Effect of accessible immobilized NAD(+) concentration on the bioaffinity chromatographic behavior of NAD(+)-dependent dehydrogenases using the kinetic locking-on strategy.

In preparation for studies aimed at establishing the relationship between immobilized NAD(+) concentration and the concentration of soluble locking-on ligand required to promote biospecific adsorption of NAD(+)-dependent dehydrogenases to immobilized NAD(+) derivatives (the "locking-on" strategy), two approaches were evaluated for varying substitution levels: (i) suitable dilution of the affinity matrix with unsubstituted Sepharose 4B and (ii) direct coupling of the required ligand concentration to the inert matrix. The latter approach was found to be the preferable strategy for evaluation of the locking-on tactic because it produced a more homogeneous distribution of immobilized NAD(+) concentration. Affinity chromatographic studies using S(6)-linked NAD(+) derivatives synthesized to various substitution levels showed that the total accessible immobilized NAD(+) concentration has a direct effect on the locking-on behavior of pyridine nucleotide-dependent dehydrogenases. The one-chromatographic-step bioaffinity purification of l-lactate dehydrogenase (L-LDH, EC 1.1.1.27) from bovine heart illustrates the potential of the locking-on strategy for protein purification applications.     

Squamous cell carcinoma in rudd Scardinius erythrophthalmus: histopathology, ultrastructure, and transmission

The histopathology and ultrastructure of a skin neoplasm in rudd Scardinius erythrophthalmus (L.) are described and the neoplasm is diagnosed as squamous cell carcinoma. In the early stage, tumour cells appear singly or in clusters close to the epidermis; then tumour cells are seen in the dermis as epithelial pearls/nests, cords or sheets with central keratin; and with further development of the neoplasm, tumour cells appear as large sheets and masses, enclosing extensive keratinous formations and foci of necrotic cells, infiltrating deeper into the dermis and penetrating the muscle layers. Increasing vascularity and inflammation are associated with all stages of progression. In some cases metastases are observed in the viscera. Electron microscopic examination shows that the neoplastic cells are joined by desmosomes; the cytoplasm contains abundant mitochondria and rough endoplasmic reticulum. The neoplasm was successfully transmitted experimentally to healthy rudd when tumour cells were inoculated subcutaneously. Eight of 19 surviving test fish developed tumour growth at the site of tumour cell injection and/or in a corresponding site on the opposite of the body. One of these 8 fish, which developed a neoplasm, also showed a microscopic internal tumour in the viscera. One of the 19 test fish showed a microscopic tumour in the spleen, even though no skin tumour was visible in this fish. No tumours were found in control fish. In contrast, intraperitoneal injection of tumour cells into healthy rudd did not result in transmission of the neoplasm.     

Fasciola hepatica cathepsin L-like proteases: biology,function, and potential in the development of first generation liver fluke vaccines

Fasciola hepatica secretes cathepsin L proteases that facilitate the penetration of the parasite through the tissues of its host, and also participate in functions such as feeding and immune evasion. The major proteases, cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) are members of a lineage that gave rise to the human cathepsin Ls, Ks and Ss, but while they exhibit similarities in their substrate specificities to these enzymes they differ in having a wider pH range for activity and an enhanced stability at neutral pH. There are presently 13 Fasciola cathepsin L cDNAs deposited in the public databases representing a gene family of at least seven distinct members, although the temporal and spatial expression of each of these members in the developmental stage of F. hepatica remains unclear. Immunolocalisation and in situ hybridisation studies, using antibody and DNA probes, respectively, show that the vast majority of cathepsin L gene expression is carried out in the epithelial cells lining the parasite gut. Within these cells the enzyme is packaged into secretory vesicles that release their contents into the gut lumen for the purpose of degrading ingested host tissue and blood. Liver flukes also express a novel multi-domain cystatin that may be involved in the regulation of cathepsin L activity. Vaccine trials in both sheep and cattle with purified native FheCL1 and FheCL2 have shown that these enzymes can induce protection, ranging from 33 to 79%, to experimental challenge with metacercariae of F. hepatica, and very potent anti-embryonation/hatch rate effects that would block parasite transmission. In this article we review the vaccine trials carried out over the past 8 years, the role of antibody and T cell responses in mediating protection and discuss the prospects of the cathepsin Ls in the development of first generation recombinant liver fluke vaccines.     

Caspase proteolysis of the cohesin component RAD21 promotes apoptosis

Caspases are a conserved family of proteases that play a critical role in the execution of apoptosis by cleaving key cellular proteins at Asp residues and modifying their function. Using an expression cloning strategy we recently developed, we isolated human RAD21/SCC1/MCD1 as a novel caspase substrate. RAD21 is a component of the cohesin complex that holds sister chromatids together during mitosis and repairs double-strand DNA breaks. Interestingly, RAD21 is cleaved by a caspase-like Esp1/separase at the onset of anaphase to trigger sister chromatid separation. Here, we demonstrate that human RAD21 is preferentially cleaved at Asp(279) by caspases-3 and -7 in vitro to generate two major proteolytic products of approximately 65 and 48 kDa. Moreover, we show that RAD21 is specifically proteolyzed by caspases into a similarly sized 65-kDa carboxyl-terminal product in cells undergoing apoptosis in response to diverse stimuli. We also demonstrate that caspase proteolysis of RAD21 precedes apoptotic chromatin condensation and has important functional consequences, viz. the partial removal of RAD21 from chromatin and the production of a proapoptotic carboxyl-terminal cleavage product that amplifies the cell death signal. Taken together, these findings point to an entirely novel function of RAD21 in the execution of apoptosis.     

