35S-sulfate Uptake Research Articles

Real-time imaging of 35S-sulfate uptake in a rape seed plant

Abstract We present real-time images of 35S-sulfate uptake in a rapeseed plant visualized by the system we developed. In the leaves of rapeseed plants, 35S accumulated in higher amounts and more rapidly in the more developed leaves. This real-time imaging system can be used to visualize the movement of both 35S and 32P in the same plant. In the pods of rapeseed, images of 35S show that 35S accumulated mostly in the terminal parts; on the other hand 32P, when applied as 32P-phosphoric acid, accumulated in the middle part of the pods.

Hormonal Regulation of Sodium/Sulfate Co-Transport in Renal Epithelial Cells

Abstract. Serum sulfate concentrations are elevated in infants, young children, and pregnant women due, at least in part, to increased renal sulfate reabsorption. Little is known about the effects of hormones, particularly those involved in growth, development, and pregnancy, on renal sulfate reabsorption. The objective of this investigation was to examine the effects of growth hormone (GH), insulin-like growth factor 1 (IGF-1), progesterone (PG), and 17β-estradiol (EST) on renal sodium/sulfate co-transport. 35S-sulfate uptake was determined in Madin-Darby canine kidney (MDCK)/NaSi-1 cells (MDCK cells that have been stably transfected with rat sodium/sulfate co-transporter (NaSi-1) cDNA) and in opossum kidney (OK) cells. NaSi-1 mRNA was determined by RT-PCR and protein levels by ELISA. GH (0.1 nM) significantly increased the sodium/sulfate co-transport in MDCK/NaSi-1 cells up to 35%. IGF-1 induced a concentration-related stimulation of the sodium/sulfate co-transport with a maximal response observed at 1000 nM (59% increase). Sodium-dependent sulfate uptake was significantly increased when cells were preincubated with 10 nM PG, 10 nM EST, or 10 nM PG/10 nM EST up to 41%, 46%, or 39%, respectively. OK cells exhibited endogenous sodium-dependent sulfate transport; significantly increased sodium/sulfate co-transport was also observed in OK cells that were preincubated with GH, IGF-1, and PG/EST, although not with EST alone. The NaSi-1 mRNA and NaSi-1 protein levels were significantly increased in MDCK/NaSi-1 cells treated with 0.1 nM GH, 100 nM IGF-1, 10 nM PG, and/or 10 nM EST compared with control. These results suggest that the increased renal sulfate reabsorption that occurs in neonates, young and pregnant humans, and animals could be mediated by the increased steady-state levels of NaSi-1 mRNA produced by the higher plasma concentrations of GH, IGF-1, or PG/EST.

Growth factors and fibrochondrocytes in scaffolds

Four growth factors, transforming growth factor-β1 (TGF-β1), platelet-derived growth factor-AB (PDGF-AB), insulin-like growth factor I (IGF-I), and basic fibroblastic growth factor (bFGF), were tested at different concentrations for their effects on extracellular matrix (ECM) production in three-dimensional cultures of meniscal fibrochondrocytes. Cells from New Zealand white rabbits were seeded on poly-glycolic acid (PGA) scaffolds and were stimulated with growth factors for three weeks. 3H-proline and 35S-sulfate labels were used to measure uptake of collagen and glycosaminoglycan (GAG) components, respectively. Biochemical assays were performed to measure the total collagen, GAG, and DNA present in the scaffolds at the end of the study. TGF-β1 (10 and 100 ng/ml) stimulated both 3H-proline and 35S-sulfate uptake, showing a dose-dependent response for both and a temporal response for 35S-sulfate uptake. IGF-I (5 ng/ml) and bFGF (25 and 100 ng/ml) showed increases in 3H-proline uptake by the third week of growth factor addition. PDGF-AB did not show notable increases in uptake. Because TGF-β1 (10 and 100 ng/ml) had visibly denser scaffolds, as evidenced by gross microscopy, at 100×, and the strongest uptake responses to both 35S-sulfate and 3H-proline, it appears to be the most effective growth factor for use in scaffold-based approaches to tissue engineer the knee meniscus.

