An immunohistochemical study of MAP2 and clathrin in gerbil hippocampus after cerebral ischemia

Changes in MAP2 and clathrin immunoreactivity were studied in gerbil hippocampus after transient cerebral ischemia. MAP2 immuno-reactivity decreased significantly by 1 h in the subiculum-CA1 and CA2 areas which correspond to reactive change, while no decrease was observed in CA1 until day 4. Before the initiation of delayed neuronal death, MAP2 immunoreactivity was not changed in CA1. On the other hand clathrin immunoreactivity increased in the pyramidal cell layer of CA1 by 3 h after ischemia and remained high for 2 days. Clathrin immunoreactivity in the pyramidal cell layer of CA1 diminished after delayed neuronal death. The transient change of clathrin was noted especially in CA1 in the period prior to delayed neuronal death. These results imply an abnormal change in clathrin turnover after ischemia, which may participate in the pathogenesis of delayed neuronal death.     

Altered genetic response to beta-adrenergic receptor activation in late passage C6 glioma cells.

Previous studies have demonstrated variability in the phenotype of rat C6 glioma cells. In the present study, we compared morphology, growth rate, and beta-adrenergic regulation of gene expression in early (P39-47) and late (P55-90) passage C6 cells. Morphological changes were observed in five independently derived, late passage populations. In four of the five, the untreated cells were more polygonal than the fibroblast-like parental cells, and only a small fraction exhibited process outgrowth after dbcAMP treatment. Untreated cells from the fifth late passage population had longer cytoplasmic processes than parental cells and responded to dbcAMP with further process outgrowth. All late passage populations had shorter generation times than the parental cells. In early passage cells, treatment with the beta-adrenergic agonist, isoproterenol (IPR), resulted in an increase in c-fos mRNA and a decrease in c-jun mRNA (Gu-bits RM, Yu H: J Neurosci Res, 30:625-630, 1991). Both of these immediate early gene responses were irreversibly lost between P50 and P55. Additional differences in basal or IPR-induced mRNA levels were observed for beta-APP, GFAP, NGF, and PPE, but not for a number of other mRNAs. These results are discussed in relationship to previously described differences in the ability of early and late passage C6 cells to accumulate cAMP (Mallorga P, et al.: Biochim Biophys Acta 678:221-229, 1981).     

Effect of prostaglandin E1 on graft function of kidneys from living related donors

Prostaglandin E₁ (PGE₁) was used in renal transplant recipients with living related donors. The drug was given intravenously from day 1 to day 7 after transplantation at a dose of 40 µg/kg twice a day. A total of 45 patients were studied divided into two groups: 25 patients were treated with PGE₁ (group B) and the remaining 20 patients did not receive the drug (group A). In group B, 24-h creatinine clearance (Ccr) was 66 ± 12.8 ml/min compared with 40.3 ± 13.4 ml/min in group A on the fifth postoperative day (P < 0.05). Urinary levels of N-acetyl-β-d -glucosaminidase (NAG) and serum levels of platelet factor 4 (PF4) in group B were significantly lower than in group A. On the fourth postoperative day, the urinary excretion of thromboxan B₂ (TxB₂) in group A was higher than in group B, but not significantly (5.1 ± 3.0 ng/day and 2.8 ± 1.1 ng/day, respectively). Acute rejection occurred in four patients in group B and in 10 patients (40%) in group A. The percentage of Leu2a-positive lymphocytes in group B was higher than in group A. We conclude that postoperative administration of PGE₁ improves graft function in kidneys from living related donors.     

