Direct mitogenic effects of insulin, epidermal growth factor, glucocorticoid, cholera toxin, unknown pituitary factors and possibly prolactin, but not androgen, on normal rat prostate epithelial cells in serum-free, primary cell culture.

Selective nutritive conditions were used to isolate normal epithelial cells from fibroblasts in primary cell cultures prepared from adult rat prostate. The pure population of normal epithelial cells proliferated at an exponential rate on a simple polystyrene substratum with doubling times of 35 to 50 hr for 10 to 12 days in the absence of high epithelial cell density, other cell types, or added extracellular matrix elements. Optimization of the nutritive environment allowed direct analysis of the hormone:growth factor requirements for sustained proliferation of the isolated epithelial cells in serum-free medium. An in situ videometric method was used to assay the effect of over 30 known hormones and growth factors on proliferation of the prostate epithelial cell population. The results revealed direct mitogenic effects of insulin, epidermal growth factor, glucocorticoid, cholera toxin, one or more unidentified factors from bovine pituitary, and possibly prolactin. No direct mitogenic effect of androgen on isolated prostate epithelial cells could be demonstrated. Radioimmunoassay of androgen in the primary cultures showed that endogenous androgen was about 34 pM on Day 1 of culture and thus probably too low to mask a response to exogenous androgen. Deletion of any single active growth factor did not reveal an androgen response. The results demonstrate a multihormonal control of normal prostate epithelial cell maintenance and proliferation without the direct participation of androgen.

Design strategies and performance of custom DNA sequencing primers.

This study surveyed strategies of sequencing primer selection and evaluated primer performance in automated DNA sequencing. We asked participants to relate their preferred primer design strategies to identify primer characteristics that are considered most important in sequencing primer design. The participants preferred primers of 18-24 nucleotides (nt), 39%-58% G + C, a melting temperature (Tm) of 53 degrees-65 degrees C with a 1-2 nt 3' GC clamp, hairpin stems of less than 2-3 bp, homopolymeric runs of less than 4-5 nt, primer dimers of less than 3-4 bp and secondary priming sites of less than 3-4 bp. We provided a 300-bp test sequence and asked participants to submit sequences of 1-3 optimal sequencing primers. Submitted primers ranged from 17-24 nt and largely conformed to the preferred parameters. Submitted primers were distributed across the test sequence, although some sites were disfavored. Surprisingly, approximately 45% of the primers were selected "manually", more than by any software package. Each of 69 submitted and 95 control primers, distributed at 3-bp intervals across the test sequence, were synthesized, purified and tested using a Model 377 PRISM DNA Sequencer with dichlororhodamine dye terminator reagents (dRhodamine dye terminators). Approximately half of the control primers were also tested using rhodamine dye terminator reagents ("old" rhodamine dye terminators). The results indicated that primer physico-chemical characteristics thought to have a strong impact on sequencing performance had surprisingly little effect. Thus, primers with high or low percent G + C or Tm, strong secondary priming scores or long 3' homopolymeric stretches yielded excellent sequences with the dRhodamine dye terminator reagents, although these characteristics had a stronger effect when the old rhodamine reagents were used. The old rhodamine reagents gave sequences with a similar average read length, but the number of errors and ambiguities or "N's" was consistently higher. Moreover, the effects of the primer physico-chemical characteristics were also more evident with the old rhodamine dyes. We conclude that under optimal sequencing conditions with highly pure template and primer, many of the commonly applied primer design parameters are dispensable, particularly when using one of the new generation of sequencing reagents such as the dichlororhodamine dye terminators.

Differential display RT PCR of total RNA from human foreskin fibroblasts for investigation of androgen-dependent gene expression.

Male sexual differentiation is a process that involves androgen action via the androgen receptor. Defects in the androgen receptor, many resulting from point mutations in the androgen receptor gene, lead to varying degrees of impaired masculinization in chromosomally male individuals. To date no specific androgen regulated morphogens involved in this process have been identified and no marker genes are known that would help to predict further virilization in infants with partial androgen insensitivity. In the present study we first show data on androgen regulated gene expression investigated by differential display reverse transcription PCR (dd RT PCR) on total RNA from human neonatal genital skin fibroblasts cultured in the presence or absence of 100 nM testosterone. Using three different primer combinations, 54 cDNAs appeared to be regulated by androgens. Most of these sequences show the characteristics of expressed mRNAs but showed no homology to sequences in the database. However 15 clones with significant homology to previously cloned sequences were identified. Seven cDNAs appear to be induced by androgen withdrawal. Of these, five are similar to ETS (expression tagged sequences) from unknown genes; the other two show significant homology to the cDNAs of ubiquitin and human guanylate binding protein 2 (GBP-2). In addition, we have identified 8 cDNA clones which show homologies to other sequences in the database and appear to be upregulated in the presence of testosterone. Four of these clones again are similar to ETS from unknown genes. Three differential expressed sequences that appear to be upregulated in the presence of testosterone show significant homology to the cDNAs of L-plastin and one to the cDNA of testican. This latter gene codes for a proteoglycan involved in cell social behavior and therefore of special interest in this context. The results of this study are of interest in further investigation of normal and disturbed androgen-dependent gene expression.

