Established method of chondroitin sulphate extraction from buffalo (Bubalus bubalis) cartilages and its identification by FTIR.

A study was conducted for extraction of chondroitin sulphate (CS) from buffalo tracheal, nasal and joint cartilages. CS was extracted from cartilages using 0.25% papain digestion, dialyzed, precipitated with 10% TCA and finally lyophilized to dry powder. Dimethylmethylene blue assay was performed to estimate the quantity of CS extracted. Identification of extracted CS was performed with SDS-PAGE and Fourier transforms infrared spectroscopy (FTIR). SDS-PAGE analysis of extracted CS revealed similar electrophoretic pattern to that of standard and the molecular weight ranged from 5 to 20 kDa. FTIR spectra of extracted CS revealed presence of characteristic peaks of -CONH vibration of amide group, coupling of C-O stretching vibration, S=O stretching vibrations and -C-O-S molecules confirms the CS moiety. It can be concluded that extraction method adopted could efficiently be utilized for the extraction of CS from buffalo by-products like tracheal, nasal and joint cartilages.

Mechanistic Studies on the Self-Assembly of PLGA Patchy Particles and Their Potential Applications in Biomedical Imaging.

Currently, several challenges prevent poly(lactic-co-glycolic acid) (PLGA) particles from reaching clinical settings. Among these is a lack of understanding of the molecular mechanisms involved in the formation of these particles. We have been studying in depth the formation of patchy polymeric particles. These particles are made of PLGA and lipid-polymer functional groups. They have unique patch-core-shell structural features: hollow or solid hydrophobic cores and a patchy surface. Previously, we identified the shear stress as the most important parameter in a patchy particle's formation. Here, we investigated in detail the role of shear stress in the patchy particle's internal and external structure using an integrative experimental and computational approach. By cross-sectioning the multipatch particles, we found lipid-based structures embedded in the entire PLGA matrix, which represents a unique finding in the PLGA field. By developing novel computational fluid dynamics and molecular dynamics simulations, we found that the shear stress determines the internal structure of the patchy particles. Equally important, we discovered that these particles emit a photoacoustic (PA) signal in the optical clinical imaging window. Our results show that particles with multiple patches emit a higher PA signal than single-patch particles. This phenomenon most likely is due to the fact that multipatchy particles absorb more heat than single-patchy particles as shown by differential scanning calorimetry analysis. Furthermore, we demonstrated the use of patchy polymeric particles as photoacoustic molecular probes both in vitro and in vivo studies. The fundamental studies described here will help us to design more effective PLGA carriers for a number of medical applications as well as to accelerate their medical translation.

A causal link from ALK to hexokinase II overexpression and hyperactive glycolysis in EML4-ALK-positive lung cancer.

A high rate of aerobic glycolysis is a hallmark of malignant transformation. Accumulating evidence suggests that diverse regulatory mechanisms mediate this cancer-associated metabolic change seen in a wide spectrum of cancer. The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion protein is found in approximately 3-7% of non-small cell lung carcinomas (NSCLC). Molecular evidence and therapeutic effectiveness of FDA-approved ALK inhibitors indicated that EML4-ALK is a driving factor of lung tumorigenesis. A recent clinical study showed that NSCLC harboring EML4-ALK rearrangements displayed higher glucose metabolism compared with EML4-ALK-negative NSCLC. In the current work, we presented evidence that EML4-ALK is coupled to overexpression of hexokinase II (HK2), one of the rate-limiting enzymes of the glycolytic pathway. The link from EML4-ALK to HK2 upregulation is essential for a high rate of glycolysis and proliferation of EML4-ALK-rearranged NSCLC cells. We identified hypoxia-inducible factor 1α (HIF1α) as a key transcription factor to drive HK2 gene expression in normoxia in these cells. EML4-ALK induced hypoxia-independent but glucose-dependent accumulation of HIF1α protein via both transcriptional activation of HIF1α mRNA and the phosphatidylinositol 3 kinase-AKT pathway to enhance HIF1α protein synthesis. The EML4-ALK-mediated upregulation of HIF1α, HK2 and glycolytic metabolism was also highly active in vivo as demonstrated by fluorodeoxyglucose-positron emission tomography imaging of xenografts grown from EML4-ALK-positive NSCLC cells. Our data reveal a novel EML4-ALK-HIF1α-HK2 cascade to enhance glucose metabolism in EML4-ALK-positive NSCLC.

SU-D-217A-04: Evaluation of the Spatial Concordance Between the Intratumoral Patterns of 18F-FLT and 18F-FDG Uptake in a Small Animal Tumor Model.

