Injury surveillance in agriculture.

Information on agricultural trauma is limited and difficult to find. Planning for effective prevention strategies and evaluation is compromised by lack of a good surveillance system. Several agencies and organizations have provided some data. Although their summation is at best an approximation of the real situation, a critical review of current data bases is presented. The literature is also reviewed attempting to characterize agricultural trauma. This characterization was classified into: 1) case descriptions, 2) reviews of general articles on the hazards of farming, and 3) descriptive surveys of agricultural injuries. A summary of the available literature still leaves a rather superficial understanding of the entire injury picture. A new approach to surveillance is necessary to overcome past deficiencies. A combined modality approach is suggested, utilizing on-site survey, mail survey, telephone interviewing, and medical record verification. Trial applications of two such systems in Minnesota are described.

Developmental changes in aldehyde dehydrogenases from mouse tissues.

The postnatal development of aldehyde dehydrogenase (AHD) isozymes from C57BL/6J mouse tissues was examined using agarose-IEF zymogram methods. Mitochondrial isozymes (AHD-1 and AHD-5) were present throughout, increasing to high levels in liver, kidney and stomach by weaning (3 weeks). These activities remained high subsequently, except for kidney AHD-5, which decreased significantly after week 4. The appearance of the cytosolic isozymes was tissue specific and time dependent: liver AHD-2 was undetected until day 21, and increased subsequently; stomach AHD-4 was first observed at day 5, increasing to adult levels by day 21; AHD-6 was active in neonatal kidney and stomach extracts, but was undetected after day 8; and AHD-7 was observed in liver and kidney extracts from day 16. These results supported previous proposals for multiple genes encoding aldehyde dehydrogenases in the mouse, based upon the distinct developmental profiles for the liver, kidney and stomach isozymes.

Mutations downstream of the polyadenylation site of a Xenopus beta-globin mRNA affect the position but not the efficiency of 3' processing.

In order to identify nucleotide sequences required for efficient and accurate polyadenylation of mRNA precursors, we have constructed a series of mutations in the X. laevis beta 1-globin gene and analyzed transcripts produced upon microinjection into Xenopus oocytes. Small deletion and linker replacement mutations, which lie in the region from 8 to 39 bp downstream of the AATAAA sequence and which effectively remove previously identified second components of the polyadenylation signal, do not greatly reduce the efficiency of processing, but in some cases alter the precise site of cleavage. We conclude that sequences downstream of the polyadenylation site affect the position of 3' RNA processing, but have minimal effects on its efficiency.

Polyadenylation of the Xenopus beta 1 globin mRNA at a downstream minor site in the absence of the major site and utilization of an AAUACA polyadenylation signal.

We show that adult reticulocytes of Xenopus laevis produce two forms of the beta 1 globin mRNA that differ in their site of polyadenylation. The minor site of polyadenylation is located 46 nucleotides downstream of the major site and is used in approximately 1% of mRNA molecules. A fusion gene was constructed containing the promoter from the thymidine kinase gene of herpes simplex virus fused to the protein coding, 3'-noncoding and 3'-flanking sequences of the X. laevis beta 1 globin gene. When injected into the nuclei of Xenopus oocytes, transcripts of this fusion gene were accurately and efficiently spliced and polyadenylated. The proportion of fusion gene transcripts terminating at the major and minor polyadenylation sites after injection into oocytes was approximately similar to that found in reticulocytes. When the AATAAA sequence element upstream from the major site was deleted, the minor site was used with a high (greater than 90%) efficiency. Therefore, by comparing the ratio of polyadenylation at the major and minor sites, it is possible to determine the effect of sequence alterations at the major site. In a construct where the AATAAA polyadenylation signal was changed to AATACA a high proportion (35%) of transcripts continued to be polyadenylated at the major site. This suggests a surprisingly high degree of flexibility in the precise polyadenylation signal.

Alcohol dehydrogenase in the mouse epididymis. Genetic variation and cellular localization.

The cellular localization of alcohol dehydrogenase (ADH) in the mouse epididymis was investigated using differential substrate specificities and genetic variation as a means of distinguishing these enzymes histochemically in tissue sections. ADH-C2 exhibited high activity in BALB/c epididymis and was observed as a discrete zone within duct epithelial cells near the nuclei. This isozyme exhibited no detectable activity in C57BL/6J epididymis extracts or histochemical sections.

The organization of the tadpole and adult alpha globin genes of Xenopus laevis.

Adult erythrocytes of X. laevis contain six electrophoretically resolvable globin polypeptides while tadpole erythrocytes contain four polypeptides, none of which comigrates with an adult protein. We show that three of the adult proteins are alpha globin polypeptides (alpha 1, alpha 2, alpha 3) and three are beta globin polypeptides (beta 1, beta 2, beta 3). We find that a tadpole alpha globin gene (alpha T1) is linked to the major adult locus in the sequence 5'-alpha T1-alpha 1-beta 1-3' with 5.2 kb separating alpha T1 from alpha 1. Another tadpole alpha globin gene (alpha T2) is linked to the minor adult locus in the sequence 5'-alpha T2-alpha 2-beta 2-3' with 10.7 kb separating alpha T2 from alpha 2. These linkage relationships are consistent with the major and minor loci having arisen by tetraploidization but the different separation of larval and adult globin genes at the two loci indicates the occurrence of some additional chromosomal rearrangement. Two alternative models are presented.