The chondrogenic potential of periosteum decreases with age

Periosteum contains undifferentiated mesenchymal stem cells that possess the potential for chondrogenesis during cartilage repair and in fracture healing. With aging, the chondrogenic potential of periosteum declines significantly. An organ-culture model was used to investigate the relationship between the chondrogenic potential of periosteum and aging. A total of 736 periosteal explants from the proximal medial tibiae of 82 rabbits, aged 2 weeks to 2 years, were cultured in agarose suspension conditions conductive for chondrogenesis, and analyzed using histomorphometry, collagen typing, wet weight measurement, 3H-thymidine and 35S-sulfate uptake, autoradiography, and PCNA immunostaining. The rabbits were skeletally mature by 6 months and stopped increasing in weight by 12 months. Chondrogenesis declined significantly with age ( P<0.0001) and was maximal in the 1.5–2 month-old rabbits. Explants from the 6 month-old rabbits formed 50% less cartilage, and by 12 months chondrogenesis reached a steady state minimal level. In parallel with this decrease in chondrogenic potential similar decreases were measured in 3H-thymidine uptake ( P<0.0001), 35S-sulfate uptake ( P=0.0117), as well as the thickness ( P<0.0001) and the total number of cells in the cambium layer of the periosteum ( P<0.0001). Autoradiography with 3H-thymidine and PCNA immunostaining confirmed the measured decrease in proliferative activity in the cambium layer where the chondrocyte precursors reside, although the percentage of proliferating cells did not change significantly with age. The most dramatic change was the marked decrease (87%) in the thickness and total cell number in the cambium layer of the perisoteum between the 2 and 12 month-old rabbits ( P<0.05). These data confirm a decline in the chondrogenic potential of periosteum with aging. Thus, one possibility for improving cartilage formation by periosteal transplantation after skeletal maturity would be to stimulate an increase in the total number of cells in the chondrocyte precursor pool early during chondrogenesis.

Hormonal regulation of sodium/sulfate co-transport in renal epithelial cells.

Serum sulfate concentrations are elevated in infants, young children, and pregnant women due, at least in part, to increased renal sulfate reabsorption. Little is known about the effects of hormones, particularly those involved in growth, development, and pregnancy, on renal sulfate reabsorption. The objective of this investigation was to examine the effects of growth hormone (GH), insulin-like growth factor 1 (IGF-1), progesterone (PG), and 17beta-estradiol (EST) on renal sodium/sulfate co-transport. 35S-sulfate uptake was determined in Madin-Darby canine kidney (MDCK)/NaSi-1 cells (MDCK cells that have been stably transfected with rat sodium/sulfate co-transporter (NaSi-1) cDNA) and in opossum kidney (OK) cells. NaSi-1 mRNA was determined by RT-PCR and protein levels by ELISA. GH (0.1 nM) significantly increased the sodium/sulfate co-transport in MDCK/NaSi-1 cells up to 35%. IGF-1 induced a concentration-related stimulation of the sodium/sulfate co-transport with a maximal response observed at 1000 nM (59% increase). Sodium-dependent sulfate uptake was significantly increased when cells were preincubated with 10 nM PG, 10 nM EST, or 10 nM PG/10 nM EST up to 41%, 46%, or 39%, respectively. OK cells exhibited endogenous sodium-dependent sulfate transport; significantly increased sodium/sulfate co-transport was also observed in OK cells that were preincubated with GH, IGF-1, and PG/EST, although not with EST alone. The NaSi-1 mRNA and NaSi-1 protein levels were significantly increased in MDCK/NaSi-1 cells treated with 0.1 nM GH, 100 nM IGF-1, 10 nM PG, and/or 10 nM EST compared with control. These results suggest that the increased renal sulfate reabsorption that occurs in neonates, young and pregnant humans, and animals could be mediated by the increased steady-state levels of NaSi-1 mRNA produced by the higher plasma concentrations of GH, IGF-1, or PG/EST.

