Exploration of new drug delivery pathways: I. Mechanism of folate uptake in cultured human cells. II. Role of nuclear localization signal peptides in the delivery of oligonucleotide into reconstituted nuclei

The roles of the folate receptor and an anion carrier in the uptake of 5- methyltetrahydrofolate (5-MeH_4folate) were studied in cultured human (KB) cells using radioactive 5-MeH_4folate. Binding of the 5-MeH_4folate was inhibited by folic acid, but not by probenecid, an anion carrier inhibitor. The internalization of 5-MeH_4folate was inhibited by low temperature, folic acid, probenecid and methotrexate. Prolonged incubation of cells in the presence of high concentrations of probenecid appeared to inhibit endocytosis of folatereceptors as well as the anion carrier. The V_(max) and K_M values for the carrier were 8.65 ± 0.55 pmol/min/mg cell protein and 3.74 ± 0.54µM, respectively. The transport of 5-MeH4folate was competitively inhibited by folic acid, probenecid and methotrexate. The carrier dissociation constants for folic acid, probenecid and methotreate were 641 µM, 2.23 mM and 13.8 µM, respectively. Kinetic analysis suggests that 5-MeH_4folate at physiological concentration is transported through an anion carrier with the characteristics of the reduced-folate carrier after 5-MeH_4folate is endocytosed by folate receptors in KB cells. Our data with KB cells suggest that folate receptors and probenecid-sensitive carriers work in tandem to transport 5-MeH_4folate to the cytoplasm of cells, based upon the assumption that 1 mM probenecid does not interfere with the acidification of the vesicle where the folate receptors are endocytosed.

Oligodeoxynucleotides designed to hybridize to specific mRNA sequences (antisense oligonucleotides) or double stranded DNA sequences have been used to inhibit the synthesis of a number of cellular and viral proteins (Crooke, S. T. (1993) FASEB J. 7, 533-539; Carter, G. and Lemoine, N. R. (1993) Br. J. Cacer 67, 869-876; Stein, C. A. and cohen, J. S. (1988) Cancer Res. 48, 2659-2668). However, the distribution of the delivered oligonucleotides in the cell, i.e., in the cytoplasm or in the nucleus has not been clearly defined. We studied the kinetics of oligonucleotide transport into the cell nucleus using reconstituted cell nuclei as a model system. We present evidences here that oligonucleotides can freely diffuse into reconstituted nuclei. Our results are consistent with the reports by Leonetti et al. (Proc. Natl. Acad. Sci. USA, Vol. 88, pp. 2702-2706, April 1991), which were published while we were carrying this research independently. We also investigated whether a synthetic nuclear localization signal (NLS) peptide of SV40 T antigen could be used for the nuclear targeting of oligonucleotides. We synthesized a nuclear localization signal peptide-conjugated oligonucleotide to see if a nuclear localization signal peptide can enhance the uptake of oligonucleotides into reconstituted nuclei of Xenopus. Uptake of the NLS peptide-conjugated oligonucleotide was comparable to the control oligonucleotide at similar concentrations, suggesting that the NLS signal peptide does not significantly enhance the nuclear accumulation of oligonucleotides. This result is probably due to the small size of the oligonucleotide.

Studies of temperature sensitive mutants of polyoma virus

Polyoma virus can undergo two different types of interactions with susceptible cells; one type of interaction leads to the production of new infectious virus and eventual cell death while the other leads to a neoplastically transformed cell which is able to continue to divide under conditions that inhibit the multiplication of uninfected normal cells. In order to study the viral genes involved in both of these virus-cell interactions the isolation of temperature sensitive mutants of polyoma virus was undertaken.

Two strains (TS-a, TS-b) which were temperature sensitive in their plaque forming ability at 38.5˚C, but not at 31.5˚C, were isolated from a mutagenized stock of the polyoma wild type virus (PY). TS-a was studied in further detail.

