Intermittent drying of bioproducts--an overview

Unlike the conventional practice of supplying energy for batch drying processes at a constant rate, newly developed intermittent drying processes employ time-varying heat input tailored to match the drying kinetics of the material being dried. The energy required may be supplied by combining different modes of heat transfer (e.g. convection coupled with conduction or radiation or dielectric heating simultaneously or in a pre-selected sequence) in a time-varying fashion so as to provide optimal drying kinetics as well as quality of the bioproduct. This is especially important for drying of heat-sensitive materials (such as foods, pharmaceutical, neutraceutical substances, herbs, spices and herbal medicines). Intermittent heat supply is beneficial only for materials which dry primarily in the falling rate period where internal diffusion of heat and moisture controls the overall drying rate. Periods when little or no heat is supplied for drying allow the tempering period needed for the moisture and heat to diffuse within the material. As the moisture content increases at the surface of the biomaterial during the tempering period, the rate of drying is higher when heat input is resumed. It is possible to control the heat input such that the surface temperature of the product does not exceed a pre-determined value beyond which thermal damage of the material may occur. This process results in reduction in the use of thermal energy as well as the mass of air used in convective drying. Thus, the thermal efficiency of such a process is higher. The quality of the product, as such color and ascorbic acid content, is also typically superior to that obtained with a continuous supply of heat. However, in some cases, there will be a nominal increase in drying time. In the case of microwave-assisted and heat pump drying, for example, the capital cost of the drying system can also be reduced by drying in the intermittent mode.

This paper provides an overview of the basic process, selected results from experiments and mathematical models for a variety of biomaterials dried in a wide assortment of dryers. It begins with a classification of intermittent drying processes that may be applied e.g. time-varying temperature, air flow rate, operating pressure as well as heat input by different modes and in different temporal variations. The beneficial effects of improving the quality of dried bioproducts by different intermittent processes are also included and discussed.     

Effect of piperine on pentobarbitone induced hypnosis in rats

Piperine (1-peperoyl piperidine), a major alkaloid isolated from Piper nigrum Linn, potentiated pentobarbitone sleeping time in dose dependant manner, with peak effect at 30 min. Blood and brain pentobarbitone levels were higher in piperine treated animals. Piperine treatment in rats, treated chronically with phenobarbitone, significantly potentiated pentobarbitone sleeping time, as compared to the controls. There was no alteration in barbital sodium sleeping time. It is possible that, piperine inhibits liver microsomal enzyme system and thereby potentiates the pentobarbitone sleeping time.     

Autonomous and non-autonomous traits mediate social cooperation in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

In the trishanku (triA ⁻ ) mutant of the social amoeba Dictyostelium discoideum, aggregates are smaller than usual and the spore mass is located mid-way up the stalk, not at the apex. We have monitored aggregate territory size, spore allocation and fruiting body morphology in chimaeric groups of (quasi-wild-type) Ax2 and triA ⁻ cells. Developmental canalisation breaks down in chimaeras and leads to an increase in phenotypic variation. A minority of triA ⁻ cells causes largely Ax2 aggregation streams to break up; the effect is not due to the counting factor. Most chimaeric fruiting bodies resemble those of Ax2 or triA ⁻ . Others are double-deckers with a single stalk and two spore masses, one each at the terminus and midway along the stalk. The relative number of spores belonging to the two genotypes depends both on the mixing ratio and on the fruiting body morphology. In double-deckers formed from 1:1 chimaeras, the upper spore mass has more Ax2 spores, and the lower spore mass more triA ⁻ spores, than expected. Thus, the traits under study depend partly on the cells’ own genotype and partly on the phenotypes, and so genotypes, of other cells: they are both autonomous and non-autonomous. These findings strengthen the parallels between multicellular development and behaviour in social groups. Besides that, they reinforce the point that a trait can be associated with a genotype only in a specified context.     

