10. Improving nitrogen fertilizer efficiency in lowland rice in tropical Asia

Rice production in Asia must increase 2.2–2.8% annually to keep abreast of increasing population. Greater fertilizer use and crop intensification together with varietal improvement and investment in irrigation will all contribute to increased rice supply. Because fertilizer and input prices have risen faster than the price of rice, increasing fertilizer N efficiency will be a major challenge for rice researchers and farmers. Greater fertilizer N efficiency may be achieved through improved timing and application methods, and particularly through better incorporation of basal fertilizer N without standing water. Other promising alternative practices are use of N-efficient rice varieties, hand or machine deep placement of urea supergranules, and use of slow release N fertilizers. Research challenges include development of placement machines for prilled urea and identification of cost-efficient nitrification and urease inhibitors. Under the present resource-scarce situation in many tropical Asian countries, several complementary practices must be followed to supplement inorganic N sources. Fertilizer supplies and proper price support should be maintained and wherever possible increased, and appropriate fertilizer materials and application methods must be devised to increase N use efficiency in lowland rice, so that increasing rice requirements are fulfilled.     

Pathogenicity of equine herpesvirus: in vivo persistence in equine tissue macrophages of herpesviuus type 2 detected in monolayer macrophage cell culture

Equine macrophages from the mammary glands of a yearling filly and an 18-year-old barren nonlactatind mare formed cell monolayers in continuous cultures. There was absence of viral cytopathic effect (CPE) in early cell culture passages. The cells from the early cell culture passages having no CPE failed to show evidence of virus or viral antigen by electron microscopic and immunofluorescence studies. Foci of CPE first appeared in the monolayer cell cultures from the filly and the mare in the 3rd and the 4th serial passages respectively, and the CPE increased on subsequent serial passages. Equine herpesvirus type 2 (EHV 2) cultures showing CPE. The macrophage monolayer culture established from the mare produced CPE foci more consistently than did the culture from the filly, and they were more numerous. Conversely, the peripheral blood mononuclear cells from the filly on cocultivation with an equine embryo kidney monolayer cell culture produced more EHV 2 CPE foci than did those from the mare. This study indicated that tissue macrophages may be a site for latent and persistent herpesvirus infections in horses.     

Toxic effect of puromycin on erythrocyte membranes which is unrelated to inhibition of protein synthesis

Exposure of rabbit or human erythrocytes to concentrations of puromycin as low as 7 x 10(-4)M for 2 hr causes damage to the cell membrane, as evidenced by increased susceptibility of the cells to hyposmotic lysis, increased cell rigidity, and ultrastructural changes consistent with severe membrane damage. Puromycin causes a concentration-dependent internalization of the erythrocyte membrane, resulting in vacuolization of the cells, at concentrations between 7 x 10(-4) M and 10(-2) M. Since the erythrocyte does not synthesize protein, the data indicate that puromycin has a direct toxic effect on erythroid cell membranes which is unrelated to its action in inhibiting the synthesis of protein.     

Carcinoembryonic antigen (CEA) in ovarian cancer: factors influencing its incidence and changes which occur in response to cytotoxic drugs

We report the serum levels of carcinoembryonic antigen (CEA) in 109 patients with ovarian cancer. Histology, degree of differentiation, and clinical stage influenced the incidence of positive CEA. Although CEA was significantly raised in patients with a variety of tumours, the highest incidence (77 per cent) was found in those with serious cystadenocarcinoma. Nearly all (94 per cent) of the poorly differentiated tumours were associated with a positive CEA result. Serial CEA levels provided a useful guide to management during cytotoxic chemotherapy, rapidly falling levels indicating a favourable tumour response which was reflected clinically. However, only two-thirds of tumours were associated with detectable CEA levels in serum, day-to-day variations in individual serum levels occurred, and CEA levels tended to fall paradoxically during terminal illness. The significance of persistently low levels in the apparent absence of disease was uncertain.     

