Detection of N-(hexanoyl)lysine in the tropomyosin 1 protein in N-methyl-N'-nitro-N-nitrosoguanidine-induced rat gastric cancer cells

N^ε-(Hexanoyl)lysine, formed by the reaction of lysine with n-6 lipid hydroperoxide, is a lipid peroxidation marker during the initial stage of oxidative stress. The aim of the present study is to indentify N^ε-(hexanoyl)lysine-modified proteins in neoplastic transformed gastric mucosal cells by N-methyl-N''-nitro-N-nitrosoguanidine, and to compare the levels of these proteins between gastric mucosal cells and normal gastric cells. Much greater fluorescence of 2-[6-(4''-hydroxy)phenoxyl-3H-xanthen-3-on-9-yl]benzoic acid, an index of the intracellular levels of reactive oxygen species, was observed for gastric mucosal cells compared to normal gastric cells. N^ε-(Hexanoyl)lysine-modified proteins were detected by SDS-PAGE or two-dimensional electrophoresis and Western blotting using anti-N^ε-(hexanoyl)lysine polyclonal antibody, and a protein band of between 30–40 kDa was clearly increased in gastric mucosal cells compared to normal gastric cells. Two N^ε-(hexanoyl)lysine-modified protein spots in gastric mucosal cells were identified as the tropomyosin 1 protein by mass spectrometry using a MASCOT search. The existence of N^ε-(hexanoyl)lysine modification in tropomyosin 1 was confirmed by Western blotting of SDS-PAGE-separated or two-dimensional electrophoresis-separated proteins as well as by the immunoprecipitation with anti-tropomyosin 1 antibody. These data indicate that N^ε-(hexanoyl)lysine modification of tropomyosin 1 may be related to neoplastic transformation by N-methyl-N''-nitro-N-nitrosoguanidine in gastric epithelial cells.     

Acid hydrolases in amniotic fluid and chorionic villi at various stages of pregnancy

A study was made at various stages of pregnancy of five acid hydrolases which occur in amniotic fluid and chorionic villi and which are relevant to serious storage disorders.In amniotic fluid β-galactosidase and α-mannosidase decreased moderately towards term, while β-glucosidase decreased markedly. N-Acetyl-β-glucosaminidase and β-glucuronidase were relatively unchanged.In chorionic villi N-acetyl-β-glucosaminidase, β-galactosidase, and α-mannosidase were substantially decreased towards term, while β-glucosidase was unchanged and β-glucuronidase markedly increased.In both amniotic fluid and chorionic villi the enzyme pattern was approximately the same as that found in liver in a previous study.The findings suggest that these enzyme assays might be useful in the diagnosis of inborn errors prenatally by using amniotic fluid, and early postnatally by using chorionic villi.     

