Effect of formulation factors on in vitro permeation of moxifloxacin from aqueous drops through excised goat, sheep, and buffalo corneas

The purpose of this investigation was to evaluate the effect of formulation factors on in vitro permeation of moxifloxacin from aqueous drop through freshly excised goat, sheep, and buffalo corneas. Aqueous isotonic ophthalmic solutions of moxifloxacin hydrochloride of different concentrations (pH 7.2) or 0.5% (wt/vol) solutions of different pH or 0.5% solutions (pH 7.2) containing different preservatives were made. Permeation characteristics of drug were evaluated by putting 1 mL formulation on freshly excised cornea (0.50 cm²) fixed between donor and receptor compartments of an all-glass modified Franz diffusion cell and measuring the drug permeated in the receptor (containing 10 mL bicarbonate ringer at 37°C under stirring) by spectrophotometry at 291 nm, after 120 minutes. Statistical analysis was done by one-way analysis of variance (ANOVA) followed by Dunnett’s test. Increase in drug concentration in the formulation resulted in an increase in the quantity permeated but a decrease in percentage permeation. Increase in pH of the solution from 5.5 to 7.2 increased drug permeation, indicating pH-dependent transport. Compared with control formulation, moxifloxacin 0.5% (wt/vol) solution (pH 7.2) containing disodium edetate (EDTA) (0.01% wt/vol) produced significantly (P<.05) higher permeation with all the corneas. Formulation with benzyl alcohol significantly (P<.05) increased permeation with buffalo cornea compared with its control. Presence of benzalkonium chloride (BAK) (0.01% wt/vol) and EDTA (0.01% wt/vol) in the formulation increased permeation to the maximum with all the corneas. The results suggest that moxifloxacin 0.5% ophthalmic solution (pH 7.2) containing BAK (0.01%) and EDTA (0.01%) provides increased in vitro ocular availability through goat, sheep, and buffalo corneas. Published: February 10, 2006 Formerly College of Pharmacy, University of Delhi, Pushp Vihar, Sector III, New Delhi-110017, India     

Pan‐Cancer analyses of Necroptosis‐Related genes as a potential target to predict immunotherapeutic outcome

Necroptosis is a unique programmed death mechanism of necrotic cells. However, its role and specific mechanism in cancer remain unclear, and a systematic pan‐cancer analysis of necroptosis is yet to be conducted. Thus, we performed a specific pan‐cancer analysis using The Cancer Genome Atlas and Genotype‐Tissue Expression databases to analyse necroptosis expression in terms of cancer prognosis, DNA methylation status, tumour mutative burden, microsatellite instability, immune cell infiltration in different types of cancer and molecular mechanisms. For the first time, we explored the correlation between necroptosis and immunotherapy prognosis. Thus, our study provides a relatively comprehensive understanding of the carcinogenicity of necroptosis in different types of cancer. It is suggested that necroptosis can be used to evaluate the sensitivity of different patients to immunotherapy and may become a potential target for tumour immunotherapy.     

Transformation and growth related changes in levels of nuclear and cytoplasmic proteins antigenically related to mammalian beta-galactoside-binding lectin

Immunofluorescence and immunoblotting experiments, using a monoclonal antibody to the 13 kDa mammalian beta-galactoside-binding lectin have shown that human lymphocytes contain nuclear and cytoplasmic proteins of apparent molecular masses of 130, 80, 65 and 13 kDa that are antigenically related to the lectin and whose levels and patterns of expression change in association with transformation, or after stimulation with mitogens. These observations, together with the finding that the myeloid cell line K562 is also rich in the 130 kDa component, whereas the mature granulocytes of normal donors and of patients with chronic myeloid leukaemia are lacking in all of the immunoreactive forms, raise the possibility that this family of lectin-related proteins may be components of growth regulatory systems that are variously elicited in the transformed and stimulated cells.     

