Importance of glycolipid synthesis for butyric acid-induced sensitization to shiga toxin and intracellular sorting of toxin in A431 cells.

The human epidermoid carcinoma cell line A431 becomes highly sensitive to Shiga toxin upon treatment with butyric acid. This strong sensitization (> 1000-fold) is accompanied by an increase in the fraction of cell-associated toxin transported to the Golgi apparatus and to the endoplasmic reticulum (ER). Furthermore, our previous work showed that the length of the fatty acyl chain of Gb3, the Shiga toxin receptor, also was changed (longer fatty acids). We have not investigated the importance of this change by testing whether glycolipid synthesis is required for the changed intracellular sorting and the toxin sensitivity. We demonstrate here that inhibition of glycosphingolipid synthesis by inhibition of N-acyltransferase with fumonisin B1, by inhibition of glucosylceramide synthetase by PDMP or PPMP, or by inhibition of serine palmitoyl transferase by beta-fluoroalanine, inhibited the butyric acid-induced change in sensitivity and the increase in the fraction of cell-associated Shiga toxin transported to the Golgi apparatus and the ER. The block in butyric acid-induced sensitization caused by beta-fluoroalanine could be abolished by simultaneous addition of sphinganine or sphingosine. Thus, the data suggest that the fatty acyl chain length of glycosphingolipids is important for intracellular sorting and translocation of Shiga toxin to the cytosol.     

The specification of metameric order in the insectCallosobruchus maculatus Fabr. (Coleoptera)

Summary Mechanically dividing an insect egg into anterior and posterior fragments results in a segment gap (Sander 1976), a loss of non-terminal segments in the constricted region. By altering the stage and duration of constriction, we produced different types of egg fragments in the pea beetleCallosobruchus. The patterns formed by these fragments suggest the existence of interactions between anterior and posterior egg regions that influence segment patterning and placement. Segments in excess of the numbers expected on the basis of permanent constrictions were produced in fragments when: (1) the constriction was released before cellularization occurred and (2) in addition the complementary fragment degenerated. Apparently the degenerating fragment induced the formation of excess segments in the developing fragment. Differences in the time and extent of excess segment formation in anterior versus posterior fragments suggest an asymmetric distribution of prerequisites for segment formation. This conclusion is consistent with our finding that a partial reversal of segment sequence (double abdomen formation) can be induced only in posterior fragments by a degenerating fragment, but not in anterior fragments (see companion paper).The formation of excess segments shows that the segment gap observed after permanent separation cannot be due to non-specific damage, caused by the process of constriction as such, to the egg or to localized putative segment precursors.     

Cytologic evidence for three human X-chromosomal segments escaping inactivation

Summary Early replication of prometaphasic human sex chromosomes was studied with the bromodeoxyuridine (BrdU)-replication technique. The studies reveal that two distal segments of Xp, including bands Xp 22.13 and Xp 22.3, replicate early in S-phase and therefore may not be subject to random inactivation. Furthermore, the replication of these distal segments of Xp occurs synchronously with those of the short arm of the Y chromosome including bands Yp 11.2 and Yp 11.32. These segments of Xp and Yp correspond well to the pairing segment of the X and Y chromosomes where a synaptonemal complex forms at early pachytene of human spermatogenesis. The homologous early replication of Yp and the distal portion of Xp may be interpreted as a remnant left untouched by the differentiation of heteromorphic sex chromosomes from originally homomorphic autosomes. A third early replicating segment is situated on the long arm of the X chromosome and corresponds to band Xq 13.1. This segment may be correlated with the X-inactivation center postulated by Therman et al. (1979).     

Distribution and behavior of sludge in upflow reactors for anaerobic treatment of wastewater

A model has been developed and experimentally checked for the physical behavior of sludge in the blanket in upflow reactors. The model is based on the mass balance for the sludge in the blanket, and can be used to predict the distribution of sludge in an upflow reactor in relation with the gas production, sludge settling characteristics, and the linear fluid velocity in the reactor. The quantitative values of the transport factors that are a measure of the efficiency of the transport of sludge by the fluid streams occurring in the reactor were determined experimentally in reactors of 30- and 200-m(3) volumes. As this was done for wastewater containing lower fatty acids as the main organic pollutants and for sludge with good settling characteristics, the predictive value of the model is limited. It may be used for the second (methane forming) step of anaerobic treatment of wastewater.     

Genetic alleviation of the self-fertilization complication when hybridizing monoecious Gelidium vagum

Fundamental genetic studies were initiated for the monoecious red alga Gelidium vagum. Color and sterility mutants were isolated and characterized to provide genetic tools, initially to identify hybrid plants when they occurred in crosses, and secondarily to eliminate self-fertilization altogether. When fertility phenotypes were scored, rapid onset of reproduction in culture was favored by long day-length, moderately high irradiance levels from fluorescent lights, warm temperature and the addition of Tris buffer to the medium. A recessive green mutant (designated grn 1) was characterized and used in subsequent crosses to allow a clear distinction between non-hybrid (green) and hybrid (red) offspring. Additional color mutants and a variety of reproductive mutants were also isolated and characterized. Male-sterile mutants had phenotypes ranging from apparently normal plants to those that produced no spermatia. Female-sterile mutants also included a variety of phenotypes, some plants having post-fertilization malfunctions during the development of the carposporophyte. Only a fraction of the sterility mutations have been phenotypically or genetically characterized, but some are straightforwardly inherited as stable, nuclear, single-gene defects. From the genetic recombination pattern, one female-sterile mutant may be loosely linked (39 cMorgans) to the grn 1 marker gene. Male sterility very effectively eliminated selfing without affecting the production of carpospores in crosses, thereby overcoming one of the most serious genetic difficulties in working with this monoecious species.     

