Synthesis and characterization of a novel DTPA polymerized hemoglobin based oxygen carrier

OBJECTIVE: The purpose of this study was to prepare a novel polymerized hemoglobin (Hb) based oxygen carrier (HBOC) designed to minimize Hb induced hypertension, while employing a simple and inexpensive method of preparation. Cyclic-diethylenetriaminepentaacetic acid (DTPA) was used to polymerize stroma free Hb (SF-Hb). METHODS: SF-Hb was isolated from red blood cells and reacted with DTPA at a constant concentration, pH, and duration. Low molar mass fractions (<100 kDa) were removed using ultrafiltration. Reactions and subsequent ultrafiltration steps were determined to be reproducible by analyzing molar mass, colloid osmotic pressure and oxygen affinity. Finally, a model of 50% exchange transfusion (ET) in rats was used to evaluate the blood pressure response to DTPA polymerized SF-Hb (Poly-DTPA-Hb). RESULTS: Poly-DTPA-Hb demonstrated a number averaged molar mass of 128.7 kDa and a weighted average of 223.0 kDa. Oxygen binding equilibrium indicated high oxygen affinity (P50 = 5.1+/-0.01 mm Hg) and little cooperativity (n = 1.4). Poly-DTPA-Hb and a control DTPA polymerized human serum albumin (Poly-DTPA-HSA) unexpectedly caused acute hypotension during the period of ET in rats (mean arterial pressure approximately 45% less than baseline). Hypotension occurring over the period of ET was determined to be mediated by calcium binding to protein associated DTPA. This effect was attenuated by the addition of calcium chloride (CaCl2) to the Poly-DTPA protein preparations. CONCLUSIONS: Cyclic DTPA anhydride can be used to create cross-linked and polymerized hemoglobin, using a simple and inexpensive process. However, the addition of CaCl2 to the preparation appears to be required to prevent calcium chelation and subsequent hypotension during infusion.     

Anti-tumor efficacy of interleukin-2-activated killer cells in human neuroblastoma ex vivo

We studied the ex vivo sensitivity of continuously cultured neuroblastoma cells from 3 different patients towards interleukin-2-induced cell-mediated cytotoxicity. A mean (+/- SD) target cell lysis (4 h 51Cr release) of 49 +/- 11, 46 +/- 8, and 32 +/- 11% in SMS-SAN, LA-N-1, and SK-N-BE2 cell lines, respectively, was achieved when neuroblastoma cells were co-cultured at an effector-to-target (E:T) ratio of 50:1 with peripheral blood mononuclear cells (PBMC) that had been preincubated for 4 days in the presence of recombinant interleukin-2 (rIL-2; 100 U/ml). Under identical conditions, 93 +/- 9% of Daudi cells (a standard target for rIL-2-activated killer cells) were lysed. Preincubation of rIL-2-induced PBMC cultures in the presence of irradiated neuroblastoma targets (LA-N-1, SK-N-BE2) resulted in a significant cytolytic augmentation. At E:T ratios of 50:1 and 10:1, day-4 rIL-2/LA-N-1-stimulated PBMC produced 69 +/- 7 and 41 +/- 11% lysis of LA-N-1 cells, as compared to 46 +/- 8 and 22 +/- (mean +/- SD) 7% lysis by untargeted PBMC that were preincubated with rIL-2 (100 U/ml) in the absence of LA-N-1 target cells (p less than 0.05). Co-incubation of rIL-2-induced PBMC preparations with irradiated LA-N-1 and SK-N-BE2 cells, respectively, did not significantly enhance the cytolytic activity against other neuroblastoma targets and the standard Daudi cell line (p greater than or equal to 0.3).(ABSTRACT TRUNCATED AT 250 WORDS)     

Divergent effects of RIP1 or RIP3 blockade in murine models of acute liver injury