Changes in proteoglycans of intervertebral disc in diabetic patients. A possible cause of increased back pain.

Characterization of the analytic profile of proteoglycans in the intervertebral discs at L4-L5 of nondiabetic (n = 5) and diabetic (n = 5) age-matched subjects. The discs used were discarded material from operations. To clarify the reason for the higher risk of disc prolapse in diabetic patients. The pathogenesis of diabetes results from a combination of neurologic dysfunctions and a yet undefined metabolic failure, which leads to an abnormal proteoglycan profile. The following methods were used to determine the proteoglycan profile: the measurement of 35S-sulfate uptake per gram wet tissue into sulfated glycosaminoglycan using fresh tissue explants; extraction of proteoglycans by 4 M guanidinium chloride containing protease inhibitors, with further purification by ultracentrifugation on cesium chloride buoyant density gradient under dissociative conditions; total uronic acid and protein contents in the various gradient fractions; assessing the length of sugar side chains of isolated 35Sulfate-glycosaminoglycan molecules by separation of the glycosaminoglycan molecules on a Sepharose 6B-CL column; and paper chromatography of the final digest products of glycosaminoglycan molecules obtained by chondroitinase ABC, a glycosaminoglycan-degrading enzyme. The findings show that discs from normal nondiabetic subjects have 15 times the rate of 35Sulfate incorporation into glycosaminoglycan molecules than do discs of diabetic patients. The proteoglycans of diabetic patients are banded at a lower buoyant density, indicating a lowered glycosylation rate and a lower number of sugar side chains per core protein. In discs of diabetic patients, there is a slight increase in the chain length of chondroitin sulfate. Further analysis of the glycosaminoglycan chains showed a decreased amount of keratan sulfate, compared with that in nondiabetic subjects. However, the total uronic acid content of the disc tissues and the ratio of uronic acid to protein of each fraction were unchanged in diabetic patients versus that in control subjects. Discs in patients with diabetes have proteoglycans with lower buoyant density and substantially undersulfated glycosaminoglycan, which with the specific neurologic damage in these patients, might lead to increased susceptibility to disc prolapse.

Human articular cartilage storage in cell culture medium: guidelines for storage of fresh osteochondral allografts.

The viability of transplanted articular cartilage is one of the determinants of outcome following the transplantation of osteochondral allografts. Disappointing results from cryopreservation have led to the practice of fresh transplantation of articular segments, especially for posttraumatic defects. To date, no studies have demonstrated in vitro viability rates for refrigerated human cartilage awaiting transplantation. This study evaluates the effects of storage on the viability of human chondrocytes using cell culture medium. Human articular cartilage obtained from notchplasties was stored for up to 48 hours in cell culture medium. Radioactive 35S-sulfate uptake was determined at 0, 24, and 48 hours, as a measure of protein, synthesis, and chondrocyte viability. Specimens were stored at 4 degrees C in culture medium. Results showed an average decrease in 35S-sulfate uptake of 0.8% at 24 hours and 6.4% at 48 hours, indicating a high level of chondrocyte viability after refrigeration. Because transplantation typically is performed within 24 hours of tissue harvest, it appears that nearly 100% of chondrocytes should survive fresh transplantation.

THE EFFECTS OF PRENATAL PROTEIN-ENERGY MALNUTRITION ON OSSIFICATION OF FETAL RAT BONES: A BIOCHEMICAL STUDY

In fetal rats whose dams were fed a low-protein diet, 35S sulfate uptake into the growth plate of the long bone and rib was higher than in the control group. The elution pattern of guanidine-HCl extract in gel chromatography revealed that the malnourished group had more high molecular weight proteoglycans in the dissociative condition and a larger aggregated portion in the associative condition than did the control group; however, the same chondroitin-sulfate chain size existed. Calcium content did not differ in both groups. Aggregated proteoglycan or a high molecular weight proteoglycan that existed in the malnourished group probably played an inhibitory role in calcification. Prenatal protein-energy malnutrition may delay the change of proteoglycan character, which could affect mineralization of fetal bones.

