Hyperosmolar Medium Research Articles

Coordinate regulation between the nuclear receptor peroxisome proliferator-activated receptor-γ and cyclooxygenase-2 in renal epithelial cells

The peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors involved in lipid metabolism and glucose utilization, in cell growth, differentiation and apoptosis, and in the regulation of pro-inflammatory genes expression such as cyclooxygenase-2 (COX-2). PPARγ is the main isoform in the renal inner medulla where it is believed to possess nephroprotective actions. In this kidney zone, COX-2 acts as an osmoprotective gene and its expression is modulated by changes in interstitial osmolarity. In the present work we evaluated whether hyperosmolar-induced COX-2 expression is modulated by PPARγ in renal epithelial cells MDCK subjected to high NaCl medium. The results presented herein show that ligand-activated PPARγ repressed COX-2 expression. But more important, the present findings show that hyperosmolar medium decreased PPARγ protein and increases the PPARγ phosphorylated form, which is inactive. ERK1/2 and p38 activation precedes PPARγ disappearance and induced-COX-2 expression. Therefore, the decrease in PPARγ expression is required for hyperosmotic induction of COX-2. We also found that PGE2, the main product of COX-2 in MDCK cells, induced these changes in PPARγ protein. Our results may alert on the long term use of thiazolidinediones (TZD) since they could affect renal medullary function that depends on COX-2 for cellular protection against osmotic stress.

Trefoil Factor Family Peptide 3 (TFF3) is Upregulated Under Experimental Conditions Similar to Dry Eye Disease and Supports Corneal Wound Healing Effects In Vitro

To elucidate the role of trefoil family peptide (TFF) 3 at the ocular surface under conditions similar to dry eye disease (DED) and in tears of patients suffering from DED. Trefoil family peptide 3 levels in tear samples from non-Sjögren's DED patients with moderate dry eye were analyzed by ELISA and compared with tears from healthy volunteers. A human corneal epithelial (HCE) cell line was treated with proinflammatory cytokines IL-1β and TNF-α, hyperosmolar medium, or scratching for up to 24 hours. Trefoil family peptide 3 gene expression and protein biosynthesis were analyzed by RT-PCR, immunofluorescence, and ELISA. Migration and proliferation of HCE cells under recombinant (r) human (h) trefoil factor family peptide 3 (TFF3) stimulation were investigated by scratching and bromodeoxyuridine (BrdU) proliferation assays. Tears of patients suffering from DED contained significantly higher TFF3 levels than tears from healthy volunteers. Stimulation of HCE cells with proinflammatory cytokines, culture under hyperosmolar conditions, or scratching resulted, with the exception of hyperosmolar conditions, in an increase in TFF3 expression and elevated secretion level of TFF3. Cell proliferation decreased and cell migration increased after 24-hours stimulation with rhTFF3. These results suggest that inflammatory factors or ocular surface damage as they occur in DED, lead to an increase of TFF3 tear film concentration, whereas hyperosmolarity does not. Our data underline a potential role for TFF3 as a candidate therapeutic for the ocular surface damage observed in DED.

Effect of Nerve Growth Factor on the In Vitro Induction of Apoptosis of Human Conjunctival Epithelial Cells by Hyperosmolar Stress

To evaluate the effects of nerve growth factor (NGF), which is activated during inflammatory episodes of ocular diseases, on the apoptotic response in cultured human primary conjunctival epithelial cells (pHCECs). Levels of NGF transcripts and NGF protein in pHCEC grown in medium with normal osmolarity (307 mOsm/L) or hyperosmolar medium (350, 400, and 450 mOsm/L) were determined using RT-PCR or ELISA, respectively. To assess apoptosis, pHCEC were cultured in normal or 400 mOsm/L hyperosmolar medium with neutralizing anti-NGF antibody or recombinant human NGF for 6 hours before analysis by flow cytometry. Levels of Bcl-xL, Bax, phosphorylated JNK, and cleaved caspase-3 were detected using Western blotting. Levels of the inflammatory cytokine IL-6 was analyzed using ELISA. Hyperosmolar conditions increased NGF levels in cultured pHCECs. Hyperosmolarity and exposure to neutralizing anti-NGF antibody significantly increased the number of apoptotic cells. Addition of recombinant human NGF to 400 mOsm/L medium decreased the number of apoptotic cells, reduced the expression of phosphorylated JNK, Bax, and cleaved caspase-3 and increased the expression of Bcl-xL. Levels of IL-6 were increased by hyperosmotic stress but decreased by exposure to recombinant human NGF. Hyperosmolarity induces apoptosis of pHCECs by activating JNK signaling. Increased levels of NGF under hyperosmolar conditions may contribute, at least in part, to the reduced apoptosis of pHCECs and may be beneficial in recovering conjunctival damage due to chronic hyperosmolar stress.

