Selective secretion of protein kinase C isozymes by thrombin-stimulated human platelets.

The protein kinase C (PKC) was secreted from thrombin-stimulated human platelets in a time- and dose-dependent manner. The PKC specific inhibitors Ro31-8220 (0.05 microM) and GF 109203X (0.5 microM) totally inhibited the secreted kinase activity. Western blot analysis of the secretory components showed reactivity to PKCalpha, PKCbetaII, and PKCdelta antibodies, but not to PKCbetaI, and p42/44 MAPK, although they were present in lysed platelets. The fractionation of platelets secreted components showed that PKC activity increased in both soluble and microparticle fractions after thrombin treatments. This is the first report demonstrating that activated human platelets selectively secrete protein kinase C isozymes. Protein kinase C secreted by platelets in this unique manner may have an extracellular role in the plasma, and may regulate cellular functions, including remodeling of vascular endothelial cells.

Ethanol inhibition of phagocytosis and superoxide anion production by microglia.

Ethanol consumption has been associated with aberrant immune responses resulting in increased susceptibility to infection including opportunistic infections of the central nervous system. We have investigated the effects of chronic ethanol treatment on phagocytosis and production of superoxide anion by microglia. Phagocytosis of radio-labeled opsonized E. coli was markedly suppressed by treating microglia with ethanol. The unstimulated synthesis of superoxide anion was not altered by ethanol treatment of microglia, but ethanol treatment effectively suppressed phorbol-12 myristate-13 acetate-stimulated microglia superoxide anion production. The results indicate that ethanol inhibition of microglia function may play a role in increased susceptibility for central nervous system infections, particularly in immunocompromised subjects.

Ethanol-induced apoptosis in human HL-60 cells.

The mechanisms responsible for aberrant immune function associated with chronic ethanol use remain obscure, but a decrease in monocyte numbers is often reported for individuals who chronically abuse ethanol. We investigated, using human HL-60 promyelocytic cell line, the possibility that ethanol induces apoptosis which contributes to decreased monocyte numbers. Characteristic features of apoptosis were observed 4 days after ethanol treatment, as documented by increased DNA fragmentation; enhanced expression of phosphatidylserine, an early marker of apoptosis; and the appearance of a hypodiploid apoptotic cell population identified by flow cytometry analysis of the cell cycle. Treatment with the protein kinase C inhibitor, GF 109203X, potentiated ethanol-induced apoptosis. Direct induction of human HL-60 cell apoptosis by ethanol and potentiation of ethanol-induced apoptosis by inhibiting protein kinase C provides a partial explanation for the cytotoxic effects of ethanol on hematopoietic progenitor cells and establishes a link between inhibiting protein kinase C activity and ethanol-induced apoptosis.

Negative and positive regulation of astrocyte DNA synthesis by ethanol.

Ethanol suppression of astrocyte mitogenesis is well recognized but ethanol, under some conditions, has also been shown to stimulate astrocyte proliferation. This study addressed the role of protein kinase C and other mitogenic factors as mechanisms responsible for the bidirectional effects of ethanol on astrocyte DNA synthesis. Ethanol treatment inhibited astrocyte DNA synthesis both at 4 hr (short term) and 24 hr (long term) in serum free medium. In contrast, when the medium contained serum, ethanol was less effective in inhibiting DNA synthesis at 4 hr and treatment with ethanol for 24 hr increased DNA synthesis. Protein kinase C activity was increased in cells treated with ethanol for either 4 or 24 hr. Ethanol inhibition of DNA synthesis in serum free medium was not reversed by down regulating protein kinase C. In contrast, downregulating protein kinase C activity by continuous treatment with phorbol myristic acetate partially reversed the effect ethanol had on DNA synthesis. Also, directly inhibiting protein kinase C with H-7 in cells maintained and treated in the presence of serum abolished the stimulatory effect ethanol had on DNA synthesis. It appears that the negative regulation of astrocyte DNA synthesis by ethanol occurs by protein kinase C and serum independent mechanisms whereas adaptive or stimulatory effects of ethanol on astrocyte DNA synthesis requires the interaction of protein kinase C with other factors present in serum.

Molecular variants of epidermal growth factor in malignant astrocytoma.

