Diabetic hepatosclerosis: another diabetes microvascular complication?

BACKGROUND: Liver disease in diabetes is common and is frequently the result of hepatic steatosis. Diabetic hepatosclerosis is a relatively recent description of sinusoidal fibrosis, without steatosis, observed in liver biopsies of people with diabetes presenting with cholestasis. Its association with other microvascular complications suggests it is a form of hepatic diabetic microangiopathy. CASE REPORT: We report the case of a 50-year-old woman with longstanding Type 1 diabetes, complicated by nephropathy resulting in cadaveric renal transplant, retinopathy, gastroparesis and neuropathy with slowly healing ulceration to her right foot. She was noted to have deranged liver function tests: alanine aminotransferase, 162 IU/l; bilirubin, 44 IU/l; alkaline phosphatase, 5279 IU/l (isoenzymes; bone 1029 IU/l, liver 4250 IU/l); γ-glutamyl transferase, 662 IU/l. A non-invasive liver screen did not reveal the cause of the cholestasis. A liver biopsy demonstrated sinusoidal fibrosis without evidence of steatosis and thus a diagnosis of diabetic hepatosclerosis was made. Comparison with a biopsy performed 11 years previously at a different trust due to elevated alkaline phosphatase levels revealed slow progression of the sinusoidal fibrosis. DISCUSSION: This case describes the longest reported clinical course of diabetic hepatosclerosis, spanning 11 years, in which time the patient did not develop evidence of cirrhosis or portal hypertension. It is difficult to estimate the clinical relevance of this condition because little is known regarding its clinical course and effect on morbidity and mortality. Identified patients should undergo low-intensity, long-term follow-up to improve understanding of its clinical sequelae and relevance.

Diabetes and cardiovascular disease: pathophysiology of a life-threatening epidemic.

Diabetes is associated with the development of premature cardiovascular disease (CVD), which relates to the clustering of risk factors such as dyslipidaemia, hypertension, obesity and hyperglycaemia in the presence of insulin resistance. In addition, diabetes is associated with an inflammatory and pro-thrombotic environment, exacerbating the development of atherothrombosis. Insulin resistance and hyperglycaemia both contribute to the development of endothelial cell dysfunction and increased oxidative stress, culminating in accelerated atherosclerosis. Clot formation and function are also directly affected by insulin resistance and hyperglycaemia, with increased levels of coagulation factors and anti-fibrinolytic proteins and a fibrin network that is more resistant to lysis, coupled with increased platelet activation.It is well recognised that the intensification of glycaemic control leads to a reduction in microvascular complications in type 1 and type 2 diabetes; however, the same is less clear with macrovascular disease. Several randomised studies have attempted to address the effect of short-, medium- and long-term glycaemic control on cardiovascular outcomes, with mixed results. The overall interpretation of these trials suggests that intensive glycaemic control in patients with a relatively short duration of diabetes, without very poor control and with no CVD, might be safe and associated with fewer cardiovascular events.This review will summarise the effects of hyperglycaemia on the development of atherothrombosis and examine key cardiovascular outcome trials following intensive glucose control.

Progression to steroid autonomy in S115 mouse mammary tumor cells: role of DNA methylation.

Although monoclonal in origin, mammary tumors acquire a marked heterogeneity of cell phenotypes, including a mixture of steroid hormone-sensitive cells and insensitive cells. We describe here long-term studies on the effects of androgen withdrawal on cloned androgen-responsive S115 mouse mammary tumor cells as a model system to investigate mechanisms by which tumor cells lose their steroid sensitivity. In the prolonged absence of androgen, the cells lost hormone-sensitive parameters reproducibly, including loss of proliferative response, saturation density response, cell morphology response, and mouse mammary tumor virus long terminal repeat (MMTV-LTR)-related RNA. These experiments have demonstrated that when deprived of hormone in the long term, a clone of responsive cells gives rise reproducibly to a population of unresponsive cells in an ordered series of phenotypic changes. At the time when the cells lost all androgen response in terms of cell biology and MMTV-LTR-RNA, increased methylation of MMTV-LTR sequences in the DNA was detected. Thereafter recovery of androgen sensitivity has not been achieved in any of these parameters. The possible role of de novo DNA methylation in the progression to androgen autonomy of S115 cells is discussed.

