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Mitochondrial DNA isolated from white blood cells was investigated in families suffering from Leber's hereditary optic neuropathy. A recently described mutation at nucleotide position 11778 was present in 5 out of 12 families and heteroplasmic mitochondrial DNA was observed in 2 of these 5 families. A rapid shift in genotype was found in one of the families with heteroplasmy: the grandmother had 60 percent mitochondrial DNA mutated at nucleotide position 11778, the mother 55 percent, and the two sons at least 95 percent. These data indicate that the number of mitochondrial DNA molecules transmitted to the progeny passes a developmental bottleneck, as previously proposed to occur in bovine oogenesis.

This paper describes the analysis, by a highly sensitive and specific enzyme immunoassay (EIA), of AcSDKP, a tetrapeptide recently isolated from fetal calf bone marrow and subsequently purified and identified which substantially inhibits entry into cycle of hematopoietic pluripotent stem cells (CFU-S). This molecule has a marked protective effect in mice during anticancer chemotherapy with phase-specific drugs and plays an essential role in maintaining CFU-S out of cycle in normal mice. Using acetylcholinesterase-AcSDKP conjugate as tracer, rabbit specific antiserum and 96-well microtiter plates coated with a mouse monoclonal anti-rabbit IgG antibody, this EIA allows detection of AcSDKP at 15 fmol levels with a coefficient of variation less than 10% in the 50-500 fmol range. When combined with high-performance liquid chromatography, this assay clearly reveals the presence of this peptide in normal human white blood cells whereas in supernatant from cultured lymphocytes and in plasma the immunoreactive material is distinct from standard AcSDKP.

We developed a competition binding assay for estimation of the intracellular inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) and optimalized it for the measurement of the Ins(1,4,5)P3 content of human blood leucocytes. The present method is considerably cheaper and requires five times fewer cells than the commercial Ins(1,4,5)P3 kit. The mean Ins(1,4,5)P3 content of human blood monocytes, granulocytes, and lymphocytes amounted to 3.3 +/- 1.2 microM, 3.1 +/- 1.4 microM, and 4.6 +/- 1.5 microM, respectively. After stimulation with formyl-methionyl-leucyl-phenylalanine (f-MLP) the Ins(1,4,5)P3 content of human granulocytes and monocytes increased 2-3 times within 10 sec and then gradually decreased, returning to basal values at 60 sec. Lymphocytes did not respond to f-MLP with an increase in their Ins(1,4,5)P3 content.

The presence and amount of the anti-inflammatory protein lipocortin 1 was determined in plasma and peripheral blood leucocytes by a highly specific, enzyme-linked immunosorbent assay. Within 120 min of a single intravenous dose of 100 mg hydrocortisone, the intracellular concentrations of lipocortin 1 in peripheral monocytes in 7 of 8 healthy men increased by a median of 225% (range 129-507%) compared with pretreatment levels, and mononuclear cell-surface lipocortin increased by a median of 224% (range 76-483%). Placebo injections had no effect. There was no increase at any time in free plasma or polymorph-associated lipocortin. In 3 of 4 subjects, induction of lipocortin was also observed when whole unseparated blood was incubated in vitro after steroid administration, but cells which had first been isolated and purified were refractory to such induction. Thus rapid changes in the concentration of an active anti-inflammatory protein can occur in man after normal therapeutic doses of hydrocortisone.

Consuming substantial quantities of n-3 fatty acids reduces atherogenesis in experimental models of atherosclerosis. The mechanisms of this beneficial effect remain uncertain. Monocyte-derived tissue macrophages are associated with atherogenesis, and inhibition of monocyte inflammatory activity could, hypothetically, be helpful in preventing atherosclerosis. We observed that stimulated human monocyte and/or macrophage production of superoxide and the occurrence of monocyte chemiluminescence, two indices of monocyte inflammatory activity, were significantly reduced by the ingestion of 6 g n-3 fatty acids/d for 6 wk. This effect was associated with a reduction of stearic and arachidonic acids whereas eicosapentaenoic and docosahexaenoic acid concentrations rose significantly. These results indicate that modest dietary n-3 fatty acid supplementation can reduce stimulated human-monocyte free-radical production and may impair the capability of macrophages derived from monocytes to promote oxidation of low-density-lipoprotein cholesterol and associated cellular toxicity.

