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Biomedical subjects

K C Gupta

Publications and source records attributed to K C Gupta.

At least 55 records · Page 3Linked to original sources
PubMed1993

Criteria for in vitro alternatives for the eye irritation test.

A proposal encompassing considerations and criteria for the development of in vitro alternatives to the eye irritation test has been developed and is presented here. Two factors need to be considered initially in developing an alternative test. The first is to determine whether the alternative assay is to be used as a screen or as a replacement for the eye irritation test. Less stringent acceptance criteria are required for an assay used as a screen than for that used as a replacement test. A screen is a preliminary test for the assessment of eye irritation. It is used for making preliminary decisions or establishing the direction for further testing. Screens answer fewer and less complex questions than a replacement test would, since the results from screens are usually confirmed by more definitive testing. A replacement test, however, must provide the same answers as in vivo methods for the assessment of eye irritation and must provide data for making a definitive toxicological assessment of eye irritation. The second factor to be considered is knowledge of the in vivo assay intended to be replaced. This knowledge should include the procedural aspects of the test and the regulatory information it provides. The following may be considered as criteria for in vitro tests used as screens or as replacements for the eye irritation test in rabbits: rationale (there should be a clear statement regarding the rationale for the use of a particular test in relation to the availability of other tests); relevance (the in vitro endpoint should have biological or physiological relevance to the effect to be detected in vivo); and validational (intralaboratory as well as interlaboratory validation must be conducted).

Animal Testing Alternatives↗
PubMed1993

Screening procedures for eye irritation.

Screens aid in identifying some severe irritants or corrosives and eliminating them from consideration for in vivo eye irritation testing. Products may be evaluated for ocular irritation potential in a stepwise progression as follows: (1) products at pH extremes of 2 or below or of 11.5 or above may be considered to be ocular irritants; (2) based on chemical structure-activity considerations, some products may be judged to have ocular irritation potential; (3) validated and accepted in vitro systems may possibly be used as a screen in the future; (4) when a test material demonstrates severe acute dermal toxicity (lethality at < or = 200 mg/kg body weight), further testing for either dermal or ocular irritation may not need to be undertaken; (5) if a substance shows a primary dermal irritation index of 5 or above, it may be considered to be an ocular irritant; (6) materials that are not removed from consideration based on these proposed screens may then be considered for testing for ocular irritation in rabbits under accepted procedures. In a survey given to participants in the workshop, a high percentage believed that screens should be used. However, opinions on the use of the individual screens varied between the different interested groups attending, with the possible future use of in vitro screens for specific product lines having the highest percentage of agreement (57-100%).

Animal Testing Alternatives↗
PubMed1993

Use of ophthalmic topical anaesthetics.

Pretreatment of the eyes of rabbits with a topical anaesthetic can be viewed as a refinement of the test for eye irritation. It reduces pain at the time of test-material administration, decreases animal distress and permits easier application of the test agent to the eye. In some cases, however, use of an anaesthetic either alone or in combination with the test substance may alter ocular responses or provide little benefit. Although anaesthetic pretreatment may result in decreased pain at the time of test-compound administration, it does not affect possible pain after the effects of the anaesthetic have dissipated. Some anaesthetics are themselves irritating to eyes. In addition, anaesthetics reduce blinking and tearing, thereby maintaining the test-material concentration at the surface of the eye longer. Corneal permeability may also be increased with pretreatment use of an anaesthetic, and may bring the test agent into contact with more structures of the eye. Some anaesthetics delay healing after ocular injury. All of these varied effects may result in increased irritation to the eye. Overall, pretreatment with anaesthetics has usually resulted in a tendency for slightly higher irritation scores; eye irritancy classification is usually unaffected.

Anesthetics, Local↗
PubMed1993

The use of low-volume dosing in the eye irritation test.