TS-a grown at 31.5˚C was found to be indistinguishable from PY in a number of physical characteristics including the heat sensitivity of the completed viral components. TS-a was inhibited in its ability to produce infectious virus in mouse cells when incubated at 38.5˚C; this inhibition could be overcome by infection with high multiplicities.

The nature of the intracellular temperature sensitive step of TS-a was analysed to some degree. It was found that this step occurs after uncoating of the infecting virus particles and about the time of new viral DNA synthesis. New infectious viral DNA does not appear to be made at the nonpermissive temperature; in contrast noninfectious capsids are made at 38.5˚C, but in amounts smaller than a full yield, such as made by TS-a at 31.5˚C or by PY at both the high and low temperature.

TS-a has also been found to be temperature sensitive in its transforming ability in vitro. Cells transformed at 31.5˚C by TS-a retain their transformed characteristics upon cultivation at 38.5˚C. Thus the temperature sensitive function seems to be important for the initiation of transformation, but not essential for the maintenance of the transformed state. TS-a also appears to be temperature sensitive in the production of tumors in newborn hamsters.

Effects produced in GaAs by MeV ion bombardment

Presented in the first part of this thesis is work performed on the ionizing energy beam induced adhesion enhancement of thin (~ 500 Angstrom) Au films on GaAs substrates. The ionizing beam, employed in the present thesis, is the MeV ions (i.e., ¹⁶O, ¹⁹F, and ³⁵Cl), with energies between 1 and 20 MeV. Using the "Scratch" test for adhesion measurement, and ESCA for chemical analysis of the film-substrate interface, the native oxide layer at the interface is shown to play an important role in the adhesion enhancement by the ionizing radiation. A model is discussed which explains the experimental data on the the dependence of adhesion enhancement on the energy which was deposited into electronic processes at the interface. The ESCA data indicate that the chemical bonds (or compounds), which are responsible for the increase in the thin film adherence, are hydroxides rather than oxides.

In the second part of the thesis we present a research performed on the radiation damage in GaAs crystals produced by MeV ions. Lattice parameter dilatation in the surface layers of the GaAs crystals becomes saturated after a high dose bombardment at room temperature. The strain produced by nuclear collisions is shown to relax partially due to electronic excitation (with a functional dependence on the nuclear and electronic stopping power of bombarding ions). Data on the GaAs and GaP crystals suggest that low temperature recovery stage defects produce major crystal distortion. The x-ray rocking curve technique with a dynamical diffraction theory analysis provides the depth distribution of the strain and damage in the MeV ion bombarded crystals.

Structural and optical properties of a-plane ZnO thin films synthesized on γ-LiAlO2 (302) substrates by low pressure metal-organic chemical vapor deposition

Nonpolar a-plane (1120) ZnO thin films have been fabricated on gamma-LiAlO2 (302) substrates via the low-pressure metal-organic chemical vapor deposition. An obvious intensity variation of the E-2 mode in the Raman spectra indicates that there exhibits in-plane optical anisotropy in the a-plane ZnO thin films. Highly-oriented uniform grains of rectangular shape can be seen from the atomic force microscopy images, which mean that the lateral growth rate of the thin films is also anisotropic. It is demonstrated experimentally that a buffer layer deposited at a low temperature (200 degrees C) can improve the structural and optical properties of the epilayer to a large extent. (c) 2007 Elsevier B.V. All rights reserved.

Effect of gibbing on protein hydrolysis during the ripening of salted herring at 4°C in polypropylene barrels

Effect of gibbing process on the protein hydrolysis in terms of free alpha amino nitrogen (FAN) content during the ripening of barrel salted herring at low temperature (4°C) was investigated. For this purpose North Sea herring (Clupea harengus) from north-east British coast was salted in polypropylene barrels and allowed to ripen at 4°C. This process of barrel salting was carried out for whole fish in one batch and gibbed fish in another batch. The investigation was performed by using new salt and used salt in separate barrels for each batch of experimental fish. Results of the present study show that protein hydrolysis was significantly higher in the ripened salt-herring produced from whole fish which was found to have more characteristic sensory properties than those produced from gibbed fish. Similar result (proteolysis) was obtained when the investigation was repeated for the spent herring although the spent herring fails to produce a ripened product with the desired characteristic sensory attributes, compared to those of pre-spawning herring.