A Prospective Study on the Gastrografin Contrast Radiology in the Management of Small Bowel Obstruction

Objective: To investigate the value of the hyperosmolar water-soluble contrast medium Gastrografin® in choosing which patients with small-intestine obstruction, caused by post-operative adhesions, to treat conservatively. Summary Background Data: Post-operative abdominal adhesions represent the main aetiological factor for intestinal obstruction. The mortality rate from intestinal obstruction has been quoted at 5 – 10% . The most effective way to reduce the morbidity and mortality caused by post-operative adhesions is, therefore, through early diagnosis and appropriate treatment. Methods: A prospective Gastrografin® study was performed in patients with small-intestine obstruction from June 2010 to June 2011. Results: Fifty-eight patients with adhesive intestinal obstruction received Gastrografin® via a nasogastric tube. Direct abdominal radiographs were taken after 4 and 8 h. Thirty-nine patients (67.2%) had Gastrografin® in the right colon within 8 h and were considered to have partial obstruction. These patients commenced oral feeding even though abdominal radiographs revealed gas–fluid levels. In the remaining 19 patients (32.7%), Gastrografin® was not observed in the right colon within 8 h: five of the 19 patients (26.3%) were successfully treated conservatively; 14 of the 19 patients (73.7%) developed toxic signs and underwent surgery, with obstruction resulting from adhesive bands being confirmed at operation. Conclusions: Conservative treatment can be recommended for patients in whom contrast medium is observed in the right colon within 8 h following administration, regardless of the presence of obstruction signs. Absence of contrast medium in the right colon within 8 h cannot, however, be considered an indication for surgery.

Water-Soluble Contrast Agents in Small Bowel Obstruction: A Useful Discriminator

AJR 2012; 199:W783 0361–803X/12/1996–W783 © American Roentgen Ray Society Water-Soluble Contrast Agents in Small Bowel Obstruction: A Useful Discriminator We read with great interest the review article, “Small Bowel Obstruction,” by Mullan et al. [1] in the February 2012 issue of the AJR. The article provides a practical overview of a frequently occurring situation in abdominal diagnostic radiology. However, we disagree with the authors on a significant topic: namely, the usefulness of abdominal radiography after ingestion of a water-soluble contrast agent. We would like to suggest the use of water-soluble contrast agents not only for diagnostic but also for therapeutic purposes. This practice may eliminate the need for surgery and decrease the length of hospital stays. Over the past decade, the use of watersoluble contrast agents in small bowel obstruction caused by adhesions has been the subject of several randomized controlled trials published in surgical journals. Its use has therefore been incorporated in the diagnostic workup of patients who present with small bowel obstruction suspected to be due to adhesions. Consequently, we receive an increasing number of requests for a conventional abdominal radiography in patients who have ingested a water-soluble contrast agent. The appearance of contrast medium in the colon within 24 hours after ingestion of a water-soluble contrast agent predicts resolution of the obstruction without the need for surgery with high sensitivity (0.97), specificity (0.96), and receiver operating characteristic value (0.98) [2]. The administration of watersoluble contrast agents significantly reduced the length of hospital stay. A reduction in the need for surgery is claimed by some authors [3, 4] but denied by others [2]. Figure 1 shows that contrast medium has progressed to the jejunum at 8 hours after administration and the ascending colon at 24 hours. The proposed therapeutic mechanism of water-soluble contrast agents is related to their hyperosmolarity (approximately six times that of extracellular fluid). This promotes shifting of fluid into the bowel lumen, which decreases the pressure gradient over the obstruction, which distends collapsed bowel segments, dilutes the contents, and facilitates passage of water that is attracted to the hyperosmolar contrast medium [3]. Water-soluble contrast agents are relatively safe to use, even if a perforation occurs. Barium contrast material does not appear to be a useful alternative. The use of barium holds risks because it may lead to complete obstruction by decreasing the viscosity of bowel contents. Moreover, in case of perforation, leakage of barium to the abdominal cavity is potentially lethal [3]. In conclusion, the use of water-soluble contrast agents can be of diagnostic and therapeutic effect in cases of adhesive small bowel obstruction. Hein P. Stallmann Jaap Borstlap Thomas L. Bollen St. Antonius Hospital, Nieuwegein, The Netherlands

