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Tenosynovial giant cell tumor is a relatively common benign proliferation affecting the articular and periarticular soft tissues. Cytologic findings on smears obtained by fine-needle aspiration are rather characteristic and include a mixture of oval or polygonal mononuclear cells showing vacuolation and/or pigment deposition along with a population of multinucleated giant cells. Separation from other giant cell lesions including true giant cell tumor of bone, chondroblastoma, aneurysmal bone cyst, and granulomatous inflammation must be made. Careful attention to the cytologic findings and correlation with clinical and radiographic data should result in the appropriate diagnosis of tenosynovial giant cell tumor in most cases.

We have isolated cDNA clones that encode a novel human gene related to agouti. Sequence analysis of this gene, named ART, for agouti-related transcript, predicts a 132-amino-acid protein that is 25% identical to human agouti. The highest degree of identity is within the carboxyl terminus of both proteins. Like agouti, ART contains a putative signal sequence and a cysteine rich carboxyl terminus, but lacks the region of basic residues and polyproline residues found in the middle of the agouti protein. Both agouti and ART contain 11 cysteines, and 9 of these are conserved spatially. ART is expressed primarily in the adrenal gland, subthalamic nucleus, and hypothalamus, with a lower level of expression occurring in testis, lung, and kidney. The murine homolog of ART was also isolated and is predicted to encode a 131-amino-acid protein that shares 81% amino acid identity to humans. The mouse was found to have the same expression pattern as human when assessed by RT-PCR. Examination by in situ hybridization using mouse tissues showed localized expression in the arcuate nucleus of the hypothalamus, the median eminence, and the adrenal medulla. In addition, the hypothalamic expression of ART was elevated approximately 10-fold in ob/ob and db/db mice. ART was mapped to human chromosome 16q22 and to mouse chromosome 8D1-D2. The expression pattern and transcriptional regulation of ART, coupled with the known actions of agouti, suggests a role for ART in the regulation of melanocortin receptors within the hypothalamus and adrenal gland, and implicates this novel gene in the central control of feeding.

Background: As a part of multimodality therapy, many patients with tumors of the trunk receive radiation therapy. The major morbidity of this therapy is often secondary to incidental radiation damage to tissues adjacent to treatment areas., Methods: We detail our use of saline breast implants placed in polyglycolic acid mesh sheets to displace visceral and solid organs away from the radiation field., Results: Analysis of CT scans and dose volume histograms reveal that this technique successfully displaces uninvolved organs away from the radiation fields, thereby minimizing the radiation dose to such organs and tissues., Conclusion: We believe this is a safe and efficacious method to prevent radiation damage to visceral and solid organs adjacent to trunk tumor sites.

Purpose. The prognosis, treatment principles and prediction of clinical outcome of patients with chondrosarcoma currently rest on histologic grading which is somewhat ambiguous due to difficulty in pathologic interpretation of this neoplasm. Immunohistochemistry, flow cytometry and oncogene/tumor suppressor gene expression have been examined as alternative indices to predict the biologic behavior of these tumors. Because of partial successes obtained with flow cytometry and because of the improvement in predicting recurrence offered by examining the S-phase fraction, we undertook the current study to determine if expression of specific regulators of the cell cycle would act as prognostic indicators for these patients.Subjects/methods. We examined archival pathologic specimens from 39 patients with at least 2 years' clinical follow-up for the presence of p53, Rb, src and MIB-1 by immunohistochemistry and correlated this with clinical histories and incidence of recurrence.Results. While Rb, p53 and src gene products were identified to a variable extent in these specimens, there was no prognostic significance to their expression. In contrast, MIB-1, an epitope expressed only during semiconservative replication and an accepted marker of cell proliferation, served as a significant prognostic indicator. MIB-1 staining was present in 14.5% of tumor cells in all specimens (range 0-59%). When MIB-1 staining was examined with respect to disease recurrence, there was a statistically significant association between staining and histologic grade (p < 0.05) as well as event-free survival (p < 0.02). Comparing survival curves stratified by MIB-1 expression, there was a significant decrease in event-free survival associated with increasing MIB-1 indices (p < 0.003). Covariates that were associated with event-free survival include histologic grade (p = 0.025) and stage (Musculoskeletal Tumor Society) (p = 0.014). There was no statistical association with patient age (p = 0.15), tumor size (p = 0.47), tumor histology (p = 0.62) or anatomic location (p = 0.316).Discussion. These results indicate that determination of the proliferation index by MIB-1 immunostaining may serve as a useful adjunct to current histopathologic classification. Patients with a high proliferation index may benefit from established adjuvant therapies or experimental approaches including immunotherapy or biologic modulation.

The adverse prognostic impact of tumor hypoxia has been demonstrated in human malignancy. We report the effects of radiotherapy and hyperthermia (HT) on soft tissue sarcoma oxygenation and the relationship between treatment-induced changes in oxygenation and clinical treatment outcome. Patients receiving preoperative radiotherapy and HT underwent tumor oxygenation measurement pretreatment after the start of radiation/pre-HT and one day after the first HT treatment. The magnitude of improvement in tumor oxygenation after the first HT fraction relative to pretreatment baseline was positively correlated with the amount of necrosis seen in the resection specimen. Patients with <90% resection specimen necrosis experienced longer disease-free survival than those with > or = 90% necrosis. Increasing levels of tumor hypoxia were also correlated with diminished metabolic status as measured by P-31 magnetic resonance spectroscopy.

