Kidney ischaemia-reperfusion injury and polyribosome structure.

BACKGROUND: Inhibition of protein synthesis and polyribosome disaggregation are the early events in cell injury provoked by various pathogenic mechanisms, including energy depletion. Polyribosome disaggregation might be expected to occur during ischaemia-reperfusion injury due to ischaemic energy depletion, but also due to detrimental effects of reactive oxygen species on various macromolecules and cellular structures. METHODS: Mouse kidney ischaemia-reperfusion injury was provoked by temporary clamping of the renal artery. The polyribosome sedimentation pattern was analyzed by sucrose density centrifugation of kidney postmitochondrial supernatant. RESULTS AND CONCLUSIONS: Ischaemia for 5 min in the mouse kidney provoked polyribosome disaggregation and an increase of monomer ribosome fraction which was augmented during 10-360 min of reperfusion. Recovery of polyribosome aggregates appeared between 6 and 24 h of reperfusion. Cycloheximide pretreatment prevented only polyribosome disaggregation caused by ischaemia and not that caused by reperfusion. This indicates different mechanisms of polyribosome disaggregation during ischaemia and reperfusion. It probably occurs in the former due to inhibition of initiation of translation, resulting in accumulation of unprogrammed monomer ribosomes, and in the latter due to the splitting of mRNA and breakdown of polyribosomes. Reperfusion did not increase ribonuclease activity in kidney cytosol, but increased the tissue concentration of malonaldehyde, indicating an augmentation in oxygen free radical generation. Possibly these may have caused a non-enzymatic breakdown of polyribosomes. However, pretreatment with allopurinol did not prevent polyribosome breakdown during ischaemia-reperfusion injury.

Mutual predictive value of c-erbB-2 overexpression and various prognostic factors in ductal invasive breast carcinoma.

AIMS AND BACKGROUND: Breast carcinoma is a heterogeneous disease, the prognosis of which correlates with various prognostic factors. The aim of this study was to assess the prognostic significance of c-erbB-2 overexpression in breast carcinoma patients in association with other known prognostic factors. METHODS & STUDY DESIGN: The relationship between immunohistochemical expression of the c-erbB-2 oncoprotein and various established prognostic factors such as tumor size, axillary node status, estrogen and progesterone receptor status, DNA ploidy, proliferation index, cathepsin D expression and histological grade in invasive ductal breast carcinoma is presented in this study. RESULTS: Of the 93 ductal invasive carcinomas 22 (23.7%) were grade I, 51 (54.8%) grade II, and 20 (21.5%) grade III, and the majority (78: 83.9%) were 2-5 cm in diameter. Tumor metastases were identified in one or more lymph nodes in 55 (59.1%) patients, the remaining 38 (40.9%) patients being lymph node negative. According to the DNA histograms 40 (43.0%) tumors were aneuploid and 53 (57.0%) were diploid, and the majority of tumors had more than 4% of cells in the S phase of the cell cycle (83.9%). Expression of c-erbB-2 as shown by immunohistochemical intense membrane staining was present in 49 (52.7%) tumors. Cathepsin D-positive cytoplasmic granular staining and cathepsin D-positive stromal macrophages were found in 60 (64.5%) and 72 (77.4%) tumors, respectively. Univariate analysis showed that overall survival correlated significantly with axillary lymph node involvement and with estrogen and progesterone receptor status for each of the receptors separately and for their coexpression, and only marginally with c-erbB-2 overexpression. In mulitivariate analysis only axillary lymph node metastases and coexpression of estrogen and progesterone receptors were found to be independent and significant prognostic factors. CONCLUSIONS: When patients were stratified according to c-erbB-2 expression it was shown that those with c-erbB-2 overexpression and grade II tumors, tumor size greater than 2 cm, high content of aneuploid cells and cathepsin D-positive stromal macrophages had a shorter long-term survival than c-erbB-2 negative patients.

Prognostic significance of cell cycle parameters in infiltrative ductal breast carcinoma.

Flow-cytometric DNA analysis was performed retrospectively from paraffin-embedded blocks in 158 consecutive ductal infiltrative breast carcinoma patients grades I-III. Normal breast tissue was used as control. Tumor proliferative activity, cell ploidy, and DNA index (DI) were related to age of patients, histological grade of tumor, tumor size, axillary lymph node status, estrogen and progesterone receptor status, menopausal status,TNM clinical classification, and survival. There was a significant association between DNA aneuploidy and a high cellular proliferative activity, increased DI, poor differentiation of tumor, primary tumor size, number of positive lymph nodes, and postmenopausal state. Increased proportion of cells in S-phase was associated with positive lymph node status and higher number of positive lymph nodes. The cell cycle parameters had no prognostic value either for overall survival of disease-free survival of the patients.