Regulation of neurotransmitter enzyme by quisqualate subtype glutamate receptors in cultured cerebellar and hippocampal neurons

The possible involvement of ionotropic and metabotropic quisqualate (QA) receptors in neuronal plasticity was studied in cultured glutamtergic cerebellar or hippocampal cells in terms of the specific activity of phosphate-activated glutaminase, an enzyme important in the synthesis of the putative neurotransmitter pool of glutamate. When cerebellar of hippocampal neurons were treated with QA, it elevated the specific activity of glutaminase in a dose-dependent manner. The half-maximal effect was obtained at about 0.1 μM, the maximum increase was at about 1 μM, but levels higher than 10 μM QA produced progressive reduction in glutaminase activity. In contrast, QA had little effects on the activities of lactate dehydrogenase and aspartate aminotransferase and the amount of protein, indicating that the increase in glutaminase was relatively specific. The QA-mediated increase in glutaminase was mimicked by the ionotropic QA receptor agonist -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; EC₅₀, about 0.5 μM), but not by the metabotropic QA receptor agonist trans-(±)-1-aino-cyclopentyl-1,3,dicarboxyalte (t-ACPD; up to 0.5 mM). The specific ionotropic QA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) inhibited QA- and AMPA-mediated increases in glutaminase activity in a dose-dependent manner, whereas other glutamate receptor antagonists, -2-amino-5-phosphonovalerate, γ- -glutamyl aminomethyl sulphonic acid and γ- -glutamyl diethyl ester were ineffective. The elevation of neurotransmitter enzyme was Ca²⁺-dependent. The increase in Ca²⁺ influx essentially through the activation of L-type voltage-operated Ca²⁺ channels, and not the mobilization of internal Ca²⁺ stores, was responsible for these QA receptor-mediated long-term plastic changes in hippocampal and cerebellar neurons.     

Breast Cancer Genes

Abstract: The identification of familial breast cancer genes heralds an era of directed breast cancer treatment. Currently, two hereditary breast cancer genes have been identified, BRCA-1 and BRCA-2 . Although accounting for only approximately 5% of all breast cancers, they are being used to identify women with germ-line alterations that are at high risk of developing breast or ovarian cancer. With the identification of such genes comes a need for consideration of the ethical issues associated with testing. These genes are also being examined from a biochemical standpoint encompassing both their biological roles and biochemical pathways in which they reside. Such studies are likely to lead to novel breast cancer therapies.     

Expression of CD44V2 in transitional cell carcinoma of the urinary bladder and in urine

CD44 is the principal cell surface receptor for hyaluronate. Variant forms of the receptor, produced by alternative splicing, have been found to be associated with tumor progression in a variety of cancers. Based on investigations at the RNA level, it has recently been proposed that expression of CD44 variant V2 was present in urothelial cancer but not in normal urothelium. Since a distinctive marker for urothelial cancer would be extremely useful, frozen sections of normal urothelium and urothelial cancer were examined for expression of standard CD44 and CD44V2. Frozen sections of specimens of 35 patients with transitional cell carcinoma of the bladder, 16 specimens of normal bladder and 5 ureters were examined. Immunohistochemical staining was performed using a polyclonal antibody to CD44V2 (PAB CD44V2), a monoclonal antibody to CD44V2 (MAB CD44V2) and a monoclonal antibody to CD44S (MAB CD44S). CD44V2 and CD44S were also measured in lysates of urine sediments from 21 patients by enzyme-linked immunoabsorbent assay (ELISA). All investigated transitional cell carcinomas expressed CD44V2. There was no differentiation between invasive and noninvasive carcinoma. CD44V2 was also expressed in normal urothelium. Standard CD44 was expressed by the transitional cell carcinoma, normal urothelium, musculature and interstitial tissue. The amount of CD44V2 and CD44S in lysates of urine sediments is not correlated to diagnosis. In contrast to investigations at the RNA level, CD44V2 on the protein level seems not to be a distinctive marker for urothelial cancer. Therefore, CD44V2 will not be a useful diagnostic marker for detection of transitional cell carcinoma.     

