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The effects were investigated of purified subsynaptic fractions on the efflux of radioactivity from a plain synaptic vesicle fraction which had incorporated [3H]dopamine. About 50% of the radioactivity incorporated into the plain vesicles (120 microgram protein) was liberated on exposure to purified synaptic membranes (30 microgram protein). The synaptic membrane-dependent efflux appeared to depend on both adenosine triphosphate and divalent cations, especially Ca2+. Of the sub-cellular fractions used, the heavy microsomal fraction showed the same effects as the synaptic membrane fraction. Purified synaptic junctions exhibited the strongest stimulating effects: the efflux was 2 times greater than that observed with synaptic membranes. The stimulating effects of myelin were less than one-seventh of those of synaptic junctional fraction. These observations may indicate that the transmitters are liberated by interaction of vesicle membrane with synaptic membrane in the presence of ATP and divalent cations.
Total body irradiation (TBI) is used as preparative regimen prior to bone marrow transplantation (BMT). Since there are more long-term survivors, follow up studies are important. We have performed a retrospective analysis of growth for 49 children, who had undergone treatment with cyclophosphamide and TBI before BMT. Of these patients 26 received single fraction (SF) TBI as a dose of 900-1000 cGy, whereas 23 received fractionated (FF) TBI as a total dose of either 1200 cGy divided in six fractions or 1440 cGy divided in eight fractions over 3 days. Half of the patients in the SF-TBI group, and 9 in the FF-TBI group had received low-dose cranial irradiation prior to TBI. In all groups a decrease in height SDS was observed. By evaluating the major factors leading to growth impairment the influence of cranial irradiation, which was demonstrable in the 1st year after TBI, could not be shown after 3 years. At this time growth was significantly more impaired in the SF group with a mean height SDS of -0.9 (+/- SD 0.9) compared to a mean height SDS -0.22 (1.02) in the FF group (P < 0.05). Measurement of segmental proportions showed a significant difference in SDS for sitting height in comparison to SDS for subischial leg length, irrespective of the TBI regimen. This was already evident 1 year after TBI and decreased during the following years. Twenty four of the patients (17 in the single fraction and 7 in the fractionated TBI group) were treated with growth hormone, but demonstrated an inappropriate response with absent catch-up growth in their legs.(ABSTRACT TRUNCATED AT 250 WORDS)
A series of experimental studies has been carried out to assess the relationship between the effectiveness of tumor control, normal tissue reaction and median survival following different radiation fractionation schedules on the solid tumor model 3924A in the ACI rat. The total radiation dose of 7500 rad and the dose per fraction of 250 rad were held constant. Tumor control and life expectancy improved with 250 rad given continuously as multiple fractions per day (MFD) (1, 2 and 3 fractions per day over 30-, 15- and 10-day periods, respectively). However, the maximum acute skin reaction surrounding the tumor was greater for MFD given continuously than for single daily fractions. We have also completed studies of the therapeutic effectiveness of MFD given intermittently at 11-day intervals. Tumor control following MFD given intermittently was comparable to that following the daily fraction schedule given continuously, and the normal tissue reaction was acceptable at a total radiation dose of 7500 rad. Experimental and clinical results in head and neck cancer indicate that large total radiation doses in the order of 7000 rad given as continuous MFD are associated with unacceptable normal tissue reaction. However, these clinical and experimental results also indicate that large total radiation doses can be given as MFD intermittently and effectively used in cancer management. The marked superiority of alternating chemotherapy and radiotherapy in obtaining a tumor cure rate of greater than or equal to 50% in our experimental system and the superiority of alternating chemotherapy and radiotherapy in extensive Hodgkin's disease (complete response rate--87%) over chemotherapy alone, radiotherapy alone, or the two modalities given together or as split course provides the clinical and experimental basis for the development of more effective protocols in the treatment of tumors responsive to chemotherapy and radiotherapy. A complete response rate of 78% in patients with head and neck cancer, and a complete response rate of 89% in patients with limited small cell carcinoma of the lung using alternating chemotherapy and radiotherapy indicate that it is now possible to proceed rapidly in clinical protocol design for these two additional tumors responsive to both chemotherapy and radiotherapy.