Correlation of two methods for determination of cathepsin D in breast carcinoma (immunohistochemistry and ELISA in cytosol).

We have studied the correlation of two methods (immunohistology and ELISA in cytosol) of cathepsin D (CD) determination in breast carcinoma patients. Fifty six specimens of tumor tissue were collected consecutively, and CD expression in tumor tissue and tissue macrophages was determined by standard immunohistochemistry using the aNCL-CDm anti-cathepsin D mouse monoclonal antibody (Novocastra Laboratories Ltd., Newcastle, UK). Additionally, CD concentration was determined by ELISA in cytosol of the same breast carcinoma specimens. CD positivity was correlated with tumor size, histological grade of tumor, and the cytosol progesterone adn estrogen receptor concentrations. There was no statistically significant correlation between examined parameters and either CD positivity by immunohistochemistry or cytosol CD concentration. The correlation between CD expression in tumor cells of breast carcinoma by immunohistochemistry and cytosol CD positivity was not found either. However, there was a significant association between abundance of CD positive stromal macrophages and cytosol CD concentration in all histological tumor types (p < 0.05). CD positive macrophages were abundant in most of cytosol CD positive specimens. These results suggest that breast cancer cytosol CD concentration is the cumulative result of CD content in both carcinoma cells and stromal macrophages.

The effect of acute hypothermia and serum potassium concentration on potassium cardiotoxicity in anesthetized rats.

We examined the effects of hypothermia on serum K+ concentration and the interaction of body temperature and K+ load on cardiac toxicity in anesthetized rats. Serum K+ concentration significantly decreased to 2.61 +/- 0.13, 2.59 +/- 0.19 and 2.39 +/- 0.14 mmol/l at 31.0 degrees C, 28.0 degrees C and 25.0 degrees C, respectively, from the control value of 2.80 +/- 0.15 mmol/l at 37.0 degrees C. We used a 300% increase in baseline QRS duration as evidence of cardiac toxicity. Serum K+ concentrations of 4.95 +/- 0.12, 4.71 +/- 0.10, 4.45 +/- 0.14 and 4.07 +/- 0.14 mmol/l resulted in cardiac toxicity at 37.0 degrees C, 31.0 degrees C, 28.0 degrees C, and 25.0 degrees C, respectively. These data indicate that the level at which an elevation of serum K+ concentration causes cardiac toxicity diminishes with progressive hypothermia. We conclude that hypothermia induces hypokalemia, possibly through redistribution, and that the myocardium appears to be more sensitive to the toxic effects of K+ as hypothermia deepens.

Effects of acute hypothermia and beta-adrenergic receptor blockade on serum potassium concentration in rats.

BACKGROUND AND METHODS: We hypothesized that beta-adrenergic receptor blockade would result in an increase in serum potassium concentration in hypothermic rats given a potassium load compared to non-beta-blocked, hypothermic, potassium-loaded rats. To test this hypothesis, we investigated the interaction between body temperature and beta-adrenergic receptor blockade on serum potassium concentrations in ureter-ligated rats with and without potassium loading. To achieve this goal, we performed three experiments. In the first experiment, serum potassium concentrations were determined in 16 rats as they were continuously cooled from 37 degrees C to 22 degrees C. In the second experiment, 12 ureter-ligated rats were cooled to 31 degrees C, after which they were rewarmed to 37 degrees C. Serum potassium concentrations were determined before and after cooling and on rewarming. Twelve other ureter-ligated rats were cooled to 31 degrees C, then given a potassium load until their serum potassium concentrations returned to their baseline values, after which they were rewarmed to 37 degrees C. Serum potassium concentrations were determined before and after cooling, during the potassium infusion, and on rewarming. In the third experiment, 14 rats were pretreated with propranolol and 14 rats served as controls. Half of the rats in each of these two groups were kept at 37 degrees C and half were cooled to 25 degrees C. All rats were then given a 690-mumol potassium chloride infusion. Serum potassium concentrations were determined before and after the potassium infusion. RESULTS: The rats developed hypokalemia with cooling, which spontaneously resolved in the rats without supplementation on rewarming to 37 degrees C. The hypothermic hypokalemic rats that had their serum potassium concentrations corrected to normothermic status (2.93 +/- 0.17 mmol/L) had marked increases in serum potassium concentrations (4.22 +/- 0.15 mmol/L) on rewarming. In the normothermic rats, potassium loading after beta-adrenergic receptor blockade resulted in even higher serum potassium concentrations (5.65 +/- 0.36 mmol/L) compared with non-beta-blocked rats given equal potassium loads (4.6 +/- 0.4 mmol/L). However, in hypothermic (25 degrees C) rats given the same potassium load, there was no difference in serum potassium concentrations in beta-blocked (6.5 +/- 0.35 mmol/L) and non-beta-blocked rats (6.63 +/- 0.3 mmol/L). CONCLUSIONS: These results suggest that acute hypothermia causes a decrease in serum potassium, probably secondary to redistribution, which is reversible on rewarming. Supplementation of potassium during hypothermia can cause a significant increase in serum potassium concentration on rewarming. Blocking beta-adrenergic receptors with propranolol did not effect hypothermia-induced hypokalemia, suggesting that the beta-adrenergic mechanism may not be functional in hypothermia.

