Urine and fecal excretion patterns of dairy cows divergent for milk urea nitrogen breeding values consuming either a plantain or ryegrass diet.

Environmental degradation has been attributed to inefficient nitrogen utilization from pastoral dairy production systems. This degradation has especially been associated with the urine patch, which has been identified as a key component of nitrate leaching to waterways. However, a lack of information exists regarding the pattern of urination events and individual urination characteristics across the day, which would help inform strategic management decisions. The aim of this study was therefore to evaluate and report the patterns and characteristics of fecal and urination events throughout the day for cows divergent for milk urea nitrogen breeding values (MUNBV) on either a plantain [Plantago lanceolata L. (PL)] or ryegrass [Lolium perenne L. (RG)] diet as ways to reduce environmental impact. Sixteen multiparous lactating Holstein Friesian × Jersey cows divergent for MUNBV were housed in metabolism crates for 72 h, with all excretion events captured and analyzed. Cows selected as low for MUNBV consistently had a 65.2-kg lower urinary urea nitrogen (UUN) load (kg/ha) than high MUNBV cows for all hours of the day when consuming RG. The association between lower urinary urea loading rates and less N leaching implies a reduced environmental impact from low MUNBV cows consuming RG. When cows consumed PL, regardless of MUNBV, they had on average a 137.5-kg (UUN/ha) lower loading rate compared with high MUNBV cows on RG and a 72.2-kg (UUN/ha) lower loading rate compared with low MUNBV cows consuming RG across the day. Cows on PL also exhibited a different diel pattern of UUN load compared with cows consuming RG. Differences in the diel pattern of N excreted in feces were also detected based on MUNBV and by diet, with low MUNBV cows excreting on average 3.06 g more N in feces per event for the majority of the day compared with high MUNBV cows when consuming RG. Lower UUN loading rates and more N excreted in feces indicate a potentially lower environmental impact from low MUNBV cows when consuming RG compared with high MUNBV cows. The use of the PL diet also resulted in lower UUN loading rates and greater levels of N excreted in feces compared with RG, therefore also indicating its ability to reduce environmental impact compared with RG.

Effect of cane use on bone marrow lesion volume in people with medial tibiofemoral knee osteoarthritis: randomized clinical trial.

OBJECTIVE: To evaluate effects of daily cane use for 3 months on medial tibiofemoral bone marrow lesion (BML) volumes in people with medial tibiofemoral osteoarthritis (OA). DESIGN: In this randomized controlled trial (RCT), 79 participants with medial tibiofemoral OA were randomized to either a cane group (using a cane whenever walking) or control group (not using any gait aid) for 3 months. The cane group received a single training session by a physiotherapist, using a biofeedback cane to teach optimal technique and body weight support and motor learning principles to facilitate retention of learning. The primary outcome was change in total medial tibiofemoral BML volume (per unit bone volume) measured from magnetic resonance imaging (MRI) at 3 months. Secondary outcomes were BML volumes (per unit bone volume) of the medial tibia and femur, and patient-reported outcomes of overall knee pain, knee pain on walking, physical function, perceived global symptom changes and health-related quality of life. MRI analyses were performed by a blinded assessor. RESULTS: Seventy-eight participants (99%) completed the primary outcome. Mean (standard deviation) daily cane use was 2.3 (1.7) hours over 3 months. No evidence of between-group differences was found for change in total medial tibiofemoral BML volume (mean difference: -0.0010 (95% confidence intervals: -0.0022, 0.0003)). Most secondary outcomes showed minimal differences between groups. CONCLUSION: Daily use of a cane during walking for 3 months aiming to reduce knee joint loading did not change medial tibiofemoral BML volumes compared to no use of gait aids. CLINICAL TRIAL REGISTRATION: Australian New Zealand Clinical Trial Registry (ACTRN12614000909628).

Reliability and measurement error of the Osteoarthritis Research Society International (OARSI) recommended performance-based tests of physical function in people with hip and knee osteoarthritis.

