Synthesis Of Other Proteins Research Articles

Developmentally regulated expression of c‐Fos and c‐Jun in the brainstem auditory nuclei of Gallus domesticus is modified by prenatal auditory enrichment

Recognition of mother's voice by human neonates and behavioral responses of birds and animals to sounds experienced prenatally emphasize the role of sensory inputs in auditory system development. Spontaneous and experience driven neural activity influence the neural circuits' refinement in developing brain. However, cellular mechanisms endowing plasticity for such structural refinement during critical developmental periods are less understood. Sensory stimulation induces fluctuating expression of transcription factors (TFs) of Fos, Jun, and Krox families in the related brain nuclei to activate genes to synthesize proteins such as those needed for cytoskeletal structures, ion channels, and regeneration. To understand the cellular mechanism of response to prenatal auditory stimulation, we studied the expression of c-Fos and c-Jun in brainstem auditory nuclei, nucleus magnocellularis, and nucleus laminaris of the domestic chick. The chick brainstems, five each of E8 (embryonic day 8), E12, E16, E20, and posthatch day 1 were processed for immunohistochemistry as well as Western blotting and quantified using image analysis systems. In controls, c-Fos and c-Jun expression in both the nuclei was developmentally up-regulated. Reduced c-Fos expression and increase in c-Jun was temporarily observed between E12-16. In the stimulated groups, c-Fos expression was elevated while c-Jun showed a reduction matched to controls. This diametrically opposing pattern of c-Fos and c-Jun expression in response to stimulation is indicative of cell survival. Thus the expression of TFs in the auditory nuclei shows a relationship beyond a simple stimulation-activity-expression. While developmental signals control the expression of TFs, extra sensory stimulation modulates their expression to possibly support neuronal survival and enhance synthesis of other proteins.

Insulin stimulates placental leucine aminopeptidase/oxytocinase/insulin-regulated membrane aminopeptidase expression in BeWo choriocarcinoma cells

Placental leucine aminopeptidase (P-LAP), a cystine aminopeptidase that is identical to insulin-regulated membrane aminopeptidase, hydrolyzes oxytocin, which results in the loss of oxytocin activity. We previously isolated genomic clones containing the human P-LAP promoter region, which included two sites homologous to the 10-bp-insulin responsive element (IRE) that was identified on the phosphoenolpyruvate carboxinase gene. We therefore postulated that insulin regulates P-LAP expression via these IREs and investigated this notion using BeWo choriocarcinoma trophoblastic cells cultured in the presence of insulin. Insulin increased P-LAP activity in a time- and dose-dependent manner. Physiological concentrations of insulin at 10 −7 M exhibited the most potent effect on P-LAP activity. Western blotting demonstrated that 10 −7 M insulin increased P-LAP protein levels. Semi-quantitative RT-PCR and Southern blotting showed that insulin also increased P-LAP mRNA, which was abrogated by prior exposure to cycloheximide. Luciferase assay did not reveal any regulatory regions within 1.1 kb upstream of the P-LAP gene that could explain the insulin-induced P-LAP mRNA accumulation. These findings indicate that insulin induces P-LAP expression in trophoblasts, and that it acts via de novo synthesis of other proteins, which partially contradicts our initial hypothesis.

Wound‐induced PAL activity is suppressed by heat‐shock treatments that induce the synthesis of heat‐shock proteins

Wounding lettuce leaves induces the de novo synthesis of phenylalanine ammonia‐lyase (PAL, EC 4.3.1.5), the accumulation of phenolic compounds, and subsequent tissue browning. A brief heat‐shock at 45°C reduces the rise in wound‐induced PAL, the accumulation of phenolic compounds, and tissue browning. The activity of PAL measured 24 h after wounding and the content of phenolic compounds (absorbance of methanol extract at 320 nm) measured 48 h after wounding was highly correlated (R2 > 0.90) in tissue developing the normal wound response and in tissue subjected to 0–180 s of heat‐shock after wounding. The synthesis of a unique set of proteins called heat‐shock proteins (hsps) is induced by these heat‐shock treatments. Western‐blot analyses of proteins isolated from wounded and heat‐shocked Iceberg and Romaine lettuce mid‐rib leaf tissue was done using antibodies against hsp 23. Only those heat‐shock treatments that were effective at inducing the synthesis of hsp 23 were effective in reducing the activity of PAL induced by wounding and the subsequent accumulation of phenolic compounds. Hsps induced in non‐wounded, whole leaves by exposure to 45°C for 150 s did not significantly interact with PAL previously synthesized in non‐heat‐shocked wounded leaves to limit its activity. The preferential synthesis of hsps over that of wound‐induced PAL, rather than the presence of hsps, may be responsible for the ability of a heat‐shock treatment to reduce the wound‐induced increase in PAL activity. Our results support this novel concept, and the possibility that heat‐shock treatments can have significant physiological effects on the response of the tissue to other stresses, not because of the specific genes they induce or repress, or the products they cause to be synthesized, but by their secondary action of influencing the synthesis of other proteins (e.g. PAL) by the suppression of non‐hsps protein synthesis.

