Diagnostic performance of cytology for assessment of hepatic lipid content in dairy cattle.

The objective of our study was to characterize the diagnostic performance of cytology for assessing hepatic lipid content (HLC) in dairy cows by comparing microscopic evaluation of lipid vacuolation in touch imprint slide preparations of liver biopsies with quantitative measurement of triglyceride concentration ([TG]; mg/mg of wet weight) in paired biopsy samples. Our study also sought to compare the diagnostic performance of liver cytology, plasma nonesterified fatty acid concentration ([NEFA]), and plasma ß-hydroxybutyrate concentration ([BHB]) derived from a measurement performed on whole blood, for assessing HLC. Chemical extraction of TG from liver tissue remains the gold standard for quantifying HLC, largely because available blood tests, although useful for detecting some types of pathology, such as increased lipid mobilization, ketosis, or hepatocellular injury, are nonspecific as to etiology. Veterinary practitioners can sample bovine liver for cytological evaluation in a fast, minimally invasive, and inexpensive manner. Thus, if highly predictive of HLC, cytology would be a practical diagnostic tool for dairy veterinarians. In our study, liver biopsy samples from Holstein cows (219 samples from 105 cows: 52 from cows 2 to 20 d prepartum, 105 from cows 0 to 10 d in milk, 62 from cows 18 to 25 d in milk) were used to prepare cytology slides and to quantify [TG] using the Folch extraction method followed by the Hantzch condensation reaction and spectrophotometric measurement. An ordinal scale (0-4) based on amount of hepatocellular cytoplasm occupied by discrete clear vacuoles was used by 3 blinded, independent observers to rank HLC in Wright-Giemsa-stained slides. Interobserver agreement in cytology scoring was good. Corresponding plasma [NEFA] and [BHB] measurements were available for 187 and 195 of the 219 samples, respectively. Liver [TG] correlated more strongly with cytology score than with NEFA or BHB, and receiver operating characteristic curve analysis showed that cytology had better diagnostic performance than either NEFA or BHB for correctly categorizing [TG] at thresholds of 5, 10, and 15%. Hepatic lipidosis in high-producing dairy cows is of major clinical and economic importance, and this study demonstrates that cytology is an accurate means of assessing HLC. Additional work is indicated to evaluate the diagnostic utility of liver cytology.

Nonregenerative anemia: mechanisms of decreased or ineffective erythropoiesis.

In veterinary medicine, anemia without an appropriate compensatory hematopoietic response is termed nonregenerative. Nonregenerative anemia is a common clinical entity, occurring as a result of diminished or ineffective erythropoiesis in association with many types of pathology. This article reviews nonregenerative anemia in domestic animals, emphasizing mechanisms of disease, and also covers other conditions associated with nonregenerative anemia in people. Many aspects of nonregenerative anemia in animals are worthy of further investigation, from molecular mechanisms of disease to epidemiologic impacts.

Oclacitinib (APOQUEL(®)) is a novel Janus kinase inhibitor with activity against cytokines involved in allergy.

Janus kinase (JAK) enzymes are involved in cell signaling pathways activated by various cytokines dysregulated in allergy. The objective of this study was to determine whether the novel JAK inhibitor oclacitinib could reduce the activity of cytokines implicated in canine allergic skin disease. Using isolated enzyme systems and in vitro human or canine cell models, potency and selectivity of oclacitinib was determined against JAK family members and cytokines that trigger JAK activation in cells. Oclacitinib inhibited JAK family members by 50% at concentrations (IC50 's) ranging from 10 to 99 nm and did not inhibit a panel of 38 non-JAK kinases (IC50 's > 1000 nM). Oclacitinib was most potent at inhibiting JAK1 (IC50 = 10 nM). Oclacitinib also inhibited the function of JAK1-dependent cytokines involved in allergy and inflammation (IL-2, IL-4, IL-6, and IL-13) as well as pruritus (IL-31) at IC50 's ranging from 36 to 249 nM. Oclacitinib had minimal effects on cytokines that did not activate the JAK1 enzyme in cells (erythropoietin, granulocyte/macrophage colony-stimulating factor, IL-12, IL-23; IC50 's > 1000 nM). These results demonstrate that oclacitinib is a targeted therapy that selectively inhibits JAK1-dependent cytokines involved in allergy, inflammation, and pruritus and suggests these are the mechanisms by which oclacitinib effectively controls clinical signs associated with allergic skin disease in dogs.