OBJECTIVE: To estimate the reliability and measurement error of performance-based tests of physical function recommended by the Osteoarthritis Research Society International (OARSI) in people with hip and/or knee osteoarthritis (OA). DESIGN: Prospective repeated measures between independent raters within a session and within-rater over a week interval. Relative reliability was estimated for 51 people with hip and/or knee OA (mean age 64.5 years, standard deviation (SD) 6.21 years; 47% females; 36 (70%) primary knee OA) on the 30s Chair Stand Test (30sCST), 40m Fast-Paced Walk Test (40mFPWT), 11-Stair Climb Test (11-step SCT), Timed Up and Go (TUG), Six-Minute Walk Test (6MWT), 10m Fast-Paced Walk Test (10mFPWT) and 20s Stair Climb Test (20sSCT) using intra-class correlation coefficients (ICC). Absolute reliability was calculated using standard error of measurement (SEM) and minimal detectable change (MDC). RESULTS: Measurement error was acceptable (SEM < 10%) for all tests. Between-rater reliability was: optimal (ICC > 0.9, lower 1-sided 95% CI > 0.7) for the 40mFPWT, 6MWT and 10mFPWT; sufficient (ICC >0.8, lower 1-sided 95% CI > 0.7) for 30sCST, 20sSCT; unacceptable (lower 1-side 95% CI < 0.7) for 11-step SCT and TUG. Within-rater reliability was optimal for 40mFPWT, and 6MWT; sufficient for 30sCST and 10mFPWT and unacceptable for 11-step SCT, TUG and 20sSCT. CONCLUSIONS: The 30sCST, 40mFPWT, 6MWT and 10mFPWT, demonstrated, at minimum, acceptable levels of both between and within-rater reliability and measurement error. All tests demonstrated sufficiently small measurement error indicating they are adequate for measuring change over time in individuals with knee/hip OA.

A 9 kDa antifreeze protein from the Antarctic springtail, Gomphiocephalus hodgsoni.

A 9 kDA antifreeze protein (AFP) was isolated and purified from the Antarctic springtail, Gomphiocephalus hodgsoni. By combining selective sampling procedures and a modified ice affinity purification protocol it was possible to directly isolate a single AFP protein without recourse to chromatographic separation techniques. Mass spectrometry identified a single 9 kDa component in the purified ice fraction. Intramolecular disulphide bonding was suggested by the presence of 12 cysteine residues. The specific amino acid composition is unique, particularly with regard to the presence of histidine (11.5%). But it also shows noticeable commonalities with insect AFPs in the abundance of cysteine (13.8%), while simultaneously hinting, through the presence of glycine (11.5%), that the metabolic building blocks of AFPs in Collembola may have a phylogenetically-determined component.

Ras effectors.

The search for proteins which interact with the active GTP-bound form of Ras in order to transmit signals for proliferation, differentiation and oncogenesis has been a long one. Now there are several strong candidates for Ras effectors that include protein kinases, lipid kinases and guanine nucleotide exchange factors. Structural information on how one Ras-binding domain in an effector interacts with Ras.GTP has recently been obtained. Recent data show that transformation by Ras oncoproteins requires the activation of several signal transduction pathways, including those which transmit signals via members of the Rho family of GTPases.

Evaluation of PEETER V1.0 urine sensors for measuring individual urination behavior of dairy cows.

Due to environmental concerns around N leaching and NO2 emissions from intensive pastoral dairying systems, there has been an increase in research focused on mitigation strategies and on-animal technologies to evaluate urination behavior of grazing dairy cows. Nitrogen leaching and NO2 emissions are associated with urine nitrogen loading onto pasture, which is a function of urine nitrogen concentration and urine volume per urination event. The PEETER V1.0 urine sensor (Lincoln University, Christchurch, New Zealand) is a promising on-animal measurement technology; however, it has yet to be validated in vivo. The objective of this work was to validate the PEETER V1.0 urine sensor's estimations of individual urination events (i.e., urine volume). We fitted 15 Holstein-Friesian × Jersey lactating dairy cows (506 ± 35 kg of live weight, body condition score of 3.75 ± 0.25, and 150.4 ± 20.7 d in milk) with individual PEETER V1.0 sensors and placed them in metabolism crates for 72 h. Every urination event (n = 480) was collected manually and compared with the urine volume estimated by the PEETER V1.0 sensor to determine precision and accuracy using Lin's concordance correlation coefficient (CCC). The CCC is calculated as a function of the Pearson's correlation (precision) and bias correction factor (Cb; Cb = 1 is perfect), and it demonstrates how far the values of the 2 methods are from perfect agreement (accuracy; i.e., a 45° line). The mean urination event volume (mean ± standard deviation) was 2.7 ± 0.94 and 2.6 ± 0.92 L for the actual and PEETER V1.0 sensor, respectively. The PEETER V1.0 sensor showed excellent precision (r = 0.90) with near-perfect accuracy (Cb = 1.00), and the CCC value was high (CCC = 0.90), indicating excellent agreement. Based on these results, the PEETER V1.0 urine sensor provides estimates that are precise and accurate. We conclude that the PEETER V1.0 sensor can be used to evaluate urination behavior of grazing dairy cows.