Stability of insulin mixtures in disposable plastic insulin syringes.

The miscibility of short and long acting porcine, human and bovine insulins has been investigated using HPLC. Soluble insulin is rapidly lost from solution when mixed with both isophane and zinc insulin, although the mechanism may be different in these two cases. The time for up to 50% or more to be lost is often less than 2 min. The percentage lost is dependent upon the ratio of the mixture and the type and origin of the insulin. In the case of soluble: zinc mixtures it is greatest for porcine and least for bovine, in the case of soluble: isophane mixtures it was least for human and greatest for bovine. The greater the original amount of soluble insulin present the less the loss. The results help explain recent clinical reports that mixtures of soluble and zinc insulins fail to achieve satisfactory control of post-prandial blood sugar levels.

Potential role of HBGF (FGF) and TGF-beta on prostate growth.

We review in this paper the role of heparin-binding growth factor (HBGF*) or fibroblast growth factor (FGF*), rat prostate cancer cells produce TGF-beta, IGF-II* and OGF*. Of these growth factors, TGF-beta and unknown labile factor with 19 kDa are the most probable candidates responsible for osteoblastic bony metastasis of prostate cancer. In vitro experiments suggest that TGF-beta modulates cell detachment of prostate cancer cells together with nutritional factors. HBGF-dependent growth of the prostate tumor epithelial cells is free from inhibition by TGF-beta, whereas normal prostate epithelial cells are sensitive to TGF-beta inhibition. Transfection experiments suggest that HBGF-2 (basic FGF) might be closely related to the malignant growth of prostate cancer, in addition to tumor angiogenesis.

Canine distemper viral inclusions in blood cells of four vaccinated dogs.

Four cases of canine distemper were detected by the presence of numerous cytoplasmic inclusions in various circulating blood cells. Fluorescent antibody techniques and electron microscopy confirmed the identity of the viral inclusions. The cases occurred in the same geographic area and within a short time span. All four dogs had been vaccinated against canine distemper, but stress or other factors may have compromised their immune status. The possibility of an unusually virulent virus strain was also considered.

Comparison of commercially available target enrichment methods for next-generation sequencing.

Isolating high-priority segments of genomes greatly enhances the efficiency of next-generation sequencing (NGS) by allowing researchers to focus on their regions of interest. For the 2010-11 DNA Sequencing Research Group (DSRG) study, we compared outcomes from two leading companies, Agilent Technologies (Santa Clara, CA, USA) and Roche NimbleGen (Madison, WI, USA), which offer custom-targeted genomic enrichment methods. Both companies were provided with the same genomic sample and challenged to capture identical genomic locations for DNA NGS. The target region totaled 3.5 Mb and included 31 individual genes and a 2-Mb contiguous interval. Each company was asked to design its best assay, perform the capture in replicates, and return the captured material to the DSRG-participating laboratories. Sequencing was performed in two different laboratories on Genome Analyzer IIx systems (Illumina, San Diego, CA, USA). Sequencing data were analyzed for sensitivity, specificity, and coverage of the desired regions. The success of the enrichment was highly dependent on the design of the capture probes. Overall, coverage variability was higher for the Agilent samples. As variant discovery is the ultimate goal for a typical targeted sequencing project, we compared samples for their ability to sequence single-nucleotide polymorphisms (SNPs) as a test of the ability to capture both chromosomes from the sample. In the targeted regions, we detected 2546 SNPs with the NimbleGen samples and 2071 with Agilent's. When limited to the regions that both companies included as baits, the number of SNPs was ∼1000 for each, with Agilent and NimbleGen finding a small number of unique SNPs not found by the other.

Iliac artery-ureteral fistula developing after dilatation and stent placement.

A fistula formed between the common iliac artery and the ureter following balloon dilatation of the distal ureter and stent placement in the presence of an ileal conduit. Possible causes are discussed.