Cartilage sulfation inhibitor from rat liver: Partial characterization of properties and biologic action

Cartilage sulfation (somatomedin) inhibitors (CSI) from rat liver produce reversible inhibition of cartilage growth. After gel filtration Sephadex G-200, CSI appear to have MW ∼100,000 and they are urea- and trypsin-labile factors. To explore further the mechanism of CSI action, we used the chick pelvic rudiment bioassay and studied the effect of CSI on the incorporation on 35S-sulfate (proteoglycan synthesis), 14C-leucine (protein synthesis), 3H-uridine (RNA synthesis), and 3H-thymidine (DNA synthesis). Normal rat serum (NRS) significantly stimulated the incorporation of all four isotopes, as expected. After a 24-hour incubation, CSI significantly blunted cartilage stimulation by NRS regarding total isotope uptake (1), 35S-sulfate (NRS, 96 ± 8 mcg/100 mg cartilage dry weight; NRS + CSI, 48 ± 4, mean ± SEM, n = 29, P < .05); and (2) 14C-leucine (NRS, 2,089 ± 172 cpm/mg dry weight; NRS + CSI, 1,102 ± 141, n = 18, P < .05); and (3) 3H-uridine (NRS, 6,711 ± 832 cpm/mg; NRS + CSI, 3,227 ± 425 cpm/mg, n = 18, P < .05); but not (4) 3H-thymidine (NRS, 3,540 ± 620 cpm/mg; NRS + CSI 3,249 ± 285, n = 19). The inhibition of 35S-sulfate and 14C-leucine uptake by CSI was dose-dependent and reversible. For 35S-sulfate, uptake by cartilage incubated with CSI alone for 40 hours was 13 ± 3 μg/100 mg; with CSI for 16 hours then fresh medium with NRS for 24 hours uptake was 39 ± 12, P < .05. Similarly, for 14C-leucine, uptake with CSI alone for 40 hours was 22.8 ± 5.4 × 10 3 cpm/mg; with CSI 16 hours, then NRS 24 hours, uptake was 59.9 ± 9.3 × 10 3, P < .05. We conclude that partially purified CSI profoundly inhibit the incorporation into chick cartilage of 35S-sulfate, 14C-leucine, 3H-uridine, but not 3H-thymidine. CSI action is dose-dependent and reversible. The data suggest that CSI can exert broad antianabolic effects on normal cartilage.

Altered glycosaminoglycan production by HL-60 cells treated with 4- methylumbelliferyl-beta-D-xyloside

Glycosaminoglycans, mainly chondroitin 4-sulfate, are located in the primary granules of human myeloid cells. These polyanionic carbohydrates are believed to play an important role in leukocyte maturation and function. To study the effect of altered chondroitin sulfate metabolism on human promyelocytic leukemia cells, we have treated HL-60 cells with 4-methylumbelliferyl-beta-D-xyloside. beta-D- Xylosides initiate the synthesis of free chondroitin sulfate chains. Cytochemical studies of treated cells demonstrated a marked increase in cytoplasmic granules stained with cationic dyes. This was confirmed by radiolabeled precursor incorporation studies that demonstrated a 344% increase in 35S-sulfate uptake into glycosaminoglycans associated with the cells and a 39% increase in incorporation into glycosaminoglycans released into the media. Chromatographic analyses of these glycosaminoglycans from treated cells demonstrated that the newly formed chondroitin sulfate chains were not attached to protein core and were of shorter length, but of greater charge density than chondroitin sulfate produced by control cells. Thus, beta-D-xyloside appears to alter the protein linkage, chain length, and sulfation of chondroitin sulfate produced by HL-60 cells, and these changes are morphologically evident. These biochemically altered cells may provide important information concerning the role of these macromolecules in myeloid development.

