An analysis of the spectra of genetic activity produced by known or suspected human carcinogens

For 24 agents classified by the International Agency for Research on Cancer as known or suspected human carcinogens, we previously catalogued the qualitative genetic bioassay data available in the literature. In the present analysis, dose information, where available, was added to this data base: either the lowest effective dose (LED) or the highest ineffective dose (HID) was recorded for each agent and bioassay system. Bioassay systems were organized according to classes of genetic activity and subdivided by the phylogenetic level of the test organism. For each compound, the quantitative results in the test systems were represented by computer-generated bar graphs ('genetic activity spectra'). The x-axis unit values corresponded to the 100 different test systems, and the y-axis values were the logarithmically transformed LED or HID values. Statistical methods and pattern-recognition techniques were used to evaluate the genetic activity spectra. Spectra were compared among agents grouped according to target-organ specificity. In addition, the spectra of all possible pairs of compounds were compared to identify compounds displaying qualitatively or quantitatively similar genetic activity. Chemically similar compounds frequently produced similar spectra of genetic activity, and it was possible to identify the most appropriate test systems for some classes of compounds. As the data base for human carcinogens is enlarged, analysis of genetic activity spectra may contribute to our understanding of the structure-activity relationships and mechanisms of action of these agents.     

Activity profiles of developmental toxicity: design considerations and pilot implementation

The available literature was searched for quantitative test results from both in vitro and in vivo assays for developmental toxicity for five model compounds: cyclophosphamide, methotrexate, hydroxyurea, caffeine, and ethylenethiourea. These compounds were chosen on the basis of their extensive utilization in a variety of assay systems for developmental toxicity as evidenced by their representation in the ETIC database (each generally has 100-500 citations encompassing multiple test systems). Nine cellular-based assays, six assays using whole embryos in culture, as well as Segment II and abbreviated exposure tests for mammalian test species are included in the database. For each assay, the critical endpoints were identified, each of which was then provided a three-letter code, and the criteria for extraction of quantitative information were established. The extracted information was placed into a computerized reference file and subsequently plotted such that the qualitative (positive/negative) and quantitative (e.g., IC50, highest ineffective dose (HID), lowest effective dose (LED] results across all test systems could be displayed. The information contained in these profiles can be used to compare qualitative and quantitative results across multiple assay systems, to identify data gaps in the literature, to evaluate the concordance of the assays, to calculate relative potencies, and to examine structure-activity relationships.     

Antimutagenicity profiles for some model compounds

The concept of activity profile listings and plots, already applied successfully to the display of mutagenicity data, has been modified for application to antimutagenicity data. The activity profiles are bar graphs that have been organized in two general ways: for antimutagens that have been tested in combination with a given mutagen and for mutagens that have been tested in combination with a given antimutagen. Doses from both the mutagen and the antimutagen are displayed and plotted together with results on enhancement or inhibition of mutagenic activity. The short-term tests that have been used extensively to identify mutagens and potential carcinogens are increasingly being used to identify antimutagens and potential anticarcinogens. Three model mutagens, N-methyl-N'-nitro-N-nitrosoguanidine, aflatoxin B1 and benzo[a]pyrene, and 4 model antimutagens, butylated hydroxyanisole, butylated hydroxytoluene, glutathione and disulfiram, were selected from the data surveyed in the published literature. It is not clear at the present time whether the inhibition of carcinogen-induced mutation is a good indicator of anticarcinogenic properties, and further research is needed. Nevertheless, the activity profiles are useful for the assessment of the available antimutagenesis data by providing rapid visualization of considerable dose information and experimental results.     

Discovery of potent, selective and orally bioavailable triaryl-sulfonamide based PTP1B inhibitors

A novel series of pTyr mimetics containing triaryl-sulfonamide derivatives (5a-r) are reported as potent and selective PTP1B inhibitors. Some of the test compounds (5o and 5p) showed excellent selectivity towards PTP1B over various PTPs, including TCPTP (in vitro). The lead compound 5o showed potent antidiabetic activity (in vivo), along with improved pharmacokinetic profile. These preliminary results confirm discovery of highly potent and selective PTP1B inhibitors for the treatment of T2DM.     

