Adrienne J. Grant

Accelerated endocytosis and incomplete catabolism of radical-damaged protein

Native bovine serum albumin (BSA) was endocytosed and degraded at a steady rate by resident peritoneal murine macrophages with barely detectable amounts remaining within the cells. Radical-damaged BSA was endocytosed and degraded up to 2.5-fold more rapidly than native BSA, but some radical-damaged BSA accumulated within the cells in a time-dependent manner. The extent of accumulation increased in parallel with that of radical damage. Thus, some radical-damaged BSA was processed less efficiently than native BSA. Such inefficient catabolism of radical-damaged proteins may contribute to certain diseases such as atherosclerosis.

Inefficient Degradation of Oxidized Regions of Protein Molecules

We have previously shown that the intracellular half-life of endocytosed oxidized albumin is much longer than that of native albumin. We now report that the regions of oxidized albumin which contain oxidation products (carbonyls and fluorophores), are less readily released as small degradation products by cell-free proteolysis than is the molecule overall. We deduce that oxidized moieties in the polypeptide chain can confer localized resistance to enzymatic proteolysis. Such resistance to proteolysis may account for the intracellular accumulation of some endocytosed oxidized protein which we have previously observed.

Hypothesis: A damaging role in aging for reactive protein oxidation products?

This paper discusses our knowledge of protein oxidation and its relationship to aging. It also outlines new observations from our laboratories concerning reactive species produced during protein oxidation, and proposes that these may inflict damage on other molecules, and hence contribute to the progression of aging. Whereas it has previously been difficult to see how relatively inert protein oxidation products could possibly have any causal role in aging, the detection of these novel reactive species implies a potentially significant role.

Heterotrophy on ultraplankton communities is an important source of nitrogen for a sponge-rhodophyte symbiosis.

SUMMARY Grazing on ultraplankton by the sponge partner of an invertebrate/algal symbiotic association can provide enough particulate organic nitrogen to support the nitrogen needs of both partners. The previously unknown natural diet of the sponge in the Haliclona–Ceratodictyon association consists of bacteria and protozoans, which are rich sources of nitrogen. Retention of ultraplankton varied with season and time of day. During the winter there was an order of magnitude more nitrogen taken up than in summer. Time of day during each season also affected the amount of ultraplankton retained. In summer retention was higher at night whereas the opposite was true during winter. Overall, the Haliclona–Ceratodictyon association is able to meet its metabolic nitrogen demands through grazing on the naturally occurring water column community.

Inhibition of algal photosynthesis by a symbiotic coral

When symbiotic dinoflagellate algae (Symbiodinium sp.), isolated from the scleractinian coral Plesiastrea versipora were incubated in homogenized host tissue, carbon fixation was usually lower than in seawater only (43 cases of of 54), and this inhibition occurred throughout the year. Algae incubated in homogenized host tissue fixed a mean of 78.84% ± 22.6% of the amount fixed by algae in seawater (n = 54, range 33–142%). The difference between rates of fixation in seawater (mean ± SD of 61.300 ± 22.71 nmol C/105 cells) and in homogenates (mean ± SD of 47.377 ± 21.42 nmol C/105 cells) in individual experiments, was highly significant (p<0.0001, paired t-test). Homogenized host tissue from populations of P. versipora separated by 4000 km inhibited photosynthesis in algae from both their own and the distant coral population. Inhibition of photosynthesis was not observed when isolated algae from the anemone, Aiptasia pulchella were incubated in homogenate from P. versipora. An inhibitor of photosynthesis was isolated from a low molecular weight fraction of homogenized host tissue of P.versipora by cation-exchange chromatography and further purified by HPLC. This is the first natural inhibitor of photosynthesis that has been found in an invertebrate with symbiotic algae.

Elastase activities of human bladder cancer cell lines derived from high grade invasive tumours

Elastase activities in intact human bladder cancer cell lines, established from three patients, were measured using a fluorogenic substrate highly specific for elastase, under conditions of physiological pH and ionic strength. This method allowed separation of cell-associated from secreted enzyme activity. As secreted elastase accounted for only 8% of the total, we concluded that the elastases were present at the cell surface. Inhibition studies using extracts of cell-surface elastases showed them to be serine proteinases which were also inhibited by alpha 1-antitrypsin. Partially purified fractions showing the highest specific activity towards the fluorogenic substrate hydrolysed insoluble elastin thus confirming the presence of elastases. This is the first time that elastase activity has been demonstrated in human bladder cancer cells and may represent a mechanism involved in tumour invasion.

