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Background: Continuous respiratory monitoring can support integrated care for chronic obstructive pulmonary disease (COPD) patients, by coupling them with remote clinical personnel who triage patients in coordination with their health care providers. When deploying such services, there remains uncertainty surrounding outcomes when at-risk patients are proactively identified and escalated for provider evaluation. This study presents findings from a service deployed in a real-world COPD cohort by analyzing the clinical interventions made during in-person and telehealth pulmonary outpatient visits following remote escalations., Methods: A single-center, retrospective, observational study of real-world COPD patients at a multisite pulmonary practice was conducted. Patients who were enrolled in a continuous respiratory monitoring service for at least one year and were seen by a provider within 7 days of an escalation by the service (N=168) were included. To evaluate the potential impact of these escalations on provider and patient burden, medical charts from outpatient visits were manually reviewed and grouped into 6 categories based on the clinical action(s) taken by the provider., Results: A total of 245 outpatient visits occurred from 168 patients within 7 days of escalation. Of the 245 visits, 206 (84.1%) resulted in clinical intervention and 163 (66.5%) resulted in treatment consistent with acute exacerbations of COPD. A total of 1.6% of the outpatient visits resulted in referral to the emergency department., Conclusion: Provider encounters occurring following the escalation of a patient from a continuous respiratory monitoring service consistently resulted in that provider administering a treatment to the escalated patient., (JCOPDF © 2024.)

Background: It is unclear whether persistent inhaled steroid exposure in chronic obstructive pulmonary disease (COPD) patients before coronavirus disease 2019 (COVID-19) is associated with hospitalization risk., Objective: Our objective was to examine the association between persistent steroid exposure and COVID-19-related hospitalization risk in COPD patients., Study Design and Methods: This retrospective cohort study used electronic health records from the Kaiser Permanente Northern California health care system (February 2, 2020, to September 30, 2020) for patients aged ≥40 years with COPD and a positive polymerase chain reaction test result for COVID-19. Primary exposure was persistent oral and/or inhaled steroid exposure defined as ≥6 months of prescriptions filled in the year before the COVID-19 diagnosis. Multivariable logistic regression was performed for the primary outcome of COVID-19-related hospitalization or death/hospice referral. Steroid exposure in the month before a COVID-19 diagnosis was a covariate., Results: Of >4.3 million adults, 697 had COVID-19 and COPD, of whom 270 (38.7%) had COVID-19-related hospitalizations. Overall, 538 (77.2%) were neither exposed to steroids in the month before COVID-19 diagnosis nor persistently exposed; 53 (7.6%) were exposed in the month before but not persistently; 23 (3.3%) were exposed persistently but not in the month before; and 83 (11.9%) were exposed both persistently and in the month before. Adjusting for all confounders including steroid use in the month before, the odds ratio for hospitalization was 0.77 (95% confidence interval 0.41-1.46) for patients persistently exposed to steroids before a COVID-19 diagnosis., Interpretation: No association was observed between persistent steroid exposure and the risk of COVID-19-related hospitalization in COPD patients., (JCOPDF © 2022.)

Background: Cannabis use among reproductive-aged Canadians is increasing, but our understanding of its impacts on fertility, pregnancy, and breast milk is still evolving. Despite the availability of many web-based resources, informed decision-making and patient counseling are challenging for expectant families and providers alike., Objective: We aimed to conduct a scoping review of publicly available web-based Canadian resources to provide information on the effects of cannabis on fertility, pregnancy, and breast milk., Methods: Following PRISMA-ScR (Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews), we systematically searched 8 databases between January 1, 2010, and November 30, 2020, and web pages of 71 Canadian obstetrical, government, and public health organizations. We included English resources discussing the effects of cannabis on fertility, pregnancy, breastfeeding, or the exposed fetus and infant. Epidemiological characteristics, readability, and content information were extracted and summarized., Results: A total of 183 resources met our inclusion criteria. Resources included content for public audiences (163/183, 89.1%) and health care providers (HCPs; 31/183, 16.9%). The resources were authored by national-level (46/183, 25.1%), provincial or territorial (65/183, 35.5%), and regional (72/183, 39.3%) organizations. All provinces and territories had at least one resource attributed to them. The majority (125/183, 68.3%) were written at a >10 grade reading level, and a few (7/183, 3.8%) were available in languages other than English or French. The breadth of content on fertility (55/183, 30.1%), pregnancy (173/183, 94.5%), and breast milk or breastfeeding (133/183, 72.7%) varied across resources. Common themes included citing a need for more research into the effects of cannabis on reproductive health and recommending that patients avoid or discontinue cannabis use. Although resources for providers were consistent in recommending patient counseling, resources targeting the public were less likely to encourage seeking advice from HCPs (23/163, 14.1%)., Conclusions: Canadian resources consistently identify that there is no known safe amount of cannabis that can be consumed in the context of fertility, pregnancy, and breastfeeding. Areas of improvement include increasing readability and language accessibility and encouraging bidirectional communication between HCPs and patients., International Registered Report Identifier (irrid): RR2-10.1136/bmjopen-2020-045006., (©Ayni Sharif, Kira Bombay, Malia S Q Murphy, Rebecca K Murray, Lindsey Sikora, Kelly D Cobey, Daniel J Corsi. Originally published in JMIR Pediatrics and Parenting (https://pediatrics.jmir.org), 19.10.2022.)