The Draize rabbit eye test was developed to provide a method for assessing the irritation potential of materials that might come in contact with human eyes. The method involves the instillation of 0.1 ml of a test liquid (100 mg solid) into the conjunctival sac of an animal's eye. A refinement of the Draize test is the low-volume eye test in which 0.01 ml of a substance is placed directly on the cornea of the eye. Studies indicate that the low-volume method provides a better correlation to human eye irritation experience for some substances. The Interagency Regulatory Alternatives Group (IRAG) proposes that the low-volume eye test can be used to substantiate the irritancy of suspect severe ocular irritants that have not been eliminated by various pre-eye test 'screens'. A substance testing positive by the low-volume method can be classified as an irritant; one that tests negative will require further testing by the use of the 0.1-ml volume procedure. For all other definitive testing, the Draize test (0.1 ml) should be used. Results from a questionnaire distributed at the IRAG workshop showed that many workshop participants thought that the low-volume test should be used as an eye irritation screening procedure.

Animal Testing Alternatives↗
PubMed1993

Number of animals for sequential testing.

US regulatory agencies have used six animals in eye irritation tests. Analyses of eye irritation tests on pesticides (n = 48), consumer products and cosmetics (n = 53), Marzulli and Ruggles database (n = 139), and cleaning products and ingredients (n = 30) have greatly extended previous investigations of the merit of reducing animal sample size in the eye test. Given the existing scoring system for positive animal responses (corneal opacity > or = 1, iritis > or = 1, conjunctival redness > or = 2 and conjunctival chemosis > or = 2), the accuracy of the classification systems currently used by these agencies was determined. The US Consumer Product Safety Commission, US Food and Drug Administration, and US Occupational Safety and Health Administration use a classification system by which a substance is designated as an irritant when at least four of six animals give a positive response. This decision rule leads to a very high accuracy of at least 99% with essentially no false positive and false negative judgments. In contrast, the system used by the US Environmental Protection Agency pesticide program, in which only one or more of six treated animals result in an irritant decision, has an accuracy of only 50-80% with very high false positive rates. Analyses indicated that test sample size could be reduced to three and still preserve very good accuracy, whereas two-animal and one-animal tests did not give satisfactory responses. A two-stage test, in which two animals are tested and evaluated in the first stage before the need for testing one more animal in the second stage is determined, also demonstrated good operating characteristics. Both the one-stage/three-animal test and the two-stage test deserve consideration.

Animals↗
PubMed1993

Scoring for eye irritation tests.

Scoring of the rabbit eye test and the resulting evaluation and classification should provide useful information about the likelihood that a test material may cause injury on contact with the human eye. When an animal test is necessary, a rabbit eye test based on the following characteristics is proposed for deriving the maximum information from the fewest animals. The ocular effects of interest should include corneal opacity, iritis and conjunctival redness. Animals should be scored for each ocular effect at 24, 48 and 72 hr after the test substance is administered. If an animal is negative at all three scoring times, it can be removed from the test at 72 hr. If it shows a positive effect at a scoring time but the lesion clears at 72 hr, it can be removed at 72 hr. If it shows a positive effect that does not clear at 72 hr, it should be scored again on day 7 when the test ends. However, if an animal shows severe effects at one or more scoring times, it can be removed from the test at 72 hr. An animal is positive if any one of the following criteria is observed at 24, 48 or 72 hr: corneal opacity of 1 or above, iritis of 1 or above, or conjunctival redness of 2 or above. Severe ocular effects (noted at 24, 48 or 72 hr) that may endanger sight deserve special recognition for the classification of chemicals and include corneal opacity of 3 or above, or iritis of 2. This proposal is consistent with the opinions of the majority of respondents who attended the Workshop on Updating Eye Irritation Test Methods, Proposals for Regulatory Consensus. The most notable exception was the suggestion by respondents to add conjunctival chemosis as one of the scoring parameters.

Animals↗
PubMed1993

An eye irritation test protocol and an evaluation and classification system.