Unlabelled: The purpose of this study was to investigate the potential role of FDG-PET in the monitoring of neoadjuvant therapy of soft-tissue and musculoskeletal sarcomas., Methods: Nine patients were studied. Neoadjuvant therapy consisted of either chemotherapy or combined radiotherapy and hyperthermia. The FDG-PET studies were obtained, when possible, prior to therapy, 1-3 wk after commencement of therapy, and prior to surgery after completion of neoadjuvant therapy. In two patients, all three studies were completed. The remainder of patients underwent one or two studies at varying timepoints., Results: In tumors treated with combined radiotherapy and hyperthermia, well-defined regions of absent uptake developed within responsive tumors, correlating pathologically with necrosis. Following treatment, a peripheral rim of FDG accumulation was found to correlate pathologically with the formation of a fibrous pseudocapsule. In tumors treated with chemotherapy, FDG accumulation decreased more homogeneously throughout the tumor, in responsive cases. Despite 100% tumor cell kill in some patients, persistent tumor FDG uptake was observed which correlated pathologically with uptake within benign therapy-related fibrous tissue. Significant FDG accumulation was also observed at the site of an uncontaminated incisional biopsy., Conclusion: These initial results demonstrate changes in tumor accumulation of FDG during and after neoadjuvant therapy; these changes are dependent on the type of neoadjuvant therapy administered. Prominent FDG accumulation was observed in benign tissues both within and adjacent to the treated tumor.

Oligodendrocyte responses in vitro to platelet-derived growth factor (PDGF) include proliferation, survival, migration, and changes in cell morphology and molecular expression. Studies of mixed glial cultures established that astrocytes secrete PDGF; thus astrocytes are considered to be key regulators of oligodendrocyte development in vitro. We previously demonstrated PDGF alpha receptor mRNA expression by oligodendrocyte progenitors and preoligodendrocytes during postnatal development of rat cerebral cortex. In the present study, we have mapped the spatial and temporal expression of PDGF A-chain ligand mRNA and alpha receptor mRNA to determine if the cell-cell interactions that form the basis for PDGF regulation of oligodendrocyte development in vitro are also present in vivo. By in situ hybridization (ISH) we demonstrate that at embryonic day 17 (E17) cells expressing receptor mRNA (PDGFR alpha +) are initially in the subventricular zone, at a distance from cells expressing ligand mRNA (PDGF+) in the cortical plate. By E20 PDGFR alpha + cells are found throughout the corpus callosum and cortical gray matter. PDGF+ cells are restricted to the cortical plate prenatally and only appeared in the corpus callosum postnatally. Combined immunocytochemistry and ISH demonstrated the PDGF+ cells colocalized with neurofilament, but not with GFAP. These data establish that PDGF is expressed by neurons during PDGFR alpha + oligodendrocyte progenitor migration from the subventricular zone to the corpus callosum and gray matter. Furthermore, neurons continue to express PDGF during the generation and differentiation of appropriate numbers of oligodendrocytes needed to myelinate axons as the nervous system matures.

Acute hematogenous salmonella osteomyelitis is rare among immunocompetent adults. In this study, the authors reported an unusual case of salmonella enteriditis osteomyelitis of the humerus complicated by methicillin-resistant Staphylococcus aureus superinfection and eventual chronic osteomyelitis in an immunocompetent host. Resection of the humeral head and a significant portion of the humeral shaft coupled with numerous surgical debridements and intravenously administered antibiotics led to resolution of symptoms. This case provides a rare example of acute hematogenous osteomyelitis complicating a closed fracture and demonstrates the difficulty associated with eradication of these specific organisms while emphasizing the principle of aggressive surgical debridement in cases of chronic osteomyelitis.

The presence of pathologic fracture in osteosarcoma raises concerns of tumor dissemination by the fracture hematoma and has been considered a contraindication to limb salvage surgery. Because this is a theoretical concern, there are little clinical data available in the literature on which to base treatment of these patients. Eighteen patients with osteosarcoma who sustained a pathologic fracture and had a minimum of 24 months of followup were reviewed retrospectively. Surgical treatment included nonoperative therapy, amputation, and limb salvage groups. Patients who refused surgical intervention (2) had a uniformly poor outcome. Patients who underwent amputation (6) had no local recurrences and 33% developed metastases. Patients who underwent limb salvage (10) experienced 3 local recurrences and 6 distant recurrences. Although the distant recurrence rate for patients undergoing amputation was no different from the rate for those undergoing limb salvage, the difference in local tumor control approached statistical significance. All patients who developed local recurrence died. Surgical treatment needs to be individualized and based on factors such as fracture displacement, stability, radiographic and histologic response to chemotherapy, and the perceived ability to resect the fracture hematoma completely.