Cyanine dye labeling reagents containing isothiocyanate groups

New isothiocyanate derivatives of cyanine dyes were synthesized as fluorescent covalent labeling reagents for proteins and other biomolecules. These dyes have maximum absorbance in the red and near infrared regions of the spectrum, have high extinction coefficients and have adequate quantum yields. Incorporating two alkyl sulfonate groups in the dye structures increases their water solubility, which is beneficial for labeling biological molecules in aqueous solution. Reactivities of proteins with these new cyanines are similar to their reactivities with fluorescein isothiocyanate. These new labeling reagents are complementary to the fluorescein and rhodamine reagents, expanding the possibilities of multicolor analyses. Sheep anti-mouse-IgG antibody was labeled with a pentamethine cyanine dye (CY5.8-ITC) and used with a fluoresceinated antibody as a second reagent for detecting human T-cell subsets by flow cytometry.     

Monte Carlo simulations of ionic channel selectivity

Monte Carlo simulations have been developed to study the selectivityof ionic channels in biological membranes. The channel is consideredto be in either of two possible states: (i) densely packed withions, the ions moving in single file in one direction, or alternatively,(ii) sparsely packed, where holes could appear at any particulartime thereby allowing bidirectional movement of ions. The twomodels enable us to envisage a quantitative flux of permeableions in the presence of smaller sized ions, taking into considerationtheir concentrations in the bulk solutions, the ion-channelinteractions and probability with which they fill up the channel.The programs are written in FORTRAN-77 (MS-FORTRAN) for an IBM-compatiblepersonal computer. From the simulation results we observe anenzymatic function of the channel and also note that the smallersized ions tend to block the movement of permeable ions. Thesimulations represent a technique for visualization of the factorsthat decide ionic permeability and help in manipulating theireffects with ease and speed which would otherwise involve intricateexperimental setups. Received on September 7, 1990; accepted on January 14, 1991     

