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Biomedical subjects

F H Wezeman

Publications and source records attributed to F H Wezeman.

At least 19 recordsLinked to original sources
PubMed2001

Bone marrow triglyceride accumulation and hormonal changes during long-term alcohol intake in male and female rats.

BACKGROUND: Chronic alcohol consumption may influence the metabolism of adipocytes, the most abundant stromal cell phenotype in bone marrow, and promote bone marrow triglyceride accretion. METHODS: Male and female rats 35 days old were fed the Lieber-De Carli liquid diet containing 36% of the calories as alcohol and were compared with pair-fed rats given an isocaloric liquid diet in which maltose-dextrin substituted for the calories supplied by alcohol. Other control rats were fed chow ad libitum. The rats were maintained on these diets for 64 days, after which the femurs were recovered and examined. RESULTS: End weights of male and female alcohol-fed rats were significantly lower than both control groups. Femur diaphyseal bone marrow triglyceride levels were significantly increased in alcohol-fed male and female rats compared with both control groups. Femur bone marrow cavity diameters were significantly increased and cortical thickness was significantly decreased by alcohol in both males and females. Serum insulin levels were significantly decreased by alcohol only in female rats compared with the ad libitum but not the pair-fed control group, and insulin-like growth factor-1 levels were significantly reduced in male and female rats given the alcohol diet compared with both controls. Male testosterone and female estradiol levels remained unchanged. Male estradiol levels were significantly increased by alcohol compared with both controls, and female progesterone levels were significantly reduced by alcohol compared with pair-fed rats. Whereas female leptin levels were unchanged by alcohol, male leptin levels were significantly increased by alcohol compared with pair-fed rats. CONCLUSIONS: Hormonal and growth factor changes during chronic alcohol consumption accompany triglyceride accumulation in diaphyseal bone marrow and may parallel the effects of alcohol on mesenchymal stem cells and the balance between osteogenic and adipogenic lineages and their cellular progenies.

Alcohol Drinking↗
PubMed2000

Developmental toxicity of valproic acid during embryonic chick vertebral chondrogenesis.

STUDY DESIGN: Valproic acid (2-n-propylpentanoic acid, VPA), an anticonvulsant drug, was studied for its effects on cartilage matrix gene expression using dot blot hybridization with cDNA probes during early chondrogenesis in the developing lumbar spine. OBJECTIVES: To determine the noncytotoxic effects of VPA on vertebral chondrocytes at various stages of embryonic chick spine development. SUMMARY OF BACKGROUND DATA: Clinical and epidemiologic studies have indicated that maternal use of valproic acid during early pregnancy causes an increased risk for spina bifida. METHODS: The sequence of chondrogenesis in the chick lumbar region was determined for stages HH23-32 and stage-correlated with matrix gene expression by dot blot hybridization analysis using cDNA probes for type II collagen, type IX collagen, and cartilage-specific aggrecan core protein. RESULTS: The mesenchymal stage of lumbar chondrogenesis in the chick spine occurs from HH23-26, the prechondrogenic stage between HH27-28, and the chondrogenic period from HH29-32. Stabilization of the cartilage phenotype in the lumbar region is evident at HH-29. Type IX collagen and aggrecan core protein mRNA levels were significantly reduced (P < 0.01) after 48 hours of incubation of the lumbar spines from HH29-31 in the presence of 200 microg/mL and 300 microg/mL valproic acid. Lumbar spines cultured for an additional 24 hours after removal of valproic acid showed only an isolated instance of escape from the inhibitory action of 200 microg/mL valproic acid for type IX collagen at HH31. CONCLUSIONS: Valproic acid significantly alters cartilage matrix gene expression during embryonic lumbar vertebral chondrogenesis. The alteration in gene expression for critical matrix proteins during vertebral chondrogenesis may be related to mechanisms underlying the failure of neural arch development in lumbar spina bifida.

Abnormalities, Drug-Induced↗
PubMed2000

Alendronate administration and skeletal response during chronic alcohol intake in the adolescent male rat.