The history and fishery potential of Kisoro Minor Lakes part two: Preliminary survey of Lakes Mutanda and Mulehe fisheries 

The fisheries of Lakes Mutanda and Mulehe during 1998/9 were mainly at subsistence scale and only few fishers operated at irregular intervals. The commercial catch records between 1963 to 1999 showed that Lake Mulehe was landing more fish than Lake Mutanda despite the fact that Lake Mutanda (26.4 km2) was bigger than Lake Mulehe (4.11 cm2). The constant decline of catches was due to irregular restocking and applying low stocking densities of fry. However, restocking should consider using species that withstand low temperature (15-240C) in the district. These include Oreochromis niloticus (Nile tilapia), Macropterus salmoides (Black bass), and Cyprinus carpio (Common carp). Most of these species have either disappeared or declined to very low levels. Due to lack of commercial fish species for harvest, the fishers by 1998/9 resorted to harvesting the haplochromines, Clarias carsoni and edible frogs (Xenopus kigesiensis) as alternative resources. Experimental studies have shown the need and techniques to enhance fish production on these two lakes.

Incidence of epizootic ulcerative syndrome (EUS) in freshwater fishes in the endemic area of Punjab, Pakistan

Incidence of Epizootic Ulcerative Syndrome (EUS) has been recorded for the first time in freshwater fishes in the endemic area of Punjab, Pakistan. Survey of private fish farms, hatchery and natural water bodies was conducted in a radius of 14 Km from around river Ravi near Lahore (Punjab Province) Pakistan. Out Of 1628 fishes belonging to 18 genera, 517 fishes of 10 genera were found affected with EUS. The incidence of EUS in culturable fishes was higher in Cirrhina mrigala (15.4%) moderate in Catla cat/a (13.3%) and lower in Labeo rohita (5.0%). Exotic fish, Chinese carp Ctenoparyngodon idella and Hypophthalmicthys molitrix were not affected with EUS. In non-culturable fishes the incidence of EUS was highest in Channa punctatus (72.8%) moderate in by C. straitus (65.45%) and comparatively lower Puntius ticto (43.7%). A slow growing temperature sensitive Saprolegnia spp. was isolated from all of EUS infected fish species. Aeromonas spp. and Pseudomonas spp. were isolated from the diseased fishes. Ectoparasites viz. Lernaea, Argulus and Triclwdina spp. were also isolated from the skin and gills of infected fish species. The disease was more severe in water having low alkalinity (70 mg/1), hardness (75 mg/1) and low temperature of 10-12 °C.

Effects of gamma radiation on nutritional and microbial quality of Pampus chinensis (Euphrasen 1788)

To evaluate the efficiency of gamma radiation in combination with low temperature Chinese pomfret, Pampus chinensis were preserved by the treatment of different doses of gamma radiation (3, 5 and 8 KGy) at freezing temperature (-20°C) during 90 days of storage period. Quality assessments for fish were carried out at an interval of 15 days during the storage period. Quality assessments were done by organoleptic, chemical (Total Volatile Nitrogen, TVN and Trimethylamine, TMA) and microbiological (Total Bacterial Count, TBC and Total Mould Count, TMC) evaluation. From the analysis of all parameters, maximum shelf-life was observed for irradiated (8 KGy) sample. It remained acceptable up to 75 days and that was the highest duration among 4 types of samples.

