Reversible bacteriophage resistance by shedding the bacterial cell wall

Phages are highly abundant in the environment and pose a major threat for bacteria. Therefore, bacteria have evolved sophisticated defence systems to withstand phage attacks. Here, we describe a previously unknown mechanism by which mono- and diderm bacteria survive infection with diverse lytic phages. Phage exposure leads to a rapid and near-complete conversion of walled cells to a cell-wall-deficient state, which remains viable in osmoprotective conditions and can revert to the walled state. While shedding the cell wall dramatically reduces the number of progeny phages produced by the host, it does not always preclude phage infection. Altogether, these results show that the formation of cell-wall-deficient cells prevents complete eradication of the bacterial population and suggest that cell wall deficiency may potentially limit the efficacy of phage therapy, especially in highly osmotic environments or when used together with antibiotics that target the cell wall.     

Characterization of an electrogenic sodium/glucose cotransporter in a human colon epithelial cell line

In this study, we have characterized the Na/glucose transporter in polarized monolayers formed by the clonal human colon carcinoma cell line HT-29-D4. Isotopic tracer flux measurements show that differentiated HT-29-D4 cells possess a sodium-dependent α-methyl-D-glucopyranoside (AMG) uptake that is competed for by increasing concentrations of D-glucose, D-galactose, and phlorizin. This transport is exclusively localized on the apical side of the epithelium. Kinetic data demonstrate the existence of a single Michaelian sodium-dependent AMG transporter with a Km of 1.2 ± 0.12 mM and a Vmax of 3.24 ± 0.25 nmol/mg of protein per min. Hill analysis reveals a coefficient of 1.9 ± 0.03, consistent with at least two sodium ions involved in AMG transport. Interestingly, the cotransporter function is not modulated by glucose in the culture medium. Transepithelial electrical parameter measurements show that the transepithelial potential difference (Vt) is glucose dependent and phlorizin sensitive. Antibodies directed against a peptide of the rabbit intestinal glucose cotransporter (Ser402-Lys420) recognize, in western blot experiments, the characteristic bands of the cotransporter on a crude membrane preparation of differentiated HT-29-D4 cells and react strongly with the apical domain of the monolayer in immunofluorescence experiments. We conclude that HT-29-D4 cells express the sodium/glucose cotransporter SGLT1 at their apical membrane and that this transporter generates the basal transepithelial potential difference. © 1995 Wiley-Liss, Inc.     

Chemical synthesis and characterization of Pi1, a scorpion toxin from Pandinus imperator active on K+ channels.

Pi1 is a 35-residue toxin cross-linked by four disulfide bridges that has been isolated from the venom of the chactidae scorpion Pandinus imperator. Due to its very low abundance in the venom, we have chemically synthesized this toxin in order to study its biological activity. Enzyme-based proteolytic cleavage of the synthetic Pi1 (sPi1) demonstrates half-cystine pairings between Cys4-Cys25, Cys10-Cys30, Cys14-Cys32 and Cys20-Cys35, which is in agreement with the disulfide bridge organization initially reported on the natural toxin. In vivo, intracerebroventricular injection of sPi1 in mice produces lethal effects with an LD50 of 0.2 microgram per mouse. In vitro, the application of sPi1 induces drastic inhibition of Shaker B (IC50 of 23 nM) and rat Kv1.2 channels (IC50 of 0.44 nM) heterologously expressed in Xenopus laevis oocytes. No effect was observed on rat Kv1.1 and Kv1.3 currents upon synthetic peptide application. Also, sPi1 is able to compete with 125I-labeled apamin for binding onto rat brain synaptosomes with an IC50 of 55 pM. Overall, these results demonstrate that sPi1 displays a large spectrum of activities by blocking both SK- and Kv1-types of K+ channels; a selectivity reminiscent of that of maurotoxin, another structurally related four disulfide-bridged scorpion toxin that exhibits a different half-cystine pairing pattern.     

Safety and efficacy of human amniotic membrane in primary pterygium surgery

Grafts made from human amniotic membrane are used to prevent recurrence of pterygium after excision. The success of the procedure can be affected by the quality of preparation and preservation of the grafts. We prospectively evaluated the safety and efficacy of cryopreserved amniotic membrane prepared at the research tissue bank of the Biotechnology Research Center in Tripoli, Libya, and used as adjunct therapy in primary pterygium excision. Twenty-six patients (15 males and 11 females) aged 21–78 years and indicated for primary pterygium excision were transplanted at the Tripoli Eye Hospital with the amniotic membrane grafts. Sixteen patients (62 %) were available for all three follow-up visits scheduled at 1, 3 and 6 months post-surgery. By the third visit, two patients (12.5 %) developed granuloma and three (18.8 %) had pterygium recurrence. The grafts were used after cryopreservation for ≤180 days or >180 days, but statistical analysis showed that the complications were not associated with the length of storage. Moreover, the high rate of complications in this study was not caused by use of cryopreserved AM. In conclusion, locally produced cryopreserved AM is safe as an adjunct therapy for treatment of primary pterygium excision.     

