Two novel matrix proteins isolated from articular cartilage show wide distributions among connective tissues

Two proteins of Mr = 58,000 and 59,000, respectively, were purified from 4 M guanidinium chloride extracts of articular cartilage by dissociative CsCl-density gradient centrifugation followed by gel chromatography on Sephadex G-200 and ion exchange chromatography on DEAE-cellulose. The two proteins differ in ionic properties and only the one with Mr = 59,000 bound to the ion exchanger. Although the two proteins showed dissimilar peptide patterns after proteolysis, their amino acid composition was similar, with very high contents of leucine and aspartic acid/asparagine. The two proteins showed no cross-reactivity in radioimmunoassays. By use of these assays, the proteins were demonstrated in extracts of most connective tissues, with high contents of about 0.1% of tissue wet weight determined in several types of cartilage. Among the non-cartilage connective tissues, tendon and sclera had the highest contents of the proteins, i.e. about 0.1% of the tissue wet weight. Bone extracts, on the other hand, contained insignificant amounts of the proteins. Only the Mr = 59,000 protein was detected in serum, its concentration being about 33 micrograms/l. Both proteins were shown to be localized in the extracellular matrix of cartilage, predominantly in the territorial matrix, by using indirect immunofluorescence.     

Sequential Hormonal Changes in the Postpartum Dairy Cow

Peripheral plasma levels of 15-keto-13,14-dihydro-PGF2α, progesterone, Cortisol, LH and prolactin were studied in 6 primiparous postpartum dairy cows. The cows were followed by hormone measurements and clinical examinations from parturition until pregnancy was established. Blood was collected 3 times per day. The cervix, uterus and ovaries were examined by rectal palpation at 6–10 days intervals. The cows were observed for signs of oestrus twice daily and were additionally teased with a bull to provoke standing heat. Four cows had a normal parturition and dropped their fetal membranes shortly afterwards. (NR group). The remaining 2 retained their fetal membranes for more than 24 h following parturition (RFM group). One out of 6 cows showed standing oestrus at the first ovulation, 4 animals were in oestrus at the second ovulation and all cows showed signs of oestrus at the third ovulation. Although the length of the first luteal phase varied from 9 to 22 days a corpus luteum was in all cases palpated. The secretion of progesterone during the first luteal phase was terminated by a PGF2α release. A significant difference in 15-keto-13,14-dihydro-PGF2α levels between the 2 groups was found on days 0–4 (2.39 vs 6.87 nmol/1 at Ρ < 0.06). Postpartum prostaglandin F2α release as reflected by the level of 15-keto-13,14-dihydro-PGF2α lasted shorter in the NR group than in the RFM group (15–17 vs 21 days). Significant positive correlations between 15-keto-13,14-dihydro-PGF2α and Cortisol as well as between prolactin and Cortisol during the first 24 days postpartum were noted only in cows having normal parturition. The most pronounced daily prolactin variations occurred during the second luteal phase (NR group), when a significant difference between the times 8.00, 12.00 and 15.00 was recorded (14.7, 31.5 and 19.7 μg/l, respectively). Moreover, a partial negative correlation between log value of prolactin and arithmetical value of LH was found in these cows only during the first luteal phase after parturition.     

Immunocytochemical demonstration of tissue-type plasminogen activator in endocrine cells of the rat pituitary gland

We immunocytochemically stained rat pituitary glands using antibodies against plasminogen activators of the tissue type (t-PA) and the urokinase type (u-PA). A large population of endocrine cells in the anterior lobe of the gland displayed intense cytoplasmic immunoreactivity with anti-t-PA. In some areas of the intermediate lobe we found a weak staining, and we observed weakly staining granular structures in the posterior lobe. Controls included absorption of the antibodies with highly purified t-PA. In addition, SDS PAGE followed by immunoblotting of pituitary gland extracts revealed only one band with an electrophoretic mobility similar to that of t-PA when stained with anti-t-PA IgG. No u-PA immunoreactivity was detected in the rat pituitary gland. Sequential staining experiments using antibodies against growth hormone and t-PA demonstrated that the t-PA-immunoreactive cells constitute a large subpopulation of the growth hormone-containing cells. These findings represent the first direct evidence for the presence of t-PA in cell types other than endothelial cells in the intact normal organism. In this article we discuss the implications of the results for a possible role of t-PA in the posttranslational processing of prohormones.     

Reduction of postischemic edema with hyperbaric oxygen

In recent years, reports have shown positive effects of hyperbaric oxygen (HBO) treatment in posttraumatic circulatory insufficiency of the extremities. A tourniquet model for temporary ischemia was used to examine such treatment in rats. The circulation of the rat hindlimb was interrupted for 3 hours, while the contralateral uninjured leg served as control. There was a significant (p less than 0.001) postischemic edema in the tourniquet leg up to 48 hours after restoration of circulation. One group of animals received treatment with hyperbaric oxygen at 2.5 atmospheres absolute (ATA) for 45 minutes after release of the tourniquet. This significantly reduced (p less than 0.001) the postischemic edema, and the reduction persisted for 40 hours after the last treatment. It is concluded that hyperbaric oxygen reduces postischemic edema. Hyperbaric oxygen may therefore be useful as an adjuvant in the treatment of acute ischemic conditions when surgical repair alone fails or is not sufficient to reverse the ischemic process.     