Necroptosis is a recently described Caspase 8-independent method of cell death that denotes organized cellular necrosis. The roles of RIP1 and RIP3 in mediating hepatocyte death from acute liver injury are incompletely defined. Effects of necroptosis blockade were studied by separately targeting RIP1 and RIP3 in diverse murine models of acute liver injury. Blockade of necroptosis had disparate effects on disease outcome depending on the precise etiology of liver injury and component of the necrosome targeted. In ConA-induced autoimmune hepatitis, RIP3 deletion was protective, whereas RIP1 inhibition exacerbated disease, accelerated animal death, and was associated with increased hepatocyte apoptosis. Conversely, in acetaminophen-mediated liver injury, blockade of either RIP1 or RIP3 was protective and was associated with lower NLRP3 inflammasome activation. Our work highlights the fact that diverse modes of acute liver injury have differing requirements for RIP1 and RIP3; moreover, within a single injury model, RIP1 and RIP3 blockade can have diametrically opposite effects on tissue damage, suggesting that interference with distinct components of the necrosome must be considered separately.The etiologies of acute liver injury are diverse and its overall public health burden is considerable. Liver injury from acetaminophen (APAP) overdose is the most common cause of death from over-the-counter drugs and is the leading cause of acute liver failure in the developed world.^{1, 2, 3} Hepatic dysfunction from autoimmune hepatitis has a prevalence of 10–20/100 000.^{4, 5} Other etiologies of acute liver failure include idiosyncratic reaction to medications such as tetracycline, severe viral or alcoholic hepatitis, acute fatty liver of pregnancy, and idiopathic causes. Clinical complications resulting from liver failure include hepatic encephalopathy, impaired protein synthesis, and coagulopathies. Moreover, there are no effective means to reverse liver failure once advanced disease sets in – regardless of etiology – and transplantation frequently remains the only option for survival.⁶Concanavalin-A (ConA) is a lectin derived from the jack-bean plant with a unique ability to induce hepatitis in a well-described murine model of acute hepatic injury. ConA stimulates mouse CD4⁺ T-cell subsets to mediate insult to hepatocytes. The resulting cytokine release can further lead to recruitment and activation of innate inflammatory mediators, which perpetuate an insidious cycle of inflammation and hepatocellular injury.^{7, 8, 9}APAP is a widely used analgesic and antipyretic. Although usually considered safe at therapeutic doses, at higher doses APAP causes acute liver failure characterized by centrilobular hepatic necrosis.^{1, 10} At therapeutic doses, >90% of APAP is metabolized by glucuronidation and sulphation and its metabolites are excreted via the renal system. Of the remaining APAP, roughly 2% is excreted intact in the urine, and approximately 8% is metabolized by the cytochrome P450 system to N-acetyl-p-benzo-quinone imine (NAPQI), which is highly reactive.^{11, 12} Hepatic glutathione (GSH) then induces the formation of a safely excretable APAP-protein adduct. However, at toxic doses of APAP, GSH becomes depleted and NAPQI is able to exert harmful effects by forming covalent bonds with mitochondrial proteins, inhibiting the Ca²⁺-Mg²⁺-ATPase and inducing mitochondrial dysfunction.^{1, 2} This disturbance leads to a decrease in ATP synthesis, disruption of cellular membrane, and eventually hepatocyte death.¹³Although GSH depletion and the resulting toxic metabolites are prerequisites for APAP hepatotoxicity, there is evidence that the severity of liver injury may depend on subsequent participation of innate immunity.^{10, 14, 15, 16} In particular, APAP-induced injury has been reported to be contingent on activation of the NLRP3 inflammasome via DAMPs released from injured hepatocytes. Inflammasome activation cleaves Caspase 1 inducing IL-1β release and galvanizing intrahepatic neutrophils and inflammatory monocytes, which exacerbate injury.¹⁷ However, alternate studies using transgenic mice suggest that NLRP3 inflammasome is largely dispensable for APAP toxicity.¹⁸ Thus the role of inflammasome activation in APAP toxicity is controversial and may be dependent on the precise experimental conditions or strain of mice employed.Apoptosis and necrosis are classically understood processes of cell death that denote either organized Caspase 8-dependent programmed cell death or non-programmed disorganized death, respectively. In contrast to necrosis, which leads to the release of DAMPs and sustains inflammation, apoptosis produces cell fragments called apoptotic bodies, which phagocytic cells are able to engulf before the contents of the cell can spill out onto the surrounding space and activate innate immunity. ‘Necroptosis'' is a recently described Caspase 8-independent method of cell death that denotes organized cellular necrosis. Necroptosis requires the co-activation of RIP1 and RIP3 kinases. Both in vitro and in vivo investigations have suggested that APAP can induce cellular demise via necrosis or Caspase 8-dependent apoptosis, which is determined, in part, by ATP availability from glycolysis.¹⁹ Zhang et al.²⁰ recently confirmed that RIP1 is necessary in APAP-induced necroptosis. Similarly, Takemoto et al.²¹ showed that RIP1 inhibition protects against reactive oxygen species (ROS)-induced hepatotoxicity in APAP-induced acute liver injury. Further, a recent report suggested that selective inhibition of RIP3 using the anticancer drug Dabrafenib alleviates APAP injury.²²In the ConA model of acute liver injury, experiments using apoptosis-resistant mice expressing mutant FADD revealed that ConA alone induced primarily necrotic cell death, whereas ConA combined with d-galactosamine induced apoptosis and necrotic cell death.²³ Zhou et al.²⁴ reported that Necrostatin-1 (Nec-1) prevents autoimmune hepatitis in mice via RIP1- and autophagy-related pathways. Another recent report investigated the role of RIP1, RIP3, and PARP-1 in murine autoimmune hepatitis. This study found that in cases where death of mouse hepatocytes is dependent on TRAIL and NKT cells, PARP-1 activity was positively correlated with liver injury and hepatitis was prevented both by RIP1 or PARP-1 inhibitors.²⁵ Our goal in the current study was to investigate, in parallel, the effects of RIP1 and RIP3 blockade in diverse models of acute liver injury. Our work suggests that modulating necroptosis may have divergent effects, depending on the etiology of hepatic injury and the specific component of the necrosome being targeted.     

