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Four different experimental designs were selected to study, whether glycosylation of hemoglobin alters susceptibility against proteolytic degradation. We compared the resistance of different hemoglobin fractions against degradation by pure proteolytic enzymes, by liver cell culture, by live organ culture and the resistance of in vitro glycosylated vs. "non-glycosylated" hemoglobin solutions. The hemolysates were characterized by high performance liquid chromatography. The degree of glycosylation of hemoglobin was further estimated by the thiobarbituric-acid assay. From the data presented it is concluded, that the glucose moiety linked to the N-terminal valine and lysine residues of the alpha- and beta chain of the hemoglobin exerts a protective effect against proteolytic cleavage. This could explain the extended lag phase in response of glycosylated hemoglobin to an improved metabolic control in diabetic patients and the discrepancy between the rate of synthesis and the rate of catabolism.

Two newborn infants with congenital toxoplasmosis despite serological testing during pregnancy were observed at our premature and neonatal intensive care unit within a short time of one another. In one case the counter-regulatory late first test, performed only in the 26th week, was positive with a high titre. The immediately recommended treatment was not carried out. Apart from the signs of congenital generalized infection, the newborn infant already manifested chorioretinitis and encephalitis. In the second case the initially serologically-negative pregnant women became infected only during the 35th to 36th week of gestation, around the time of the third serological examination. The child was born with slight signs of general infection, but without CNS involvement. Immediate postnatal treatment resulted in complete cure. This shows that such rare cases which can no longer be detected by serological testing can be treated postnatally with good results.

In order to look for the position of amyloid P in the macromolecular connective tissue and extracellular matrix system, we performed binding studies involving affinity chromatography. Binding studies revealed the strong binding of fibronectin to amyloid P (S-AP). The fibronectin-amyloid P linkage was dissociated after elution with 2 M urea. Heparan sulfate, a major glycosaminoglycan of the extracellular matrix, showed strong binding to S-AP, which was dissociated at 3 M urea. Laminin, collagen type I and type IV, reduced and alkylated glomerular basement membranes as well as the glycosamino-glycans hyaluronic acid and chondroitin-4-sulfate failed to bind to S-AP. Our binding studies show that amyloid P can react strongly with extra cellular matrix proteins and can help to explain the presence of amyloid P in normal connective tissue.

The role of histamine in physiological and pathological states has not been fully elucidated. In the pathological state histamine release can be induced by several mechanisms. The most frequent events are IgE-mediated release, complement-mediated release and release by chemical agents. During inflammation histamine is set free and contributes to the course of inflammatory processes, either enhancing or inhibiting inflammatory reactions. In order to look for a target in the glomerulum we examined H2 receptor binding to isolated glomerular cells using 3H-cimetidine as ligand. Two binding sites were detected, a low affinity site with a kD = 6.3 microM and a high affinity site exhibiting a kD = 0.18 microM. These findings are comparable to properties of 3H-cimetidine receptor binding to brain membranes.

Though there is sufficient evidence that fibronectin is an integral extracellular matrix protein of the normal human kidney, the distribution and the interaction with structural proteins of the kidney have not been resolved. There are disagreements between investigators whether fibronectin is a component of the glomerular basement membrane or linked to it. We have been examining the interaction between the glomerular basement membrane and its components type IV collagen and laminin and fibronectin. Applying affinity chromatography we detected significant interaction between collagen type IV and laminin, and fibronectin on the other hand. The noncovalent interaction between collagen type IV and fibronectin was reversible after elution with 2 M urea, the association between laminin and fibronectin was even stronger and reversible applying 3 M urea for elution. The glomerular basement membrane-fibronectin interaction reflected the laminin pattern though minor binding sites at 1 M urea and 2 M urea could be detected. These interaction studies showed the chemical and thermodynamical possibilities and probability of the glomerular basement membrane and fibronectin linkage.

