Analysis of the activity of promoters from two photosynthesis-related genes psaF and petH of spinach in a monocot plant, rice.

The subunit III of photosystem I and ferredoxin-NADP(+)-oxidoreductase are encoded by nuclear genes, namely psaF and petH. The activity of their promoters from spinach has been evaluated in transgenic tobacco earlier. Evaluation of the activity of these Dicotyledoneae-specific promoters has been carried out in a monocot system (i.e. rice) by transient gene expression system, based on electroporation-mediated gene delivery into protoplasts from leaves and roots. It has been found that various promoter deletions show higher activity in leaf protoplasts and elements for quantitative response are widely distributed. Transgenic rice has also been produced with a petH promoter and gus reporter gene construct. Although petH promoter is a weak promoter in comparison to the 35S promoter, it expresses well in green tissues and could be useful for plant genetic engineering.

Nucleotide sequence of psbQ gene for 16-kDa protein of oxygen-evolving complex from Arabidopsis thaliana and regulation of its expression.

The psbQ gene encoding a 16-kDa polypeptide of the oxygen-evolving complex of photosystem II has been isolated from Arabidopsis thaliana and characterized. The gene consists of a 28 nucleotide long leader sequence, two introns and three exons encoding a 223-amino-acid precursor polypeptide. The first 75 amino acids act as a transit peptide for the translocation of the polypeptide into the thylakoid lumen. Expression studies show that the gene is light-inducible and expresses only in green tissues with high steady-state mRNA levels in leaves. Using this gene as a probe, restriction fragment length polymorphism between two ecotypes, Columbia and Estland, has also been detected.

Alternative approach and evaluation of three potency assay methods for testing adsorbed tetanus toxoid.

Thirteen batches of adsorbed Tetanus Toxoid (TT) from different manufacturers were tested for potency by three different methods viz: (i) An Antibody Induction Method (AIM) developed in mice: (ii) WHO lethal challenge in mice; and (iii) Conventional Antibody Induction (I.P). Method in guinea pigs. The potency results obtained in AIM, by serological evaluation of immunized mice were found identical and correlated significantly with those obtained by WHO recommended lethal challenge test in mice. The potency data obtained in the present study was found comparable with other studies. An AIM in mice thus offers an alternative to lethal challenge tests and can replace guinea pig model. Out of 107 serum samples obtained from immunized guinea pigs in the conventional antibody induction method, 90% samples contained more than 4 units of tetanus antitoxin per ml. End point titres of 42 serum samples belonging to 5 batches of TT also showed much higher tetanus antitoxin content when determined by TN test. The potency data obtained thus suggest revision of the minimum requirement in Indian Pharmacopoeia which is too low and which may be increased as indicated by the present study.

Quantitation of tetanus and diphtheria antitoxins in mouse sera by indirect haemagglutination.

Serum samples obtained from 75 groups of mice immunized with various doses of adsorbed tetanus vaccine, adsorbed diphtheria-tetanus vaccine and adsorbed diphtheria-tetanus-pertussis vaccine were titrated for tetanus antitoxin content by an in-vitro indirect haemagglutination (IHA) and by toxin neutralization test (TN) in mice. From these serum samples of 49 groups of mice which were immunized with combined vaccine containing diphtheria toxoid were titrated for their diphtheria antitoxin content by IHA and by i.d. toxin neutralization test (TN) in guinea pigs. Good correlations were found between the estimates obtained by in-vitro IHA and in vivo TN tests in both tetanus and diphtheria antitoxin titrations. The minimum level of tetanus or diphtheria antitoxin detectable by IHA was 0.00039 IU/ml. It is concluded that IHA is a simple, sensitive and reproducible alternative test which can replace the animal TN tests for the estimation of tetanus and diphtheria antitoxins and could reliably be used in the potency assay of tetanus and diphtheria toxoids of combined vaccines based on antibody induction in mice.

An antibody induction method in mice for the potency assays of diphtheria and tetanus components in combined vaccines.

Eleven batches of Adsorbed Diphtheria-Tetanus (DT) vaccines and thirteen batches of Adsorbed Diphtheria-Pertussis-Tetanus (DTP) vaccines were tested for the potency of diphtheria and tetanus components by an Antibody Induction Method (AIM) developed in mice. The potency results obtained were found comparable and did not show any statistically significant difference with those obtained by WHO recommended lethal challenge tests for diphtheria in guinea pigs and for tetanus in mice. AIM in mice is more economical as both diphtheria and tetanus components of combined vaccine can be tested in the same experiment and the procedure also eliminates the use of guinea pigs required in the lethal challenge/conventional tests. The data obtained while testing tetanus component by the conventional antibody induction (IP) method in guinea pigs suggests that minimum requirements laid down in i.p. is too low which may be fixed as at least 3 out of 9 guinea pig sera and should contain > or = 4 units of tetanus antitoxin per ml.