The results are presented of a five-year prospective trial of 411 patients with breast carcinoma treated by mastectomy and post-operative radiotherapy. Twice or thrice weekly dose-fractionation techniques were used, i.e. 12 fractions in 28 days and six fractions in 18 days. The results show that, in terms of survival and control of local disease, the two techniques are very similar. The early and late radiation effects on the normal tissues up to five years are similar and acceptable. Dose levels thought to be biologically equivalent on the basis of experience at St Thomas's Hosiptal were selected. It is stressed that there is a narrower margin of normal tissue tolerance when six fractions over 18 days are used, that it is essential to treat all fields on each treatment occasion and that the total maximum tissue dose of 3600 R is not exceeded. Attention is drawn to the fact that the dose for six fractions, derived from the NSD formula which would be predicted to be equivalent to the 12-fraction dose is, in fact, over 10% higher than the dose used here and likely to produce unacceptable late radiation changes.
In the present study fractionation of the Cerastes cerastes cerastes snake venom by gel filtration on Sephadex G-75 gave 14 protein fractions. Phospholipase PLA2 activity is not uniformly correlated with the lethality to mice in regard to all venom fractions. F11 which is the richest in PLA2 activity is less toxic than F3, which contains a small amount of PLA2, and F12 is the lowest in lethality and PLA2 activity. Treatment of Ehrlich ascites-bearing mice with two i.p. injections of the most lethal fraction (F3) or a non-lethal fraction (F4) resulted in a significant antitumor activity demonstrated by an increase in the mean survival time of the animals (22.5 and 27.9 days) and in the tumor inhibition ratio of tumor growth (T/C% 139 and 172, respectively), compared to tumor-bearing controls. The cytotoxic activity of F3 and F4 against Ehrlich ascites carcinoma cells might be due to the presence of a cytotoxin rather than to the direct cytolytic effect of the PLA2 because the non-lethal F4 is free from PLA2. Treatment of Swiss albino mice with two i.p. injections of F3 or F4 at the adopted dose levels produced no detrimental side effects demonstrated by the insignificant changes in the tested serum and liver parameters. Treatment of the tumor-bearing mice with the same venom fractions significantly modulated all of the studied biochemical parameters in the serum and liver tissues, compared to normal controls.
Bovine kappa-casein was fractionated at pH 8.0 on DEAE-Sepharose with an NaCl gradient, followed by DEAE-cellulose chromatography using a decreasing pH gradient from pH 6.0 to 4.5. At least ten components could be identified, each differing in N-acetylneuraminic acid (NeuAc) and/or phosphorus content. Two components appeared to be multiply-phosphorylated, but did not contain NeuAc. The possible significance of this finding in relation to the mode of phosphorylation and glycosylation in vivo is discussed. A carbohydrate-free fraction as well as two NeuAc-containing fractions were compared in their substrate behaviour towards the action of the milk-clotting enzyme chymosin at pH 6.6 and 30 degrees C. To this end the trichloroacetic acid-soluble reaction products were analysed by high-performance gel-permeation chromatography. In order of increasing carbohydrate content the kcat. values found ranged from 40 to 25 s-1 and the Km values from 9 to 3 microM; the overall substrate properties of these components as reflected by the kinetic parameter kcat./Km ranged from 5 to 8 microM-1 X S-1. Irreversible polymerization of the carbohydrate-free fraction brought about a more-than-2-fold increase in Km, the kcat. value remaining virtually constant. The kcat./Km found for the cleavage of whole kappa-casein at pH 6.6 was of the same magnitude as the kcat./Km found for the polymerized carbohydrate-free fraction (i.e. about 3 microM-1 X S-1). No indication of substrate inhibition was found for the carbohydrate-free fraction.
