Assembly of DNA with histones and nonhistone chromosomal proteins in vitro

We have examined, by protein binding assays, thermal denaturation, and circular dichroism, the possible effects of histones on nonhistone chromosomal protein (NHCP) interactions with DNA. For these studies, we have fractionated mouse Krebs II chromosomal proteins into three discrete fractions: Mo, 5 M urea-soluble NHCP; M1, 5 M urea-1 M NaCl-soluble NHCP from 5 M urea-extracted chromatin; and M3, 5 M urea-3 M NaCl-soluble chromosomal proteins from 5 M urea-1 M NaCl-extracted chromatin. These fractions contain heterogeneous populations of NHCP, and were found to differentially affect histone binding to DNA by methods of reconstitution, or by direct binding of M0, M1, or M3 to urea-salt reconstituted DNA with histones. M0 was found to exert a unique effect on the thermal denaturation and circular dichroic spectra of DNA-histone complexes. M0 from Krebs II chromatin was also found to complete for DNA sites in the presence of M0 from mouse liver chromatin. In addition, in 5 M urea, pH 8.0, histone binding to DNA reached saturation at 1.85 mg/mg of DNA, higher than the in vivo ratio of 1.00 mg/mg of DNA. Saturation of histone binding to DNA occurred only in the presence of 5 M urea, resulting in a reduction of nonspecific histone-histone interactions on DNA.     

Interactions between the subfractons of calf thymus H1 and nonhistone chromosomal proteins HMG1 and HMG2

The nonhistone chromosomal proteins, HMG1 and HMG2, interact with the various subfractions of calf thymus H1 with a high degree of specificity. Subfractions 1b and 2 interact very strongly with HMG1 to form heterodimers. In contrast, subfractions 3a and 3b interact much more weakly. HMG2 interacts with 3a and 3b but shows no detectable complexing with 1a and 2.     

A comparative study of guinea pig and rat tracheal reactivity

Species differences in behavior and responses of guinea pig (GPT) and rat (RT) tracheal smooth muscle to electrical field stimulation (EFS) and chemicals were investigated under semi-isometric conditions. Indomethacin augmented and papaverine reduced the contractions elicited by EFS in both tissues. They relaxed the GPT and did not affect the tone decline in the RT. Experiments with different load, cartilage content, thread and rubber strips suggest the role of elastic elements in passive elongation of the RT. This was independent of muscarinic, adrenergic, histaminergic and serotoninergic receptors, nitric oxide, eicosanoids and metabolic processes. The second response on repeated administration of acetylcholine was augmented and that of serotonin attenuated in both GPT and RT. Upon repeated elevation of [K+]o the tissues responded in an opposite manner. Further data are provided on similarities (modulation of cholinergic transmission by prostaglandins, sensitization to acetylcholine and desensitization to serotonin) and differences (innervation, responses to repeated elevation of [K+]o, presence/absence of prostaglandin-regulated basal tone and spontaneous activity) in reactivity of GPT and RT.     

A comparative study of the chymotrypsin-like activity of the rat liver multicatalytic proteinase and the ClpP from Escherichia coli

