases were focussed at pH 5.5, 5.6, 5.65 and 7.15. Similar enzymes have been identified in epididymis, kidney, liver and pancreas. These results indicate that 1) at present no specific substrate for prostatic secretory or lysosomal acid phosphatases is available and 2) that no prostate-specific prostatic acid phosphatase (PAP) exists in the rat ventral prostate.Supported by the Deutsche Forschungsgemeinschaft (Au 48/6)     

Cell wall-lytic activity in Chlorella fusca

The soluble fraction of homogenates of synchronous Chlorella fusca was tested for carbohydrate-lyzing activities. With isolated cell walls and -1,4-mannan or carboxymethyl cellulose as substrates, a sharp increase in activity occurred shortly before release of the daughter cells followed by a decline during release. The lytic activities were partially purified by ammonium sulphate precipitation and analyzed by gel filtration on a calibrated column. Apparent molecular weights were 27,000 for cell wall autolysin(s) and -1,4-mannanase, 36,000 for carboxymethyl cellulase and 70,000 for another -1,4-mannanase. Incubation of isolated cell walls with an enzyme preparation purified by ammonium sulphate precipitation resulted in release of up to 70% of the cell wall carbohydrate as monosaccharide, predominantly mannose and glucose. The carbohydrate released in vivo into the culture medium shortly before and during liberation of the daughter cells consisted largely of polymeric material with rhamnose, fucose and mannose as main constitutents. Upon poisoning the cells with NaN₃ or carbonyl cyanide p-trifluoromethoxy-phenylhydrazone, however, a monosaccharide fraction consisting of mannose and glucose was predominant in the medium. It is suggested that the major products of cell wall lysis in vivo are monosaccharides which are rapidly taken up and metabolized by the developing daughter cells in an energy-dependent manner.     

Targeted ablation of gonadotrophs in transgenic mice depresses prolactin but not growth hormone gene expression at birth as measured by quantitative mRNA detection

We previously reported that transgenic ablation of gonadotrophs results in impaired development of cells immunostainable for prolactin (PRL) but not of cells immunostainable for growth hormone (GH) or proopiomelanocortin (POMC) in pituitary of newborn mice. The question remained whether this reduction in PRL protein is a reflection of reduced PRL mRNA expression, or whether this regulation is only situated at the translational level. We therefore generated a new series of transgenic mice in which gonadotrophs were ablated by diphtheria toxin A targeting, and analyzed hormone mRNA levels instead of hormone protein around the day of birth. Pituitary mRNA expression levels of luteinizing hormone- (LH), PRL and GH were quantified using real-time TaqMan RT-PCR. Of the 13 transgenic mice obtained, 8 showed a clear-cut reduction (ranging from 62 to 98%) in LH mRNA levels. PRL mRNA values were significantly reduced in the transgenic mice (p=0.0034), while GH mRNA expression was unaffected (p=0.93). An additional observation was that female newborn mice produce 5 times more LH mRNA than male mice whereas no sex difference was observed for expression levels of PRL and GH mRNA. Moreover, in the wild-type mice, LH mRNA expression was 20-fold higher than GH mRNA expression which in turn was 500- to 1,000-fold higher than PRL mRNA expression, suggesting a low expression level of the PRL gene at birth. In conclusion, the present data support the hypothesis that embryonic development of PRL gene expression is stimulated by gonadotrophs.     

Beziehungen Zwischen Chlorophyllgehalt und Chloroplastenstruktur in Einer Chlorina-Mutante von Arabidopsis Thaliana (L.) Heynh