Ambiguity hindering self-management and prevention of osteoporosis in post-menopausal women.

As osteoporosis relies largely on self-managed prevention and adherence to long-term treatment regimens, it is imperative that those at risk understand the disease that they are attempting to prevent. Ambiguity regarding osteoporosis and reluctance to take anti-osteoporosis medication (AOM) as well as calcium was noted in Australian post-menopausal women. This may lead to underestimating women's own risk of osteoporosis and fracture. INTRODUCTION: Fragility fractures caused by osteoporosis have been known to inflict significant personal and financial burden on individuals and society. As treatment of osteoporosis relies largely on self-managed prevention and adherence to long-term AOM regimens, it is imperative that women have a sound understanding of the disease that they are attempting to prevent. Much can also be gained from qualitatively exploring the level of osteoporosis knowledge particularly in post-menopausal women who are at greater risk of osteoporosis and fractures. This study thus aims to determine what post-menopausal Australian women know about osteoporosis and osteoporosis prevention. METHOD: Six focus group sessions, using purposive sampling, were conducted with 23 female participants (mean age 68 years (range 62-83)). Women responded to a series of open-ended questions regarding their knowledge about osteoporosis. The audiotaped focus groups were transcribed verbatim and analysed using a thematic analysis framework. RESULTS: Three key themes were identified: ambiguity about the nature of osteoporosis, ambiguity about osteoporosis prevention and reluctance to take AOM and calcium. CONCLUSION: Ambiguity associated with risk and prevention may provide women with a false sense of security that they are adequately acting to prevent the disease. Underestimation of their risk of osteoporosis and fracture as well as reluctance associated with AOM may be barriers to osteoporotic fracture prevention.

"Even though I am alone, I feel that we are many" - An appreciative inquiry study of asynchronous, provider-to-provider teleconsultations in Turkana, Kenya.

Non-physician clinicians (NPCs) in low and middle-income countries (LMICs) often have little physical proximity to the resources-equipment, supplies or skills-needed to deliver effective care, forcing them to refer patients to distant sites. Unlike equipment or supplies, which require dedicated supply chains, physician/specialist skills needed to support NPCs can be sourced and delivered through telecommunication technologies. In LMICs however, these skills are scarce and sparsely distributed, making it difficult to implement commonly used real-time (synchronous), hub-and-spoke telemedicine paradigms. An asynchronous teleconsultations service was implemented in Turkana County, Kenya, connecting NPCs with a volunteer network of remote physicians and specialists. In 2017-18, the service supported over 100 teleconsultations and referrals across 20 primary healthcare clinics and two hospitals. This qualitative study aimed to explore the impact of the telemedicine intervention on health system stakeholders, and perceived health-related benefits to patients. Data were collected using Appreciative Inquiry, a strengths-based, positive approach to assessing interventions and informing systems change. We highlight the impact of provider-to-provider asynchronous teleconsultations on multiple stakeholders and healthcare processes. Provider benefits include improved communication and team work, increased confidence and capacity to deliver services in remote sites, and professional satisfaction for both NPCs and remote physicians. Health system benefits include efficiency improvements through improved care coordination and avoiding unnecessary referrals, and increased equity and access to physician/specialist care by reducing geographical, financial and social barriers. Providers and health system managers recognised several non-health benefits to patients including increased trust and care seeking from NPCs, and social benefits of avoiding unnecessary referrals (reduced social disruption, displacement and costs). The findings reveal the wider impact that modern teleconsultation services enabled by mobile technologies and algorithms can have on LMIC communities and health systems. The study highlights the importance of viewing provider-to-provider teleconsultations as complex health service delivery interventions with multiple pathways and processes that can ultimately improve health outcomes.

A unified call to action from Australian nursing and midwifery leaders: ensuring that Black lives matter.