A comparative study of the chymotrypsin-like activity of the purified recombinant ClpP protease and the multicatalytic proteinase from rat liver is presented. The peptidase activity of both enzymes has been analyzed with several synthetic fluorogenic peptides, containing either aromatic or nonpolar amino acids in their P1 position. The respective Vmax, Km, and Vmax/Km were calculated from kinetic experiments. The substrate specificity of the multicatalytic proteinase, as expressed by Vmax/Km values, indicate the following substrate preference: N-Suc-IIW-MCA > N-Suc-LY-MCA > N-Suc-LLVY-MCA > or = N-Suc-AAF-MCA > N-Cbz-GGL-beta-NA > Glut-GGF-beta-NA > FPAM-4-MNA. In the case of the ClpP the order of preference is: N-Suc-LY-MCA > N-Suc-IIW-MCA > N-Suc-LLVY-MCA > or = N-Suc-AAF-MCA > or = N-Cbz-GGL-beta-NA > FPAM-4-MNA (where: N-Suc, N-succinyl-; MCA, 7-amido-4-methyl coumarin; beta-NA, beta-naphthylamide; N-Cbz, N-benzyloxycarbonyl-; 4-MNA, 4-methoxy-beta-naphthylamide; Glut, glutaryl. This similar substrate specificity is further supported by the lack of activity of both enzymes against SY-MCA and N-Suc-AAPF-MCA (known substrates of chymotrypsin), by very reduced activity against N-Suc-AAA-MCA and by no significant activity against LG-beta-NA. The results of mixed substrate experiments have shown that all the peptides that are substrates seem to be hydrolyzed by a single class of chymotrypsin-like site in both enzymes. The substrate specificity studies suggest a possible evolutionary relationship between the catalytic component of the ClpP of Escherichia coli and the multicatalytic proteinase chymotrypsin-like catalytic component. This conclusion is further supported by other circumstantial evidence: the fact that affinity-purified anti-ClpP antibodies cross-react with two polypeptide components of the rat liver multicatalytic proteinase complex, presented here and also shown previously; the known resemblance of both structures at the electron microscope level; and their reported role in the degradation of NH2-end rule substrates.     

Oxidative metabolism in a rat hepatoma (N13) and isolated rat hepatocytes: a flow cytometric comparative study

Recently, we have developed a new and fast kinetic method for assessing mitochondrial membrane potential by flow cytometry, based on the quantitation of the initial rate of rhodamine 123 (Rh123) uptake by living cells. This test has proved suitable to detect metabolic and toxic effects on mitochondria. To characterize energy metabolism in a rat hepatoma cell line (N13), we applied this method to assess several metabolic pathways that eventually generate mitochondrial membrane potential. Using this approach, we found that N13 hepatoma cells retain an oxidative capacity comparable with that observed in isolated hepatocytes under the same conditions. These results show that this cell line may represent an adequate biological model to perform metabolic and toxicological studies in vitro.     

A comparative study of spirochaetes from the porcine alimentary tract

Strains of Treponema hyodysenteriae capable of inducing swine dysentery in specific pathogen-free pigs were compared with other spirochaetes from the porcine alimentary tract by biochemical and serological tests and by electrophoresis of their proteins. Carbohydrate fermentation and esculin hydrolysis were similar in all the spirochaetes. Indole was produced by T. hyodysenteriae and by some of the other spirochaetes. Analysis of the fatty acids produced from glucose showed a difference between T. hyodysenteriae and other spirochaetes only in the amount of n-butyric acid produced. The indirect fluorescent antibody test showed extensive cross-reactions between all the spirochaetes unless antisera were first absorbed. A microtitre agglutination test and a growth-inhibition test were both more specific; strains of T. hyodysenteriae could be distinguished from the other spirochaetes using unabsorbed sera. Both tests revealed some antigenic heterogeneity among strains of T, hyodysenteriae. The cell proteins of a single strain of T. hyodysenteriae gave an electrophoretic pattern distinct from those of the other spirochaetes. Two of the six spirochaetes not associated with swine dysentery, PWS/B and PWS/C, were indistinguishable serologically and electrophoretically. The other four strains were serologically distinct from one another and from PWS/B and PWS/C. Only two of these spirochaetes were examined electrophoretically, but each gave a different pattern from PWS/B and PWS/C. The diversity observed among spirochaetes not associated with swine dysentery indicates that their suggested inclusion in a single species, T. innocens, may prove to be unjustified.     

