Properties of microsomal acyl coenzyme A reductase in mouse preputial glands

Synthesis of long-chain fatty alcohols in preputial glands of mice is catalyzed by an NADPH-dependent acyl coenzyme A (CoA) reductase located in microsomal membranes; sensitivity to trypsin digestion indicates that the reductase is on the cytoplasmic side of the membrane. Results with pyrazole and phenobarbital demonstrate the reaction is not catalyzed by a nonspecific alcohol dehydrogenase or an aldehyde reductase. Acyl-CoA reductase activity is sensitive to sulfhydryl and serine reagent modification, is stimulated by bovine serum albumin, and produces an aldehyde intermediate. The activity is extremely detergent sensitive and cannot be restored even after removal of the detergents. Phospholipase C or asolectin treatment does not release the acyl-CoA reductase from microsomal membranes, but causes a significant decrease in the activity recovered in the membrane pellet. Glycerol does not solubilize the reductase activity, nor does 3.0 m NaCl; however, the combination of glycerol and 3.0 m NaCl did release about 50% of the acyl-CoA reductase from the microsomal pellet. Substrate concentration curves obtained in the presence or absence of bovine serum albumin show significant differences in enzyme activities. The reductase is sensitive to the concentration of palmitoyl-CoA and is progressively inhibited at levels beyond the critical micellar concentration of the substrate. The apparent K_m for acyl-CoA reductase is 14 μm; however, the maximum velocity varies with the concentration of albumin used. Expression of enzyme activity in delipidated microsomes requires specific phospholipids, which suggests that in vivo regulation of acyl-CoA reductase activity could be achieved through modifications in membrane lipid composition.     

SCE evaluations in human lymphocytes after G0 exposure to mitomycin C Lack of expression of MMC-induced SCEs in cells that have undergone greater than two in vitro divisions

We conducted a series of experiments designed to determine whether DNA damage induced in G₀ lymphocytes by mitomycin C (MMC) would be expressed as sister-chromatid exchanges during the second and third post-treatment cell cycles. Lymphocytes from normal donors were exposed to MMC for 2 h prior to culture in the presence of phytohemagglutinin. MMC-treated and control cells were subsequently exposed to bromodeoxyuridine (BrdUrd) for the entire culture period (i.e. 48 h or 72 h) or for the terminal 24 h of 72-h cultures. We observed a 3–4-fold increase in SCEs in MII metaphases from lymphocytes treated with MMC and cultured in the presence of BrdUrd for the entire culture period. In contrast, in replicate cultures of MMC-treated lymphocytes that were exposed to BrdUrd for the terminal 24 h only, the SCE frequency in uniformly harlequinized metaphases was not significantly different from that observed in control cultures. We interpret these data as providing evidence that MMC-induced lesions (or alterations) in the DNA of G₀ lymphocytes are probably expressed as SCEs during the first period of mitogeninduced DNA synthesis, and that these lesions do not persist and give rise to SCEs in subsequent cell divisions.     

Increased sister-chromatid exchange in bone-marrow cells of mice exposed to whole cigarette smoke

Male Wistar rats were fed diets of varying selenium content in order to obtain selenium-deficient and selenium-supplemented rats. After 5-6 weeks on the respective diet, the rats were used to investigate how selenium influences the effect of dimethylnitrosamine (DMN) on some liver enzymes and related reactions. The selenium-dependent glutathione peroxidase activity in postmicrosomal supernatant from liver was about 1% in selenium-deficient rats as compared to selenium-supplemented rats or rats fed a standard diet. The highest DMN-demethylase activity was observed in postmitochondrial supernatant from selenium-deficient rat liver, and the lowest in selenium-supplemented rats. No dietary effect was observed on hepatic microsomal cytochrome P450 levels. C-Oxygenation of N,N-dimethylaniline (DMA) was not affected by the selenium level. On the other hand, selenium deficiency seemed to reduce N-oxygenation of DMA. The mutagenicity of DMN in Chinese hamster V79 cells after metabolic activation by the isolated perfused rat liver, was approximately doubled when selenium-deficient livers were used as compared to selenium-supplemented livers and livers from rats fed a standard diet. A negative correlation between DMA-N-oxygenation and mutagenicity from DMN was observed, whereas no correlation between DMA-C-oxygenation and mutagenicity from DMN was found.     

