Aggregate-formation by Clostridium butyricum

Summary Clostridium butyricum was grown in a glucose-limited chemostat culture at a dilution rate of 0.1 h^–1 at pH 6.0. With 0.9% w/v input glucose in the medium the cells were found to grow in suspension and glucose was fermented completely to acetate and butyrate. An increase in the input concentration of glucose resulted in increased concentrations of end-products, but not all extra glucose was consumed. It could be demonstrated that this was due to a lowering of the maximal growth rate by elevated levels of butyric acid. However, prolonged growth in the presence of high glucose concentrations led to an increase in biomass. This was caused by the selection of a variant that was less sensitive to butyrate. This variant was able to form aggregates in an anaerobic gas-lift reactor at high dilution rates. Inoculation of these aggregates in a conventional chemostat culture with high glucose input resulted in an aggregated culture that remained stable for at least 6 months, and in which all glucose was consumed. Whether the organisms grew in suspension or in aggregates was found to be determined by the concentration of butyrate. The isolation of aggregate-forming variants from chemostat cultures leads to a very simple and new type of immobilization technique.Offprint requests to: G. R. Zoutberg     

Replacement of potassium ions by ammonium ions in different micro-organisms grown in potassium-limited chemostat culture

The biomass concentration extant in potassiumlimited cultures of either Klebsiella pneumoniae or Bacillus stearothermophilus (when growing at a fixed temperature and dilution rate in a glucose/ammonium salts medium) increased progressively as the medium pH value was raised step-wise from 7.0 to 8.5. Because the macromolecular composition of the organisms did not vary significantly, this increase in biomass could not be attributed to an accumulation of storage-type polymers but appeared to reflect a pH-dependent decrease in the cells' minimum K⁺ requirement. Significantly, this effect of pH was not eviden with cultures in which no ammonium salts were present and in which either glutamate or nitrate was added as the sole nitrogen source; however, it was again manifest when various concentrations of NH₄Cl were added to the glutamate-containing medium. This suggested a functional replacement of K⁺ by NH ₄ ⁺ , a proposition consistent with the close similarity of the ionic radii of the potassium ion (1.33 Å) and the ammonium ion (1.43 Å). At pH 8.0, and with a medium containing both glutamate (30 mM) and NH₄Cl (100 mM), cultures of B. stearothermophilus would grow without added potassium at a maximum rate of 0.7 h^-1. Under these conditions the cells contained maximally 0.1% (w/w) potassium (derived from contaminating amounts of this element in the medium constituents), a value which should be compared with one of 1.4% (w/w) for cells growing in a potassiumlimited medium containing initially 0.5 mM K⁺. Qualitatively similar findings were made with cultures of K. pneumoniae; and whereas one may not conclude that NH ₄ ⁺ can totally replace K⁺ in the growth of these bacteria, it can clearly do so very extensively.     

The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes

The alpha-like globin gene cluster in rabbits contains embryonic zeta- globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.      

Cytochemical evaluation of gamma-glutamyl-transpeptidase activity in cervical smears

The cervicovaginal smears of 43 patients attending an outpatient service for early cancer detection were cytochemically studied for the presence of gamma-glutamyl-transpeptidase (GGT) in epithelial cells. This was done in order to evaluate such an enzyme phenotype as a marker for cancer development. The results showed that 70% of the 38 patients with a cytologic diagnosis of "inflammatory" or preneoplastic/neoplastic conditions had GGT-positive cells in their smears. None of the five cytologically normal cases showed any epithelial cells with GGT activity. Although most of the GGT-positive cells were metaplastic, some morphologically normal, dysplastic or neoplastic cells also expressed the enzyme. The data suggest that cytochemically detectable transpeptidase activity appears whenever alterations of the normal epithelial microenvironment occurs, but is not necessarily linked to the carcinogenic process. Therefore, cytochemically GGT-positive cells should not be used as an indicator of neoplastic transformation of the cervical epithelium.     

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

POLYMORPHISM IN THE DESMID XANTHIDIUM REGULARE AND ITS TAXONOMIC IMPLICATIONS

This paper refers to a case of polymorphism in the desmid genus Xanthidium Ehr. It is based on material from Lake Dais Irmaios, the main body of water in the Zoological and Botanical Garden in Recife, Pernambuco, northeastern Brazil, collected at 4 different times of the year during 1967 and 1968. A detailed examination of almost 1300 specimens showed an enormous variety in form of Xanthidium regulare Nordst., X. fragile Borge, and X. pseudoregulare Borge, thus allowing the authors to draw the following conclusions: (1) the name X. regulare Nordst. should be retained until further and more detailed studies on form variation within the species are available; (2) the names X. regulare Nordst. var. asteptum Nordst. in Borge, X. regulare Nordst. var. sexangulare Grönbl., X. regulare Nordst. var. sexangulare Grönbl. f. robustior Grönbl., X. fragile Borge, X. fragile Borge forma, and X. fragile Borge var. depauperatum Borge should be considered synonymous, all referring to a single variety of X. regulare Nordst., var. asteptum Nordst. in Borge emend. C. Bic. & L. M. Carv.; (3) X. pseudoregulare Borge must be treated as a variety of X. regulare Nordst. and must be called X. regulare Nordst. var. pseudoregulare (Barge) C. Bic. & L. M. Carv. Finally, a key is given to the 3 varieties of X. regulare Nordst. proposed in the present paper.     