The modification of haemoglobin A by glucose to haemoglobin A Ic is a classical example for a nonenzymatic glucosylation (n. e. glu.) reaction of a protein. It has been suggested, that various proteins are subjected to this process during hyperglycemia phases, if the protein meets certain steric, electrostatic and biochemical characteristics. In the present report n. e. glu. of haemoglobin, serum protein, collagen, basement membrane protein and alkaline phosphatase is discussed. It is suggested that n. e. glu. might influence protein function and forms thus the biochemical basis for specific complications of diabetes mellitus in an outside pregnancy.

The proteases trypsin, alpha-chymotrypsin and papain were incubated with glucose for a period of 10 days at 37 degrees C and activity was tested in comparison to the enzymes incubated with the puffer solution only without glucose addition. Papain additionally was incubated for 10 days at 37 degrees C with the carbohydrates galactose, sucrose, lactose, glucosamine, galactosamine and mannosamine. While trypsin and chymotrypsin showed no change in enzymatic activity after incubation with glucose, the activity of papain was reduced by 70% to 90% (mean 84%). Incubation with galactose also inhibited papain activity but to a lesser extent (25% to 60%, mean 43%). Incubation with the other carbohydrates failed to inhibit papain activity. The mechanism inferred is nonenzymatic glucosylation of papain possibly as ketoamine linkage at the lysine residues situated close to the active site of papain causing steric or allosteric hindrance of the papain activity. The serine hydrolases trypsin and chymotrypsin without lysine residues near their active sites revealed unchanged activity after incubation with glucose.

Non-enzymatic galactosylation has been investigated by in vitro incubation of red cell haemolysate, a HbAo-preparation and of GBM of healthy children. The effects of non-enzymatic galactosylation of haemoglobin has been studied by high pressure liquid chromatography, the effects of GBM galactosylation by immunoelectrophoresis. Subsequently, the occurrence of elevated values for HbAIa-c and GSP was evaluated in 14 galactosaemic children (11 transferase deficiency, 3 galactokinase deficiency), as well as urinary acid glycosaminoglycae excretion and GBM immunoelectrophoretic mobility in 6 of these 14 children measured. The results were compared to the respective values of healthy control children. After exclusion of significant non-enzymatic glucosylation by measuring postprandial blood glucose values the galactosaemic children showed significantly increased values for HbAIa-c (8.85 +/- 2.0% versus 7.7 +/- 0.3%; p less than 0.02), for GSP (0.43 +/- 0.13 mmol 5-HMF/mg protein versus 0.32 +/- 0.07 mmol 5-HMF/mg protein; p less than 0.005) as well as for urinary acid glycosaminoglycane excretion (45.3 +/- 23.4 micrograms/mg kreatinine versus 9.9 +/- 2.3 micrograms/mg Kreatinine; p less than 0.01). 3 out of the 6 children showed alpha 1-immunoelectrophoretic mobility of GBM antigens which was found also after incubation of GMB with galactose. The other 3 children had alpha 2-immobility, which was found in the healthy controls as well as in the control incubations. The impact of galactose on increased non-enzymatic glycosylation in children with galactosaemia as well as the significance of this finding for diagnostic purposes or for clarifying pathophysiological aspects of the disease remains to be studied further.

Risk of premature birth was evaluated retrospectively in 610 women in the first days after delivery using the prematurity risk score published by Thalhammer 1973. The calculated risk of premature birth was compared than prospectively to postpartal morbidity of the newborn infants as determined by duration of hospital stay, incidence of respiratory distress syndrome, need of artificial ventilation and mortality. A positive correlation could be found between risk of premature birth and postpartal morbidity of the newborn infants especially in small premature infants with birthweights of 2000 grams and less. The same correlation existed also in two groups of infants out of two different obstetric clinics which showed the same distribution of prenatal risks and the same prenatal care frequencies. It clearly becomes evident that infants with the same prenatal risks but good prenatal care during pregnancy had much lower hospital stays, lower respiratory distress frequencies and lower mortality rates than babies delivered from pregnancies badly cared for. These prenatal care related differences in postpartum morbidity again were much more evident in infants out of lower birth weight classes.

