[Neonatal suppurative parotitis]. / Eitrige Parotitis bei einem Neugeborenen.

This report describes the case of a 10 days old newborn of a diabetic mother with gestosis, who developed erythema and swelling of the right cheek. A diagnosis of acute suppurative parotitis due to S. aureus was made. Following this case report the most important facts of epidemiology, pathogenesis, clinical manifestation, diagnostics, and therapy of suppurative parotitis are discussed.

Anti-CD4 monoclonal antibody treatment in acute and early chronic antigen induced arthritis: influence on macrophage activation.

OBJECTIVE: To investigate the indirect effects of anti-CD4 treatment on the functions of macrophages (CD4(-) in mice) in the acute and early chronic phase of mouse antigen induced arthritis (AIA). METHODS: C57BL/6 mice with AIA were treated intraperitoneally with the anti-CD4 mAb GK1.5 or control rat IgG on days -1, 0, 1, 3, 5, and 7. Proinflammatory cytokines (IL1 beta, IL6, and TNF alpha) were quantified by sandwich ELISA in joint extracts, serum, and supernatants of ex vivo stimulated spleen/lymph node cells or peritoneal macrophages (+LPS/IFN gamma). Nitric oxide (NO) levels in supernatants of ex vivo stimulated peritoneal macrophages were measured by the Griess reaction. Proteolytic activity in joint homogenates was analysed by gelatin, casein, and elastin zymography, and substrate assays. RESULTS: Anti-CD4 treatment significantly reduced joint swelling in acute (days 3, 5) and early chronic AIA (day 7) and diminished inflammation and destruction scores in late chronic AIA (day 21). On day 3, anti-CD4 treatment significantly reduced IL6 levels in all compartments. IL1 beta was reduced in joint extracts, unaffected in serum or cells from lymphoid organs, and increased in stimulated peritoneal macrophages. TNF alpha was significantly increased in the joints, decreased in serum, and otherwise unchanged. NO production by stimulated peritoneal macrophages was significantly reduced by anti-CD4 treatment. Lower activity of matrix metalloproteinases and neutrophil elastase was seen in joint extracts of anti-CD4 treated animals than in IgG treated AIA controls. CONCLUSION: CD4(+) T cell directed treatment had strong local and systemic effects on macrophages. These indirect effects may contribute to the reduction of destructive mediators/joint destruction in AIA.

Anti-CD4 monoclonal antibody treatment in acute and early chronic antigen-induced arthritis: influence on T helper cell activation.

To examine the effects of anti-CD4 mAb treatment in acute and chronic antigen-induced arthritis (AIA), C57BL/6 mice were treated intraperitoneally either with the depleting anti-CD4 mAb GK1.5 or with rat-IgG (control) on Days -1, 0, 1, 3, 5, and 7. Arthritis was monitored by assessment of joint swelling and histological evaluation in the acute (Day 3) and the chronic phase (Day 21) of AIA. To determine the effects on cellular immune responses, in vivo T-cell reactivity (delayed type hypersensitivity; DTH) was measured, as well as protein levels of TH1- (IL-2, IFN-gamma) and TH2-cytokines (IL-4, IL-10) in joint extracts and supernatants of ex vivo stimulated spleen and lymph node cells. The humoral immune response was analysed by measuring serum antibodies against methylated bovine serum albumine (mBSA) and extracellular matrix proteins. Treatment with GK1.5 reduced swelling, inflammation, and destruction of the arthritic joint. Unexpectedly, the effects were even more pronounced in the acute than in the chronic phase. The anti-inflammatory effect was accompanied by a diminished DTH against the arthritogen mBSA and a decrease of TH1-cytokine production in spleen and pooled body lymph nodes, whereas the TH2-cytokine production in these organs was unchanged and the humoral immune response was only moderately reduced. There was a failure of depleting CD4+ T-cells in the joint, reflected also by unchanged local cytokine levels. Therefore, systemic rather than local effects on the TH1/TH2 balance appear to underlie the therapeutic efficacy of anti-CD4 treatment in AIA.