A study has been made of granulated metrial gland (GMG) cells and adjacent layer 1 cytotrophoblast in the labyrinthine placenta of the mouse. In those cellular associations in which both cell types appeared healthy with the light microscope, examination with the electron microscope identified features which may form part of the early stages in the interaction which can occur between GMG cells and some layer 1 trophoblast. These included microvillous processes from each cell forming an interdigitating network, a polarization of mitochondria in a GMG cell with interdigitating microvilli, platelets, and some GMG cells which appeared to form a specialized cell junction with a small lymphocyte. The latter observation indicates that a tripartite functional relationship may exist between lymphocytes, GMG cells and layer 1 labyrinthine trophoblast. In a quantitative analysis of GMG cells in the maternal blood spaces of placentae no significant differences in the numbers of GMG cells present in these spaces or involved in an interaction with layer 1 labyrinthine trophoblast were detected between inbred and outbred pregnancies. These results suggest that histocompatibility antigens are not significant factors in determining the frequency of interactions between GMG cells and trophoblast.

Human alpha interferon produced in Sendai virus-stimulated leukocytes was purified by immunosorbent affinity chromatography and subjected to subtype analysis. All subtypes in leukocyte culture supernatant were confirmed in purified preparation. One of these subtypes was reactive in Western blotting with antibody specific to alpha II (omega). N-terminal amino acid sequence analysis of this particular subtype showed addition of two amino acids to the N-terminus of alpha II predicted through cDNA studies. The apparent cleavage site for leader sequence of alpha II was different from the site common to all other alpha subtypes. This is the first report on the isolation of natural alpha II and its unique N-terminal amino acid sequence.

The relationship between ascorbic acid status and the urinary excretion of hydroxyproline was examined in 11 healthy male subjects fed an ascorbic acid-deficient diet for 14 wk while in a metabolic unit. The diet provided 5 mg ascorbic acid/d and was supplemented with ascorbic acid to give intakes of 65 mg/d (2 wk), 5 mg/d (4 wk), 605 mg/d (3 wk), 5 mg/d (4 wk), and an average 375 mg/d (1 wk). The urinary excretion of hydroxyproline increased by an average of 16% and 30% after the first and second depletion periods, respectively, and decreased to baseline values after supplementation with normal or high doses of vitamin C. Significant (p less than 0.05) inverse correlations were found between urinary hydroxyproline and plasma, red cell, and leukocyte ascorbic acid. These results show that urinary hydroxyproline excretion increases during human vitamin C deficiency but that this effect is not strong enough to provide a reliable marker of mild vitamin C deficiency.

AIDS in children is usually caused by vertical transmission of human immunodeficiency virus type 1 (HIV-1). Aborted eight-week fetal and placental tissue from HIV-1 positive and negative (enzyme-linked immunosorbent assay and Western blot) women was analysed by immunocytochemistry and in-situ hybridisation. Maternal decidual leucocytes, villous trophoblastic derivatives, villous mesenchymal cells, and embryonic blood cell precursors in tissues from seropositive patients all stained for HIV-1 antigen and hybridised for nucleic acids. These observations suggest that a cytological pathway for vertical transmission of HIV-1 is established by eight weeks gestational age.