Left ventricular volume and ejection fraction were measured in 22 survivors of acute myocardial infarction by means of two-dimensional echocardiography and using a Simpson's rule algorithm. Ten of the 22 patients experienced complications. For the group as a whole, there were no significant trends in left ventricular volume and ejection fraction between the first and third days and the third month after infarction. In the subgroups with uncomplicated and complicated infarction, there were trends towards increasing and decreasing ejection fractions, respectively, which failed to attain statistical significance, however. The difference in ejection fraction between both subgroups had become significant at 3 months; 55.2 +/- 11.1% in uncomplicated v. 41.3 +/- 6.9% in complicated cases (P less than 0.01). Individual changes in ejection fraction falling outside the limits of reproducibility of the method as assessed previously were observed between day 1 and day 3 in only 2 patients with uncomplicated and in 2 patients with complicated infarction. Between day 1 and 3 months such changes occurred in 8 patients with uncomplicated infarction (upward in 5 and downward in 3), and in 8 patients with complicated infarcts (upward in 3 and downward in 5). We conclude that changes in ejection fraction as measured by two-dimensional echocardiography tend to correlate with complications.
The glycoproteins of porcine zonae pellucidae have been fractionated into three families (PZP1-3) by gel filtration HPLC [Nakano et al. (1987) Biochem. Int. 14, 417-423]. However, they still comprise heterogeneous molecular species differing in electric charge. We found that sulfate, but not phosphate, is contained in PZP1-3 by a simple and rapid method for microanalysis of the anionic groups. These families were efficiently separated into many fractions by anion-exchange HPLC. When elution was performed by stepwise increase in NaCl concentration in 8 M urea/20 mM Tris-HCl, pH 8.0, a single distinctive peak emerged for each step. The analyses of amino acids, monosaccharides, and anions of the eight separated fractions of the major family, PZP3, showed that larger amounts of sulfated lactosamine linked to the constituent proteins are present in the fractions that are eluted later: the chain length and/or the chain number of these polylactosamines and the sulfate content increased with stepwise increase in NaCl concentration. Composition analyses also revealed that twice as much N-glycolylneuraminic acid is present as N-acetylneuraminic acid in all fractions. The contents of these sialic acids in the fractions slightly increased in the order of elution. These results together with those of the analyses of endo-beta-galactosidase digests showed that the charge heterogeneity of the porcine zona proteins is due mainly to differences in the amount of sulfated lactosamine, which is predominantly distributed in the non-reducing regions of the sugar chains.
We compared the pharmacological actions of the high and low molecular mass fractions of Sho-saiko-to using a murine immunologically induced liver injury model to estimate the roles of these fractions in the expression of the pharmacological action. In a Bacillus Calmette-Guerin (BCG)/lipopolysaccharide (LPS)-induced liver injury model, Sho-saiko-to and both of its fractions significantly reduced the increases in the aminotranseferase levels in serum. They also reduced the increase in the nitric oxide (NOx) level in serum. On the other hand, Sho-saiko-to and its high molecular mass fraction suppressed the increase in plasma NOx level in an LPS-induced endotoxin shock model but its low molecular mass fraction did not. These results suggest the possibility that both fractions act hepatoprotectively in a different manner. We believe that these results can help to elucidate the mechanism of action of ingredients in Sho-saiko-to.
The objective of this study was to compare the number of recovered spermatozoa, in different parts of the uterine horn and oviduct in gilts, after insemination with fractionated (experiment) and non-fractionated (control) liquid stored semen. The number of spermatozoa and volume of backflow was also investigated. Twenty three cross-bred gilts were used in the study. They were divided into 2 groups, a control group (non-fractionated liquid stored semen, n=10) which were inseminated with 100 ml of liquid stored semen containing 3,000 million spermatozoa per dose and an experimental group (fractionated liquid stored semen, n=10) which were inseminated with 50 ml of liquid stored semen, with 3,000 million spermatozoa per dose and followed by another 50 ml of semen dilutor (Beltsville Thawing Solution, BTS). Thereafter, backflow semen was collected and measured every 15 min for a period of 1 hr. Three or 12 hr after insemination, 5 gilts from each group had the uterus, the horn of the uterus, the oviducts and the ovaries removed under general anaesthesia. The horn of uterus and the oviducts were seperated by ligation into 6 segments. All 6 segments were flushed with BTS to collect all spermatozoa within the segment. Recovered spermatozoa were counted, using a haemocytometer and the volume recorded. It was seen that the percentage of spermatozoa in the backflow semen in the experimental group was less than in the control group. The difference was not significant in the gilts that were operated on 3 hr after insemination, the mean number of spermatozoa in the uterine horn and the utero-tubular junction (UTJ) was more in the experimental than in the control group, but less in the isthmus and the ampulla of the oviduct. The gilts which were operated on 12 hr after insemination, had relativity more ovulating gilts in the control group than in the experimental group (3 of 4 gilts compare to 3 of 5 gilts). The control group had more spermatozoa in the oviduct than the experimental group, but less in UTJ and in the horn of the uterus. Again the difference was not significant. It can be concluded that fractionated (experimental) or non-fractionated (control) insemination of semen with the same number of spermatozoa provides no significant difference in the number of spermatozoa either in the horn of the uterus, the UTJ or the oviduct of gilts.