Transient expression of gus gene in intact seed embryos of Indica rice after electroporation-mediated gene delivery.

Two-day-old germinating intact seed embryos of Oryza sativa variety Basmati 370 were electroporated with a view to examine suitability of this system for gene delivery. The experiments were done with a plasmid having gus gene under the control of CaMV 35S promoter. Spectrofluorophotometric GUS assay revealed high activity of the introduced gene when embryos were given three electrical pulses at 1600 V cm(-1) and 100 µF capacitance with a pulse length of 75 ms. Additionally, histochemical localization of GUS activity in seedlings and various organs such as leaves, coleoptiles and roots was also done. Expression of GUS activity was studied up to 15 days and found to be organ-specific, thereby showing that embryos can indeed serve as efficient recipient system. Use of cycloheximide revealed that GUS activity appears as a result of early protein synthesis after electroporation and is substantially stable in vivo.

Developmental and light-dependent cues interact to establish steady-state levels of transcripts for photosynthesis-related genes (psbA, psbD, psaA and rbcL) in rice (Oryza sativa L.).

The steady-state transcript levels for psbA, psbD, psaA and rbcL are low in dark-grown rice seedlings as compared to those grown in light. Following seed germination, they accumulate in an age-dependent manner, in dark as well as light, reaching a maximal level on the 7th or 8th day, before a slow decline sets in. But transcripts for psbA and psbD continue to maintain relatively-high levels even after 10 days of growth in light. Exposure of 5-day-old dark-grown seedlings to light results in an approximately 25-60-fold increase in transcripts during a period of 72 h, followed by a decrease. An analysis of data from both lines of investigation reveals that the developmental programme increases the transcript levels for psbA, psbD, psaA and rbcL by about 10-, 2.3-, 7.0- and 8.0-fold, respectively, between 5-8 days after germination and it is independent of light. At the same time, exposure of the seedlings to light during this period further enhances transcript levels by 5-, 11.4-, 6.6- and 7.8-fold, respectively. Thus, both developmental and light-dependent cues contribute to establish steady-state levels of transcripts for the chloroplast genes investigated.

Identification of 3',5'-cyclic AMP in axenic cultures of Lemna paucicostata by high-performance liquid chromatography.

Nucleotides were purified from the extract of aseptically grown Lemna paucicostata 6746 by low-pressure column chromatography and thin-layer chromatography. In this partially purified sample, a compound has been identified, using a highly sensitive HPLC-fluorometric detection method, with chromatographic properties and substrate specificity for cyclic nucleotide phosphodiesterase similar to authentic 3',5'-cyclic AMP. The level of cAMP has been estimated to be 70-80 pmol/g fresh wt.

Testing of adsorbed diphtheria-pertussis-tetanus (DPT) vaccine for freedom from abnormal toxicity.

A total of 112 samples of adsorbed DPT vaccine were tested in mice for freedom from abnormal toxicity by three procedures: (a) single human dose by intraperitoneal route; (2) single human dose by subcutaneous route; and (3) half the single human dose by intraperitoneal route. The percentage of samples passing by the three methods was 25, 90 and 54.5, respectively. Method 3, which is being recommended now by the World Health Organization, is supported by the results of this study to be neither very stringent nor very lenient to pass the adsorbed DPT vaccine for freedom from abnormal toxicity.

Differentiation in explants from mature leguminous trees.

Stem and petiole explants, obtained from mature trees, ofAlbizzia lebbeck,Cassia fistula andC.siamea callused and differentiated shoot-buds and later shoots on B5 medium supplemented with either 0.5 mg/l IAA + 1 mg/l BAP or BM + 2 mg/l NAA + 0.5 mg/l BAP. The stem explants were more responsive than the petiole explants. InA.lebbeck, the IAA substituted medium favoured differentiation from both types of explants. However, inC.fistula, the type of explants rather than the medium composition had an overriding influence on shoot differentiation since those from petiole hardly responded in either medium. It has been possible to obtain plantlets from bothA.lebbeck andC.fistula under conditions conducive to rooting. Plantlets ofA.lebbeck have also been successfully transferred to the field.

Development of a mouse model to estimate the potency of the diphtheria toxoid component of diphtheria-tetanus and diphtheria-tetanus-pertussis vaccines.

A mouse model to estimate the potency of the diphtheria toxoid component in diphtheria-tetanus vaccines and diphtheria-tetanus-pertussis vaccines has been developed as an alternative to the conventional method of testing in guinea-pigs. Optimal conditions with regard to dose, route and period of immunization have been standardized. The maximum levels of antitoxin were detected five weeks after vaccination and the s.c. route was found to be optimal. Potency data have been compared with other studies in mouse models and with those obtained by the conventional method in guinea-pigs.

Floral Induction in a Photoperiodically Insensitive Duckweed, Lemna paucicostata LP6 : Role of Glutamate, Aspartate, and Other Amino Acids and Amides.