Release of soluble factors from lymph nodes containing mycobacterial granulomas and their effect on fibroblast function in vitro

A study has been made of the action of culture supernatants from guinea pig lymph nodes containing mycobacterial granulomas on protein and DNA synthesis of homologous fibroblast cultures. Supernatants from both the Bacillus Calmette-Guerin (BCG) and Mycobacterium leprae granulomas release soluble nondialysable factors in vitro which stimulate [¹⁴C]proline and [¹⁴C]leucine incorporation by fibroblasts and depress their [³H]thymidine uptake. These supernatants did not show any detectable migratory inhibitory activity in vitro. On the other hand, supernatants from sensitized lymphocytes incubated with purified protein derivative (positive migratory inhibitory activity) had no effect on fibroblast function. Thus, the effect of granuloma supernatants is unlikely to be due to lymphokines. However, supernatants from dinitrofluorobenzene-sensitized lymph nodes also showed a stimulation of [¹⁴C]proline incorporation into total protein synthesised by fibroblasts and depressed the [³H]thymidine uptake. Furthermore, supernatants from live BCG organisms in culture on addition to fibroblasts enhanced their [³H]thymidine uptake in vitro. It would appear therefore that fibroblast activation in lymph nodes containing mycobacterial granulomas could result from the release of soluble factors of lymphocyte origin rather than from cells of the mononuclear phagocyte system. These factors appear to be independent of classical lymphokines that act on macrophages in vitro. An additional factor may be derived from the mycobacteria themselves.     

Expression of embryonic hemoglobin genes in mice heterozygous for α-thalassemia or β-duplication traits and in mice heterozygous for both traits

Hemoglobins of mouse embryos at 11.5 through 16.5 days of gestation were separated by electrophoresis on cellulose acetate and quantitated by a scanning densitometer to study the effects of two radiation-induced mutations on the expression of embryonic hemoglobin genes in mice. Normal mice produce three kinds of embryonic hemoglobins. In heterozygous α-thalassemic embryos, expression of EI (x₂y₂) and EII (α₂y₂) is deficient because the x- and α-globin genes of one of the allelic pairs of Hba on chromosome 11 was deleted or otherwise inactivated by X irradiation. Simultaneous inactivation of the x- and α-globin genes indicates that these genes must be closely linked. Reduced x- and α-chain synthesis results in an excess of y chains that associate as homotetramers. This unique y₄ hemoglobin also appears in β-duplication embryos where excess y chains are produced by the presence of three rather than two functional alleles of y- and β-globin genes. In double heterozygotes, which have a single functional allele of x- and α-globin genes and three functional alleles of y- and β-globin genes, synthesis of α and non-α chains is severely imbalanced and half of the total hemoglobin is y₄. Mouse y₄ has a high affinity for oxygen, P₅₀ of less than 10 mm Hg, but it lacks cooperativity so is inefficient for oxygen transport. The death of double heterozygotes in late fetal or neonatal life may be due in large part to oxygen deprivation to the tissues.     

Novel features of hamster pinealocyte ultrastructure

Pineal glands from young adult hamsters (Mesocricetus auratus Water-house) kept on a 12 : 12 light : dark photoperiod were examined by electron microscopy. Ultrastructural features of nuclei, mitochondria, and centrioles that have not been reported previously are presented in this paper. Of particular interest is the presence of modified centrioles in normal hamster pinealocytes.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP: I. Antigen-induced uptake of nucleotides and nucleosides by lymphocytes