A technique for the separation of transcriptionally active and inactive nucleosomes by mercury affinity chromatography has been applied to study the nucleosomal distribution of DNA sequences from the GAL1, ACT1, HIS4, MAT alpha, and HMRa genes of yeast. In mammalian cells, the method has been shown to separate active from inactive nucleosomes and to fractionate the active nucleosomes into two classes, one retained on the mercury column because of salt-labile associations with certain thiol-reactive non-histone proteins, and the other bound by covalent linkage of the cysteine 110 thiol groups of histone H3 molecules to the mercurated support. The first class of nucleosomes is elutable in 0.5 M NaCl; the second is displaced by 10 mM dithiothreitol (DTT) (Walker, J., Chen, T. A., Sterner, R., Berger, M., Winston, F., and Allfrey, V.G. (1990) J. Biol. Chem. 265, 5736-5746). We show that, in wild-type yeast cells, in which histone H3 lacks cysteinyl residues, very little DNA and a negligible complement of nucleosomes appear in the DTT-eluate, confirming the requirement for the H3-thiols in the mercury-binding reaction. Moreover, the DTT-eluted fraction is seriously deficient in the actively transcribed GAL1, ACT1, HIS4, and MAT alpha DNA sequences. Site-directed mutagenesis was employed to create an H3 gene containing a cysteine codon in place of the alanine codon at position 110 of the yeast H3 amino acid sequence. A strain was constructed containing the mutant histone H3 gene instead of the normal H3 gene. Subsequent fractionations of the mutant nucleosomes by mercury-affinity chromatography revealed a characteristic nucleosome peak in the DTT-eluted fraction. Its content of transcribed GAL1, ACT1, and HIS4 DNA sequences was 20- to 500-fold higher than that of the corresponding DTT-eluted fraction of wild-type yeast. Although this result is in accord with the finding that, in mammalian cells, the thiol groups of histone H3 become accessible when nucleosomes "unfold" during transcription, we find that nucleosomes containing the GAL1 DNA sequences of the yeast H3-mutant also bind to the mercury column when that gene is not being expressed. We conclude that many yeast nucleosomes are maintained in a "primed," potentially active state, possibly due to the very high constitutive levels of acetylation of the core histones. However, the nucleosomes of the HMRa gene, which is not expressed in a MAT alpha yeast strain, are virtually absent from the DTT-eluted nucleosome fractions of the H3-mutant cells, indicating that prolonged silencing of the gene is accompanied by compaction and loss of H3-thiol reactivity of its nucleosomes.