Androgen regulation by the long terminal repeat of mouse mammary tumor virus.

Mouse mammary tumor virus (MMTV) has long been implicated in mouse mammary carcinogenesis, and it is now well established that the long terminal repeat (LTR) contains regulatory sequences responsible for glucocorticoid-mediated induction of viral RNA. However, we have demonstrated previously that androgens as well as glucocorticoids can regulate MMTV RNA in the S115 mouse mammary tumor cell line. To determine if androgens act directly on the LTR in these cells, plasmids were constructed with the MMTV LTR joined to the coding sequences of genes not normally expressed in the cells. Following transfection of these chimeric genes into S115 cells, we show that the expression of the genes is regulated by both androgens and glucocorticoids. Furthermore, hormonal regulation is also conferred by the LTR on the neighboring guanine phosphoribosyltransferase (gpt) gene. Thus, androgens can act on the LTR of MMTV when the appropriate receptors are present in the cells, and this interaction can influence the expression of additional adjacent genes.

High-dose oral colecalciferol loading in obesity: impact of body mass index and its utility prior to bariatric surgery to treat vitamin D deficiency.

Obesity is associated with lower vitamin D levels compared with normal weight subjects, and if levels are not replaced prior to bariatric surgery, this can increase fracture risk as bone density typically falls post-operatively. We analysed the effect of body mass index (BMI) on vitamin D levels in response to 300 000 IU of colecalciferol in patients with vitamin D deficiency (<30 nmol L-1 ). Patients were grouped according to their BMI as normal weight (20-24.9 kg m-2 ), overweight (25-29.9 kg m-2 ), obese class I (30-34.9 kg m-2 ) and obese class II and above (>35 kg m-2 ). The records were retrospectively analysed to investigate the effects of BMI on vitamin D (total 25-hydroxy vitamin D [25(OH)D]), serum Ca2+ and parathyroid hormone (PTH) levels at 6, 12, 26 and 52 weeks compared with baseline. Compared with normal weight subjects, overweight and obese patients achieved lower mean peak total 25(OH)D levels (6 weeks post-loading), which was most significant in the class II and above group (mean total 25(OH)D levels 96.5 ± 24.2 nmol L-1 and 72.42 ± 24.9 nmol L-1 , respectively; P = 0.003). By 26 weeks, total 25(OH)D levels fell in all groups; however, there was now a significant difference between the normal weight subjects and all other groups (mean total 25(OH)D levels 84.1 ± 23.7 nmol L-1 ; 58 ± 20 nmol L-1 , P = 0.0002; 62.65 ± 19.2 nmol L-1 , P = 0.005; 59.2 ± 21 nmol L-1 , P = 0.005, respectively). Far fewer patients in the overweight and obese groups maintained levels above the recommended level of 75 nmol L-1 52 weeks post-loading (93%; 20%, P = 0.0003; 23%, P = 0.01; and 14%, P = 0.001, respectively). Alternative regimes for the treatment of vitamin D deficiency are needed in overweight and obese patients, especially those in whom bariatric surgery is planned.

Differential effects of steroid hormones on parameters of cell growth.

Steroid hormones affect the growth of many tumor cells both in vivo and in vitro. Growth of cells in vitro can be studied as either anchorage dependent (monolayer culture) or anchorage independent (suspension culture), and in each case, steroid hormones can alter log-phase proliferation rate, saturation density, or cell morphology. Results presented here demonstrate that different steroid hormones can have different effects on each of these parameters in breast cancer cells. In androgen-responsive cells, glucocorticoid and androgen both stimulated fibroblastic morphology and saturation density in monolayer and growth in suspension, but glucocorticoid inhibited log-phase proliferation rate while androgen stimulated it. By transfection experiments, it has been possible to separate the androgen-regulated stimulation of cell proliferation from the androgen-regulated cell morphology changes indicating that the two parameters are not totally interdependent. The same separation, however, was not achieved for glucocorticoid regulation. Further separation of hormone effects was achieved also during the natural course of progression to steroid autonomy. In particular, the androgen and glucocorticoid stimulations of growth in suspension were separated. The experiments described here carry important messages for the design and interpretation of any experiment aimed at elucidating molecular events involved in steroid-mediated cell growth in culture.

