A high percentage yield of tyrosine hydroxylase-positive cells from rat E14 mesencephalic cell culture.

In the ventral mesencephalon of the E14 rat fetus, 90% of the dopaminergic, tyrosine hydroxylase positive (TH+) cells are localized in 1.0 mm3 of tissue. This same ventral mesencephalic region also contains 90% of the dopamine content of the E14 ventral brainstem (2.2 +/- 0.3 nmol/mg protein). When cells were prepared for culturing from this localized area, and plated at a density of 2.5 x 10(5) cells/cm2, 17-21% of the cells were TH+, at 4 and 12 h, and at 1, 5, 7 and 10 days after plating. The percentage of TH+ cells was also 17-21% when examined at 4 h, 12 h or 5 days after plating at densities ranging from 7.8 x 10(3) to 2.5 x 10(5) cells/cm2. However, cell survival at a density of less than 6.2 x 10(4) cells/cm2 was poor after 5 days in culture. Based on the degree of neurite elongation and complexity, cell maturation appeared to be complete at 5 days in culture (DIV5), and appeared to be maintained at this level up to DIV10. By DIV14, neurite retraction was evident, and the cells were more rounded. These signs may indicate the inception of senescence in the cultures. A benztropine-sensitive, concentration-dependent dopamine uptake mechanism was demonstrated in the cultures at DIV7, and DA could be released from preloaded cells using 50 mM K+. Five morphological subtypes of TH+ cells were identified in the cultures. This primary culture of the ventral mesencephalic, dopaminergic area, with a high percentage of TH+ cells, is suitable for use in acute biochemical and cellular studies, between DIV 5 and DIV10.     

Matrix Vesicles Promote Mineralization in a Gelatin Gel

Extracellular matrix vesicles (MVs) are associated with initial calcification in a variety of tissues, but the mechanisms by which they promote mineralization are not certain. In this study, MVs isolated from fourth passage rat growth plate chondrocyte cultures were included within a gelatin gel into which calcium and phosphate ions diffused from opposite ends. In this gel, apatite formation occurs by 3.5 days in the absence of mineralization promoters, allowing measurement of the ability of different factors to ``nucleate' apatite before this time or to assess the effects of molecules which modulate the rate and extent of mineral deposition. Mineral ion accumulation and crystal type are assayed at 5 days. In this study, MV protein content in the central band of a 10% gelatin gel was varied by including 100 μl of a Tris-buffered solution containing 0–300 μg/ml MV protein. There was a concentration-dependent increase in mineral accretion. Whereas 10 μg MV protein in the gel did not significantly promote apatite formation as compared with vesicle-free gels, 20 and 30 μg MV protein in the gel did promote apatite deposition. Inclusion of 10 mM β-glycerophosphate in the gels, along with MVs, did not significantly increase apatite formation despite the demonstrable alkaline phosphatase activity of the MVs. In contrast, MVs at all concentrations significantly increased apatite accumulation when proteoglycan aggregates or ATP, inhibitors of apatite formation and proliferation, were included in the gel. Slight increases in calcium, but not phosphate accumulation, were also noted when an ionophore was included with the MVs to facilitate Ca ion transport into the vesicles. FT-IR analysis of the mineral formed in the vesicle-containing gels revealed the presence of a bone-like apatite. These data suggest that MVs facilitate mineralization by providing enzymes that modify inhibitory factors in the extracellular matrix, as well as by providing a protected environment in which mineral ions can accumulate. Received: 28 January 1996 / Accepted: 9 August 1996     

Needle tract recurrences after closed biopsy for sarcoma: Three cases and review of the literature

Background: Percutaneous closed needle biopsy of musculoskeletal neoplasms has gained in popularity. However, it remains controversial whether or not to resect the needle tract for fear of a local recurrence. A single published case report exists, noting the lone tract recurrence of an extremity skeletal osteosarcoma. Methods: We report on three additional individuals who demonstrated that tract local recurrences may occur after a closed needle biopsy for nonosteosarcoma, nonextremity sarcomas. For perspective, the world literature is reviewed to identify tract recurrences for other malignancies and the results of needle biopsy in musculoskeletal neoplasms. Results: Eighty-nine percent of needle tract local recurrences occur when carcinomas are subjected to biopsy, as reported in the literature. Forty-seven cases since 1950 are described representing essentially all tumor types. The nature of musculoskeletal neoplasms makes closed biopsy more difficult than for softer, more homogeneous, and easier to access neoplasms. Conclusions: Local recurrences of sarcoma may occur in closed needle biopsy tracts. Strong consideration should be given to open biopsy and tract resection.     

Brain neurons and glial cells express Neu differentiation factor/heregulin: a survival factor for astrocytes.

Neu differentiation factor (NDF, also called heregulin) was isolated from mesenchymal cells on the basis of its ability to elevate phosphorylation of ErbB proteins. Earlier in situ hybridization analysis showed that NDF was transcribed predominantly in the central nervous system during embryonic development. To gain insights into the role of NDF in brain we analyzed its distribution by immunohistochemistry and in situ hybridization. Late-gestation (day 17) rat embryos displayed high NDF immunoreactivity in both motor (e.g., putamen) and limbic (e.g., septum) regions. Lower levels of the factor were exhibited by adult brain, except for the cerebellum, where NDF expression was increased postnatally. Both neurons and glial cells were identified by immunohistochemistry as NDF-producing cells (e.g., pyramidal neurons in the cerebral cortex and glial cells in the corpus callosum). By establishment of primary cultures of rat brain cells we confirmed that NDF was expressed in neurons as well as in astrocytes. In addition, by using such primary cultures we observed that NDF treatment exerted only a limited mitogenic effect, which was accompanied by significant acceleration of astrocyte maturation. Furthermore, long-term incubation with the factor specifically protected astrocytes from apoptosis, implying that NDF functions in brain as a survival and maturation factor for astrocytes.     

Parental occupation and birth outcome in an agricultural community

The general birth outcome and prevalence of specific birth defects was investigated within an agricultural community through the review of birth records in a major hospital in Imperial County, California. Of all singleton births (N = 2 463) occurring within a four-year period, 990 or 40.2% involved offspring with one or both parent(s) who were agricultural workers. The progeny of agricultural and nonagricultural workers were similar with regard to sex ratios, prevalence of low birth-weight infants, stillbirth rate, minor and major malformation rates, and prevalence of neonatal deaths. Limb reduction defects, however, occurred more frequently among offspring of agricultural workers (5.05 per 1 000 total births versus 2.19 per 1 000 total births, rate ratio = 2.3). Furthermore, the prevalence of limb reduction defects among agricultural workers was 3- to 14-fold higher than available United States rates (0.36-1.65 per 1 000 total births). Findings from our study suggest that agricultural communities and, in particular, agricultural workers may be at excess risk of producing a child with a limb reduction defect.