Surgical implantation of transmitters into fish

Although the Animal Welfare Act does not cover poikilotherms, individual institutions and policies and legal requirements other than the Animal Welfare Act (e.g., the US Public Health Service and the Interagency Research Animal Committee's Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training) require the review of projects involving fish by institutional animal care and use committees (IACUCs). IACUCs may, however, lack the knowledge and experience to evaluate fish projects judiciously, especially when the projects are in field settings. Surgeries involving implantation of transmitters and other instruments into the coelom, which now comprise a very common research tool in the study of free-ranging fishes, are examples of surgeries that use a broad spectrum of surgical and anesthetic techniques, some of which would not be considered acceptable for similar work on mammals. IACUCs should apply the standards they would expect to be used for surgeries on homeotherms to surgeries on fish. Surgeons should be carefully trained and experienced. Surgical instruments and transmitters should be sterile. Regulations and laws on the use of drugs in animals should be followed, particularly those concerned with anesthetics and antibiotics used on free-ranging fish. Exceptions to surgical procedures should be made only when circumstances are extreme enough to warrant the use of less than optimal procedures.     

193 nm photolysis of aromatic and aliphatic dipeptides in aqueous solution: dependence of decomposition quantum yield on the amino acid sequence

The values of molar absorption coefficient and quantum yields of photodecomposition and peptide bond scission were determined for a number of aromatic and aliphatic dipeptides under 193 nm laser irradiation in neutral argon-saturated aqueous solution. Under these conditions we could show that no dependence of the dipeptide decomposition quantum yield on the sequence of amino acid residues exists, neither for aromatic dipeptides nor for aliphatic ones.     

Radiogenic initiation of thyroid cancer: a common cellular event

A transplantation system for clonogenic cells in rat thyroid was used, which allows quantitative evaluation of both the acute cytotoxicity and the late neoplastic effects of ionizing radiation at the cellular level in vivo. We have obtained direct experimental evidence that radiogenic initiation of neoplasia in vivo is a common cellular event, and that cell number influences the expression of initiation. Ten per cent of those graft sites which had received 26 clonogens surviving 5 Gy developed carcinomas, while 4 per cent of those which received 26 unirradiated clonogens developed carcinomas. By comparison, 26 per cent of the sites that were inoculated with 411 surviving irradiated clonogens developed carcinomas while none of the 38 transplant sites that received 411 unirradiated clonogens developed carcinomas. Total tumour incidence (carcinomas plus adenomas) followed the same pattern.     

Synthesis of a highly substituted N(6)-linked immobilized NAD(+) derivative using a rapid solid-phase modular approach: suitability for use with the kinetic locking-on tactic for bioaffinity purification of NAD(+)-dependent dehydrogenases

This study is concerned with further development of the kinetic locking-on strategy for bioaffinity purification of NAD(+)-dependent dehydrogenases. Specifically, the synthesis of highly substituted N(6)-linked immobilized NAD(+) derivatives is described using a rapid solid-phase modular approach. Other modifications of the N(6)-linked immobilized NAD(+) derivative include substitution of the hydrophobic diaminohexane spacer arm with polar spacer arms (9 and 19.5 A) in an attempt to minimize nonbiospecific interactions. Analysis of the N(6)-linked NAD(+) derivatives confirm (i) retention of cofactor activity upon immobilization (up to 97%); (ii) high total substitution levels and high percentage accessibility levels when compared to S(6)-linked immobilized NAD(+) derivatives (also synthesized with polar spacer arms); (iii) short production times when compared to the preassembly approach to synthesis. Model locking-on bioaffinity chromatographic studies were carried out with bovine heart l-lactate dehydrogenase (l-LDH, EC 1.1.1.27), bakers yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) and Sporosarcinia sp. l-phenylalanine dehydrogenase (l-PheDH, EC 1.4.1.20), using oxalate, hydroxylamine, and d-phenylalanine, respectively, as locking-on ligands. Surprisingly, two of these test NAD(+)-dependent dehydrogenases (lactate and alcohol dehydrogenase) were found to have a greater affinity for the more lowly substituted S(6)-linked immobilized cofactor derivatives than for the new N(6)-linked derivatives. In contrast, the NAD(+)-dependent phenylalanine dehydrogenase showed no affinity for the S(6)-linked immobilized NAD(+) derivative, but was locked-on strongly to the N(6)-linked immobilized derivative. That this locking-on is biospecific is confirmed by the observation that the enzyme failed to lock-on to an analogous N(6)-linked immobilized NADP(+) derivative in the presence of d-phenylalanine. This differential locking-on of NAD(+)-dependent dehydrogenases to N(6)-linked and S(6)-linked immobilized NAD(+) derivatives cannot be explained in terms of final accessible substitutions levels, but suggests fundamental differences in affinity of the three test enzymes for NAD(+) immobilized via N(6)-linkage as compared to thiol-linkage.     

OVARIAN INFLUENCE ON POLLEN TUBE GROWTH,AS INDICATED BY THE SEMIVIVO TECHNIQUE

With the semivivo technique, pollen tubes grow from the bases of cut styles. The length of pollen tubes protruding from such styles, or growing through intact styles, is greatly enhanced by allowing initial pollen tube growth to occur before the ovary is removed. The presence of other floral parts has no apparent effect in this study. This improved semivivo method provides an abundant supply of pollen tubes that may show fewer artifacts than do those produced with in vitro growth. It also suggests that the ovary plays an important role in pollen-style interactions.