Cloning, Expression, and Affinity Purification of RecombinantShigella flexneriInvasion Plasmid Antigens IpaB and IpaC

Shigella flexneriand related enteropathogenic bacteria are important agents of bacillary dysentery, a potentially life-threatening illness for children in underdeveloped regions of the world. Onset of shigellosis stems fromS. flexneriinvasion of colonic epithelial cells, leading to localized cell death and inflammation. Invasion plasmid antigens (Ipa) B, C, and D are three secreted proteins encoded by the large virulence plasmid ofS. flexnerithat have been implicated as essential effectors of this cell invasion process. These proteins are expressed as part of theipaoperon and are among the major targets of the host immune response to shigellosis. Biochemical characterization of the Ipa invasins has been complicated by the fact they have not been purified in the quantities needed for detailedin vitroanalysis. Here we describe the first cloning, expression, and extensive purification of IpaB and IpaC fusion proteins fromEscherichia colifor use in dissecting of the protein biochemistry ofS. flexneripathogenesis. A variety of approaches were used to prepare significant quantities of these proteins in their soluble forms, including the use of different host cell lines, modification of bacterial growth conditions, and the use of alternative plasmid expression vectors. Now that these Ipa proteins are available in a highly pure form, it will be possible to initiate studies on their important biological and immunological properties as well as their recruitment into high-molecular-weight protein complexes. Together with IpaD (purified as part of a previous study), these purified proteins will be useful for: (a) exploring properties of the host immune response toS. flexneriinvasion, (b) elucidating the specific biochemical properties that lead to pathogen internalization, (c) analyzing the importance of specific Ipa protein complexes in host cell invasion, and (d) monitoring, or perhaps even augmenting, the efficacy of live oral vaccines in human trials.     

Depolarization of the Electrogenic Transmembrane Electropotential of Zea mays L. by Bipolaris (Helminthosporium) maydis Race T Toxin, Azide, Cyanide, Dodecyl Succinic Acid, or Cold Temperature

The transmembrane electrical potential of root cells of Zea mays L. cv. W64A in a modified 1× Higinbotham solution was partially depolarized by semipurified toxin obtained from Bipolaris (Helminthosporium) maydis race T. At a given toxin concentration depolarization of Texas cytoplasm cells was much greater than for normal cytoplasm cells. This observation correlated directly to the differential host susceptibility to the fungus. The time course and magnitude of depolarization were dependent on toxin concentration; at high concentration the electropotential difference change was rapid. Cortex cells depolarized more slowly than epidermal cells indicating that the toxin slowly permeated intercellular regions. Toxin concentrations which affected electropotential difference were of the same magnitude as those required to inhibit root growth, ion uptake, and mitochondrial processes.

Azide, cyanide, and cold temperature (5 C) gave the same partial depolarization as did the toxin. Dodecyl succinic acid caused complete depolarization. These and other data indicate that one of the primary actions of the toxin is to inhibit electrogenic ion pumps in the plasmalemma.

     

Glucose tolerance factor: an essential dietary agent

Glucose tolerance factor (GTF), the biologically active form of chromium, is an essential dietary agent that potentiates the action of insulin and thereby functions in regulating carbohydrate metabolism. Dietary trends that show increased consumption of more highly processed foods may be leading to deficiencies of'GTF and chromium in man.     

Schistosoma haematobium and S. japonicum: analysis of the ribosomal RNA genes and determination of the "gap" boundaries and sequences