This study was performed to explore the relationship between tumor oxygenation and treatment outcome in human soft tissue sarcoma. Twenty-two patients with nonmestastatic, high-grade, soft tissue sarcomas underwent preoperative irradiation and hyperthermia and pretreatment measurement of tumor oxygenation. The 18-month actuarial disease-free survival was 70% for patients with tumor median oxygen pressure (pO2) values of >10 mm Hg but only 35% for those with median pO2 values of <10 mm Hg (P=0.01). There were eight treatment failures; the first site of recurrence was lung in all patients. Median pO2 was 7.5 mm Hg for metastasizing tumors versus 20 mm Hg for nonmetastasizing tumors (P=0.03). Potential mechanisms and implications for clinical trial design are discussed.

Tenascin-C is an oligomeric glycoprotein of the extracellular matrix that is expressed in a variety of processes including development, tissue remodeling, wound healing, cell adhesion/antiadhesion, and cell/matrix interactions. Tenascin has recently been acknowledged as a component of the extracellular matrix of articular cartilage, but its function remains unclear. In this study, bovine articular chondrocytes were grown in alginate beads for 35 days to examine the kinetics of tenascin synthesis and incorporation into de novo extracellular matrix. During the culture period, 6 harvest days were established in which culture medium was recovered, alginate beads were dissociated with an EDTA solution, and chondrocytes were collected and lysed by sonication. Total DNA determination performed on the cell lysates demonstrated chondrocyte survival and proliferation. Western blotting performed on the medium, EDTA/alginate, and lysate samples demonstrated the production of both the 220 and 320 kDa tenascin size variants and their differential compartmentalization within the culture system. Tenascin was incorporated into the alginate bead matrix at a constant rate of 3.8 micrograms/day. The 320 kDa variant was produced in higher quantity, but the 220 kDa fragment was twice as likely to be incorporated into the de novo matrix. Methylene blue/acid fuchsin staining and tenascin immunohistochemistry demonstrated the incorporation of tenascin into a progressively expanding matrix surrounding the chondrocytes. The results suggest a role for tenascin in the assembly of the chondrocyte matrix and as a soluble mediator of chondrocytes with possible diverse functions for the tenascin size variants.

Giant cell tumor of bone has been shown to behave more aggressively when located in the wrist and hand. Although nearly 4% of giant cell tumors arise in the foot and ankle, biological features specific to this location have not been identified. In our experience with more than 300 cases of giant cell tumor, 12 arose in the foot and ankle and were followed for more than 2 years. These included nine females and three males ranging in age from 15 to 52 years (mean age, 29.5 years). All patients presented with pain of 5.0 months' mean duration and 9 of 12 tumors demonstrated aggressive radiographic features, including bone erosion and destruction; five had either invasion of a joint or a soft tissue mass present. Unlike the hand, where metacarpal and phalangeal lesions are common, no tumors arose in the forefoot and nine of the tumors were present in the ankle region. Four patients were treated with resection (no recurrence), two with curettage and cement packing (one recurrence), and six with curettage and autologous bone graft (two recurrences), which resulted in an overall recurrence rate of 25%. None of the recurrent tumors have returned after additional treatment, which consisted of curettage and cement packing in two cases and resection in one case. Five tumors (four primary, one recurrent) were treated with local resection and reconstruction with no major complications and with no amputations performed. Thus, giant cell tumors of the foot and ankle can be treated with local procedures, which result in recurrence rates similar to those found in more common locations.

Chondrocytes in the growth plate progress in an orderly fashion from resting through proliferating to hypertrophic cells. In the region of hypertrophic chondrocytes, the cartilage is invaded by capillary loops and endochondral ossification is initiated. It is currently believed that growth factors may regulate the proliferation and maturation of chondrocytes and the synthesis of extracellular matrix in the growth plate. The ordered sequence of proliferation and differentiation observed in the growth plate provides a unique opportunity to study the role of acidic fibroblast growth factor, basic fibroblast growth factor, and transforming growth factor-beta 1 in the regulation of these processes. In this study, expression of the mRNA of these growth factors was examined using total RNA extracted from the physis and epiphysis of rat tibias. Transforming growth factor-beta 1 mRNA was detected by Northern hybridization. Expression of the genes encoding acidic and basic fibroblast growth factors was demonstrated by polymerase chain reaction amplification. In addition, using polyclonal antibodies against these growth factors, we localized them by immunohistochemical analysis. Strong intracellular staining with a predominantly nuclear pattern was observed in chondrocytes from the proliferating and upper hypertrophic zones. In contrast, chondrocytes in the resting zone stained only faintly for the presence of these growth factors. Some chondrocytes in the resting zone adjacent to the proliferating zone stained with these antibodies, and the antibodies also stained cells in the zone of Ranvier, which regulates latitudinal bone growth. Lastly, the location of transforming growth factor-beta 1 was examined further with use of a polyclonal antipeptide antibody specific for its extracellular epitope.(ABSTRACT TRUNCATED AT 250 WORDS)