Placental anticoagulant protein-I (PAP-I), a member of the lipocortin protein family, is a potent in vitro anticoagulant whose in vivo function is unknown. Very low levels of PAP-I were present in plasma of normal volunteers (0 to 5 ng/ml) and in randomly chosen plasma specimens from hospitalized patients (0 to 28 ng/ml). Review of selected hospital records did not reveal any single clinical entity that correlated with plasma levels. PAP-I was also found in amniotic fluid (12 to 107 ng/ml) and in conditioned medium of cultured endothelial cells (49 +/- 20 ng/ml). Gel filtration experiments showed that PAP-I was intact and uncomplexed in plasma and amniotic fluid. The protein was fairly abundant intracellularly: 4080 +/- 2560 ng/mg total protein in cultured umbilical vein endothelial cells; 178 +/- 109 ng/mg in platelets; 564 +/- 384 ng/mg in leukocytes; and 8.4 +/- 4.3 ng/mg in erythrocytes. The levels of PAP-I increased in platelet-rich plasma after stimulation of platelets with arachidonic acid but not after stimulation with ADP, epinephrine, thrombin, ristocetin, or collagen. These data suggest that PAP-I probably does not function as a circulating natural anticoagulant in normal persons.

Cerebral MRI and CT findings were compared with various cerebrospinal fluid (CSF) parameters and clinical features in 75 patients with multiple sclerosis (MS). There were positive correlations between CSF albumin as a parameter of blood-brain barrier function and morphological parameters, namely the number of nonperiventricular foci and periventricular involvement demonstrated by MRI and ventricular enlargement as shown by CT. Apart from positive correlations between the number of nonperiventricular foci and CSF levels of IgA and IgG, including the IgG synthetic rate, no other correlations were found between CSF parameters (leucocyte count and immunoglobulin concentrations) and morphological findings. The unremitting-progressive type of MS was distinguished from the relapsing-remitting form by a lower CSF leucocyte count and a higher degree of disability, but there was no difference between the morphological findings in the two forms. Positive correlations were found between degree of disability and both periventricular involvement in the MRI and CT evidence of ventricular enlargement. In terms of correlations with the duration of disease, cerebral MRI proved to be far superior to CSF evaluations.

The automated continuous flow system for the extraction and fluorimetric analysis of histamine based on the principle of Shore et al. (1959) has been improved. With lower consumption of reagents and further simplification of the working conditions, histamine can be determined quantitatively in a routine fashion in aqueous samples, with or without protein content, up to a concentration of approximately 0.1 ng/ml. The rate of analysis is 30 samples/h.

An anion-exchange high-performance liquid chromatography method has been used to quantitate the intracellular purine and pyrimidine nucleotides in extracts of pure lymphocytes, monocytes, neutrophils, eosinophils, erythrocytes, and platelets isolated from the blood of healthy human donors. For accurate and reproducible measurements of the nucleotide profiles in different types of pure leukocytes, the cell suspensions have to be free of platelets and erythrocytes. Incubation of the purified leukocytes for 1 h at 0 degrees C did not alter the nucleotide concentrations but reduced the interdonor variation to 10%. Incubation of purified lymphocytes for 1 h at 37 degrees C caused considerable changes in the relative concentrations of the adenine, guanine, uracil, and cytosine nucleotides. During this incubation the cell viability, the cell number, and the ATP:ADP ratio decreased. Incubation of monocytes and granulocytes for 1 h at 37 degrees C caused considerable loss of cells and/or cell death. For erythrocytes and platelets reproducible nucleotide concentrations were obtained after extraction of freshly isolated cells. During storage of erythrocytes, both at 0 degrees C and at 37 degrees C, a decrease in the ATP:ADP ratio was detected. In all cell types the predominant nucleotides were purine nucleotides, especially adenosine triphosphate. The relative concentrations of the adenine, guanine, uracil, and cytosine nucleotides were very reproducible per cell type and appeared to be characteristic for each cell type. The total nucleotide content was nearly the same for all cell types except erythrocytes, when expressed per microgram of protein. The described methods for purification and storage of blood cells will be useful for comparison of blood cells from healthy donors with those of patients, for example, leukemia patients, in which deviations of the purine and pyrimidine metabolic enzymes have already been described.