Analgesic and anti-inflammatory activity of the proanthocyanidin shellegueain A from Polypodium feei METT.

Analgesic and antiinflammatory activity of proanthocyanidin isolated from Polypodium feei roots has been tested using acetic acid-induced writhing and carrageenan-induced paw edema methods, respectively. The compound at doses of 50 and 100 mg/kg significantly decreased writhing responses of mice induced by 0.7 % acetic acid along the 60 min test in a dose-dependent manner. The compound at a dose of 100 mg/kg gave the percent protection of 76.23 higher than that of acetylsalicylic acid (59.84 %) at a dose of 50 mg/kg. In the antiinflammatory test, this compound caused significant inhibition of the rats' plantar edema induced by 1 % of carrageenan, but this activity was observed only at a higher dose (200 mg/kg). These findings suggest that proanthocyanidin of P. feei roots might have analgesic and antiinflammatory activity, and its mechanism of action might be due to the inhibition of prostaglandin biosynthesis, because the proanthocyanidin fraction had an inhibitory effect on cyclooxygenase, but not on 5-lypoxygenase enzymes.     

Coagulanolide, a withanolide from Withania coagulans fruits and antihyperglycemic activity

One new withanolide, (17S,20S,22R)-14alpha,15alpha,17beta,20beta-tetrahydroxy-1-oxowitha-2,5,24-trienolide) named coagulanolide (4) along with four known withanolides 1-3 and 5 have been isolated from Withania coagulans fruits and their structures were elucidated by spectroscopic techniques. The compounds 1-5 showed significant inhibition on postprandial rise in hyperglycemia post-sucrose load in normoglycemic rats as well as streptozotocin-induced diabetic rats. The compound 5 also showed significant fall on fasting blood glucose profile and improved the glucose tolerance of db/db mice. Further compound 5 showed antidyslipidemic activity in db/db mice. The median effective dose of the compound 5 was determined to be around 25 mg/kg in streptozotocin-induced diabetic rats, which is comparable to the standard antidiabetic drug metformin. Our results provide further support to explain the traditional use of W. coagulans as antihyperglycemic cum antidyslipidemic agent by the traditional medical practitioners.     

Paradoxical effects of learning the Morris water maze on adult hippocampal neurogenesis in mice may be explained by a combination of stress and physical activity

Studies in rats that assessed the relation of hippocampus-dependent learning and adult hippocampal neurogenesis suggested a direct regulatory effect of learning on neurogenesis, whereas a similar study in mice had not found such causal link. We here report a substantial decrease of BrdU-positive cells and other measures of adult hippocampal neurogenesis in mice trained in the hidden (HID) or cued version (VIS) of the Morris water maze as compared to untrained animals (CTR). Particularly, cells on advanced stages of neuronal development contributed to this decrease, whereas earlier progenitors (type 2 cells) were not diminished in HID, but were diminished in VIS as compared to CTR. The differential regulation of type 2 cells in HID and VIS may have been caused by a different degree of physical activity, given that a time-yoked control group did not differ from HID, and type 2 cells reportedly constitute the proliferative dentate gyrus population that primarily responds to physical activity. The decrease of hippocampal neurogenesis by water maze training was reversible by pre-exposing animals to the water maze prior to training, suggesting that stress associated with training may have caused the acute downregulation of adult neurogenesis. We propose that in mice the Morris water maze does not provide a pure enough learning stimulus to study the presumed effects of 'learning' on adult neurogenesis. In addition, however, our data show that physical activity that is intricately linked to many cognitive tasks in rodents might play an important role in explaining effects of learning on cellular hippocampal plasticity.     