Severe adhesive small bowel obstruction

Adhesive small bowel obstruction is a frequent cause of hospital admission. Water soluble contrast studies may have diagnostic and therapeutic value and avoid challenging demanding surgical operations, but if bowel ischemia is suspected, prompt surgical intervention is mandatory. A 58-year-old patient was operated for extensive adhesive small bowel obstruction after having had two previous laparotomies for colorectal surgery, and had a complex clinical course with multiple operations and several complications. Different strategies of management have been adopted, including non-operative management with the use of hyperosmolar water soluble contrast medium, multiple surgical procedures, total parenteral nutrition (TPN) support, and finally use of antiadherences icodextrin solution. After 2 years follow-up the patient was doing well without presenting recurrent episodes of adhesive small bowel obstruction. For patients admitted several times for adhesive small bowel obstruction, the relative risk of recurring obstruction increases in relation to the number of prior episodes. Several strategies for non-operative conservative management of adhesive small bowel obstruction have already addressed diagnostic and therapeutic value of hyperosmolar water soluble contrast. According to the most recent evidence-based guidelines, open surgery is the preferred method for surgical treatment of strangulating adhesive small bowel obstruction as well as after failed conservative management. Research interest and clinical evidence are increasing in adhesions prevention. Hyaluronic acid-carboxycellulose membrane and icodextrin may reduce incidence of adhesions.

Regulatory volume increase (RVI) and apoptotic volume decrease (AVD) in U937 cells in hypertonic medium

Changes in intracellular water, K+ and Na+ of U937 cells incubated in hyperosmolar medium supplemented with 200 mM sucrose have been studied. Cells were stained with acrydine orange, ethydium bromide, APOPercentage dye, which marks the phosphatidyl serine distribution on the plasma membrane; and FLICA polycaspase fluorescent dye. It was found that cell shrinkage produced by direct osmotic effect induced both a regulatory volume increase and apoptotic volume decrease. The regulatory volume increase dominated at the early stage, whereas apoptotic volume decrease prevailed at the later stage. Therefore, U937 cells were capable of triggering apoptosis and apoptotic volume decrease, despite the unimpaired regulatory volume increase response, and the current opinion that the dysfunction of the regulatory volume increase is a prerequisite for apoptosis and apoptotic volume decrease (Subramanyam et al., 2010) should be revised. It is concluded that the apoptotic volume decrease plays a significant role in preventing osmotic lysis in apoptotic cells, rather than in initiating apoptosis.

Osmoregulation in Lilium pollen grains occurs via modulation of the plasma membrane H+ ATPase activity by 14-3-3 proteins.

To allow successful germination and growth of a pollen tube, mature and dehydrated pollen grains (PGs) take up water and have to adjust their turgor pressure according to the water potential of the surrounding stigma surface. The turgor pressure of PGs of lily (Lilium longiflorum) was measured with a modified pressure probe for simultaneous recordings of turgor pressure and membrane potential to investigate the relation between water and electrogenic ion transport in osmoregulation. Upon hyperosmolar shock, the turgor pressure decreased, and the plasma membrane (PM) hyperpolarizes in parallel, whereas depolarization of the PM was observed with hypoosmolar treatment. An acidification and alkalinization of the external medium was monitored after hyper- and hypoosmotic treatments, respectively, and pH changes were blocked by vanadate, indicating a putative role of the PM H(+) ATPase. Indeed, an increase in PM-associated 14-3-3 proteins and an increase in PM H(+) ATPase activity were detected in PGs challenged by hyperosmolar medium. We therefore suggest that in PGs the PM H(+) ATPase via modulation of its activity by 14-3-3 proteins is involved in the regulation of turgor pressure.