Alendronate is an aminobisphosphonate that inhibits bone resorption in osteoporotic humans and rats but does not induce osteomalacia. Several bisphosphonates, including alendronate, also have direct positive actions on osteoblasts, bone formation, and mineralization. We studied the effects of alendronate on skeletal development in adolescent male rats during chronic alcohol intake. Four groups of age- and weight-matched male Sprague-Dawley rats (35 days of age) were fed the Lieber-DeCarli diet containing 36% of calories as EtOH (E), the EtOH diet plus 60 mg/kg alendronate (EA) every other day intraperitoneally (ip), an isocaloric diet (I), or the isocaloric diet plus 60 mg/kg alendronate (IA) every other day ip. Body weight, femur length, serum levels of osteocalcin (OC), insulin-like growth factor 1 (IGF-1), testosterone, and luteinizing hormone (LH); femur distal metaphyseal and middiaphyseal bone mineral density (BMD) and tibial metaphyseal gene expression for alpha-1-type I collagen (Col I), OC, and bone alkaline phosphatase (AP); and femur strength by four-point bending to failure were measured after 28 days of feeding and alendronate injections. Serum alcohol levels at death were 156 +/- 13 mg/dl (E) and 203 +/- 40 mg/dl (EA). Alendronate given to alcohol-fed rats increased metaphyseal BMD by more than 3-fold over rats fed alcohol alone. Alendronate given to isocaloric pair-fed rats increased metaphyseal BMD by more than 2.5-fold over rats fed the isocaloric diet alone. Cortical BMD was reduced by alcohol but was increased by alendronate. Alcohol consumption reduced serum IGF-1 levels, and alendronate increased IGF-1 levels in alcohol-fed rats. Serum OC, testosterone, and LH were unaffected by alcohol and alendronate. Quantitative dot blot hybridization using rat complementary DNA (cDNA) probes and normalization against 18S subunit ribosomal RNA (rRNA) levels revealed no changes in tibial metaphyseal gene expression for type I collagen, osteocalcin, or alkaline phosphatase. Alcohol significantly reduced the biomechanical properties of the femurs that were partially compensated by alendronate. Chronic alcohol consumption uncouples formation from ongoing resorption, and resorption is inhibited by alendronate. However, alendronate's positive effects on osteoblast-mediated mineralization during chronic alcohol consumption point to the potential use of bisphosphonates in the treatment of decreased bone formation secondary to alcohol-induced diminished osteoblast function.

Alcoholism↗
PubMed1999

Chronic alcohol consumption during male rat adolescence impairs skeletal development through effects on osteoblast gene expression, bone mineral density, and bone strength.

BACKGROUND: The effect of chronic alcohol ingestion on bone formation is mediated through its direct actions on osteoblasts. The affected population of mature osteoblasts declines in both number and function resulting in decreased cancellous bone volume and cortical bone strength. Although the mechanism of action on osteoblasts is unknown, alcohol alters osteoblast gene expression and matrix synthesis. METHODS: Male rats consuming alcohol (EtOH) daily for 60 days from 35 days of age until 95 days of age (unrecovered group) were compared to rats switched to a regular diet of rat chow without EtOH for an additional 90 days (recovered group). The effects of chronic dietary EtOH on skeletal development during adolescence were examined in the unrecovered and recovered rats by hormonal analysis, bone mineral density determination, bone histomorphometry, metaphyseal gene expression for osteoblast-specific proteins, and biomechanical analysis. RESULTS: The unrecovered EtOH imbibing rats weighed less than their paired isocaloric-fed and ad libitum mates. Statistically significant reductions occurred in femur lengths in the unrecovered EtOH-fed group compared to controls. Serum testosterone levels were significantly decreased by EtOH consumption but returned to higher normal levels during the recovery period. Serum insulin-like growth factor-1 (IGF-1) levels were unaffected by EtOH. Serum osteocalcin levels in the unrecovered EtOH-fed group were higher than those in the recovered group but EtOH intake did not elevate the unrecovered levels compared to isocaloric or ad libitum control rats. Quantitative computed tomography (QCT) determination of bone mineral density (BMD) revealed a statistically significant reduction only in the distal femur metaphysis in the unrecovered EtOH-fed rats. BMD increased during recovery in the distal femur metaphysis and femur mid-cortex. Image analysis of midsagittal sections of the proximal tibial metaphysis of unrecovered rats revealed reductions in cancellous area, trabecular cellularity and thickness, and increased trabecular separation. Cortical widths were significantly reduced by chronic EtOH consumption. These changes remained statistically significant at the end of the recovery period. Four-point biomechanical testing of femurs from EtOH-fed and control unrecovered groups revealed significant reductions in cortical strength, energy-to-failure, and stiffness. These cortical characteristics returned to normal values with abstinence. Tibial metaphyseal alpha-1 type I collagen and osteocalcin mRNA expression levels were significantly elevated above the paired isocaloric control levels after 60 days of EtOH consumption. Metaphyseal alkaline phosphatase mRNA levels remained unaltered by EtOH consumption in the unrecovered group. After 90 days of abstinence alpha-1 type I collagen and alkaline phosphatase gene expression levels remained significantly elevated over the isocaloric and ad libitum control levels (collagen) and the isocaloric control value (alkaline phosphatase). However, metaphyseal osteocalcin mRNA levels declined to normal levels during abstinence. CONCLUSIONS: Chronic consumption of EtOH during the peripubertal period of skeletal growth leads directly to decreased metaphyseal and cortical bone mediated through effects on osteoblasts. Removal of EtOH from the diet is accompanied by incomplete restoration of normal bone metabolism during skeletal growth.