Heparin-binding growth factor/prostatropin attenuates inhibition of rat prostate tumor epithelial cell growth by transforming growth factor type beta.

Normal rat prostate epithelial cell growth requires both epidermal growth factor and heparin-binding growth factor/prostatropin. In contrast, epithelial cells derived from the transplantable Dunning R3327H rat tumor require either epidermal growth factor or heparin-binding growth factor/prostatropin. Transforming growth factor type beta inhibited normal epithelial cell growth. Transforming growth factor beta inhibited epidermal growth factor-dependent growth of tumor epithelial cells, independent of epidermal growth factor concentrations. Transforming growth factor beta increased the effective dose of heparin-binding growth factor type 1 required to support tumor epithelial cell growth by 10-fold but saturating levels of heparin-binding growth factor type 1 (290 pM) completely attenuated the inhibitory effect of transforming growth factor beta. These results suggest that prostate tumor epithelial cells may escape the inhibitory effect of transforming growth factor beta as a consequence of alteration of the concurrent requirement for both epidermal growth factor (or homologues) and heparin-binding growth factors.

The effect of size and fabric weight of protective coveralls on range of gross body motions.

This study evaluated the effects of garment size and fabric weight on range-of-motion (ROM). Ten male subjects performed a series of twelve gross body movements while wearing each of nine similarly styled coveralls. The coveralls were undersized, appropriately sized, and oversized, and were constructed from three different weights of poly/cotton fabric. A balanced 3 x 3 repeated measures experimental design was used, along with a seminude control condition. ROM was measured with a two-arm manual goniometer. Garment size significantly affected (p < .05) ROM for all movements except shoulder extension and trunk lateral flexion. Compared to seminude ROM, undersized garments reduced the mean ROM by as much as 24% in the case of hip flexion. Fabric weights on ROM were significant for shoulder extension and elbow, hip, knee, and shoulder horizontal flexion. Fabric weight affected ROM less than garment size. Interaction effects between fabric weight and size generally were not significant. These results demonstrate that undersized garments can measurably reduce the wearer's movement capability. Providers of protective clothing should ensure that garments are not undersized and should consider the benefits of oversizing against possible safety and wearer acceptance problems.

Outpatient angiography and interventional radiology: safety and cost benefits.

A three-year study of the safety and cost benefits of outpatient angiography and interventional radiology was undertaken. Of 140 ambulatory patients (17% of all procedures) no delayed complications after discharge were encountered; the estimated cost savings was $20,886. This protocol appears to be both safe and cost-effective.

Different hormonal requirements for androgen-independent growth of normal and tumor epithelial cells from rat prostate.

The proliferation of isolated normal prostate epithelial cells from rat and man is androgen-independent and requires cholera toxin, insulin, dexamethasone, epidermal growth factor (EGF) and one or more polypeptide factors that are concentrated in bovine neural tissue. The active agents in the neural tissue extract are heparin-binding polypeptides (prostatropins), the predominant form of which has a molecular weight of 17400 and an acetylalanine at the aminoterminus. Prostatropins supported a half-maximal increase in normal prostate epithelial cell number at 50 picomolar. The proliferation of primary and serially-cultured epithelial cells from androgen-responsive Dunning R3327 rat prostate tumors was also androgen-independent, but exhibited dramatic alterations in response to hormones that stimulated normal cell proliferation. At low cell density, androgen-independent growth of isolated tumor-derived epithelial cells was independent on cholera toxin, was stimulated by dexamethasone, required insulin and either EGF or prostatropin. The presence of either EGF or prostatropin masked the response to the other factor. In the absence of EGF, purified prostatropins supported a half-maximal increase in tumor cell number at 7 picomolar. Endogenous production of EGF-like and prostatropin-like factors or both was suggested by the reduced requirement for EGF and prostatropin at high prostate tumor cell density. These results suggest that anti-hormonal therapies against prostate tumor growth should be based on intervention with the activity of insulin (or insulin-like factors) or simultaneous intervention with both EGF and prostatropin (or their homologues).

Modified nutrient medium MCDB 151, defined growth factors, cholera toxin, pituitary factors, and horse serum support epithelial cell and suppress fibroblast proliferation in primary cultures of rat ventral prostate cells.

Nutrient medium WAJC 401 containing 5 micrograms/ml insulin. 10 ng/ml EGF, 10 ng/ml cholera toxin, 50 micrograms/ml pituitary extract, 1 microgram/ml prolactin, 1 microM dexamethasone, and 5% horse serum supports the rapid proliferation of rat ventral prostate epithelial cells in primary culture. The same medium suppresses the growth of prostate fibroblasts.