p21ras couples the T cell antigen receptor to extracellular signal-regulated kinase 2 in T lymphocytes.

It has previously been shown in T cells that stimulation of protein kinase C (PKC) or the T cell antigen receptor (TCR) induces the rapid accumulation of the active guanosine triphosphate-bound form of p21ras. These stimuli also induce the activation of extracellular signal-regulated kinase 2 (ERK2), a serine/threonine kinase that is rapidly activated via a kinase cascade in response to a variety of growth factors in many cell types. In this study, we show that p21ras is a component of the TCR signaling pathway that controls ERK2 activation. In the human Jurkat T cell line, transient expression of constitutively active p21ras induces ERK2 activation, measured as an increase in the ability of an ERK2-tag reporter protein to phosphorylate myelin basic protein. Thus, constitutively active p21ras bypasses the requirement for PKC activation or TCR triggering to induce ERK2 activation. In addition, activation of PKC or the TCR produces signals that cooperate with activated p21ras to stimulate ERK2. Conversely, expression of a dominant negative mutant of ras, Ha-ras N17, blocks ERK2 activation after TCR stimulation, indicating that endogenous p21ras function is necessary for the TCR-stimulated ERK2 activation. Taken together, these results demonstrate that the activation of p21ras is both necessary and sufficient to induce ERK2 activation in T cells.

Ice-active proteins from New Zealand snow tussocks, Chionochloa macra AND C. rigida.

The ice active protein profile of New Zealand snow tussocks Chionochloa macra and C. rigida consisted of ice nucleation activity but no antifreeze or recrystallization inhibition activity. The ice nucleation activity was similar in the two species, despite them being collected at different altitudes and at different times. The activity is intrinsic to the plant and is associated with the surface of the leaves. Snow tussocks collect water from fog. Nucleation sites on the surface of their leaves may aid the efficiency of this process.

Grazing dairy cows with low milk urea nitrogen breeding values excrete less urinary urea nitrogen.

There is an increasing pressure on temperate pastoral dairy production systems to reduce environmental impacts, coming from the inefficient use of N by cows in the form of excessive urinary N excretion and subsequent N leaching to the waterways and NO2 emissions to the atmosphere, these impacts have spurred research into various mitigation strategies, which have so far overlooked animal-based solutions. The objectives of this study were first, to investigate the relationship between MUN breeding values (MUNBV) and urinary urea N (UUN) concentrations and total excretion in grazing dairy cows; and secondly, to evaluate such a potential relationship in the context of different sward compositions and stage of lactation. Forty-eight multiparous, lactating Holstein-Friesian dairy cows genetically divergent for MUNBV were strip-grazed on either a ryegrass-white clover (24 cows) or ryegrass, white clover and plantain sward (24 cows), during both early and late lactation. Cows were fitted with Lincoln University PEETER sensors to evaluate urination behaviour by measuring frequency and volume of urination, as well as daily urine excretion. Urine and faeces were sampled for urea N content. Milk yield and composition were measured for individual cows in both periods. There was a positive relationship between MUNBV and MUN (R2 = 0.67, P ≤ 0.05), with MUN decreasing 1.61 ± 0.19 mg/dL per unit decrease in MUNBV across both sward types and stages of lactation. Urinary urea N concentration decreased 0.67 ± 0.27 g/L (R2 = 0.46, P ≤ 0.05) per unit decrease of MUNBV, with no effect on urine volume or frequency (number of urination events per day), which resulted in a 165.3 g/d difference in UUN excretion between the animal with the highest and the lowest MUNBV. At the same milk yield, percentage of protein in milk increased by 0.09 ± 0.03 (R2 = 0.61, P ≤ 0.05,) per unit decrease in MUNBV. Our results suggest that breeding and selecting for dairy cows with low MUNBV can reduce urinary urea N deposition onto pasture and consequently the negative environmental impact of pastoral dairy production systems in temperate grasslands. Moreover, reducing MUNBV of dairy cows can potentially increase farm profitability due to greater partitioning of N to milk in the form of protein.