In assays of leukocyte function it is desirable to correlate a specific measurement, e.g., the release of enzymes, the production of metabolites or the secretion of cytokines with the amount of cellular material. Many authors use either the protein or the DNA content of the cells as a reference. Specific measurements of cellular activity are often expressed as a ratio of activity/protein or activity/DNA. This communication describes a dye-binding assay which allows the determination of total protein of the same cell sample which was first used for the quantitation of a specific cellular function. The microplate assay described is an adaptation of the Coomassie blue dye-binding assay without the need of a separate step to lyse the cells. The test is useful for both adherent and non-adherent cells and has a detection limit of less than 500 ng BSA or 5000 leukocytes.

The possibility of quantitative determination of human leukocyte interferon using FITC-labeled anti-interferon antibody was studied. Anti-interferon immunoglobulin of high specific activity was obtained as a result of long-term immunization of a donkey with human leukocyte interferon preparation and subsequent fractionation and immunoadsorption steps. This immunoglobulin was labeled with FITC and then used for titration of human leukocyte interferon by the fluorescence inhibition immunoassay. The titres of different preparations of human leukocyte interferon in the immunoassay were comparable with the titres of the same preparations detected by the inhibition of a cytopathic effect of VSV.

The binding of [1,6-3H]2,3,7,8-tetrachlorodibenzo-p-dioxin ([3H]TCDD) in human leukocyte cytosol has been studied using electrofocusing in polyacrylamide gel. One single peak of bound [3H]TCDD is found after completed focusing with an isoelectric point of 6.0. The binding capacity for 1.5 nM [3H]TCDD in the leukocyte cytosol was completely saturated by incubation in the presence of 150 nM unlabelled 2,3,7,8-tetrachlorodibenzofuran. [3H]TCDD also binds to a single binding species in human serum and plasma which focuses at pH 5.2 and which is not saturated by the addition of a 100-fold excess of unlabelled 2,3,7,8-tetrachlorodibenzofuran to the incubation. The levels of specific binding of [3H]TCDD in leukocytes from healthy blood donors ranged from 0 to 50 fmol/mg cytosolic protein.

A quantitative method for the determination of coenzyme Q10 (CoQ10) in human blood has been devised which allows recovery of essentially 100% of the CoQ10. The use of whole blood rather than plasma includes the CoQ10 in white cells. The method utilizes TLC instead of saponification to fractionate lipid impurities, because CoQ10 is sensitive to saponification, and utilizes CoQ11 as an internal standard which is advantageous over CoQ9 and a synthetic quinone. The final step of HPLC frequently reveals a peak with a retention time like that of CoQ9 which, being less than that of CoQ10, can be near other peaks of impurities.

Extensive studies in animal models indicate that subclinical ascorbic acid deficiency impairs the conversion of cholesterol to bile acid, elevates plasma cholesterol levels, and predisposes to development of cholesterol cholelithiasis. The present study was designed to see if this is also true in man. Five normal volunteers were hospitalized in a metabolic ward and placed on a controlled diet containing 3-4 mg of ascorbic acid each day. Ascorbic acid supplementation was given as follows: control period I (days 1-33), 75 mg/day; deficient period (days 34-96), 0 mg/day; and repletion period (days 97-101), 1000 mg/day. In addition, three of the subjects were studied during a second control period (days 102-139) during which they were given 75 mg/day of ascorbic acid. Ascorbate levels at the end of both control periods were 0.87-1.34 mg/dl in plasma and 19.4-29.5 micrograms/10(8) cells in leukocytes. At the end of the deficient period these levels were 0.09-0.15 mg/dl in plasma and 6.2-10.0 micrograms/10(8) cells in leukocytes, levels approaching those seen in scurvy. There was no effect of ascorbic acid deficiency on plasma cholesterol and triglycerides; plasma cholesterol in high, very low, and low density lipoprotein fractions; biliary lipid composition and saturation index of gallbladder bile; synthesis, fractional turnover, or pool size of either cholic or chenodeoxycholic acids; output of fecal acid or neutral sterols; and fecal sterol balance. Total bile acid pool size calculated by the one-sample technique was reduced 11% in the deficient period compared to control period I (P less than 0.005), and increased to 98.7% of the baseline levels in control period II. However, total bile acid pool calculated by the Lindstedt method did not change during deficiency. These data demonstrate that short-term subclinical ascorbic acid deficiency near the scorbutic range has no significant effect on bile acid and cholesterol metabolism in man.