Zusammenfassung In einer chlorina-Mutante (ch ₃) von Arabidopsis thaliana war der Gesamtchlorophyllgehalt auf etwa 1/10 der Menge in der grünen Normalform vermindert und zugleich das Verhältnis der Chlorophylle a/b wesentlich erhöht. Die parallel dazu durchgeführte elektronenmikroskopische Analyse der Chloroplasten zeigte eine starke Reduktion des Membrankörpers und vor allem eine Hemmung der Granadifferenzierung, die in bestimmten Stadien zur Ausbildung völlig abnormer Schichtstapel führte. In Abhängigkeit von der Entwicklung sowie von Umweltbedingungen änderten sich Chlorophyllgehalt und Chloroplastenstruktur in der Mutante in verschiedener Richtung. Diese Änderungen durch endogene und exogene Faktoren wurden vergleichend untersucht, um Einblick in die Beziehungen zwischen Pigmentmangel und Strukturstörung der Chloroplasten zu gewinnen.Das charakteristisch abweichende a/b-Verhältnis (d.h. der relative Chlorophyll b-Mangel) in der Mutante normalisierte sich unter bestimmten Versuchsbedingungen auf zweierlei Weise: 1. Unter dem Einfluß einer Dauerbeleuchtung erhöhte sich mit der Gesamtchlorophyllmenge auch der relative Chlorophyll b-Gehalt, offenbar weil eine Schwellenkonzentration von Chlorophyll a überschritten und damit eine normale Synthese von Chlorophyll b (aus Chlorophyll a?) möglich wurde. 2. Bei Kultur von Keimlingen unter 8:16 Std Licht-Dunkelwechsel oder bei alten, ausbleichenden Rosettenblättern beruhte die Normalisierung des a/b-Verhältnisses auf einem relativ schnelleren Abbau von Chlorophyll a.Im elektronenmikroskopischen Querschnitt nahmen Anzahl, Differenzierung und Schichtung der Thylakoide mit steigender Gesamtchlorophyllmenge zu, während die Membranen bei abnehmendem Chlorophyllgehalt abgebaut wurden. Jedoch ließ sich keine Beziehung der Chloroplastenstruktur zum absoluten oder relativen Chlorophyll b-Gehalt erkennen. Daß Chlorophyll b für den Aufbau einer normalen Chloroplastenfeinstruktur tatsächlich nicht nötig ist, ergab die elektronenmikroskopische Analyse einer anderen, Chlorophyll b-freien Arabidopsis-Mutante (ch ¹), in deren Chloroplasten Membrankörper mit typischen Granastapeln nachgewiesen werden konnten.

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Relations between chlorophyll content and chloroplast fine structure in a Chlorina mutant of Arabidopsis thaliana (L.) Heynh

Summary In a chlorina mutant (ch ₃) of Arabidopsis thaliana the total chlorophyll content was reduced to about 1/10 the amount of the green wild-type. At the same time the ratio of chlorophyll a/b was markedly increased. The electron microscopic analysis of the chloroplasts showed a considerable reduction in the membrane formation. Especially an inhibition of the grana differentiation was apparent, leading in some stages to an entirely abnormal thylakoid stacking. Depending on developmental as well as environmental conditions, chlorophyll content and chloroplast structure varied in the mutant in a different manner. These changes induced by endogenous and exogenous factors were comparatively investigated in order to reveal the relations between pigment deficiency and structural disturbances in the chloroplasts.The characteristically deviating a/b-ratio (i.e. the relative chlorophyll b deficiency) in the mutant was normalized under particular experimental conditions in two different ways: 1. Under the influence of continous illumination the relative chlorophyll b content increased together with the total amount of chlorophyll, obviously because a threshold of concentration of chlorophyll a was exceeded and thus a normal synthesis of chlorophyll b (from chlorophyll a?) became possible. 2. In cultures of seedlings under an 8:16 hr light-dark-succession as well as in old bleaching rosette leaves the normalization of the a/b-ratio was due to a relatively more rapid destruction of chlorophyll a.In electron microscopic cross sections the number, differentiation, and stacking of the thylakoids increased with an increasing total amount of the chlorophylls, while with decreasing chlorophyll content the membranes evidently disintegrated. However, no relation between chloroplast differentiation and absolute or relative chlorophyll b content could be established. Indeed, the proof that chlorophyll b is actually not necessary for the formation of a normal chloroplast structure was furnished by the electron microscopic analysis of another, chlorophyll b-less mutant (ch ¹), in the chloroplasts of which a membrane differentiation with typical grana piles could be demonstrated.

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Für die Gewährung eines Stipendiums an den ersten Autor, das diese Arbeit ermöglichte, sei der Alexander von Humboldt-Stiftung, Bad Godesberg, verbindlichst gedankt.     

Clonal heterogeneity in delayed decrease of plating efficiency of irradiated HeLa cells

The clonogenic potential of progeny of irradiated HeLa cells was studied at different times after single doses of 4–12 Gy. The dose-dependent decrease in plating efficiency that was observed resembled the effect termed delayed lethal mutation by Seymour et al. (1986). The effect decreased with time after irradiation. Individual clones of irradiated and non-irradiated cells were isolated, expanded and replated 5 weeks after irradiation, i.e., after between 200000 and 1000 000 progeny had formed from the individual parent cell. The plating efficiency of progeny of unirradiated cells did not vary much, whereas clonal progeny of irradiated cells had plating efficiencies ranging from 3% to 76%. The plating efficiency was not related to the cell number in the original clone.     