MAP kinase regulation--the oncogene connection.

The mitogen-activated protein kinase (MAP kinase) family of cytoplasmic serine/threonine protein kinases is activated by a wide range of extracellular stimuli. In this review we focus on the accumulating evidence that proteins encoded by proto-oncogenes and oncogenes are either involved in the regulation of the MAP kinase pathway or are its targets.

Requirement of Ras-GTP-Raf complexes for activation of Raf-1 by protein kinase C.

Receptor tyrosine kinase-mediated activation of the Raf-1 protein kinase is coupled to the small guanosine triphosphate (GTP)-binding protein Ras. By contrast, protein kinase C (PKC)-mediated activation of Raf-1 is thought to be Ras independent. Nevertheless, stimulation of PKC in COS cells led to activation of Ras and formation of Ras-Raf-1 complexes containing active Raf-1. Raf-1 mutations that prevent its association with Ras blocked activation of Raf-1 by PKC. However, the activation of Raf-1 by PKC was not blocked by dominant negative Ras, indicating that PKC activates Ras by a mechanism distinct from that initiated by activation of receptor tyrosine kinases.

Inheritance of proliferative breast disease in breast cancer kindreds.

Previous studies have emphasized that genetic susceptibility to breast cancer is rare and is expressed primarily as premenopausal breast cancer, bilateral breast cancer, or both. Proliferative breast disease (PBD) is a significant risk factor for the development of breast cancer and appears to be a precursor lesion. PBD and breast cancer were studied in 103 women from 20 kindreds that were selected for the presence of two first degree relatives with breast cancer and in 31 control women. Physical examination, screening mammography, and four-quadrant fine-needle breast aspirates were performed. Cytologic analysis of breast aspirates revealed PBD in 35% of clinically normal female first degree relatives of breast cancer cases and in 13% of controls. Genetic analysis suggests that genetic susceptibility causes both PBD and breast cancer in these kindreds. This study supports the hypothesis that this susceptibility is responsible for a considerable portion of breast cancer, including unilateral and postmenopausal breast cancer.

Ice-active proteins and cryoprotectants from the New Zealand alpine cockroach, Celatoblatta quinquemaculata.

Celatoblatta quinquemaculata is moderately freezing tolerant. We have investigated low and high molecular weight compounds that may be associated with its survival. Glycerol and trehalose were identified as potential cryoprotectants, with trehalose at the higher concentration. Trehalose was at its highest concentration in late autumn, during the periods sampled. Water contents declined with time and were significantly lower in late autumn than in late summer. No thermal hysteresis activity was detected in haemolymph or in extracts of the head, muscles and the fat body. Extracts of the Malpighian tubules showed an hexagonal crystal growth form, as did those of the gut tissue and gut contents. The gut tissue had high levels of thermal hysteresis (approximately 2 degrees C) and the gut contents somewhat lower levels (approximately 0.6 degrees C). Recrystallization inhibition activity mirrored that of thermal hysteresis, with activity absent in the haemolymph or fat body cells but present in the gut tissues and contents. Activity was reduced by heating and was associated with a molecule >14kDa in size. These findings suggest an antifreeze protein is involved. In fed animals, ice nucleation is likely to start in the gut. Gut cells have a much greater resistance to freezing than do fat body or Malpighian tubule cells. The antifreeze protein may enable this tissue to survive freezing stress by inhibiting recrystallization.

MAP kinase kinase kinase, MAP kinase kinase and MAP kinase.

Signal transduction pathways that respond to external signals through the MAP kinase family of protein kinases are involved in diverse responses in eukaryotic cells. MAP kinases are one element in a series of kinases that serve to connect the plasma membrane with cytoplasmic and nuclear events. MAP kinases have the unusual feature that their activation requires threonine and tyrosine phosphorylation carried out by a dual specificity protein kinase. Recent advances have shown that in two MAP kinase pathways (the mating response pathway in the fission yeast Schizosaccharomyces pombe, and receptor tyrosine kinase signalling), the small GTP binding protein ras p21 links membrane events to kinase pathway activation.