Multiple sensitivities of mammary tumor cells in culture.

Cloned cell lines derived from the androgen-responsive Shionogi 115 mouse mammary carcinoma, when cultured in the presence of 3.5 X 10(-8) M testosterone, retain their responsiveness to testosterone and 5alpha-dihydrotestosterone and exhibit fibroblast-like morphology; their growth is poorly regulated by cell density. Dexamethasone at 10(-8) M inhibits proliferation of these cells by 30% but stimulates them by 235% at 10(-6) M in the temporary absence of testosterone. Cell growth is little affected by serum concentration. When cultured for 3 to 4 weeks in testosterone-free medium, however, the cells lose their androgen responsiveness and retain the inhibitory but not the stimulatory response to dexamethasone. They also show an increased sensitivity to serum and increased density regulation and change to an epithelial morphology. It is suggested that the loss of sensitivity to androgens, which does not result from absence of androgen receptor, is related in a complex way to the increased sensitivities to serum and density regulation.

Correlation of growth properties and morphology with hormone responsiveness of mammary tumor cells in culture.

A cell line derived from the androgen-responsive Shionogi 115 mouse mammary carcinoma is being used to investigate the changes in cell function accompanying steroid removal from steroid-responsive breast tumor cells. Shiongi 115 mouse mammary carcinoma cells become androgen unresponsive after two weeks of culture in the absence of testosterone although androgen receptors are still present in the cytoplasm and nucleus. Androgen-responsive cells are fibroblastic in appearance, have a low serum requirement, and can proliferate in suspension culture. Unresponsive cells, however, show a flattened morphology, are serum sensitive, and are anchorage dependent. As the androgen receptor mechanism of the unresponsive cells is intact, loss of sensitivity may be caused by a postreceptor defect. To determine the nature of this defect, the temporal relationships of changes in hormone responsiveness, morphology, serum sensitivity, and growth properties have been examined over a period of seven weeks following removal of testosterone from Shionogi 115 mouse mammary carcinoma cell cultures. Responses to testosterone and anchorage independence are both lost over a period of one to two weeks while serum sensitivity increases more slowly during the same period. The most rapid and least reversible change is seen in morphology. It is suggested that hormone responsiveness is related to properties of the cytoskeleton and cell membrane which influence growth rates and multiple sensitivities of tumor cells.

Effect of estradiol on human breast cancer cells in culture.

Conditions are described for growing and maintaining the estradiol sensitivity of the human breast cancer cell line ZR-75-1 both in monolayer and suspension cultures. Either newborn calf or fetal calf serum can be used in the culture medium, but an effect of estradiol on growth of the cells was only observed reproducibly if the serum was first treated with dextran-charcoal. Sulfatase treatment of the sera prior to dextran-charcoal treatment did not decrease cell growth in the absence of added estradiol, indicating that estrogen sulfates are unlikely to contribute to cell growth in dextran-charcoal-treated sera. In monolayer cultures, estradiol increased both the growth rate and final saturation density of the cells for each individual plating density tested in a dose-dependent manner with maximal stimulation occurring between 10(-10) and 10(-8) M estradiol. Estradiol also markedly increased the ability of the cells to grow both in suspension and semisolid Methocel cultures. In suspension, the cells grew as tight balls which clustered together to give small organoid-like structures reaching diameters of 4 mm and composed of an outer shell of living cells containing a central cavity of necrotic cells. In the absence of estradiol in both monolayer and suspension, the cells went through a limited and constant number of divisions and then stopped, such that the final cell number was determined by the initial plating density. In the presence of estradiol, this block was removed such that in monolayer cultures the final cell number was independent of plating density. A major loss of estradiol response was found if the cells were grown for 7 to 14 days in the absence of estradiol. This loss of response appeared to be due to a loss of ability to grow rather than to selective cell death within the population.

Effects of estradiol and tamoxifen on human breast cancer cells in serum-free culture.