PURPOSE: PET imaging allows for the visualization of tumor microenvironment and identification of aggressive or radioresistant tumor subvolumes that can be targeted with an escalated radiation dose. Multiple PET tracers have been developed for visualization of different aspects of tumor microenvironment; however, the spatial distribution of tracers in tumors is equally affected by tumor tissue viability and tracer delivery limitations. Given these issues and the low resolution associated with PET imaging, two different PET tracers can produce very similar images. Therefore, it is important to demonstrate that a novel PET tracer does provide additional useful information to that obtained with other tracers. This study investigates the added value of performing 18F-FLT PET imaging as well as 18F-FDG imaging. METHODS: Head and neck tumor xenografts grown in nude mice were used to study intratumoral tracerdistributions. 18F-FDG and 18F-FLT PET images were obtained on subsequent days using a small animal PET/CT. Pinnacle 9 was used to deformably register the CT image from the FLT PET/CT to the FDG PET/CT image set. The generated deformation was applied to the FLT PET image to achieve an unbiased FLT to FDG PET image registration. The Pearson correlation coefficient between FDG and FLT was calculated voxel- by-voxel within a tumor contour. Overlap analysis of thresholded tracer distributions was carried out by comparing Dice similarity coefficients. RESULTS: Both SQ20B and FaDu tumors showed a moderate voxel-by-voxel correlation between FDG and FLT intratumoral patterns of uptake with an average rho value of .56 and .63 respectively (range .37-.76) despite significant differences in tumor morphology. The average volumes under thedice coefficient surface for SQ20B and FaDu tumors were not significantly different. CONCLUSIONS: Despite being equally affected by the issues of tracer delivery, necrosis and PET resolution, FDG and FLT PET images displayed an observable difference at clinically relevant thresholds.

Quantum dots for live cells, in vivo imaging, and diagnostics.

Research on fluorescent semiconductor nanocrystals (also known as quantum dots or qdots) has evolved over the past two decades from electronic materials science to biological applications. We review current approaches to the synthesis, solubilization, and functionalization of qdots and their applications to cell and animal biology. Recent examples of their experimental use include the observation of diffusion of individual glycine receptors in living neurons and the identification of lymph nodes in live animals by near-infrared emission during surgery. The new generations of qdots have far-reaching potential for the study of intracellular processes at the single-molecule level, high-resolution cellular imaging, long-term in vivo observation of cell trafficking, tumor targeting, and diagnostics.

Quantum dots for molecular imaging and cancer medicine.

Extract: The past few decades have witnessed technical advances that have introduced cell biologists and physicians to a new, dynamic, subcellular world where genes and gene products can be visualized to interact in space and time and in health and disease. The accelerating field of molecular imaging has been critically dependent on indicator probes which show when and where genetically or biochemically defined molecules, signals or processes appear, interact and disappear, with high spatial and temporal resolution in living cells and whole organisms. For example, the use of radionuclide tracers combined with 3-dimensional (3-D) imaging systems such as Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) are now helping clinicians to characterize the molecular status of tumors deep within patients. Other types of imaging probes rely on the bioluminescence and fluorescence of genetically encoded proteins (originally found in fireflies and jellyfish, respectively) or entirely synthetic fluorochromes, or a combination of both. New powerful biological fluorescence microscopes provide the ability to study single molecules within single cells. Multiphoton confocal microscopy has been developed to allow for the capturing of high-resolution, 3-D images of living tissues that have been tagged with highly specific fluorophores.

MicroPET imaging of Cre-loxP-mediated conditional activation of a herpes simplex virus type 1 thymidine kinase reporter gene.

Site-specific recombination tools such as the Cre-loxP system are used to create animal models where conditional gene deletion/activation studies are required. In the current proof of principle study, we have demonstrated that a PET reporter gene (PRG), the herpes simplex virus type 1 thymidine kinase (HSV1-tk), can be made to remain silent and can be activated by Cre-loxP-mediated recombination in cell culture and in living mice. An adenovirus carrying a silent HSV1-tk was tail-vein injected (1 x 10(9) PFU) in six transgenic mice that express Cre recombinase in their liver (Cre+) and in four control mice (Cre-). The liver-specific expression of the PRG in Cre+ mice was detected in the microPET following injection of the reporter probe, 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine ([(18)F]-FHBG). The [(18)F]-FHBG accumulation in the liver in terms of percent-injected dose per gram of tissue was 7.72+/-1.13 for the Cre+ mice and 0.10+/-0.02 for the Cre- mice (P<0.05) 48 h after adenoviral injection. These results were further validated by quantitative RT-PCR, western blotting and by in vitro assays for herpes simplex virus type 1 thymidine kinase enzyme activity. Thus by using the Cre-loxP system it is possible to modulate a PRG and noninvasively monitor the extent of Cre-loxP-mediated gene activation by imaging in a microPET scanner.