EGF triggers neuronal differentiation of PC12 cells that overexpress the EGF receptor.

BACKGROUND: Mitogen-activated protein (MAP) kinase is the central component of a signal transduction pathway that is activated by growth factors interacting with receptors that have protein tyrosine kinase activity. The stimulation of PC12 phaeochromocytoma cells with nerve growth factor leads to the sustained activation and nuclear translocation of the p42 and p44 isoforms of MAP kinase and induces the differentiation of these chromaffin cells to a sympathetic-neuron-like phenotype. In contrast, stimulation with epidermal growth factor induces a transient activation of p42 and p44 MAP kinases without pronounced nuclear translocation and does not trigger cell differentiation. We have examined whether the differential activation of MAP kinases forms the basis of the differential response of the cells to the two factors. RESULTS: By overexpressing either wild-type or mutant receptors for epidermal growth factor in PC12 cells, we found that p42 and p44 MAP kinase activity remains elevated for longer in cells that overexpress receptors than in untransfected cells. Epidermal growth factor promotes both a striking nuclear translocation of p42 MAP kinase and the differentiation of the overexpressing cells. CONCLUSIONS: Our results strongly suggest that the distinct effects of nerve growth factor and epidermal growth factor on PC12 cell differentiation can be explained by differences in the extent and duration of activation of p42 and p44 MAP kinases in response to the two factors, without invoking a signal transduction pathway specific to nerve growth factor.

Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2.

BACKGROUND: Mitogen-activated protein (MAP) kinases (or extracellular signal regulated kinases; Erks) and stress-activated protein (SAP) kinases mediate cellular responses to a wide variety of signals. In the Erk MAP kinase pathway, activation of MAP kinases takes place in the cytoplasm and the activated enzyme moves to the nucleus. This translocation to the nucleus is essential to MAP kinase signalling because it enables the kinase to phosphorylate transcription factors. Whether components of the pathway mediated by the SAP kinase p38 change their cellular location on activation is not clear; we have therefore studied the cellular localisation of components of this pathway before and after stimulation. RESULTS: The p38 SAP kinase substrate MAP-kinase-activated protein kinase-2 (MAPKAP kinase-2) contains a putative nuclear localisation signal which we show is functional and required for activation by a variety of stimuli. Following phosphorylation of MAPKAP kinase-2, nuclear p38 was exported to the cytoplasm in a complex with MAPKAP kinase-2. Export of MAPKAP kinase-2 required phosphorylation by p38 but did not appear to require the kinase activity of MAPKAP kinase-2. The p38 activators MKK3 and MKK6 were present in both the nucleus and the cytoplasm, consistent with a role in activating p38 in the nucleus. CONCLUSIONS: In the p38 SAP kinase pathway, MAPKAP kinase-2 serves both as an effector of p38 by phosphorylating substrates and as a determinant of cellular localisation of p38. Nuclear export of p38 and MAPKAP kinase-2 may permit them to phosphorylate substrates in the cytoplasm.

Ras signalling is required for inactivation of the tumour suppressor pRb cell-cycle control protein.

Ras proteins act as molecular switches, responding to signals by entering the active GTP-bound, rather than the inactive GDP-bound, state. The inhibition of normal Ras proteins by microinjection of neutralizing antibody or expression of dominant-negative mutants has shown that Ras signalling is required for growth factors to stimulate DNA synthesis [1] [2], but the link between Ras and the cell-cycle machinery is not clear. Regulation of the phosphorylation state of the retinoblastoma protein (pRb), the product of the tumour suppressor gene Rb, is a key event in the progression of cells from G1 phase into S phase. In growth-arrested or early G1 cells, pRb is hypophosphorylated and binds to transcription factors of the E2F family [3]. These pRb-E2F complexes act to suppress gene transcription required for entry into DNA synthesis either by preventing E2F from stimulating transcription or by actively repressing transcription [4]. During G1, cyclin-dependent kinases (CDKs) become activated and phosphorylate pRb at multiple sites, leading to the dissolution of pRb-E2F complexes and gene transcription [5]. Here, we have tested the hypothesis that Ras signalling is required for the inactivation of pRb. A neutralizing antibody directed against p21Ras was microinjected into cells derived from mutant mouse embryos that lack Rb or CDK inhibitors (CDKIs). Cells without pRb or the p16 CDKI were more resistant to the inhibitory effects of the anti-Ras antibody. DNA synthesis in some tumour cell lines was completely resistant to the anti-Ras injection, indicating that p21Ras is required for pRb inactivation but also has other functions in cell-cycle progression.