Background: During the COVID-19 pandemic, the University of Virginia adult cystic fibrosis (CF) center transitioned from in-person clinical encounters to a model that included interdisciplinary telemedicine. The pandemic presented an unprecedented opportunity to assess the impact of the interdisciplinary telemedicine model on clinical CF outcomes., Research Question: What are the clinical outcomes of a care model that includes interdisciplinary telemedicine (IDC-TM) compared with in-person clinical care for patients with CF during the COVID-19 pandemic?, Study Design and Methods: Adults with CF were included. The prepandemic year was defined as March 17, 2019, through March 16, 2020, and the pandemic year (PY) was defined as March 17, 2020, through March 16, 2021. Patients were enrolled starting in the PY. Prepandemic data were gathered retrospectively. Telemedicine visits were defined as clinical encounters via secured video communication. Hybrid visits were in-person evaluations by physician, with in-clinic video communication by other team members. In-person visits were encounters with in-person providers only. All encounters included previsit screening. Outcomes were lung function, BMI, exacerbations, and antibiotic use. FEV 1 percent predicted, exacerbations, and antibiotic use were adjusted for the effect of elexacaftor/tezacaftor/ivacaftor treatment., Results: One hundred twenty-four patients participated. One hundred ten patients were analyzed (mean age, 35 years; range, 18-69 years). Ninety-five percent had access to telemedicine (n = 105). Telemedicine visits accounted for 64% of encounters (n = 260), hybrid visits with telemedicine support accounted for 28% of encounters (n = 114), and in-person visits accounted for 7% of encounters (n = 30). No difference in lung function or exacerbation rate during the PY was found. BMI increased from 25 to 26 kg/m 2 (t 100 = -4.72; P < .001). Antibiotic use decreased from 316 to 124 episodes (z = 8.81; P < .0001)., Interpretation: This CF care model, which includes IDC-TM, successfully monitored lung function and BMI, identified exacerbations, and followed guidelines-based care during the pandemic. A significant decrease in antibiotic use suggests that social mitigation strategies were protective., Trial Registry: ClinicalTrials.gov; No.: NCT04402801; URL: www., Clinicaltrials: gov., (Copyright © 2021 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.)

This study aimed to consider childhood poverty in relation to a count measure of adverse childhood experiences (ACEs) as a predictor of adult health outcomes and to determine whether associations are sensitive to how childhood poverty is operationalized. A sample of 10,784 adult residents was derived using data 2014-2015 Wisconsin annual Behavioral Risk Factor Survey data, derived from the Centers for Disease Control and Prevention (CDC) Behavioral Risk Factor Surveillance System (BRFSS). Adult health outcomes (health risk behaviors, general health problems, chronic health problems, and depression) were predicted using a more conservative and severe indicator of childhood poverty, and authors tested whether observed associations were attenuated by the inclusion of an ACE count variable. Findings showed that severe indicators of childhood poverty are associated with general and chronic health problems as well as adult depression. These associations are attenuated, but remain intact, when ACEs are included in regression models. Using the CDC BRFSS data for Wisconsin, the study showed that associations between childhood poverty and adult health are sensitive to the way in which childhood poverty is operationalized. The relationship between childhood poverty and other ACEs is complex and thus warrants treating the former as a distinct childhood adversity rather than an item in an ACE summary score., (© 2021 National Association of Social Workers.)

Asthma is one of the most common underlying diseases in women of reproductive age that can lead to potentially serious medical problems during pregnancy and lactation. A group of key stakeholders across multiple relevant disciplines was invited to take part in an effort to prioritize, strategize, and mobilize action steps to fill important gaps in knowledge regarding asthma medication safety in pregnancy and lactation. The stakeholders identified substantial gaps in the literature on the safety of asthma medications used during pregnancy and lactation and prioritized strategies to fill those gaps. Short-term action steps included linking data from existing complementary study designs (US and international claims data, single drug pregnancy registries, case-control studies, and coordinated systematic data systems). Long-term action steps included creating an asthma disease registry, incorporating the disease registry into electronic health record systems, and coordinating care across disciplines. The stakeholders also prioritized establishing new infrastructures/collaborations to perform research in pregnant and lactating women and to include patient perspectives throughout the process. To address the evidence gaps, and aid in populating product labels with data that inform clinical decision making, the consortium developed a plan to systematically obtain necessary data in the most efficient and timely manner., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

The science of glycobiology has made rapid strides in the past few decades. A recent development has been the introduction of glyconutrients as nutritional supplements for health support and disease management. This article reviews the basic and clinical science of glyconutrients and presents a brief perspective on the impact of the field of glycomics.

The history of the International Court of Justice (ICJ) is summarized, with a discussion of some of its earlier Advisory Opinions. The Advisory Opinion on the legality of nuclear arms is considered in the light of the principles of international humanitarian law and a review of nuclear weapons effects. The present government's position on nuclear weapons as outlined in the Strategic Defence Review (which ignores the issue of legality) is examined critically.

Airway smooth muscle (ASM) mass appears to be increased in the bronchi of patients with chronic severe asthma. Although the precise mechanisms that induce these changes are unknown, increases in ASM mass are caused, in part, by ASM cell proliferation. After allergen challenge in rats, it has been possible to demonstrate an increase in ASM mass by morphometric techniques. To examine whether hyperplasia is involved in ASM cell growth in vivo, we investigated whether repeated allergen challenges in sensitized Brown Norway rats stimulated DNA synthesis in airway epithelial and ASM cells. Animals that were actively sensitized to ovalbumin (OA) received either three aerosolized OA or saline challenges at 5-day intervals. DNA synthesis was measured by indirect immunohistochemical techniques with an anti-bromodeoxyuridine (BrdU) antibody. OA inhalations increased ASM mass as determined by morphometry and also induced DNA synthesis in both airway epithelial and ASM cells in the airways of sensitized animals compared with saline-challenged control animals. ASM mass was increased in large- and medium-sized airways but not in small airways. However, the number of BrdU-positive ASM cells normalized to basement membrane length was also greater in the large- and medium-sized airways compared with that in the small airways. When the number of BrdU-positive epithelial cells was normalized to basement membrane length, there was no difference among airway sizes and the number of BrdU-positive epithelial cells. These data suggest that DNA synthesis is induced in both airway epithelial and ASM cells after inhalational antigen challenge.

In a variety of diseases including asthma, inflammation causes microvascular leakage and activates thrombin. In addition to cleaving fibrinogen to fibrin, thrombin may have other important cellular effects. Because airway inflammation and vascular permeability are important determinants of airway hyperreactivity, we have studied the effects of thrombin on airway smooth muscle. Using cultured human airway smooth muscle cells, we have examined whether alpha-thrombin can evoke calcium responses, phosphoinositide turnover, or cell proliferation. We have demonstrated that alpha-thrombin does increase cytosolic calcium and phosphoinositide hydrolysis in a dose- and time-dependent manner that may be inhibited by pretreating cells with r-hirudin. In addition, we have shown that thrombin stimulates airway smooth muscle cell proliferation. By contrast, bradykinin, which evoked comparable increases in cytosolic calcium and phosphoinositide turnover, did not stimulate airway smooth muscle cell growth. We conclude that thrombin effectively increases cytosolic calcium and induces PI hydrolysis and, in addition, is capable of stimulating airway smooth muscle cell growth. However, the lack of an effect of bradykinin on cell growth suggests that increases in calcium and PI turnover alone will not induce airway smooth muscle cell proliferation. We suggest that alpha-thrombin may be important in the pathogenesis of both increased airway resistance as well as the structural changes seen as a consequence of chronic asthma.