Age Factors↗
PubMed1998

Morphological foundations of precartilage development in mesenchyme.

Hyaline cartilage is archetypic for the appendicular skeleton and the vertebral column. It arises from pluirpotential mesenchymal ancestor cells that remain morphologically undifferentiated prior to a localized cell aggregation in specific regions destined to undergo chondrogenesis. The critical ultrastructural studies of limb bud mesenchymal differentiation prior to, during, and after aggregation were largely completed during the 1970s. These studies accurately and reproducibly described the changes in the cells and matrix with reference to the developmental stages of the embryonic chick and mouse. Collectively, the morphological literature concerning mouse and chick chondrogenesis is in fundamental agreement on the timing and sequence of cell and matrix changes. The morphological observations are foundational and are now extensively correlated with the molecular events of cartilage differentiation.

Animals↗
PubMed1997

Tibial segmental defect repair: chondrogenesis and biomechanical strength modulated by basic fibroblast growth factor.

BACKGROUND: The effect of recombinant human basic fibroblast growth factor (bFGF) on cartilage development and bone biomechanical strength during healing of a tibial segmental defect was studied in the rat. Two reports on the effect of basic FGF administration during fracture healing and several reports on the effects of acidic FGF have documented different responses of callus cartilage to this important growth factor. This is the first report of the effect of bFGF on cartilage formation in the healing of a grafted segmental defect in the rat. METHODS: The tibiae of 80 male rats underwent segmental resection of the mid-diaphyseal region. One-half of this group consisted of controls that received insertion of an intramedullary wire with a coralline hydroxyapatite graft and Gelfoam without bFGF. The tibiae of the other half were treated identically but had the Gelfoam impregnated with 1 microgram bFGF. Animals were killed at 2, 4, and 8 weeks postoperatively. Histological sections were stained with toluidine blue to differentiate the cartilage. Areas of metachromatically stained extracellular matrix, cell areas, cell size, and cellularity were quantified by using image analysis. Unfixed treated and control tibiae were tested for bone failure strength by using four-point bending on an Instron machine. RESULTS: Control bone failure strength was significantly greater than bFGF-treated bones at 2 weeks and energy-to-failure was significantly decreased in treated bones at 2 weeks. Although strength increased with time in all groups, treated groups at 4 and 8 weeks did not differ from controls. Basic FGF treatment promoted an increase in the development of normal hyaline cartilage and vasculogenesis at 2 weeks as compared with controls. Total cartilage declined over time in all groups. Average cell size and cell number did not change with either treatment or time. Bone formation and healing was equivalent in treated and control groups at 8 weeks. CONCLUSIONS: The results indicate that bFGF released directly and initially but not continuously exerts a transient positive effect on hyaline cartilage formation at the expense of repair site strength and does not accelerate healing.

Animals↗
PubMed1997

Immunohistochemical localization of fibroblast growth factor-2 in normal and brachymorphic mouse tibial growth plate and articular cartilage.

Epiphyses of the proximal tibiae of 7-week-old normal and homozygous recessive brachymorphic mice (bm/bm) were immunostained using a monoclonal antibody to basic fibroblast growth factor to determine its expression in growth plate cartilage, osteoblasts on the surfaces of the primary spongiosa and articular cartilage. In the normal growth plate the immunoreactive factor was present in chondrocytes of the proliferating and upper hypertrophic zones but absent from lower hypertrophic chondrocytes. Immunostaining was present only in the territorial extracellular matrix immediately adjacent to the chondrocytes of the proliferating and upper hypertrophic zones. Osteoblasts of the primary spongiosa stained heavily in normal mice. Strong staining was observed in intermediate zone articular chondrocytes. Cells in the superficial layer of articular cartilage were unstained. The extracellular matrix of the articular cartilage was completely free of immunostaining. In contrast, the reduced size of bm/bm growth plates was accompanied by significantly reduced staining intensity in proliferating and upper hypertrophic chondrocytes, and staining was absent from the territorial extracellular matrix of all zones of the bm/bm growth plate. Osteoblasts of the primary spongiosa of bm/bm mice stained less than those of normal mice. Articular cartilage chondrocytes in the intermediate zone stained with less intensity in bm/bm mice, and the cells of the superficial layer were unstained. The extracellular matrix of bm/bm articular cartilage was completely free of staining. Brachymorphic epiphyseal growth plate and articular chondrocytes, and osteoblasts in the primary spongiosa, express reduced amounts of immunoreactive fibroblast growth factor-2. This phenotypical characteristic may be associated with abnormal endochondral ossification and development of bone in brachymorphic mice.