Chromatin condensation modulates access and binding of nuclear proteins

The condensation level of chromatin is controlled by epigenetic modifications and associated regulatory factors and changes throughout differentiation and cell cycle progression. To test whether changes of chromatin condensation levels per se affect access and binding of proteins, we used a hypertonic cell treatment. This shift to hyperosmolar medium increased nuclear calcium concentrations and induced a reversible chromatin condensation comparable to the levels in mitosis. However, this condensation was independent of mitotic histone H3 serine 10 phosphorylation. Photobleaching and photoactivation experiments with chromatin proteins—histone H2B-GFP and GFP-HP1α—before and after induced chromatin condensation demonstrated that hypercondensation reduced their dissociation rate and stabilized their chromatin binding. Finally, measuring the distribution of nucleoplasmic proteins in the size range from 30 to 230 kDa, we found that even relatively small proteins like GFP were excluded from highly condensed chromatin in living cells. These results suggest that structural changes in condensed chromatin by themselves affect chromatin access and binding of chromatin proteins independent of regulatory histone modifications.—Martin, R. M., Cardoso, M. C. Chromatin condensation modulates access and binding of nuclear proteins.

Genotyping, physiological features and proteolytic activities of a potentially pathogenic Acanthamoeba sp. isolated from tap water in Brazil

Acanthamoeba spp., known to cause keratitis and granulomatous encephalitis in humans, are frequently isolated from a variety of water sources. Here we report for the first time the characterization of an Acanthamoeba sp. (ACC01) isolated from tap water in Brazil. This organism is currently being maintained in an axenic growth medium. Phylogenetic analysis based on SSU rRNA gene sequences positioned the new isolate in genotype T4, closest to the keratitis-causing isolate, A. polyphaga ATCC 30461 (∼99% similarity). Acanthamoeba ACC01 and A. polyphaga 30461 both grew at 37 °C and were osmotically resistant, multiplying in hyperosmolar medium. Both isolates secreted comparable amounts of proteolytic enzymes, including serine peptidases that were optimally active at a near neutral/alkaline pH and resolved identically in gelatin gels. Incubation of gels at pH 4.0 with 2 mM DTT also indicated the secretion of similar cysteine peptidases. Altogether, the results point to the pathogenic potential of Acanthamoeba ACC01.

Lactobacillus acidophilus 74-2 and Butyrate Induce Cyclooxygenase (COX)-1 Expression in Gastric Cancer Cells

Cyclo-oxygenase (COX) profile predicts prognosis of gastric cancer; COX-2 positive tumors are more often aggressive, and COX-2 suppression is protective against gastric cancer. In contrast, COX-1 suppression is harmful to the intestinal mucosa. The COX-1, COX-2, and COX-1ir expression profiles were measured with real-time PCR in primary (AGS) and metastatic (NCI-N87) gastric adenocarcinoma cell lines treated with butyrate, hyperosmolar medium, and, in the case of NCI-N87, cell-free supernatants of probiotic bacteria Lactobacillus acidophilus 74-2 and Bifidobacterium lactis 420. The cell lines showed differences in the profile when treated with either hyperosmolar medium or butyrate. In NCI-N87 COX-2 expression was higher but only COX-1 expression was significantly upregulated by butyrate. Similarly to butyrate, the cell-free supernatant of L. acidophilus 74-2 upregulated COX-1, while COX-2 expression remained unchanged. COX-1ir, including COX-3, was upregulated by probiotics and osmotic stress. In conclusion, consumption of L. acidophilus 74-2 could be beneficial for the expression of cytoprotective COX-1.