Action potentials activate voltage-dependent calcium channels and attendant increases in cytosolic calcium concentration ([Ca2+]i) in many excitable cells. The role of these channels in the regulation of [Ca2+]i in nonspiking cells that do not depolarize to membrane potentials sufficient to activate a substantial fraction of the available current is less clear. Measurements of the peak activation and steady-state inactivation of L-type calcium currents have predicted the existence of a noninactivating current window over a voltage range where channel inactivation is incomplete. The degree to which such small currents might regulate [Ca2+]i, however, has not been established. Here we demonstrate a "calcium window" in nondialyzed, quiescent smooth muscle cells over a small voltage range near the resting membrane potential. Sustained depolarizations in this voltage range, but not to more positive potentials, resulted in sustained rises in calcium, despite the fact that macroscopic inward currents were < 2 pA. The calcium window corresponded well with the predicted window current determined under the same conditions; the peak of the calcium window occurred at -30 mV, with steady-state rises in [Ca2+]i in some cells at -50 mV. Steady-state rises in [Ca2+]i following depolarization were completely blocked by nisoldipine and were augmented and shifted to more negative potentials by BAY K8644. Voltage-dependent calcium channels thus regulate steady-state calcium levels in nonspiking cells over a voltage range where macroscopic currents are only barely detectable. This voltage range is bounded at negative potentials by calcium channel activation and at more positive potentials by channel inactivation.

Fc receptors for immunoglobulins are found on many cells and are important in host defense. We transfected Fc gamma RIIIA, present on macrophages and natural killer (NK) cells, into COS-1 cells to study its role in phagocytosis and calcium mobilization in the absence of other Fc gamma receptors. Human Fc gamma RIIIA-alpha (CD16) was cotransfected with its associated chains, either Fc gamma RIIIA gamma or zeta. Both gamma and zeta were observed to induce a phagocytic signal, but gamma was at least sixfold more effective than zeta. Conservative substitution by phenylalanine of either one of the two cytoplasmic tyrosine residues in the gamma chain resulted in markedly diminished phagocytosis and calcium mobilization. Tyrphostin 23, an inhibitor of tyrosine kinases, reversibly inhibited phagocytosis. Further, in vitro kinase assays with the wild type and mutant gamma chains demonstrated that the wild type gamma chain, but not the mutant gamma chains, is phosphorylated. These results suggest that the cytoplasmic tyrosine residues and tyrosine phosphorylation are required for Fc gamma RIIIA to mediate two signal transduction events: phagocytosis and calcium mobilization.

Rat C-reactive protein (CRP) is a pentameric glycoprotein composed of five apparently identical monomers, two of which form a disulfide-linked dimer (Rasosouli, M., Sambasivam, H., Azadi, P., Dell, A., Morris, H. R., Nagpurkar, A., Mookerjea, S., and Murray, R. K. (1992) J. Biol. Chem. 267, 2947-2954). In this study, the nature of the oligosaccharide chain of rat CRP was investigated by fast atom bombardment-mass spectrometry (FAB-MS), and general features of its biosynthetic pathway were also analyzed. FAB-MS, electrospray-mass spectrometry, and linkage analysis demonstrated that each monomer of rat CRP contained one oligosaccharide chain, predominantly a disialylated biantennary structure, attached to Asn-128. The biosynthesis of rat CRP was studied by immunoprecipitation of CRP synthesized in vitro and by cultured hepatocytes. The results revealed that each monomer of rat CRP was synthesized individually as a single-chain precursor with a cleavable signal sequence. The translocated species was sensitive to digestion by endoglycosidase H (endo H), indicating that it possessed a high mannose oligosaccharide. Rat CRP acquired the ability to bind to phosphorylcholine-Sepharose and to form the dimeric and oligomeric species prior to acquiring resistance to endo H. Studies using tunicamycin revealed that the N-linked oligosaccharide present in rat CRP was not required for formation of its dimeric component, oligomerization, ability to bind to phosphorylcholine, or secretion. The non-glycosylated rat CRP, however, was still able to bind to phosphorylcholine-Sepharose and to be secreted by hepatocytes.

Previous studies have demonstrated a dihydropyridine-insensitive, receptor-activated calcium influx pathway in cultured human airway smooth muscle (ASM) cells. To further define the biophysical characteristics of this pathway, the relationship between membrane potential and cytosolic free calcium ([Ca2+]i) was studied with the combined methods of the patch-clamp technique and single cell calcium imaging. The nystatin perforated-patch method was used to maintain normal intracellular calcium buffering and receptor-activated signal transduction processes in voltage-clamped cells. Single voltage-clamped human ASM cells responded to exposure to histamine (200 microM) with an initial transient rise in [Ca2+]i followed by a secondary sustained elevation that was dependent on extracellular calcium. Before agonist activation, step changes in holding potential produced only slight changes in [Ca2+]i, whereas, after activation, cells developed a sustained rise in [Ca2+]i that showed a large variation as a function of membrane potential. Depolarization from -80 to 0 mV caused a fall in the steady-state [Ca2+]i to basal levels or slightly below. Repolarization to -80 mV caused the redevelopment of the sustained phase of the calcium response. When calcium was removed from the extracellular fluid by the addition of a stoichiometric excess of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), the voltage dependence of the sustained phase was abolished. In a series of experiments, agonist addition evoked a 54-fold increase in the voltage dependence of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat C-reactive protein (CRP) is unique among mammalian CRPs in being a glycoprotein and in containing a covalently linked dimer in its pentameric structure. To investigate these features, cDNA clones encoding rat CRP were isolated from an expression library, and the primary structure of the protein was derived. Taken along with the results of Northern blotting, we conclude that a single mRNA of approximately 2,500 nucleotides codes for a precursor of rat CRP with a signal sequence of 19 amino acids and a polypeptide of 211 amino acids, the latter sharing extensive homology with human, rabbit, and mouse CRPs. The deduced sequence agreed with results obtained from partial microsequencing and mapping by fast atom bombardment-mass spectrometry. Two potential sites for N-glycosylation (Asn-128 and Asn-147) and a C-terminal heptapeptide (Leu-205 to Ser-211, containing two cysteines at positions 208 and 209) were unique to rat CRP. The protein was also shown to be composed of five apparently identical monomers, two of which form a dimer linked by two interchain disulfide bonds involving Cys-208 and Cys-209. These same cysteines form an intrachain disulfide bond in the other three monomers. The primary structure of rat CRP and the basis of dimer formation have, therefore, been elucidated.