Purpose: The improved survival in patients with Ewing's sarcoma over the past two decades has placed increased importance on achievement of local disease control. Ewing's sarcoma that arises in the pelvis has been recognized to have a worse prognosis than that in the appendicular skeleton, and the role of surgical resection in these cases remains controversial. The current study attempts to identify a benefit to surgical resection in these patients., Methods: We retrospectively examined 39 patients who presented with Ewing's sarcoma in a pelvic location, all of whom were treated systemically with chemotherapy. Twenty patients received radiation only as a means of local control, and 19 underwent resection with or without radiation therapy. The patients were evaluated with end points of disease-free survival and overall survival for a minimum of 24 months and a mean of 58 months., Results: There was an even distribution among patients who underwent surgical resection for local control as compared with those who received only radiation therapy with respect to age, site, date of treatment, and stage of disease. Despite uncontrolled biases including tumor size and response to chemotherapy that would be expected to favor patients who undergo resection, surgery in addition to or in substitution for radiation therapy did not result in a statistically significant increase in disease-free survival or overall survival. Local disease control was comparable between those who underwent resection and those who did not: three patients in each group developed a local recurrence., Conclusion: Currently, morbidity of surgical resection should be weighed against the efficacy and secondary complications of radiation therapy in the decision-making process for local disease control. The issue of whether overall survival and local disease control is improved in patients who undergo surgical resection remains controversial and may require a prospective randomized trial to be answered definitively.

The clinicopathologic features of calcific myonecrosis are presented from results of an examination of 3 cases of this rare syndrome and review of the literature. Calcific myonecrosis is a painful, expansile, calcified mass that develops in muscle several decades after lower extremity trauma that typically has been associated with vascular injury. Plain radiographs show a well-defined and heavily calcified mass replacing the leg musculature. The calcifications are present in a thin, linear pattern and are organized around the periphery of the lesion. Smooth erosion of the adjacent bone may be present, whereas magnetic resonance imaging shows a heterogeneous signal with enhancement limited to the periphery of the mass. Pathologic features consist of a centrally cystic mass arising in muscle filled with friable, tan to dark red, soft debris. The cyst walls are firm and fibrous and contain many needle-like, elongated, calcified shards of necrotic tissue composed of hypocellular fibrous tissue with focal aggregates of hemosiderin-laden macrophages. The cyst contents are composed of necrotic skeletal muscle and acellular amorphous debris containing many cholesterol crystals, fibrin, and recent hemorrhage, including focal aggregates of organizing thrombus. The pathologic findings suggest that calcific myonecrosis might expand with time by virtue of recurrent intralesional hemorrhage into a chronic calcified mass that eventually becomes symptomatic. Surgical intervention is associated with a high rate of complication, particularly in cases in which intralesional procedures have been done.

In the treatment of soft tissue sarcomas, hyperthermia has been demonstrated to enhance tumor necrosis from radiation therapy. The current study reports the clinical course of patients treated with this neoadjuvant therapy regimen. Forty-four patients with deep, undisturbed, nonmetastatic, high grade soft tissue sarcomas completed a neoadjuvant treatment protocol with combined hyperthermia and radiation therapy followed by wide surgical resection. Negative surgical margins were obtained in 40 patients. There was one local recurrence, thus yielding a local control rate of 97.5%. All other failures were either through regional lymphatic spread or pulmonary metastasis. As a group, the patients at 36 months had a 72% overall and a 58% disease-free survival. The most common pathologic diagnosis was malignant fibrous histiocytoma (MFH), which demonstrated a 36-month survival of 52% vs. 82% for others (P = 0.02). Tumor size was not prognostically significant for disease free or overall survival (P = 0.13). Those patients with surgical margins < 1 cm had a significantly lower disease-free survival and overall survival in a multivariate analysis (P = 0.02 and P = 0.006, respectively). Overall survival did not correlate with either the number of hyperthermia treatments received or the amount of tumor necrosis. Although this neoadjuvant protocol results in excellent local control rates, overall survival rates are comparable to adjuvant therapy employing radiation alone.

Alveolar soft-part sarcomas are rare and seldom considered in the differential diagnosis of a soft-tissue mass. Thus, early clinical recognition can be elusive. The authors have identified several clinical and radiographic features of alveolar soft-part sarcoma, emphasizing the importance of magnetic resonance imaging in the preoperative diagnostic and staging workup. Accurate diagnosis and treatment of this unusual tumor requires clinical suspicion and clinicopathologic correlation with appropriate radiographic studies. If the clinical or radiographic interpretation is equivocal, early biopsy is essential to differentiate alveolar soft-part sarcoma from arteriovenous malformation.

During the years 1985 to 1989, 82 patients were included in the soft tissue sarcoma protocol. Preoperative irradiation (50-54 Gy) was performed in all patients before tumor extirpation. Microwave hyperthermia was performed in conjunction with radiation in patients who had gross tumor remaining after initial biopsy. Primary closure with vascularized tissue (flaps) in lieu of conventional wound closure by skin approximation led to less complications (19% versus 51%), fewer secondary procedures for wound closure (10% versus 35%), shorter average hospitalization (15 versus 48 days) and greater limb salvage rate (97% versus 91%). The authors conclude that vascularized tissue (flaps) for primary wound closure in irradiated tissue leads to improved wound healing, and should be considered the procedure of choice for heavily irradiated soft tissue sarcoma defects.

