Curcumin induced oxidative stress attenuation by N-acetylcysteine co-treatment: a fibroblast and epithelial cell in-vitro study in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic Pulmonary Fibrosis (IPF) is a fatal lung disease of unknown etiology with only two federally approved drug options. Given the complex molecular pathogenesis of IPF involving multiple cell types and multiple pathways, we explore the effects of a potential antifibrotic and antioxidant drug combination. Curcumin is a polyphenolic compound derived from turmeric with significant biological activity including a potential antifibrotic capacity. N-acetylcysteine (NAC) is a precursor to the antioxidant glutathione. To advance our understanding of these molecules, and to identify a clinical application, we present a small number of focused experiments that interrogates the effect of curcumin and NAC on pathways relevant to IPF in both fibroblasts and epithelial cells. METHODS: Primary epithelial cell and fibroblasts isolated from patients with IPF were challenged with a combination treatment of NAC and curcumin. Evaluation of the antifibrotic potential and effect on oxidative stress was performed through QPCR gene expression analysis and functional assays including scratch tests, viability assays, and measurement of induced reactive oxygen species. RESULTS: We demonstrate that curcumin alone does have antifibrotic potential, but that effect is accompanied by proapoptotic increases in oxidative stress. Coupled with this, we find that NAC alone can reduce oxidative stress, but that epithelial cell viability is decreased through this treatment. However, co-administration of these two molecules decreases oxidative stress and maintains high cell viability in both cell types. In addition, this co-treatment maintains an antifibrotic potential. CONCLUSIONS: These findings suggest a novel application for these molecules in IPF and encourage further exploration of this potential therapeutic approach.

Perfusion alters free zinc levels in the rodent brain.

BACKGROUND: Fixation of brain tissue is a common practice which allows preservation of tissue and aids in preventing structural and chemical abnormalities. However, fixation procedures may disrupt the levels of biometals such as zinc when compared to tissue that is fresh-frozen. Thus, we sought to determine if any differences in free-zinc levels exist between perfused and fresh-frozen tissue. Zinc is an essential biometal critical for cellular communication and memory and exists in both bound and free forms; the latter playing critical roles in synaptic communication. New method: C57BL/6 J mice were divided into two water types: those given lab water and those given water supplemented with 10 ppm zinc carbonate. Perfusion was carried out with 4% paraformaldehyde on half of the animals in each water group to assess the impact on levels of free Zn as measured through Zinpyr-1 fluorescence. RESULTS: There were significant differences in Zn fluorescence values between Zn-supplemented and lab water groups as well as between perfused and fresh-frozen tissues in the dentate gyrus and CA3 regions of the hippocampus, regions critical in learning & memory. Comparison with existing methods: These results show that when determining a method for euthanasia, any future histological techniques involving assessment of metal content should first be considered. CONCLUSIONS: Researchers must be cautious with the way in which tissue is collected and treated since this can lead to misleading conclusions when linking changes in behavior and relative levels of trace metals.

JP-8 jet fuel-induced DNA damage in H4IIE rat hepatoma cells.

We investigated the genotoxicity of middle distillate jet fuel, Jet Propulsion 8 (JP-8), on H4IIE rat hepatoma cells in vitro. DNA damage was evaluated using the comet (single cell gel electrophoresis) assay. Cells were exposed for 4h to JP-8 (solubilized in ethanol (EtOH) at 0.1% (v/v)) to concentrations ranging from 1 to 20microg/ml. Exposure to JP-8 resulted in an overall increase in mean comet tail moments ranging from 0.74+/-0.065 (0.1% EtOH control) to 3.13+/-0.018,4.36+/-0.32,5.40+/-0.29,7.70+/-0.52 and 11.23+/-0.77 for JP-8 concentrations 3, 5, 10, 15 and 20microg/ml, respectively. Addition of DNA repair inhibitors hydroxyurea (HU) and cytosine arabinoside (Ara-C) to cell culture with JP-8 resulted in accumulation of DNA damage strand breaks and increase in comet tail length. Inclusion of 4mM HU and 40microM Ara-C with 3, 5, 10 and 20microg/ml JP-8 concentrations resulted in increased mean tail moments to 5.94+/-0.43,10.12+/-0.72,17.03+/-0.96,and29.25+/-1.55. JP-8, in the concentrations used in this study, did not result in cytotoxicity or significant apoptosis, as measured using the terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-X nick end labeling (TUNEL) assay. These results demonstrate that relevant exposures to JP-8 result in DNA damage to H4IIE cells, and suggest that DNA repair is involved in mitigating these effects.