1. Fluorescence measurements of intracellular calcium concentrations ([Ca2+]i) were made on cultured human airway smooth muscle cells using the dye Fura-2. The response to either histamine (100 microM) or bradykinin (1 microM) was biphasic, with a transient increase in [Ca2+]i followed by a sustained [Ca2+]i increase lasting many minutes. The average steady-state (plateau) [Ca2+]i following agonist activation was 267 +/- 5 nM, whereas the average basal [Ca2+]i was 148 +/- 4 nM. 2. The sustained rise in [Ca2+]i required the continued presence of either histamine or bradykinin and was dependent on extracellular Ca2+. The magnitude of the transient rise in [Ca2+]i was not dependent on extracellular Ca2+. Sustained, receptor-activated rises in [Ca2+]i were rapidly abolished by chelation of extracellular Ca2+, or addition of non-permeant polyvalent cations, whereas these agents had minor effects in the absence of agonist. These data indicate that the sustained increase in [Ca2+]i was dependent on receptor-activated Ca2+ influx. 3. Receptor-activated Ca2+ influx was not affected by treatment with organic Ca2+ channel antagonists (nifedipine (10 microM), nisoldipine (10 microM) or diltiazem (10 microM] or agonists (Bay K 8644 (500 nM to 10 microM) or Bay R 5417 (500 nM]. The magnitude of the sustained rise was also not affected by pre-treatment with ouabain (100 microM) indicating little involvement of Na(+)-Ca2+ exchange in the influx mechanism. 4. Receptor-activated Ca2+ influx could be completely inhibited by several polyvalent cations (Co2+, Mn2+, Ni2+, -Cd2+ or La3+). Quantitative estimates of the potency of block were obtained for Ni2+ and La3+. These measurements indicate that the pKi for Ni2+ was 3.6 and for La3+ was 3.5. 5. Both Mn2+ and Co2+ ions caused a time-dependent quench of intracellular Fura-2; however, permeation of neither ion was increased following receptor activation, indicating that the influx pathway is not permeable to these cations. 6. Fura-2 was used to monitor the rate of Ba2+ entry into airway smooth muscle cells by monitoring the Ca(2+)-Fura-2 and Ba(2+)-Fura-2 isosbestic points as well as the 340 and 380 nm signals. Cell activation did not increase the rate of Ba2+ entry indicating that the Ca2+ influx pathway was poorly permeant to Ba2+ ions. Ba2+ (2 mM) was able to inhibit Ca2+ entry as shown by its effects on the Ba(2+)-independent, Ca(2+)-dependent wavelength (371 nm). 7. The voltage dependence of Ca2+ influx was examined before and after agonist-induced activation. The effect of KCl-induced depolarization prior to cell activation was to cause a slight increase in [Ca2+]i.(ABSTRACT TRUNCATED AT 400 WORDS)

The neutral glycolipids and gangliosides of sheep thymus and of sheep peripheral lymphocytes were compared. The patterns of both of these major classes of glycolipids were more complex in thymus than in the lymphocytes. The incorporation of radioactivity from D-[1-14C]galactose into the individual glycolipids of control and concanavalin A-stimulated sheep peripheral lymphocytes was also studied. A marked enhancement of incorporation into trihexosylceramide and an alteration of the pattern of incorporation into gangliosides were noted in the mitogen-treated cells. The results suggest that significant alterations of glycolipid composition and metabolism may occur during at least certain stages of lymphocyte differentiation.

1. The patterns of phosphopolypeptides produced by endogenous phosphorylation in vitro of rough- and smooth-membrane fractions of the microsomal fraction of mouse liver were studied by radioautographic analysis of sodium dodecyl sulphate/polyacrylamide-gel electrophoretograms. 2. A minimum of 17 polypeptides of both rough- and smooth-microsomal-membrane fractions were phosphorylated by using [gamma-(32)P]-ATP as the phosphate donor; only minor differences in phosphorylation pattern between the two membrane fractions were detected. 3. Phosphorylation in vitro by [gamma-(32)P]ATP was markedly stimulated by Mg(2+), but not by cyclic AMP, cyclic GMP or Ca(2+). The phosphorylation of certain polypeptides was preferentially stimulated by Mg(2+). Addition of cyclic AMP resulted in a decrease in the amount of (32)P detected in one polypeptide of mol.wt. approx. 56000, present in both the rough- and smooth-membrane fractions. 4. [gamma-(32)P]GTP was found to be a relatively poor donor of (32)P as compared with [gamma-(32)P]ATP. However, incubation of rough- and smooth-membrane fractions with this compound resulted in the phosphorylation of one polypeptide of mol.wt. approx. 96000 that was scarcely or not at all phosphorylated by [gamma-(32)P]ATP. 5. Under the conditions of incubation used, appreciable incorporation of (32)P from [gamma-(32)P]ATP occurred into products migrating at the front of the electrophoretograms; these products were identified as being principally comprised of 1-phosphatidylinositol 4-phosphate. Incorporation of (32)P into this lipid was also markedly stimulated by Mg(2+). 6. The overall results show that a considerable number of polypeptides of the rough- and smooth-microsomal-membrane fractions of mouse liver may be phosphorylated in vitro and indicate that the enzymes responsible are principally non-cyclic AMP-dependent protein kinases.