ATPase-dependent energy spilling by the ruminal bacterium,Streptococcus bovis

When the ruminal bacterium Streptococcus bovis was grown in batch culture with glucose as the energy source, the doubling time was approximately 21 min and the rate of bacterial heat production was proportional to the optical density (1.72 W/g protein). If exponentially growing cultures were treated with chloramphenicol, there was a decline in heat production, but the rate was greater than 0.30 W/g protein even after growth ceased. Since there was no heat production after glucose depletion, this growth-independent energy dissipation (spilling) was not simply due to endogenous metabolism. Stationary cells which were washed and incubated in nitrogen-free medium containing an excess of glucose produced heat at a rate of 0.17 W/g protein. Monensin and tetrachlorosalicylanilide (TCS), compounds which facilitate an influx of protons, caused a more than 2-fold increase in heat production. Dicyclohexylcarbodiimide (DCCD) virtually eliminated growth-independent heat production regardless of the mode of growth inhibition. Because DCCD had little effect on the glucose phosphotransferase system, it appeared that the combined action of proton influx and the membrane bound F₁F₀ proton ATPase was responsible for energy spilling.Abbreviations DCCD dicyclohexylcarbodiimide - TCS tetrachlorosalicylanilide     

Evaluation of monitoring approaches and effects of culture conditions on recombinant protein production in baculovirus-infected insect cells

The baculovirus infection process ofSpodoptera frugiperda (Sf9) insect cells in oxygen-controlled bioreactors in serum-free medium was investigated using a recombinantAutographa californica (AcNPV) virus expressing -galactosidase enzyme as a model system. A variety of monitoring techniques including trypan blue exclusion, fluorescent dye staining, oxygen uptake rate (OUR) measurements, and glucose consumption were applied to infected cells to determine the best way of evaluating cell integrity and assessing the course of baculovirus infection. The metabolism of newly-infected cells increased 90% during the first 24 hours, but as infection proceeded, and cells gradually succumbed to the baculovirus infection, the cytopathic effect of the baculovirus on the cells became evident. Oxygen and glucose uptake rate measurements appeared to more accurately assess the condition of infected cells than conventional trypan blue staining, which tended to overestimate cell viability in the mid stages of infection. The optimal harvest time varied, depending on which technique — SDS-PAGE, chromogenic (ONPG) or fluorometric (C₁₂FDG) — was used to monitor -galactosidase production. Specific -galactosidase production was found to be insensitive to a wide range of culture dissolved oxygen tensions, whereas resuspending cells in fresh medium prior to infection increased volumetric productivity approximately two-fold (800,000 units -galactosidase/ml) compared to cultures infected in batch mode and allowed successful infections to occur at higher cell densities.Abbreviations ONPG ortho-phenyl 2--D-galactopyranoside - OUR oxygen uptake rate (-mol O₂/liter/hour) - q_glucose specific glucose uptake rate (mg glucose/10⁶cell/hour) - q_glutamine specific glutamine uptake rate (mg glutamine/10⁶cell/hour) - qO₂ specific oxygen uptake rate (-mol O₂/10⁶cell/hour) - MOI virus multiplicity of infection (viral plaque forming units/cell)     

Differential accumulation of mRNAs encoding extracellular and intracellular PR proteins in tomato induced by virulent and avirulent races of Cladosporium fulvum

Tomato leaves infected by the fungal pathogen Cladosporium fulvum contain several types of intracellular and extracellular pathogenesis-related (PR) proteins. Previously, we reported the purification and serological characterization of five extracellular PR proteins: P2, P4, P6, a chitinase and a -1,3-glucanase [22, 23]. Here we describe the purification of a basic intracellular 33 kDa -1,3-glucanase and the isolation and characterization of cDNA clones encoding the two extracellular P14 isomers P4 and P6, the extracellular acidic -1,3-glucanase and a basic 35 kDa -1,3-glucanase, different from the purified 33 kDa protein. Southern blot analysis demonstrated that tomato PR proteins are not encoded by large gene families, as is the case in tobacco. The number of genes corresponding to each protein was estimated to vary between one and three. A northern blot analysis indicated that the mRNAs for the extracellular PR proteins (P4, P6 and acidic -1,3-glucanase) accumulate to similar levels in compatible and incompatible tomato-C. fulvum interactions, although the maximum level of expression is reached much faster in the incompatible interaction. On the other hand, the mRNA for the basic 35 kDa -1,3-glucanase is induced rapidly to high levels in both interactions, but declines in time to background levels only in the incompatible interaction. The relevance of this difference in relation to plant defence is discussed.     