Genetic analyses of cis-acting sequences controlling expression of human immunodeficiency virus type 1 coreceptor-CCR5 gene in rabbits and CXCR4 gene in monkeys.

INTRODUCTION: Human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus all use chemokine receptors (CCR5, CXCR4, and minor receptors) to gain entry into a susceptible cell and establish infection successfully by way of membrane fusion. Many such chemokine receptors that can act as entry cofactors under in vitro conditions have been identified, but the roles of CCR5 and CXCR4 chemokine receptors in infection, tropism, and pathogenesis have been studied in greater detail. The promoter region of CCR5 gene is quite polymorphic in humans, and mutations that affect the progression of HIV-1 have been identified. STUDY DESIGN/METHODS: We studied the nature of mutations in the CCR5 promoter region in rabbits. Large number of mutations, deletions, substitutions, and point mutations were observed all along the 400 base pair region of the promoter. RESULTS: We show that rabbit CCR5 promoter possesses features common to both humans and monkeys and lacks the second highly polymorphic region B in the CCR5 promoter that was previously identified in monkeys. Besides providing important evolutionary information, our findings can directly make an impact on the known expression levels of CCR5 protein that can modulate the progression of HIV-1 in rabbits. The CXCR4 promoter of monkeys showed polymorphisms that were largely caused by single nucleotide changes when compared with humans. CONCLUSIONS: This distinctly different evolutionary pattern suggests a more important role for chemokine receptor-CCR5 in the host defense.

Noninvasive optical imaging of firefly luciferase reporter gene expression in skeletal muscles of living mice.

The ability to monitor reporter gene expression noninvasively offers significant advantages over current techniques such as postmortem tissue staining or enzyme activity assays. Here we demonstrate a novel method of repetitively tracking in vivo gene expression of firefly luciferase (FL) in skeletal muscles of mice using a cooled charged coupled device (CCD) camera. We first show that the cooled CCD camera provides consistent and reproducible results within +/-8% standard deviation from mean values, and a detection sensitivity (range tested: 1 x 10(4) - 1 x 10(9) plaque form-ing units (pfu)) of 1 x 10(6) pfu of E1-deleted adenovirus expressing FL driven by a cytomegalovirus promoter (Ad-CMV-FL). The duration and magnitude of adenoviral mediated (1 x 10(9) pfu) FL gene expression were then followed over time. FL gene expression in immunocompetent Swiss Webster mice peaks within the first 48 hours, falls by 98% after 20 days, and persists for >150 days. In contrast, FL activity in nude mice remains elevated for >110 days. Finally, transduced Swiss Webster and nude mice were sacrificed to show that the in vivo CCD signals correlate well with in vitro luciferase enzyme assays (r(2)=0.91 and 0.96, respectively). Our findings demonstrate the ability of the cooled CCD camera to sensitively and noninvasively track the location, magnitude, and persistence of FL gene expression. Monitoring of gene therapy studies in small animals may be aided considerably with further extensions of this technique.

Short-term REM sleep deprivation effect on temperature rhythm of rats.

Circadian rhythm of body temperature (CRT) is altered in endogenous depression and many psychiatric disorders. Even the sleep pattern is disrupted. Sleep deprivation alleviates symptoms in depression. The present study was conducted to find the role of noradrenergic innervation to the pineal gland in bringing about the effect of REM sleep deprivation (REMSD) on the CRT. Adult male Wistar rats (n = 12) divided into 2 groups were used for the study. The group I rats (n = 6) underwent superior cervical ganglionectomy and the group II rats (n = 6) were sham ganglionectomised. After recovery rats were given REMSD for 48 hours. The CRT was measured in three periods of the study i.e. basal, post operative and post REMSD. The results indicated REMSD increased the Amplitude and Mesor of the CRT in both the groups which was shortlasting and reversible thus suggesting non sympathetic mediation of the pineal in bringing about the circadian rhythm alteration due to REMSD.

Superior cervical ganglionectomy leads to dampening of amplitude of temperature rhythm in rats.

The circadian rhythm of body temperature (CRT) is a robust marker of the endogenous pacemaker function and is one of the most frequently studied rhythms. Melatonin, the main secretion of the pineal gland seems to have more of a thermomodulatory role in controlling the body temperature than having a direct role in thermoregulation. The sympathetic innervation to the pineal via the superior cervical ganglion determines the melatonin secretion, and superior cervical ganglionectomy (SCGx) decreases the secretion of melatonin. The present study was conducted on the Wistar rats (n = 12) to determine the role of melatonin in modulation of CRT. Adult male rats were either ganglionectomised (n = 6) or sham ganglionectomised (n = 6). Rectal temperature was recorded for CRT analysis. Cosinar analysis of the temperature record was done to get the acrophase, amplitude and mesor. Our results show that SCGx decreases the amplitude to the rhythm but has no effect on the mesor of the rhythm. Our study confirms that melatonin has little role to play in the thermoregulation and its role is mainly in thermomodulation.