As previously shown by two dimensional thin-layer chromatography L-azetidine-2-carboxylic-acid (L-Az) is incorporated into type I skin collagen instead of proline when 3 week old mice are fed with a 0,1% solution of L-Az orally. Ultrastructural investigations did not reveal significant changes in collagen periodicity and on fibril diameter. The collagen fibrils of the upper papillary dermis seemed to be packed more densely, sometimes only one electron dense lamina was seen instead of basal lamina and plasma membrane. The glycosaminoglycane-induced fibrillogenesis was not changed in contrary to the collagen-heat-gelation fibrillogenesis at 37 degrees C, where no gel aggregation could be seen. The reconstruction of native fibres from collagen solutions was disturbed too, several finer precipitated fibrils being detectable. On infrared spectroscopy significant differences in absorption spectra were detected. Correlating with previous results of reduced tensile strength and normal melting point of L-Az collagen we can conclude that L-Az might cause rather intermolecular than intramolecular disturbances of crosslinking.

The collagenase inhibiting effect of erythromycin already observed in vitro was demonstrated also after oral administration of the drug in vivo in one child with epidermolysis bullosa letalis and one child with epidermolysis bullosa dystrophica. Despite inhibition of skin collagenase activity during administration of the drug the frequency of bullous eruptions and healing process of affected skin areas remained unchanged. This suggests no direct causal relation existing between skin collagenase activity and epidermolysis bullosa. Whether increased collagenase activity reflects a secondary reaction of the organism cannot be concluded from this study.

In order to correlate biochemical changes of the hair with physical properties we present a model for the examination techniques. L-azetidine was incorporated into the hair keratin complex and the resulting mechanoelastic properties were determined using the ultramicrohardness testing system on scanning electron microscopy. Structure was investigated by X-ray diffraction and incorporation of L-azetidine was detected by thin-layer chromatography. This system could possibly be introduced for examination of hair changes in humans. 8 white mice, 3 weeks of age, were given L-azetidine-2-carboxylic acid in water (0.1 g/100 ml) as only source of fluid over a period of 5 weeks. They had free access to dry mouse cake only. 8 animals of the same strain, who had free access to tap water and mouse cake and were kept under the same conditions, served as controls. After 5 weeks, the animals were sacrificed and hair was obtained for analyses. 2 dimensional thin-layer chromatography of hair hydrolyzed with 6N HCl at 105 degrees C for 12 hours revealed 2 additional spots in the chromatographic pattern in the experimental animals in comparison with the control group. 1 of the spots was identified as L-azetidine-2-carboxylic acid, while the second spot was possibly a degradation product of L-azetidine on acid hydrolysis at a high temperature. Hair of the animals was put into Mark capillaries and subjected to X-ray diffraction, which showed a markedly disordered orientation of keratin. Impression studies using scanning electron microscopy revealed a remarkably reduced elasticity of hair with incorporated L-azetidine. These findings may be explained on the basis of qualitative or quantitative changes in the helical structure of the keratin complex of hair, which is responsible for the elastic properties, whereas the globular matrix is responsible for the firmness of the hair.

The serum concentrations of thyrotropin (TSH), thyroxine (T4), trijodothyronine (T3) and of 3,3',5', trijodothyronine (reverse T3, rT3) were followed up during the first 4 weeks of life in 5 healthy premature infants and in 6 premature infants with hyaline membrane syndrome and artificial ventilation. TSH and T4 concentrations remained unchanged in both groups. T3 levels increased during the observation period and were significantly lower in the sick prematures on days 1 and 3 (p less than 0,02). T3 and the rT3/T3 ratio was increased in healthy and in the sick prematures, the sick prematures showing higher values, and decreased in both groups during the first week of life. We found no significant correlation between TSH and the peripheral thyroid hormone levels as well as no relation between the respiratory compliance-indicating degree of lung maturity-and the T3 or T4 concentrations. Postpartal T3 or T4 serum levels were correlated significantly with the duration of artificial ventilation. Our data indicate that a "low T3 syndrome" was present in all prematures after birth and that respiratory distress syndrome increased conversion of T4 to rT3. From our results we cannot conclude that substitution therapy with thyroid hormones may be useful in premature infants with the hyaline membrane syndrome.