Seasonal dynamics in the occurrence and abundance of Pseudo-nitzschia species in the Maheshkhali channel of the Bay of Bengal, Bangladesh

Occurrence and abundance of Pseudo-nitzschia spp. at the mouth of the Maheshkhali channel of the Bay of Bengal, Bangladesh were studied. Plankton and water samples were collected monthly from the sub-surface water during high tide at daytime from June 2000 to May 2001. Four species of Pseudo-nitzschia, namely P. pungens, P. pseudodelicatissima, P. delicatissima and P. australis were identified and among them the first three were most commonly encountered and they varied seasonally. Pseudo-nitzschia delicatissima was the dominant species during the autumn and winter months, whereas P. pungens was dominant during the summer and spring months. Pseudo-nitzschia pseudodelicatissima exhibited its highest abundance level during the summer. Surface water temperature, salinity, nitrate-nitrogen (N03-N) and phosphate-phosphorus (P04-P) were recorded and their relationship with the occurrence and abundance of Pseudo-nitzschia species were studied. At the mouth of the Maheshkhali channel, Pseudo-nitzschia cell density was highest in late autumn (November) when highest salinity (35 o/oo) and P04-P (3.2 mg/l) concentrations and low temperature (23 °C) were recorded.

Production of freshwater giant prawn using over-wintered juveniles for enhancement of farm income and reduction of rural poverty in Bangladesh

Freshwater giant prawn, Macrobrachium rosenbergii fry produced during late season can not withstand low temperature thus the prawn culture programme during winter is hampered. To overcome this problem, late season (August-September) prawn juveniles (0.9-6.8 g) were stocked at a density of 1.43 to 3.57/square meter in 350-476 square-meter ponds in Pabna and Mymensingh districts during October 2000 and cultured till May 2001. Monthly average water temperature during the winter months (December-February) varied from 16 to 22 °C and gradually increased to 32 °C in May. The prawn fry showed fast growth rate and attained an average weight of 60-70 g within eight months including three winter months. Growth compensation was observed during summer months. Survival rate was 60-79%. After extrapolation of the present growth rate more than 1,600 kg/ha production can be achieved in better-managed ponds. Extrapolated cost of production was Tk. 268,000 and 200,000 Tk./ha in two best ponds, sale value was Tk. 644,9146 [sic] and 528,466 and gross profit was Tk. 376,000-410,000, suggesting a higher economic feasibility of farming freshwater prawn with over-wintered juveniles.

Studies on arsenical creosote as a wood preservative for marine structures. Part 2. Observation on leaching, corrosion and resistance to borer attack

A detailed study on arsenical creosote with reference to leaching, corrosion and anti-borer properties was carried out. Results showed that aging had very little effect on the preservative which suggested better fixation of the preservative into the wood. Corrosion of mild steel, galvanised iron, aluminium-magnesium alloy (M57S) and copper panels in the preservative was found to be negligible. Normal creosote and low temperature creosote of Regional Research Laboratory, Hyderabad, both fortified with arsenic trioxide resisted borer damage on wooden panels for a period of over five months in the port of Cochin. The performance of low temperature creosote fortified with arsenic was found to be equally satisfactory when compared to normal creosote fortified in the same manner. A loading of 208.6 Kgs/ml³ for Haldu (Adina cordifolia) and 138 Kgs/m³ for Mango (Mangifera indica) in the case of normal creosote and 177 Kgs/m³ for Mango the case of RRL creosote were found to be sufficient for treating the wood.

Effects of gamma radiation and freezing on the chemical and sensory changes in shrimp Penaeus monodon (Fabricius, 1978)

Gamma radiation (3, 6 and 9 kGy) in combination with low temperature (-20°C) were applied to retain the quality and shelf-life of shrimp, Penaeus monodon for a longer period. The quality was assessed by monitoring the chemical (TVN, TMA) and sensory changes in irradiated and non-irradiated (control) samples. Among chemical indicators of spoilage, total volatile nitrogen (TVN) values for irradiated shrimps were found to be 2.26, 2.18 and 1.57 mg N/100g of sample at 3, 6 and 9 kGy respectively after 90 days whereas for non-irradiated samples it was found 2.45mg N/100 g of sample. Trimethylamine (TMA) value for non-irradiated samples after 90 days were found 2.30mg N/100 g sample whereas that for irradiated shrimps at 3, 6 and 9 kGy were found to be 2.10, 2.08 and 1.98 mg N/100 g sample respectively. The sensory scores of control sample were gradually decreased with the progress of storage period. From this study, it was clear that gamma radiation in combination with low temperature showed shelf-life extension (90 days) in each dose of radiation used but during the use of 9 kGy radiation, P. monodon showed best quality.