ZR-75-1 human mammary cancer cells can be grown in serum-free medium in an estradiol- and tamoxifen-sensitive manner. Growth occurs both in monolayer and suspension culture, although serum is necessary during the initial plating of the cells in monolayer culture. Optimal stimulation of proliferation by estradiol occurs at 10(-8) and 10(-9) M. Tamoxifen alone weakly stimulated growth in a dose-dependent manner up to 10(-7) M, but inhibition was observed at 10(-6) M. This inhibition was not seen if the cells were cultured in 10% dextran:charcoal-treated fetal calf serum. This difference is ascribed to tamoxifen binding components in the serum. In the absence of serum, tamoxifen (10(-6) M) abolished the proliferative effect of estradiol at all concentrations of estradiol below 10(-7) M. Higher concentrations of estradiol partially overcame the tamoxifen effect. Consistent antiestrogenic effects of tamoxifen required an estradiol:tamoxifen ratio of 1:1000.

Effect of estrogen and progestin treatments on endometria from postmenopausal women.

PIP: This report describes experiments designed to answer several important questions about the biochemistry of estrogen-stimulated postmenopausal endometrium; in particular; how much estrogen enters the endometrium and the biological effectiveness of that estrogen in women receiving different forms of postmenopausal estrogenic therapy. To this end, nuclear and cytoplasmic estradiol receptor (ER) and cytoplasmic progesterone receptor (PR) were measured in curettage samples of endometria from women receiving, either sequentially or cyclically, Premarin, Harmogen, Progynova, or mestranol at either low or high doses. Cyclical treatment with estrogen alone was compared with sequential therapy with 3 weeks of estrogen plus 1 week of estrogen plus 1 week of estrogen plus northisterone. No difference in any of the receptor levels was found in samples obtained during the 3rd week of any of the 4-week treatment cycles. For 2-3 weeks of a treatment cycle, the receptor levels were similar to those seen in premenopausal, proliferative-phase endometrium, suggesting that postmenopausal endometrial cells are subjected to a very potent estrogenic stimulus for a considerable period. Norethisterone ingestion for 1 week decreased both the amount of nuclear ER and the percentage of total cellular ER that were in the nuclear fraction. Estradiol dehydrogenase was also induced by the progestin. The presence of this enzyme could result in lowered nuclear ER levels which were seen during the progestogenic phase of the treatment schedule. Nuclear ER was lower during Week 3 than Week 2 of estrogen treatment. In addition, PR was negatively correlated with nuclear ER in postmenopausal tissues obtained in Week 3 in contrast to positive correlations seen in premenopausal samples. Possibly a 3-week treatment with estrogen leads to a refractory condition. When receptor levels of normal, cystic hyperplastic, typical hyperplastic, and atypical hyperplastic tissues were compared, the endometria that had been returned to normal histology suggested that cells in atypical hyperplastic endometrial cells may be more estrogen sensitive than other types of endometrial tissues.^ieng

Changes in microfilament and focal adhesion distribution with loss of androgen responsiveness in cultured mammary tumor cells.

The actin-containing microfilaments, microtubules, and fibronectin expression of Shionogi 115 mouse mammary tumor cells were visualized by indirect immunofluorescence microscopy. Also studied was the focal adhesion distribution as revealed by interference reflection microscopy and the ability of the cells to grow in suspension culture. All these parameters were documented for androgen-responsive and -unresponsive cells grown in culture, as well as the transition of androgen-responsive to -unresponsive cells when deprived of androgen. The androgen-unresponsive cells had extensive and prominent microfilament bundles together with focal adhesions on the lower cell surface and also showed strict anchorage dependence for growth. In contrast, microfilament bundles and focal adhesions were absent from androgen-responsive cells, which furthermore had the ability to grow in suspension culture. Differences were also apparent in fibronectin expression, the androgen-unresponsive cells having more of this glycoprotein detectable on their surfaces than the androgen-responsive cells. The androgen-responsive and -unresponsive cells had similar microtubule arrays, however. During the transition from the androgen-responsive to the androgen-unresponsive phenotype, the androgen-responsive cells gradually took on the characteristics of androgen-unresponsive cells as judged by cellular morphology or the presence of focal adhesions and microfilament bundles. At intermediate stages in this process, characteristics of both cell types were visible in the cell populations. However, at the stage where all androgen-responsive characteristics were lost, the cells were no longer androgen sensitive. The loss of androgen responsiveness in Shionogi 115 mouse mammary tumor cells is correlated with changes at the cell membrane and the microfilament component of the cytoskeleton.