Spot 42 RNA Regulates Polycistronic Translation

The Escherichia coli galactose operon is regulated by the adenosine 3′,5′-monophosphate (cAMP) receptor protein (CRP) and is transcribed as a polycistronic message. However, the translation of the proteins encoded by the polycistronic message [UDP-galactose-4-epimerase (GalE), galactose-1-phosphate uridyl transferase (GalT), and galactokinase (GalK)] differs under different metabolic conditions, so-called "discoordinate expression." Decreased cAMP or glucose as a carbon source results in decreased amounts of GalK and GalT; whereas GalE is more abundant. Spot 42 RNA is transcribed from the spot forty-two ( spf ) gene, which is inhibited by cAMP through the CRP. Overexpression of spf inhibited synthesis of GalK, selectively, having little effect on the synthesis of other proteins from the galactose operon. In cells deficient for CRP , the expression of spf and GalK was reduced. This effect on GalK was abolished if the cells were also deficient for spf . Spot 42 RNA bound to the galK region of the polycistronic galactose operon message. Thus, Spot 42 RNA mediates the inhibition of GalK translation to produce discoordinate gene expression from a polycistronic RNA in response to changes in metabolic status in E. coli . T. Møller, T. Franch, C. Udesen, K. Gerdes, P. Valentin-Hansen, Spot 42 RNA mediates discoordinate expression of the E. coli galactose operon. Genes Dev. 16 , 1696-1706 (2002). [Abstract] [Full Text]

Dendritic spine structural anomalies in fragile-X mental retardation syndrome.

Fragile-X syndrome is the most common single-gene inherited form of mental retardation. Morphological studies suggest a possible failure of the synapse maturation process. Cerebral cortical spine morphology in fragile-X syndrome and in a knockout mouse model of it appears immature, with long, thin spines much more common than the stubby and mushroom-shaped spines more characteristic of normal development. In human fragile-X syndrome there is also a higher density of spines along dendrites, suggesting a possible failure of synapse elimination. While variously misshapen spines are characteristic of a number of mental retardation syndromes, the overabundance of spines seen in fragile-X syndrome is unusual. Taken with evidence of neurotransmitter activation of the synthesis of the fragile-X protein (FMRP) at synapses in vitro and evidence for behaviorally induced FMRP expression in vivo, and with evidence compatible with a role for FMRP in regulating the synthesis of other proteins, it is possible that FMRP serves as an 'immediate early protein' at the synapse that orchestrates aspects of synaptic development and plasticity.

The effect of continuously applied cyclic mechanical loading on the fibronectin metabolism of articular cartilage explants.

Articular cartilage serves primarily as a load-bearing material able to regulate its own metabolic activity in response to the mechanical stimuli applied. Fibronectin plays a critical role in the organization and function of the cartilage extracellular matrix. The purpose of this study was to investigate systematically the effect of load magnitude, frequency and duration of loading on the synthesis, content and release of fibronectin and proteins by mature bovine articular cartilage explants using a novel mechanical loading system. Increasing the load magnitude, as well as the duration of loading, inhibited the synthesis and content of fibronectin and proteins; the fibronectin synthesis was more specifically affected than the overall protein synthesis indicating that fibronectin is more responsive to pressure than synthesis of other proteins. Reducing the load frequency did not modulate the inhibitory effect of a given cyclic stress on synthesis and content of fibronectin and proteins even though explants were more compressed. The release of endogenous fibronectin was significantly reduced independent of the applied loading protocols when compared with unloaded controls. This study demonstrates that the magnitude and the duration of loading influences the degree of inhibition of fibronectin and protein synthesis, while loaded explants possess an elevated but limited capacity to bind fibronectin. Compared with other studies, our present results show that the applied load function in particular has a profound effect on the metabolism of chondrocytes.