Internal organization of the major adult alpha- and beta-globin genes of X. laevis.

We describe the isolation of two recombinant lambda phages, each containing genomic DNA fragments encoding both the major adult alpha- and beta-globin mRNAs of X. laevis. The DNA fragment in the two clones have restriction maps which indicate that they are each derived from a different member of the pair of alleles present in the heterozygote used as the source of DNA for cloning. The characterization of these two clones by restriction mapping, R looping and DNA sequencing shows that the alpha 1- and beta 1-globin genes lie in the orientation separated by 7.7 kb of DNA. There are two introns in the alpha 1-globin gene and two in the beta 1-globin gene, and they interrupt the genes at exactly the same positions as the introns found in all known mammalian alpha- and beta-globin genes. The exon sequences proximal to the introns show a much higher degree of homology with mammalian sequences than the sequences distal to intron/exon junctions, and the introns in the beta 1-globin gene of X. laevis are very similar in length to the corresponding introns in the beta-globin genes of several mammals and the chicken.

Antibody-mediated neutralization of virus is abrogated by mycoplasma.

The ability of a mouse mammary tumor cell line to abrogate antibody neutralization of vesicular stomatitis virus was shown to be due to the presence of mycoplasma. The mycoplasma was isolated from the cell line and typed as Mycoplasma orale. Colonies of this mycoplasma were used to deliberately infect cell cultures which then gained the capacity to reactivate antibody-neutralized virus. The extent of the reactivation depended on the source of neutralizing antiserum. Other species of mycoplasma were tested and were found to reactivate neutralized virus, indicating that this may be a general phenomenon of mycoplasma contamination.

Effect of testosterone propionate on ABP levels in rats hypophysectomised at different ages using individual sampling.

The effect of age at hypophysectomy on the response of the regressed rat testis to testosterone propionate (TP) and FSH with respect to androgen-binding protein (ABP) levels was studied in individual animals. All treatments were begun 30 days after surgery. Treatment of rats 35, 45, 55 and 75 days of age at surgery with TP (1 mg/260 g for 25 days) significantly increased the level of ABP in the testes of animals in all age groups except those hypophysectomized at 35 days of age. TP treatment did not significantly elevate epididymal levels of ABP above those found in untreated rats in any age group. In animals hypophysectomized at 100 days of age, acute treatment (3 days) with FSH (150 and 300 mug/day) significantly increases the ABP levels per testis and per epididymis. Similar treatment with 750 mug TP/day did not result in a statistically significant increase in testicular ABP. No synergism between the two hormones was noted under the conditions described. Significant restoration of testicular ABP levels per mg protein was achieved with 1 mg TP/day by 5 days of treatment. Treatment of hypophysectomized adult rats with FSH raised the epididymal/testicular ratio of ABP to about 40% of that found in intact rats while comparable treatment with TP (750 mug/day for 3 days or 1 mg/day for 10 days) only slightly affected the ratio. It is postulated that FSH may facilitate ABP transport to the epididymis in addition to affecting its production by the testis.

Differential effect of phosphonoacetic acid on the expression of Epstein-Barr viral antigens and virus production.

The effects of phosphonoacetic acid on cell growth, expression of Epstein-Barr virus antigens, and virus production in human and marmoset lymphoblastoid cell lines have been studied. The drug had no significant effect at concentrations up to 100 mug/ml on cell growth or total cell DNA synthesis. Higher doses induced not only a drastic decrease in DNA synthesis and cell grwoth, but also a dramatic cell enlargement. Immunofluorescence studies showed that greater than or equal to 30 mug/ml of phosphonoacetic acid inhibited viral capsid antigen synthesis without affecting the expression of the nuclear antigen or the spontaneous and 5-iodo-2'-deoxyuridine-induced early antigens. Production of transforming Epstein-Barr virus was also blocked.

Some properties of androgen-binding activity in rat testis.

High-affinity (Ka approximately equal to 5 X 10(8) M-1 for testosterone) androgen-binding activity in rat testis was shown to have a rapid dissociation rate constant (t1/2 = 3 min, 0 degrees C, 30% glycerol buffer) using dextran-coated charcoal to separate bound from free hormone. Because of this fact, exchange of endogenous and labeled hormone was complete in the assay incubation time (16 h, 0 degrees C) and Scatchard plots of the high-affinity binding data were shown to measure total as contrasted to available sites. The binding was highly specific for androgens. Polyacrylamide gel electrophoresis separated high-affinity androgen-binding protein (Rf 0.54) from albumin (Rf 0.62). Binding site estimates under saturating conditions or by Scatchard analysis of electrophoresis data utilizing [3H]dihydrotestosterone agreed reasonably well with estimates made by the charcoal technique using [3H]testosterone.

Induction of growth in resting fibroblastic cell cultures by Ca++.