IN VITRO SYNTHESIS AND SECRETION OF LYSOZYME BY MONONUCLEAR PHAGOCYTES

Pure cultures of three types of mononuclear phagocytes—mouse peritoneal macrophages, unstimulated or after thioglycollate stimulation, and human monocytes—synthesize and secrete large amounts of lysozyme in vitro. The macrophage lysozyme is indistinguishable from authentic lysozyme in its ability to lyse M. lysodeikticus, inhibition by specific antisera, a similar size of 14,000 and cationic charge. Lysozyme secretion in culture is characterized by a large net increase in total lysozyme, 4–20-fold in 3 h, 75–95% of which is in the medium, and its continued extracellular accumulation over at least 2 wk in culture. Lysozyme is the major ¹⁴C-labeled protein secreted into the medium by both unstimulated and thioglycollate-stimulated macrophages and the 0.75–1 µg produced per 1 x 10⁶ cells/day represents 0.5–2.5% of the total cell protein. Lysozyme is a cell-specific marker for mononuclear phagocytes and the PMN, which contains preformed enzyme, since it is absent in lymphoid cells and a variety of fibroblast and epithelioid cell lines. Lysozyme production is also a useful measure of mononuclear phagocyte cell number. The rate of lysozyme production and secretion is remarkably constant for all cell types under a variety of culture conditions. Production by the mouse macrophage increases threefold on the 2nd day in culture and then remains linear with time. Production is optimal at a relatively low serum concentration, but can be maintained, in the absence of serum, in lactalbumin hydrolysate or, at a reduced level in basal media. The production and secretion of lysozyme are independent of the production of macrophage acid hydrolases. Net increase and secretion of lysozyme occur under conditions where acid hydrolases like N-acetyl β-glucosaminidase, β-glucuronidase, β-galactosidase, and cathepsin D are neither accumulated nor secreted. Massive phagocytosis of latex particles has no effect on lysozyme production and secretion. Lysozyme production can be rapidly inhibited by treatment with cycloheximide (0.4 µg/ml) whereas inhibition of its production by colchicine (10^–6 M) occurs only after a lag period of more than 8 h, and is probably due to a secondary effect. These results show that mouse macrophages provide a simple in vitro system to measure lysozyme secretion and its control. These studies also indicate the possible importance of mononuclear phagocytes in the secretion of a variety of biologically active products and in the modification of their environment.     

Biochemical activities of lysosomal acid hydrolases in leukemic cells

The biochemical activities of 8 lysosomal acid hydrolases in leukemic cells from 48 patients were examined. Characteristic alterations were found in α-mannosidase, β-galactosidase and N-acetyl-β-glucosaminidase activities of leukemic cells. The level of α-mannosidase activity was much higher in myelo(mono)genous leukemias (AML, AMoL, AMMoL, CML and CMMoL) than in lymphogenous ones (ALL, T-cell leukemia, hairy cell leukemia and CLL) without exception. The β-galactosidase activity also differed as a result of α-mannosidase, except in T-cell leukemia. In T-cell leukemia it was within the range of normal lymphocytes, but in the other lymphogenous leukemias it was significantly below normal. N-acetyl-β-glucosaminidase activity in myelo(mono)genous leukemic cells was above the range of normal granulocytes. The changes in these enzyme levels were consistent. The lymphocytic or myelocytic nature of three cases of acute undifferentiated leukemia could be determined by enzyme studies. In two cases it was lymphocytic and in one it was myelocytic. The enzymatic abnormalities were also found in morphologically mature neutrophils from patients with not only chronic types (CML, CMMoL) but also acute types (AMoL, AMMoL) of leukemias, and were similar to those of their respective leukemic cells. Analysis of lysosomal enzymes (at least three of those mentioned above), can elucidate one of the biochemical properties of leukemic cells and may be valuable in the differentiation of leukemias.     

Requirement for alpha-L-fucose on the macrophage membrane receptor for MIF

α-L-fucose abolishes the activity of guinea pig migration inhibitory factor (MIF) on the macrophages. Other sugars such as α-D-glucose, β-D-galactose, α-L-rhamnose, methyl-α-D-mannoside, and N-acetyl-β-D-glucosamine had no effect. Theabolition of MIF activity by α-L-fucose was reversible. When macrophages were incubated with α-L-fucosidase, a glycosidase which splits terminal α-L-fucose from oligosaccharides, the macrophages no longer responded to MIF. On the other hand, MIF incubated with α-L-fucosidase was still active. These experiments strongly suggest that α-L-fucose comprises an essential part of a macrophage membrane receptor for MIF.     

New α-galactosidase-inhibiting aminohydroxycyclopentanes

A set of cyclopentanoid α-galactosidase ligands was prepared from a partially protected ω-eno-aldose via a reliable (2 + 3)-cycloaddition protocol with slightly modified conditions. The obtained N-benzylisoxazolidine ring was selectively opened and the configuration of the hydroxymethylgroup was inverted. Consecutive deprotection provided an aminocyclopentane, which was N-alkylated to furnish a set of potential α-galactosidase inhibitors. Their glycosidase inhibitory activities were screened with a panel of standard glycosidases of biological significance.