Certain immunological parameters in subacute cold stress.

The effect of subacute cold swimming stress on the immune system of albino rats was investigated. Subacute cold stressed animals showed an increase in total WBC count, eosinophils and basophils. Phagocytic index and avidity index were also increased indicating hyperactive phagocytic process. On the other hand NBT reduction and soluble immune complex levels decreased significantly in stressed animals. There were no significant changes in the weight of the lymphoid organs.

Oral hydrocortisone pharmacokinetics: a comparison of fluorescence and ultraviolet high-pressure liquid chromatographic assays for hydrocortisone in plasma.

Three fasted, male subjects received single 10-, 30-, and 50-mg oral doses of hydrocortisone tablets on separate occasions. Endogenous hydrocortisone was suppressed by giving 2 mg of dexamethasone 9 hr prior to dosing. Plasma samples obtained serially for 8 hr after hydrocortisone dosing were assayed by reversed-phase high-pressure liquid chromatography (HPLC) with UV detection and by normal-phase HPLC with fluorescence detection of the dansylhydrazine derivative of hydrocortisone. The two assay methods yielded equivalent plasma hydrocortisone concentrations. Metabolite interference was absent in both assay methods. Drug concentrations in plasma from all three doses of hydrocortisone were described by one-compartment open-model kinetics, with first-order absorption and elimination, and an absorption lag time. Mean Cmax values of 199, 393, and 419 ng/ml were obtained at 1.0, 1.0, and 1.7 hr following the 10-, 30-, and 50-mg doses, respectively. Hydrocortisone was cleared from plasma with an elimination half-life of approximately 1.5 hr. Within the dosage range studied, plasma levels of hydrocortisone were related, but not directly proportional, to dose size. This apparent lack of proportionality may be due to reduced drug availability or altered distribution with increasing dose.

Simple high-performance liquid chromatographic assay for norethindrone--mestranol in combination tablets.

A simple, sensitive, and specific high-performance liquid chromatographic procedure was developed to assay norethindrone--mestranol combination tablets. The method involves a chloroform extraction of a single pulverized tablet. After centrifugation, and aliquot of the supernate was injected into a modular high-performance liquid chromatograph. The effluent from the silica column was monitored serially with a fixed-wavelength UV detector (254 nm) for norethindrone quantitation and a fluorescence detector (230 nm for excitation and 280 nm cutoff filter for emission) for mestranol quantitation. Progesterone was used as an internal standard. The method was employed successfully in content uniformity studies of several brands of commercially available tablets.

Suppression of endogenous hydrocortisone with dexamethasone.

A newly developed high-pressure liquid chromatographic method was used to study the optimum dosage regimen needed to suppress endogenous hydrocortisone. Nine volunteers were randomly placed in three groups. Each group received 1 mg of dexamethasone at 11 pm (Treatment A), 2 mg of dexamethasone at 11 pm (Treatment B), or 1 mg at 11 pm and an additional 1 mg at 6 am the following day (Treatment C). Analysis of multiple blood samples obtained the day before and the day after drug administration showed suppression in all three groups. Although the duration and extent of this suppression varied, adequate suppression to permit bioavailability studies was observed for Treatments B and C.

Tissue distribution of retinol and its metabolites after administration of double-labelled retinol.

The tissue concentrations and distribution of radioactivity present in retinol and its metabolites were investigated in vitamin A-deficient rats 24h after injection of physiological doses (10mug) of [6, 7-14C2, 11,12-3H2] retinol. The highest concentration of radioactivity was observed in the adrenals, followed by kidney, spleen, liver, intestine and blood. The total radioactivity was greatest in urine, followed in descending order by liver, kidney, blood and intestine. The 14C/3H ratios of crude light-petroleum extracts in the liver, intestines, lungs, heart and faeces were similar to the ratio of the injected retinol dispersion. However, the 14C/3H ratios in the adrenals, kidney, spleen, blood, brain and urine were quite different from that of injected retinol. Alumina chromatography of the kidney and intestinal extracts demonstrated that retinol and retinyl palmitate are the principal forms of vitamin A present. However, alumina chromatography of the liver extract did not reveal the presence of retinol but yielded a major compound with a low 14C/3H ratio. That this compound was not retinol was shown by its inability to react with ethanolic HC1 to yield anhydroretinol. The distribution of radioactivity in ether-soluble, acidic and water-soluble fractions of urine indicated that most of the radioactivity was present in the acidic and water-soluble fractions. The 14C/3H ratios in ether-soluble and acidic fractions were higher than that of injected retinol, whereas in the water-soluble fraction the ratio was similar to the injected material.