The present study has measured EGF levels in primary brain tumor tissues. EGF levels were measured by specific radioimmunoassay (RIA) and further analyzed by reversed-phase high performance liquid chromatography (RP-HPLC) followed by RIA and radioreceptor binding. The levels of EGF-like immunoreactivity (EGF-LI) in astrocytoma-IV tumors were fourfold greater than those in normal brain tissues. In astrocytoma-II and astrocytoma-III tumors, however, levels of EGF-LI were not different from those in normal brain. HPLC analysis of extracts from normal brain tissue and astrocytoma-II showed one peak of EGF-LI that coeluted with standard human EGF (retention time 22 min). Interestingly, EGF-LI in extracts of astrocytoma-IV tumors eluted in two distinct peaks with retention times of 24 and 26 min (Astro-A and Astro-B, respectively). Materials in both Astro-A and Astro-B peaks reduced the specific binding of [125I]hEGF to EGF receptors in human placental membranes. These studies demonstrate elevated levels of EGF-LI in malignant astrocytoma, but not in benign tumors. Furthermore, two different EGF-like molecules that are different from native EGF are present in malignant astrocytoma.

Hexamethylene bisacetamide-induced differentiation of Friend virus-transformed murine erythroleukemia cells is associated with parallel changes in casein kinase II and guanine nucleotide exchange factor activities.

In mammalian cells, the guanine nucleotide exchange factor (GEF, eIF-2B) plays a major role in the regulation of initiation of protein synthesis. It catalyzes the exchange of eukaryotic chain initiation factor (eIF)-2-bound GDP for GTP and facilitates the recycling of eIF-2 during polypeptide chain initiation. We used the Friend virus-transformed murine erythroleukemia (MEL) cell system to elucidate the translational regulatory processes that occur during growth and hexamethylene bisacetamide (HMBA)-induced cell differentiation. GEF activity is increased during growth and decreased during MEL cell differentiation, and this parallels the overall changes in protein synthesis during this period. Inhibition of GEF activity in induced cells may occur indirectly by phosphorylation of the alpha-subunit of eIF-2. However, the decrease in GEF activity in induced cells cannot be reversed by increasing the concentration of eIF-2-GDP added as a substrate in the GEF assay. This is diagnostic for the presence of eIF-2 alpha(P)-GDP in cell lysates and suggests that regulation of GEF activity may occur by one or more mechanisms other than eIF-2(alpha) phosphorylation. We have previously shown that the activity of GEF may be influenced directly by phosphorylation with casein kinase II (CK-II) of the 82-kD subunit of the factor. CK-II activity parallels the changes in GEF activity and the rate of protein synthesis during growth and differentiation of MEL cells. Addition of 1mM spermidine, a stimulator of CK-II but not of purified GEF, in induced MEL cell extracts enhances both CK-II and GEF activities approximately 48 and 32%, respectively. The results presented suggest that the inhibition of protein synthesis during MEL cell differentiation may be linked to the decreased CK-II and GEF activities.

Inhibition of rabbit reticulocyte guanine nucleotide exchange factor activity by heparin and its reversal by polyamines.

We have previously demonstrated that phosphorylation of the 82-kDa subunit of the guanine nucleotide exchange factor, eIF-2B, by casein kinases I and II stimulates eIF-2B activity. In the present report, we show that heparin binds to eIF-2B (KD approximately 0.3 microM) and inhibits the GDP/GTP exchange activity of eIF-2B. The effect is dose dependent, with half-maximal inhibition (IC50) achieved at 0.03 microM heparin. Although spermidine alone does not stimulate or inhibit guanine nucleotide exchange, it reverses the inhibitory effect of heparin up to 90 percent (IC50 = 0.22 mM). ATP and NADPH which interact with the 65 and 55-kDa subunits also partially relieve heparin inhibition of nucleotide exchange, and the effects of ATP and NADPH are additive. By using a gel overlay technique, we demonstrate that 125I-heparin binds to the 82, 65 and 55-kDa subunits of eIF-2B.

Effects of Celastrus paniculatus on passive avoidance performance and biogenic amine turnover in albino rats.