Cytokinesis in the green alga Eremosphaera viridis: Plasmalemma formation from “open membranes”

Summary Cytokinesis in the unicellular chlorococcalean alga Eremosphaera viridis de Bary has been investigated by electron microscopy of thin sections. The new plasmalemmata of the daughter cells in this organism form centrifugally within a phycoplast. Unlike other cell division systems each new plasmalemma is formed, not by the fusion of vesicles, but rather by the fusion of open membranes which are characteristically heavily stained. Measurements of these open membranes reveal that they are 11 nm thick with a central 4,5 nm unstained portion. The possible origin of these open membranes as burst-open vesicles has been suggested from the presence of intensely straining vesicles in the vicinity of the cell equator. Calculations of vesicle and open membrane surface areas support this contention.     

Further studies on a unique T-tubular acid phosphatase in avian skeletal muscle

Summary Whith the unique observation, using conventional cytochemistry, of acid phosphatase reaction production in the T-tubules of the posterior latissimus dorsi muscle of the chicken, the possibility of andocytosis of lysosomal enzymes by muscle cells came into question. After testing the substrate specificity of this T-tubular phosphatase, it was clear that the enzyme was not 5-nucleotidase for a typical lysosomal acid phosphatase. The T-tubular enzyme hydrolysed glucose 6-phosphate and -glycerophosphate at pH 5.0 but not cytidine-5-monophosphate which was hydrolysed by dense bodies and autophagic vacuoles. The cytochemical evidence points to a mique phosphatase present on mucle cell membranes which apparently does not belong to the vacuolar apparatus of skeletal muscle and is not 5-nucleotidase.     

Distribution of pectic polysaccharides throughout walls of suspension-cultured carrot cells

Summary A monoclonal antibody (2 F 4) recognizing a conformational epitope of polygalacturonic acid was used for immunogold localization of pectins in walls of suspension-cultured carrot (D. carota L.) cells at the electron microscopic level. In microcolonies of young or mature cells, polygalacturonic acid was essentially located on the middle lamella material expanded at three-way junctions between cells or lining intercellular spaces but was not found in primary walls. Middle lamellae far from junction zones and intercellular spaces were not recognized. Largely esterified pectic polymers, only detected by the 2 F 4 antibodies after on-grid de-esterification treatment by pectin methyl esterases, were present within all primary cell walls. Golgi bodies and associated vesicles were also labeled by the 2 F 4 antibodies only after de-esterification treatment, which indicates that pectic polymers are synthesized and secreted in a highly esterified form. A decrease of pectin esterification, which results probably from an in situ enzymatic de-esterification of the pectic polymers of the primary walls, was observed in senescent cells. These results are discussed in relation to biochemical analyses showing changes of the methyl ester content of pectins during the cell-wall growth.     

True cellulase production by Clostridium thermocellum grown on different carbon sources

Summary Clostridium thermocellum produced different levels of true cellulase (Avicelase) depending on the carbon source used for growth. In defined medium with fructose, the cellulase titer was seven times higher than with cells growing on cellobiose and four times higher than cells growing with glucose. During the lag phase on fructose, the differences were even more dramatic, i.e. 60 times higher than in cells growing on cellobiose and 40 times that of cells lagging or growing in glucose. In an attempt to detect factors that might contribute to these differences, we considered intracellular ATP, chemical potential (pH), electrical potential (Y), proton motive force (p), growth rate, and rates of uptake of inorganic phosphate and sugars. We noted a direct correlation between cellulase production and intracellular ATP levels and an inverse relationship of cellulase production with Y and p values. It thus appears that cellulase is best produced by cells high in ATP and low in Dp and its electrical component DY. There was no obvious relationship between the cellulase titer and the other parameters. Although the physiological significance of such correlations is unknown, the data suggest that further investigation is warranted.