Cardiotoxicity is the main obstacle to the use of high-dosage adriamycin in chemotherapy. It is difficult to decide whether or not treatment should be continued when the cardiac function -- irrespective of the method by which it is evaluated -- is at the lower limit of normality. Some authors consider that chemotherapy can be pursued as long as the shortening fraction of the echocardiographic diameter remains within normal limits in relation to the end-systolic constraint. We have established the limits of normality of this relationship before chemotherapy in 53 patients with normal cardiovascular system. We conclude that the end-systolic constraint essentially depends on the end-systolic diameter, so that the results provided by the study of the shortening fraction end-systolic constraint relationship are qualitatively the same as those of the shortening fraction-end-systolic diameter relationship, which is much easier to obtain. It seems to us that the criteria of cardiotoxicity after administration of adriamycin 300 mg/m2 are: (1) shortening fraction lower than 25 p. 100; (2) ventricular dilatation (end-systolic diameter greater than 40 mm) without associated valve disease; (3) reduction of the shortening fraction (whatever its value) in relation to the end-systolic diameter by more than 2 standard deviations on the regression slope of the correlation; (4) more than 25 p. 100 reduction of the shortening fraction after administration of adriamycin 300 mg/m2, betraying a high sensitivity to the cardiotoxic effects of the drug. Such individual sensitivity, studied in 25 patients, seemed to vary widely from one subject to another and to be independent of the initial status.(ABSTRACT TRUNCATED AT 250 WORDS)
Fractionation of an extract of pertussis microbes was carried out with the aid of gel-filtration on Sephadex G-100, ion-exchange chromatography on DEAE-cellulose, and preparative electrophoresis. Fractions differing in serological, immunogenic activity and the content of antigenic components were isolated. In using the method of gel-filtration of sefadex G-100 the greatest serological, immunogenic and histamine-sensitizing activity was possessed by the high-molecular fraction containing 8 of 11 antigenic components detected in the initial extract. The antigenic components were distributed into 5 fractions by the ion exchange chromatography on DEAE-cellulose. The greatest serological activity was possessed by fractions exiting from the column at the 0.01--0.04M interval of the phosphate buffer concentration. A method of preparative electrophoresis from the pertussis microbes extract was applied and two fractions were isolated from the anode and the cathode zones, each containing 4 antigenic components only, but possessing serological and immunogenic activity and having no histamine-sensitizing properties.
The content of N-acetylneuraminic acid (ANA) was measured in the water-soluble and membrane fractions of the visual, locomotory, auditory and sensomotory divisions of rat brain cortex as well as in glycoproteins and glycolipids of the membrane fraction. The content of ANA in the water-soluble fraction of the visual, locomotory, auditory and sensomotory analyzers of brain cortex was 0.55, 0.56, 0.55 and 0.65 mkg/mg of dry weight, respectively. The ANA content in the membrane fraction was 9.2, 8.8, 8.2 and 8.4 mkg/mg of dry weight, respectively. The ANA content in the membrane fraction of glycoproteins was 2.8, 2.4, 2.2 and 2.8 mkg/mg of dry weight, respectively., that in the membrane fraction glycolipids--5.2, 7.2, 5.9 and 5.5 mkg/mg of dry weight, respectively. The ANA content in the membrane glycolipids of the locomotory division of rat brain cortex differed significantly from that of all the other divisions.
YBA, a Moloney virus-induced leukemia in CBA mice, and a relatively weak immunogenic tumor, was screened for the presence of immunogenic antigens. The tumor was subjected to homogenization and subcellular fractionation on sucrose gradients; the immunogenic subcellular fractions underwent further separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The immunogenicity of the subcellular fractions and the SDS-PAGE-isolated molecular species were tested by (their) subcutaneous injection into syngeneic mice and examination of their splenocytes examined against tumor cell and normal cell targets by the chromium release cell-mediated lympholysis assay. Tumor cell homogenates were also separated by SDS-PAGE and tested for immunogenicity without prior fractionation. Splenocytes from mice that had received injections of certain SDS-PAGE-isolated epitopes derived from YBA tumor homogenate or its light and heavy subcellular fractions generated effective cytotoxic responses against YBA target cells after 6 days in vitro cultivation. In contrast, intact YBA tumor cells or non-separated tumor homogenates failed to induce an efficient cytotoxic response. The effector cells induced with the immunogenic SDS-PAGE-isolated epitopes of YBA tumor were specific, since they cytolysed the homologous target cells more efficiently than unrelated target cells or syngeneic normal cells. The activity of these effector cells was affected by varying the effector: target ratio. Augmentation of the cytotoxic responses was obtained when the splenocytes of mice immunized with SDS-PAGE-isolated epitopes of YBA tumor were restimulated in vitro, with the homologous neoplastic cells. Immunogenic SDS-PAGE epitopes were isolated from YAC tumor also (YAC is a Moloney-induced tumor of A mice). The effector cells induced with these separated epitopes were characterized as thymus-derived cells and not as natural killer cells. The results suggest that (1) the molecular repertoire of YBA and YBA tumors contain immunogens that can induce a specific antitumor cell-mediated response; (2) the isolated molecular species injected are more efficient immunogens than the entire, unseparated homogenate sample or a dose of 10(8) intact inactivated tumor cells; and (3) the gel matrix may be responsible for the enhanced cell-mediated response induced against the weakly immunogenic tumor.