Functional ionotropic glutamate receptors emerge during terminal cell division and early neuronal differentiation of rat neuroepithelial cells.

Ionotropic glutamate receptors mediate fast forms of excitatory synaptic transmission in mature neurons and may play critical roles in neuronal development. However, the developmental stage at which neuronal cells begin to express functional receptors and their roles in lineage progression remain unclear. In the present study, neural precursor cells were isolated from the cortical neuroepithelium of embryonic day 13 rats, and rapidly expanded in serum-free medium in response to basic fibroblast growth factor. RT-PCR revealed the presence of mRNAs encoding AMPA(A), AMPA(C), KA(1), KA(2), NMDA(1), and NMDA(2D) subunits after 3 days in culture. The functional expression of AMPA/kainate and NMDA receptors was investigated using Ca(2+) imaging and whole-cell patch-clamp recording techniques in cells pulse-labeled with bromodeoxyuridine (BrdU) for 1-4 hr. The recorded cells were then double-immunostained for BrdU incorporation and neuron-specific beta-tubulin (TuJ1). The results show that AMPA/kainate and NMDA induced increases in cytosolic Ca(2+) and inward currents only in differentiating neurons. In contrast, proliferating (BrdU(+)TuJ1(-)) cells failed to respond to any ionotropic glutamate receptor agonists. Interestingly, Ca(2+) imaging revealed that a subpopulation of BrdU(+)TuJ1(+) cells also responded to AMPA, indicating the emergence of functional ionotropic AMPA/kainate receptors during terminal cell division and the earliest commitment to neuronal cell lineage. These in vitro results were supported by flow cytometric sorting of AMPA-responsive cells pulse-labeled with BrdU for 1 hr in vivo, which revealed that functional AMPA receptors appear in BrdU(+)TuJ1(+) cells under physiological conditions and may play a role in terminal cell division.

Acetylcholine stimulates cortical precursor cell proliferation in vitro via muscarinic receptor activation and MAP kinase phosphorylation.

Increasing evidence has shown that some neurotransmitters act as growth-regulatory signals during brain development. Here we report a role for the classical neurotransmitter acetylcholine (ACh) to stimulate proliferation of neural stem cells and stem cell-derived progenitor cells during neural cell lineage progression in vitro. Neuroepithelial cells in the ventricular zone of the embryonic rat cortex were found to express the m2 subtype of the muscarinic receptor. Neural precursor cells dissociated from the embryonic rat cortical neuroepithelium were expanded in culture with basic fibroblast growth factor (bFGF). reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the presence of m2, m3 and m4 muscarinic receptor subtype transcripts, while immunocytochemistry demonstrated m2 protein. ACh and carbachol induced an increase in cytosolic Ca2+ and membrane currents in proliferating (BrdU+) cells, both of which were abolished by atropine. Exposure of bFGF-deprived precursor cells to muscarinic agonists not only increased both cell number and DNA synthesis, but also enhanced differentiation of neurons. These effects were blocked by atropine, indicating the involvement of muscarinic ACh receptors. The growth-stimulating effects were also antagonized by a panel of inhibitors of second messengers, including 1,2-bis-(O-aminophenoxy)-ethane-N,N,N', N'-tetraacetic acid (BAPTA-AM) to chelate cytosolic Ca2+, EGTA to complex extracellular Ca2+, pertussis toxin, which uncouples certain G-proteins, the protein kinase C inhibitor H7 and the mitogen-activated protein kinase (MAPK) inhibitor PD98059. Muscarinic agonists activated MAPK, which was significantly inhibited by atropine and the same panel of inhibitors. Thus, muscarinic receptors expressed by neural precursors transduce a growth-regulatory signal during neurogenesis via pathways involving pertussis toxin-sensitive G-proteins, Ca2+ signalling, protein kinase C activation, MAPK phosphorylation and DNA synthesis.

Investigation of in vitro toxicity of jet fuels JP-8 and Jet A.