The effects of 20 amino acids and two amides were studied on the flowering of a photoperiodically insensitive duckweed, Lemna paucicostata LP6. Alanine, asparagine, aspartate, cystine, glutamate, glutamine, glycine, lysine, methionine, proline, serine, and threonine induced flowering under a photoperiodic regime of 16 hours light and 8 hours darkness. Among these, glutamate and aspartate were found to be the most effective for flower induction. These acids could initiate flowering even at 5 x 10(-7) molar level, though maximal flowering (about 80%) was obtained at 10(-5) molar. Change in the photoperiodic schedule or the pH of the nutrient medium did not influence glutamate- or aspartate-induced flowering. The low concentrations at which glutamate and aspartate are effective suggests that they may have a regulatory role rather than simply acting as metabolites.

The reduction of animal usage by the application of indirect haemagglutination in the potency testing of diphtheria toxoid in combined vaccines.

Eight Adsorbed Diphtheria-Tetanus vaccines and 13 Diphtheria-Tetanus-Pertussis vaccines made by four different manufacturers were tested for the potency of the diphtheria components in guinea-pigs by the method of British Pharmacopoeia (1973). Two-hundred-and-ten guinea-pig sera consisting of ten sera related to each vaccine sample thus obtained were titrated for diphtheria antitoxin by indirect haemagglutination (IHA) and the conventional toxin neutralization (TN) tests. Statistical analysis of the results showed a good correlation between the titres obtained with the two tests. The potencies of the diphtheria components of various vaccines calculated from the antitoxin content of the respective guinea-pig sera titrated by the IHA test correlated significantly with the potencies obtained from the antitoxin content titrated by the routinely used TN test. The use of IHA in place of the TN test thus offers as an alternative that permits a reduction in animal usage.

A comparison of the indirect haemagglutination test with the toxin neutralization test for the estimation of diphtheria antitoxin in mouse sera.

Serum samples from 42 groups of mice immunized for different immunization periods with various doses of Adsorbed Diphtheria-Tetanus Vaccine, Adsorbed Diphtheria-Tetanus and Pertussis Vaccine and a standard diphtheria toxoid were assayed for their diphtheria antitoxin content by indirect haemagglutination (IHA) and by toxin neutralization (TN) tests. A very good correlation of 0.91 was obtained between the results of the two methods. There was no statistically significant difference between the IHA and the TN titres obtained. Adsorption with sheep red cells and treatment of the sera with 2-mercaptoethanol had no effect on the IHA titres. The minimum level of antitoxin detectable by the IHA test was 0.00039 IU ml-1. IHA proved to be a sensitive, specific and reproducible method which can be used reliably for the assay of diphtheria antitoxin in mouse sera.

Further observations on the stability of cholera vaccine at different temperatures.

The stability of cholera vaccine samples held at 4-8 and 20-25 degrees C for 2 1/2 years was studied with regard to the number of organisms, total nitrogen content and relative antigenicity. After this length of time, the percentage loss in the number of organisms averaged 10.7 at 4-8 degrees C and 22.7 at 20-25 degrees C. The maximum loss in number of organisms took place during the first six months and in the next two years there was very little loss in the number of organisms. The total nitrogen content after 2 1/2 years remained almost unaltered at these temperatures. All the ten samples had acceptable relative antigenicity for both the Inaba and Ogawa serotypes after exposure at 4-8 degrees C for 2 1/2 years, while at 20-25 degrees C only two samples for the Inaba serotype and one sample for the Ogawa serotype retained antigenicity within acceptable limits after the same time. The loss of antigenicity after 2 1/2 years at 20-25 degrees C in comparison with antigenicity at 4-8 degrees C ranged from 41.5 to 83.1% (mean 61.9%) for the Inaba serotype and from 47.3 to 80.3% (mean 65.1%) for the Ogawa serotype. From this study on the cholera vaccine manufactured at the Central Research Institute, Kasauli, it is concluded that the shelf life of cholera vaccine may be increased from 18 months to at least 24 months, when the vaccine is stored under refrigeration.

Role of Catecholamines in Promotion of Flowering in a Short-Day Duckweed, Lemna paucicostata 6746.

l-Epinephrine, l-norepinephrine, and l-isoproterenol substantially promote flowering under a photoperiodic regime of 8 hours light and 16 hours darkness in Lemna paucicostata 6746 when grown on the modified Bonner-Devirian medium devoid of ethylenediaminetetraacetic acid. If catecholamines are provided to plants at 10(-4) molar level prior to transferring them to the short-day regime, they not only induce more floral primordia but also significantly improve flower development and sustain the flowers for a longer period. Propranolol (10(-4) molar), a beta-adrenergic blocking agent, partially suppresses flowering and the inhibition of flowering is relieved by catecholamines.