<Emphasis Type="Italic">Fusarium sacchari</Emphasis>: A Cause of Exogenous Fungal Endophthalmitis: First Case Report and Review of Literature

A case of exogenous fungal endophthalmitis due to Fusarium sacchari is being reported. It has never been reported in this clinical entity before; hence, to the best of our knowledge, this is first report of F. sacchari as a cause of exogenous fungal endophthalmitis and also the first time when this fungus has been isolated from an Indian patient.     

Production of feed enzymes (phytase and plant cell wall hydrolyzing enzymes) by<Emphasis Type="Italic">Mucor indicus</Emphasis> MTCC 6333: Purification and characterization of phytase

The production of phytase and associated feed enzymes (phosphatase, xylanase, CMCase, alpha-amylase and beta-glucosidase) was determined in a thermotolerant fungus Mucor indicus MTCC 6333, isolated from composting soil. Solid-substrate culturing on wheat bran and optimizing other culture conditions (C and N sources, level of N, temperature, pH, culture age, inoculum level), increased the yield of phytase from 266 +/- 0.2 to 513 +/- 0.4 nkat/g substrate dry mass. The culture extract also contained 112, 194, 171, 396, and 333 nkat/g substrate of phosphatase, xylanase, CMCase, beta-glucosidase and alpha-amylase activities, respectively. Simple 2-step purification employing anion exchange and gel filtration chromatography resulted in 21.9-fold purified phytase. The optimum pH and temperature were pH 6.0 and 70 degrees C, respectively. The phytase was thermostable under acidic conditions, showing 82% residual activity after exposure to 60 degrees C at pH 3.0 and 5.0 for 2 h, and displayed broad substrate specificity. The Km was 200 nmol/L and v(lim) of 113 nmol/s per mg protein with dodecasodium phytate as substrate. In vitro feed trial with feed enzyme resulted in the release of 1.68 g inorganic P/kg of feed after 6 h of incubation at 37 degrees C.     

Characterization of protein transacetylase from human placenta as a signaling molecule calreticulin using polyphenolic peracetates as the acetyl group donors

We have earlier shown that a unique membrane-bound enzyme mediates the transfer of acetyl group(s) from polyphenolic peracetates (PA) to functional proteins, which was termed acetoxy drug: protein transacetylase (TAase) because it acted upon several classes of PA. Here, we report the purification of TAase from human placental microsomes to homogeneity with molecular mass of 60 kDa, exhibiting varying degrees of specificity to several classes of PA confirming the structure-activity relationship for the microsome-bound TAase. The TAase catalyzed protein acetylation by a model acetoxy drug, 7,8-diacetoxy-4-methyl coumarin (DAMC) was established by the demonstration of immunoreactivity of the acetylated target protein with anti-acetyl lysine antibody. TAase activity was severely inhibited in calcium-aggregated microsomes as well as when Ca2+ was added to purified TAase, suggesting that TAase could be a calcium binding protein. Furthermore, the N-terminal sequence analysis of purified TAase (EPAVYFKEQFLD) using Swiss Prot Database perfectly matched with calreticulin (CRT), a major microsomal calcium binding protein of the endoplasmic reticulum (ER). The identity of TAase with CRT was substantiated by the observation that the purified TAase avidly reacted with commercially available antibody raised against the C-terminus of human CRT (13 residues peptide, DEEDATGQAKDEL). Purified TAase also showed Ca2+ binding and acted as a substrate for phosphorylation catalyzed by protein kinase C (PKC), which are hallmark characteristics of CRT. Further, purified placental CRT as well as the commercially procured pure CRT yielded significant TAase catalytic activity and were also found effective in mediating the acetylation of the target protein NADPH cytochrome P-450 reductase by DAMC as detected by Western blot using anti-acetyl lysine antibody. These observations for the first time convincingly attribute the transacetylase function to CRT. Hence, this transacetylase function of CRT is designated calreticulin transacetylase (CRTAase). We envisage that CRTAase plays an important role in protein modification by way of acetylation independent of Acetyl CoA.