Successful treatment of gut-caused halitosis with a suspension of living non-pathogenic Escherichia coli bacteria--a case report.

UNLABELLED: In up to 90% of cases, severe halitosis is a result of gastrointestinal or orolaryngeal problems. This case study reports on a girl with bad breath caused by increased formation of malodorous intestinal gases (halitosis), which could be successfully treated with a suspension of living non-pathogenic bacteria Escherichia coli. CONCLUSION: in unclear cases of bad breath, an increased formation of intestinal gases should also be considered.

Pancreatic elastase 1 in feces of preterm and term infants.

BACKGROUND: Determination of fecal pancreatic elastase 1 (E1) is a reliable and noninvasive test of exocrine pancreatic function. Adult reference values of greater than 200 microg E1/g feces do not seem to be applicable to early infancy because of immature pancreatic function. Because reference values for infants do not exist, the current study was aimed to define reference values for preterm and term infants up to 12 months of age. METHODS: The authors measured pancreatic E1 concentration in feces of 148 infants up to 12 months of age. Infants with known bowel or pancreatic disorders were excluded from the study. RESULTS: The authors found that 96.8% of all children had E1 concentrations greater than an adult lower limit after 2 weeks of life, independent of gestational age. Up to 48 hours after birth, none of the preterm infants had an E1 concentration of greater than 30 microg/g meconium, whereas 43% of the term infants had normal adult values. CONCLUSIONS: The adult reference value for pancreatic E1 of greater than 200 microg/g feces can be applied to infants older than 2 weeks, independent of gestational age, birth weight, and the type of nutrition.

An evolutionarily conserved tripartite tryptophan motif stabilizes the prodomains of cathepsin L-like cysteine proteases.

Cathepsin L-like cysteine proteinases contain an evolutionarily highly conserved alpha-helical motif in the proregion. This is called the ER(F/W)N(I/V)N motif according to the conserved amino acids along one side of the helix. We studied the function of this motif using site-directed mutagenesis experiments of human procathepsin S. We replaced each of these amino acids with alanine and constructed deletion mutants lacking parts of the helix. All mutants were expressed in HEK 293 cells, but only one, W52A, was not processed to mature cathepsin S, nor was it phosphorylated or secreted into the culture medium. W52 is part of the hydrophobic core in the propeptide region of cathepsin S comprising two additional tryptophan residues, W28 and W31, also conserved among cathepsin L-like cysteine peptidases. Replacement of the latter with alanine led to consequences similar to those with the W52A mutation. Recombinant propeptides containing mutations of one of the three tryptophan residues were three orders of magnitude less effective as inhibitors of mature cathepsin S than the wild-type propeptide. The results point to a dominant role of the respective hydrophobic stack in the proper folding, transport and maturation of procathepsin S and related cathepsin L-like cysteine proteinases.

The half-life of human procathepsin S.

Two processes, synthesis and degradation, contribute to the intracellular concentration of a protein. As most malignant tumors or tumor cell lines show elevated levels of proteinases, we studied the half-life of a cysteine proteinase, procathepsin S, in order to determine whether tumor cells can regulate their cathepsin concentration via changing the degradation rate of the enzyme. The following procathepsin S species were examined: wild-type procathepsin S in macrophages, recombinant procathepsin S in human embryonic kidney cells (HEK 293 cells), recombinant nonglycosylated procathepsin S in HEK 293 cells, wild-type procathepsin S in the established nonsmall cell lung carcinoma cell line 97TM1. The half-lives of both wild-type procathepsins S expressed in macrophages and in HEK 293 cells were 1 h, whereas that of procathepsin S in the tumor cell line was 2 h. Nonglycosylated procathepsin S was not processed. The degradation of mature cathepsin S proceeded with a half-life of 16-18 h. All cell lines studied secreted substantial amounts of procathepsin S into the culture medium. No further maturation of secreted procathepsin S has been observed in the culture medium. We suggest a disturbed sorting mechanism in tumor cells.