Autophagy in pancreatic cancer: An emerging mechanism of cell death

Pancreatic cancer, the fourth leading cause of cancer-related death in the United States, is resistant to current chemotherapies. Therefore, identification of different pathways of cell death is important to develop novel therapeutics. Our previous study has shown that triptolide, a diterpene triepoxide, inhibits the growth of pancreatic cancer cells in vitro and prevents tumor growth in vivo. However, the mechanism by which triptolide kills pancreatic cancer cells was not known, hence, this study aimed at elucidating it. Our study reveals that triptolide kills diverse types of pancreatic cancer cells by two different pathways; it induces caspase-dependent apoptotic death in some cell lines and death via a caspase-independent autophagic pathway in the other cell lines tested. Triptolide-induced autophagy requires autophagy-specific genes, atg5 or beclin 1 and its inhibition results in cell death via the apoptotic pathway, whereas inhibition of both autophagy and apoptosis rescues triptolide-mediated cell death. Our study shows for the first time that induction of autophagy by triptolide has a pro-death role in pancreatic cancer cells. Since triptolide kills diverse pancreatic cancer cells by different mechanisms, it makes an attractive chemotherapeutic agent for future use against a broad spectrum of pancreatic cancers.Key words: pancreatic cancer, triptolide, apoptosis, caspase-3Pancreatic adenocarcinoma is one of the most lethal human malignancies. It is the fourth leading cause of cancer-related death in the United States. The five-year survival rate for pancreatic cancer is estimated to be <5% due to its aggressive growth, metastasis and resistance to radiation and most systemic chemotherapies. Hence, efforts are ongoing to understand the pathobiology of pancreatic cancer to develop innovative and effective therapies against it. A promising candidate for future therapeutic use against pancreatic cancer is a diterpene triepoxide, triptolide. Our previous studies show that triptolide inhibits the growth of pancreatic cancer cells in vitro and prevents tumor growth in vivo. Since the mechanism by which triptolide kills pancreatic cancer cells was not known, we decided to elucidate it.The K-ras, p53, p16 and DPC4 genes are the most frequently altered genes in pancreatic adenocarcinoma. In this study we have used diverse pancreatic cancer cell lines, MiaPaCa-2, Capan-1, S2-013 and S2-VP10 cells, which have mutations in all the above-mentioned genes and BxPC-3 and Hs766T cells, which have mutations in the p53, p16 and DPC4 genes, but have a wild-type K-ras gene. The treatment of all the cell lines with triptolide results in a significant time- and dose-dependent decrease in cell viability, independent of cell cycle arrest. After treatment with triptolide, only MiaPaCa-2, Capan-1 and BxPC-3 cells show an increase in the apoptosis parameters: cytochrome c release from mitochondria into the cytosol, caspase-3 activation and phosphatidylserine externalization. In contrast to this, S2-013, S2-VP10 and Hs766T cells show an induction of autophagy: an increase in LC3-II levels (by immunoblotting and immufluorescence), increase in acridine orange-positive cells, inhibition of the PtdIns3K/Akt/mTOR pathway and induction of the ERK1/2 pathway. Also, none of the cell lines tested show necrosis as evidenced by the absence of the release of lactate dehydrogenase. These results indicate that triptolide induces apoptosis in MiaPaCa-2, Capan-1 and BxPC-3 cells, whereas it induces autophagy in S2-013, S2-VP10 and Hs766T cells.Since the role of autophagy in cancer was controversial we investigated whether triptolide-induced autophagy has a prosurvival or a pro-death role. As autophagy-associated cell death is independent of caspase-3, we tested the effect of triptolide on pancreatic cancer cells in the absence of caspase-3. Treatment of cells with triptolide post-caspase-3 knockdown shows a significant rescue of cell viability only in MiaPaCa-2, but not S2-013 or S2-VP10 cells. This indicates that in contrast to MiaPaCa-2, triptolide-mediated cell death in S2-013 and S2-VP10 cells is independent of caspase-3. Next, we tested the role of autophagy in triptolide-mediated cell death in pancreatic cancer cells. In spite of a knockdown of autophagy-specific genes (atg5 and beclin 1), treatment of S2-013 and S2-VP10 cells with triptolide show a significant decline in cell viability, which is comparable to the cells treated with triptolide in the presence of autophagy genes. Subsequently we show that death in the absence of autophagy-specific genes is due to the utilization of an alternate cell death pathway, apoptosis. Furthermore, in the absence of both autophagy-specific and apoptosis-specific genes, triptolide-mediated cell death is rescued in S2-013 and S2-VP10 cells. Thus, these results confirm that triptolide-induced autophagy has a pro-death role in S2-013 and S2-VP10 cells and that these cells do not have a defect in the apoptotic machinery; however, they respond to triptolide by activating the autophagic pathway instead of the apoptotic pathway. Our studies also reveal the presence of a crosstalk between the two cell death pathways, apoptosis and autophagy, in pancreatic cancer cells.In conclusion, our study shows for the first time that triptolide induces autophagy in pancreatic cancer cells. It sheds light on the fundamental question as to whether autophagy is protective or causes cell death, proving convincingly that induction of autophagy causes cell death of some pancreatic cancer cells. Although a basal level of autophagy is necessary to maintain cellular homeostasis, its prosurvival role can be switched into a cell death mechanism if the amplitude of autophagy increases above a threshold level which is incompatible with viability, as seen in S2-013, S2-VP10 and Hs766T cells after triptolide treatment. Furthermore, there exists a crosstalk between apoptosis and autophagy in S2-013 and S2-VP10 cells; either both pathways function independently to kill the cells, with autophagy being the preferred pathway or autophagy antagonizes apoptosis and hence apoptosis is seen only after inhibiting autophagy. Although there is no direct correlation between the selection of cell death pathway in response to triptolide and the genotype of the cell lines, the choice of autophagic cell death pathway could depend on the metastatic potential of the cells; S2-013, S2-VP10 and Hs766T cell lines being more metastatic than the others, which merits further investigation. In conclusion, the ability of triptolide to induce cell death in diverse pancreatic cancer cells by either mechanism makes it an attractive chemotherapeutic agent against a broad spectrum of pancreatic cancers.     