The effect of temperature and protein synthesis on the renaturation of firefly luciferase in intact H9c2 cells.

A mild increase in temperature that does not exert an effect on tolerance development or synthesis of heat shock proteins (Hsps) in control cells can stimulate these processes when applied to cells that have previously been heat shocked. To study the underlying mechanism of this effect, H9c2 cells were stably transfected with the gene encoding firefly luciferase (Luc). Heat-shock-induced inactivation of Luc and its subsequent reactivation is frequently used as a model for cellular protein denaturation and renaturation. Luc reactivation was determined following a damaging heat shock (43 or 44 degrees C for 30 min) in cells that were subsequently exposed to either control temperatures (37 degrees C) or various mild hyperthermic conditions (from 38.5 to 41.5 degrees C for 1 h). To prevent changes in Luc activity consequent to new synthesis of Luc, Luc reactivation was monitored in the presence of cycloheximide, an inhibitor of protein synthesis. The results showed that reactivation of Luc was inhibited when heat-treated cells were post-treated under mild hyperthermic conditions. The observed increase in Hsp synthesis under mild hyperthermic post-heat shock conditions therefore appears to be the result of an increase in the period during which denatured proteins are present. In addition, we studied Luc reactivation in the absence of protein synthesis inhibitors. This condition led to much higher Luc activity. By estimating half-life times of Luc, the contribution of new Luc synthesis in this recovery could be determined, and only partially explained the observed increase in Luc reactivation after heat shock. Thus the synthesis of other proteins must be important for the renaturation of heat-damaged proteins.

Broad Spectrum Thiopeptide Recognition Specificity of theStreptomyces lividans TipAL Protein and Its Role in Regulating Gene Expression

Microbial metabolites isolated in screening programs for their ability to activate transcription of the tipA promoter (ptipA) in Streptomyces lividans define a class of cyclic thiopeptide antibiotics having dehydroalanine side chains ("tails"). Here we show that such compounds of heterogeneous primary structure (representatives tested: thiostrepton, nosiheptide, berninamycin, promothiocin) are all recognized by TipAS and TipAL, two in-frame translation products of the tipA gene. The N-terminal helix-turn-helix DNA binding motif of TipAL is homologous to the MerR family of transcriptional activators, while the C terminus forms a novel ligand-binding domain. ptipA inducers formed irreversible complexes in vitro and in vivo (presumably covalent) with TipAS by reacting with the second of the two C-terminal cysteine residues. Promothiocin and thiostrepton derivatives in which the dehydroalanine side chains were removed lost the ability to modify TipAS. They were able to induce expression of ptipA as well as the tipA gene, although with reduced activity. Thus, TipA required the thiopeptide ring structure for recognition, while the tail served either as a dispensable part of the recognition domain and/or locked thiopeptides onto TipA proteins, thus leading to an irreversible transcriptional activation. Construction and analysis of a disruption mutant showed that tipA was autogenously regulated and conferred thiopeptide resistance. Thiostrepton induced the synthesis of other proteins, some of which did not require tipA.

Oxygen‐dependent electron flow influences photosystem II function and psbA gene expression in the cyanobacterium Synechococcus sp. PCC 7942

During acclimated growth in Synechococcus sp. PCC 7942 a substantial proportion of the electrons extracted from water by photosystem II ultimately flow back to oxygen. This flow increases rapidly under high light, which allows Synechococcus to maintain photosystem II centers largely open, even under excessive excitation. The electron flow to oxygen with increasing light accounts for the progressive discrepancy between the light response curve of measured oxygen evolution, and the light response curve of photosystem II activity estimated from fluorescence measures. In cells under anoxia this flexible electron sink is lost and photosystem II centers suffer partial closure at the growth light intensity, with closure becoming more severe under excess light. As predicted from earlier work this PSII closure results in rapid loss of psbAI message, encoding the D1:1 protein of PSII, and induction of psbAII/AIII encoding the alternate D1:2 protein. The changes in the mRNA pool are not, however, reflected at the protein level, and D1:1 remains in the thylakoid membranes. There is no accumulation of D1:2, despite some continued synthesis of other proteins. PSII closure, therefore, results in repression of psbAI and induction psbAII/AIII expression, but D1:1/D1:2 exchange is blocked by anoxia, downstream from transcription. D1:1 protein and PSII activity are quite stable under anoxia and moderate illumination. Nevertheless, upon recovery under oxygenic conditions, the existing D1:1 is lost from the membranes, resulting in a transient drop in PSII activity. This suggests that under normal conditions the cells use oxygen to facilitate preemptive turnover of D1 proteins.