Animals↗
PubMed1994

Anesthetic agents modify tissue proteinase inhibitor content and tumor behavior.

Anesthetic agents may modify the tissue content of low molecular weight proteinase inhibitors (mol wt less than 50,000) and affect the colonization and proliferation of B16-F10 melanoma cells in lungs. Lungs of female mice exposed to halothane in oxygen had significantly greater low molecular weight proteinase inhibitor activity than those from unexposed female mice. The amount of activity in male mice similarly exposed did not differ from unexposed controls. Lungs of exposed female mice also had greater activity as compared with males similarly exposed. No differences occurred in identical experiments with ketamine. High-performance liquid chromatography revealed that the inhibitor in all groups of both sexes shared a major peak with a molecular weight similar to that of the trypsin inhibitor aprotinin. Female mice bearing B16-F10 melanoma cells and exposed to halothane in oxygen had significantly more small lung tumor nodules than tumor-bearing female mice injected with ketamine. However, the total number of nodules did not differ between the two groups. These observations support the hypothesis that stimulation of proteinase inhibitory activity by halothane in oxygen may be responsible for an inhibition of tumor cell proliferation, resulting in smaller tumor nodules and no effect on the incidence of colonization.

Animals↗
PubMed1990

Osteoblast low-molecular-weight proteinase inhibitor. I. Isolation and characterization of activity from osteoblastic cells and bone.

Isolated mouse calvarial cells having phenotypic characteristics of osteoblasts, mouse parietal bone segments, mouse serum, and control mouse lung fibroblasts were extracted in NaCl and ultrafiltered to recover final concentrates having nominal molecular weights between 50,000 and 1000 daltons. Final concentrates of osteoblasts and bone but not of serum or control fibroblasts were positive for the inhibition of trypsin degradation of fibrin. Osteoblast final concentrates inhibited trypsin hydrolysis of the synthetic substrate p-tosyl-L-arginine methyl ester. Osteoblast and bone final concentrates comigrated with Trasylol but were electrophoretically distinct from alpha 1-antiproteinase. Final concentrates of osteoblast and bone extracts did not inhibit tadpole collagenase using the [3H]glycine-labeled diffuse chick collagen fibril assay. High-performance liquid chromatography (HPLC) of osteoblast final concentrates after purification using immobilized trypsin affinity chromatography revealed the presence of a major peak that was positive for the inhibition of trypsin. Molecular weight determination by HPLC indicated that the inhibitor(s) range in nominal molecular weight from 4300 to 5100 daltons. The presence of low-molecular-weight serine proteinase inhibitory activity in bone suggests its participation in the regulation of bone resorption through the regulation of enzyme activation of collagenase, and possibly its role in defense against bone matrix enzymatic degradation during tumor cell invasion.

Animals↗
PubMed1987

Cytochemical localization of tartrate-resistant acid phosphatase, alkaline phosphatase, and nonspecific esterase in perivascular cells of cartilage canals in the developing mouse epiphysis.