Human ultrafiltrated leukocyte extracts (MW less than 5000) were fractionated by Sephadex G-10 column chromatography and the effects of these fractions on leukocyte random locomotion were investigated in vitro. Fr-4, one of these fractions, had significant leukocyte random locomotion inhibitory activity, independent of the presence of mononuclear leukocytes. This inhibitory activity was not due to cytotoxic effects on leukocytes. As seen by scanning electron microscopy, the number of cell surface pseudopods on leukocytes incubated with Fr-4 was reduced. Fr-4A, one of three fractions separated from Fr-4 by Sephadex G-25 column chromatography, significantly inhibited leukocyte random locomotion. Fr-4A contained numerous components, one of which was identified as 2-deoxyribose, on the basis of thin-layer chromatography. Biologically 2-deoxyribose showed an inhibitory effect on leukocyte locomotion and a reduction of the extrusion of pseudopods on the surface of leukocytes, at the range of assayed concentrations. This inhibitory activity is probably derived from 2-deoxyribose.

An enzymatic method for determining red cell glycogen was developed and its recovery and precision ascertained. Initial red cell glycogen values in 24 healthy volunteers ranged from 11-60 micrograms/g haemoglobin which compares with 20-105 micrograms/g haemoglobin previously reported. More thorough removal of leucocytes suggested however that variations in the determined red cell glycogen were due to glycogen derived from leucocytes. In 34 red cell preparations with varying leucocyte numbers, the glycogen concentration correlated positively with leucocyte count (r = +0.81, p less than 0.001), whereas glycogen concentration showed poor correlation with red cell count (r = -0.30, 0.1 greater than p greater than 0.05). The results question the reported presence of glycogen in normal red cells and suggest that contamination by leucocytes has been responsible for the previously reported normal range.

Marked sensitivity of tumor tolerance to blood glucose level is demonstrated in a mouse model of human breast cancer. A theory is cited that explains the high association of hyperglycemia with malignancy, especially breast cancer, via glycemic modulation of cellular immunity. Three groups of BALB/C mice were injected with an aggressive mammary tumor and placed on three dietary regimens designed to produce three different glycemic levels. Mortalities 70 days after injection were 16 of 24 hyperglycemic mice, 8 of 24 normoglycemic, and 1 of 20 hypoglycemic (chi-square p less than .005). Taken together with other experiments and human data discussed briefly, this result suggests that glycemic modulation of tumor tolerance should be evaluated in human trials.

Studies in animal models suggest that ascorbic deficiency impairs T-cell-mediated immunity. We studied five normal volunteers hospitalized on a metabolic unit and consuming a strictly controlled diet deficient in ascorbic acid I) after a 5-wk control period of ascorbic acid supplementation (75 mg/day) and 2) after a 9-wk period of no supplementation. Three of the subjects were restudied after a 5-wk period of ascorbic acid supplementation after the deficient period. At the end of both control periods ascorbic acid levels in plasma ranged from 0.9 to 1.3 mg/dl and in leukocytes from 19 to 30 microgram/10(8) cells. At the end of the deficient period levels of ascorbic acid in plasma ranged from 0.09 to 0.15 mg/dl and in leukocytes from 6.2 to 10 microgram/10(8) cells, levels at or below those frequently found in frank scurvy. None of the T-cell parameters tested including mitogen responsiveness to phytohemagglutinin and percentage of T-cells bearing receptors for IgM (helper cells) and IgG (suppressor cells) was different in the deficient period compared to the control periods. One patient with spontaneous scurvy (plasma ascorbic acid 0.07 mg/dl, leukocytic ascorbic acid 4.9 microgram/10(8) cells) was studied at the time of admission and after vigorous ascorbic acid repletion. All T-cell parameters after repletion were unchanged from admission. We conclude that in man ascorbic acid deficiency, even at the scorbutic level, does not alter T-cell numbers or impair in vitro T-cell function.