Chickpea Defensive Proteinase Inhibitors Can Be Inactivated by Podborer Gut Proteinases1

Abstract Developing chickpea (Cicer arietinum L.) seeds 12 to 60 d after flowering (DAF) were analyzed for proteinase inhibitor (Pi) activity. In addition, the electrophoretic profiles of trypsin inhibitor (Ti) accumulation were determined using a gel-radiographic film-contact print method. There was a progressive increase in Pi activity throughout seed development, whereas the synthesis of other proteins was low from 12 to 36 DAF and increased from 36 to 60 DAF. Seven different Ti bands were present in seeds at 36 DAF, the time of maximum podborer (Helicoverpa armigera) attack. Chickpea Pis showed differential inhibitory activity against trypsin, chymotrypsin, H. armigera gut proteinases, and bacterial proteinase(s). In vitro proteolysis of chickpea Ti-1 with various proteinases generated Ti-5 as the major fragment, whereas Ti-6 and -7 were not produced. The amount of Pi activity increased severalfold when seeds were injured by H. armigera feeding. In vitro and in vivo proteolysis of the early- and late-stage-specific Tis indicated that the chickpea Pis were prone to proteolytic digestion by H. armigera gut proteinases. These data suggest that survival of H. armigera on chickpea may result from the production of inhibitor-insensitive proteinases and by secretion of proteinases that digest chickpea Pis.

Basal Synthesis of Heat Shock Protein 70 Increases with Age in Rat Kidneys

The heat shock protein (HSP) system is a mechanism of cell defense induced by stress, constitutively expressed during basal conditions and essential to the maintenance of cellular integrity. Acutely induced HSP synthesis decreases with aging, but the effect of age on the basal expression of HSP70 has not been specifically addressed so far. The aim of this work is to study the age-dependent basal concentrations of HSP70 mRNA in rat kidneys. In 8 young (2–3 months), 6 adult (6–11 months) and 6 old male Wistar rats (22–27 months), steady-state concentrations of HSP70 and γ-actin mRNA and of rRNA were measured. Pentosidine was measured by HPLC. The basal, unstimulated HSP70 mRNA is increased in young and old rats compared with adult subjects [young: 182% of adult levels (100–299), old: 167% of adults (142–209); p < 0.005]. The amount of pentosidine increases with age (young: 0.6 ± 0.1; adult: 1.65 ± 0.15; old: 2.3 ± 0.3 pmol/mg of protein; p < 0.0001). It seems likely that different mechanisms are responsible for the increased HSP70 basal synthesis in both the young and old animals. The prevalence of anabolic activity can trigger the increased basal production of HSP70 in young rats. The accumulation of posttranslational modified proteins, documented by pentosidine, can chronically enhance HSP70 synthesis in aged animals. The suppression of the synthesis of other proteins accompanying HSP-selective production might contribute to the impairment of specific cell functions in aging.

The lom gene of bacteriophage λ is involved in Escherichia coli K12 adhesion to human buccal epithelial cells

The role of the lom gene of bacteriophage λ in adhesion of Escherichia coli to human buccal epithelial cells (HBC) was studied testing the adherence of λ lom + and λ lom::Tn phoA E. coli lysogens. λ lom + prophage increased 50% E. coli adhesion. This effect was not observed with λ lom::Tn phoA. These results suggest that the normal Lom protein participates directly in adhesion or regulates the synthesis of other protein(s), which may be involved in adhesion.