Addition of N⁶, O²′-dibutyryl cAMP (DbcAMP) to keyhole limpet hemocyanin (KLH)-primed rabbit lymph node cells for 1 hr, followed by its removal and the addition of KLH, had no effect on the subsequent antibody response, whereas addition of KLH for 1 hr followed by DbcAMP resulted in a 100% enhancement of antibody synthesis. Addition of cholera enterotoxin (CT), which rapidly and irreversibly binds to lymphocytes and activates adenylate cyclase, either before or after the addition of antigen, elevated the antibody response by 100%. These results suggested that some antigen-induced event(s) may be required for DbcAMP to exert its enhancing effects on the antibody response. The effect of KLH on the uptake of DbcAMP by KLH-primed lymph node cells was investigated. One and one hundred micrograms of KLH, which induce optimal and supraoptimal antibody synthesis, respectively, promoted maximal uptake of DbcAMP. This induced uptake was first detectable about 12 hr after addition of KLH, and it peaked during 24–48 hr of culture. DbcAMP uptake induced by a brief exposure of KLH (0–1 hr) was equivalent to that observed with long-term KLH addition (0–24 hr). KLH-induced DbcAMP uptake required KLH-reactive lymphocytes and represented active transport. Antibody to rabbit T lymphocytes inhibited this antigen-induced uptake. The mitogens concanavalin A (Con A) (T cells) and goat anti-rabbit Fab' (anti-Fab') (B cells) also stimulated DbcAMP uptake, as did human serum albumin (HSA) and myoglobulin (Mb) when added to homologously primed cells, indicating the generality of the phenomenon. [³H]DbcAMP entered the cells as di- or monobutyryl cAMP with about 40% metabolized to 5′AMP. This uptake could be competitively inhibited by other adenine or guanine nucleotides and nucleosides.     

Hydration of cis- and trans-epoxymethyl stearates by the cytosolic epoxide hydrase of mouse liver.

Man is exposed to epoxides of fatty acids from a number of sources, yet their degradative metabolism is not well understood. In mouse liver the 100,000 g supernatant or the cytosolic fraction is the most active fraction in hydrating $\text{cis}$- and $\text{trans}$-epoxymethyl stearates with the oxirane ring opening in a $\text{trans}$ manner to give the corresponding $\text{threo}$ and $\text{erythro}$ diols, respectively. Hydration was also observed in the microsomal, nuclei and cell debris, and mitochondrial fractions in decreasing order of specific activity.     

Tyrosine protein kinase activity in the DMBA-induced rat mammary tumor: inhibition by quercetin

Tyrosine protein kinase activity was measured in membranes from DMBA-induced mammary tumors, with Angiotensin II as substrate. The apparent Km for the peptide was 3.3 mM. This enzymatic activity is inhibited by Ca+2; Mn+2 can replace Mg+2 with an increase in the Km for ATP from 47 /microM to 172 microM. The enzymatic activity was not affected by cyclic AMP but was inhibited in dose dependent manner by quercetin, a bioflavonoid which is known to inhibit proliferation of malignant cells in vitro.     

Human duodenal gland (Brunner's gland) mucus glycoprotein analysis

A mucus glycoprotein of the duodenal gland is characterized. The glycoprotein was isolated from a water-soluble homogenate fraction of the submucosal tissue of the most proximal part of the small intestine, containing the duodenal gland, and was purified from contaminating protein by two sequential equilibrium-centrifugation steps in CsCl density gradients. Structural analysis of the purified glycoprotein showed two regions in the protein core: one part characterized by the presence of essentially all of the cysteine residues and another by the presence of most of the serine and threonine. Carbohydrate was found linked to the latter part. Rat (H. L. Smits, P. J. M. van Kerkhof, and M. F. Kramer (1982) Biochem. J. 203, 779-785.) and human duodenal gland mucus glycoprotein show homology in chemical composition. Both glycoproteins have a relatively high protein content and contain little sulfate and no neuraminic acid. In man the mucus glycoprotein, however, has a higher content of serine plus threonine, a lower content of N-acetylglucosamine, a slightly higher content of fucose, and a lower molar ratio of N-acetylgalactosamine relative to serine plus threonine.     

Identification of immunogens of Mycoplasmapneumoniae by protein blotting

Proteins of $\text{Mycoplasma}\text{pneumoniae}$ were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose sheet by blotting. Sera obtained from infected hamsters and immunized rabbits were then incubated with the nitrocellulose strips. Proteins which are capable of eliciting antibodies were detected by indirect immunoradioautography using ¹²⁵I-labeled antisera against hamster or rabbit IgG. Antibodies to seven immunogens were demonstrated in the sera of hamsters infected with $\text{M.}\text{pneumoniae}$ by inhalation, while many more proteins were found to be capable of stimulating antibodies in rabbits immunized parenterally with mycoplasmas. This method should prove useful for identifying those components of a microorganism which elicit antibody responses and contribute to the protective state.     