Identification of a novel composite transposable element, Tn5280, carrying chlorobenzene dioxygenase genes of Pseudomonas sp. strain P51.

Analysis of one of the regions of catabolic plasmid pP51 which encode chlorobenzene metabolism of Pseudomonas sp. strain P51 revealed that the tcbA and tcbB genes for chlorobenzene dioxygenase and dehydrogenase are located on a transposable element, Tn5280. Tn5280 showed the features of a composite bacterial transposon with iso-insertion elements (IS1066 and IS1067) at each end of the transposon oriented in an inverted position. When a 12-kb HindIII fragment of pP51 containing Tn5280 was cloned in the suicide donor plasmid pSUP202, marked with a kanamycin resistance gene, and introduced into Pseudomonas putida donor plasmid pSUP202, marked with a kanamycin resistance gene, and introduced into Pseudomonas putida KT2442, Tn5280 was found to transpose into the genome at random and in single copy. The insertion elements IS1066 and IS1067 differed in a single base apir located in the inner inverted repeat and were found to be highly homologous to a class of repetitive elements of Bradyrhizobium japonicum and distantly related to IS630 of Shigella sonnei. The presence of the catabolic genes tcbA and tcbB on Tn5280 suggests a mechanism by which gene clusters can be mobilized as gene cassettes and joined with others to form novel catabolic pathways.     

Glucosylceramide is synthesized at the cytosolic surface of various Golgi subfractions

In our attempt to assess the topology of glucosylceramide biosynthesis, we have employed a truncated ceramide analogue that permeates cell membranes and is converted into water soluble sphingolipid analogues both in living and in fractionated cells. Truncated sphingomyelin is synthesized in the lumen of the Golgi, whereas glucosylceramide is synthesized at the cytosolic surface of the Golgi as shown by (a) the insensitivity of truncated sphingomyelin synthesis and the sensitivity of truncated glucosylceramide synthesis in intact Golgi membranes from rabbit liver to treatment with protease or the chemical reagent DIDS; and (b) sensitivity of truncated sphingomyelin export and insensitivity of truncated glucosylceramide export to decreased temperature and the presence of GTP-gamma-S in semiintact CHO cells. Moreover, subfractionation of rat liver Golgi demonstrated that the sphingomyelin synthase activity was restricted to fractions containing marker enzymes for the proximal Golgi, whereas the capacity to synthesize truncated glucosylceramide was also found in fractions containing distal Golgi markers. A similar distribution of glucosylceramide synthesizing activity was observed in the Golgi of the human liver derived HepG2 cells. The cytosolic orientation of the reaction in HepG2 cells was confirmed by complete extractability of newly formed NBD-glucosylceramide from isolated Golgi membranes or semiintact cells by serum albumin, whereas NBD-sphingomyelin remained protected against such extraction.     

Linear relation between rate and thermodynamic force in enzyme-catalyzed reactions

Starting from enzyme kinetics, it is shown that generally a linear rather than a proportional relationship exists between rate and free energy changes in biochemical processes. In the derivation the boundary condition of constant substrate plus product is used, which is appropriate for many cellular systems. An example is the ADP plus ATP concentration in mitochondrial oxidative phosphorylation, as is illustrated experimentally.     

A monolayer study of the reaction of trinitrobenzene sulphonic acid with amino phospholipids.

The reaction of trinitrobenzene sulphonic acid with amino phospholipids, and in particular phosphatidylethanolamine has been studied by the monolayer technique. Injection of trinitrobenzene sulphonic acid under a monolayer of amino phospholipid results in an increase in surface pressure. The rate and extent of the pressure change is greatly affected by the initial surface pressure, the fatty acid composition of the lipid, and the presence of other non-reactive lipids, especially negatively charged phospholipids. The extent of the reaction was measured with 32P-labelled phospholipids isolated from Bacillus subtilis. Only about 80% of the phosphatidylethanolamine in the monolayer could be converted to its trinitrophenyl derivative. In the presence of negatively charged phospholipids such as cardiolipin or phosphatidylglycerol, a further 20% decrease in the trinitrophenylation of phosphatidylethanolamine was found. The pressure increase occurring during trinitrophenylation could also be correlated with the extent of the reaction by comparison of the force-area curves of pure phosphatidylethanolamine, its trinitrophenyl derivative and mixtures of both compounds. The data may offer an explanation for the observation that incomplete labelling of amino phospholipids frequently occurs in natural membranes and furthermore indicate that the use of chemical labelling techniques in the study of lipid asymmetry in biological membranes must be approached with great caution.