How does p21ras transform cells?

Oncogenic forms of p21ras are found in a wide range of human tumors. However, the mechanism by which p21ras transforms remains obscure. Genetic evidence has identified a domain of p21ras that is involved with interaction with an effector molecule required for transformation. Two proteins, GAP and the tumor suppressor NF1, interact with p21ras in this region but it is an unresolved puzzle whether either of these is the an unresolved puzzle whether either of these is the effector. After interaction with an effector, two downstream events--activation of protein kinase C and another pathway--are necessary for induction of DNA synthesis by oncogenic p21ras; however, morphological transformation does not require activation of protein kinase C.

Tyrosine 763 of the murine granulocyte colony-stimulating factor receptor mediates Ras-dependent activation of the JNK/SAPK mitogen-activated protein kinase pathway.

The receptor for granulocyte colony-stimulating factor (G-CSF) can mediate differentiation and proliferation of hemopoietic cells. A proliferative signal is associated with activation of the ERK mitogen-activated protein kinase (MAPK) pathway. To determine whether other MAPK pathways are activated by G-CSF signalling, we have investigated activation of JNK/SAPK in cells proliferating in response to G-CSF. Here we show that G-CSF and interleukin-3 activate JNK/SAPK in two hemopoietic cell lines. The region of the G-CSF receptor required for G-CSF-induced JNK/SAPK activation is located within the C-terminal 68 amino acids of the cytoplasmic domain, which contains Tyr 763. Mutation of Tyr 763 to Phe completely blocks JNK/SAPK activation. However, the C-terminal 68 amino acids are not required for ERK2 activation. We show that activation of JNK/SAPK, like that of ERK2, is dependent on Ras but that higher levels of Ras-GTP are associated with activation of JNK/SAPK than with activation of ERK2. Two separate functional regions of the G-CSF receptor contribute to activation of Ras. The Y763F mutation reduces G-CSF-induced Ras activation from 30 to 35% Ras-GTP to 10 to 13% Ras-GTP. Low levels of Ras activation (10 to 13% Ras-GTP), which are sufficient for ERK2 activation, require only the 100 membrane-proximal amino acids. High levels of Ras-GTP provided by expression of oncogenic Ras are not sufficient to activate JNK/SAPK. An additional signal, also mediated by Tyr 763, is required for activation of JNK/SAPK.

Plasma membrane-targeted ras GTPase-activating protein is a potent suppressor of p21ras function.

Although p21ras is localized to the plasma membrane, proteins it interacts with, such as the GTPase-activating proteins (GAPs) ras GAP and neurofibromin (NF1), are not, suggesting that one function of p21ras GTP may be to target such proteins to the plasma membrane. To investigate the effects of targeting ras GAP to the plasma membrane, ras C-terminal motifs sufficient for plasma membrane localization of p21ras were cloned onto the C terminus of ras GAP. Plasma membrane-targeted ras GAP is growth inhibitory to NIH 3T3 fibroblasts and COS cells. This growth inhibition correlates with GAP catalytic activity, since the plasma membrane-targeted C-terminal catalytic domain or the GAP-related domain of neurofibromin is inhibitory, whereas the similarly targeted N-terminal domain is not. Moreover, the inhibition is abrogated by the inactivating mutation L902I, which abolishes ras GAP catalytic activity. Coexpression of oncogenic mutant ras rescues cell viability, but the majority of rescued colonies are phenotypically untransformed. Furthermore, in focus assays, targeted ras GAP suppresses transformation by oncogenic mutant ras, and in reversion assays, targeted ras GAP can revert cells transformed by oncogenic mutant ras. Neither the targeted or nontargeted N-terminal domain nor the L902I mutant of ras GAP has any transforming activity. These data demonstrate that ras GAP can function as a negative regulator of ras and that plasma membrane localization potentiates this activity. However, if ras GAP is involved in the effector functions of p21ras, it can only be part of the effector complex for cell transformation.