Regression of tumors in mice vaccinated with professional antigen-presenting cells pulsed with tumor extracts

Vaccination with tumor extracts circumvents the need to identify specific tumor rejection antigens and extends the use of active immunotherapy to the vast majority of cancers, in which specific tumor antigens have not yet been identified. In this study we examined the efficacy of tumor vaccines comprised of unfractionated tumor material presented by professional antigen-presenting cells (APC): dendritic cells (DC) or macrophages (M phi). To enhance the relevance of these studies for human patients we used 2 poorly immunogenic murine tumor models and evaluated the effectiveness of the vaccination protocols in tumor-bearing animals. APC (in particular DC) pulsed with unfractionated extracts from these "poorly immunogenic" tumors were highly effective in eliciting tumor-specific cytotoxic T lymphocytes. A measurable CTL response could be detected after even a single immunization with tumor extract-pulsed DC. DC or M phi pulsed with tumor extract were also effective vaccines in tumor-bearing animals. In the murine bladder tumor (MBT-2) model a modest extension of survival and 40% cure rate was seen in the animal groups immunized with DC or M phi pulsed with MBT-2 tumor extract. DC or M phi pulsed with B16/F10.9 tumor extract were also remarkably effective in the B16 melanoma lung metastasis model, as shown by the observation that treatment with APC caused a significant reduction in lung metastases. Cumulatively, the CTL and immunotherapy data from the two murine tumor systems suggest that APC (in particular DC) pulsed with unfractionated cell extracts as a source of tumor antigen may be equally or more effective than genetically modified tumor vaccines.     

In vitro culture of hamster ovarian primary interstitial cells: effect of serum

The function of ovarian interstitial cells has been largely addressed using rat theca-interstitial cell culture. However, this preparation is primarily enriched with theca and secondary interstitial cells, which make it difficult to address selectively the function of the primary interstitial cells. We have developed an in vitro culture of hamster ovarian primary interstitial cells. Cells were isolated from postnatal hamster ovaries by collagenase digestion and purified over a Percoll gradient. The preparation contained 90% viable, pure interstitial cells, which anchored to the plastic and glass culture surface in the presence of fetal bovine serum. Cell proliferation was noted in the presence of serum dosages higher than 0.2%; however, reduction of serum concentration to 0.1% or complete serum starvation did not affect cell viability but almost completely abolished cell proliferation as determined by [3H]thymidine incorporation, labeling index, and DNA content of the culture. All cells exhibited active 3beta-hydroxysteroid dehydrogenase and P450 side chain cleavage immunoreactivity, which corresponded to basal progesterone and androstenedione accumulation. Replacement of serum to starving cells resulted in the induction of the "S" phase and "M" phase specific cyclins, and resumption of cell proliferation. Our results indicate that hamster primary interstitial cells can be cultured in vitro as a monolayer, and the anchorage and proliferation of these cells depend on serum supplement; however, a viable monolayer can be maintained for several days without serum. This model will be useful for addressing the mechanisms of differentiation of ovarian interstitial cells.     

Novel chemically modified oligonucleotides provide potent inhibition of P-glycoprotein expression

One major form of multiple drug resistance (MDR) to cancer therapeutic agents is mediated by overexpression of P-glycoprotein, a membrane ATPase that serves as a drug efflux pump. In humans, this protein is the product of the MDR1 gene. We have used chemically modified antisense oligonucleotides to reduce expression of P-glycoprotein in multidrug-resistant fibroblasts and colon carcinoma cells. Although several types of oligonucleotides were tested, compounds having a phosphorothioate backbone and a methoxyethoxy (ME) group at the 2' position of the ribose ring proved to have the greatest potency. Thus, phosphorothioate 2'-ME oligonucleotides directed against either the AUG codon region or the stop codon region of the MDR1 message produced substantial (50-70%) inhibition of P-glycoprotein expression at concentrations of < or = 50 nM. In addition, such treatment resulted in augmented drug uptake as measured by flow cytometry. Unmodified phosphorothioate compounds of the same sequence were active only in the micromolar range. We also tested the ability of several potential delivery agents to enhance the pharmacological effectiveness of anti-MDR1 oligonucleotides. Both commercial Lipofectin, a well known liposomal transfection agent, and a liposomal preparation based on a novel "facial amphiphile" were effective in enhancing their pharmacological effects of antisense oligonucleotides. A Starburst dendrimer, a type of cationic polymer, was also effective in oligonucleotide delivery. This report emphasizes that significant improvements in antisense pharmacology can be made through judicious use of both chemical modifications of oligonucleotides and appropriate delivery systems.