Primary cultures of airway smooth muscle cells were exposed to histamine, and intracellular free calcium transients were measured by the calcium-sensitive dye fura-2. Stimulation with 100 microM histamine resulted in a rise in intracellular calcium from an unstimulated level of 178 +/- 25 to 497 +/- 154 nM Ca2+ (SE; n = 14) and a return to base-line free calcium concentration within 1 min of stimulation. Pretreatment of cells with the H1 receptor blocker pyrilamine (2.5 microM) abolished the response; however, the calcium transient was not altered by pretreatment with the H2 blocker cimetidine (50 microM), by chelation of external calcium, or by pretreatment with 2 mM Co2+ or 5 microM nifedipine. Activation of protein kinase c by 200 nM phorbol 12-myristate 13-acetate (PMA) resulted in no detectable rise in cytosolic calcium but completely blocked the release of internal calcium by histamine. We conclude that 1) histamine causes a transient rise of cytosolic calcium in airway smooth muscle, 2) the rise in cytosolic calcium is mediated by H1 receptor coupling that triggers release of internal calcium stores, and 3) activation of protein kinase c blocks the histamine-induced release of intracellular calcium.

Synthesis of a relatively rare glycoprotein (germin) signals the onset of growth in germinating wheat embryos. Germin mRNA (1075 nucleotide residues) has an 85-residue 5'-untranslated sequence, a 69-residue sequence that can encode a 23-residue signal-peptide sequence, a 603-residue sequence that can encode a 201-residue mature-protein sequence, and a 318-residue 3'-untranslated sequence that begins with a UAA-terminator codon, ends with a 63-residue polyadenylate tract, and has three polyadenylation (and other, related) signals (AAUAAN etc.). One polyadenylation signal is just 9 nucleotides from the polyadenylation site, the shortest stretch of nucleotides yet found between polyadenylation signal and site in any animal or plant mRNA. The mature-protein coding sequence in germin mRNA contains an unusually high proportion (87%) of G + C in the third positions of its codons. The amino acid sequence of germin does not have extensive internal homologies or repetitions, and it is not characterized by regions of unusually high charge density, as is nucleoplasmin, another water-soluble homopentameric protein with otherwise closely related structural properties. Germin does, however, contain a stretch of 34 uncharged amino acid residues and these may possibly mediate its homopentameric structure and its remarkable resistance to enzymic proteolysis. In view of a possible association of germin with cellular membranes, the most interesting relatedness of the germin sequence to the sequences of other proteins is an 80% homology between a decapeptide sequence in mature germin and a decapeptide sequence in Escherichia coli glycerol-3-phosphate acyltransferase. The relation of germin-gene structure to overall gene regulation during early plant growth is discussed.

Cytosolic calcium is a key determinant of the contractile state of airway smooth muscle (ASM). To investigate the mechanisms by which histamine affects cytosolic calcium, we measured changes in inositol 1,4,5-trisphosphate (IP3) following the addition of histamine to cultured canine ASM cells. The effect of phorbol 12-myristate 13-acetate (PMA) on IP3 formation was investigated under conditions previously shown to abolish histamine-induced calcium release. In both intact cells and ASM membranes, histamine produced a significant increase in IP3 formation, which was inhibited by PMA. The site of this blockade was investigated by examining the effect of PMA on guanine nucleotide-stimulated IP3 formation and on phosphoinositide-specific phospholipase C (PI-PLC) activity in ASM membranes. Guanine nucleotide-stimulated IP3 formation was inhibited by PMA pretreatment. Membrane-associated PI-PLC activity was also decreased, an effect that was not due simply to a shift in the calcium sensitivity of the enzyme. We conclude that in cultured canine ASM cells, PMA blocks histamine-induced IP3 formation and that this inhibition is caused, in part, by a postreceptor site of action of protein kinase C, possibly via a direct effect on PI-PLC.

Evidence is presented that a distinctive type of glutathione-S-transferase (GSHTase-P), and a cytosolic polypeptide of Mr 52,000 (P-52), each appearing in greatly increased amounts in hepatocyte nodules during liver carcinogenesis in the rat, are so far indistinguishable. The probable identity of the two polypeptides was established with the use of SDS polyacrylamide gel electrophoresis and Western blot techniques with purified GSHTase-P and P-52 and their respective antibodies and by comparison of the sequence of the first 26 N-terminal amino acids. Since the enzyme and the polypeptide are each considered to be the best available early markers for hepatocyte nodules, as putative precancerous lesions, their probable identity makes them attractive cellular components for in depth studies on their transcriptional and translational regulation and their use in new approaches to the sequential analysis of liver carcinogenesis.

1-O-Alkyl-2-O-acyl-3-O-beta-D-(3'-sulfatoxygalactopyranosyl)-sn- glcerol (SGG) and its nonsulfated analog (GG) are the major glycolipids of rat testis. Aspects of the biosynthesis and metabolism of these two lipids have been investigated by determining their specific activities at various times after injection of [16-14C]palmitic acid, [1-14C]cetyl alcohol, and D-[1-14C]galactose into the testes of the adult rats. Evidence was obtained from studies with each of these three radioactive compounds that is consistent with the interpretation that GG exhibits a precursor relationship to SGG in vivo. The turnover time of GG, as estimated from the use of each of the three precursors, ranged between 21 and 69 h. In contrast, a slow increase of radioactivity in SGG was observed following injection of each of the three precursors, a plateau value being reached between 72 and 168 h. The radioactivity in the acyl, alkyl, and galactosyl moieties of SGG thereafter remained quite constant for another 21 days. Small amounts of monoalkylmonoacylglycerol were detected in rat testis. Radioactive studies indicated that this compound could be a precursor of GG and (or) monoalkyldiacylglycerol, another lipid that was also detected in rat testis. The results are consistent with the concept that the synthesis of SGG occurs primarily at an early stage of spermatogenesis and that the various moieties of this lipid exhibit almost complete metabolic stability during the subsequent complex stages of this process.

The brain of adult rats were analyzed for the presence of 35-SO4-containing glycolipids following intraventricular injection of Na2-35SO4. Radiochromatographic analyses revealed the presence of two minor 35-SO4-containing glycolipids, in addition to sulfogalactosylceramide. One of these two minor sulfolipids was isolated and tentatively identified as a 1-O--alkyl-2-0-acyl-3-(3'-sulfogalactosyl)-glycerol, a compound recently demonstrated to be the major glycolipid of mammalian testis. The alkyl and acyl compositions of the compound from rat brain are more heterogeneous than those from rat testis. The non-sulfated form of the galactoglycerolipid was also detected in rat brain. The amount of the sulfogalactoglycerolipid in rat brain is 0.19 mumol per gram wet weight, approximately one-third of the amount in rat testis (per gram wet weight), and is approximately one-fifteenth that of sulfogalactosylceramide in rat brain. The possible significance of the common occurrence in brain and testis of sulfated and non-sulfated galactolipids is discussed.