The Systemic Inflammatory Response to Cardiopulmonary Bypass

The Systemic Inflammatory Response to Cardiopulmonary Bypass

Expression of Src-like Adapter Protein mRNA Is Induced by All-trans Retinoic Acid

By using a differential display method, specific bands were selected from ladder PCR products derived from ATRA-dependent differentiated U937 cells, in comparison with those of untreated U937. By screening the cDNA library of ATRA-dependent differentiated U937 cells with one of the PCR products, we cloned the src-like adapter protein (SLAP). Northern blot analysis of U937 cells with or without ATRA treatment indicated that the SLAP mRNA was clearly induced by ATRA. The induction was inhibited by the addition of cycloheximide, indicating that ATRA acted indirectly through synthesis of other proteins. The SLAP mRNA was induced in HL60 and NB-4 but not in K562 or THP-1. Interestingly, these cells in which SLAP mRNA was induced by ATRA all showed ATRA-dependent cell differentiation. The relationship between SLAP and cell differentiation is unclear, but SLAP may transduce a signal for cell differentiation.

Effect ofl-lysine-α-oxidase on the synthesis of proteins associated with autoimmune pathologies in HIV infection

In HIV-infected MT-4 cells,l-lysine-α-oxidase, an inhibitor of HIV reproduction, selectively inhibits the synthesis of histone proteins, including 18K antigen (H2B histone). This protein is the target for autoimmune suppression of helper T cells. The enzyme has a low effect on the synthesis of other proteins and DNA. In addition to 18K antigen,l-lysine-α-oxidase suppresses the synthesis of high-molecular-weight proteins (270, 220, 140, 100, 75, 70, and 60 kD).

Developmental and Morphogenetic Gene Regulation inHaliotis rufescensLarvae at Metamorphosis

The planktonic larva of the red abalone, Haliotis rufescens, must develop competence and recognize an appropriate exogenous morphogenetic cue before it can settle and metamorphose. The molecular mechanisms underlying the development of larval competence and the induction of metamorphosis in this and other marine invertebrates are only now starting to be understood. In Haliotis, some of the morphogenetic cues, chemosensory receptors, and receptor-mediated signal transduction pathways controlling the induction of metamorphosis from the planktonic larva to the benthic post-larva have been identified. To ascertain how these initial signaling pathways regulate the global changes that occur at metamorphosis, we have investigated the expression of two classes of genes: (i) those that are expressed but not thought to be functionally required in the veliger larva, and (ii) those that exhibit marked changes in tissue-specific expression induced at metamorphosis. Expression of the genes encoding a larval chymotrypsin and a muscle-specific tropomyosin reveal two distinct developmental pathways governing gene expression and metamorphosis in Haliotis; changes in the synthesis of other proteins and in morphology support this conclusion. One of these pathways, termed the morphogenetic pathway, is linked directly to the processes of settlement and metamorphosis; the induction of tropomyosin in the adult columellar muscle progenitors 48 hr after induction of metamorphosis provides the best and most direct evidence at the molecular level for the existence of this pathway in Haliotis. The other pathway, which we have termed the anticipatory (or preparatory) pathway, is first activated prior to or at the attainment of larval competence, and is induced again at metamorphosis. The induction of expression of a chymotrypsin gene in migrating digestive amoebocytes in the larva, and the depletion of tropomyosin mRNA from cells of the larval retractor muscle, provide molecular evidence for this program. We review here the patterns of expression of these genes, and suggest that the uncoupling of these pathways reflects an adaptation of Haliotis larval development to meet the future demands of benthic life.

Regulation of human Gc (vitamin d—binding) protein levels: Hormonal and cytokine control of gene expression in vitro

Studies were performed in Hep3B hepatocytes to better elucidate the mechanisms regulating circulating levels of human group—specific component (Gc). We measured changes in Gc messenger RNA (mRNA) synthesis and levels of secreted protein resulting from treatment of hepatocytes with cytokines and hormones known to influence synthesis of other proteins of hepatic origin. We particularly focused on compounds known to be prototypic stimulants during the acute phase response. Interleukin-6 (IL-6) and dexamethasone were shown to increase Gc mRNA approximately twofold while transforming growth factor beta (TGFβ) decreased Gc mRNA in a dose-dependent fashion by up to fivefold. The effects on secreted Gc protein levels were similar. These results indicate that Gc protein appears to be regulated differently than the other members of this gene family, albumin and alpha-fetoprotein (AFP), which are negative acute phase reactants. In addition, these contrasting effects on Gc synthesis of IL-6 and dexamethasone and of TGFβ suggest that high basal levels of Gc synthesis may be maintained during the acute phase response.