Tricyclic antidepressant drugs bind to histamine receptors in rodent and other mammalian tissues. However, a readily accessible, normal human tissue for studies of these drugs has not been available. We showed that freshly isolated human leukocytes can be used for this purpose. Following isolation by dextran sedimentation, leukocytes were incubated for 30 sec. in histamine concentrations from 1.0 nM to 1.0 mM. A dose-related increase in intracellular cyclic AMP was observed. When the cells were preincubated for 10 min. in the tricyclic, nortriptyline, and then challenged with histamine, the concentration of histamine needed for a comparable cyclic AMP increase was elevated 100-fold over non-preincubated cells. These results indicate that the tricyclic drug interacts with histamine receptors on leukocytes, and that these cells may be used for biochemical and clinical studies of these drugs.

Plasma cholesterol, plasma and white blood cell ascorbic acid concentrations were estimated in 30 untreated, 32 salbutamol-treated patients with bronchial asthma and in 57 normal controls. Treated and untreated asthmatics had significantly higher plasma cholesterol but lower plasma and white cell ascorbic acid levels than normal controls. A negative correlation was found between white cell ascorbic acid level and plasma cholesterol concentration in both treated and untreated asthmatics. These findings are discussed.

We report the establishment of a radioimmune assay for human alpha-interferon using highly purified alpha-interferon labeled with iodine-125, a purified rabbit immunoglobulin directed against human interferon and the Cowan strain of Staphylococcus aureus. Radiolabeling conditions used do not change the antigenicity of interferon molecules. The regression of counts bound upon log. dose was found to be linear down to 10 units/ml of alpha-interferon (6-7 X10-12 M). This assay was specific for alpha-interferons derived from human peripheral blood leukocytes and from a continuous line of lymphoblastoid cells. No cross-reaction was found with either human beta-interferon or murine interferon. Neither human serum nor plasma interfered with the assay. Correlation between biological assay and radioimmune assay was found to be significant.

Glycosphingolipids carrying carbohydrate sequences recognized by antibodies and lectins can be detected on thin layer chromatograms using an avidin-biotin enzyme system (ABC reagents). This same method can be used to detect glycosphingolipids blot-transferred from thin layer chromatograms to nitrocellulose. This method has certain advantages over the original radioimmunoassay method, including development of positive bands in minutes after incubation with the substrate, avoidance of handling hazardous radioactive materials and stability of reagents. We have demonstrated the usefulness of this method for immunostaining glycosphingolipids with both monoclonal and polyclonal anti-carbohydrate antibodies. These reagents have previously been used to detect carbohydrate antigens in tissues and isolated cells and now it is possible to use the same reagents for the detection of glycosphingolipid antigens on chromatograms.

A cDNA clone for human transferrin was identified from a human liver cDNA library by pre-screening with different ss-cDNA probes against length-fractionated liver mRNAs, positive hybridization-selection and nucleotide sequence analysis. The insert was of 1 kb, encoding human transferrin from aminoacid 403 through the COOH terminus, with a 3' non coding region of 166 nucleotides. This insert hybridized with a single major mRNA species of about 2.4 kb and several genomic DNA restriction fragments. Hybridization of the Southern blots with different parts of the transferrin insert and at different stringences suggest that the various bands observed correspond to splice sites inside one gene rather than to hybridization to several related genes. Finally, a single or a low number of transferrin gene copies seem to exist in the human genome.