Monoclonal antibodies against a component related to soluble estrogen receptor.

Mice were immunized with 1 to 3 micrograms of cytoplasmic estrogen receptor fragment purified from human myometrium by affinity chromatography. Two RE-antibody-secreting clones were detected from one fusion that were capable of precipitating cytosol RE. Monoclonal antibody D5 (subclass IgG1) reacts with an antigen that is related to RE from immunoprecipitation studies but which can be separated from the hormone binding unit. In the presence of anti-mouse serum, D5 precipitates labeled human cytoplasmic RE complexes from breast tumor, fibroid, myometrial, and endometrial preparations but does not react with nuclear RE from human endometrium or cytoplasmic RE from other species tested. Conversely, antibody C3 (Class IgM) precipitates human cytoplasmic RE and nuclear RE complexes as well as labeled cytoplasmic RE from rat and calf uterus and chick oviduct. Neither antibody reacts with progesterone receptor or androgen receptor from human breast tumor, SHBG from human plasma, or rat alpha-fetoprotein. With D5, steroid labeling of cytoplasmic RE at 25 degrees increased the RE immune complex precipitated. D5 precipitates molybdate-stabilized RE from myometrial cytosol when labeled at 25 degrees but not at 4 degrees. C5 precipitates molybdate-stabilized RE whether cytosol was steroid-labeled at 4 degrees or 25 degrees. For D5, optimal precipitation of RE from human breast tumor was observed when cytosol was steroid-labeled at 25 degrees in buffers of pH range 5 to 6. Immunochemical studies indicate that D5 is associated with a Mr 29,000 component in RE-positive cytosols. Electrofocusing and sucrose density gradient analysis confirmed that D5 antigen is a non-hormone-binding component related to cytosolic RE from breast tumor and myometrium.

Immunoradiometric studies with monoclonal antibody against a component related to human estrogen receptor.

A human specific monoclonal antibody (D5) raised against a Mr 36,000 cytosolic estrogen receptor component (RE) partially purified from human myometrium was used to develop a simple, rapid, and sensitive solid-phase immunoradiometric assay (IRMA) for the reactive antigen in tissue cytosols from breast tumors, myometrium, endometrium, and endometrial carcinomas. The IRMA did not detect antigen in RE-negative cytosols from human breast tumors and endometrial carcinomas. RE-positive cytosols from chick oviduct and calf and rat uteri failed to produce an IRMA response. A pilot study indicated a significant correlation (P less than 0.001) between D5 IRMA value and RE sites in breast tumors assayed by [3H]estradiol binding sites. The presence of D5 antigen was dependent on the presence of cytosolic RE but not progesterone receptor. RE-positive patients age 50 years and over demonstrated significantly higher D5 assay values than did patients under 50 years. The data suggest that the D5 antigen is a component of the estrogen receptor or coordinately regulated with the receptor in human cells and that the assay method may have clinical use.

Histochemical studies with an estrogen receptor-related protein in human breast tumors.

The histochemical characteristics of a Mr 29,000 phosphoprotein related to estradiol receptor are described in a large series of human breast tumors. The antigen was detected with a monoclonal antibody (D5) raised against partially purified human myometrial estradiol receptor. An indirect immunoperoxidase method was used with methacarn-fixed, wax-embedded sections. Quantitation of staining and its reproducibility are described. Results with trucut biopsies agree with those obtained with larger tumor sections. Normal breast is infrequently positive. Histochemical staining is higher in invasive carcinoma than in normal breast with ductal carcinoma in situ adjacent to infiltrating tumors exhibiting intermediate values. Furthermore, most in situ carcinomas have a heterogeneous staining pattern. About 20% of invasive tumors also exhibit heterogeneity. No simple correlation is seen between staining and histological grade. There are more low-staining tumors in young (less than 50 yr old) patients than in older women. Staining correlates with levels of cytosol estradiol receptor but not cytosol progesterone receptor. However, cytosol estradiol receptor-negative, cytosol progesterone receptor-positive tumors tend to have positive Mr 29,000 phosphoprotein levels. Positive staining is associated with a higher response rate to hormone therapy (50%). None of the negative tumors responded to hormone treatment. With these patients, comparison of histochemical assay for Mr 29,000 phosphoprotein and [3H]estradiol binding assays indicated that the former was at least as good as the latter assay in predicting hormone response. About 20% of cytosol estradiol receptor-positive tumors have low Mr 29,000 phosphoprotein, and such tumors have poor response to hormone treatment.