Subarterial ventricular septal defect in an infant with sudden unexpected death: cause or coincidence?

A case of unexpected death in a 3-month-old infant with a subarterial ventricular septal defect (VSD) is described. Although VSDs in medically stable infants are not usually associated with fatal outcome, this case and review of the literature suggest that a small percentage of such patients may have an increased risk of sudden death.

Isolation and characterization of a mouse homolog of the Drosophila segment polarity gene dishevelled.

In the Drosophila embryo dishevelled (dsh) function is required by target cells in order to respond to wingless (wg, the homolog of Wnt-1), demonstrating a role for dsh in Wnt signal transduction. We have isolated a mouse homolog of the Drosophila dsh segment polarity gene. The 695-amino-acid protein encoded by the mouse dishevelled gene (Dvl-1) shares 50% identity (65% similarity) with dsh. Similarity searches of protein and DNA data bases revealed that Dvl-1 encodes an otherwise novel polypeptide. While no functional motifs were identified, one region of Dvl-1 was found to be similar to a domain of discs large-1 (dlg), a Drosophila tumor suppressor gene. In the embryo, Dvl-1 is expressed in most tissues, with uniformly high levels in the central nervous system. From 7.5 days postcoitum Dvl-1 is expressed throughout the developing brain and spinal cord, including those regions expressing Wnt-1 and En. Expression of Dvl-1 in adult mice was found to be widespread, with brain and testis exhibiting the highest levels. The majority of Dvl-1 expression in the adult cerebellum is in the granular cell layer, similar to the pattern seen for engrailed-2 (En-2). Throughout postnatal development of the brain Dvl-1 is highly expressed in areas of high neuronal cell density.

Prostatic binding protein, polyamine, and DNA synthesis in rat ventral prostate cells.

Prostatic binding protein (PBP), polyamine, and DNA synthesis were examined in primary cultures of rat ventral prostate cells. Soon after aggregates of prostate cells were placed in culture, PBP synthesis fell dramatically and DNA synthetic activity in the epithelial cells increased. The amount of polyamines that were labeled with [3H] from [3H] ornithine fell transiently, but rose again at or before the peak of DNA synthesis. Individual 3H-labeled polyamines in cells and medium were dansylated and separated by thin-layer chromatography. The ratio of [3H] Spermidine plus [3H] Spermine to [3H] Putrescine in the culture medium declined as DNA synthesis increased. Ornithine decarboxylase (ODC) activity fell dramatically along with PBP synthesis even as DNA synthesis and 3H-labeled polyamines increased in the prostate-cell cultures. These results support others that suggest that high ODC activity in prostate epithelial cells is a correlate of prostate epithelial cell function rather than proliferation. However, prostate epithelial cells retain the capacity to synthesize significant levels of polyamines from ornithine (especially Putrescine) during proliferation even when ODC activity is low.

The androgen-dependent rat prostate protein, probasin, is a heparin-binding protein that co-purifies with heparin-binding growth factor-1.

Rat prostate extracts contain an abundant 20-22 kilodalton heparin-binding protein with near identical chromatographic properties, but only 0.2-1% of the mitogenic activity, of bovine brain heparin-binding growth factor-1 (acidic fibroblast growth factor). Amino terminal amino acid sequence (met-met-thr-asp-lys-asn-leu-lys-lys-lys-ile-glu-gly-asn-trp-arg-thr-val -tyr- leu-ala-ala-ser-?-val-glu-lys-ile-asn-glu-gly-ser-pro) and immunochemical analysis revealed that the protein is identical to the androgen-dependent protein "probasin".

Blood pressure in schoolchildren measured under standardized conditions.

The blood pressures of 6346 children who were between the ages of seven and 17 years were measured under standardized conditions. Blood pressures were similar in prepubertal boys and girls. After puberty, the systolic blood pressures of the girls remained unchanged whereas those of the boys continued to rise. The difference between the fourth and fifth Korotkoff sounds was 2 mm at both the 50th and 95th percentiles. The systolic blood pressure was observed to fall over a 10-min period by 4-9 mmHg at the 50th percentile and 9-15 mmHg at the 95th percentile. Over the same period of time, the diastolic blood pressure was seen to fall by 2-3 mmHg and 3-6 mmHg at the 50th and 95th percentiles, respectively. There was no further fall in blood pressure after 10 min. The conditions and timing of measurement are important in blood-pressure evaluation and may explain the differences in blood pressure that have been reported for different populations.