Focus on the research utility of intravascular ultrasound - comparison with other invasive modalities

Intravascular ultrasound (IVUS) is an invasive modality which provides cross-sectional images of a coronary artery. In these images both the lumen and outer vessel wall can be identified and accurate estimations of their dimensions and of the plaque burden can be obtained. In addition, further processing of the IVUS backscatter signal helps in the characterization of the type of the plaque and thus it has been used to study the natural history of the atherosclerotic evolution. On the other hand its indigenous limitations do not allow IVUS to assess accurately stent struts coverage, existence of thrombus or exact site of plaque rupture and to identify some of the features associated with increased plaque vulnerability. In order this information to be obtained, other modalities such as optical coherence tomography, angioscopy, near infrared spectroscopy and intravascular magnetic resonance imaging have either been utilized or are under evaluation. The aim of this review article is to present the current utilities of IVUS in research and to discuss its advantages and disadvantages over the other imaging techniques.     

A simple HPLC method for plasma level monitoring of mitotane and its two main metabolites in adrenocortical cancer patients

Mitotane (o,p'-DDD or (1,1-dichloro-2-[o-chlorophenyl]-2-[p-chlorophenyl]ethane, DDD) is the drug of choice for non-resectable and metastatic adrenocortical carcinomas (ACC). Measurement of mitotane and metabolites, o,p'-DDE (1,1-dichloro-2-[p-chlorophenyl]-2-[o-chlorophenyl]ethene, DDE) and o,p'-DDA (1,1-[o,p'-dichlorodiphenyl] acetic acid, DDA) provides a better understanding of mitotane pharmacokinetics and pharmacodynamics. We have developed a simple, robust and efficient high performance liquid chromatography (HPLC) method to measure mitotane and its two main metabolites, DDE and DDA. The method involves a single ethanol extraction of mitotane, DDE, DDA, and an internal standard (int std) p,p'-DDD (1,1-dichloro-2,2-bis(p-chlorophenyl)ethane) with an extraction efficiency of 77-88%. All compounds are measured simultaneously using a reversed-phase phenyl HPLC column with an isocratic elution of mobile phase at a flow rate of 0.6 ml/min followed by UV detection at λ 226 nm. Inter and intra-day validation demonstrates good reproducibility and accuracy. Limits of quantitation are 0.2 μg/ml for mitotane and DDE, and 0.5 μg/ml for DDA. The method has been evaluated in plasma from 23 patients on mitotane therapy, revealing DDA concentrations 1-18 times higher than the parent compound.     

Evidence of the formation of direct covalent adducts of primaquine, 2-tert-butylprimaquine (NP-96) and monohydroxy metabolite of NP-96 with glutathione and N-acetylcysteine

2-tert-Butylprimaquine (NP-96) is a novel quinoline anti-malarial compound with superior therapeutic profile than primaquine (PQ). Moreover, it is the first 8-aminoquinoline that is established to be devoid of methemoglobin toxicity. The purpose of the present study was to investigate covalent adduct formation tendency of PQ, NP-96 and their phase I metabolites with glutathione (GSH) and N-acetylcysteine (NAc). For the same, the two compounds were incubated in human and rat liver microsomes in the presence of trapping agents and NADPH. In a control set, NADPH was excluded, while a blank was also studied that was devoid of both NADPH and microsomes. The components in the reaction mixtures were initially separated on a C-18 column (250 mm×4.6mm, 5 μm) using a mobile phase composed of acetonitrile and 10 mM ammonium acetate in a gradient mode. The samples were then subjected to LC-MS(n) and LC-HR-MS analyses, and data were collected in full scan MS, data dependent MS/MS, targeted MS/MS, neutral loss scan (NLS) and accurate mass (MS/TOF) modes. In a significant finding, both PQ and NP-96 themselves showed potential to bind covalently with GSH and NAc, as adducts were observed even in the control and blank incubations. Intense peaks corresponding to covalent adduct of mono-hydroxy metabolite of NP-96 with GSH and NAc were also detected in NADPH supplemented reaction solution.     

A spectrophotometric method for calmodulin assay

The activity of calmodulin as an activator of cAMP phosphodiesterase was assayed. AMP was hydrolyzed by 5'-nucleotidase, and the adenosine formed was measured by both liquid scintillation counting and spectrophotometry at 265 nm. Calmodulin activities measured by the two methods were equivalent, indicating that spectrophotometric assay of calmodulin can be used in place of the isotopic method.     