Simple biochemical method to assess progestin effects on human endometrial DNA synthesis and its application to endometrial carcinoma.

Endometria of normal histology from postmenopausal women receiving either estrogen or estrogen plus a progestin have been analyzed for nuclear estradiol receptor, epithelial DNA synthesis, isocitric dehydrogenase, and estradiol dehydrogenase activities. Epithelial DNA synthesis correlated positively with nuclear estradiol receptor and negatively with both the dehydrogenases; this result was obtained regardless of whether the enzyme activity was related to the protein or DNA content of the samples. Thus, either of the dehydrogenases might provide an index of progestin effects on proliferative activity in endometrial carcinomata. Provera administered in vivo had no effect on either dehydrogenase activity in soluble estradiol receptor-poor carcinomata, whereas both dehydrogenase activities were high in some but not all soluble estradiol receptor-rich tumors. The enzyme activities in Provera-treated tumors have been compared with those in normal epithelium and endometrium from postmenopausal women taking estrogen plus progestin. The activities of both dehydrogenases were lower in soluble estradiol receptor-rich carcinomata than in either endometrium or epithelium from estrogen plus progestin-primed, normal postmenopausal women. This may indicate suboptimal progestin effects in the patients with carcinoma, and potential reasons for this are discussed.

Histochemical studies with a monoclonal antibody raised against a partially purified soluble estradiol receptor preparation from human myometrium.

A monoclonal antibody (D5) raised against affinity-purified cytosol estradiol receptor (REC) from human myometrium has been used to stain human tissues by means of an indirect immunoperoxidase method. Good staining was obtained with ethanol-, glutaraldehyde-, or Carnoy's-fixed material but not with formalin or Bouin's fixation. Cytoplasmic staining of human breast tumors exhibited a highly significant correlation (P less than 0.001) with REC assayed by conventional estradiol-binding assay provided that allowance was made for both staining intensity and cellularity of the tumor; no correlation existed with soluble progesterone receptor content. Both patient age and tumor differentiation influenced staining patterns in the same way as did REC content. Cultured REC-positive human breast tumor cell lines (MCF-7, ZR-75-1, and CA-2) showed positive staining as did cultured epithelium from human milk. Epithelia in normal breast and fibroadenoma exhibited variable staining that rarely reached the intensity seen in REC-positive tumor cells. The staining patterns of human normal endometrium, myometrium, fallopian tube, ectocervix, endocervix, and ovary and neoplastic endometrium and ovary are described. In every situation thus far examined only cytoplasmic staining has been observed.

Physicochemical properties of dipalmitoyl phosphatidylcholine after interaction with an apolipoprotein of pulmonary surfactant.

We studied the interaction between an apolipoprotein of pulmonary surfactant and the principal lipid found in this material, dipalmitoyl phosphatidylcholine. The apolipoprotein was extracted from canine surfactant and purified to greater than 90% homogeneity. The apolipoprotein was mixed for 16 h at room temperature with dipalmitoyl phosphatidylcholine dispersed in a buffer containing 0.1 M NaCl and 3mM CaCl2. Unbound lipid, unbound protein, and recombinants of lipid and protein were separated by density gradient centrifugation. 71% of the apolipoprotein was found associated with dipalmitoyl phosphatidylcholine. In comparable experiments using bovine plasma albumin about 13% of the albumin was recovered with the lipid. The physicochemical state of the lipid in the apolipoprotein-lipid complex was modified after binding of the protein. A distinct phase transition at 42 degrees C could no longer be detected, and the rate of adsorption to an air-liquid interface of the apolipoprotein-lipid complex was greater than that of the lipid alone. Surface tension vs. surface area isotherms of the dipalmitoyl phosphatidylcholine-apolipoprotein materials, however, were similar to those exhibited by pure dipalmitoyl phosphatidylcholine. The results suggest a physiological role for this apolipoprotein. It may bind to dipalmitoyl phosphatidylcholine under conditions expected in vivo, and may modify the physical properties of the aggregated dipalmitoyl phosphatidylcholine to form domains of lipid in a liquid-crystalline array. The complex dipalmitoyl phosphatidylcholine and apolipoprotein would have the physical properties necessary for its physiological function, allowing it to absorb to the alveolar interface and reduce its surface tension to less than 10 dynes/cm. Dipalmitoyl phosphatidylcholine, by itself, is in a gel-crystalline array below its phase transition temperature (42 degrees C) and would be incapable of effecting these actions.