A comparative study of metastatic patterns of ductal and lobular carcinoma of the breast from two matched series (376 patients)

Five studies comparing metastatic patterns of lobular and ductal carcinoma have reported conflicting results. These results are based on the evaluation of heterogeneous groups, without taking clinical differences into account. The aim of this clinical study is to compare metastatic patterns of matched ductal and lobular carcinomas. One hundred eighty-eight lobular carcinomas were matched with 188 ductal carcinomas who showed identical ages, menopausal status, TNM, and dates of treatment. Hepatic, lung and brain metastases are more frequent in ductal carcinoma. Metastases to the gastrointestinal system, gynecologic organs, and peritoneum are more characteristic of lobular carcinoma. Life table analysis of overall and disease free survival shows no significant differences. The metastatic patterns of ductal and lobular carcinomas are different, and physicians should be aware that the classical staging of the breast cancer is not fit to lobular carcinoma. fit to lobular carcinoma. Could we define patients presenting high metastatic risks? Could we use CA. 125, sonography, laparoscopy in the staging? Should we change the whole therapeutic approach of lobular carcinoma?     

Two highly specialized histone H1 proteins are the major chromosomal proteins of the sperm of the sea anemone Urticina (Tealia) crassicornis

The sperm nuclear basic proteins (SNBPs) from a sea anemone (Urticina crassicornis) have been isolated and characterized for the first time. They consist of two sperm-specific members of the histone H1 family with Mr 22,700 and 24,600. They amount to about 60--70% of the total chromosomal sperm proteins. Their amino acid composition and the primary structure of their trypsin-resistant core indicate a strong relation to histone H5 from the nucleated erythrocytes of birds and amphibians as well as to other high sperm-specific H1-like (PL-I) proteins from phylogenetically distant groups. The major presence of histone H1-like protein in the sperm of an organism belonging to such a low phylogenetic group provides experimental support to the hypothesis that SNBPs may all have evolved from a primitive histone precursor.     

Exocytotic proteins in enterochromaffin-like (ECL) cells of the rat stomach

Proteins participating in vesicular docking and fusion have been identified in the nervous system. Such proteins appear to be important for the molecular regulation of exocytosis also in non-neuronal cells. The enterochromaffin-like (ECL) cells of the gastric acid-secreting (oxyntic) mucosa secrete histamine and chromogranin A-derived peptides, such as pancreastatin. Using immunohistochemistry, we have examined whether the ECL cells of the rat stomach, identified with antibodies to histidine decarboxylase (HDC, the histamine-forming enzyme), express the same exocytotic proteins as neurons. The ECL cells displayed immunoreactivity for synaptophysin, synaptotagmin III, vesicle-associated membrane protein-2 (VAMP-2), cysteine string protein (CSP), vesicular monoamine transporter-2 (VMAT-2), synaptosomal-associated protein of 25 kDa (SNAP-25), syntaxin, and Munc-18, but not for synaptotagmin I/II and VAMP-1. Synaptophysin and VMAT-2 could be detected not only in the ECL cells, but also in a population of HDC-negative cells. The demonstration of synaptotagmin III in only a limited number of ECL cells suggests the existence of a subpopulation of ECL cells. The results show that several exocytotic proteins, previously identified in neurons, are present in rat stomach ECL cells. Hence, proteins engaged in vesicular docking and in the fusion of granule/vesicle membrane with plasma membrane seem to exist in both neurons and endocrine cells.     

A regional study of developing rat brain: the accumulation and distribution of proteolipid proteins

The accumulation and distribution of proteolipid proteins in rat brain and selected brain regions (cerebellum, cerebral cortex, basal ganglia, and hippocampus) were studied during early postnatal development. In whole brain an eightfold increase of proteolipid was observed between ten and 33 days after birth. This was reflected in the separate regions examined where the proteolipid protein content increased six- to ten-fold during the same period. The basal ganglia and cerebral cortex contributed the greatest amount to the total proteolipid present. However, at 28-33 days the greatest concentration (mg/g tissue) was observed in the basal ganglia and hippocampus. When the proteolipid protein preparations were examined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, distinctive, heterogeneous patterns for each brain region were obtained. Proteolipid from basal ganglia (the region richest in white matter) consisted primarily of two major protein bands with apparent molecular weights of approximately 21,500 and 26,000. Both of these bands dramatically increased in quantity during myelination, and the larger protein coelectrophoresed with isolated myelin proteolipid protein. Both bands were also found present in proteolipid preparations from the other brain regions but in varying amounts relative to the total. The data suggest that the increase in proteolipid observed during this developmental period was due in large measure to the accumulation of myelin-specific proteolipids, but also that a significant proportion of the increase was due to the accumulation of nonmyelin components.     

