Investigation on Processing Variables for the Preparation of Fluconazole-Loaded Ethyl Cellulose Microspheres by Modified Multiple Emulsion Technique

Fluconazole-loaded ethyl cellulose microspheres were prepared by alginate facilitated (water-in-oil)-in-water emulsion technology and the effects of various processing variables on the properties of microspheres were investigated. Scanning electron microscopy revealed spherical nature and smooth surface morphology of the microspheres except those prepared at higher concentration of emulsifiers and higher stirring speeds. The size of microspheres varied between 228 and 592 μm, and as high as 80% drug entrapment efficiency was obtained depending upon the processing variables. When compared up to 2 h, the drug release in pH 1.2 HCl solution was slower than in pH 7.4 phosphate buffer saline solution. However, this trend was reversed at high shear conditions. The microspheres provided extended drug release in alkaline dissolution medium and the drug release was found to be controlled by Fickian-diffusion mechanism. However, the mechanism shifted to anomalous diffusion at high shear rates and emulsifier concentrations. The aging of microspheres did not influence the drug release kinetics. However, the physical interaction between drug and excipients affected the drug dissolution behaviors. X-ray diffractometry (X-RD) and differential scanning calorimetry (DSC) analysis revealed amorphous nature of drug in the microspheres. Fourier transform infrared (FTIR) spectroscopy indicated stable character of fluconazole in the microspheres. The stability testing data also supported the stable nature of fluconazole in the microspheres. The fluconazole extracted from 80% drug-loaded formulation showed good in vitro antifungal activity against Candida albicans. Thus, proper control of the processing variables involved in this modified multiple emulsion technology could allow effective incorporation of slightly water soluble drugs into ethyl cellulose microspheres without affecting drug stability.     

Fine needle aspiration cytology of Hodgkin's lymphoma: A cytohistologic correlation study from a cancer center in Kuwait

OBJECTIVE: To assess the diagnostic accuracy offine needle aspiration cytology (FNAC) in the diagnosis of Hodgkin's lymphoma (HL). STUDY DESIGN: We selected all the cases in which a cytologic diagnosis of HL, suggestive of or suspicious for HL, or HL as the prime differential diagnosis was offered on FNAC. These cases were correlated with histopathologic follow-up. Cases of primary HL diagnosed on cytology but without histopathology were excluded from the study. RESULTS: Histopathologic follow-up was available in 46 cases. Of these, 42 were correctly diagnosed as HL, and there was a discorrelation in 4 cases, comprising 3 cases of non-HL (T-cell-rich B-cell lymphoma [TCRBCL]-2, anaplastic large cell lymphoma-1) and 1 case of metastatic carcinoma. Overall accuracy was 91.3%. In 14 cases, the cytologic features were diagnostic ofrecurrence; hence, no histopathologic examination was done. No follow-up was available for the remaining 19 cases, which were excluded from the study. CONCLUSION: FNAC is very useful for rapid and accurate approach to the diagnosis of recurrent and most cases of primary HL. Because of morphologic similarities, it is difficult to differentiate HL from anaplastic large cell lymphoma and TCRBCL on FNAC. It is advisable to request a histopathologic examination in all cases of primary HL.     

Modulator-induced interference in functional cross talk between the substrate and the ATP sites of human P-glycoprotein