The influence of thirty-four adamantane, protoadamantane, and homoadamantane derivatives on the phase transition characteristics of the bilayer in dipalmitoyl lecithin liposomes has been determined by differential scanning calorimetry. Each of these compounds induces a broadening of the phase transition profile of the lipid bilayer that is dependent upon the concentration of the solute and its molecular structure. The concentration--response curves obtained for these solutes suggest that the cage compound derivatives modify the phase properties and under some conditions may induce a phase separation in the doped bilayer. The relative activity sequences obtained for the compounds examined cannot be accounted for by simple considerations of lipid/water partition coefficients, substitution constants based on free energy relationships, or the relative polarities or sizes of substituent groups. The observations are consistent with the hypothesis that the position and orientation of a solute within the bilayer are critical factors in determining its relative potency. The position of a solute within the bilayer is significantly controlled by the presence of polar substituents and by the relative geometric relationships of these groups. For a given substituent group, the shape and size of the hydrocarbon cage becomes increasingly important. It is apparent that seemingly minor modifications in the structure of a solute can significantly alter its influence on the phase transition behavior of a bilayer.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to examine the polypeptide patterns of rat liver rough and smooth endoplasmic reticulum (ER) membrane fractions stripped of ribosomes. Approximately 67 polypeptides were resolved from the rough ER membrane fraction. The polypeptide pattern of the smooth ER membrane fraction was similar to that of the rough ER membrane fraction, but exhibited substantially lower amounts of some seven polypeptides. Three of these polypeptides, of apparent molecular weights 63,000, 65,000, and 87,000, were of particular interest, as they could not be ascribed to contamination of stripped rough ER membrane fractions by residual ribosomal polypeptides. Conditions of treatment with low concentrations of trypsin were established that markedly diminished the capacity of the stripped rough ER membrane fraction to bind ribosomes in vitro and that also effected a partial detachment of ribosomes from nonstripped rough ER membranes; the results of electrophoretic analyses of rough ER membrane fractions treated in these manners are described. Comparison of the polypeptide patterns of guinea pig, mouse, and rabbit liver ER membrane fractions with rat liver ER membrane fractions revealed considerable variations in the distribution of the polypeptides of 63,000, 65,000, and 87,000 molecular weight among the ER membrane fractions of these species. The combined results of these studies indicate that the polypeptide of 87,000 molecular weight, although particularly sensitive to attack by trypsin, is not involved in the binding of ribosomes to the rough ER membrane fraction. Studies by others (cf. Kreibich, G., Grebenau, R., Mok, W., Pereyra, B., Rodriguez-Boulan, E., and Sabatini, D. D. (1977) Fed. Proc. 36, 656) have implicated the polypeptides of 63,000 and 65,000 molecular weight in this process. The patterns of phosphorylated polypeptides of rough and smooth ER membrane fractions of rat and mouse liver were also examined, using labeling in vivo with sodium [32p]phosphate or in vitro with [gamma-32P]ATP. Approximately 25 phosphorylated components were resolved by electrophoresis in the ER membrane fractions of both species. Evidence is presented that suggests that the great majority of these components are phosphopolypeptides. Differences were noted in the patterns of phosphorylation produced by in vivo and in vitro labeling; minor differences were also observed between the patterns of phosphorylation of the rough and smooth ER membrane fractions in either situation. The overall results afford an indirect approach toward evaluating the possible involvement of specific rough ER membrane polypeptides in ribosome-binding and reveal that liver ER membranes contain a substantially greater number of phosphorylated polypeptides thatn previously reported.

Binding of purified cholera toxin to cell surface receptors has been visualized by an indirect immunofluorescence procedure. Normal nucleated cells from blood, bone marrow and lymphoid tissues, express these receptors with the possible exception of erythroid precursors. Cells from patients with chronic lymphoid or myeloid leukaemias have a normal receptor expression but acute leukaemic cells showed a marked deficiency in cholera toxin binding. Insertion of purified Gm ganglioside into membranes of acute leukaemic cells provided cellular binding sites for the toxin.

We report the development of a nontransformed line of human airway smooth muscle cells retaining smooth muscle-specific contractile protein expression and physiological responsiveness to agonists implicated in inflammatory airway diseases. Specific responses to histamine, leukotrienes, bradykinin, platelet-activating factor, substance P, and thromboxane analogues are demonstrated as well as functional coupling to beta-adrenergic receptors. The cell line was characterized using indirect immunofluorescence, as well as electrophoretic separation and immunoblot analysis of smooth muscle-specific actin. Functional responses were assessed by measurements of cytosolic calcium and stimulation of adenosine 3',5'-cyclic monophosphate production. The cells retain their responsiveness over many population doublings and should be a useful model to examine specific receptor-effector mechanisms, as well as the effects of neurohumoral agents on the regulation of airway smooth muscle growth and differentiation.

In order to help determine whether alterations of the profiles of glycosphingolipids occur consistently in human tumours, the neutral glycosphingolipids and gangliosides of nine lung tumours (one adenocarcinoma, four squamous cell, two mixed adeno-squamous cell, one large cell and one oat-cell carcinomata) were analysed. The control tissue consisted of adjacent lung; it contained neutral glycosphingolipids corresponding in properties to glucosyl-, lactosyl-, globotriaosyl- and globotetraosyl-ceramides. All of the tumours also contained these four neutral glycosphingolipids. However, in addition, five of the tumours (two of the squamous, the large cell and the two mixed adeno-squamous cell carcinomata) contained neutral glycosphingolipids corresponding in properties to lactotriaosyl- and neolactotetraosyl-ceramides; these same tumours also exhibited higher amounts of lactosylceramide than the other tumours analysed. Both of the two former neutral glycosphingolipids and very substantial amounts of the latter neutral glycosphingolipid were detected in pneumonic lung and in polymorphonuclear leucocytes; it thus appears possible that these particular compounds were derived from these latter cells rather than from the tumour cells. The ganglioside patterns of the tumours were almost equivalent in complexity to that exhibited by the control lung tissue. This study shows that the profiles of two major classes of glycosphingolipids (neutral glycosphingolipids and gangliosides) occurring in lung tumours are almost as complex as those of the parent tissue, a finding in contrast with the notably simplified patterns of these lipids found in many cancer cells grown in vitro. It also suggests that when lactotriaosyl- and neolactotetraosyl-ceramides and high amounts of lactosylceramide are detected in human tumours, the possibility must be considered that these compounds are derived from polymorphonuclear leucocytes.