Nurses and midwives of Australia now is the time for change! As powerfully placed, Indigenous and non-Indigenous nursing and midwifery professionals, together we can ensure an effective and robust Indigenous curriculum in our nursing and midwifery schools of education. Today, Australia finds itself in a shifting tide of social change, where the voices for better and safer health care ring out loud. Voices for justice, equity and equality reverberate across our cities, our streets, homes, and institutions of learning. It is a call for new songlines of reform. The need to embed meaningful Indigenous health curricula is stronger now than it ever was for Australian nursing and midwifery. It is essential that nursing and midwifery leadership continue to build an authentic collaborative environment for Indigenous curriculum development. Bipartisan alliance is imperative for all academic staff to be confident in their teaching and learning experiences with Indigenous health syllabus. This paper is a call out. Now is the time for Indigenous and non-Indigenous nurses and midwives to make a stand together, for justice and equity in our teaching, learning, and practice. Together we will dismantle systems, policy, and practices in health that oppress. The Black Lives Matter movement provides us with a 'now window' of accepted dialogue to build a better, culturally safe Australian nursing and midwifery workforce, ensuring that Black Lives Matter in all aspects of health care.

Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting.

The VPS34 gene product (Vps34p) is required for protein sorting to the lysosome-like vacuole of the yeast Saccharomyces cerevisiae. Vps34p shares significant sequence similarity with the catalytic subunit of bovine phosphatidylinositol (PI) 3-kinase [the 110-kilodalton (p110) subunit of PI 3-kinase], which is known to interact with activated cell surface receptor tyrosine kinases. Yeast strains deleted for the VPS34 gene or carrying vps34 point mutations lacked detectable PI 3-kinase activity and exhibited severe defects in vacuolar protein sorting. Overexpression of Vps34p resulted in an increase in PI 3-kinase activity, and this activity was specifically precipitated with antisera to Vps34p. VPS34 encodes a yeast PI 3-kinase, and this enzyme appears to regulate intracellular protein trafficking decisions.

Understanding the Relationship Between Male Gender Socialization and Gender-Based Violence Among Refugees in Sub-Saharan Africa.

Gender-based violence (GBV) in humanitarian emergencies is progressively recognized as a global public health problem. Detrimental gender norms influence male perpetrated GBV against women, and social and structural contexts of forced migration and camp resettlement contribute to problematic gender norm development. The review sought to elucidate the dynamics that link gender socialization among male youth in sub-Saharan Africa with violent sexual behaviors. Two concepts were explored: (1) male gender socialization in sub-Saharan Africa related to GBV perpetration patterns and (2) the effect of forced migration on male socialization and GBV. We reviewed articles using a standard systematic review methodology, searching academic databases for peer-reviewed articles, and contacting experts for gray literature. Our initial search identified 210 articles. We manually reviewed these, and 19 met the review inclusion criteria. We identified 20 variables from the first concept and 18 variables from the second. GBV perpetration by male youth is positively associated with social pressures as well as cultural and religious beliefs. Amid forced migration, personal, societal, and cultural preexisting gender inequalities are often amplified to encourage GBV perpetration. The literature revealed aspects of culture, language, role modeling, religion, and the context of violence as important factors that shape young men's perspectives regarding the opposite sex and gender relations as well as sexual desires and dominance. Overall, though, literature focusing on male socialization and GBV prevention is limited. We made recommendations for future studies among refugee male youth in order to better understand these relationships.

Topical anaesthesia and intravenous cannulation success in paediatric patients: a randomized double-blind trial.

BACKGROUND: It is not known whether the choice of topical anaesthetic influences the likelihood of successful i.v. cannulation in the paediatric population. The null hypothesis of this study was that no difference exists in the initial success rate of cannulation between two commonly used topical anaesthetics. METHODS: A randomized double-blind trial conducted on patients between the age of 12 months and 12 yr presenting to a tertiary hospital emergency department. Patients requiring cannulation were randomized to either 4% amethocaine gel (AnGEL) or 5% lidocaine and prilocaine in a 1:1 emulsion (EMLA). The primary endpoint was success of initial attempt at i.v. cannulation. RESULTS: One hundred and seventy-seven patients were analysed of 203 enrolled. The success rate of AnGEL (73/97, 75%) and EMLA (59/80, 74%) did not significantly differ (chi2(1) 0.05, P=0.82). CONCLUSIONS: No difference exists in the cannulation success rates between the two anaesthetics. The choice of topical anaesthetic in paediatric cannulation should be based on other factors such as cost, time to anaesthesia, efficacy of the agent, and adverse effect profile.

Evaluation of four commercial enzyme-linked immunosorbent assays for the diagnosis of bovine paratuberculosis in Chilean dairy herds.