Altered Glycosaminoglycan Production by HL-60 Cells Treated With 4-Methylumbelliferyl-ß-D-Xyloside

Glycosaminoglycans, mainly chondroitin 4-sulfate, are located in the primary granules of human myeloid cells. These polyanionic carbohydrates are believed to play an important role in leukocyte maturation and function. To study the effect of altered chondroitin sulfate metabolism on human promyelocytic leukemia cells, we have treated HL-60 cells with 4-methylumbelliferyl-beta-D-xyloside. beta-D-Xylosides initiate the synthesis of free chondroitin sulfate chains. Cytochemical studies of treated cells demonstrated a marked increase in cytoplasmic granules stained with cationic dyes. This was confirmed by radiolabeled precursor incorporation studies that demonstrated a 344% increase in 35S-sulfate uptake into glycosaminoglycans associated with the cells and a 39% increase in incorporation into glycosaminoglycans released into the media. Chromatographic analyses of these glycosaminoglycans from treated cells demonstrated that the newly formed chondroitin sulfate chains were not attached to protein core and were of shorter length, but of greater charge density than chondroitin sulfate produced by control cells. Thus, beta-D-xyloside appears to alter the protein linkage, chain length, and sulfation of chondroitin sulfate produced by HL-60 cells, and these changes are morphologically evident. These biochemically altered cells may provide important information concerning the role of these macromolecules in myeloid development.

Human fetal cartilage response to plasma somatomedin activity in relation to gestational age.

The response of human fetal costal cartilage (gestational age 12--25 weeks) to plasma containing somatomedin activity has been examined in vitro. Uptake of both 3H-thymidine and 35S-sulfate into the cartilage was increased; the increase in 35S-sulfate uptake was relatively constant (mean = 198% of basal, N = 25) throughout the period studied; however, the increase in 3H-thymidine uptake varied with gestational age, reaching a maximum of about 200% at 15--17 weeks and decreasing thereafter. Such a change in cartilage sensitivity to somatomedins could be an important feature in the pattern of skeletal growth of the human fetus during gestation. The data do not confirm that human cartilage is most sensitive to somatomedins during intrauterine life.

Sulfate Metabolism in Rat Calvaria Cultured Under Vitamin A Deficient Conditions

The metabolism of sulfated glycosaminoglycans (GAG) in bone has been found to be altered in vitamin A deficiency. A neonatal rat calvarium model system was used to determine if these changes are related to catabolic defects. Two day old rat pup calvaria were cultured in media containing serum from A- or A+ rats and radiolabeled sulfate or glucosamine. The incorporation of 35S-sulfate into the GAG fraction of calvaria cultured with A- serum for 48 hours was significantly increased compared to the values found in calvaria cultured with A+ serum (A-, 1,970 +/- 300; A+, 940 +/- 177; X +/- SD). The uptake of 35S-sulfate into the GAG fraction of calvaria cultured with A- serum showed continuous increase over 96 hours,whereas, 35S-sulfate uptake leveled off after 24 hours in the A+ group. There was also a significant increase in [14C]glucosamine uptake into the GAG fractions of calvaria cultured with A-serum (A-, 1,966 +/- 537; A+, 1,662 +/- 244; X +/- SD). To determine if the alteration in metabolism of sulfated GAG was in the biosynthetic or degradative pathways, a chase study was performed in which the calvaria were prelabeled with 35SO4. The rate of tissue loss of 35S-sulfate was lower in the total digests and the GAG fractions from calvaria cultured with A- serum than those cultured with A+ serum. Thus, the alteration in the metabolism of the sulfated GAG resulting from A- culture conditions seems to be defect in the degradative process.