The glycolipids of human testis and sperm have been compared. Both adult testis and the sperm exhibited remarkably complex, but generally similar, patterns of glycolipids. In particular, both contained appreciable amounts of the sulfogalactosylmonoalkylmonoacylglycerol, recently shown to be the principal glycolipid of the testis and sperm of a number of animals. In contrast, immature (prebuteral) human testis did not contain this compound. To extend knowledge on the possible distribution of sulfogalactosylmonoalkylmonoacylglycerol in the testes of other chordates, we have also analysed the glycolipids of the testes of a number of birds and fish. None of the testes from these species contained the above compound. Instead, sulfogalactosylceramide was found to be a major glycolipid of the testis of mature fowl, duck and skate-fish and sulfogalactosylglucosylceramide of the testis of mature salmon and trout. Immature duck testis contained only a trace of sulfogalactosylceramide. These studies reveal intriguing differences between the sulfatides of various chordates, lend support to the concept that sulfatides increase markedly in testis at a specific stage of spermatogenesis and suggest an important role for sulfatides in testicular and spermatozoal function.

Plasma membrane and bile canalicular membrane fractions were prepared from rat liver using NaHCO3, NaHCO3--CaCl2, and K2HPO4-KH2PO4 buffers (all at pH 7.4). The amount (expressed as milligrams protein per gram liver) of plasma membrane fraction exceeded the amount of bile canalicular membrane fraction using each of these three media; the use of NaHCO3-CaCl2 afforded a substantially higher yield of both types of membranes. The two membrane fractions exhibited complex patterns of polypeptides (greater than 30) on sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. Several reproducible differences in polypeptide patterns were observable between the two membrane fractions; in particular, components possibly corresponding to the heavy chain of myosin and to action were prominent in the bile canalicular membrane fraction. The effects of incubation in the above three buffers and in Tris--HCl (pH 7.4) on the polypeptide patterns of both types of membrane were studied. Many polypeptides were released from each type of membrane in all of these media. Differential effects on the polypeptide patterns of either type of membrane fraction were observed among the various buffers. In terms of minimizing loss of polypeptides, in general, NaHCO3--CacCl2 appeared to be the best buffer and Tris--HCl the worst buffer. The significance of these results for the preparation and storage of liver cell plasma membrane fractions is briefly discussed.

The use of L-[35S]methionine (500-700 Ci/mmol (1 Ci = 37 GBq) for labelling the polypeptides of liver rough (R) and smooth (S)endoplasmic reticulum (ER) membrane fractions in vivo was studied. Adult mice were injected intraperitoneally with 400 muCi of the isotope and killed at various times (2'min to 24 h) thereafter. RER and SER fractions were prepared, stripped of ribosomes, and treated with Triton X-100 to remove intravesicular contents. Sufficient radioactivity was present in individual aliquots (75 microgram protein) of the ER membrane fractions to permit their analysis by fluorography after separation by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. By 3 min, although the majority of the labelled components were of intravesicular origin, some 12 membrane polypeptides were labelled in the RER fraction (including one corresponding in migration to cytochrome P-450); some 6 of these latter polypeptides were labelled to a lesser degree in the SER membrane fraction at this time. By 5 min, the patterns of radioactive polypeptides of the RER and SER fractions (including both membrane and intravesicular components) were identical. By 7 min, some 28 labelled membrane polypeptides were detectable in the total microsomal membrane. Analysis of the 24-h samples revealed that all the membrane polypeptides seen by staining with Coomassie blue were visualised by fluorography. Other studies revealed the applicability of the approach used for producing highly labelled cell sap and serum proteins. The overall results demonstrate the suitability of L-[35S]methionine administered in vivo for producing mouse liver ER membrane polypeptides of relatively high radioactivity and are consistent with a rapid conversion of RER to SER by ribosome detachment or membrane flow.

The activity of acetyltransferase was detected in the microsomal fraction of rat liver by incubation with [3H]acetyl-CoA and by analyses using sodium dodecyl sulfate - polyacrylamide gel electrophoresis. Endogenous membrane proteins of relatively high molecular weight were found to serve as substrates. Optimal conditions for assay of the enzyme were defined. A deacetylase activity was also detected, which was inhibited by 2 mM ethylenediaminetetraacetic acid. Further subfractionation disclosed that the acetyltransferase activity was most enriched in the Golgi fraction, in which its specific activity was some ninefold greater than in the total homogenate. The radioactive labelling of Golgi-associated proteins observed was relatively intense, exceeding that of histone and ribosomal proteins in the homogenate. Analysis of the acetylated Golgi fraction by two-dimensional electrophoresis revealed approximately 90 radioactive polypeptides. Various treatments demonstrated that a minimum of 80% of the incorporated radioactivity was present as derivatives of N-acetylneuraminic acid, principally N-acetyl-9-mono-O-acetylneuraminic acid (Neu5,9Ac2). The sialic acid O-acetyltransferase activity detected is thus probably identical to that reported by Varki and Diaz; the intense labelling of proteins reflects the ability of Golgi apparatus fractions to take up and concentrate acetyl-CoA. Protein-bound radioactive Neu5,9Ac2 was also detected in the medium of hepatocytes incubated with N-[3H]acetylmannosamine, demonstrating that these cells synthesize certain proteins containing acetylated sialic acids, some of which may be secreted. The data confirm that the Golgi apparatus is a major site of acetylation of protein-bound sialic acids in rat liver in vitro and provide new information showing that many glycoproteins undergo this particular type of modification.