The regulation of the T-lymphocytes precursor pool by a humoral factor released by the thymus.

Studies in congenitally athymic nude mice grafted with syngeneic thymus enclosed or not in a cell-impermeable Millipore chamber show that the thymus secretes one or several humoral factors controlling the size of the pool of early T-lineage cells produced by the bone marrow.     

Determination of simultaneous turnover of serotonin,dopamine and norepinephrine in the telencephalon of unrestrained behaving rats

Rats received an injection of tritiated tryptophan and tyrosine via a chronic indwelling jugular catheter, and at 60 and 90 minutes post-injection, were killed by near-freezing. The three biogenic monoamines were separated by ion exchange and thin layer chromatographyand were quantitated with respect to radioactivity and content. The decline in specific activities can be used to calculate rate constants and turnover of serotonin, dopamine and norepinephrine concurrently in unrestrained behaving animals.     

Positive correlation between the levels of natural killer cells and the in vivo resistance to syngeneic tumor transplants as influenced by various routes of administration of Corynebacterium parvum bacteria.

Depending on the route of administration, heat-killed Corynebacterium parvum (C. parvum) bacteria caused an increase or decrease of natural killer (NK)-cell activity in mice. We used a syngeneic tumor with known susceptibility to NK lysis in vitro. The tumor was administered to mice whose NK levels had been increased or decreased by previous inoculations of C. parvum bacteria. A positive correlation between changes in NK-cell activity as measured in vitro and changes in tumor resistance as measured in vivo was observed. Additional evidence was provided in support of the view that NK cells may play an important role in resistance to tumor growth. The route of administration of C. parvum was considered important for protection against tumor growth.     

Regulation of peroxo-bridge formation between cobalt(II)-carnosine complexes by histidine: Possible involvement in polycythemia

The mechanisms by which histidine stabilizes the cobalt(II)-carnosine complex from oxidation to cobalt(III) in aqueous solution are investigated with ¹H-nmr, laser Raman, and Fourier transform-infrared spectroscopy. Histidine has at least three effects on the cobalt(II)-carnosine complex. First, over the concentration range of at least 5 to 250 mM, histidine stabilizes the cobalt(II)-carnosine complex from oxidation by excluding solvent molecules from the equatorial coordination positions. Second, at the upper end of this concentration range, histidine reduces the strained nonplanarity of the equatorial coordination positions around the cobalt(II) ion that results from tridentate chelation by carnosine. Bidentate ligation by histidine causes the carnosine to bind as a bidentate ligand also. Third, bidentate ligation of two carnosine molecules to the equatorial coordination positions of Co(II) ion places the β-alanyl residues inthe vicinity of the two axial coordination positions and thereby inhibits the binding of molecular oxygen. Substitution of a molecule of histidine for one of these two carnosine molecules makes an axial coordination position available for binding oxygen. The first two effects are expected to stabilize the cobalt(II) ion from rapid oxidation, whereas the third effect is expected to give long-term stability of the peroxo-bridged complex. Since bidentate ligation of histidine is favored over monodentate ligation only when the concentration of Co(II) ion is not limiting and is inhibited by high concentrations of carnosine in the same solution, the results presented provide a possible explanation for the observation that the stability of the Co(II) complexes toward oxidation and their ability to bind molecular oxygen depend on both the relative and absolute concentrations of Co(II) ion, carnosine, and histidine in solution. Furthermore, these results provide additional support to the suggestion that the high activity of carnosinase in kidney is involved in part in regulation of the oxygen sensor in this organ.     