EFFECTS OF FREE AMINO ACIDS ON THE ISOLATED SYMBIOTIC ALGAE OF THE CORAL PLESIASTREA VERSIPORA (LAMARCK) : ABSENCE OF A HOST RELEASE FACTOR RESPONSE

Abstract Symbiotic algae incubated in host tissue homogenate of the coral Plesiastrea versipora for 2 h in the light released at least four and a half times as much photosynthetically fixed carbon (range 13.8±3.1 to 158±9.5 nmol C/10 6 algae) as algae incubated in seawater (range 1.4±0.3 to 10.8±0.6 nmol C/10 6 algae) indicating the presence of ‘host release factor’. When algae were incubated in a low molecular weight fraction of homogenate containing partially purified ‘host release factor’ they also released more carbon (range 62.2±3.7 to 279±11.4 nmol C/10 6 algae) than algae incubated in seawater. This low molecular weight fraction contained free amino acids. We tested the hypothesis that the free amino acids in this fraction were responsible for ‘host release factor’ activity. Algae incubated in a mixture of free amino acids equivalent to those found in this fraction, released more fixed carbon (range 2.4±0.3 to 25.2±0.2 nmol C/10 6 algae) than algae incubated in seawater but in each experiment, release was much lower than when algae were incubated in host tissue homogenate. These data indicate that the stimulation of release of photosynthetically fixed carbon from the symbiotic algae of Plesiastrea versipora incubated in partially purified host release factor is not primarily due to the presence of free amino acids. We are continuing further studies to determine the exact nature of the active compound.

Enhanced Enzymatic Degradation of Radical Damaged Mitochondrial Membrane Components

The location of a protein (soluble or membrane-bound) influences the extent of oxidative damage caused by free radicals. It has been established that after radical attack, soluble proteins can become more susceptible to hydrolysis by individual proteinases than native proteins. We have now examined the hydrolytic susceptibility following radical attack of a protein that is located within a membrane environment, mitochondrial monoamine oxidase (MAO). After exposure to oxygen radicals generated by gamma irradiation, hydrolysis of sub-mitochondrial particles (SMP) containing MAO was increased in three respects. First, the generation of small fragments of MAO by the proteinases elastase and trypsin, was enhanced. Second, the generation by these enzymes and by phospholipase A2 of non-sedimentable membrane fragments containing MAO was also increased. Third, autolysis of SMP was enhanced. Hence, proteins located within membranes may become more susceptible to enzymatic degradation following oxidative damage.

A symbiosome membrane is not required for the actions of two host signalling compounds regulating photosynthesis in symbiotic algae isolated from cnidarians

In many cnidarians, symbiotic algae live within host-derived symbiosomes. We determined whether a symbiosome membrane alters the response of isolated symbiotic algae to two signalling compounds that regulate algal carbon metabolism. Host release factor (HRF), which stimulates photosynthate release, and photosynthesis inhibiting factor (PIF), which inhibits photosynthetic carbon fixation, are found in homogenised tissue of the scleractinian coral Plesiastrea versipora. Compared with seawater controls, photosynthate release from isolated algae incubated in P. versipora homogenate for 2 h in the light was: 6 to 19-fold higher from its own algae (free of symbiosomes); 19 to 32-fold higher from Zoanthid robustus algae (within symbiosomes) and 3 to 24-fold higher from Z. robustus algae (free of symbiosomes); and from cultured algae (free of symbiosomes) was seven-fold higher from Montipora verrucosa and four-fold higher from Cassiopeia xamachana. Incubation of algae in P. versipora homogenate inhibited photosynthesis by: 33-49% in P. versipora algae; 29-47% in Z. robustus algae (regardless of whether or not the symbiosome was present); and 25% in M. verrucosa algae. In C. xamachana algae, photosynthesis increased. We conclude that the symbiosome is not essential for, yet does not block, the effects of HRF and PIF.

MALIGNANCY-RELATED CHARACTERISTICS OF WILD TYPE AND DRUG-RESISTANT CHINESE HAMSTER OVARY CELLS

&NA; Chinese hamster ovary (CHO) cell lines are a very popular cell model for a wide range of studies but are often misused experimentally as a substitute for normal cells. Although CHO was originally derived from normal tissue, the cell lines studied here, including the parental wild type, have many characteristics which indicate that they have undergone malignant transformation. Biological properties associated with malignancy were investigated in this study on wild type CHO cells and 4 drug resistant sublines, EOT, Col R‐22, Pod R11‐6, and Vin R‐1. We report evidence of tumorigenicity in experimental animals, invasive capacity, in vivo and in vitro, protease release by 2 of the cell lines, features related to drug resistance in the mutant sublines, and numerical and structural chromosomal abnormalities.