The in vitro cytotoxicity and electrophysiological toxicity of Jet Propulsion-8 (JP-8 jet fuel) on four cell types: H4IIE liver cell line, NIH Swiss 3T3 cell line, neuroblastoma x glioma NG108-15 cells, and embryonic hippocampal neurons were investigated. H4IIE cells exposed to Jet A (a commercial fuel) and JP-8 demonstrated identical toxicity with an IC50 of 12.6 +/- 0.4 micrograms/ml for the two fuels. Comparison of H4IIE and NIH/3T3 toxicity to JP-8 revealed that NIH/3T3 cells were more sensitive to JP-8 than H4IIE cells, with an IC50 8.5 +/- 0.1 micrograms/ml. JP-8 exposure for the hippocampal neurons proved to be highly toxic (IC50 of < 2 micrograms/ml), while in contrast, the NG108-15 cells were much less sensitive. Electrophysiological examination of NG108-15 cells showed that administration of JP-8 at 1 microgram/ml did not alter significantly any of the electrophysiological properties. However, exposure to JP-8 at 10 micrograms/ml during a current stimulus of +46 pA decreased the amplitude of the action potential to 83 +/- 7% (n = 4), the rate of rise, dV/dtMAX to 50 +/- 8% (n = 4), and the spiking rate to 25 +/- 11% (n = 4) of the corresponding control levels. These results demonstrate JP-8 induced cytotoxic varies among cell types. The possible mechanisms underlying these observations are presented.

Overview of expression of matrix metalloproteinases (MMP-17, MMP-18, and MMP-20) in cultured human cells.

We recently described the cell type distribution of several matrix metalloproteinases (MMP-1 through MMP-16). In this report we extend this study by analysis of three recently described MMPs. PCR primers for MMP-17, MMP-18, and MMP-20 were optimized for use in RT-PCR. The results demonstrate one or more cell lines or tissue that express mRNA for each of these newly described MMPs.

Kir 4.1 channel expression in neuroblastomaxglioma hybrid NG108-15 cell line.

To study a possible involvement of inwardly rectifying K+ 4.1 (Kir 4. 1) channels in neural cell development, RT-PCR, immunocytochemistry and whole-cell patch-clamp techniques were used to assess expression of Kir 4.1 channels in proliferating and differentiated NG108-15 cells. RT-PCR revealed co-expression of Kir 4.1 and rat ether-a-go-go-related gene (R-ERG) mRNAs in both proliferating and differentiated cells. The relative Kir 4.1 mRNA concentration increased markedly as cells progressed from undifferentiated to differentiated cells. Kir 4.1-immunoreactivity was barely detectable in undifferentiated cells, but clearly detected in differentiated cells, indicating that Kir 4.1 gene and protein expressions are developmentally regulated. However, corresponding Kir 4.1 current could not be detected in differentiated cells using whole-cell patch-clamp recording. The 'silent' channel/receptor, often found in tumor cells, may carry genetic defects, which prevent functional expression of the channel. NG108-15 may serve as unique model for studying the relationship between the expression of an ion channel gene and the electrophysiological phenotype it encodes.

A role for inwardly rectifying K+ channels in differentiation of NG108-15 neuroblastoma x glioma cells.

The whole-cell patch-clamp technique was used to assess the current carried by inwardly rectifying K+ channels (K(ir)) and the resting membrane potential (RMP) during long-term culture of NG108-15 cells. Culture of this cell line in serum-free medium triggers differentiation of a type I, neuron-like cell type followed by an eventual predominance of a type II, proliferative cell type. NG108-15 K(ir) currents, which strongly resemble currents carried by human ether-a-go-go related gene (HERG) K+ channels, exhibited significantly smaller current density for the more depolarized undifferentiated cells in growth media (GM) and type II cells compared to the neuron-like type I cells. Detailed examination of the transition from undifferentiated GM cells to type I cells revealed a shift in the voltage dependence of K(ir) activation which paralleled the more hyperpolarized RMP, neurite outgrowth, and biochemical differentiation characteristic of type I cells. Reverse-transcription polymerase chain reaction experiments using primers for the rat variant of HERG, RERG, revealed a a nearly twofold increase in RERG mRNA as cells differentiate from GM to type I, a finding entirely consistent with the increased K(ir) current density derived from patch-clamp recordings. Administration of CsCl(5 mM) blocked K(ir) currents and depolarized the RMP of type I cells. Furthermore, culture of NG108-15 cells in serum-free medium but with CsCl added significantly prevented neurite extension, an effect which was entirely reversible upon subsequent removal of CsCl. In contrast, other K+ channel inhibitors (4-aminopyridine and tetraethylammonium), at concentrations without marked effects on K(ir), failed to affect neurite extension. These results suggest an important role of the K(ir) channels in determining the RMP and triggering morphological differentiation of the cell line.