Oestrogen and progesterone receptors in primary breast cancer: a population study.

The results of a single centre study on cytoplasmic oestrogen (ER) and progesterone (PR) receptors and their combinations in primary breast cancer of 1957 patients from various parts of Croatia are presented. The frequency of ER+ tumours and tumour mean ER concentration were higher in patients over 50 years of age, while PR frequency and concentration were similar in patients over and under 50. The ER concentration was positively correlated with the age of patients, but the age-related increase in ER concentration appeared between 50 and 70 years of age. The pattern of receptor coexistence was age related. The frequency of ER+PR+ and ER+PR- increased and that of ER-PR+ and ER-PR- tumours decreased with the age of patients. The concentrations of ER and PR were higher in ER+PR+ than in ER+PR- or ER-PR+ tumours, respectively. When the patients were divided into groups under and over 50 years of age these differences appeared only in the latter group, while in the former the concentrations of ER were similar in ER+PR+ and in ER+PR- tumours, and the concentration of PR was higher in ER+PR+ than in ER-PR+ tumours. These data suggest a biological difference between breast cancers with various receptor combinations, as well as a difference in pathogenesis of the receptor negative and positive breast cancer.

Sex steroid receptors in intracranial tumors.

Estrogen (ER), progestin (PGR), and androgen (AR) receptors were assayed in cytosols prepared from 38 various intracranial tumors. The receptors were in the following proportions (number of receptor-positive/number of tumors examined): meningiomas were positive for PGR (4/6) and AR (2/5); glioblastomas were also positive for PGR (3/21) and AR (7/21); astrocytomas were positive only for PGR (4/5); and oligodendrogliomas only for AR. In two hamartomas AR was present, while in one chordoma both PGR and AR were present. In this latter tumor ER were not assayed due to insufficient material. The receptors were present in concentrations between 10 and 20 fmol/mg protein. Exceptions were two meningiomas and a chordoma with a high concentration of PGR and AR. Our results support the notion that a proportion of intracranial tumors contains sex steroid receptors, and some of these tumors might be hormonally dependent.

Androgen receptors in normal and neoplastic laryngeal tissue.

Androgen cytoplasmic (ARc) and nuclear (ARn) receptors were analyzed in normal and cancerous laryngeal tissue obtained from male and female patients. In men, neither ARc nor ARn were detectable in most of the normal larynx tissue specimens, while either ARc, ARn, or both were present in eight of 16 laryngeal carcinomas. In women, all three normal and two of four malignant laryngeal tissue specimens possessed ARc without ARn. The presence of androgen receptors in some laryngeal carcinomas shows that these tumors are possibly hormone sensitive, and hormonal therapy should be considered in treating these tumors.

Effects of hypoglycemia on rat brain polyribosome sedimentation pattern.

Insulin-induced hypoglycemia provokes polyribosome disaggregation and accumulation of monomeric ribosomes in the brain of rats with hypoglycemic paresis and coma. The extent of brain polyribosome disaggregation depends on the decrease of blood glucose concentration, and in comatose animals on the duration of hypoglycemia. Cycloheximide prevents the disaggregation of brain polyribosomes induced by hypoglycemia, indicating that hypoglycemia affects brain protein synthesis, decreasing the rate of initiation relative to the rate of elongation of polypeptide chain synthesis.

Lymphocyte reactivity to mitogens and tumor sex steroid receptors in breast cancer patients.

In 60 breast cancer patients in stages I and II the blastogenic transformation of peripheral blood lymphocytes after phytohaemagglutinin (PHA) pokeweed mitogen (PWM) and concanavalin A (Con A) stimulation were assayed and estrogen (ER) and progesterone (PgR) receptor concentrations in tumor cytosol were measured. A negative correlation between lymphocyte reactivity to the mitogens and tumor steroid receptors concentration was found. The lymphocyte response to the mitogens in the patients with ER-PgR-tumors (R-) was significantly higher than in those with tumors either ER+PgR-or ER-PgR+ (R+) or ER+PgR+ (R++). There was also a negative correlation between lymphocyte response to PHA and either ER or PgR concentrations in the tumors. These results suggest that the presence of steroid receptors in tumor cells may be associated with the depression of immunological reactivity in breast cancer patients.