A concise and robust synthesis of new cyclopentanoid competitive inhibitors of α-galactosidases related to Fabry''s disease and other α-galactosidase related disorders.     

Identification of the radicals formed in the reactions of some endogenous photosensitizers with oleic acid under the UVA irradiation

Electron spin resonance measurements were performed for the reactions of some endogenous photosensitizers (flavin mononucleotide or flavin adenine dinucleotide or folic acid or β-nicotinamide adenine dinucleotide or β-nicotinamide adenine dinucleotide phosphate or pyridoxal-5''-phosphate or urocanic acid) with oleic acid under the ultraviolet light A irradiation using α-(4-pyridyl-1-oxide)-N-tert-butylnitrone as a spin trap reagent. Of the endogenous photosensitizers, prominent electron spin resonance signals (α^N = 1.58 mT and α^Hβ = 0.26 mT) were observed for the reaction mixture of flavin mononucleotide (or flavin adenine dinucleotide or folic acid), suggesting that radical species form in the reaction mixtures. Singlet oxygen seems to participate in the formation of the radicals because the electron spin resonance peak heights increased for the reactions in D₂O to a great extent. A high performance liquid chromatography-electron spin resonance-mass spectrometry was employed to identify the radicals formed in the reactions of the endogenous photosensitizers (flavin mononucleotide or flavin adenine dinucleotide or folic acid) with oleic acid under the ultraviolet light A irradiation. The high performance liquid chromatography-electron spin resonance-mass spectrometry analyses showed that 7-carboxyheptyl and 1-(3-carboxypropyl)-4-hydroxybutyl radicals form in the reaction mixture of flavin mononucleotide (or flavin adenine dinucleotide or folic acid).     

Administration of PPAR��/�� agonist reduces copper-induced liver damage in mice: possible implications in clinical practice

In this study we investigated if peroxisome proliferator-activated receptor β/δ activation protects from copper-induced acute liver damage. Mice treated with copper had significant body weight loss, serum alanine aminotransferase increase, modest changes in liver histology, increase of tumor necrosis factor α and macrophage inflammatory protein 2 mRNA and 8-hydroxy-2''-deoxyguanosine. Mice treated with copper and peroxisome proliferator-activated receptor β/δ agonist GW0742 had significantly less body weight loss, less serum alanine aminotransferase increase, less tumor necrosis factor α, macrophage inflammatory protein-2 and 8-hydroxy-2''-deoxyguanosine upregulation than copper treated mice. The opposite effect was observed in mice treated with copper and peroxisome proliferator-activated receptor β/δ antagonist GSK0660. In vitro, copper induced reactive oxygen species, which was lower in cells treated with GW0742 or transfected with peroxisome proliferator-activated receptor β/δ expression vector; together, transfection and GW0742 had an additive reactive oxygen species-reducing effect. Copper also upregulated Fas ligand and Caspase 3/7 activity, effects that were significantly lower in cells also treated with GW0742. In conclusion, peroxisome proliferator-activated receptor β/δ activation reduced copper-induced reactive oxygen species, pro-inflammatory and acute phase reaction cytokines in mice liver. Peroxisome proliferator-activated receptor β/δ agonists could become useful in the management of copper-induced liver damage.     

A Bicyclic 1-Deoxygalactonojirimycin Derivative as a Novel Pharmacological Chaperone for GM1 Gangliosidosis