Charge and acidity compensation during proton-sugar symport in Chlorella: The H+-ATPase does not fully compensate for the sugar-coupled proton influx

This study was undertaken in order to demonstrate the extent to which the activity of the plasmalemma H⁺-ATPase compensates for the charge and acidity flow caused by the sugar-proton symport in cells of chlorella vulgaris Beij.. Detailed analysis of H⁺ and K⁺ fluxes from and into the medium together with measurements of respiration, cytoplasmic pH, and cellular ATP-levels indicate three consecutive phases after the onset of H⁺ symport. Phase 1 occurred immediately after addition of sugar, with an uptake of H⁺ by the hexoseproton symport and charge compensation by K⁺ loss from the cells and, to a smaller degree, by loss of another ion, probably a divalent cation. This phase coincided with strong membrane depolarization. Phase 2 started approximately 5 s after addition of sugar, when the acceleration of the H⁺-ATPase caused a slow-down of the K⁺ efflux, a decrease in the cellular ATP level and an increase in respiration. The increased respiration was most probably responsible for a pronounced net acidification of the medium. This phase was inhibited in deuterium oxide. In phase 3, finally, a slow rate of net H⁺ uptake and K⁺ loss was established for several further minutes, together with a slight depolarization of the membrane. There was hardly any pH change in the cytoplasm, because the cytoplasmic buffering capacity was high enough to stabilize the pH for several minutes despite the net H⁺ fluxes. The quantitative participation of the several phases of H⁺ and K⁺ flow depended on the pH of the medium, the ambient Ca²⁺ concentration, and the metabolic fate of the transported sugar. The results indicate that the activity of the H⁺-ATPase never fully compensated for H⁺ uptake by the sugar-symport system, because at least 10% of symport-caused charge inflow was compensated for by K⁺ efflux. The restoration of pH in the cytoplasm and in the medium was probably achieved by metabolic reactions connected to increased glycolysis and respiration.Abbreviations DMO dimethyloxazolidinedione - EDTA ethylcnediaminetetraacetic acid - p.c. packed cell volume     

Characterization of liposomes coated with S-layer proteins from lactobacilli

The stability of liposomes coated with S-layer proteins from Lactobacillus brevis and Lactobacillus kefir was analyzed as a previous stage to the development of a vaccine vehicle for oral administration. The interactions of the different S-layer proteins with positively charged liposomes prepared with soybean lecithin or dipalmitoylphosphatidylcholine were studied by means of the variation of the Z potential at different protein-lipid ratios, showing that both proteins were able to attach in a greater extent to the surface of soybean lecithin liposomes. The capacity of these particles to retain carboxyfluorescein or calcein by exposure to bile salts, pancreatic extract, pH change and after a thermal shock showed that both S-layer proteins increased the stability of the liposomes in the same magnitude. The non-glycosylated protein from L. brevis protects more efficiently the liposomes at pH 7 than those from L. kefir even without treatment with glutaraldehyde.     

Intracellular pH regulatory mechanism in human atrial myocardium: functional evidence for Na(+)/H(+) exchanger and Na(+)/HCO(3)(-) symporter

Intracellular pH (pH(i)) exerts considerable influence on cardiac contractility and rhythm. Over the last few years, extensive progress has been made in understanding the system that controls pH(i) in animal cardiomyocytes. In addition to the housekeeping Na(+)-H(+) exchanger (NHE), the Na(+)-HCO(3)(-) symporter (NHS) has been demonstrated in animal cardiomyocytes as another acid extruder. However, whether the NHE and NHS functions exist in human atrial cardiomyocytes remains unclear. We therefore investigated the mechanism of pH(i) recovery from intracellular acidosis (induced by NH(4)Cl prepulse) using intracellular 2',7'-bis(2-carboxethyl)-5(6)-carboxy-fluorescein fluorescence in human atrial myocardium. In HEPES (nominally HCO(3)(-)-free) Tyrode solution, pH(i) recovery from induced intracellular acidosis could be blocked completely by 30 microM 3-methylsulfonyl-4-piperidinobenzoyl, guanidine hydrochloride (HOE 694), a specific NHE inhibitor, or by removing extracellular Na(+). In 3% CO(2)-HCO(3)(-) Tyrode solution, HOE 694 only slowed the pH(i) recovery, while addition of HOE 694 together with 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (an NHS inhibitor) or removal of extracellular Na(+) inhibited the acid extrusion entirely. Therefore, in the present study, we provided evidence that two acid extruders involved in acid extrusion in human atrial myocytes, one which is HCO(3)(-) independent and one which is HCO(3)(-) dependent, are mostly likely NHE and NHS, respectively. When we checked the percentage of contribution of these two carriers to pH(i) recovery following induced acidosis, we found that the activity of NHE increased steeply in the acid direction, while that of NHS did not change. Our present data indicate for the first time that two acid extruders, NHE and NHS, exist functionally and pH(i) dependently in human atrial cardiomyocytes.     