This study was undertaken to determine whether a mRNA for glial fibrillary acidic protein (GFAP) was present in increased amounts as a response to injury and, if so, how was its temporal expression related to the demonstration of GFAP by immunocytochemical techniques. A cerebral freeze-injury was produced in mice and at intervals thereafter the animals were anesthetized, perfused with formalin and histological sections of the brain through the injured area were prepared. A riboprobe for GFAP mRNA labeled with S35 and an immunocytochemical probe for GFAP were utilized to localize mRNA and GFAP immunoreactivity, respectively. For mRNA studies, the histological slide exposed to either sense or antisense probe was overlaid with x-ray film or dipped in photographic emulsion. The developed film was quantitated by digital image analysis. Emulsions were examined by dark-field microscopy. The results indicate that mRNA for GFAP is increased in the cortex in the environs of the injury by 6 hours, becomes maximal at 4-5 days, and is present in increased amounts up to 14 days. The message is enhanced in the adjacent cortex, the subpial region, the adjacent corpus callosum and in the ipsilateral and contralateral callosal radiations. This pattern of enhancement follows the distribution of post-injury edema. Glial fibrillary acidic protein is demonstrable at 24-48 hours after injury. Thus, there is a rapid response of the astrocyte to injury with increased mRNA expression that is followed by expression of GFAP immunoreactivity.

Rotary subluxation of the scaphoid has not been previously reported in the English-language literature as a factor causing persistent or recurrent carpal tunnel syndrome. This report describes a sixty-seven-year-old man with persistent carpal tunnel syndrome. X-ray films showed a scapholunate gap and the scaphoid maintained a flexed position. At surgery the median nerve was found to be fixed to the undersurface of the transverse carpal ligament on the lateral side and was being compressed by the distal pole of the scaphoid.

Chondrogenesis can occur during a bone repair process, which is related to several growth factors. Transforming growth factor beta 1 (TGF-beta 1) downregulates the expression of type II collagen by chondrocytes in vitro, but injection of TGF-beta 1 into the periosteum in vivo increases type II collagen mRNA levels and initiates chondrogenesis. We examined the effect of TGF-beta 1 on collagen gene expression in a bovine periosteum-derived cell culture system to evaluate its direct effect on the periosteum. Cultured cells expressed alkaline phosphatase and collagen pro alpha 1(I) and pro alpha 1(II) mRNAs. A low level of type II collagen synthesis was demonstrated by immunoprecipitation. TGF-beta 1 had no effect on periosteal cell proliferation. Expression of collagen pro alpha 1(I) mRNA did not change with TGF-beta 1 treatment, but alkaline phosphatase mRNA showed a dose-dependent decrease. Expression of collagen pro alpha 1(II) mRNA was stimulated 2.7-fold by TGF-beta 1. TGF-beta 1 also caused a 2.6-fold increase in type II collagen synthesis by immunoprecipitation. These findings indicate that TGF-beta 1 is an enhancer of the expression of the chondrocyte phenotype of the periosteal cells and suggest that TGF-beta 1 is important in initiating and promoting cartilage formation in vivo.

In this report a rapid, simple and economical means of preparing a cDNA probe of specified length, sequence and specific activity is described. The process involves the use of a polymerase chain reaction to incorporate radiolabelled nucleotides into a single stranded or double stranded cDNA sequence. A pair of oligonucleotide primers are synthesized, flanking the sense and antisense strands of a selected sequence. The primers are then used with a cloned DNA fragment or a cellular source of RNA or DNA as a template to amplify the specific gene sequence. The sequence to be used as a probe is selected from the known sequence using free energy calculations of the secondary structure. The calculation of free energy predicts regions of stable secondary structure which may hinder transcription and thus are to be avoided. By selecting the distance between primers the probe length can be controlled to allow adequate probe permeability into tissue samples. The specific region of the gene sequence can be chosen to differentiate between closely related sequences by avoiding areas of homology. Altering the concentration of a radiolabelled nucleotide allows direct control of probe specific activity. The use of asymmetric PCR allows the preferential generation of an antisense single stranded cDNA sequence for a higher sensitivity in the detection of low abundance mRNA. This report highlights the advantage of this technique in generating probes for in situ hybridization. However, any technique that relies on homology for detection of sequences, such as Northern and Southern blotting could also utilize this technique.