Sites of sulfatation in the chondrocytes of the articular cartilage of the rabbit

35S sulfate uptake by the articular cartilage chondrocytes, from biopsies of rabbit, have been studied by high resolution autoradiography. The Golgi apparatus, rough endoplasmic reticulum, cytosol, cytoplasmic membrane and extracellular space were considered as cell compartments in the quantitative analysis of the autoradiograms. The results obtained show: 1) a high activity of radiosotope incorporation in the Golgi apparatus; 2) a fast rhythm of transfer of the substances labelled in the Golgi apparatus to the cell membrane; 3) significant labelling of the rough endoplasmic reticulum, throughout the experiment. It is concluded: 1) The grains observed in the rough endoplasmic reticulum show a significant radioisotope uptake on this level, and this evidence some sulfotransferase activity. 2) The high 35S sulfate uptake level which is observed in the Golgi apparatus demonstrates that the highest sulfotransferase enzyme activity is located in this cell area, thus showing that the "early" sulfation that began in the rough endoplasmic reticulum was completed by a "late" sulfation in the Golgi apparatus. It is here that complete chondromucoprotein building takes place before being excreted. 3) The high transfer level of the labelled substances from the Golgi apparatus shows that the sulfated product secretion for building the cartilage matrix takes place rapidly since a great label increase can be already observed at the beginning of the chase period in the outer surrounding area of the chondrocyte membrane.

Ultrastructural Autoradiographic Study of the Uptake and Intracellular Localization of 35S-Sulfate by Developing Human Neutrophils

AbstractThe uptake and intracellular localization of 35S-sulfate was studied in developing neutrophils from normal human bone marrow by means of ultrastructural autoradiography in order to identify the pathway and sites of sulfate incorporation into glycosaminoglycans present in selected granule populations in these cell forms. Marrow cells were pulse labeled for 10 min in vitro at 37°C with 200 μCi/ml Na235SO4 in either phosphate buffer (pH 7.4) or RPMI 1640 and cold chased for intervals up to 4 hr. Autoradiographic data indicated that promyelocytes involved in primary (azurophilic) granule synthesis showed a striking and preferential uptake of labeled sulfate with localization of silver grains over the Golgi and formative granules, while 35S-sulfate uptake by myelocytes engaged in secondary (specific) granule formation was minimal. A second period of 35S-sulfate uptake occurred during the final stages of neutrophil maturation extending from the metamyelocyte into the segmented neutrophil stage. 35S-sulfate concentrated in the Golgi and in condensing saccules arising from the concave Golgi face and was subsequently localized in small round to oblong (tertiary) granules formed in these cells. Labeled sulfate uptake by metamyelocytes and segmented neutrophils was appreciably less than noted for neutrophilic promyelocytes. Thus, autoradiography using 35S-sulfate permitted the delineation of three distinct phases of neutrophil maturation. Two of these phases involved the uptake of labeled sulfate which became incorporated into distinct types of cytoplasmic granules reported to contain sulfated glycosaminoglycans, viz., primary granules in promyelocytes and tertiary granules formed during late maturational stages of the neutrophilic leukocyte.

Effect of ascorbic acid on arylsulfatase activities and sulfated proteoglycan metabolism in chondrocyte cultures.

A correlation between increased arylsulfatase activities and decreased sulfated proteoglycan content in human osteoarthritic articular cartilage suggested a possible interrelationship between these parameters. Since we had previously shown that ascorbate caused a decrease in levels of arylsulfatase A and B activities in normal chondrocyte cultures, the validity of the above relationship was examined by measuring the effect of vitamin C on the biosynthesis and distribution of 35S-labeled proteoglycans and arylsulfatase A and B activities in cell extracts of chondrocytes derived from normal and osteoarthritic tissue. Arylsulfatase A and B activities were found to be reduced in the presence of ascorbic acid in all normal and osteoarthritic cell lines examined when measured 3, 6, 10, and 13 days after the introduction of the vitamin in the culture medium. Acid phosphatase activity, on the other hand, was found to be elevated in the presence of ascorbate. The inhibitory effect by ascorbic acid on arylsulfatase activities could be reversed by withdrawing the vitamin from the nutrient medium. Addition of EDTA to the cell extracts before assay also reversed the inhibiton. Sulfated proteoglycan biosynthesis as reflected in 35S-sulfate uptake per milligram of DNA was significantly increased in the presence of ascorbic acid. The distribution of the newly synthesized molecules between the cell layer and medium fractions was altered. In the presence of ascorbate, more deposition into the cell layer of newly synthesized macromolecules occurred. These data suggest an inverse relationship between arylsulfatase activities and the stability of the newly synthesized sulfated proteoglycans in the extracellular matrix.