Overview of matrix metalloproteinase expression in cultured human cells.

The matrix metalloproteinases (MMP) have been implicated in tumor invasion and metastasis both by immunohistochemical studies and from the observation that specific metalloproteinase inhibitors block tumor invasion and metastasis. Oligonucleotide primers for thirteen MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16) were optimized for use in RT-PCR. A semi-quantitative RT-PCR assay was used to determine the pattern of MMP mRNA expression in 84 normal and transformed or carcinogen transformed human cell lines and strains derived from different tissues. The results demonstrate one or more cell lines which express thirteen members of the MMP family. In addition, various oncogene transfected human fibroblast cell strains were analyzed for MMP expression. We confirm that over-expression of the H-ras oncoprotein correlates with up-regulation of MMP-9 and demonstrate that over-expression of v-sis also up-regulates MMP-9. A cell line immortalized following myc expression was found to up-regulate MMP-7, MMP-11 and MMP-13. Inappropriate expression of several MMP mRNAs was detected in breast, prostate, bone, colon and oral tumor derived cell lines. Identification of at least one cell line expressing each of thirteen MMPs and the observation of oncogene induced expression of several MMPs should facilitate analysis of the transcriptional mechanisms controlling each MMP.

Immunofluorescence microscopy and flow cytometry characterization of chemical induction of latent Epstein-Barr virus.

The effects of chemical induction of latent Epstein-Barr virus (EBV) with 12-O-tetradecanoyl phorbol-13-acetate (TPA) and n-butyrate on cell viability and induction of latent EBV in Raji and X50-7 B lymphocytes, indicated by expression of the diffuse component of the EBV early antigen (EA-D), were measured by visual immunofluorescence microscopy (of both viable and nonviable cells) and fluorescence-activated cell sorter (FACS) flow cytometry (of viable cells only). Cell viability at 4 days decreased moderately for treated Raji cells (9 to 37%, compared to 55 to 69% for untreated cells) and markedly for X50-7 cells (1-32% compared to 35-44% in untreated cells). The highest EA-D levels in viable cells occurred in Raji cells treated with both TPA and n-butyrate and untreated X50-7 cells. TPA and n-butyrate acted synergistically to induce latent EBV, resulting in increased levels of EA-D production in Raji cells and cell death in X50-7 cells. Methodological differences including the ability to detect antigen in only viable cells by FACS flow cytometry accounted for the higher levels of EA-D observed by FACS analysis compared to the levels observed by immunofluorescence microscopy. FACS analysis may be more objective and reproducible than immunofluorescence microscopy for the detection of EBV induction and also permits viral protein expression to be distinguished in the subpopulation of viable cells.

RT-PCR without RNA isolation.

Reverse transcription-PCR (RT-PCR) has traditionally required time-consuming RNA extraction and purification. This report demonstrates that one can completely avoid the RNA extraction step in RT-PCR by basing the comparison of samples on cell number rather than micrograms of total RNA. A new method for lysing cells while preserving RNA is described. RT-PCR is carried out (i) by rapidly freezing cells in the presence of ribonuclease inhibitor (RNase inhibitor) plus dithiothreitol and (ii) by using extracts of 250 or fewer cells directly in the RT-PCR assay. Aldolase mRNA, extracted by freeze-thawing cells in the presence of RNase inhibitor, was found to be stable at 42 degrees C for over three hours. Since the RT step can be completed within 1 h, there is minimal degradation of mRNA. This simple procedure avoids the use of harsh reagents, which may inhibit enzymes involved in RT-PCR, and produces results virtually identical to methods that employ guanidinium thiocyanate and phenol for RNA extraction. Optimized conditions for each parameter of the procedure are described that permit amplification of mRNA from as few as four cells.