We have previously shown by immunocytochemistry in rat primary glial cultures that transferrin (Tf) is an early developmental marker for oligodendrocytes. The present work addresses the issue of Tf gene expression and synthesis by neural cells in vitro. For this purpose, we used rat embryonic neuronal cultures and newborn glial cultures of astrocytes and oligodendrocytes. Cultured fibroblasts and C6 glioma cells were used as negative controls. We found that Tf mRNA is present in oligodendrocytes, astrocytes, and neurons. However, oligodendrocytes and astrocytes, but not neurons, were shown to synthesize and secrete Tf. Neither fibroblasts nor C6 glioma cells expressed detectable amounts of Tf mRNA. Tf mRNA levels in astrocyte cultures appeared to be under hormonal control since hydrocortisone markedly reduced message levels. These results show that both astrocytes and oligodendrocytes can synthesize and secrete Tf under cell culture conditions. However, epigenetic factors, such as hydrocortisone, may repress the expression of Tf in astrocytes in vivo.

The morphological and immunocytochemical identification and characterization of the myelin-forming cell, the oligodendrocyte, have defined a model system for developmental studies. The myelin-deficient (md) rat mutant lacks myelin in the central nervous system and fails to express the normal developmental increase in oligodendroglial and myelin markers, apparently as a consequence of a point mutation in the proteolipid protein gene. In the present work, we compared the developmental pattern of primary glial cultures derived from newborn md rat brains to those derived from wild-type animals. Brain cell suspensions were prepared from each rat pup and cultured separately. We found by immunocytochemical and enzymatic analyses for the various markers that the developmental cascade of oligodendroglial marker expression is delayed, oligodendrocytes failing to mature compared to normal cultures. However, a partial recovery of marker expression was observed in md-derived cultures as compared to development previously reported in the intact md animals. We suggest that the partial recovery of the sequential expression of oligodendroglial markers may be due to a supportive environment provided to the oligodendrocyte progenitor cell (1) by tissue culture conditions or (2) by the absence of the blood-brain barrier in contrast to its presence in the intact animal.

To identify and assess the consequences of the mutation in myelin-deficient (md) rats, the myelin proteolipid protein (PLP) gene and its expression were studied in md rats. Southern blots of the PLP gene demonstrated that no major deletions or insertions have occurred in this gene. In addition, the mutation in this gene does not result in a splicing defect in the RNAs, since all exons are represented in md PLP RNAs. These data are consistent with results in another laboratory indicating that a point mutation in the PLP gene in md rats results in a single amino acid alteration in the protein. To elucidate the molecular mechanisms producing reduced levels of PLP, myelin basic protein (MBP) and glycerol phosphate dehydrogenase (GPDH) mRNAs, and their corresponding proteins in md rats, in vitro transcription assays were performed. Transcription of the PLP gene in nuclei isolated from 23-day-old md rat brains was dramatically reduced relative to normal tissue. Thus, the single amino acid alteration in this protein alters the regulation of transcription of this gene. In contrast, the transcriptional activities of the MBP and GPDH genes in md rats were indistinguishable from normal animals. Thus, the lower level of MBP and GPDH mRNA and protein in md rats relative to normal results from a posttranscriptional event.

Oligodendrocyte development and myelinogenesis, both in vivo and in vitro, are characterized by the sequential and coordinate expression of markers which participate in the differentiation of oligodendrocytes as a prerequisite for myelination. The myelin deficient (md) rat shows greatly reduced mRNA expression for several oligodendrocyte markers: glycerol phosphate dehydrogenase (GPDH), myelin basic protein (MBP) and proteolipid protein (PLP). Brain GPDH mRNA levels are initially equivalent in md and unaffected littermates, but the mutant rats fail to display the normal developmental increase in gene expression. Immunostaining of brain tissue sections also reveals decreased expression of these oligodendrocyte markers. The number of oligodendrocytes containing GPDH-like immunoreactivity is reduced in mutant rats, and in general these cells appear morphologically less complex with shorter processes. However, the intensity of staining in many oligodendrocytes appears equivalent to that observed in unaffected rats. Expression of the neuronal marker, glutamic acid decarboxylase, and the astrocyte markers, glutamine synthetase and glial fibrillary acidic protein, are largely unaffected at either the mRNA or protein level. Mixed glial cultures prepared from the brains of neonatal male md rats possess fewer oligodendrocytes compared to cultures derived from unaffected littermates, and the temporal sequence of marker development is delayed. Although an abnormality in the PLP gene is suspected in the md rat, these findings document profound deficits in many oligodendrocyte gene products.

Extrinsic optical probes have become important tools for monitoring membrane potential, with probes now available for many tissue or cell suspension systems. In each case that has been studied in detail, it seems that the mechanism involves a shift in the equilibrium population of the probe from one chemical environment to another in response to the transmembrane potential; the environments perturb the probe's spectrum differently. As this indirect mechanism involves a redistribution of dye between chemical environments that are likely to vary if a given probe is transferred from one membrane to another, a potential probe that is effective and calibrated for all membrane systems has not been realised. We present here evidence for a direct response of a probe chromophore to the electric field across membrane systems. The results suggest it might be possible to develop a universal set of membrane probes.