Rabbit haptoglobin is a tetrameric protein consisting of two nonglycosylated alpha and two glycosylated beta chains, the latter being joined to the former and the former to each other by disulfide linkages. We describe here the results of studies that analyzed the biosynthetic pathway of rabbit haptoglobin by using cultured hepatocytes incubated with L-[35S]cysteine. The initial form of haptoglobin detected in hepatocytes exhibited Mr = 46,000, was glycosylated, and corresponded in migration to the initial species formed when the mRNA for rabbit haptoglobin was translated using the reticulocyte lysate system coupled with dog pancreatic microsomes. This one-chain intermediate was rapidly cleaved into a glycosylated form of the beta chain and into the mature alpha chain, these chains being joined by disulfide linkages. Dimerization also occurred rapidly, forming a tetrameric precursor of haptoglobin. Several other intracellular glycosylated forms of the beta chain were detected subsequently, representing intermediates formed during oligosaccharide processing prior to secretion of mature haptoglobin. Addition of tunicamycin (5 micrograms/ml) inhibited glycosylation of the initial form of haptoglobin detected, but subsequent proteolytic processing into alpha and beta chains still occurred. Our results showed that the pathway of biosynthesis of rabbit haptoglobin closely resembles that reported for rat haptoglobin ( Hanley , J. M., Haugen , T. H., and Heath, E. C. (1983) J. Biol. Chem. 258, 7858-7869).

The purpose of this study was to establish when alterations of the electrophoretic patterns of the polypeptides and phosphopolypeptides of the microsomal membrane fraction of the livers of rats become observable after initiation of acute hepatic ischemia. Ischemia was initiated by clamping the vascular supply to the left and median lobes of the livers of adult male rats. The animals were killed at various times thereafter (up to 6 h, and in certain instances, 24 h) and microsomal membrane fractions were prepared from each. The patterns of the polypeptides and phosphopolypeptides of these fractions were analysed by sodium dodecyl sulfate--polyacrylamide gel electrophoresis, using staining with Coomassie blue to analyse the polypeptides and radioautography to analyse 32P-labelled phosphopolypeptides. Alterations of the polypeptide pattern were apparent in the fractions from animals killed at 4 h and after; prior to this time point, subtle alterations, at most, could be distinguished. Effects of acute ischemia on the pattern of phosphopolypeptides of the microsomal membrane fraction were studied after phosphorylation in vivo (produced by intraperitoneal injection of [32P]phosphoric acid) and in vitro (using [gamma-32P]ATP as phosphate donor). No marked changes in the phosphopolypeptide pattern produced by phosphorylation in vivo were observed until 6 h after clamping, by which time a diminution of the radioactivity in the majority of the phosphopolypeptides was evident. However, noteworthy alterations of the pattern of phosphopolypeptides produced by phosphorylation in vitro were observable in the membrane fractions from animals subjected to 2 h of ischemia. Overall the study provides a base line delineating the time sequence during which alterations of the electrophoretic patterns of the polypeptides and phosphopolypeptides of rat liver microsomal membranes become evident following the onset of acute hepatic ischemia and reveals that gross alterations of the polypeptide patterns of these membranes and of certain other subcellular fractions are not an early occurrence following this severe type of injury. The possible utility of the application of phosphorylation in vitro for detecting early alterations in membrane structure following cell injury is suggested.

Induction of hepatic microsomal mono-oxygenase species after administration of various xenobiotics is a well-documented phenomenon. To examine the number and specific species of rat liver microsomal membrane polypeptides involved in such responses, we have used sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to analyse microsomal fractions from animals treated with a number of important xenobiotics. The following are the principal points to have emerged from this study. 1. A minimum of twelve electrophoretically distinct patterns of induction of haemopolypeptides and other polypeptides could be distinguished after administration, either singly or in certain combinations, of phenobarbital, 3-methylcholanthrene, polychlorinated biphenyls, 2-acetylaminofluorene, safrole (or isosafrole), pregnenolone-16 alpha-carbonitrile and ethanol. The patterns consisted of various permutations of the amounts of eight polypeptides of 47000-56000 mol.wt., of which at least three were haemopolypeptides. The possible identities of these polypeptides, which included species of cytochrome P-450, cytochrome P-448 and epoxide hydratase, are discussed. 2. Agents (3-methylcholanthrene, benzo[a]-pyrene, polychlorinated biphenyls, 2,3,7,8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone) that result in the induction of cytochrome P-448 caused a marked increase in two polypeptides of 54000 and 56000 mol.wt., whereas safrole and isosafrole induced only the former polypeptide. 3. Administration of 2-acetylaminofluorene resulted in the induction of two polypeptides; evidence is presented that suggests that one of these is a species of epoxide hydratase [cf. Levin, Lu, Thomas, Ryan, Kizer & Griffin (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3240-3243] ANd that the other may be a novel haemopolypeptide. 4. The overall results emphasize the complexity of the responses exhibited by rat liver microsomal fractions to the administration of xenobiotics.

A previous study from our laboratory (L.C. Eriksson et al., Biochem. Biophys. Res. Commun., 117: 740-745, 1983) revealed that a cytosolic polypeptide of approximate Mr 21,000 (designated P-21) was markedly elevated in amount in hepatocyte nodules induced by six different regimens. The molecular weight of this polypeptide, subsequently revised to approximately 26,000, was redesignated P-26 and was identified (T.H. Rushmore et al., Biochem. Biophys. Res. Commun., 143: 98-103, 1987) as a subunit of a placental form of glutathione S-transferase (K. Sato et al., Gann 75: 199-202, 1984), also named glutathione S-transferase 7-7 (H. Jensson et al., FEBS Lett., 187: 115-120, 1985). We describe here a convenient method for purifying relatively large amounts of P-26 from hepatocyte nodules involving the sequential use of affinity chromatography on S-hexyl glutathione-Sepharose 4B, CM-Sephadex, and DEAE-Sephacel. Evidence is presented that P-26 exists as a dimer of approximate Mr 52,000 (P-52). Analyses by two-dimensional electrophoresis have indicated that the subunits of Mr 26,000 may consist of five separate charged isomers. Investigations using appropriate antisera and analysis by amino acid sequencing have provided additional confirmation that P-52 is probably identical to rat placental glutathione S-transferase. Antibodies to P-52 are proving to be useful as a marker of new cell populations that appear regularly during hepatocarcinogenesis.

The cytosolic polypeptides of hepatocyte nodules in six models of liver carcinogenesis were analysed by SDS-polyacrylamide gel electrophoresis and their patterns compared with these of control and variously treated livers. The amount of a polypeptide of Mr 21,000 was about tenfold elevated in the cytosol of five of the six types of nodules and moderately elevated in the sixth. Certain other polypeptides, particularly one of Mr 26,000, also varied in amount, so that all of the nodules analysed could be distinguished from liver by their electrophoretic patterns. Some possible identities of the two polypeptides are discussed. Their study may have mechanistic as well as diagnostic importance.