The hst gene was originally identified as a transforming gene in DNAs from stomach cancers and a noncancerous portion of stomach mucosa by transfection assays using NIH3T3 cells (1,2). Subsequently, the hst gene obtained directly from leukocyte DNA of a leukemia patient was sequenced (3,4). Here, cosmid clones containing the hst gene were isolated directly from normal human leukocyte DNA and from T361-2nd-1 cells, a secondary transformant of NIH3T3 cells induced by transfection of DNA from a stomach cancer. All clones containing the hst gene from these different sources transformed NIH3T3 cells with similar efficiency. Restriction map of the hst gene from normal leukocyte DNA was identical with that from leukocyte DNA of a leukemia patient, while the hst gene from T361-2nd-1 cells was rearranged at the 168th nucleotide upstream of the TATA box.

Immunohistochemical techniques have been used to investigate the expression of fetal trophoblast antigens and the maternal leucocytic response in molar pregnancy and choriocarcinoma. The antigenic phenotype of morphologically defined trophoblast populations in complete, partial and invasive moles was analogous to that in normal pregnancy. All trophoblast phenotypes described in normal pregnancy were also identified in choriocarcinoma, suggesting that extensive differentiation into heterogeneous subgroups occurs in malignant trophoblast. The maternal leucocytic infiltrate in molar pregnancy consisted of T lymphocytes and class II MHC-positive macrophages. CD2-positive, CD3-negative lymphocytes were identified in molar decidua but not in uterine tissue in choriocarcinoma. Similarly, endometrial granulocytes were present in molar decidua but not in choriocarcinoma; these cells were associated with decidualization rather than with fetal trophoblast.

Activator proteins for sphingolipid hydrolases (saposins) are small acidic, heat-stable glycoproteins that stimulate the hydrolysis of sphingolipids by lysosomal enzymes. The molecular mass of each stimulator is about 10 kDa, but glycosylated forms of higher mass exist too. The distribution and developmental changes in two saposins and their precursor proteins were studied with the aid of monospecific antibodies against saposin-B and saposin-C. They show a wide distribution in rat organs and forms intermediate between saposin and prosaposin (the precursor protein containing four different saposin units) could be seen. The amount of saposin and the degree of processing from prosaposin are quite different in different tissues. The saposins are the dominant forms in spleen, lung, liver, and kidney, while skeletal muscle, heart, and brain contain mainly precursor forms. In human blood, leukocytes contain mainly saposin, while plasma contains mainly precursor forms and platelets show many forms. Their subcellular distribution was studied using rat liver. The saposins of approximately 20 kDa are dominant in the light mitochondrial, mitochondrial, and microsomal fractions, following the distribution of the activity of a lysosomal marker enzyme. The nuclear fraction exhibits bands corresponding to non-glycosylated saposin. The soluble fraction contained much precursor forms. A developmental study of rat brain showed that the concentration of saposin precursors increased with age.

A new method has been developed for the determination of amsacrine (AMSA) in human nucleated hematopoietic cells. In order to prevent efflux during the cell separation procedure, white blood cells (WBCs) were separated from red blood cells by dextran sedimentation, leaving the WBCs in their natural environment. After cell counting, pelletting the cell suspension and correcting for the admixture of supernatant, AMSA was extracted from the WBCs and determined by high-performance liquid chromatography. Linearity of extraction was observed up to 40.10(6) cells. The inter-assay variation was 4.7%. Plasma and cellular concentrations were measured in five patients at the end of a 3-h infusion of 100 mg/m2 AMSA. A pharmacokinetic study of plasma and cellular AMSA concentrations up to 19 h after infusion was carried out. AMSA concentrations in WBCs correlated well with the plasma levels (n = 20, r = 0.967) with an accumulation factor compared to the plasma concentration of 2.6-9.8 in the patients studied. The method described is useful for studying cellular pharmacokinetics of AMSA in man.