Lysosomal β-galactosidase (β-Gal) deficiency causes a group of disorders that include neuronopathic GM₁ gangliosidosis and non-neuronopathic Morquio B disease. We have previously proposed the use of small molecule ligands of β-Gal as pharmacological chaperones (PCs) for the treatment of GM₁ gangliosidosis brain pathology. Although it is still under development, PC therapy has yielded promising preclinical results in several lysosomal diseases. In this study, we evaluated the effect of bicyclic 1-deoxygalactonojirimycin (DGJ) derivative of the sp²-iminosugar type, namely 5N,6S-(N′-butyliminomethylidene)-6-thio-1- deoxygalactonojirimycin (6S-NBI-DGJ), as a novel PC for human mutant β-Gal. In vitro, 6S-NBI-DGJ had the ability to inhibit the activity of human β-Gal in a competitive manner and was able to protect this enzyme from heat-induced degradation. Computational analysis supported that the rigid glycone bicyclic core of 6S-NBI-DGJ binds to the active site of the enzyme, with the aglycone N′-butyl substituent, in a precise E-orientation, located at a hydrophobic region nearby. Chaperone potential profiling indicated significant increases of enzyme activity in 24 of 88 β-Gal mutants, including four common mutations. Finally, oral administration of 6S-NBI-DGJ ameliorated the brain pathology of GM₁ gangliosidosis model mice. These results suggest that 6S-NBI-DGJ is a novel PC that may be effective on a broad range of β-Gal mutants.     

Immunological memory in mice. I. Physical separation and partial characterization of memory cells for different immunoglobulin classes from each other and from antibody-producing cells

Plaque forming cells (PFC) of different immunoglobulin classes producing antibodies against sheep erythrocytes were separated according to their buoyant densities by means of equilibrium centrifugation in a stepwise BSA gradient. In the period of 7–10 days after immunization γM PFC are markedly enriched in fractions of low density and relatively depleted in fractions of high density. The distribution of total γG PFC shows less enrichment in the lower density fractions and less depletion in the higher density fractions. The density profile for γG_2a PFC is even flatter, with a significant difference (depletion) relative to the unseparated spleen cells only in the highest density fraction. The density gradient distributions of cells able to transfer an adoptive immune response of the various immunoglobulin classes are markedly different from the PFC distribution. Cells obtained 7–10 days after immunization able to transfer an IgM response are present in the same proportions across the density gradient, whereas memory cells for γG_2a obtained at this time are markedly enriched in fractions of low density and virtually depleted from high density fractions. With increasing time after primary immunization, the γG_2a memory cells increase progressively in density and by 6 weeks the higher and lower density fractions have the same proportions of γG_2a memory cells. The total γG (mainly γG₁) memory cells by 7–10 days show slight enrichment in low density fractions and no depletion in high density fractions. The conclusions were reached that (a) memory for γG₁ develops earlier than memory for γG_2a and (b) that memory for anti-SRBC antibodies of different classes is carried in separate cells. When gradient fractions enriched for PFC and memory cells for all classes were completely depleted of PFC using glass bead columns, the ability of this fraction to transfer memory for all classes was not diminished. This shows that memory cells are not identical with cells secreting antibodies.     

Detection of Fabry hemizygotes and heterozygotes by measurement of -galactosidase in urine

The determination of α-galactosidase in urine can be used as a simple method for the diagnosis of Fabry hemizygotes. The activity of this enzyme was related to that of N-acetyl-β-glucosaminidase. The ratio N-acetyl-β-glucosaminidase/α-galactosidase in urine was relatively constant in any one individual. In the control group, the mean value of this ratio was 7.4 (range 1.2–20.5). In Fabry hemizygotes (n = 6) the ratio was 50 or higher. Three types of carriers could be recognized, with high (n = 1), intermediate (n = 2) and normal (n = 3) values, so that with this procedure some of the carriers are detected.     

Adenovirus-mediated E2-EPF UCP Gene Transfer Prevents Autoamputation in a Mouse Model of Hindlimb Ischemia

