Encapsulation of bacterial cells in cytoprotective ZIF-90 crystals as living composites.

Exploiting metal-organic frameworks (MOFs) as selectively permeable shelters for encapsulating engineered cells to form hybrid living materials has attracted increasing attention in recent years. Optimizing the synthesis process to improve encapsulation efficiency (EE) is critical for further technological development and applications. Here, using ZIF-90 and genetically engineered Escherichia coli (E. coli) as a demo, we fabricated E. coli@ZIF-90 living composites in which E. coli cells were encapsulated in ZIF-90 crystals. We illustrated that ZIF-90 could serve as a protective porous cage for cells to shield against toxic bactericides including benzaldehyde, cinnamaldehyde, and kanamycin. Notably, the E. coli cells remained alive and could self-reproduce after removing the ZIF-90 crystal cages in ethylenediaminetetraacetic acid, suggesting a feasible route for protecting and prolonging the lifespan of bacterial cells. Moreover, an aqueous multiple-step deposition approach was developed to improve EE of the E. coli@ZIF-90 composites: the EE increased to 61.9 ± 5.2%, in contrast with the efficiency of the traditional method (21.3 ± 4.4%) prepared with PBS buffer. In short, we develop a simple yet viable strategy to manufacture MOF-based living hybrid materials that promise new applications across diverse fields.

Habitat-related mtDNA polymorphism in the stored-bean pest Callosobruchus chinensis (Coleoptera: Bruchidae).

The genetic diversity of populations of the azuki bean beetle, Callosobruchus chinensis (Linnaeus) from natural, pre-harvest and post-harvest sites, was investigated to understand population structure and gene flow. A 522-bp fragment of the mitochondrial gene COI was sequenced for eight populations of C. chinensisfrom Japan, Korea and Taiwan collected from different habitats. Six haplotypes were detected, one of which, U1, occurred most frequently and widely. The following hypotheses were tested as a cause of the wide distribution of haplotype U1; (i) topographical separation (by national boundaries), (ii) host plant species, and (iii) habitat type (natural, pre-harvest crop, or post-harvest storage). Categorization of collection sites by country or by host species did not yield differences in the occurrence of haplotype U1, but habitat type did. Populations utilizing cultivated post-harvest hosts that were mass stored were highly likely to be the common haplotype, whereas host plants in natural habitats away from agriculture were utilized by populations with locally characteristic haplotypes. Sampling of commercial beans for quarantine and export purposes indicated that gene flow in C. chinensis was largely unidirectional into Japan at the present time.

Anion effects on fluid absorption from rat jejunum perfused in vivo.

Perfusion of rat jejunal segments in vivo with an isotonic, HCO3-free SO4-Ringer solution resulted in low rates of net sodium (JNanet) and water absorption. When the perfusion fluid was changed to one containing 25 mM Na2SO3, JNanet increased from 4.7 +/- 1.2 to 11.6 +/- 1.5 (SE) mumol X cm-1 X h-1 (P less than 0.001). This increased absorption was accompanied by comparable increases in chloride and water absorption, occurred without a detectable change in potential difference across the perfused segment, and was readily reversed on reinstitution of perfusion with SO4-Ringer. Perfusion with SO3-Ringer had no effect on electrolyte absorption from terminal segments of rat ileum. Addition of L-phenylalanine stimulated absorption from SO4-Ringer perfusate but not from SO3-Ringer perfusate. Addition of 25 mM NaHCO3 to SO4-Ringer perfusate caused parallel increases in JNanet and JHCO3net; when 25 mM NaHCO3 was added to SO4-Ringer perfusate that also contained 25 mM NaSCN, the same increase in JHCO3net occurred but was not associated with any increase in JNanet. These results indicate a potent effect of SO2-3 and HCO-3 to stimulate JNanet from rat jejunum but not from ileum. These anion effects on intestinal transport in vivo resemble their effects on ATPase activity of brush-border fractions from small intestine in vitro and raise the possibility that these effects on ion transport could be mediated through the changes in brush-border ATPase activity, which are brought about by exposure to these anions, although other explanations are also possible.

Anion-stimulated phosphohydrolase activity of intestinal alkaline phosphatase.