The accuracy of 4 commercial enzyme-linked immunosorbent assays (ELISAs) for diagnosis of bovine paratuberculosis was compared using sera from 53 Mycobacterium avium subsp. paratuberculosis (MAP) fecal culture-positive dairy cows (cases) and sera from 345 dairy cattle resident in 11 fecal culture-negative herds on 2 consecutive occasions 1 year apart (controls). The specificity of all 4 ELISA kits was >99%, and their diagnostic sensitivity ranged from 30.2% to 41.5%. Pairwise comparison of ELISAs found no significant differences (McNemar's chi-square test > 0.05), and assay agreement for categorical assay interpretation (positive or negative) was high (>98%) with kappa values ranging from 0.84 to 0.95. Receiver operating characteristic (ROC) curve analysis and the corresponding area under the ROC curves indicate that kit B had the highest overall accuracy. Thus, all 4 ELISA kits for bovine paratuberculosis had comparable accuracy when tested on Chilean dairy cattle, with kit B having a slight statistical advantage based on ROC area under the curve analysis. This suggests that any of the 4 kits could be appropriate for herd certification and for paratuberculosis control programs on Chilean dairy cattle.

Evaluation of dimethyl sulphoxide effects on initial response to endotoxin in the horse.

REASONS FOR PERFORMING STUDY: Endotoxaemia is one of the most severe and ubiquitous disease processes in horses. Although dimethyl sulphoxide (DMSO) is used clinically in horses, there is no study indicating its efficacy in endotoxaemic horses. HYPOTHESIS: DMSO ameliorates the clinical response to i.v. lipopolysaccharide (LPS) administration. METHODS: Eighteen horses were assigned randomly to one of 4 groups: Normosol-LPS (0.2 mug/kg bwt, i.v.); DMSO (1 g/kg bwt, i.v.)-saline; high-dose DMSO (1 g/kg bwt, i.v.)LPS; low-dose DMSO (20 mg/kg bwt, i.v.)-LPS. Horses participating in the DMSO-saline group were later assigned randomly to one of the LPS groups. Data for physical parameters, white blood cell counts, plasma TNF-alpha, and blood lactate and glucose concentrations were examined for the effect of treatment using a repeated-measures mixed-model ANOVA. A value of P<0.05 was considered significant. RESULTS: Endotoxaemia occurred in all horses receiving LPS, as indicated by the clinical score, physical parameters, haemoconcentration and leucopenia. High-dose DMSO ameliorated the effect of LPS on fever. DMSO, at either dose, but did not have a significant effect on LPS-induced changes in all other evaluated parameters. CONCLUSIONS: In this study, DMSO had minimal effects on clinical signs of induced endotoxaemia in horses. The effects were manifested by amelioration of LPS-induced fever.

The interaction of dietary fibers and cholesterol upon the plasma lipids and lipoproteins, sterol balance, and bowel function in human subjects.

To identify any metabolic effects of dietary fiber upon cholesterol metabolism in man, six adult volunteer subjects were fed eucaloric cholesterol-free formula diets, with and without added dietary fiber for two 4-wk periods. A large quantity of dietary fiber was fed, some 60 g of plant cell wall material (or 16 g of crude fiber) derived from corn, beans, bran, pectin, and purified cellulose. This provided about five times the fiber intake of the typical American diet. The addition of fiber to the cholesterol-free diet did not change either the plasma cholesterol level (171+/-21 mg/dl, SEM, to 167+/-18) or the triglyceride (103+/-39 to 93+/-27 mg/dl). The excretion of both endogenous neutral steroids and bile acids were unchanged with fiber (505+/-41 to 636+/-75 mg/day and 194+/-23 to 266+/-47 mg/day, respectively.) However, total fecal steroid excretion was increased 699+/-29 to 902+/-64 mg/day, P < 0.025). With fiber, intestinal transit time was decreased (59+/-9 to 35+/-8 h, P < 0.005), and both the wet and dry stool weights were greatly increased.A second group of six subjects was fed similar diets containing 1,000 mg cholesterol derived from egg yolk. The addition of fiber to the 1,000-mg cholesterol diet did not alter either plasma cholesterol level (233+/-26 to 223+/-36 mg/dl) or triglyceride (102+/-19 to 83+/-11 mg/dl). The excretion of endogenous neutral steroids (618+/-84 to 571+/-59 mg/day), of bile acids (423+/-122 to 401+/-89 mg/day), and of total fecal steroids (1,041+/-175 to 972+/-111 mg/day) were unchanged by fiber. The absorption of dietary cholesterol was not altered when fiber was added to the 1,000-mg cholesterol diet (44.0+/-3.3 to 42.9+/-2.5%). A two-way analysis of variance utilizing both groups of subjects indicated a significant (P < 0.001) effect of dietary cholesterol upon the plasma cholesterol concentration. We concluded that a large quantity of dietary fiber from diverse sources had little or no effect upon the plasma lipids and sterol balance in man in spite of the fact that intestinal transit time and stool bulk changed greatly.