Proliferation of Peripheral Blood Mononuclear Cells Increases Riboflavin Influx

Abstract. Previously we demonstrated that proliferation of peripheral blood mononuclear cells (PBMC) causes a five-fold increase in cellular uptake of biotin; this increase is mediated by an increased number of biotin transporters on the PBMC surface. In the present study, we investigated the specificity of this phenomenon by determining whether the cellular uptake of riboflavin also increases in proliferating PBMC and whether the increase is also mediated by an increased number of transporters per cell. We characterized [3H]riboflavin uptake in both quiescent and proliferating PBMC. In quiescent PBMC, [3H]riboflavin uptake exhibited saturation kinetics and was reduced by addition of unlabeled riboflavin (P < 0.05) or lumichrome (P < 0.01). These observations are consistent with transporter-mediated uptake. [3H]Riboflavin uptake was reduced at 4°C compared with 37°C (P < 0.01) and by 2,4-dinitrophenol (P < 0.05) but not by ouabain or incubation in sodium-free medium. These data provide evidence for an energy-dependent but sodium-independent transporter. Proliferating PBMC accumulated approximately four times more [3H]riboflavin than quiescent PBMC (P < 0.05). Because both transporter affinity and transporter number per cell (as judged by maximal transport rate) were similar in quiescent and proliferating PBMC, we hypothesize that the increased riboflavin uptake by proliferating PBMC reflects only increased cellular volume. To test this hypothesis, PBMC volume was reduced using hyperosmolar medium; [3H]riboflavin uptake decreased to about 50% of isotonic controls (P < 0.01). Thus we conclude that proliferating PBMC increase cellular content of riboflavin and biotin by two different mechanisms.

Effects of Osmoprotectants on Hyperosmolar Stress in Cultured Human Corneal Epithelial Cells

Increased tear osmolarity in dry eye disease has been found to stimulate production of inflammatory cytokines and matrix metalloproteinases by ocular surface epithelial cells. Prokaryotic and mammalian organ system cells maintain normal function under hypertonic conditions by the synthesis or accumulation of osmoprotectant compounds. This study assessed the effect of osmoprotectant compounds on the activation state of mitogen-activated protein (MAP) kinases in human corneal epithelial cells incubated in hyperosmolar conditions. Human corneal epithelial cells were incubated in media of isotonic, physiological osmolarity (300 mOsm) and in hyperosmolar media (400 mOsm), in the presence and absence of osmoprotectants, including several amino acids (L-carnitine and betaine), glycerol, and the polyol erythritol. The phosphorylation (activation) states of c-Jun N-terminal kinases (JNK) and p38 MAP kinases were monitored by Western blot and bead-based immunoassays. Hyperosmolar conditions achieved by addition of sodium chloride or sucrose increased ratios of phosphorylated JNK and p38 to total JNK and p38. Compared with controls, 10 mM L-carnitine or 40 mM erythritol significantly lowered levels of activated MAP kinases in response to hyperosmolar stress. They also lowered ratios of phosphorylated to total kinases to barely detectable levels in cells cultured in isotonic media. The osmoprotectants L-carnitine and erythritol, alone or in combination, were found to protect against stress activation of corneal epithelial cells cultured in hyperosmolar media.

Hyperosmolarity-Induced Cornification of Human Corneal Epithelial Cells Is Regulated by JNK MAPK

To evaluate the effects of hyperosmolar stress on expression of cornified envelope (CE) precursors and transglutaminases (TGs) by primary cultured human corneal epithelial (PCHCE) cells and the regulatory effects of JNK MAPK on this process. Expression of CE precursors and TGs were evaluated in PCHCE cells exposed to media of increasing osmolarity (350-450 mOsM) for 24, 48, and 72 hours. JNK1 and -2 MAPKs were inhibited by addition of short interfering (si)RNA. Relative levels of mRNA transcripts and proteins were evaluated. TG activity, cell viability, and apoptosis were detected in PCHCE cells, with or without siRNA-JNKs. Exposure of PCHCE cells to hyperosmolar medium increased TG activity at 3 hours, levels of the CE precursors SPRR1b and -2a and membrane-associated TG1 mRNA at 6 hours, and tissue-type TG2 mRNA at 24 hours. Osmotic stress decreased corneal epithelial cell viability, which was due in part to stimulation of apoptosis and cornification death. Inhibiting JNK2 production by siRNA in osmotically stressed PCHCE cells prevented the stimulation of SPRR and membrane-associated TG1 production and TG activity, and improved cell viability, whereas inhibition of JNK1 prevented early apoptosis. Osmotic stress promotes production of certain CE proteins and cross-linking membrane-associated TG1 and decreases cell viability via JNK MAPK-mediated pathways. Strategies that inhibit JNK production downregulate the cornification response of PCHCE cells to osmotic stress. These findings have potential therapeutic implications for preventing cornification of the corneal epithelium in response to the hyperosmolar tear film in dry eye disease.