PLASMA PHOSPHOLIPASE A2 ACTIVITY IN CLINICAL ACUTE MYOCARDIAL INFARCTION

1. Phospholipase A2 (PLA2) cleaves phospholipids to produce a lyso-phospholipid and free fatty acid and, in view of the biological activity of the products, PLA2 may play a role in many disease states. Lyso-phospholipids and free arachidonic acid increase in ischaemic myocardium, indicating that ischaemia activates the enzyme. 2. Plasma PLA2 activity was measured in patients with acute myocardial infarction, based on the release of labelled arachidonic acid from Escherichia coli cell membrane. Fourteen males (peak serum creatine phosphokinase (CK) above twice upper normal) were studied on day 1 (within 6 h of chest pain onset), days 2-4, and days 6-9. Normal age matched males (n = 13) were also studied. 3. Plasma PLA2 in patients with uncomplicated myocardial infarction (n = 12) was, initially, 1.14 +/- 0.10 (s.e.m.) nmol/min per mL plasma, similar to that in the normal group (1.52 +/- 0.14). On days 2-4, PLA2 activity increased to 1.94 +/- 0.18 (P less than 0.001) and this activity was correlated with the earlier peak CK level (P less than 0.02). On days 6-9, PLA2 activity was 1.49 +/- 0.13 while in two patients who developed complications and underwent open-heart surgery between the last two measurements, there were further increases to 4.22 and 4.04 nmol/min per mL. 4. The increase in plasma PLA2 in uncomplicated myocardial infarction is likely to be due to release from the damaged myocardium; whether it contributes to pathophysiology is uncertain.     

Synthesis and cytotoxic activity of new alkyl[3-(2-chloroethyl)ureido]benzene derivatives

Several alkyl[3-(2-chloroethyl)ureido] (CEU) benzene derivatives were prepared as potential anticancer agents. These new compounds were readily prepared in good yields by addition of anilines to 2-chloroethylisocyanate. Their cytotoxic activity was evaluated on human breast cancer (MDA-MB-231), human colon adenocarcinoma (LoVo) and mouse lymphocytic leukemia (P388D₁) tumor cell lines. Several new CEUs were significantly more cytotoxic than the nitrogen mustard chlorambucil. The biological activity of these aromatic urea derivatives seems to be related to the nature and position of the alkyl substituents on the aromatic ring. Substitution by branched alkyl groups on position 4 of the aromatic ring led to cytotoxic molecules which are up to 5 times more potent than the standard chlorambucil.     

Expression of pS2, c-erbB-2, and cathepsin D during the menstrual cycle in human breast cancers

Background: Many studies have addressed the effect of the timing of surgery for breast cancer relative to menstrual cycle phase, with conflicting results. Explanations for the possibility that survival could be altered by the appropriate timing of breast cancer surgery in humans remain speculative. Methods: We examined the expression of three estrogen related proteins (c-erbB-2, cathepsin D, pS2) in the breast tumors from 69 premenopausal women sampled in different phases of the menstrual cycle. Data on S-phase fraction and hormone receptor expression were also analyzed. Immunohistochemical assays were used to measure the proteins of interest. S-phase fraction was determined by flow cytometry. Analyses were performed based on fraction of cells staining positive for the protein, density of stain, and a histoscore that combined both fraction of positive cells and density. Results: We found no differences in c-erbB-2, cathepsin D, hormone receptor, or S-phase levels in tumors sampled in the follicular versus luteal phase, or perimenstrual versus periovulatory phase. The exception was pS2, which was expressed at greater levels during the luteal than during the follicular phase of the cycle (p<0.01); but there was no difference in pS2 expression when the patients were classified as periovulatory versus perimenstrual. Conclusions: Our findings do not support a variation in c-erbB-2, cathepsin D, S-phase fraction, or receptor expression as an explanation for the differences in breast cancer prognosis when surgery is timed by menstrual cycle phase. The finding that pS2 (an indicator of hormone sensitivity, and possibly better prognosis) is expressed at higher levels in tumor samples during the luteal phase suggests that the biologic profile of breast tumors may vary with the menstrual cycle and that these variations deserve further study.