Interactions between SH2 domains and tyrosine-phosphorylated platelet-derived growth factor beta-receptor sequences: analysis of kinetic parameters by a novel biosensor-based approach.

The interaction between SH2 domains and phosphotyrosine-containing sequences was examined by real-time measurements of kinetic parameters. The SH2 domains of the p85 subunit of the phosphatidylinositol 3-kinase as well as of other signaling molecules were expressed in bacteria as glutathione S-transferase fusion proteins. Phosphotyrosine-containing peptides, corresponding to two autophosphorylation sites on the human platelet-derived growth factor beta-receptor that are responsible for phosphatidylinositol 3-kinase binding, were synthesized and used as capturing molecules, immobilized on a biosensor surface. The association and dissociation rate constants for binding to both sites were determined for intact p85 and the recombinant SH2 domains. High association rates were found to be coupled to very fast dissociation rates for all interactions studied. A binding specificity was observed for the two SH2 domains of p85, with the N-terminal SH2 binding with high affinity to the Tyr-751 site but not to the Tyr-740 site, and the C-terminal SH2 interacting strongly with both sites. This approach should be generally applicable to the study of the specificity inherent in the assembly of signaling complexes by activated protein-tyrosine kinase receptors.

Templating the 3D structure of conducting polymers with self-assembling peptides.

Self-assembly of a tetrapeptide covalently attached to a thiophene-based monomer produced a gel with a fibrous, porous structure. Conditions were identified in which the thiophene end groups could undergo polymerization while retaining the 3D structure, resulting in the formation of nanofibrous gels with conductivities averaging 10-4 S cm-1.

Interruption of the fragile X syndrome expanded sequence d(CGG)(n) by interspersed d(AGG) trinucleotides diminishes the formation and stability of d(CGG)(n) tetrahelical structures.

Fragile X syndrome is caused by expansion of a d(CGG) trinucleotide repeat sequence in the 5' untranslated region of the first exon of the FMR1 gene. Repeat expansion is thought to be instigated by formation of d(CGG)(n)secondary structures. Stable FMR1 d(CGG)(n)runs in normal individuals consist of 6-52 d(CGG) repeats that are interrupted every 9-11 triplets by a single d(AGG) trinucleotide. By contrast, individuals having fragile X syndrome premutation or full mutation present >54-200 or >200-2000 monotonous d(CGG) repeats, respectively. Here we show that the presence of interspersed d(AGG) triplets diminished in vitro formation of bimolecular tetrahelical structures of d(CGG)(18)oligomers. Tetraplex structures formed by d(CGG)(n)oligomers containing d(AGG) interspersions had lower thermal stability. In addition, tetraplex structures of d(CGG)(18)oligomers interspersed by d(AGG) triplets were unwound by human Werner syndrome DNA helicase at rates and to an extent that exceeded the unwinding of tetraplex form consisting of monotonous d(CGG)(18). Diminished formation and stability of tetraplex structures of d(AGG)-containing FMR1 d(CGG)(2-50)tracts might restrict their expansion in normal individuals.

Codons 12 and 13 of H-ras protooncogene interrupt the progression of DNA synthesis catalyzed by DNA polymerase alpha.

Mutagenesis of protooncogenes has been postulated to contribute to the initiation and progression of human cancer. Activating mutations in the H-ras gene are predominantly single-base substitutions and are most frequently identified at codons 12, 13, and 61. We have analyzed the effects of DNA sequence context at specific codons that are hot spots for ras mutation with respect to abnormalities in copying by purified DNA polymerase alpha, a major eucaryotic replication enzyme. Exon 1 of H-ras gene was inserted into M13 mp19, single-stranded DNA constructs were isolated, and the progression of synthesis by polymerase alpha was measured. Strong termination sites were found in codons 12 and 13. Pausing at these codons is abolished when the template is mutated at the middle base of codon 12, the same alteration that converts H-ras into an activated oncogene. Resistance of codon 12 in double-stranded constructs to digestion with restriction enzymes and computer investigation of the ras sequence suggest that these termination sites are in a region of secondary structure. The frequency of sequence alterations within DNA chains that have been extended past codons 12 and 13 was found to be < 0.01. We consider a variety of mechanisms by which the potential secondary structure involving codons 12 and 13 may contribute to the pausing of DNA polymerase alpha and to the generation of clustered mutations at this site.