Regulation of human Gc (vitamin D — binding) protein levels: Hormonal and cytokine control of gene expression in vitro

Studies were performed in Hep3B hepatocytes to better elucidate the mechanisms regulating circulating levels of human group-specific component (Gc). We measured changes in Gc messenger RNA (mRNA) synthesis and levels of secreted protein resulting from treatment of hepatocytes with cytokines and hormones known to influence synthesis of other proteins of hepatic origin. We particularly focused on compounds known to be prototypic stimulants during the acute phase response. Interleukin-6 (IL-6) and dexamethasone were shown to increase Gc mRNA approximately twofold while transforming growth factor beta (TGF beta) decreased Gc mRNA in a dose-dependent fashion by up to fivefold. The effects on secreted Gc protein levels were similar. These results indicate that Gc protein appears to be regulated differently than the other members of this gene family, albumin and alpha-fetoprotein (AFP), which are negative acute phase reactants. In addition, these contrasting effects on Gc synthesis of IL-6 and dexamethasone and of TGF beta suggest that high basal levels of Gc synthesis may be maintained during the acute phase response.

Synthesis and phosphorylation of osteopontin by avian epiphyseal growth-plate chondrocytes as affected by differentiation

The regulation of synthesis and phosphorylation of osteopontin in relation to avian epiphyseal growth-plate chondrocyte differentiation was studied in situ and in culture. Osteopontin gene expression was evaluated in the tibia growth-plate of 3-week-old chickens by in situ hybridization. The gene was expressed mainly at the lower hypertrophic zone where cartilage matrix is calcified and endochondral bone formation is initiated. Within the hypertrophic region, a poorly labeled area separated the layer of osteopontin-positive hypertrophic chondrocytes from those associated with endochondral bone formation. In culture, proliferative chondrocytes show no alkaline phosphatase activity in contrast to ascorbic acid-treated chondrocytes which display the enzyme activity. Chondrocytes not treated with ascorbic acid, exhibited lower levels of osteopontin mRNA than the treated cells. The phorbol ester TPA—an activator of protein kinase C—and to a lesser extent FGF but not EGF, stimulated osteopontin gene expression. Chondrocytes secreted low levels of phosphorylated osteopontin to the medium. EGF treatment resulted in the appearance of phosphorylated osteopontin in the medium, without affecting the synthesis of other proteins. FGF and TGF β, but not IGF-I or IGF-II, also caused phosphorylation of osteopontin. Ascorbic acid-treated chondrocytes secreted higher levels of phosphorylated osteopontin than the non-treated cells, but addition of FGF or TPA did not stimulate osteopontin phosphorylation any further. Parathyroid hormone caused a dose-dependent attenuation of osteopontin phosphorylation and inhibited the EGF-dependent osteopontin phosphorylation. The results suggest that osteopontin gene expression and phosphorylation in chondrocytes are regulated by separate mechanisms. The response to the various controlling agents varies with the state of differentiation. Both processes—the synthesis and phosphorylation of osteopontin—are under the control of local growth factors which are involved in bone growth and calcification.

Milk Protein Synthesis, Gene Expression, and Hormonal Responsiveness in Primary Cultures of Mammary Cells from Lactating Sheep

A ruminant mammary cell culture that accurately reproduces mammary function in vitro would be a valuable tool in studies of ruminant lactation, With this in mind, we have examined milk protein synthesis and secretion, milk protein mRNA abundance, and hormonal responsiveness in primary cultures of mammary acini from lecturing sheep. α- and β-casein protein synthesis, β-lactoglobulin synthesis, and α-casein, β-casein, and β-lactoglobulin secretion are maintained at high levels for 8 h in culture, but then decline to approximately 25% of maximal rates between 8 and 24 h in culture, whereas synthesis of other proteins remains unaltered. The relative abundance of α-S1-casein, β-lactoglobulin, and α-lactalbumin mRNAs similarly decline between 8 and 24 h in culture. Extracellular labeled α-casein is increased fourfold in the presence of fetal calf serum (FCS). In total, FCS alters the abundance of 47 of 68 secreted proteins detected by two-dimensional electrophoresis. However, FCS and lactogenic/galactopoietic hormones had no effect on the rate of decline of mammary function and did not promote any regaining of function when present for up to 9 days in culture. These results suggest that providing its limitations are recognized, this primary cell culture system may be useful in studying some aspects of ruminant mammary function in vitro.