E2-EPF ubiquitin carrier protein (UCP) stabilizes hypoxia-inducible factor-1α (HIF-1α) inducing ischemic vascular responses. Here, we investigated the effect of UCP gene transfer on therapeutic angiogenesis. Adenovirus-encoded UCP (Ad-F-UCP) increased the expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) in cells and mice. Conditioned media from UCP-overexpressing cells promoted proliferation, tubule formation, and invasion of human umbilical-vascular-endothelial cells (HUVECs), and vascularization in chorioallantoic membrane (CAM) assay. Ad-F-UCP increased the vessel density in the Martigel plug assay, and generated copious vessel-like structures in the explanted muscle. The UCP effect on angiogenesis was dependent on VEGF and FGF-2. In mouse hindlimb ischemia model (N = 30/group), autoamputation (limb loss) occurred in 87% and 68% of the mice with saline and Ad encoding β-galactosidase (Ad-LacZ), respectively, whereas only 23% of the mice injected with Ad-F-UCP showed autoamputation after 21 days of treatment. Ad-F-UCP increased protein levels of HIF-1α, platelet-endothelial cell adhesion molecule-1 (PECAM-1), smooth muscle cell actin (SMA) in the ischemic muscle, and augmented blood vessels doubly positive for PECAM-1 and SMA. Consequently, UCP gene transfer prevented muscle degeneration and autoamputation of ischemic limb. The results suggest that E2-EPF UCP may be a target for therapeutic angiogenesis.     

CHEMICAL BASIS FOR AN IMMUNOLOGICAL SPECIFICITY OF A STRAIN OF STAPHYLOCOCCUS AUREUS

Antisera, prepared against formalin-killed cells of Staphylococcus aureus, strain Copenhagen, agglutinated the cell walls of this strain. The agglutination was inhibited by the teichoic acid from the cell wall of this strain, by any degradation product of this teichoic acid which contained the α-acetylglucosaminyl-ribitol unit, by α-phenyl-acetylglucosaminide, and by N-acetylglucosamine, but not by a large number of other haptens related to the cell wall. In quantitative experiments, however, only 40 to 50 per cent of antibody adsorption to cell wall could be inhibited by teichoic acid or by N-acetylglucosamine. The α-acetylglucosaminyl-ribitol unit in the teichoic acid is, therefore, an important immunological determinant in the cell wall of this strain, although other immunological specificities may also exist. The cell walls were also agglutinated by heterologous antisera prepared against streptococcal Group A carbohydrate or against horse serum azophenyl-β-acetylglucosaminide. The heterologous agglutination, however, was specific for the β-acetylglucosaminyl-ribitol units in the teichoic acid.     

Glomerular and tubular damage markers are elevated in patients with diabetes

OBJECTIVE

We investigated in a cross-sectional study the levels of serum and urinary damage markers in diabetic patients (n = 94) and nondiabetic control subjects (n = 45) to study the association of glomerular (IgG), proximal tubular (kidney injury molecule [KIM]-1, N-acetyl-β-d-glucosaminidase [NAG], neutrophil gelatinase–associated lipocalin [NGAL], and cystatin C), and distal tubular (heart fatty acid–binding protein [H-FABP]) damage markers with kidney disease severity, as assessed by albuminuria and estimated glomerular filtration rate (eGFR).

RESEARCH DESIGN AND METHODS

Damage markers were measured in triplicate in fresh morning urine samples and in plasma.

RESULTS

Of the diabetic patients, 41 were normoalbuminuric, 41 were microalbuminuric, and 12 were macroalbuminuric. Urinary NAG (ninefold), NGAL (1.5-fold), and H-FABP (3.5-fold) were significantly elevated in normoalbuminuric diabetic patients compared with nondiabetic control subjects. Urinary concentrations of all markers increased per albuminuria stratum, except KIM-1. All urinary damage markers, except KIM-1, were significantly associated with albuminuria, independent of age, sex, and plasma concentrations of the corresponding biomarker (standard βs between 0.35 and 0.87; all P ≤ 0.001). All urinary damage markers, except KIM-1, were significantly associated with the eGFR in univariate models (standard βs between −0.38 and −0.21; all P < 0.04). After adjusting for age, sex, plasma concentration of the corresponding damage marker, and albuminuria, only the association of H-FABP with eGFR remained significant (standard β −0.26; P = 0.037).