Expression of the BRK tyrosine kinase in mammary epithelial cells enhances the coupling of EGF signalling to PI 3-kinase and Akt, via erbB3 phosphorylation.

A high proportion of human breast cancers, in contrast with normal mammary tissue, express the intracellular tyrosine kinase BRK. BRK expression enhances the mitogenic response of mammary epithelial cells to epidermal growth factor, and conferment of a proliferative advantage through this mechanism may account for the frequent elevation of BRK expression in tumours. Here we report that BRK expression in mammary epithelial cells, at pathologically relevant levels, results in an enhanced phosphorylation of the epidermal growth factor receptor-related receptor erbB3 in response to epidermal growth factor. As a consequence, erbB3 recruits increased levels of phosphoinositide 3-kinase, and this is associated with a potentiated activation of Akt. This effect of BRK on the regulation of phosphoinositide 3-kinase and Akt activity may account for BRK's ability to enhance mammary cell mitogenesis, and raises the possibility that breast tumours expressing BRK may acquire a resistance to pro-apoptotic signals.

Autophosphorylation promotes complex formation of recombinant hepatocyte growth factor receptor with cytoplasmic effectors containing SH2 domains.

Hepatocyte growth factor (HGF), also known as scatter factor (SF), is a polypeptide which induces motility and/or mitogenesis in epithelial cells. The receptor for HGF/SF, p190MET, is a two-chain transmembrane tyrosine kinase encoded by the MET proto-oncogene. To identify the cytoplasmic effectors involved in signal transduction we have produced the human HGF/SF receptor in insect cells (Sf9) by means of a recombinant baculovirus. Two 170-kDa forms of the receptor were synthesized in Sf9 cells: the uncleaved single-chain precursor (which is by far the more abundant) and the proteolytically processed two-chain molecule. Both receptor species are phosphorylated on tyrosine in vivo and are active kinases in vitro. The recombinant receptor binds and phosphorylates in vitro four known cytoplasmic transducers containing src homology region 2 (SH2) domains: the 85-kDa subunit of phosphatidylinositol 3-kinase (Pl 3-kinase), rasGAP, phospholipase-C gamma (PLC-gamma), and p59Fyn, a tyrosine kinase of the src family. In all cases the association is strictly dependent on tyrosine phosphorylation of the receptor, indicating that it occurs via specific interaction with the SH2 domains. These results show that the HGF/SF receptor has the sequence requirements for binding a spectrum of cytoplasmic transducers whose different combinations in target cells may result in the observed pleiotropic biological response.

Cell adhesion and acquisition of detergent resistance by the cytoskeleton of cultured chick fibroblasts.

About 30% of the proteins of adherent cultured chick embryo fibroblasts are not solubilized by the non-ionic detergent Triton X-100 and remain firmly attached to the substratum. The insoluble residue contains a considerable part of the cell's cytoskeleton and its major constituents are large external transformation-sensitive (LETS) protein, the heavy chain of myosin, a 52,000 molecular weight protein and actin. Kinetic studies reveal that cytoskeleton insolubility in Triton is acquired either concurrently with cell adhesion or very closely with it. Neither cell adhesion nor binding of the Triton cytoskeleton to the substratum require de novo synthesis of protein. In the attempt to assess the role of LETS protein in cytoskeleton attachment, we find that trypsin-detached cells rapidly acquire Triton-insoluble cytoskeleton although their LETS protein content is about 15--20% of its level in long-term cultures. Removal of the great majority of LETS molecules of adherent cultures by either urea or trypsin treatment does not affect the relative amount or composition of the anchored cytoskeletal proteins. Also, LETS protein of cultures exposed to cycloheximide for extended periods of time, is reduced to 10% of its maximum amount without much affecting the attachment and composition of the cytoskeleton. It is deduced that the great majority of LETS protein is not required for the attachment of the Triton cytoskeleton to the substratum.