The effects of an indigenous drug, Celastrus oil, extracted from the seeds of Celastrus paniculatus on learning and memory in a two compartment passive avoidance task was studied in albino rats. The effects on the contents of norepinephrine (NE), dopamine (DA) and serotonin (5-HT) in the brain and on the levels of their metabolites both in the brain and urine were also assessed. Significant improvement was observed in the retention ability of the drug treated rats compared with the saline administered controls. The contents of NE, DA and 5-HT and their metabolites in the brain were significantly decreased in the drug treated group. The urinary metabolite levels were also significantly decreased except for total 3-methoxy-4-hydroxyphenyl glycol. These data indicate that Celastrus oil causes an overall decrease in the turnover of all the three central monoamines and implicate the involvement of these aminergic systems in the learning and memory process.

A study of antitrypsin and macroglobulin levels in serum and saliva of patients with gingivitis.

Periodontal diseases are associated with chronic inflammation. The destruction of connective tissue matrix is responsible for the pathogenesis of chronic inflammatory states. The degradation of matrix is initiated extra and pericellularly by proteinases produced locally at the inflammatory site. The regulation of these proteinases are by inhibitors present in serum and extravascular tissues, and it is the proteinase/proteinase inhibitor balance that determines the progression of chronic inflammatory state. Few contradicting studies are available on changes in the levels of proteinase inhibitors in serum in periodontal disease. The occurrence of these inhibitors in saliva has not been studied in detail. The present study was aimed at measuring the Proteinase inhibitors in serum and saliva of patients with periodontal disease.

Circulating immune complexes in intracranial neoplasms.

The levels of circulating immune complexes (CIC) were assayed in the sera of 109 patients with intracranial space occupying lesions. The CIC levels were significantly increased in all the brain tumours. After treatment, the CIC levels were still significantly increased when compared to the controls but showed no change when compared to their respective pre-operative values. Further, no change was observed in the CIC levels between the malignant and benign tumour case. Moreover, in brain tumours, 90% of the CIC precipitate consisted of IgG. However, the CIC levels fail to prognosticate the process of the disease in these patients.

Phosphorylation of rabbit reticulocyte guanine nucleotide exchange factor in vivo. Identification of putative casein kinase II phosphorylation sites.

The guanine nucleotide exchange factor (GEF) is a multi-subunit protein which catalyzes the exchange of GDP for GTP in eukaryotic chain initiation factor 2. Phosphorylation of the 82-kDa subunit of GEF in vitro by casein kinase II (CK-II) is associated with a 5-fold increase in nucleotide exchange activity. However, phosphorylation of GEF in vivo has not been studied, and the kinase(s) that phosphorylate GEF have not been identified. The 82-kDa subunit of GEF was partially sequenced, and a synthetic peptide was used to generate polyclonal anti-peptide antibodies that react specifically with this subunit. To examine the phosphorylation of GEF in intact cells, the protein was isolated and purified extensively from metabolically 32P-labeled rabbit reticulocytes. Only the 82-kDa subunit was found to be phosphorylated, and on Western blots the anti-peptide antisera reacted specifically with the labeled subunit. Phosphoamino acid analysis indicated that phosphorylation occurred exclusively on Ser residues. Digestion with cyanogen bromide of in vivo labeled protein and GEF phosphorylated in vitro by CK-II produced comparable phosphopeptide maps. However, additional phosphopeptide bands were also observed with GEF derived from intact cells. Sequence analysis obtained by Edman degradation of the phosphopeptides was compared with the deduced amino acid sequence of a cloned 82-kDa subunit of GEF [Bushman, J. L., Asuru, A. I., Matts, R. L., & Hinnenbusch, A. G. (1993) Mol. Cell. Biol. 13, 1920-1932]. Putative sites of phosphorylation were identified at Ser 703 and/or 704, which contain the sequence S(P)XXD, a CK-II consensus recognition motif.(ABSTRACT TRUNCATED AT 250 WORDS)

Translational control of eukaryotic gene expression. Role of the guanine nucleotide exchange factor and chain initiation factor-2.

In mammalian cells, the guanine nucleotide exchange factor (GEF or eIF-2B) is a key regulator of polypeptide chain initiation. The exchange of GDP bound to chain initiation factor 2 (eIF-2) for GTP by GEF is a rate limiting step in protein synthesis. The multisubunit characteristics of GEF suggest that this protein is composed of several distinct structural and functional domains, and is regulated by allosteric means and by phosphorylation. The activity of GEF may be regulated indirectly by the phosphorylation state of the smallest subunit of eIF-2 (alpha-subunit). On the other hand, phosphorylation of the largest subunit of GEF (82-kD subunit) by casein kinase (CK) I or II stimulates GDP/GTP exchange. GEF contains NADPH which is required for structural integrity of the protein. Upon stimulation of cells by insulin and growth factors, allosteric activation of GEF by sugar phosphates and other effector molecules may also play an important role in the regulation of polypeptide chain initiation. In this article, recent information about structure-function relationship of eIF-2 and GEF in nucleotide exchange and the regulatory mechanisms that influence the rate of polypeptide chain initiation under various physiological and pathological conditions are presented.