Effect of Dilution of Hyperosmolar Contrast Medium on Characteristics of Pain during Hysterosalpingography

Background: Hyperosomolar contrast media such as Urografin 370 when used in concentrated form during hysterosalpingography (HSG} often provoke pelvic pain. Dilution of such contrast medium should theoretically produce little or no pain after the procedure. Objective: To compare the incidence and duration of painful side effects caused by the use of concentrate and diluted solutions of Urografin, a hyperosmolar contrast medium, during HSG. Method: A randomised double-blind study has been carried out on 140 female patients who underwent HSG for primary infertility during a six months period using an allocation list to concentrated and diluted Urografin solution. Information on the occurrence and severity of pain was obtained from the patient immediately and 48 hours after the procedure. Result: Statistically significant difference (p NQJHM Vol. 14 (1) 2004: pp. 67-69

Effect of Hyperosmolality on β-Defensin Gene Expression by Human Corneal Epithelial Cells

As human beta-defensins (hBD) are important antimicrobial peptides at epithelial surfaces, including the ocular surface, we tested the effect of hyperosmolar conditions on the expression of these peptides by human corneal epithelial cells (HCECs). Simian virus 40-transformed HCECs (n = 5) or primary cultured HCECs (n = 5) were treated with serum-free media or serum-free hyperosmolar (400-500 mOsm/kg) media for 24 hours or serum-free 500 mOsm/kg media for 12 to 48 hours. The effect of hyperosmolality on interleukin-1beta (IL-1beta)-induced hBD-2 expression was also tested. IL-6 expression was studied as a marker of IL-1beta function. Expression of hBD-1, -2, and -3 and IL-6 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). The levels of active IL-1beta (culture supernatants and cell lysates) and pro-IL-1beta (cell lysates) were detected by enzyme-linked immunosorbent assay. HCECs constitutively expressed hBD-1 and -3 but not hBD-2. Hyperosmolar media had no effect on the basal expression of hBD-1 or -3 and did not induce the expression of hBD-2. Treatment with 500 mOsm/kg media for 24 hours decreased the ability of IL-1beta to upregulate hBD-2 and IL-6 expression. Active or pro-IL-1beta was not detected in any cell culture sample. Our results suggest that the hyperosmolar environment observed in diseases such as dry eye does not alter defensin expression. However, a hyperosmolar environment may influence cytokine function in ocular surface cells and thus affect their response to injury and inflammation.

TonEBP/OREBP Is a Regulator of Nucleus Pulposus Cell Function and Survival in the Intervertebral Disc