Chondrocyte cultures express matrix metalloproteinase mRNA and immunoreactive protein; stromelysin-1 and 72 kDa gelatinase are localized in extracellular matrix vesicles.

Previous studies have shown that costochondral cartilage cell cultures produce extracellular matrix vesicles which contain metalloproteinase activity. In the present study, we examined whether two matrix metalloproteinases (MMPs) known to be present in cartilage, stromelysin-1 and 72 kDa gelatinase, are expressed by fourth passage resting zone and growth zone costochondral chondrocytes and whether they are specifically incorporated into matrix vesicles produced by the cells. We also examined whether the cells synthesize tissue inhibitor of metalloproteinase-1 and -2 (TIMP-1 and TIMP-2). Oligonucleotide primers for stromelysin-1, 72 kDa gelatinase, tissue inhibitor of metalloproteinases-1 and -2 (TIMP-1 and TIMP-2), and GAPDH were synthesized and optimized for use in the reverse transcription-polymerase chain reaction (RT-PCR). It was found that both resting zone and growth zone chondrocytes produced mRNA for both MMPs and the two TIMPs. Further, immunostaining of cell layers with antibodies to 72 kDa gelatinase and stromelysin-1 showed that both cell types produced these MMPs in culture. Substrate gel electrophoresis and Western analysis were used to characterize MMP activity in matrix vesicles, media vesicles, or plasma membranes as well as in conditioned media produced by the chondrocyte cultures. It was found that matrix vesicles but not plasma membranes or media vesicles were selectively enriched in stromelysin-1. Also, 72 kDa gelatinase was found in matrix vesicles, but to a lesser extent than seen in media vesicles. The relative activity of each enzyme detected was cell maturation-dependent. No MMP activity was detected in conditioned media produced by either cell type. The results of this study show that MMPs are expressed by resting zone and growth zone chondrocytes in culture and differentially distributed among three different membrane compartments. This suggests that, in addition to the well-known activators and inhibitors of MMP activity in the matrix, differential membrane distribution may enable more precise control over the site, rate, and extent of matrix degradation by the cell.

Regulation of matrix metalloproteinases following cellular transformation.

During progression towards malignancy, many tumor cells display changes in their repertoire of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs). The recent finding that many members of the MMPs are regulated by protooncogenes may explain the frequent observation of changes in MMP gene expression during progression of many tumor types. While studies involving enzymatic assays of MMPs are usually confined to one or a few MMPs, reverse transcription-PCR (RT-PCR) permitted the analysis of seven members of the MMP family and two members of the TIMP family in several normal and transformed cell lines. RT-PCR permitted us to confirm the observation that MMP-9 is activated following transformation and also to observe the previously unreported activation of MMP-7 in SV40-transformed cells. It has previously been found that MMP-1, -2, -3, -8, and -9 are upregulated by phorbol esters; we have found that MMP-10 is also upregulated by phorbol esters. The phorbol ester upregulation of MMP-1, -3, and -10 was found to be abolished in cells transformed by SV40 virus. Several studies have shown that MMP-1 is upregulated by an integrin-mediated signal transduction pathway. This study demonstrates that MMP-3 and MMP-10 are also regulated by integrin-mediated signal transduction and that upregulation by this pathway is abolished following SV40 transformation. In summary, the more global view of MMP expression afforded by RT-PCR indicates that MMP-1, -3, and -10 are regulated by both integrin-mediated signal transduction and phorbol esters. While fibroblasts and transformed bone cells express several members of the MMP gene family, several other cell types do not express MMPs.

Effectiveness of quality of life therapy for depression.

A bibliotherapy outcome study was conducted to assess the efficacy of Quality of Life. Therapy for depression. Sixteen clinically depressed community volunteers who showed an aptitude for and interest in bibliotherapy and were not suffering from other disorders met weekly to discuss a manual on Quality of Life Therapy. All subjects who completed treatment were reclassified as nondepressed and showed significant increases in quality of life and self-efficacy at the end of treatment. All but one subject maintained these improvements at a follow-up assessment. The potential for increasing the improvement rate of a treatment by matching patients to treatment modalities that fit their aptitudes, skills, and interests is discussed.