CONCLUSIONS

Glomerular and tubular markers are associated with albuminuria, independently of eGFR, suggesting that albuminuria reflects both glomerular and tubulointerstitial damage. Only urinary H-FABP is associated with eGFR independently of albuminuria and, therefore, may be a promising urinary damage marker to assess diabetic kidney disease.Diabetic nephropathy occurs in 20–40% of patients with diabetes and is the leading cause of chronic kidney disease and end-stage renal disease in the U.S. and many other Western countries (1,2). The onset of elevated levels of urinary albumin excretion is an early sign of diabetic nephropathy. Various studies have shown that in subjects with diabetes, microalbuminuria predicts the occurrence of macroalbuminuria and renal function decline (3). As a result, high albuminuria has become an established renal risk marker in these patients (4).Classically, albuminuria is regarded as the consequence of diabetes-induced glomerular damage. More recently, it is increasingly appreciated that the renal tubulointerstitium plays a role in the pathogenesis of diabetic nephropathy, with prolonged exposure to a variety of metabolic and hemodynamic injuring factors associated with sustained diabetes disease as contributing factors (5,6). Furthermore, persistent albuminuria secondary to glomerular lesions may be directly harmful to renal tubular cells, leading to tubular inflammation and tubulointerstitial fibrosis (7,8).Several tubular damage markers recently have been discovered. Increased levels of these markers are supposed to indicate proximal tubular damage in the case of kidney injury molecule (KIM)-1, neutrophil gelatinase–associated lipocalin (NGAL), N-acetyl-β-d-glucosaminidase (NAG), and cystatin C and distal tubular damage in the case of heart fatty acid–binding protein (H-FABP). These tubular damage markers have been extensively investigated in the field of predicting the occurrence of acute kidney injury after various nephrotoxic insults, such as ischemia during cardiac surgery, sepsis, and administration of contrast medium (9–11). Little research has been done in patients with chronic kidney disease. In this study, we investigated the serum and urinary levels of the aforementioned damage markers in diabetic patients and nondiabetic control subjects in order to investigate the relation of these markers to the severity of kidney disease as assessed by albuminuria and the estimated glomerular filtration rate (eGFR). As a secondary aim, we investigated whether these damage markers are related to eGFR independent of albuminuria.     

Alpha lipoic acid selectively inhibits proliferation and adhesion to fibronectin of v-H-ras-transformed 3Y1 cells

Here, we focused on the effects of racemic α-lipoic acid on proliferation and adhesion properties of 3Y1 rat fibroblasts and the v-H-ras-transformed derivative, HR-3Y1-2 cells. Racemic α-lipoic acid inhibited proliferation of HR-3Y1-2 but not 3Y1 cells at 0.3 and 1.0 mM. R-(+)-α-lipoic acid also inhibited proliferation of HR-3Y1-2 cells equivalent to that of racemic α-lipoic acid. In addition, racemic α-lipoic acid decreased intracellular reactive oxygen species levels in HR-3Y1 cells but not 3Y1 cells. Next, we evaluated the effects of racemic α-lipoic acid on cell adhesion to fibronectin. The results indicated that racemic α-lipoic acid decreased adhesive ability of HR-3Y1-2 cells to fibronectin-coated plates. As blocking antibody experiment revealed that β1-integrin plays a key role in cell adhesion in this experimental system, the effects of racemic α-lipoic acid on the expression of β1-integrin were examined. The results indicated that racemic α-lipoic acid selectively downregulated the expression of cell surface β1-integrin expression in HR-3Y1-2 cells. Intriguingly, exogenous hydrogen peroxide upregulated cell surface β1-integrin expression in 3Y1 cells. Taken together, these data suggest that reduction of intracellular reactive oxygen species levels by α-lipoic acid could be an effective means of ameliorating abnormal growth and adhesive properties in v-H-ras transformed cells.     