Effect of gamma radiation in combination with freezing on the microbiological changes in frozen shrimp Penaeus monodon

In this study gamma radiation (3, 6 and 9 kGy) in combination with low temperature (-20°C) were applied to retain the quality and shelf-life of shrimp, Penaeus monodon for a longer period. The quality was assessed by monitoring microbiological changes (TBC, TMC, TYC, TCC and Salmonella count) in irradiated and non-irradiated (control) samples. Among microbiological indicators of spoilage, total bacterial count (TBC) values for irradiated shrimps were found to be 1875, 1625 and 1525 cfugˉ¹ of sample at 3, 6 and 9 kGy respectively after 90 days whereas for non-irradiated samples it was found 2475 cfugˉ¹ of sample. Total moulds count (TMC) value for non-irradiated samples after 90 days were found 425 cfugˉ¹ sample whereas that for irradiated shrimps at 3, 6 and 9 kGy were found to be 275, 250 and 200 cfugˉ¹ sample respectively. Total yeast count (TYC) value for non-irradiated samples after 90 days were found 4125 cfugˉ¹ sample whereas that for irradiated shrimps at 3, 6 and 9 kGy were found to be 2850, 2150 and 1725 cfugˉ¹ sample respectively. Total coliform count and Salmonella count showed that those were absent during 90 days storage period. From this study, it was clear that gamma radiation in combination with low temperature showed shelf-life extension (90 days) in each dose of radiation used but during the use of 9 kGy radiation, Penaeus monodon showed best quality.

Seasonal variations in the activity budget of Nomascus concolor jingdongensis at Mt. Wuliang, Central Yunnan, China: Effects of diet and temperature

We studied seasonal variation in the activity budget of a habituated group of Nomascus concolor jingdongensis at Mt. Wuliang, Central Yunnan, China from March 2005 to April 2006 via scan sampling at 5-min intervals. The study site is near the northern extreme of the distribution of hylobatids, at high altitude with extreme seasonality of temperature and rainfall. During the day, feeding manifested a bimodal pattern of high activity levels in mid-morning and mid-afternoon, whereas resting reached a peak at midday, with proportionally less time used for traveling. Annually, the group spent an average of 40.0% of the time resting, 35.1% feeding, 19.9% traveling, 2.6% singing, 1.2% playing, and 1.3% in other activities. The proportion of time allocated to activities showed significant monthly variations and was influenced by the diet and temperature. Gibbons increased traveling and playing time and decreased feeding time when they ate more fruit, and they decreased traveling, singing, and playing time and increased feeding time when they ate more leaves. Moreover, when the temperature was low, the gibbons decreased time traveling and increased time resting. In summary, black-crested gibbons employed high-effort activities when they ate more fruit and energy-conservation patterns when they ate more leaves and in low temperature. Behavioral data from the site are particularly useful in understanding gibbon behavioral adaptations to different sets of ecological conditions.

Kinetic control of catalytic CVD for high-quality graphene at low temperatures.

Low-temperature (∼600 °C), scalable chemical vapor deposition of high-quality, uniform monolayer graphene is demonstrated with a mapped Raman 2D/G ratio of >3.2, D/G ratio ≤0.08, and carrier mobilities of ≥3000 cm(2) V(-1) s(-1) on SiO(2) support. A kinetic growth model for graphene CVD based on flux balances is established, which is well supported by a systematic study of Ni-based polycrystalline catalysts. A finite carbon solubility of the catalyst is thereby a key advantage, as it allows the catalyst bulk to act as a mediating carbon sink while optimized graphene growth occurs by only locally saturating the catalyst surface with carbon. This also enables a route to the controlled formation of Bernal stacked bi- and few-layered graphene. The model is relevant to all catalyst materials and can readily serve as a general process rationale for optimized graphene CVD.