Simplified purification and testing of colloidal gold probes

A novel efficient method for purifying and testing colloidal gold probes has been developed. The method consists of concentrating colloidal gold particles conjugated to IgG or protein A in dialysis bags over silica gel and purifying them by gel chromatography on small columns of Sephacryl S-400. Fractions collected are tested by paper immunocytochemical models. Comparisons to gold probes purified by conventional ultracentrifugation documents that ultrastructural staining intensities and total yield of gold probes is the same, but that the chromatographically purified gold probes are less prone to aggregation or clumping. The method has been extensively used for preparing conjugates of 5, 10 or 15 nm gold particles with antirabbit immunoglobulins but has also been exploited for preparing streptavidin-gold conjugates, protein A-gold conjugates and antirabbit immunoglobulin-silver conjugates.     

Activation and growth requirements for cytotoxic and noncytotoxic T lymphocytes

The number and nature of the "signals" required for lymphocyte activation have been so repetitively and academically discussed over the last 15 years that both the readers and the authors appear exhausted by such exercises. Yet, what may be considered to be the essential question, the basis for self-nonself discrimination, remains to be clarified. Since it has been established that clonal expansion and maturation to effector functions are brought about by polyclonally ("immunologically nonspecific") active factors, it is obvious that the crucial "step" in this context is the initial interaction of antigen with specific receptors of immunocompetent lymphocytes. This initial discriminatory event appears to proceed differently on the various cell subsets. We first deal with the mechanism of induction and growth of cytotoxic-T-lymphocyte precursors, and then discuss the inductive requirements leading to proliferation of T helper cells.     

Photosynthesis and nitrogen utilization in exponentially growing nitrogen-limited cultures of Lemna gibba

The photosynthetic performance and nitrogen utilization of Lemna gibba L. G3 adapted to limited nitrogen supply was studied. The plants were adapted to two levels of nitrogen limitation where the nitrogen addition rates were calculated to sustain relative growth rates (RGR) of 0.15 day^?1 and 0.25 day^?1, respectively. The photosynthetic performance of these cultures was compared to nitrogen-sufficient cultures with an average RGR of 0.32 day^?1. Plants transferred from nitrogen-sufficient conditions attained RGR values corresponding to the nitrogen addition rates after 6 to 10 days. Light-saturated net photosynthesis declined during adaptation according to the drop in growth rate, and a concomitant decrease in the respiration rate was recorded. The efficiency of net photosynthesis on a dry weight basis increased with increased nitrogen supply, whereas it was the same in all cultures when expressed on a chlorophyll basis. The light compensation point was unaffected by the nitrogen regime. Limited nitrogen supply resulted in an increased proportion of dry matter in the roots, which led to decreased leaf area ratios. The net assimilation rates also decreased, but not to the same extent as the leaf area ratios. Growth-limiting amounts of nitrogen were added to the cultures once daily, and the net influx of N was higher than the requirement for N, also in adapted cultures with a steady growth rate. This resulted in transient, periodic fluctuations in the NO₃^?, NH₄⁺ and amino acid pools. Also the rates of NO₃^? reduction and NH₄⁺ assimilation fluctuated as did the amino acid assimilation which paralleled NH₄⁺ assimilation. The role of flux rates over the plasmalemma and tonoplast for control of nitrogen assimilation rates are discussed.     

immunocytochemical localization of urokinase-type plasminogen activator in lewis lung carcinoma

The invasively growing and metasizing Lewis lung carcinoma consistently contained urokinase-type plasminogen activator (u-PA) enzyme activity. When investigated immunocytochemically with antibodies against u-PA, different parts of individual tumors showed a pronounced heterogeneity in staining intensity. Strong staining was found in areas with invasive growth and degradation of surrounding normal tissue, while other areas were completely devoid of staining. Immunoreactivity occurred both with a perinuclear cytoplasmic localization in tumor cells and associated with apparently extracellular material. SDS PAGE of tumor extracts, under both reducing and nonreducing conditions, followed by immunoblotting, showed only one immunocytochemically stainable band with an electrophoretic mobility corresponding to that of purified proenzyme to u-PA, while no two-chain u-PA was detected. This indicates that the major part of the activator in Lewis lung carcinoma is present as one-chain pro-u-PA.     

Synthesis of mannose oligosaccharides via reversal of the α-mannosidase reaction

Summary Three naturally occurring isomers of the disaccharideO--d-mannosyl-d-mannoside were synthesized by reversing the hydrolytic activity of jack bean -mannosidase at 75°C in a very high concentration of mannose. Higher oligosaccharides were also obtained at the later stages of the reaction. The maximum total yield of disaccharides was 37% (w/w) based on the total amount of saccharides.     

Identification of the stable free radical tyrosine residue in ribonucleotide reductase.

The small subunit of iron-dependent ribonucleotide reductases contains a stable organic free radical, which is essential for enzyme activity and which is localized to a tyrosine residue. Tyrosine-122 in the B2 subunit of Escherichia coli ribonucleotide reductase has been changed into a phenylalanine. The mutation was introduced with oligonucleotide-directed mutagenesis in an M13 recombinant and verified by DNA sequencing. Purified native and mutant B2 protein were found to have the same size, iron content and iron-related absorption spectrum. The sole difference observed is that the mutant protein lacks tyrosyl radical and enzymatic activity. These results identify Tyr122 of E. coli protein B2 as the tyrosyl radical residue. An expression vector was constructed for manipulation and expression of ribonucleotide reductase subunits. It contains the entire nrd operon with its own promoter in a 2.3-kb fragment from pBR322. Both the B1 and the B2 subunits were expressed at a 25-35 times higher level as compared to the host strain.