Effects of brain ischemia on intermediate filaments of rat hippocampus

Neurofilaments subunits (NF-H, NF-M, NF-L) and glial fibrillary acidic protein (GFAP) were investigated in the hippocampus of rats after distinct periods of reperfusion (1 to 15 days) following 20 min of transient global forebrain ischemia in the rat. In vitro [¹⁴Ca]leucine incorporation was not altered until 48 h after the ischemic insult, however concentration of intermediate filament subunits significantly decreased in this period. Three days after the insult, leucine incorporation significantly increased while the concentration NF-H, NF-M, and NF-L were still diminished after 15 days of reperfusion. In vitro incorporation of³²P into NF-M and NF-L suffered immediately after ischemia, but returned to control values after two days of reperfusion. GFAP levels decreased immediately after ischemia but quickly recovered and significantly peaked from 7 to 10 days after the insult. These results suggest that transient ischemia followed by reperfusion causes proteolysis of intermediate filaments in the hippocampus, and that proteolysis could be facilitated by diminished phosphorylation levels of NF-M and NF-L.     

Metabolic shift analysis at high cell densities

Abstract: In high cell density cultures it is virtually inevitable that the environment to which the cells are exposed is heterogeneous. Thus, with suspended cultures, individual cells are subject to temporal changes in their environment whereas with aggregated or immobilized cells, the culture can be considered as being formed by a number of subpopulations, each with its own environmental characteristics. In addition, in a high cell density environment, high concentrations of end products may negatively influence the growth rate. This may result in the selection of organisms with an altered metabolic behaviour or with a decreased sensitivity to the adverse effects of the product. We discuss the consequences of this heterogeneity with regard to carbon source metabolism in view of the ability of many bacterial species to adapt to environmental conditions. Selection of variant organisms was found to occur with Clostridium butyricum when grown for a prolonged time in a medium containing approx. I-50 mM glucose. In contrast to the original strain, these variants could sustain a high maximal growth rate in the presence of butyric acid. In addition, they had acquired the capacity to spontaneously form aggregates and were able to carry out a completely solventogenic fermentation. Heterogeneous metabolic activity in aggregated cells is demonstrated with cultures of Lactobacillus laevolacticus , an aggregateforming lactic acid bacterium that converts glucose completely to o-lactate. By using microelectrodes, we show that the fraction of metabolically active cells decreases with increasing aggregate size: in larger aggregates steep pH gradients occur with the effect that only the outer layer of the aggregate is metabolically active, i.e. contributes to lactic acid formation, whereas with smaller aggregates all cells remain active. As a result, the net specific lactic acid production rate of the population as a whole is not invariably increased with increased aggregate size.     

The effect of light levels on daily patterns of chlorophyll fluorescence and organic acid accumulation in the tropical CAM treeClusia hilariana

Sandy plains are characteristic of the coastal region of Brazil. We investigated the diel patterns of changes in organic acid levels, leaf conductance and chlorophylla fluorescence for sun-exposed and shaded plants ofClusia hilariana, one of the dominant woody species in the sandy coastal plains of northern Rio de Janeiro state. Both exposed and shaded plants showed a typical CAM pattern with considerable diel oscillations in organic acid levels. The degradation of both malic and citric acids during the midday stomatal closure period could lead to potential CO₂ fixation rates of 28 mol m^-2 s^-1 in exposed leaves. Moreover, exposed leaves exhibited large increases in total non-photochemical quenching (q_N) accompanied by a substantial decrease in effective quantum yield during the course of the day. However, these potential high rates of CO₂ fixation and the increases inqn of exposed plants were not enough to maintain the primary electron acceptor of photosystem II (q_A) in a low reduction state, similar to that of shaded plants. As a result, there was a moderate increase in the reduction state of q_A throughout the day. Most of the decline in photochemical efficiency of exposed leaves ofC. hilariana was reversible, as evidenced by the high levels of pre-dawn potential quantum yields (F_v/F_m) and their rapid recovery after sunset. However, the depletion of the organic acid pool in the afternoon resulted in an accentuated subsequent drop in F_v/F_m, suggesting that prolonged periods of water stress accompanied by high irradiance levels may expose plants ofC. hilariana in unprotected habitats to the danger of photoinhibition.     

Aerobic 2-ketogluconate metabolism of Klebsiella pneumoniae NCTC 418 grown in chemostat culture.

Klebsiella pneumoniae NCTC 418 is able to convert 2-ketogluconate intracellularly to 6-phosphogluconate by the combined action of an NADPH-dependent 2-ketogluconate reductase and gluconate kinase. Synthesis of the former enzyme was maximal under 2-ketogluconate-limited growth conditions. An instantaneous transition to a 2-ketogluconate-excess condition resulted in an acceleration of catabolism of this carbon source, accompanied by complete inhibition of biosynthesis. It is suggested that the cause of this inhibition resides in depletion of the NADPH pool due to the high rate at which NADPH is oxidized by 2-ketogluconate reductase.