Quantitative monitoring of capsular contraction around smooth and textured implants.

The purpose of this study was to assess the efficacy of textured silicone implants in reducing the incidence of capsular contracture. Each of 10 female New Zealand, albino rabbits received 2 saline-filled implants, 1 on either side of the lateral chest wall. The surface of 1 implant was smooth silicone, whereas the other implant's surface was textured silicone. Magnetic resonance imaging (MRI) scans of the implants were performed at 0, 9, 17, 26, 34, and 40 weeks after implantation. Data from the MRI scans were used to calculate the effective surface area of implants at each analysis interval. This technique provided a noninvasive method of monitoring implant contraction as a function of time. Eight rabbits completed the study. Four of 8 smooth implants developed contractures, whereas none of the textured implants developed contracture. For the 4 smooth implants that developed contractures, MRI scans calculated 72 +/- 12% contraction at 17 weeks, but the Baker palpation test detected only mild firmness. From 17 to 40 weeks, the mean percentage of contraction for these implants changed minimally, but their mean Baker score increased from mild to severe (II to IV). Quantitative data from MRI scans were much more predictive of final implant contraction than palpation (Baker test), applanation tonometry, or indentation tonometry. The latter two tests only detected the final stages of severe implant contraction.

Ribosomal association of the yeast SAL4 (SUP45) gene product: implications for its role in translation fidelity and termination.

The SAL4 gene of the yeast Saccharomyces cerevisiae encodes a novel translation factor (Sal4p) involved in maintaining translational fidelity. Using a polyclonal antibody raised against a Sal4p-beta-galactosidase fusion protein, Sal4p was shown to be almost exclusively associated with the ribosomal fraction. Even when the ribosomes were treated with 0.8 M KCl, only low levels of Sal4p were detected in the post-ribosomal supernatant, suggesting a very strong affinity between Sal4p and the ribosome. Analysis of the distribution of Sal4p in the ribosomal population revealed that it was principally associated with 40S subunits, monosomes and polysomes. Incubation in high salt concentrations (0.8 M KCl) suggested that the affinity of Sal4p for the 40S subunit was lower than that for monosomes or polysomes. The Sal4p:ribosome association was only maintained when ribosomes were prepared in the presence of the translation elongation inhibitor cycloheximide; in uninhibited cells much lower levels of Sal4p were detectable in the 'run-off' polysomes. In view of these data, and given the stoichiometry of Sal4p to individual ribosomal proteins (estimated at less than 1:20), we suggest that Sal4p plays an ancillary role in translation termination.

Inputs to spinocerebellar tract neurones located in stilling's nucleus in the sacral segments of the rat spinal cord.

Spinocerebellar neurones located in the sacral segments of the rat spinal cord have been identified electrophysiologically. The neurones studied were located 0.7-1.1 mm deep to the cord dorsum, lateral and dorsal to the central canal in the medial part of lamina VII. Neurones were identified as spinocerebellar by antidromic activation from the cerebellar surface, the lowest threshold stimulation sites being near the midline on the posterior lobe vermis (lobule VIII). Estimates of conduction velocities of the axons ranged from 15-32 m/s (mean 22.8 m/s) and are directly comparable to velocities of presumed ventral spinocerebellar tract neurones recorded in the same animals. In intact animals, activity was most strongly influenced by passive movement of the tail. Activation by proprioceptors was confirmed with nerve stimulation: all of the neurones studied were discharged by stimulation of nerves which innervate ipsilateral tail muscles. In many cases responses appeared close to the threshold of the nerve, indicating that the largest, fastest conducting afferents (group Ia muscle spindle primary afferents) were responsible for them. Latencies of EPSPs or spikes were brief and in many cases indicative of a monosynaptic connection. We conclude that this group of neurones is powerfully and monosynaptically excited by group I muscle afferents and thus resemble the cells of Clarke's column and cells of the central cervical nucleus, both of which occupy a similar location in the grey matter of more rostral segments.

A method to predict the probabilities to homozygous recessives and of heterozygotes.

Where the occurrence of homozygous recessives and heterozygotes is known in the pedigrees of prospective sires and dams, a method and appropriate tables are given to predict the minimum probability 1) that progeny will be homozygous recessive, or 2) that progeny of dominant phenotype are heterozygous.