Sorting of non-glycosylated human procathepsin S in mammalian cells.

Cathepsin S, a lysosomal cysteine protease, is synthesized as inactive precursor. It is activated in the lysosomes by a proteolytic cleavage of the propeptide. HEK 293-cells which do not express cathepsin S were transfected with cDNA of either wild type human procathepsin S or a mutant procathepsin S in which Asn of the only glycosylation site in the proregion was replaced by Gln. The cells expressed glycosylated and non-glycosylated procathepsin S, respectively. Large amounts of the precursors were secreted into the culture media by both transfectants. Secreted wild type procathepsin S contained Man-6-phosphate in the oligosaccharide chain. Wild type procathepsin S was activated in the cells but no maturation occurred in the culture media. In vitro processing of glycosylated as well as of non-glycosylated procathepsin S gave fully active enzymes thus indicating that the oligosaccharide chain was not necessary for proper folding. A reuptake of the glycosylated and non-glycosylated procathepsin S by HEK 293-cells could be observed. Small amounts of mature cathepsin S were detected in the lysosomes of the mutant transfectants. Subcellular fractionation showed non-glycosylated procathepsin S in the membrane fraction. Non-glycosylated procathepsin S was bound to the plasma membrane at 2 degrees C, suggesting an additional sorting motif in the cathepsin S molecule besides the Man-6-phosphate residue.

Fructose 2,6-bisphosphate metabolism in Ehrlich ascites tumour cells.

Cancer cell energy metabolism is characterized by a high glycolytic rate, which is maintained under aerobic conditions. In Ehrlich ascites tumour cells, the concentration of fructose 2,6-bisphosphate (Fru-2,6-P2), the powerful activator of 6-phosphofructo-1-kinase, is tenfold increased. The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), synthesizing and degrading Fru-2,6-P2, was characterized. The molecular mass is 120 kDa. The dependence of PFK-2 activity on the substrate concentrations is hyperbolic (Km for Fru-6-P = 0.09 mM; Km for ATP = 0.7 mM), while the dependence of the FBPase-2 activity on the concentrations of Fru-2,6-P2 is sigmoidal (K0.5 for Fru-2,6-P2 = 4 microM). The PFK-2/FBPase-2 activity ratio is 1. PFK-2 activity is inhibited by citrate (I0.5 = 0.17 mM) and phosphoenolpyruvate (I0.5 = 0.08 mM) but only weakly by glycerol 3-phosphate (I0.5 = 1.57 mM). In contrast to the liver enzyme, the activity of tumour PFK-2/FBPase-2 is not influenced by the action of cAMP-dependent protein kinase. The kinetic properties as well as ion-exchange chromatography pattern differ from their normal counterparts in liver and muscle. The properties are likely to contribute to the maintenance of the high glycolytic rate in these tumour cells.

Binding of fructose 2,6-bisphosphate to yeast phosphofructokinase.

Binding of Fru-2,6-P2 to yeast phosphofructokinase was investigated by ultrafiltration technique. Per mol of subunit of phosphofructokinase (M = 100,000) 0.5 moles of Fru-2,6-P2 are bound. The binding curve proceeds cooperatively (nH = 1.8 +/- 0.2). The apparent affinity constant of Fru-2,6-P2 amounts to about 2.25 +/- 0.12 microM. Fru-1,6-P2 decreases the affinity of yeast phosphofructokinase to Fru-2,6-P2. The data can be described by assuming either competition of Fru-2,6-P2 and Fru-1,6-P2 for the same binding site or conformationally mediated interactions.

Kinetic effects of fructose-1,6-bisphosphate on yeast phosphofructokinase.