The nucleus pulposus is an aggrecan-rich hydrated tissue that permits the intervertebral disc to resist compressive loads. Adaptation to loading is achieved through an elevation in disc osmolarity mediated by the numerous charged glycosoaminoglycan side chains of the aggrecan molecule. The goal of this investigation was to determine the functional role of the osmo-regulatory protein, TonEBP, in cells of the nucleus pulposus. We found that TonEBP and its downstream target genes were robustly expressed in the tissues of the disc. Above 330 mosmol/kg, cultured nucleus pulposus cells up-regulated target genes TauT, BGT-1, and SMIT; above 450 mosmol/kg, there was raised expression of HSP-70. In hypertonic media there was activation of TauT and heat shock protein-70 (HSP-70) reporter activity and increased binding of TonEBP to the TonE motif. When cells were transfected with the dominant-negative form of TonEBP (DN-TonEBP) there was suppression of TauT and HSP-70 reporter gene expression; pTonEBP enhanced reporter gene expression. Moreover, in hypertonic media, forced expression of DN-TonEBP induced apoptosis. We suppressed TonEBP using small interfering RNA technique and noted a decrease in TauT reporter activity in isotonic as well as hyperosmolar media. Finally, we report that the aggrecan promoter contains two conserved TonE motifs. To evaluate the importance of these motifs, we overexpressed DN-TonEBP and partially silenced TonEBP using small interfering RNA. Both approaches resulted in suppression of aggrecan promoter activity. It is concluded that TonEBP permits the disc cells to adapt to the hyperosmotic milieu while autoregulating the expression of molecules that generate the unique extracellular environment.

Honey dressing in pediatric burns

The medicinal properties of honey have been recognized since antiquity. Although used as an adjuvant method of accelerating wound healing from ancient times, honey has been sporadically used in the treatment of burns. Honey acts mainly as a hyperosmolar medium and prevents bacterial growth. Because of its high viscosity, it forms a physical barrier, and the presence of enzyme catalase gives honey an antioxidant property. Its high-nutrient content improves substrate supply in local environment promoting epithelialization and angiogenesis. In pediatric burn patients no exclusive study has been conducted using honey as a burn dressing. An attempt is being made to evaluate the effect of honey in the management of burns in pediatric patients.

Protein Cargo Delivery Properties of Cell-Penetrating Peptides. A Comparative Study

Application of cell-penetrating peptides for delivering various hydrophilic macromolecules with biological function into cells has gained much attention in recent years. We compared the protein transduction efficiency of four cell-penetrating peptides: penetratin, Tat peptide, transportan, and pVEC and studied the effects of various medium parameters on the uptake. Depletion of cellular energy and lowering of temperature strongly impaired the internalization of protein complexed with cell-penetrating peptides, confirming the endocytotic mechanism of peptide-mediated protein cellular transduction. Peptide-induced protein association with HeLa cells decreased 3-6-fold in energy-depleted cells. Inhibition of clathrin-dependent endocytosis by the hyperosmolar medium decreased the uptake of peptide-avidin complexes 1.5-3-fold and the removal of cholesterol from the plasma membrane 1.2-2-fold, suggesting that both clathrin-dependent and independent endocytosis were involved in peptide-induced cellular delivery of avidin. However, even under conditions of cellular energy depletion, ceasing of cellular traffic, and partial depolarization of plasma membrane, peptide-protein complexes associated with HeLa cells, as observed by FACS analysis and spectrofluorimetry. Among the studied peptides, pTat and transportan revealed higher protein transduction efficiency than penetratin or pVEC.

PhgABC , a Three-Gene Operon Required for Growth of Streptococcus pneumoniae in Hyperosmotic Medium and In Vivo

To cause disease, bacterial pathogens need to be able to adapt to the physiological conditions found within the host, including an osmolality of approximately 290 mosmol kg(-1). While investigating Streptococcus pneumoniae genes contained within pneumococcal pathogenicity island 1, we identified a three-gene operon of unknown function termed phgABC. PhgC has a domain with similarity to diacylglycerol kinases of eukaryotes and is the first described member of a family of related proteins found in many gram-positive bacteria. phgA and phgC mutant strains were constructed by insertional duplication mutagenesis and found to have impaired growth under conditions of high osmotic and oxidative stress. The compatible solutes proline and glycine betaine improved growth of the wild-type and the phgA mutant strains in hyperosmolar medium, and when analyzed by electron microscopy, the cellular morphology of the phgA mutant strain was unaffected by osmotic stress. The phgA and phgC mutant strains were reduced in virulence in models of both systemic and pulmonary infection. As the virulence of the phgA mutant strain was not restored in gp91phox(-/-) mice and the phgA and phgC mutant strains had reduced growth in both blood and serum, the reduced virulence of these strains is unlikely to be due to increased sensitivity to the respiratory burst of phagocytes but is, instead, due to impaired growth at physiological osmolality.