The human P-glycoprotein (Pgp, ABCB1) is an ATP-dependent efflux pump for structurally unrelated hydrophobic compounds, conferring simultaneous resistance to and restricting bioavailability of several anticancer and antimicrobial agents. Drug transport by Pgp requires a coordinated communication between its substrate binding/translocating pathway (substrate site) and the nucleotide binding domains (NBDs or ATP sites). In this study, we demonstrate that certain thioxanthene-based Pgp modulators, such as cis-(Z)-flupentixol and its closely related analogues, effectively disrupt molecular cross talk between the substrate, and the ATP, sites without affecting the basic functional aspects of the two domains, such as substrate recognition, binding, and hydrolysis of ATP and dissociation of ADP following ATP hydrolysis. The allosteric modulator cis-(Z)-flupentixol has no effect on [alpha-(32)P]-8-azido-ATP binding to Pgp under nonhydrolytic conditions or on the K(m) for ATP during ATP hydrolysis. Both hydrolysis of ATP and vanadate-induced [alpha-(32)P]-8-azido-ADP trapping (following [alpha-(32)P]-8-azido-ATP breakdown) by Pgp are stimulated by the modulator. However, the ability of Pgp substrates (such as prazosin) to stimulate ATP hydrolysis and facilitate vanadate-induced trapping of [alpha-(32)P]-8-azido-ADP is substantially affected in the presence of cis-(Z)-flupentixol. Substrate recognition by Pgp as determined by [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) binding both in the presence and in the absence of ATP is facilitated by the modulator, whereas substrate dissociation in response to vanadate trapping is considerably affected in its presence. In the Pgp F983A mutant, which is impaired in modulation by cis-(Z)-flupentixol, the modulator has a minimal effect on substrate-stimulated ATP hydrolysis as well as on substrate dissociation coupled to vanadate trapping. Finally, cis-(Z)-flupentixol has no effect on dissociation of [alpha-(32)P]-8-azido-ADP (or ADP) from vanadate-trapped Pgp, which is essential for subsequent rounds of ATP hydrolysis. Taken together, our results demonstrate a distinct mechanism of Pgp modulation that involves allosteric disruption of molecular cross talk between the substrate, and the ATP, sites without any direct interference with their individual functions.     

Changes in uterine protein secretion during luteal and follicular phases and detection of phosphatases during luteal phase of estrous cycle in buffaloes (Bubalus bubalis)

Changes in uterine proteins during different reproductive states and their functional significance though known in other species have not been established in buffaloes. An attempt has been made to unravel the changes in composition of buffalo uterine secretion with growth and regression of corpora-lutea during early, mid and late luteal and follicular phase of estrous cycle using gel filtration and electrophoresis techniques. Also the phosphatases activities in luteal phase uterine secretions have been studied. Gel filtration chromatography analysis revealed a protein peak in void volume of the column, the intensity of which was more in all the luteal phase samples than follicular phase samples. Alkaline phosphatase was also found eluted in the void volume. The other three uterus-specific peaks (Peaks V-VII) were detected below 13.7 kd molecular weight. There were at least five peaks of acid phosphatases activity in chromatogram. Silver staining of SDS-PAGE gel detected as many as 40 protein bands in the uterine fluid of which nine proteins were glycoproteins. Molecular weight (MW) comparison revealed the major protein band at 66 kd which could be serum albumin. Comparison of uterine proteins with serum protein bands revealed a 93.5 kd glycoprotein in buffalo serum that did not appear in uterine fluid and at least 11 uterus-specific protein bands (506, 470, 241, 114, 49, 38, 33, 26, 19.2, 16, and 14.3 kd). The 38 and 19.2 kd bands were luteal-stage specific. Intense periodic acid Schiff's (PAS) stained bands in uterine proteins compared to serum indicated glycosylation process in endometrial epithelial cells. The study suggested that buffalo uterine secretion contained mainly serum and several uterus-specific proteins of which few were luteal phase specific. Further study on characterizing the unique or most abundant proteins and defining their role in uterine functions would help to address the cause of low reproduction rate in buffaloes.     

A quantitative study of the morphological development and bacterial colonisation of the gut of the tammar wallaby Macropus eugenii eugenii and brushtail possum Trichosurus vulpecula during in-pouch development