Yeast phosphofructokinase is known to be effectively activated by fructose-2,6-bisphosphate and AMP. In the absence of the two effectors, fructose-1,6-bisphosphate activates or inhibits the enzyme according to the concentrations of the substrates and of inorganic phosphate. At cellular concentrations of the substrates, however, the effects of fructose-1,6-bisphosphate are negligible. Whereas the activation of the enzyme by AMP is not affected by fructose-1,6-bisphosphate, the latter was found to diminish strongly the activity of the fructose-2,6-bisphosphate-activated enzyme. Inorganic phosphate amplifies the activating effect of fructose-2,6-bisphosphate and augments also the deactivation of the fructose-2,6-bisphosphate-activated enzyme. The deactivating action of fructose-1,6-bisphosphate with respect to fructose-2,6-bisphosphate dominates at low concentrations of fructose-6-phosphate and high levels of ATP and might be of regulatory significance.

Binding of fructose-1,6-bisphosphate to yeast phosphofructokinase.

Binding of fructose-1,6-bisphosphate to yeast phosphofructokinase (EC 2.7.1.11) was measured in a concentration range of 5 to 200 microM of fructose-1,6-bisphosphate with the ultrafiltration technique. At saturation two molecules of fructose-1,6-bisphosphate are bound per subunit of the octameric enzyme. Two distinct types of binding sites have been observed. The high affinity sites (KH = 32.7 +/- 5 microM) exhibit a hyperbolic response in respect to the binding of fructose-1,6-bisphosphate, the low affinity sites (KL = 57.2 +/- 6 microM) show significant positive cooperativity.

Binding of 113mIn ion to human erythrocytes.

The 113mIn ion is tightly bound to hemoglobin; less than 20% of the total amount of 113mIn present in the red cells are attached to the stroma. The extraction of the heme from purified hemoglobin with HCl/acetone mixture showed 99% of the 113mIn activity in the heme fraction. In comparison to the 51Cr and 99mTc isotopes known to be bound to the beta-chain of globin only, 20 and 30%, respectively, of their activities were found in the heme fraction. Acetyl acetone is necessary for effective labelling of erythrocytes with 113mIn. Only 7% of the acetyl acetone applied were found in the cells associated with the heme. It is not involved in the binding of 113mIn to hemoglobin, but facilitates the transport of the 113mIn ions into the cells.

Inhibition of fructose 1,6-bisphosphatase from pig liver by fructose 2,6-bisphosphate.

The inhibition of pig liver fructose 1,6-bisphosphatase by fructose 2,6-bisphosphate has been investigated over a wide range of substrate concentration by measuring the release of labelled inorganic phosphate from [1-32P]fructose 1,6-bisphosphate. The activity of the enzyme can be inhibited completely by fructose 2,6-bisphosphate. The inhibiting effect is most pronounced at low substrate concentrations. The results have been analyzed in terms of a mathematical model assuming a competitive interaction of fructose 1,6-bisphosphate and fructose 2,6-bisphosphate at the catalytic site as well as a synergistic cooperation of the two hexose bisphosphates at an inhibiting allosteric site of the enzyme.

Effects of fructose 1,6-bisphosphate on the activation of yeast phosphofructokinase by fructose 2,6-bisphosphate and AMP.

Fructose 1,6-bisphosphate decreases the activation of yeast 6-phosphofructokinase (ATP:fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11) by fructose 2,6-bisphosphate, especially at cellular substrate concentrations. AMP activation of the enzyme is not influenced by fructose 1,6-bisphosphate. Inorganic phosphate increases the activation by fructose 2,6-bisphosphate and augments the deactivation of the fructose 2,6-bisphosphate activated enzyme by fructose 1,6-bisphosphate. Because various states of yeast glucose metabolism differ in the levels of the two fructose bisphosphates, the observed interactions might be of regulatory significance.

An electron microscopy study of the quarternary structure of yeast phosphofructokinase.