Recent investigations point to the nonenzymatic glycosylation as a cause of long-term complications in diabetes mellitus. We describe an enzymatic activity that cleaves glucose from the glomerular basement membrane (GBM), present in lysosomal preparations of diabetic lymphocytes. The GBM, nonenzymatically glycosylated or obtained from rats with diabetes, were incubated with enzyme preparations, separated on Sephadex G-25 and applied for glucose measurement on gas chromatography and mass spectroscopy. The lysosomal preparation of diabetic lymphocytes cleaved from rat GBM, which were nonenzymatically glycosylated 300-500 ng glucose/mg GBM protein, from diabetic rat GBM 300 ng glucose/mg GBM protein. A lysosomal preparation of normal lymphocytes failed to do so, indicating enzyme induction in the diabetic state. Control studies with the glycosylated hemoglobin AIc confirmed this finding and showed the specificity of the enzyme, as alpha-glucosidase and beta-glucosidase failed to cleave the N-glycosidic bond between glucose and the protein. The enzymatic activity can be described formally as a N-l-deoxyfructofuranosyl-glucohydrolase, which could be responsible for a potential reversibility of diabetic GBM changes.

8 mice were given L-acetidin-2-carboxylic acid orally over a period of 5 weeks. Another 8 mice served as control animals. Finally, the mice were sacrificed, their hair samples were hydrolized and applied to thin layer chromatography which revealed the incorporation of L-acetidin-2-carboxylic acid into mouse hair expressed by an additional spot. This experimental amino acid metabolic disorder served as a model for the application of infrared spectroscopy for screening hair in order to check metabolic disorders. As the multiinternal reflection-infrared spectra showed significant differences we suggest that infrared spectroscopy can be used as a noninvasive screening technique.

Two peptides, alpha-endorphin and beta-melanocyte stimulating hormone, were nonenzymatically glucosylated, degraded by several proteolytic enzymes and the susceptibility against proteolytic enzymes compared to the peptides in the native, nonglucosylated state. Glucosylation was controlled by thin layer chromatography revealing Rf differences between the native and glucosylated peptides. Quantitative estimation of peptides was performed applying high performance liquid chromatography. The results showed reduced susceptibility of nonenzymatically glucosylated peptides to the proteolytic enzymes (papain, chymotrypsin, trypsin and papain) applied.

In order to elucidate the role of L-acetidine-2-carboxylic acid (L-ac) incorporated into collagen type I of the skin instead of proline, we looked for the mechanoelastic properties of skin. Mice were orally fed with 0, 1% solution of L-ac for 5 weeks, then sacrificed, and type I collagen was extracted. Incorporation of the proline analogue (L-ac) could be shown by two dimensional thinlayer chromatography. The melting point (Tm) of type I collagen was determined by circular dichroism: 36, 5 +/- 1 degrees C for normal collagen, 37 +/- 1 degrees C for L-ac-collagen. This insignificant difference indicates that there was no alteration of the thermal stability of the collagen helix after incorporation of L-ac. Tensile strength was examined on whole strips of skin and worked out at means = 1,00 N/mm, s = 0,20 N/mm, sigma = 0,04 N/mm for normal individuals and means = 0,62 N/mm, s = 0,24 N/mm, sigma = 0,05 N/mm for L-ac-fed animals. The considerable difference could be estimated by the Wilcoxon test (p less than 0,005). As the stability of the collagen helix (shown through melting point determination) has not decreased, the reduced mechanoelastic property of tensile strength seems to be due to intermolecular rather than to intramolecular disturbances of the cross connection of the triple helix.

The effect of the airway-occlusion-technique (AOT) on the cardiac- and respiratory frequency, respiratory compliance and pO2 was studied in 5 healthy and 7 cardio-respiratory ill newborns. During a mean study duration of 10.8 minutes (11 occlusions) none of the variables mentioned changed significantly. We conclude that the non-invasive AOT, which informs about lung mechanics and respiratory control, carries no risk for the newborn infant.