Cytochemical localization of tartrate-resistant acid phosphatase (TRAP), tartrate-sensitive acid phosphatases (TSAP), alkaline phosphatase, and nonspecific esterase was used to characterize perivascular cells within cartilage canals. In the distal femoral epiphyses of 5- to 7-day-old mice, three stages of canal development can be distinguished, and at each developmental stage different perivascular cells were present with morphological characteristics of degradative cells. Vacuolated cells resembling macrophages, fibroblastic cells, and chondroclasts were present adjacent to the matrix in superficial, intermediate, and deep canals, respectively. In order to characterize these perivascular cells cytochemically, nonspecific esterase and TSAP staining was used to identify macrophages, alkaline phosphatase staining was used to identify fibroblastic cells, and TRAP staining was used to identify chondroclasts. There were no cells present in the canals at any developmental stage that were positive for TSAP or strongly positive for nonspecific esterase, placing doubt on the identity of the vacuolated cells as macrophages. Alkaline phosphatase-positive perivascular cells were present in the intermediate and deep canals adjacent to matrix containing alkaline phosphatase-positive chondrocytes. These alkaline phosphatase-positive cells were found in the same location within canals as the fibroblastic cells. Tartrate-resistant acid phosphatase was localized in chondroclasts at the tips of deep canals but was not confined exclusively to chondroclasts. Except for the very early stage of canal development prior to chondrocyte hypertrophy, TRAP-positive cells were present at the tips of superficial and intermediate canals as well as at the tips of the deep canals.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗
PubMed1987

Morphometric analysis of cartilage canals in the developing mouse epiphysis.

Cartilage canal development in the distal femoral epiphysis of 5- to 7-day-old mice can be divided into three stages as previously described [Cole and Wezeman, Am. J. Anat. 174: 119-129, 1985]. Using this model, a morphometric analysis of canal volume density at the three stages of development was performed and provided evidence that canal formation significantly exceeds epiphyseal growth. These data are consistent with initial canal formation by invasion rather than by inclusion.

Animals↗
PubMed1986

Concomitant analysis of osteoblastlike cell migration and proliferation on a serum-enriched growth surface.

Osteoblastlike cell migration and accompanying proliferation on a growth surface precoated with fetal calf serum (FCS) was quantified using a modification of the chemotactic model of Alessandri et al. (1983) and autoradiography. Culture dishes were precoated with 1%, 10%, or 100% FCS and were overlaid with agar. Three-millimeter-diameter wells were cut and first-passage osteoblastlike cells in serum-free medium were seeded into the wells. At 12 and 48 hours, outward migration was quantified by measuring (1) the distance osteoblastlike cells had migrated peripheral to the well margin, and (2) the number of osteoblast-like cells peripheral to the well margin. The data indicated that the migration of osteoblast-like cells was related to time and FCS concentration. More cells migrated a further distance at 48 hours than at 12 hours. In addition, with greater FCS concentrations, osteoblastlike cell migration increased; 3H-thymidine pulse labelling showed no incorporation of label into osteoblastlike cells at 12 hours. However, pulse labelling after 48 hours demonstrated that a small number of nuclei peripheral to the well margin were labelled. The data suggest that proliferation contributes negligibly to the population of osteoblastlike cells peripheral to the well margin. The appearance of osteoblastlike cells peripheral to the well margin is due primarily to migration.

Animals↗
PubMed1986

Retention of bone cell viability in mouse calvarial explants after cryopreservation.

Newborn mouse calvaria, cyropreserved at -196 degrees C in serum-free medium containing dimethyl-sulfoxide, were compared to unpreserved explants for bone cell viability by [3H]thymidine uptake. Other explants were studied using autoradiography to compare the histological appearance of the cryopreserved and control unpreserved explant sites of cellular localization of [3H]thymidine. After short-term cryopreservation, calvarial bone cells, including less differentiated osteoprogenitor cells, survived as indicated by their incorporation of the DNA precursor. With culture continuing for up to 24 hr after thawing and in the continuous presence of [3H]thymidine, additional labeled thymidine was incorporated, indicating that the proliferative ability of explant cells persists after cryopreservation. Cryopreserved bone explants did not, however, incorporate the same amount of labeled thymidine as did controls at each time point studied. These events, as measured quantitatively and observed by autoradiography of the tissue, indicate that newborn calvarial bone cell proliferation in vitro continues after cryopreservation. The large surface:mass ratio of the tissue and its proportionate volume of calcified matrix apparently permits it to behave as an isolated cell population with regard to the diffusion of the cryoprotectant and thermal conductivity, thus permitting the retention of explant viability.

Animals↗
PubMed1986

Responsiveness of mouse calvaria to parathyroid hormone after explant cryopreservation: 45Ca release in vitro.

Newborn mouse calvaria prelabeled with 45Ca and cryopreserved at -196 degrees C in serum-free medium containing dimethylsulfoxide were compared to unpreserved explants for response to parathyroid hormone during subsequent culture. After short-term cryopreservation followed by rapid thawing, the viable explants continued to release 45Ca to the culture medium but additions of parathyroid hormone to the medium did not cause increased bone resorption. The data suggest that cryopreservation and thawing impairs mechanisms responsible for parathyroid hormone action on bone cells.