Intestinal Targeting of Ganciclovir Release Employing a Novel HEC-PAA Blended Lyomatrix

A hydroxyethylcellulose-poly(acrylic acid) (HEC-PAA) lyomatrix was developed for ganciclovir (GCV) intestine targeting to overcome its undesirable degradation in the stomach. GCV was encapsulated within the HEC-PAA lyomatrix prepared by lyophilization. Conventional tablets were also prepared with identical GCV concentrations in order to compare the GCV release behavior from the lyomatrix and tablets. GCV incorporation (75.12%) was confirmed using FTIR, DSC, and TGA. The effect of GCV loading on the microstructure properties of the lyomatrix was evaluated by SEM, AFM, and BET surface area measurements. The in vitro drug release study showed steady and rapid release profiles from the GCV-loaded lyomatrix compared with the tablet formulation at identical pH values. Minimum GCV release was observed at acidic pH (≤40%) and maximum release occurred at intestinal pH values (≥90%) proving the intestinal targeting ability of the lyomatrix. Kinetic modeling revealed that the GCV-loaded lyomatrix exhibited zero-order release kinetics (n?=?1), while the tablets were best described via the Peppas model. Textural analysis highlighted enhanced matrix resilience and rigidity gradient (12.5%, 20 Pa) for the GCV-loaded lyomatrix compared to the pure (7%, 9.5 Pa) HEC-PAA lyomatrix. Bench-top MRI imaging was used to confirm the mechanism of GCV release behavior by monitoring the swelling and erosion rates. The swelling and erosion rate of the tablets was not sufficient to achieve rapid zero-order GCV release as with the lyomatrix. These combined results suggest that the HEC-PAA lyomatrix may be suitable for GCV intestinal targeting after oral administration.     

Differential response of stomata to air humidity in the parasitic mistletoe (Phthirusa pyrifolia) and its host,mandarin orange (Citrus resitulata)

Measurements of CO₂ and H₂O exchange rate and the calculated leaf conductance of attached leaves were conducted over a range of leaf-to-air vapour pressure difference (VPD) (1.5 to 5.5 kPa) to compare the response of the parasitic mistletoe, Phthirusa pyrifolia, with that of its host, the mandarin orange, Citrus reticulata. Seedlings of the host infected with the parasite were grown in well-watered and adequately fertilized large pots outdoors at the CIAT headquarters, Palmira, Colombia, South America. Observations of leaf anatomy of the parasite and nutrient analysis of young tissues of both the parasite and host were made. The photosynthetic rate of the host decreased linearly with increased VPD, whereas the parasite showed a constant rate. This trend coincided with similar responses in leaf conductance. Due to the insensitivity of the parasite stomata, the transpiration rate increased linearly with VPD as compared with an initial increase and then a decrease in the host transpiration rate. The higher photosynthetic rate and the closure of stomata of the host resulted in high water use efficiency as compared with that of the parasite. The parasite accumulated in its leaves more N, P, K and less Ca and Mg than the host. The significance of the host-parasite differential response to air humidity is discussed in relation to mechanism underlying stomatal sensitivity and in the context of host-parasite association.Visiting Scientist, Coordinator, and Research Assistant of the Cassava Physiology Program.     

Photosynthetic responses of cassava cultivars (Manihot esculenta Crantz) from different habitats to temperature

Maximum photosynthetic CO₂ exchange rates (P_n) of single attached leaves were determined for several cassava cultivars selected from different habitats and grown in pots outdoors at CIAT, Colombia, S.A. P_n rates were in a narrow range of 22 to 26 mol CO₂ m^–2s^–1 for all cultivars tested when measured at high photon flux density, normal air, optimum temperature and with low leaf-air vapor pressure differences. For all tested cultivars (9 cvs.), there was a broad optimum temperature for P_n between 25 to 35°C. At temperatures below and above this range P_n declined in all cultivars with P_n rates reaching 80% of maximum at 20 and 40°C. P_n temperature coefficient (Q₁₀) from 15–25°C was 1.6±0.2 across cultivars. No consistent relation existed between P_n, optimum temperature, and the original habitat.     

Influence of differences in leaf anatomy on net photosynthetic rates of some cultivars of Cassava

Gas exchange measurements and leaf anatomy of 10 cassava cultivars were conducted to study the interrelationship between the relatively high photosynthetic rates and the factors limiting internal CO₂ diffusion. The internal mesophyll surface area per unit leaf surface area (A^mes/A) and the intracellular components of CO₂ diffusion and fixation resistance (R^cellCO₂) were determined. Among the group of cultivars tested net CO₂ exchange rates were 26±2.5 mol CO₂ m^–2 s^–1 in normal air and intense light and A^mes/A ranged from 14 to 38. Estimated R^cellCO₂ ranged from 4300 to 13,000 s m^–1. The combined and compensating effects of A^mes/A and R^cellCO₂ accounted for both the high net photosynthetic rates (P_n) and the lack of large differences in P_n among cultivars.     

C3-C4 intermediate photosynthetic characteristics of cassava (Manihot esculenta Crantz)

Cassava, bean and maize leaves were fed with¹⁴CO₂ in light and the primary products of photosynthesis identified 5 and 10 seconds after assimilation. In maize, approximately three quarters of the labelled carbon was incorporated in C₄ acids, in beans about two thirds in PGA, and in cassava approximately 40–60% in C₄ acids with 30–50% in PGA. These data indicate that cassava possesses the C₄ photosynthetic cycle, however due to the lack of typical Kranz anatomy appreciable carbon assimilation takes place directly through the Calvin-Benson-Bassham cycle.