An in vivo test protocol and an evaluation and classification system for the determination of eye irritation potential of chemicals and mixtures (substances) is proposed. The protocol uses two or three rabbits and reduces distress in test animals. The test substances are classified as non-irritant, irritant or severe irritant to meet regulatory needs. They may be classified on the basis of past experience with similar compounds or mixtures. Screens such as structure-activity relationships, pH extremes, validated and accepted in vitro tests, severe dermal irritation (primary dermal irritation index > or = 5) or severe dermal toxicity (lethality at < 200 mg/kg body weight) should be used to classify irritant or severe irritant materials when one or more of the screens can provide convincing evidence. For suspected severe irritant materials, the proposed in vivo test permits the use of one rabbit and instillation of 0.01 ml (0.01 g) of the test material into the cornea. Materials that are not classified irritant or severe irritant by screens or severe irritant by one rabbit test are tested in two or three rabbits; 0.1 ml (0.1 g) is instilled into the conjunctival sac. The responses (corneal opacity, iritis and conjunctival redness) are scored according to the modified Draize scoring system at 24, 48 and 72 hr and 7 days post-instillation. A rabbit is considered positive when corneal opacity of 1 or above, iritis of 1 or above or conjunctival redness of 2 or above is present at 24, 48 or 72 hr post-instillation. The material is classified as a severe irritant when the rabbit in the one-animal test or two or more rabbits in the standard test have responses of corneal opacity of 3 or above and iritis of 2 at 24, 48 or 72 hr, or positive responses on day 7 after instillation. The material is classified as an eye irritant when two or more rabbits are positive but the responses are not severe and they clear 7 days after instillation. The material is classified as a non-irritant when no more than one rabbit is positive. The opinions expressed in this article are those of the authors and do not necessarily reflect the views of US Federal agencies.

Animals↗
PubMed1993

Solid-phase introduction and intracellular photoinduced reaction of a water-soluble meso-tetracarboxyporphine conjugated to an antisense oligodeoxyribonucleotide.

Oligonucleotide conjugated with water-soluble meso-tetra(4-carboxyphenyl) porphine (TPPC4) has been prepared by a supporting synthesis and novel solid-phase conjugation strategy. The conjugates could be used in dual fashion: i) on formation of iron complex, target DNA could be site-specifically cleaved on incubation with dithiothreitol; ii) on incubation of RR 1022 rat epithelial cell culture with non-metalized oligonucleotide TPPC4 conjugate, cytotoxic effect was detected after irradiation with laser light at 635 nm.

Animals↗
PubMed1992

Deamidation of human erythrocyte protein 4.1: possible role in aging.

The human erythrocyte membrane protein 4.1 exists in two major electrophoretic forms: 4.1a (80 Kd) and 4.1b (78 Kd). Mass spectrometry and amino acid analysis of the proteolytic peptides derived from carboxyl-terminal regions of these proteins indicate that they differ by deamidation of two aspargine residues at positions 478 and 502. Electrophoretic analysis of carboxyl-terminal peptides has shown that the mobility difference between the two polypeptides is due to the deamidation of Asn502 and not that of Asn478. This observation was confirmed by converting a congener of the protein 4.1b to 4.1a by site-directed mutagenesis of Asn502 to Asp. These results unambiguously demonstrate that deamidation of Asn502 is responsible for conversion of protein 4.1b to 4.1a. Since the conversion of protein 4.1b to 4.1a, under physiological conditions, occurs in a time-dependent manner, our study clearly shows that deamidation is an excellent marker for red blood cell aging.

Amino Acid Sequence↗
PubMed1992

Nuclear entry and nucleolar localization of the Newcastle disease virus (NDV) matrix protein occur early in infection and do not require other NDV proteins.

A large proportion of the Newcastle disease virus (NDV) matrix (M) protein is found in the nuclei of infected chicken embryo cells. Kinetic analysis indicated that much of the M protein enters the nucleus early in infection, concentrating in discrete regions of the nucleus and remaining there throughout infection. The M protein was found in localized regions of the nuclei of a variety of cell lines infected with NDV. Immunostaining for both M protein and nucleolar antigens indicated that most of these regions represent nucleoli. Moreover, this nucleolar localization of the M protein was observed in chicken embryo cells infected with 11 different strains of NDV. Only the M protein of strain HP displayed a modified pattern, concentrating in the nucleolus early in infection but in the cytoplasm late in infection. M protein transiently expressed in COS-1 cells also localized to the nucleus and nucleolus, indicating that the M protein does not require other NDV proteins for this localization.

Animals↗
PubMed1992

Isolation of human monoclonal antibodies binding to B fragment of diphtheria toxin.