Animals↗
PubMed1985

Perivascular cells in cartilage canals of the developing mouse epiphysis.

Morphological variability among perivascular cells adjacent to cartilage matrix during the elongation of canals through both uncalcified and calcified matrix has not been reported. Cartilage canals were located in distal femoral epiphyses of 5- to 7-day-old mice and identified as vascular channels arising from perichondrial surfaces along the condyles and intercondylar fossae. Three stages of canal development were identified based on the length of canals and on characteristics of chondrocytes and matrix surrounding the canals. Superficial canals terminated in uncalcified matrix of resting cartilage; intermediate canals terminated in matrix containing hypertrophic chondrocytes; deep canals terminated in calcified matrix. The ultrastructural morphology of perivascular cells in contact with the matrix varied in the three stages. Cells resembling fibroblasts and vacuolated macrophages were present adjacent to the uncalcified matrix in superficial canals. At the tips of intermediate canals, cells resembling fibroblasts were larger, contained numerous lysosomes and phagolysosomes, and were in intimate contact with the matrix. At the tips of deep canals, chondroclasts with ruffled borders and clear zones contacted the calcified matrix. The results indicate that 1) mouse epiphyses provide a suitable model for studying cartilage-canal perivascular cells, 2) calcification of cartilage matrix occurs along the course of the canal, and 3) the morphology of perivascular cells in contact with the matrix may be determined, in part, by matrix calcification.

Animals↗
PubMed1985

Multicellular tumor spheroid interactions with bone cells and bone.

In vitro coculture techniques were used to study HSDM1C1 murine fibrosarcoma multicellular tumor spheroid (HSDM1C1-MTS) interactions with mouse calvarial bone cells having osteoblastic characteristics and mouse bone explants. HSDM1C1-MTS attached to confluent bone cell monolayers and their attachment rate was quantified. HSDM1C1-MTS interaction with bone cells was further demonstrated by the release of 3H-deoxyuridine from prelabeled bone cells during coculture with multicellular tumor spheroids. HSDM1C1-MTS-induced cytotoxicity was mimicked by the addition of 10(-5) M prostaglandin E2 (PGE2) to 3H-deoxyuridine-labeled bone cells. The effects of low (10(-9) M) and high (10(-5) M) concentrations of PGE2 on bone cell proliferation were also studied. Higher concentrations of PGE2 inhibited bone cell proliferation. HSDM1C1-MTS resorbed living explants in the presence of indomethacin, suggesting that other tumor cell products may also participate in bone resorption. HSDM1C1-MTS caused direct bone resorption as measured by the significantly elevated release of 45Ca from prelabeled, devitalized calvaria. However, the growth of a confluent bone cell layer on devitalized, 45Ca-prelabeled calvaria resulted in a significant reduction in the amount of 45Ca released subsequent to the seeding of HSDM1C1-MTS onto the explants. Bone cells at the bone surface may act as a barrier against invasion and tumor cell-mediated bone resorption. Violation of this cellular barrier is achieved, in part, by tumor cell products.

Animals↗
PubMed1984

Tumor cell proteinase visualization and quantification using a fluorescent transition-state analog probe.

The fluorescent proteinase transition-state analog inhibitor, dansyl-L-argininal (DnsArgH), may be a selective probe of cysteine and serine-type proteinases in a fibrosarcoma tumor cell line (HSDM1C1). DnsArgH binds with high affinity to proteinases because of its transition-state analog properties, and on association it gives a dramatically increased fluorescent yield. The DnsArgH binding is inhibited by the serine proteinase inhibitor diisopropyl fluorophosphate and by the cysteine proteinase inhibitor p-chloromercuribenzoate. The fluorescence emission appears at its maximum steady-state yield immediately on addition of DnsArgH to the HSDM1C1 fibrosarcoma cells. The immediacy of the DnsArgH reaction supports the contention that DnsArgH binding may be to cell surface-associated proteinases. Quantification of the cell proteinase concentration, by comparison of the fluorescence yield obtained from DnsArgH interactions with bovine trypsin and papain, indicates 10(-15) to 10(-16) mol of proteinase per HSDM1C1 cell. In fluorescence microscopy, DnsArgH fluorescence appears distributed throughout the fibrosarcoma cell without association to organelles. DnsArgH fluorescence from normal fibroblast controls (IMR-90) was found to be substantially lower than in the transformed fibrosarcoma cells, supporting a hypothesis that proteinases have a role in malignancy.

Arginine↗