Blood samples were volunteered by workers in a Finnish iron foundry who were occupationally exposed to polycyclic aromatic hydrocarbons and from control subjects not known to be occupationally exposed to this class of chemical carcinogens. DNA was isolated from peripheral white blood cells and digested with micrococcal nuclease, spleen phosphodiesterase and nuclease P1. The DNA digest was then incubated with [gamma-32P]ATP and polynucleotide kinase. Aromatic adducts present in the digest that were resistant to nuclease P1 were thus 32P-labelled while unmodified nucleotides were not. The 32P-labelled adducts were resolved by t.l.c. and detected by autoradiography. Foundry workers were classified as belonging to high, medium or low exposure groups according to their exposure to airborne benzo[a]pyrene (high greater than 0.2, medium 0.05-0.2, low less than 0.05 microgram BP/m3 air). Aromatic adducts were found to be present in DNA from 3/4 samples from the high exposure group, 8/10 samples from the medium exposure group. 4/18 samples from the low exposure group and 1/9 samples from the unexposed controls. The levels of adducts found in the high and medium group samples ranged up to 1 adduct in 10(7) nucleotides but the levels formed in the low exposure group samples were not significantly different from those in unexposed controls. No differences related to the smoking habits of the subjects were observed. Most of the DNA adducts detected had chromatographic mobilities distinct from those formed when the 7,8-diol 9,10-oxide of BP reacted with DNA. The results indicate that highly-exposed individuals are more likely to contain aromatic DNA adducts in their white blood cells, but large interindividual variations were evident. In addition, multiple samples from the same subjects indicate that qualitative and quantitative changes in adduct patterns occur with time. This pilot study suggests that 32P-postlabelling may be useful in monitoring human exposure to known and to previously unidentified environmental genotoxic agents.

We have investigated the transfer of specific cell-mediated immunity (CMI) to osteosarcoma-associated antigens (OSAA) to hamsters with dialyzable leukocyte extracts (DLE) from OSAA-immunized rabbits. The transfer of specific CMI was determined by leukocyte adherence inhibition (LAI) assay and skin testing. DLE was prepared from rabbits immunized with OSAA, purified protein derivative (PPD), or fibrosarcoma cell plasma membrane preparation (FSM). Control DLE was prepared from rabbits injected with 0.85% NaCl. Significant leukocyte adherence inhibition was observed with leukocytes from hamsters that had received OSAA-specific, PPD-specific, and FSM-specific rabbit DLE, when OSAA, PPD, and FSM were used as antigens, respectively. Similarly, significant ear swelling after injection of OSAA, PPD, or FSM was observed only in hamsters that had received DLE from rabbits immunized with OSAA, PPD, or FSM, respectively. These results suggest that CMI specific for OSAA, PPD, or FSM can be transferred to normal hamsters by DLE from immunized rabbits.

A sensitive method for quantitative analysis of aspartylglucosamine as its dabsyl chloride derivative by high-performance liquid chromatography is described. Precolumn-derivatized aspartylglucosamine and internal standard (carboxymethylcysteine) are separated on a reversed-phase column with a mobile phase consisting of phosphate buffer and acetonitrile and monitored by UV-VIS detection at 436 nm. Aspartylglucosamine acts in the assay like a polar amino acid, and it can be separated from interfering substances in urine with a retention time of ca. 13 min. Its detection limit is ca. 0.3 microM in water and 0.5-1.0 microM in urine and other biological samples, which permits its quantitation in normal urine, for example. The within-day coefficient of variation is less than 4.7% and the day-to-day coefficient of variation is less than 8.3%. The present method is applicable to the direct analysis of aspartylglucosamine in body fluids and tissues without any prepurification and, in combination with automated liquid chromatography, allows rapid assay of a large number of samples in the detection of aspartylglycosaminuria. The sensitivity of the assay also allows direct quantitation of aspartylglucosamine in normal urine and leukocytes of aspartylglycosaminuria patients, and may thus be used in metabolic studies of the compound.