Homogeneous phosphofructokinase from yeast with a molecular weight of 835 000 and composed of eight subunits (four alpha and four beta subunits) was examined by electron microscopy and image computer processing. Three types of particles were seen representing different projections of the phosphofructokinase molecule. A structural model of the enzyme was developed, each of the subunits having two oblong "domains" with a length of 8 and 4.5 nm, respectively. The smaller "domain" is attached to the center of the larger one. One alpha and one beta subunit are regarded to form a heterodimer. Four heterodimers (alpha beta) are tetrahedrally assembled giving rise to point group symmetry 222.

Inorganic phosphate amplifies the effects of AMP and fructose-2,6-bisphosphate on yeast phosphofructokinase.

Inorganic phosphate is an important regulator of yeast phosphofructokinase activity. In the absence of AMP and fructose-2,6-bisphosphate the dependence of enzyme activity on the concentration of inorganic phosphate is sigmoidal. AMP and fructose-2,6-bisphosphate increase the affinity of phosphofructokinase to inorganic phosphate. At low fructose-6-phosphate concentrations inorganic phosphate amplifies the activating effect of AMP and fructose-2,6-bisphosphate. Yeast phosphofructokinase is more sensitive to ATP inhibition in the absence of inorganic phosphate than in its presence. While in the absence of inorganic phosphate a definite ATP inhibition prevails even at high levels of AMP or fructose-2,6-bisphosphate, the ATP inhibition can be relieved by the cooperation of inorganic phosphate and fructose-2,6-bisphosphate. These effects of inorganic phosphate provide an explanation for the stimulation of glycolysis under anaerobic conditions by inorganic phosphate at unchanged concentrations of AMP and fructose-2,6-bisphosphate (Lagunas and Gancedo, Eur. J. Biochem. 137, 479-483 (1983)).

Interaction of ADP and fructose-2,6-bisphosphate with phosphofructokinase-1 from yeast.

ADP was found to activate or, depending on the experimental conditions, to inhibit yeast phosphofructokinase-1. In the absence of AMP and fructose-2,6-bisphosphate ADP increases the apparent affinity of the enzyme to fructose-6-phosphate. At low ATP concentrations the maximum activity with respect to fructose-6-phosphate decreases in the presence of ADP, while at high ATP a significant increase of the maximum activity by ADP is observed. In the presence of fructose-2,6-bisphosphate and AMP only the inhibiting effect of ADP persists. The data may be interpreted in terms of a hyperbolic inhibition mechanism.

Influence of inorganic phosphate on the kinetic properties of yeast phosphofructokinase.

Yeast phosphofructokinase is effectively activated by inorganic phosphate. In the absence of other allosteric stimulators, inorganic phosphate increases the maximum activity of the enzyme only. In the presence of the activators AMP and fructose 2,6-bisphosphate inorganic phosphate causes changes in the maximum activity and the enzyme affinity to fructose 6-phosphate. Inorganic phosphate augments the sensitivity of phosphofructokinase to the activators AMP and fructose 2,6-bisphosphate and increases the respective maximum activities. The extent of activation of the enzyme by inorganic phosphate prevails at low levels of fructose 6-phosphate and high ATP concentrations.

Interaction of Cibacron blue F3G-A with yeast phosphofructokinase.

The binding of Cibacron blue F3G-A to yeast phosphofructokinase was investigated by means of ultracentrifugation. Four moles of Cibacron blue are tightly bound per subunit of phosphofructokinase (dissociation constant = 0.26 microM). This stoichiometry does not correspond to the stoichiometry of ATP binding to yeast phosphofructokinase (two moles of ATP per subunit). Moreover, 32 moles of the dye are bound per subunit of phosphofructokinase to a second class of binding sites with low affinity (dissociation constant = = 53 microM). The action of Cibacron blue on yeast phosphofructokinase cannot be explained completely in terms of its function as ATP analogue.