A comparative study of ketorolac (Toradol) and magnesium sulfate for arrest of preterm labor

BACKGROUND: We evaluated the efficacy and safety of ketorolac (Toradol). METHODS: In this prospective trial, 88 women in confirmed preterm labor at < or =32 weeks' gestation were randomized to receive magnesium sulfate given as an initial 6 g intravenous bolus followed by continuous infusion therapy (2 to 6 g/hr) or intramuscularly administered ketorolac (60 mg loading dose) followed by 30 mg every 6 hours for a maximum of 24 hours. RESULTS: The study groups were similar with respect to age, parity, cervical status, and gestational age on admission. Ketorolac was more rapid (2.71 hr+/-2.16) in the arrest of preterm labor than was magnesium sulfate (6.22 hr+/-5.65). No patient required discontinuance of either drug due to adverse effects. There was no difference in the incidence of neonatal complications between the two groups. CONCLUSION: In gestations with preterm labor at <32 weeks, ketorolac appears to be an appropriate first-line tocolytic agent.     

Tramadol (Tramal) in the treatment of rheumatic diseases-- comparative study

In order to evaluate the efficacy of centrally acting analgesics. In treating rheumatic diseases, tramadol hydrochloride (Tramal Grunental) has been administered to a group of 68 patients (36 women and 32 men), who received 100 mg twice a day during a 10-day treatment. The testing comprised 14 female patients with rheumatoid arthritis, 20 patients (7 women and 13 men) with degenerative (OA) hip and knee diseases and 34 patients (15 women and 19 men) affected by the vertebrogen painful syndrome of lumbar spine. The control group comprised 12 patients (9 women and 3 men) with rheumatoid arthritis using non-steroidal antiinflammatory drugs only, 22 patients (12 women and 10 men) with the OA of the hip and knee, using paracetamol only, and 30 patients (15 women and 15 men) affected by the vertebrogen painful syndrome of lumbar spine, also using paracetamol only. The visual analogue scale has been used in following the pain relief assessments during the therapy. It has thus been observed that the intensity of pain has not been significantly relieved with the acute rheumatic diseases (p > 0.05) in the control group either; that the significant pain relief has occurred with the degenerative (OA) rheumatic diseases (p < 0.05) but not in the control group; while the best analgetic effect of tramadol has been proved on the patients affected by the vertebrogen painful syndrome of lumbar spine (p < 0.01) but was not significant in the control group. During the therapeutic treatment 13 patients (19%), mostly the elderly, experienced side effects, manifested as nausea and the dry mouth.     

A comparative study on extraction of Fe(III) from chloride leach liquor using TBP, Cyanex 921 and Cyanex 923

A comparative study on extraction of Fe(III) from the HCl leach liquor of low grade iron ore tailings has been carried out using Tri-n-butyl phosphate (TBP), Cyanex 921 and Cyanex 923 in distilled kerosene. The percentage extraction of iron increased with increasing HCl and extractant concentrations. The extracted species in each case was found to be HFeCl₄·S. The extraction isotherms for the above extractants indicated quantitative extraction of Fe(III) in 3-stages at O:A ratio of 3:2 with TBP, and in 2-stages at O:A ratio of 1:1 with Cyanex 921 and Cyanex 923. The stripping studies of the loaded organic phases were carried out with 0.4 M HCl. The stripping isotherms indicated 2-stages at O:A ratio of 5:2 for TBP, and 3-stages at O:A ratio of 2:3 for Cyanex 921 and Cyanex 923. From the extraction studies, the extraction efficiency of the extractants for Fe(III) was in the order TBP < Cyanex 921 < Cyanex 923. Although Cyanex 923 was found to be the best extractant, the percentage stripping of Fe from the loaded Cyanex 923 was the least. The stripping of Cyanex 923 was 94.9%, but with TBP and Cyanex 921, it was 99.8% and > 99.9%, respectively.