We have determined the intragenic organization of the rRNA genes of Schistosoma haematobium and S. japonicum and found them to be similar to that of S. mansoni and other eukaryotes. An entire ribosomal repeat approximately 10 kbp in size from each species was isolated as a SalI fragment from a genomic library constructed in bacteriophage lambda. The segments encoding both the small and large rRNAs have been identified using three cloned EcoRI fragments of S. mansoni as probes. There were three EcoRI fragments (4.2, 3.0, 1.6 kbp) from S. haematobium and four EcoRI fragments (4.6, 2.3, 1.7, 1.0 kbp) from S. japonicum. As in a wide variety of organisms within the protostome phyla, the 28S rRNA in schistosomes contains a "gap" which separates it into two fragments. The length of the gap sequence in S. haematobium is 54 bases and it is identical to that in S. mansoni in both length and sequence. However, in S. japonicum the sequence is between 64-67 bases long. In each case, irrespective of the species, the gap is located at the same position within the 28S rRNA. Secondary structures of the gap sequence derived by computer analysis predict a conformation with the minimum free energy that has an UAAU tract in a hairpin loop for S. haematobium and an UAUU tract for S. japonicum.     

Insulin-secreting beta-cells possess specific receptors for interleukin-1 beta

The effect of the cytokine interleukin-1 beta on the insulin secretory responsiveness of single beta-cells (HIT-T15) was investigated. In the short-term, IL-1 beta induced a dosage-dependent stimulation of insulin release. In contrast, in the long-term, IL-1 beta, inhibited both basal and secretagogue-stimulated insulin secretion. We also demonstrate the simultaneous presence of specific high and low affinity binding sites for IL-1 beta on beta-cells. IL-1 beta, which has been implicated in the pathogenesis of insulin-dependent diabetes, may therefore mediate its opposing effects on beta-cells through a specific plasma membrane receptor.     

Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.     

Transmembrane electropotential in barley roots as related to cell type, cell location, and cutting and aging effects

Transmembrane electropotential difference (PD) was measured in whole roots of barley (Hordeum vulgare L. cvs. Compana and Himalaya). Seedlings were grown 4 to 5 days in aerated 0.5 mm CaSO(4) or a nutrient solution. Measurements of PD were made with roots bathed in CaSO(4), KCl + CaSO(4), or the nutrient solution. The following results were found. (a) There was a radial PD gradient with epidermal cells being 10 to 58 millivolts less negative than cells in the third layer of the cortex (outside to inside). There was no longitudinal PD gradient in the region 0.5 to 4 cm from the root tip, nor was there any difference between the PD of young root hairs and other epidermal cells. (b) Cell PD in excised whole roots was not detectably different from that found in roots attached to the shoot, and was unchanged for 2 hours from excision. (c) In 1-centimeter sections of root, cell PD at the freshly cut surface was depolarized by 90 millivolts from that in the intact root; cells farther than 1 millimeter from the cut surface were not depolarized. The PD of cells at the cut surface became more negative upon aging the segment in 0.5 mm CaSO(4), eventually becoming greater by -25 millivolts than that in cells of intact roots. Cells in segments to which the root tips were attached had less negative PDs after aging than those in subapical segments, indicating a possible hormonal effect. PDs in aged, excised segments are not equivalent to those in intact roots. (d) Creeping of cytoplasm over electrode tips inserted into the vacuole gave measurements of vacuole-to-cytoplasm PD of + 9 millivolts in 0.5 mm CaSO(4) and + 35 millivolts in 1 mm KCl + 0.5 mm CaSO(4). Most of the cell PD was across the plasmalemma. (e) The reducing sugar content of roots in CaSO(4) solution was greater than that of roots in the nutrient solution in which ion uptake, particularly K(+) occurred.     

Inhibition of ion accumulation in maize roots by abscisic acid

Summary An inhibition of root growth, a decrease in the amount of potassium (as ⁸⁶Rb) and phosphate (³²P) accumulation by the root, and a partial depolarization of transmembrane electropotential were observed to develop with a similar time course and to a similar extent when intact maize (Zea mays L.) roots were treated with 10^-5 M abscisic acid (ABA). Potassium uptake was inhibited by ABA when excised, low-salt roots were bathed in KCl, KH₂PO₄, or K₂SO₄. ABA did not affect the ATP content of the tissues, the activity of isolated mitochondria, nor the activity of microsomal K⁺-stimulated ATPases.