Urinary excretion of 6-hydroxymethylpterin in brain tumours.

The urinary 6-hydroxymethylpterin(Pt-6-CH2OH) excretion was determined in 87 patients with brain tumours and in 50 control patients. The Pt-6-CH2OH levels were significantly elevated in all patients with tumours. No difference was observed when malignant tumours were compared with benign neoplasms. Following therapy, the Pt-6-CH2OH levels were partially reduced when compared with control patients and their pre-operative values.

Urinary excretion of pseudouridine in patients with brain tumours.

Elevated urinary levels of modified nucleosides, especially pseudouridine (psi), have been observed in patients with a variety of malignant neoplasms. In this report, the urinary psi levels were estimated by high performance liquid chromatography in 93 patients with brain tumours and in 40 age and sex matched controls. The psi levels were found to be excreted in equal amounts in the control and the tumour groups. There was no significant difference in psi when patients with malignant diseases were compared with those with benign tumours. Following therapy, the psi levels remained unchanged when compared to the controls and their preoperative values. It is concluded that urinary psi concentration is not a useful marker for brain tumours.

Serum adenosine deaminase activity in brain tumours.

Adenosine deaminase (ADA) activity in serum was estimated in 86 patients with intracranial tumours and 40 healthy volunteers. Although high ADA concentrations in biological fluids and tumour tissues were observed in several neoplastic conditions, there was no significant difference in the ADA in sera of brain tumour patients when compared to the control values. Therefore, cell-mediated immunity probably does not play a significant role in brain tumours.

Effects of piracetam on retention and biogenic amine turnover in albino rats.

The chronic effects of orally administered 2-pyrrolidone acetamide (piracetam) on one-trial, passive avoidance task were studied in albino rats. The effects on the contents of norepinephrine (NE), dopamine (DA), and serotonin (5-HT) in the brain and on the levels of their metabolites both in the brain and urine were also assessed. Significant improvement was observed in the retention ability compared with saline-administered controls. The contents of NE, DA, and 5-HT and their metabolites in the brain were significantly decreased after piracetam administration. The urinary metabolite levels were also significantly decreased except total 3-methoxy-4-hydroxyphenyl glycol (MHPG). These data indicate that piracetam causes an overall decrease in the turnover of central monoamines. Thus, the results of this study implicate the involvement of NE, DA, and 5-HT systems in learning and memory processes. Piracetam did not exert any GABAergic effect as shown by the absence of change in the brain GABA levels.

Elevation of serum ceruloplasmin levels in brain tumours.

Serum ceruloplasmin levels have been estimated in 80 patients with various intracranial space occupying lesions and in 30 controls. The ceruloplasmin levels were significantly increased in all brain tumours except in meningiomas. After therapy, the ceruloplasmin levels were still significantly increased when compared to controls and their respective preoperative values. However, the rise in levels of ceruloplasmin in malignant tumours compared to benign was statistically not significant. It is concluded that ceruloplasmin may have a role to play as an acute phase reactant protein in brain tumours.

Evaluation of serum beta 2-microglobulin in oral cancer.

Beta 2-microglobulin (beta 2-m) low molecular mass protein was measured in serum of normal subjects and in patients with oral cancer. There was a significant increase in beta 2-m levels in oral cancer patients. The possible role of beta 2-m as a biochemical parameter and its significance in oral cancer is discussed.

Serum immunoglobulins in brain tumours.

Immunoglobulin level in the sera of 62 patients with intracranial space occupying lesions was assayed using the radial immunodiffusion method. Serum IgM levels showed a highly significant increase in all types of brain tumour when compared to controls. Serum IgG levels were also increased in benign as well as malignant cases. Serum IgA levels were high only in benign cases. Hence, increased serum Ig levels may be of prognostic value in these cases.