Experimental studies on the bridging hypothesis of anaphylaxis: haptenic determinants required to elicit immediate-type reactions in calf skin by separate or concurrent sensitization with reagins of different specificity

The present study has established, that cows suitably immunized with either DNP-edestin (DNP-Ed), di-DNP-gramicidin-J [(DNP)₂-Gram], respectively, or p-azobenzenearsonate-Ed (ABA-Ed) synthesized and secreted reaginic antibodies (IgE) into colostrum. Whereas ABA-Ed failed to elicit more than a low response, there was however a persistent and increased antibody synthesis between 10 and 56 days after priming with DNP-Ed. Bivalent and multivalent DNP haptens differing in molecular size, degree of substitution, and rigidity were compared for their effectiveness in eliciting Prausnitz-Küstner (P-K) reactions in either newborn colostrum-deprived calves or in those 4 wk of age. The sensitization with reaginic anti-DNP antibody has been accomplished either by feeding colostrum of the immunized dam or by intradermal injection of reaginic serum or colostral whey. It could be demonstrated that equimolar doses of the bivalent α,N-(ε,N-DNP-aminocaproyl-)-ε,N-DNP-L-lysine and the multivalent dinitrophenylated bovine serum albumin were equally effective in eliciting reactions in skin sites provided that a high affinity antibody was used for sensitization. By contrast, the comparatively rigid, bivalent hapten, (DNP)₂-Gram consistently failed to induce comparable reactions. Furthermore, it was clearly shown that optimal distances of determinant groups on the haptenic molecule are a prerequisite for positive P-K reactions, since α,ε,N-bis-DNP-lysine failed to induce comparable reactions. Concurrent sensitization of skin sites with reaginic anti-DNP and anti-ABA antibodies provides the final proof that cross-linking of two adjacent reaginic molecules on the mast cell surface by a bivalent hapten is required for effective elicitation of immediate-type reactions. This has been accomplished by utilizing the bivalent ε,N-DNP-α,N-[(4-hydroxy-3-azobenzenearsonic acid)-phenacetyl]-L-lysine (DNP-ABA) carrying noncross-reactive haptenic groups, which was consistently effective in eliciting P-K reactions in doubly but never in singly sensitized skin sites. It is apparent from the results that equimolar doses of monovalent haptens could completely inhibit the response to DNP-ABA. The present studies finally establish that mast cells of newborn colostrum-deprived calves lack IgE molecules on their surface. Thus, mast cells of newborn calves may be unique, to investigate the molecular mechanisms involved in immediate-type reactions more precisely.     

LYSOSOMAL ACID HYDROLASES IN MICE INFECTED WITH BCG

Experiments are reported dealing with the increase of lysosomal acid hydrolases induced by BCG infection. Acid hydrolases were determined quantitatively in peritoneal MP, liver homogenate, and plasma of normal and BCG-infected mice. A significant increase of acid phosphatase, β-glucuronidase, and cathepsin was found in MP and liver homogenate of BCG-infected mice. In plasma also a significant increase of acid phosphatase and β-glucuronidase was noticed. The results of the determination of the enzymes in centrifugally separated subcellular fractions of liver homogenate indicated clearly that the acid hydrolases associated mainly with the "large granular" fraction, which consists of mitochondria, lysosomes, and microsomes and that infection with BCG caused significant increase of the enzymes specifically in this fraction. Differences in the pattern of location among centrifugally separated fraction of liver homogenate were observed between acid phosphatase and the other two acid hydrolases. MP cultured in vitro doubled their acid phosphatases content within 24 hours, whereas β-glucuronidase rather decreased in the same cells.     

A new variant mucolipidosis: Biochemical investigations on two siblings

Biochemical studies are presented on two siblings with some features of Mucolipidosis III, but with distinctive clinical findings. Levels of β-galactosidase, -mannosidase, β-glucuronidase, N-acetyl-β-glucosaminidase and -fucosidase found in serum from these patients ranged from 10 to 100 times higher than normal. The ratio of heat stable to heat labile serum isoenzymes of N-acetyl-β-glucosaminidase is considerably greater than normal.