Phosphohydrolase activity of a highly enriched commercial preparation of calf intestinal alkaline phosphatase was stimulated in the presence of HCO3. SO4, Cl, SCN, and acetate did not stimulate hydrolysis, whereas SO3 exhibited a bimodal effect, stimulating at low (25mM) concentration but inhibiting at high (100 mM) concentration. The pH optimum of this stimulation by HCO3 or SO3 was 8.5--9.0 and was maximal at a Mg concentration of 0.5 mM. HCO3 increased the Vmax of the reaction without changing the Km for ATP. ATP, GTP, UTP, and xanthosine triphosphate were equally effective as substrates, whereas AMP and p-nitrophenyl phosphate were much less effective. Alkaline phosphatase activity was inhibited by L-cysteine and L-phenylalanine, compounds that also inhibited the HCO3-ATPase activity of the preparation. Passage of the commercial preparation through an anion-exchange column yielded a fraction with enriched alkaline phosphatase and HCO3-ATPase activities; this fraction proved to be a single protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, on isoelectric focusing, and by immunologic techniques. These studies strongly suggest that alkaline phosphatase and anion-stimulated phosphohydrolase activities are properties of the same protein in small intestine. It is possible that alkaline phosphatase may function as a HCO3-ATPase involved in intestinal absorption and secretion.

Requirement of Zn to demonstrate HCO3-stimulated ATPase activity of rat small intestinal brush border.

The existence of a membrane-bound HCO3-stimulated ATPase in intestinal mucosa is controversial. A crude brush border fraction of rat small intestinal homogenates contained HCO3-ATPase activity which was inhibited by preincubation with 3 mM EDTA. Alkaline phosphatase activity of this preparation was also inhibited in a parallel, time-dependent fashion by preincubation with EDTA. When 5 mM ZnSO4 accompanied 3 mM EDTA in the preincubation mix, preservation of both enzyme activities occurred, demonstrating a requirement of Zn for the activity of both these phosphatases. These studies support the earlier contention that HCO3-ATPase and alkaline phosphatase activities may be different properties of the same enzyme, and raise the possibility that the ATPase could play a role in intestinal ion transport. The failure to identify a membrane-bound HCO3-ATPase by other workers could be due to the exposure of EDTA which occurred in their tissue preparation.

Anion-stimulated ATPase activity of brush border from rat small intestine.

Differential centrifugation of rat small intestinal homogenates produced a crude brush border (BB) fraction that was enriched 15-fold for the marker enzymes, alkaline phosphatase and sucrase; contamination with mitochondrial enzymes, monoamine oxidase and succinate dehydrogenase, was minimal. ATP hydrolysis by this BB fraction was stimulated by addition of several anions to the incubation medium: HCO3 and Cl were equally effective in this regard, with NO3, NO2, SO4, and acetate being less stimulatory. SCN and CNO inhibited ATPase activity, whereas the divalent anion SO3 was stimulatory at low concentrations (less than 25 mM) but inhibitory at 100 mM. Maximum anion stimulation was observed at a Mg concentration of 0.5 mM, and pH optimum was 8.5. Kinetic analysis showed that HCO3 increased the Vmax without altering the Km for ATP; the Ka for this effect of HCO3 was 35 mM. This enzyme activity was completely inhibited by 20 mM L-phenylalanine, 10 mM L-cysteine, and 3 mM EDTA, compounds that also inhibited intestinal alkaline phosphatase. These results demonstrate the presence of anion-stimulated ATPase activity in rat small intestinal brush border and suggest that this activity may be related to intestinal alkaline phosphatase. The role of this enzyme in intestinal transport is not known, but could relate to the regulation of intestinal absorption and secretion.

Suppression of antidiuretic hormone secretion by clonidine in the anesthetized dog.

Studies were performed to determine the mechanism by which the antihypertensive agent clonidine increases urine flow (V). In 11 anesthetized, hydropenic dogs, i.v. administration of clonidine (30 mug/kg) increased arterial pressure from 128 +/- 4 to 142 +/- 3 mm Hg and slowed heart rate from 138 +/- 7 to 95 +/- 7 beats/min within 30 min of injection; blood pressure then fell to 121 +/- 5 mm Hg 30 to 60 min after injection, and 112 +/- 5 mm Hg in the next 30-min period. V increased from 0.36 +/- 0.09 to 0.93 +/- 0.13 Uosm ml/min and urine osmolality (Uopsm) decreased from 1378 +/- 140 to 488 +/- 82 mOsm/kg of H2O 30 to 60 min following injection (P less than 0.001). These changes were accompanied by a decrease in TcH20. This increased V was not associated with increased glomerular filtration rate (GFR) or solute excretion, and occurred in acutely denervated kidneys and kidneys protected from the initial increase in arterial pressure by constriction of a suprarenal aortic clamp. By contrast, V TcH2O and UOsm were not altered by clonidine administration in seven acutely hypophysectomized dogs receiving a constant infusion of antidiuretic hormone (ADH) (80 muU/kg/min), despite similar hemodynamic changes produced by the drug. The results suggest that clonidine increases V through inhibition of ADH release, possibly via an indirect pathway mediated by the drug's alpha-adrenergic on the circulation.