We have performed a comprehensive study of calcium tracer flux, distribution, content, and ionized cytoplasmic calcium during monocyte activation. A model of monocyte calcium was developed from 45Ca uptake and exodus curves which indicated that cell calcium was partitioned between three compartments. The magnitude of the time constants for each pool lead us to propose cellular locations for these three compartments: a surface plasma membrane pool, a cytoplasmic pool, and an organelle pool. 45Ca uptake and exodus experiments were analyzed using a nonlinear least squares fit of compartmental exchange rates and sizes. The production of superoxide was used as a reflection of the state of activation of the monocytes treated with Concanavalin A (Con A). We found that Con A-treated monocytes have an increase in the calcium exchange rate with the cytoplasmic pool from 0.04 to 0.07/min (P less than 0.05), and an increase in the size of the cytoplasmic pool from 0.08 to 0.13 pmol/cell (P less than 0.05). There were no significant changes in the exchange rates or sizes associated with either of the other two compartments. The cytoplasmic ionized calcium was measured with the fluorescent probe, Quin 2, which indicated a resting level of 83 nM free calcium in unadhered monocytes. Con A stimulation caused a doubling of the cytoplasmic free calcium to 163 nM within 45 s. This increment in cytoplasmic free calcium preceded the onset of superoxide following Con A treatment. These studies indicate that Con A binding to the plasma membrane increases the monocyte plasma membrane permeability to calcium. External calcium enters the cell at an increased rate and contributes to both internally bound and free calcium. The magnitude of the increase in free calcium is proportional to the concentration of Con A and stimulates calcium extrusion via the calcium transport ATPase. Moreover, there is an increased concentration of ionized cytoplasmic calcium which has the potential to interact with other cellular regulators that modulate cell activation and superoxide production.

We have purified unadhered human monocytes in sufficient quantities to prepare monocyte plasma membrane vesicles and study vesicular calcium transport. Monocytes were isolated from plateletpheresis residues by counterflow centrifugal elutriation. By combining this source and procedure, 7 x 10(8) monocytes of over 90% purity were obtained. The membranes, isolated on a sucrose step gradient, had an 18-fold enrichment in Na,K-ATPase, a 29-fold diminution of succinate dehydrogenase activity and were vesicular on transmission electron micrographs. The membrane vesicles loaded with oxalate accumulated calcium only in the presence of Mg and ATP. Calcium uptake did not occur if ATP was replaced by any of five nucleotide phosphates or if Mg was omitted. Calcium transport had a maximal velocity of 4 pmoles calcium/micrograms vesicle protein/min and a Km for calcium of 0.53 microM. The ionophore A23187 completely inhibited calcium accumulation while 5 mM sodium cyanide and 10 microM ouabain had no effect. A calcium-activated ATPase was present in the same plasma membrane vesicles. The calcium ATPase had a maximal velocity of 18.0 pmoles calcium/micrograms vesicle protein/min and a Km for calcium of 0.60 microM. Calcium-activated ATPase activity was absent if Mg was omitted or if (gamma - 32P) GTP replaced (gamma - 32P) ATP. Monocyte plasma membranes that were stripped of endogenous calmodulin by EGTA treatment showed a reduced level of calcium uptake and calcium ATPase activity. The addition of exogenous calmodulin restored the transport activity to that of unstripped monocyte plasma membranes. Thus, monocyte plasma membrane vesicles contain a highly specific, ATP-dependent calcium transport system and a calcium-ATPase with similar high calcium affinities.

The enzymatic activities in post-mortem rat brain kept at 4 degrees C and at 25 degrees C were determined for a number of enzymes localized in specific cell types in the central nervous system. Choline acetyltransferase (CAT), glycerol-3-phosphate dehydrogenase (GPDH), glutamine synthetase (GS), lactate dehydrogenase (LDH) and 2',3'-cyclic nucleotide phosphohydrolase (CNPase) were found to be very stable at both 4 degrees C and 25 degrees C with only slight, if any, losses of activity being seen even at periods as long as 72 hr. Glutamic acid decarboxylase (GAD) activity was less stable than that of the other enzymes. In brains kept at 4 degrees C GAD activity was stable out to 24 hr after which it began to decline rapidly to 65% of control at 72 hr. In brains kept at 25 degrees C, GAD activity was stable for 6-8 hr and then began to steadily decline to 58% of control at 24 hr and 29% of control at 72 hr. Assuming that these enzymes have similar stabilities in post-mortem human brain, the effect of post-mortem delay in processing tissues may be of lesser significance than other factors with regard to the measured enzyme activities in human brain samples.

The extracellular ionized calcium and magnesium requirements for lectin-induced lymphocyte DNA synthesis were measured in a serum-free system. The use of this system permitted measurements of the ionized calcium and magnesium concentrations with ion-selective electrodes. Maximal DNA synthesis was observed at 270 microM ionized calcium and at 100 microM ionized magnesium in phytohemagglutinin-treated lymphocytes. Lymphocyte DNA synthesis was much more sensitive to reduction of external ionized calcium than to reduction of ionized magnesium. In calcium-free medium (ionized calcium 25 microM), DNA synthesis was reduced by 90%, but in magnesium-free medium (ionized magnesium concentration 7 microM) DNA synthesis was reduced by only 30%. Fifty percent of DNA synthesis stimulated by phytohemagglutinin (PHA) and concanavalin A (Con A) was observed at external ionized calcium concentrations of 97 and 43 microM, respectively. When lymphocytes were stimulated with PHA and the external calcium was chelated with EGTA, 50% inhibition of DNA synthesis was observed at 98 microM ionized calcium. This value agreed well with the free calcium required for PHA activation of DNA synthesis (97 microM). Cytoplasmic calcium, measured with the fluorescent probe Quin 2, increased following lectin exposure if the extracellular ionized calcium concentration was greater than 80 microM. No increase in cytoplasmic calcium could be detected in lectin-treated lymphocytes below 80 microM extracellular ionized calcium, although substantial DNA synthesis was sustained.