An extremely low activity of β-galactosidase was found in fibroblasts cultured from one patient. Levels of the remaining enzymes were in the low normal range. Similarly, β-galactosidase levels were low in heart, kidney, liver, spleen and lung of one patient who died during the course of the study. Activities of the remaining enzymes were close to normal.

No excessive excretion of mucopolysaccharide was noted, however, changes in distribution of several fractions were found. Mucopolysaccharide labeled with radioactive sulfate was degraded by cultured fibroblasts at a normal rate.

In addition to clinical differences, the biochemical studies further demonstrate the uniqueness of these patients.     

SEPARATION OF TEICHOIC ACID OF STAPHYLOCOCCUS AUREUS INTO TWO IMMUNOLOGICALLY DISTINCT SPECIFIC POLYSACCHARIDES WITH α- AND β-N-ACETYLGLUCOSAMINYL LINKAGES RESPECTIVELY : ANTIGENICITY OF TEICHOIC ACIDS IN MAN

Human sera were found to contain antibodies precipitating with each of two samples of teichoic acid of Staphylococcus aureus prior to immunization; these antibodies were probably formed as a result of contact or infection with this microorganism. Injection of teichoic acid into two individuals resulted in a rise in circulating antibody to teichoic acid; a third subject probably had a primary response to α-teichoic acid. Quantitative precipitin and agar diffusion studies revealed the presence of two distinct antibodies in the sera and showed that each specimen of teichoic acid was a mixture of two polymers an α-linked N-acetylglucosaminyl-ribitol polymer and a β-linked N-acetylglucosaminyl-ribitol polymer, termed α- and β-teichoic acids respectively. The α-teichoic acid anti-α-teichoic acid system was inhibited best by α-linked glucosaminides and the β-anti-β-teichoic acid system was inhibited best by a β-linked glucosaminide. The α- and (β-teichoic acids could be separated from each other by specific precipitation under appropriate conditions and recovered from the washed specific precipitates. The existence of two distinct teichoic acid polymers raises important questions as to cell wall structure and the biosynthesis of the teichoic acids.     

Early and discrete changes in permeability of Escherichia coli and certain other gram-negative bacteria during killing by granulocytes

Rapid killing of Escherichia coli by intact or disrupted rabbit granulocytes or by granulocyte fractions was found to be accompanied by an equally rapid increase in permeability of the E.coli envelope. This increase in permeability was detected by determining entry of substances that normally do not cross E.coli''s permeability barrier, namely actinomycin D and o-nitrophenyl-β-D-galactopyranoside (ONPG), a substrate for cytoplasmic β-galactosidase. Because E.coli continue to incorporate radioactively labeled precursors into bacterial RNA and protein for at least 1 h, despite rapid killing by granulocytes, entry of actinomycin D could be measured by its inhibitory effect on macromolecular synthesis. Entry was evident within minutes after exposure to granulocytes or granulocyte fractions and is independent of pH over a range of 6.5–9.0. The effect of disrupted granulocytes or partially purified fractions on susceptibility of E.coli to actinomycin D and entry of ONPG is dose dependent. That the entry of actinomycin D and ONPG was not caused by gross destruction of the envelope is indicated by two sets of observations: (a) net influx of ⁴²K was maintained for at least 15 min, even though efflux of potassium was immediately accelerated upon addition of bactericidal concentrations of granulocyte fractions; (b) β-galactosidase did not leak out of E.coli under conditions that produce maximal inhibition by actinomycin D. Different species of gram-negative bacteria exhibited different susceptibilities to the bactericidal and permeability effects of granulocyte fractions. Thus, three strains of E.coli and one strain of Salmonella typhimurium were highly susceptible to both the bactericidal and the permeability enhancing effects of granulocyte fractions, whereas two strains of Serratia marcescens and one strain of Pseudomonas aeruginosa were resistant to both effects. Another strain of P. aeruginosa was rendered susceptible to actinomycin D without being killed and two strains of S. typhimurium remained insensitive to actinomycin D while being killed by granulocytes.