Of all the components of the culture medium, only CaCl2 induces DNA replication when added to resting cultures of Balb/c 3T3 cells. The effect is present even in a serum-free medium. Increasing the Ca++ concentration above the standard 1.8 mM in the medium of a new culture increases the total number of cells ultimately produced, without affecting the initial cell growth rate. This effect is synergistic with that of serum. The elevated Ca++ concentration also induces striking morphological changes. The Ca++ effect could not be reproduced by a Ca++ ionophore. These observations afford a new tool for studying how the various intracellular events following the addition of growth factors to resting cultures are involved in the control of cellular growth.

Hormonal influences on the level of testicular androgen binding activity: effect of FSH following hypophysectomy.

Concentrations of high affinity (Ka equals 5.5 plus or minus 0.83 times 10-8M-1) androgen binding activity in carbonextracted rat testicular supernatants have been determined by Scatchard plot analysis under a variety of hormonal situations: (a) 0-90 days following hypophysectomy; (b) 0-60 days of daily injection of NIH-FSH-P1 (150 mug) beginning 1 day after hypophysectomy; and (c) 0-60 days of daily FSH treatment beginning 30 days after hypophysectomy. The high affinity binding component declined from 0.32 pmoles/mg protein intact adults to 0.28, 0.17, and less than 0.08 (limit of detectability) pmoles/mg protein at 11, 16 and 31 days, respectively. FSH treatment beginning immediately after surgery slowed this decline, giving values of 0.30, 0.17, and 0.12 pmoles/mg protein after 11, 29, and 54 days of treatment, respectively. Expressed as pmoles per testis this represented 96, 61, and 40% of the intact control level. Similar effects on the level of androgen binding protein (Rf 0.54) were measured by steady-state polyacrylamide gel electrophoresis (PAGE). The concentration in both testis and epididymis declined gradually to nondetectable levels by 30 days after surgery. FSH treatment for 11, 29, and 54 days, respectively, resulted in 0.37, 0.38, and 0.03 pmoles of sites/mg protein in testis compared to 0.34 in intact controls and 5.7, 2.6, and 0.2 pmoles/mg protein in epididymis compared to 2.8 in intact controls. Doses of 80, 150, and 300 mug FSH/rat/day for 3 days beginning 30 days after hypophysectomy when postmeiotic elements of the germinal epithelium had degenerated caused graded increases in both testicular and epididymal levels of androgen binding protein. Prolonged FSH treatment (150 mug) under these conditions resulted in an increase in binding activity to a level of 0.12, 0.19, 0.17, and 0.20 pmoles/mg protein after 3, 11, 25, and 56 days of treatment. On a per testis basis this represented less than 20% of intact control levels. PAGE estimates were comparable except at the long treatment intervals. These results indicate that FSH treatment influences the level of androgen binding protein in adult testis and epididymis. This may reflect a direct influence on synthesis, degradation, and transport and/or indirect effects on general maintenance and responsiveness of the pertinent cell types.

Androgen binding in the testis: in vitro production of androgen binding protein (ABP) by Sertoli cell cultures and measurement of nuclear bound androgen by a nuclear exchange assay.

Androgen binding activity in the testis has two components. One component, ABP, has been shown to be produced by Sertoli cell cultures for at least 9 days in the absence of exogenously added hormones. FSH (10-100 microgram/ml) markedly enhances the secretion of ABP. MIX has a potentiating effect after long treatment intervals (7 days). In order to study the second component, intracellular androgen receptor, a nuclear exchange assay was developed. Competition for exchange activity using 3H-dihydrotestosterone was significant for a 500 fold excess of testosterone, dihydrotestosteron, progesterone, and cyproterone acetate. The exchange activity was increased 2-10 fold by prior treatment in vitro or in vivo with testosterone. Significant exchange activity was found in long-term hypophysectomized adult and immature animals and in tubule and germ cell fractions. In isolated germ cell fractions, the highest concentration of exchange activity was associated with the most mature elements. These data suggest that androgen exchange activity may exist in both Sertoli cell and germ cell fractions and suggest that the mechanism of action of androgens in the testis is quite complex.

Isolation and culture of FSH responsive Sertoli cells.

Methods for the isolation and culture of enriched populations of Sertoli cells from 20-60 day old rats are described. The identity of the Sertoli cells was verified by bright light and electron microscopy. Freshly isolated Sertoli cells specifically bound follicle stimulating hormone (FSH) but not luteinizing hormone (LH) and responded to FSH stimulation with dramatic increase in cyclic AMP level. Isolated Sertoli cells, maintained in culture for 11 days, showed no evidence of proliferation but retained their characteristic ultrastructural features and FSH binding ability. Incubation of cultured cells with FSH resulted in a significant stimulation of cyclic AMP and androgen binding protein (ABP). Since the freshly isolated or cultured cells were predominantly (greater than 80%) Sertoli cells, these results provide direct evidence that the Sertoli cells represent a primary target site for FSH activity in the testes. The culture method also provides a valuable in vitro model for the study of chronic effects of various agents on the Sertoli cell.