Effects of inhibiting protein synthesis on the secretion of surfactant by type II cells in primary culture.

Pulmonary surfactant is isolated from the alveolar lumen as a complex of lipid and protein (King, R.J., Martin, H., Mitts, D. and Holmstrom, F.M. (1977) J. Appl. Physiol. 42, 483-491). We wished to determine whether the secretion of this complex was dependent upon cellular activities associated with the synthesis of protein, and whether the pre-formed lipids of surfactant would be released from the cell even though the synthesis of newly-formed protein was inhibited. Alveolar epithelial Type II cells were isolated from adult rat lung and grown to confluency in primary culture. The synthesis and secretion of the apolipoprotein of surfactant and its principal lipid, dipalmitoyl phosphatidyl-choline, were followed by isotopic precursor techniques. The synthesis of the apolipoprotein was reduced to 14% of control by 1 . 10(-4) M cycloheximide and to 2.5% of control by 4 . 10(-4) M cycloheximide. These concentrations of cycloheximide, however, had no effect on the rate of synthesis or release of DPPC. The secretion of the apolipoprotein which had been synthesized before the addition of 1 . 10(-4) M cycloheximide was not inhibited by this compound. Cells maintained at 5 degrees C neither synthesized nor released surfactant. We conclude, therefore, that the synthesis of cellular protein is not required for the secretion of surfactant, but that the continuous generation of metabolic energy may be essential. These results, together with those of previous kinetic studies (see above references), suggest that the lipid and protein constituents of surfactant may be contained within lamellar bodies prior to their release into the extracellular environment.

Aspects of secondary and quaternary structure of surfactant protein A from canine lung.

The results of a large number of studies indicate that pulmonary surfactant contains a unique protein whose principal isoform has a molecular weight of about 30,000, and whose presence in surfactant is associated with important metabolic and physicochemical properties. This protein, SP-A, as isolated from canine surfactant, contains a domain of 24 repeating triplets of Gly-X-Y, similar to that found in collagens. These studies were undertaken to determine whether SP-A forms a collagen-like triple helix when in solution, and to describe certain aspects of its size and shape. Our experiments were done on SP-A extracted by two different methods from canine surfactant, and on SP-A produced by molecular cloning. The results from all three preparations were similar. The circular dichroism of the complete protein was characterized by a relatively large negative ellipticity at 205 nm, with a negative shoulder ranging from 215 to 230 nm. There was no positive ellipticity, and the spectrum was not characteristic of collagen. Trypsin hydrolysis resulted in a fragment with peak negative ellipticity at about 200 nm, without the negative shoulder. Further hydrolysis of this fragment with pepsin resulted in a CD spectrum similar to that of collagen. The spectrum of the collagen-like fragment was reversibly sensitive to heating to 50 degrees C, and was irreversibly lost after treatment with bacterial collagenase. SP-A migrated on molecular sieving gels with an equivalent Stokes radius of 110 to 120 A, and had a sedimentation coefficient of 14 S. Using these data we calculate a molecular weight of about 700,000. The hydrodynamic characteristics can be approximated as a prolate ellipsoid of revolution having an axial ratio of about 20. We conclude that SP-A aggregates into a complex of 18 monomers, which may form six triple-helices. The shape of the complex is considerably more globular than collagen and is not consistent with end-to-end binding of the helices to form fibrous structures.