Modulation of post-thaw sperm functions with oviductal proteins in buffaloes

A study was undertaken to determine the effects of oviductal proteins obtained from various stages of the estrous cycle on spermatozoa characteristics in buffaloes. Oviducts were collected from apparently healthy buffalo genital tracts (nonluteal and luteal stage of estrous cycle) and separated into isthmus and ampulla. Each segment of oviduct (nonluteal and luteal) was flushed with PBS (pH 7.4). The flushing obtained was centrifuged (3000 rpm; 30 min), filtered (0.2 microm) and frozen at -20 degrees C. The proteins in pooled nonluteal isthmic and ampullary and luteal isthmic and ampullary fluids were precipitated overnight using ammonium sulphate, centrifuged (10000 rpm; 30 min) and dialyzed (>10 kDa). After protein estimation, aliquots of samples containing 10 mg proteins were lyophilized in cryovials and stored in frozen form at -20 degrees C. Six pooled good-quality ejaculates collected by artificial vagina method from two Murrah buffalo bulls were utilized for the study. After fresh semen analysis, each pooled ejaculate was splited into five parts and extended in Tris-egg yolk-citrate extender (20% egg yolk; 7% glycerol), so that final dilution yielded approximately 60 million sperm cells per ml, and cryopreserved in 0.5 ml French straws (30 million sperm cells/straw) in LN(2) (-196 degrees C). Before freezing, nonluteal isthmic and ampullary and luteal isthmic and ampullary proteins were incorporated at the rate of 1mg/ml of extended semen. The equilibrated and frozen-thawed (37 degrees C for 30 s) semen was evaluated for motility, live %, acrosomal integrity percentage, bovine cervical mucus penetration test and hypo-osmotic sperm swelling test. Besides this, spermatozoa from treatment and control groups were incubated at 37 degrees C for 6 h in sperm TALP. Among the nonluteal and luteal oviductal proteins, the former maintained higher (P < 0.05) post-thaw sperm motility, live %, and acrosomal integrity than the control group. Between the isthmic and ampullary proteins, the isthmic proteins incorporated group maintained higher (P < 0.05) post-thaw sperm motility, live %, and acrosomal integrity. Similarly, higher sperm penetration distance in cervical mucus was recorded in nonluteal isthmic proteins incorporated group. But, irrespective of the stage of an estrous cycle, isthmic proteins included group maintains higher sperm membrane integrity as revealed by higher (P < 0.05) swollen sperm percentage in response to hypo-osmotic solution than the ampullary proteins included and control groups. Similarly, at any time during incubation the sperm motility and viability was higher (P < 0.05) in isthmic proteins treated group than the ampullary and control group. But, the same trend was not observed in terms of acrosomal integrity percentages. It is inferred that inclusion of oviductal proteins in the extender prior to freezing improved post-thaw semen quality. Oviductal proteins differentially affected sperm function depending upon the region of oviduct and the stage of estrous cycle at which the proteins were obtained.     

Staphylococcus aureus-derived peptidoglycan induces Cx43 expression and functional gap junction intercellular communication in microglia

Gap junctions serve as intercellular conduits that allow the exchange of small molecular weight molecules (up to 1 kDa) including ions, metabolic precursors and second messengers. Microglia are capable of recognizing peptidoglycan (PGN) derived from the outer cell wall of Staphylococcus aureus, a prevalent CNS pathogen, and respond with the robust elaboration of numerous pro-inflammatory mediators. Based on recent reports demonstrating the ability of tumor necrosis factor-alpha and interferon-gamma to induce gap junction coupling in macrophages and microglia, it is possible that pro-inflammatory mediators released from PGN-activated microglia are capable of inducing microglial gap junction communication. In this study, we examined the effects of S. aureus-derived PGN on Cx43, the major connexin in microglial gap junction channels, and functional gap junction communication using single-cell microinjections of Lucifer yellow (LY). Exposure of primary mouse microglia to PGN led to a significant increase in Cx43 mRNA and protein expression. LY microinjection studies revealed that PGN-treated microglia were functionally coupled via gap junctions, the specificity of which was confirmed by the reversal of activation-induced dye coupling by the gap junction blocker 18-alpha-glycyrrhetinic acid. In contrast to PGN-activated microglia, unstimulated cells consistently failed to exhibit LY dye coupling. These results indicate that PGN stimulation can induce the formation of a functional microglial syncytium, suggesting that these cells may be capable of influencing neuro-inflammatory responses in the context of CNS bacterial infections through gap junction intercellular communication.     

Enzymatic activity of naturally occurring 1-acylglycerol-3-phosphate-O-acyltransferase 2 mutants associated with congenital generalized lipodystrophy

Mutations in the gene encoding 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2) have been reported in patients with congenital generalized lipodystrophy (CGL). AGPAT2, a 278 amino acid protein, belongs to the acyltransferase enzyme family, and has two conserved motifs, NHX(4)D and EGTR, involved in the enzymatic activity. The AGPATs catalyze acylation of lysophosphatidic acid (LPA) to phosphatidic acid (PA) during the biosynthesis of glycerophospholipids and triglycerides from glycerol-3-phosphate. The present studies were designed to determine the enzymatic activity of AGPAT2 mutants found in CGL patients to provide a molecular explanation for the phenotype and to obtain additional information about the structure-function relationship of AGPAT2 protein. The enzymatic activities of the wild type AGPAT2 and mutants were determined in cell lysates of overexpressing Chinese hamster ovary cells by measuring the conversion of [(3)H]LPA to [(3)H]PA in the presence of oleoyl-coenzyme A. Whereas, the R68X, 221delGT, 252delMRT, D180fsX251, and V167fsX183 mutants had markedly reduced enzymatic activity (median <15% of the wild type), the mutants, 140delF, G136R, and L228P, retained median activity ranging from 15% to 40% of the wild type enzyme. However, the missense mutant, A239V, had 90% of the wild type activity. We suggest that reduction in AGPAT2 enzymatic activity underlies the loss of adipose tissue in CGL. Our observations reveal an important role of various carboxy-terminal residues in determining the enzymatic activity of AGPAT2.