Reduction-oxidation (Redox) and vascular tissue level of homocyst(e)ine in human coronary atherosclerotic lesions and role in extracellular matrix remodeling and vascular tone

Hyperhomocyst(e)inemia in patients with coronary and peripheral arterial occlusion has been demonstrated by others. Redox-state of homocyst(e)ine causes dysfunction of endothelial cells and promote growth of vascular smooth muscle cells. The role of tissue, protein bound and unbound, oxidative mixed disulfides in the development of fibrous plaque in atherosclerotic lesion is not known. Redox-state around the fibroblasts and vascular smooth muscle cells modulates the expression of extracellular matrix (ECM) components (Tyagi et al. 1996, J Cell Biochem, 61: 139-151). To determine the role of tissue homocystine in fibrotic atherosclerotic plaque development, coronary arteries were isolated from ischemic explanted hearts (n = 10). Apparently normal vascular tissue was obtained from idiopathic cardiomyopathic explanted hearts (n = 10). Tissue extract were prepared from atherosclerotic lesions and from normal arteries devoid of adventitia. Interaction of homocystine with Ellman's reagent (5, 5-dithio-bis-2-nitro benzoic acid) catalyzed by limiting amount of reducing agent (catalyst) generated change in optical density (OD) at 412 nm in dose dependent fashion. We have generated a standard curve between change at 412 nm and amount of homocystine. The change in OD at 412 nm with increasing amount (0-25 g) of homocystine demonstrated linearity. The protein-bound oxidized disulfides were precipitated by trichloroacetic acid (TCA) and free-oxidative disulfides in the supernatant were collected. The pathophysiological amount of protein-bound disulfide in atherosclerotic tissue (1.0 ± 0.2g/mg total protein) was 10 times that in normal tissue (0.1 ±0.01 g/mg, p < 0.001). The amount of free oxidative disulfide in atherosclerotic tissue (1.5 ± 0.3g/mg) was 15 times that in normal tissue (0.12 ± 0.02 g/mg, p < 0.001). To determine the role of homocystine in ECM expression, ECM collagenase activity in the presence and absence of homocystine was measured by zymography. The effect of homocysteine on collagenase activity was biphasic, increased at < [0.01 mM] and inhibited at > [0.1 mM]. To determine whether homocystine regulates vascular tone, isometric measurements were carried out using normal coronary rings. Results suggested that homocystine induced endothelial-modulated vasoconstriction in coronary vessels. Tissue oxidative disulfides and the homocystine may contribute to the development of fibrotic atherosclerotic lesions and vascular dysfunction.     

A novel fluorescence ratiometric method confirms the low solvent viscosity of the cytoplasm.

Two homologous indocyanine dyes, Cy3.18 and Cy5.18, can be used as a ratio pair for fluorometric determination of solvent viscosity. Succinimidyl ester derivatives of these dyes can be attached to inert carrier macromolecules, such as Ficoll 70, for measurement of intracellular or intravesicular solvent viscosity. When the viscosity of the solvent was varied by various methods, the fluorescence intensity ratio (Cy3/Cy5) in a mixture of Cy3.18-Ficoll 70 (Cy3F70) and Cy5.18-Ficoll 70 (Cy5F70) in solution was found to be solely a function of solvent viscosity and was insensitive to other solvent parameters such as dielectric constant, temperature, and the ability of the solvent to form hydrogen bonds. Most important, it was insensitive to the presence of large macromolecules, such as proteins, which increase the shear viscosity but have little effect on solvent viscosity. Following microinjection into the cytoplasm of living tissue culture cells, no binding of Cy3F70 or Cy5F70 to intracellular components was detected by fluorescence recovery after photobleaching. Fluorescence intensity ratio imaging of Cy3F70 and Cy5F70 in non-motile interphase CV1 and PtK1 cells showed that the solvent viscosity of cytoplasm was not significantly different from water and showed no spatial variation.