We compared the rates of change of various morphological parameters of the stomach, small intestine, caecum and colon of tammar wallabies and brushtail possums with body mass during in-pouch development. These were correlated with changes in the numbers of bacterial species in the various gut segments. In the pouch-young of both species, the wet tissue masses of all gut segments increased with body mass in a positively allometric manner (i.e. with a body mass exponent > 1), suggesting that the mass of each component was disproportionately low at birth, but increased disproportionately rapidly postnatally. However, the lengths of the wallaby stomach and small intestine scaled isometrically with respect to body mass (i.e. with a body mass exponent around 0.33), which may indicate that the shape of these components changes to the adult form during early neonatal development. Conversely, the length of the caecum and colon of both wallabies and possums scaled in a positively allometric manner with respect to body mass, showing area to volume compensation. This may indicate a more general pattern of disproportionately rapid postnatal enlargement in areas that are distal to the principal sites of neonatal digestion (i.e. the stomach). The numbers of bacterial species present in the various gastrointestinal segments of both species were low in animals aged 100 days or less but there was a significant increase in microbial diversity in the caecum of brushtail possums aged over 100 days. The possum caecum also showed the greatest rate of increase in wet tissue mass relative to body mass. It is postulated that caecal development may act as a nidus for establishment of communities of commensal microflora in the developing marsupial.     

Evidence for coordinated interaction of cyclin D3 with p21 and cdk6 in directing the development of uterine stromal cell decidualization and polyploidy during implantation.

Uterine decidualization, characterized by stromal cell proliferation, and differentiation into specialized type of cells (decidual cells) with polyploidy, during implantation is critical to the pregnancy establishment in mice. The mechanisms by which the cell cycle events govern these processes are poorly understood. The cell cycle is tightly regulated at two particular checkpoints, G1-S and G2-M phases. Normal operation of these phases involves a complex interplay of cyclins, cyclin-dependent kinases (cdks) and cdk inhibitors (CKIs). We previously observed that upregulation of uterine cyclin D3 at the implantation site is tightly associated with decidualization in mice. To better understand the role of cyclin D3 in this process, we examined cell-specific expression and associated interactions of several cell cycle regulators (cyclins, cdks and CKIs) specific to different phases of the cell cycle during decidualization in mice. Among the various cell cycle molecules examined, coordinate expression and functional association of cyclin D3 with cdk4 suggest a role for proliferation and, that of cyclin D3 with p21 and cdk6 is consistent with the development of polyploidy during stromal cell decidualization.     

Cell-type-specific expression of transforming growth factor-alpha in the mouse uterus during the peri-implantation period.

Immunohistochemistry as well as in situ and Northern blot hybridization were employed to determine temporal and cell-type-specific expression of transforming growth factor-alpha (TGF-alpha) in the mouse uterus during the peri-implantation period. The co-localization of TGF-alpha (by immunohistochemistry) with its mRNA (by in situ hybridization) in the luminal and glandular epithelia on Days 1-4 of pregnancy (Day 1 = vaginal plug) and also in many stromal cells on Days 3 and 4 indicates that these cells are the primary sites of TGF-alpha synthesis during the preimplantation period. The higher levels of TGF-alpha mRNA in total uterine RNA on Day 4, as shown by Northern blotting, is consistent with the recruitment of stromal cells expressing this gene. During the post-implantation period (Days 5-8), the co-localization of the mRNA and protein in the decidua at the implantation sites suggests that the decidualizing stromal cells synthesize TGF-alpha. Although in situ hybridization showed the presence of mRNA in embryos on Days 5-8, immunostaining was noted in the embryo only on Days 5 and 6. These results suggest that uterine and embryonic expression of TGF-alpha during the peri-implantation period could be involved in embryonic development, preparation of the uterus for implantation, and decidualization.     

Some Physiological Parameters and Seed Lipid Composition of Transgenic Tobacco Plant

To see the effects of foreign gene introduction on the physiological performance and the quality and quantity of seed lipids, we studied transgenic tobacco plant as a model system, as tobacco seeds are oil seeds. Using Agrobacterium Ti plasmid based vectors, tobacco plants cv Petit Havana were transformed by NPT II gene as selectable marker. Transformed T₀ generation plants raised in tissue culture were transferred to pots and selfed. From the seeds, T₁ generation plants were grown in pots and their physiological performance was assessed. The transgenic plants showed slightly slower rates of germination and growth. Total chlorophyll content, chlorophyll a/b ratio and specific leaf weight, however, remained unchanged. The transgenic plants also had delayed flowering. However, total protein, lipid content and fatty acid composition of lipids of seeds in transgenic plants did not show appreciable difference from the seeds from control plants. Thus the physiological cost of transgenic plant for the extra genetic load was only marginal, if any.