Stimulation of splenic T cells on syngeneic monolayers of epidermal basal cells (EBC) in mice. Regulation by Lyt 1+ and Lyt 2+ cell subsets

Syngeneic proliferative response of splenic T cells against monolayers of epidermal basal cells (EBC) was obtained with C57BL/6 and DBA/2 mice. Optimal response, as assessed by [³H]thymidine uptake, occurred on the 6th day of coculture. The level of [^3H]thymidine uptake by unseparated spleen cells was lower than by fractionated T cells from C57BL/6 mice, and null for DBA/2 mice. It was not significantly different when lymphocytes were cocultured with syngeneic or allogeneic epidermal cells. Ia antigens did not appear to be involved in the syngeneic response, since it was not prevented by pretreating stimulator monolayers with monoclonal anti-Ia^k antibody or by adding this antibody directly to the cultures. When the proliferative responses of separated Lyt 1⁺ and Lyt 2⁺ cell subsets were compared, the prominent role of Lyt 1⁺ cells was demonstrated. Enhancement of the T-cell reactivity by eliminating Lyt 2⁺ cells and suppression of the response of a constant number of Lyt 1⁺ cells by adding Lyt 2⁺ cells suggested that Lyt 2⁺ cells could suppress and modulate the Lyt 1⁺ cell proliferation.     

Identification of a rabbit B cell alloantigen with an antiserum produced by alloimmunization with thymus cells.

Alloimmunization with rabbit thymus cells resulted in an antiserum (anti-Rly) which was shown to react with rabbit lymphocytes by an indirect rosette technique. The titration curve obtained with dilutions of anti-Rly antiserum on lymph node cells revealed two plateaus indicating that the antiserum was multispecific; at low dilutions of antiserum, within the first plateau, both B and T cells were rosetted whereas at high dilutions, within the second plateau, only B cells were rosetted. The antigen detected at high dilution was designated LB-1 (lymphocyte B cell alloantigen 1). The evidence that the cells identified within the second plateau are B cells is as follows: 1) simultaneous enumeration of LB-1⁺ and Ig⁺ (B) cells by use of distinguishable erythrocytes (sheep and human) as indicator cells revealed that of the 53% rosettes observed, essentially all (51%) were mixed rosettes containing both erythrocytes whereas simultaneous enumeration of LB-1⁺ and T⁺ cells (identified by anti-T cell antiserum) showed essentially no mixed rosettes (less than 2%); 2) approximately 80% of purified Ig⁺ (B) cells were identified as LB-1⁺ cells whereas essentially no (< 1%) purified T cells could be detected as LB-1⁺; 3) the percentages of LB-1⁺ cells and Ig⁺ cells were both reciprocal to the precentages of T⁺ cells identified in various lymphoid organs except for bone marrow; 4) the removal of LB-1⁺ cells from spleen cells of rabbits immunized with sheep red blood cells resulted in a depletion (42–71%) of direct plaque forming cells (PFC). Since the percentages of bone marrow cells rosetted using anti-LB-1 antiserum (approximately 70%) was much greater than the percentage rosetted using anti-Ig (approximately 10%), it appears that the anti-LB-1 antiserum is not directed against an Ig allotype. The titration curves of the anti-Rly antiserum on peripheral blood lymphocytes of a large rabbit family suggested that the LB-1 antigen on B cells is an alloantigen probably inherited in simple Mendelian fashion. Adsorption studies indicated that the LB-1 antigen on B cells is not detectable on brain, liver, kidney or erythrocytes.     

Characterization of the stimulation of neuronal Na+, K+-ATPase activity by low concentrations of ouabain

Low concentrations (< 10^?7 M) of ouabain stimulate the activity of Na⁺, K⁺-ATPase in whole homogenates of rat brain. The magnitude of this stimulation varies from 5 to 70%. The concentrations of ouabain which induces maximal stimulation is also highly variable and ranges between 10^?9 to 10^?7 M. The ouabain stimulation disappears following 1:50 dilution and 2 h preincubation or freezing and thawing of the membranes or their treatment with deoxycholate. “Aging” of a preparation of ATPase also results in loss of its ability to be stimulated by ouabain but ouabain inhibition is preserved. No stimulation of enzyme activity by ouabain is observed in rat brain microsomal fraction. The β-adrenergic blocker propranolol does not inhibit the ouabain induced stimulation of ATPase activity. It is suggested that the stimulation of Na⁺, K⁺-ATPase activity by low concentrations of cardiac glycosides if a result of either the displacement of an endogenous ouabain-like compound from the enzyme or an indirect effect by changing membrane surrounding environment of the Na⁺, K⁺-ATPase.