Human tonsillar lymphocytes immunized in vitro with diphtheria toxoid were fused with the heteromyeloma cell line SP2/HPT. Hybrids were selected on hypoxanthine-thymidine aminopterin (HAT) medium and assayed for antibody production both by ELISA and Western blotting. Four stable hybridoma cell lines producing 3-7 micrograms/mL of human IgG1 antibodies were isolated. In Western blotting studies these antibodies show binding to the B fragment of diphtheria toxin. Cells from the subclone P2-13/G35 were injected i.p. in pristane-primed nude mice, and ascites fluid was collected after 4-6 weeks. The monoclonal antibody neutralized the cytotoxicity of diphtheria toxin in the Vero cell assay system. This human monoclonal antibody may have a potential in the immunotherapy of diphtheric disease.

Animals↗
PubMed1991

A general method for the synthesis of 3'-sulfhydryl and phosphate group containing oligonucleotides.

The syntheses are described of polymer supports useful for the synthesis of 3'-partially protected sulfhydryl, free sulfhydryl or phosphate group containing oligonucleotides. The supports are compatible with established phosphoramidite chemistry of oligonucleotide synthesis giving rise to oligonucleotides with terminal 3'-partially protected sulfhydryl, free sulfhydryl or phosphate function during final deprotection. Crosslinking of the thiol group containing oligonucleotide to sulfhydryl group specific fluorescent probes was carried out with high selectivity, in high yields under mild conditions. 3-Aminopropylated Controlled Pore Glass (CPG) was succinylated with succinic anhydride followed by the reaction with S-(2-thio-5-nitropyridyl)-2-mercaptoethanol in the presence of dicyclohexylcarbodiimide (DCC). The resultant polymer support was reacted with 4,4'-dimethoxytrityloxyalkanthiol 5(a - c) to yield the derivatized polymer supports 5(a - c). The support 5a directly leads to oligonucleotide-3'-phosphate on deprotection with ammonical DTT at 55 degrees C while the supports 5b and 5c lead to oligonucleotide-3'-thiols or partially protected 3'-sulfhydryl group containing oligonucleotides during final deprotection.

Base Sequence↗
PubMed1991

Comparative bioavailability of slow release diclofenac (Voveran SR) with enteric coated tablet and internationally used Voltaren Retard.

The objective of the study was to compare the enteric coated diclofenac sodium (Voveran), the slow release formulation developed in India (Voveran SR) and the internationally marketed formulation Voltaren Retard. Ten healthy volunteers were administered 100 mg each of the three formulations in a three-way crossover fashion. Blood samples were collected over 24 hours following administration of the drug; plasma levels of unchanged drug were determined by gas chromatography. Pharmacokinetic parameters for the three formulations were compared. The extent of the drug available from the three formulations was the same as the mean AUC values were not significantly different. Cmax and MRT values for the two slow release formulations were comparable but were significantly different from the values obtained with the enteric coated formulation. Tmax values for the two slow release formulations were similar while the enteric coated tablet had faster time to peak. Voveran SR is comparable to Voltaren Retard and has the distinct advantage of a slow release formulation in that its Cmax is much lower and levels are maintained over 12 hours and detectable upto 24 hours. This slow release formulation will offer clinical advantages of better compliance, relief of early morning symptoms and better tolerability over long term usage.

Adult↗
PubMed1990

Initiation of translation at CUG, GUG, and ACG codons in mammalian cells.

Site-directed mutants of the ACG start codon of the C' protein encoded in the polycistronic Sendai virus P/C mRNA revealed that CUG, GUG, and ACG codons initiated translation rather efficiently (10-30% of the AUG initiation) in COS-1 host cells. In addition, AUA and AUU codons initiated translation at about 5% efficiency, while UUG did not initiate translation. The sequence context of these start codons (purine residues at -3 and +4) was crucial in their recognition by the ribosome. The location of the non-AUG codons in the P/C mRNA did not play a role in its recognition by ribosomes. By using CUG, the most efficient non-AUG start codon, instead of the original ACG codon and inserting an additional upstream CUG codon in the P/C mRNA, the amount of the C' protein was increased and a novel protein was synthesized. Syntheses of an increased level of C' and the novel protein did not affect downstream initiations of the P and C proteins, suggesting that more ribosomes bind the mRNA than are actually utilized for initiation of translation.

Animals↗