Protective effects of activated vitamin D receptor on radiation‐induced intestinal injury

Radiation‐induced intestinal injury (RIII) is a common complication after radiation therapy in patients with pelvic, abdominal, or retroperitoneal tumours. Recently, in the model of DSS (Dextran Sulfate Sodium Salt) ‐induced intestinal inflammatory injury, it has been found in the study that transgenic mice expressing hVDR in IEC (Intestinal Epithelial Cell) manifest highly anti‐injury properties in colitis, suggesting that activated VDR in the epithelial cells of intestine may inhibit colitis by protecting the mucosal epithelial barrier. In this study, we investigated the effect of the expression and regulation of VDR on the protection of RIII, and the radiosensitivity in vitro experiments, and explored the initial mechanism of VDR in regulating radiosensitivity of IEC. As a result, we found that the expression of VDR in intestinal tissues and cells in mice can be induced by ionizing radiation. VDR agonists are able to prolong the average survival time of mice after radiation and reduce the radiation‐induced intestinal injury. For lack of vitamin D, the radiosensitivity of intestinal epithelial cells in mice increased, which can be reduced by VDR activation. Ensuing VDR activation, the radiation‐induced intestinal stem cells damage is decreased, and the regeneration and differentiation of intestinal stem cells is promoted as well. Finally, on the basis of sequencing analysis, we validated and found that VDR may target the HIF/PDK1 pathway to mitigate RIII. We concluded that agonism or upregulation of VDR expression attenuates radiation‐induced intestinal damage in mice and promotes the repair of epithelial damage in intestinal stem cells.     

In vivo interaction between alveolar macrophages and Cryptococcus neoformans

In vivo interactions of rabbit alveolar macrophages (AM) and Cryptococcus neoformans, a yeast pathogenic for humans, were studied. As a control, inert silica particles of a similar diameter (5–6 μm) were used. Of 16 rabbits, 6 were instilled intratracheally with fluorescein-labelled heat-killed C. neoformans, 6 with fluorescein-labelled silica particles and 4 with saline only. After 24 h, the AM were collected by lung lavage, and phagocytosis, oxidative metabolism, phagolysosomal pH and morphology were studied. The accumulated number of yeasts attached to the AM was almost the same for C. neoformans as for the silica particles. The ingested fraction of C. neoformans was even higher than that of the silica particles. Quantitative NBT reduction by the AM, reflecting their oxidative metabolism, was markedly increased by exposure to C. neoformans for 24 h. The phagolysosomal pH was on the average lower in phagolysosomes with C. neoformans than with the silica particles, although approximately 2% of the phagolysosomes with C. neoformans had neutral pH. Phagolysosomes with neutral pH was not observed for silica particles. Electron microscopy showed presence of C. neoformans in phagolysosomes of AM. The conclusion of this study is that the phagocytic activity, oxidative metabolism and phagolysosomal pH AM against C. neoformans are significant 24 h after the exposure. This revised version was published online in June 2006 with corrections to the Cover Date.     

Variation between mouse major urinary protein genes isolated from a single inbred line

We describe ten Charon 4A genomic DNA clones from BALB/c mice which include at least seven different major urinary protein (MUP) genes. We have established the orientation of all seven sequences, and have placed six of them in precise register by means of restriction site maps and Southern blot hybridization with cloned cDNA sequences. Four of the seven genomic sequences (family I sequences) form hybrids with six independent cDNA clones that have a high thermal stability and hybridize more strongly with mRNA from three inbred mouse lines. Hybrids between the remaining three genomic sequences and the cDNA clones have a lower thermal stability and hybridize less strongly with mRNA from the three inbred lines. Homologies between different cloned sequences extend over as much as 15 kb. No clone contains parts of two MUP genes, and no homology has been detected between the 3' flanking region of one MUP gene and the 5' flanking region of another.