Enhancement of bacitracin biosynthesis by branched-chain amino acids in a regulatory mutant of Bacillus licheniformis.

DL-4-Azaleucine-resistant mutant of Bacillus licheniformis azlr-1 isolated after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, was a better bacitracin producer than the parent strain. In the minimal medium, the antibiotic biosynthesis was 4 times higher in the mutant than in the parent strain and less dependent on L-leucine addition. In the complex fermentation medium, the yield was 18-20% higher in the mutant strain. Transaminase B activity measured in the crude extract revealed that the branched-chain amino acid biosynthetic enzymes were 5-10 times derepressed supplying bacitracin synthetase with enhanced quantity of isoleucine and leucine, the building units of bacitracin molecule.

High affinity binding of ochratoxin A to plasma constituents.

The binding properties of ochratoxin A to human and porcine plasma constituents were studied in vitro. Ochratoxin A was shown to bind to other plasma constituents with a considerably higher affinity than the earlier known nonspecific binding to plasma albumins. The association constant for the high affinity macromolecule (s) in human plasma was found to be 2.3 X 10(10) M-1 and for porcine plasma 0.59 X 10(10) M-1. The macromolecule (s) to which ochratoxin A binds more specifically than to albumins have not been identified, but their molecular weights were estimated by gel filtration to be 20 000.

Effects of cadmium on mouse liver polyribosome function.

The distribution of ribosomes between their various functional states (native subunits, unprogrammed ribosomes, monoribosomes and polyribosomes) was analyzed in livers of cadmium-treated (CdCl2, 20 umol/kg, l h) and control mice. The ribosomes with double-labelled RNA were separated by sucrose density-gradient centrifugation in zonal rotor, and monomeric ribosome fraction was subsequently isolated and analyzed by selective dissociation of unprogrammed ribosomes. The analysis shows that the increase in monomeric ribosomes, occurring during polyribosome disaggregation in livers of cadmium-treated mice, entirely due to the increase of the unprogrammed ribosome fraction at the expense of polyribosomes. Protein synthetic activity of polyribosomes in vivo in livers of cadmium-treated and control mice were compared by double labelling of nascent and soluble polypeptides with [14C]-and [3H]-leucine. Incorporation of radioactivity was relatively higher in nascent polypeptides and lower in soluble polypeptides in cadmium-treated as compared with control mice. The results indicate that cadmium inhibits protein synthesis in livers of mice, affecting both the rate of initiation and the rate of elongation, but decreasing the former more than the latter.

Effects of mercury bichloride on mouse kidney polyribosome structure and function.

Polyribosome sedimentation pattern and their in vitro protein synthetic ability were investigated in kidneys of mice treated with a single injection of HgCl2. Mercury bichloride, after 1 h, evokes polyribosome disaggregation, the extent of which is logarithmically correlated with the dose in the range of 2.5-20 micromoles/kg. With the dose of 2.5 micromoles/kg the effect occurs in 1 h, it is maximal between 1 h and 3 h. After 6 h polyribosomes are reaggregated. Cycloheximide pretreatment does not prevent the HgCl2 induced disaggregation of kidney polyribosomes. The cell-free system derived from kidneys of HgCl2 treated mice (10 micromoles/kg, 1 h) has a decreased protein synthetic ability. Both, in livers of mice treated with 20 micromoles/kg HgCl2 and in isolated rat's reticulocytes incubated with 20 micro M HgCl2 during 1 h there were no apparent changes in the polyribosome sedimentation patterns.

Inhibition of mitogen and specific antigen-induced human lymphocyte proliferation by cadmium.

Cadmium chloride (CdCl2) added to human lymphocyte culture inhibits the proliferative response induced by phytohaemagglutinin (PHA), pokeweed mitogen and allogenic lymphocytes in mixed lymphocyte reaction. Minimally effective concentrations of CdCl2 were 3.3, 1.6 and 1.6 microM, respectively. The inhibition was greatest when CdCl2 was added at initiation of cultures and declined if the addition of CdCl2 was postponed. The presence of CdCl2, regardless of the presence of PHA during the first 24 h of incubation suppressed the proliferative response to subsequent stimulation with PHA, indicating that cadmium affects an early step of blastogenic transformation.