Co-cultivation of confluent rat astrocyte cultures with embryonic chick neurons resulted in induction of glutamine synthetase activity in the astrocytes. This induction of glutamine synthetase in astrocytes by neurons was independent of induction by hydrocortisone and forskolin, but was dependent on the length of co-cultivation and the number of neurons present in the co-culture. Cycloheximide and actinomycin D inhibited the induction of glutamine synthetase in astrocytes by neurons, whereas cytosine arabinoside had no apparent effect. Results suggest that this induction of glutamine synthetase in astrocytes is mediated by cell contact with neurons and may represent a specific neuronal and glial interaction.

Calcium has been proposed as an intracellular second messenger for activation of secretion, phagocytosis, and the oxidative burst of neutrophils. We have examined the role of calcium in human monocyte activation. Concanavalin A (Con A)-stimulated monocytes displayed an increment in cytoplasmic ionized calcium at 31 +/- 6 s and the onset of superoxide production at 61 +/- 9 s. The increase in cytoplasmic calcium invariably preceded the onset of superoxide production. If the external calcium concentration was reduced to less than 28 nM by the addition of 10 mM EGTA, superoxide production was not diminished at 5 min; however, superoxide production decreased thereafter. The Con A-evoked increment in cytoplasmic ionized calcium was blunted upon the addition of EGTA and decreased further with time. Both the production of superoxide and the Con A-evoked increment in cytoplasmic ionized calcium displayed a 50% inhibition after 15 min of calcium depletion and were completely inhibited after 60 min. Total cell calcium fell from 0.7 to 0.5 fmol/cell, and the basal level of ionized calcium fell from 83 to 30 nM after 60 min. Histidine, a strong chelator of divalent cations other than calcium and magnesium, had no effect on monocyte superoxide production or on ionized calcium concentrations, indicating that EGTA inhibition was due to cell calcium depletion. In calcium-depleted cells, Con A did not evoke superoxide production until calcium was restored to the incubation medium. The restoration of calcium to Con A-treated, calcium-depleted monocytes permitted a rapid rise in the cytoplasmic ionized calcium, and the production of superoxide within 9 s. These data suggest that an increase in ionized cytoplasmic calcium is necessary for the activation of monocyte superoxide production by Con A. The rise in ionized calcium in response to Con A results, in part, from an internal redistribution of calcium, which is sufficient to permit superoxide generation.

Phenylalanine hydroxylase, a liver-associated enzyme, is induced markedly by glucocorticoids in two permanent rat-hepatoma cell lines. In order to gain evidence that this phenomenon also occurs in vivo, we examined the effect of adrenalectomy and/or hormone supplementation on the levels of phenylalanine hydroxylase in the livers of adult rats: glucocorticoid administration increases, and adrenal ablation reduces, the activity of the hepatic enzyme, and the diminution occurring in the latter instance is entirely prevented by concurrent hormone replacement. These results thus corroborate earlier findings from a single experiment and are consistent with the hypothesis that adrenal corticosteroid hormones participate in modulating phenylalanine-hydroxylase levels within the diploid hepatocyte.

Increases in the mRNA levels of two neuroglial markers, glutamine synthetase (EC 6.3.1.2; GS) and glycerolphosphate dehydrogenase (EC 1.1.1.8; GPDH), were observed in hydrocortisone-treated cultures of astrocytes and oligodendrocytes, respectively, and in C6 cells by Northern blot analysis and in situ hybridization. In vitro transcription assays demonstrated increased GS transcription in isolated nuclei from hydrocortisone (HC)-treated primary cultures of astrocytes and C6 cells, relative to untreated cells. This increased transcription is reflected in increased GS mRNA levels in the cytoplasm and increased levels of GS protein synthesis. Sodium butyrate (NaB) blocked the glucocorticoid-mediated increase in GS transcription in the primary cultures of astrocytes but not in C6 cells. From our earlier observations (Kumar et al: J Neurochem 43:1455-1463, 1984) we found NaB in combination with HC to increase the levels of GS mRNA and GS protein synthesis (Weingarten et al: FEBS Lett 126:289-291, 1981). We now report that NaB, alone or in combination with HC, does not increase the rate of transcription, suggesting that NaB plays a role in post-transcriptional regulation of GS in C6. In addition, we report the presence of two distinct sizes of GS mRNA, 2.9 and 1.8 kb, in the primary cultures of astrocytes and C6 cells.