35S-sulfate uptake by mouse harderian gland: Effect of serum from patients with Graves' disease

Sera from patients with Graves' disease, some having ophthalmopathy, were tested for an effect on the 35S-sulfate uptake by the mouse harderian gland and for the presence of the long-acting thyroid stimulator (LATS). For both tests a twofold concentrate of serum globulin was prepared by precipitation with ammonium sulfate. Of sera from 15 patients with ophthalmopathy, globulin from nine enhanced 35S-sulfate uptake significantly as compared with the effect of globulin from normal human serum. Serum globulin from only one of nine patients with Graves' disease without ophthalmopathy gave a similar result. The activity was found in IgG purified by anion-exchange chromatography. LATS assay of the serum globulin fraction was positive with 12 of the 24 sera but coincided with the harderian-stimulating activity in only four instances. It is concluded that there is a factor, distinct from LATS, in the gamma globulin fraction of serum of patients with ophthalmopathy of Graves' disease that stimulated radiosulfate uptake by the harderian gland of the mouse; the relevance of these data to ophthalmopathy of Graves' disease is discussed.

Studies of homologous immunization with mouse harderian gland

Exophthalmos has been reported to follow immunization of guinea pigs with homologous harderian gland. In this report we describe the effects of immunizing mice with harderian gland. We examined in particular the influence of immunization on the uptake of 35S-sulfate by harderian glands since this is thought to reflect events underlying the production of exophthalmos. Female mice were immunized with homologous harderian gland emulsified in Freund's complete adjuvant and were found to have an increase in the 6-hr 35S-sulfate uptake by the harderian gland; 35S-sulfate uptake by salivary gland and pancreas was unaffected. Injection of 10 mU thyrotropin had no effect on 35S-sulfate uptake by harderian gland of the immunized mice, in contrast to a stimulation of 35S-sulfate uptake by the pancreas of the same animals and by the harderian gland and pancreas of nonimmunized animals. There was a greater concentration of water plus lipid in harderian glands of immunized animals; glycosaminoglycans, measured as uronic acid, also were increased. Histologically the harderian glands of approximately one-third of the sensitized mice showed minimal infiltration with lymphocytes but hemagglutinating antibodies against the gland were present in about half the sera tested. These data show that the harderian gland of mice is influenced by homologous immunization; the relationship of changes in the metabolism of this gland to human exophthalmos remains speculative.

Effect of insulin in vitro on 14C-acetate and 35S-sulfate uptake into aortic mucopolysaccharides in normal and diabetic rats.

SummaryInsulin in vitro failed to stimulate the uptake of 14C-acetate or 35S-sulfate into aortic mucopolysaccharides of normal animals. Aortas from diabetic animals showed normal 14C-acetate uptake which was decreased by the addition of insulin, and a normal 35S-sulfate uptake, which was significantly increased by insulin added in vitro.

Vitamin C deficiency and connective tissue. 3. Determinations of hexosamine, uronic acid, hydroxyproline and uptake of radiosulfate (35S) in granulation tissue.

SummaryIn seven days old granulation tissue, obtained by a technique developed by Rudas, a significant decrease in the content of hydroxyproline representing the content of collagen, was demonstrated during ascorbic acid deficiency. The content of hexuronic acid, a component of acid mucopolysaccharides, was determined by the carbazole and the orcinol methods. By either method a significantly increased content of uronic acid was found during ascorbic acid deficiency. The carbazole to orcinol ratio was increased significantly. The hexosamine content, which is a measure of the content of both acid and neutral mucopolysaccharides, was significantly increased. By contrast, the 35S-sulfate uptake was similar in granulation tissue from normal and scorbutic animals. It is concluded that a ground substance with an increased, and qualitatively altered, fraction of acid mucopolysaccharides is formed in scorbutic granulation tissue.