Molecular and biochemical characterization of 2-hydroxyisoflavanone dehydratase. Involvement of carboxylesterase-like proteins in leguminous isoflavone biosynthesis

Isoflavonoids are ecophysiologically active secondary metabolites of the Leguminosae and known for health-promoting phytoestrogenic functions. Isoflavones are synthesized by 1,2-elimination of water from 2-hydroxyisoflavanones, the first intermediate with the isoflavonoid skeleton, but details of this dehydration have been unclear. We screened the extracts of repeatedly fractionated Escherichia coli expressing a Glycyrrhiza echinata cDNA library for the activity to convert a radiolabeled precursor into formononetin (7-hydroxy-4'-methoxyisoflavone), and a clone of 2-hydroxyisoflavanone dehydratase (HID) was isolated. Another HID cDNA was cloned from soybean (Glycine max), based on the sequence information in its expressed sequence tag library. Kinetic studies revealed that G. echinata HID is specific to 2,7-dihydroxy-4'-methoxyisoflavanone, while soybean HID has broader specificity to both 4'-hydroxylated and 4'-methoxylated 2-hydroxyisoflavanones, reflecting the structures of isoflavones contained in each plant species. Strikingly, HID proteins were members of a large carboxylesterase family, of which plant proteins form a monophyletic group and some are assigned defensive functions with no intrinsic catalytic activities identified. Site-directed mutagenesis with soybean HID protein suggested that the characteristic oxyanion hole and catalytic triad are essential for the dehydratase as well as the faint esterase activities. The findings, to our knowledge, represent a new example of recruitment of enzymes of primary metabolism during the molecular evolution of plant secondary metabolism.     

The genetic toxicology of putative nongenotoxic carcinogens

This report examines a group of putative nongenotoxic carcinogens that have been cited in the published literature. Using short-term test data from the U.S. Environmental Protection Agency/International Agency for Research on Cancer genetic activity profile (EPA/IARC GAP) database we have classified these agents on the basis of their mutagenicity emphasizing three genetic endpoints: gene mutation, chromosomal aberration and aneuploidy. On the basis of results of short-term tests for these effects, we have defined criteria for evidence of mutagenicity (and nonmutagenicity) and have applied these criteria in classifying the group of putative nongenotoxic carcinogens. The results from this evaluation based on the EPA/IARC GAP database are presented along with a summary of the short-term test data for each chemical and the relevant carcinogenicity results from the NTP, Gene-Tox and IARC databases. The data clearly demonstrate that many of the putative nongenotoxic carcinogens that have been adequately tested in short-term bioassays induce gene or chromosomal mutations or aneuploidy.     

The mutagenicity and DNA-damaging activity of cyclic aliphatic sulfuric acid esters.

The cyclic aliphatic sulfuric acid esters 1,2-ethylene sulfate (ESF), 1,3-propylene sulfate (PSF) and 1,3-butylene sulfate (BSF) have been tested for their mutagenic and DNA-damaging activity. Mutagenicity of the compounds was established with his-auxotrophic indicator strains of Salmonella typhimurium using the in vitro plate test and the host-mediated assay technique with mice as host animals. The DNA-damaging activity was tested in a repair test with Proteus mirabilis mutants defective in DNA repair.In the repair test with a set of P. mirabilis strains (PG713 hcr^?rec^?: PG273 hcr⁺rec⁺) PSF and BSF showed a preferential growth inhibition of the repair-defective strain suggesting DNA-damaging activity of these chemicals. No such activity was found for ESF using the same concentrations of 5 and 15 μmol/plate.All cyclic sulfates revert the tester strain TA1535 of S. typhimurium in vitro indicating their ability to induce base substitutions. Compared with the reference compounds dimethyl sulfate (DMS), diethyl sulfate (DES), 1,3-propane sulfone (PPS) and 1,4-butane sulfone (BTS) the mutagenic activity in the plate test can be described as follows: PPS > PSF > BSF > BTS > ESF > DES > DMS.Dose-response studies in the host-mediated assay with tester strain TA1950 of S. typhimurium as genetic indicator system revealed a linear dosedependency of mutagenic activity. For PPS and PSF the lowest effective dose (LED) has been established as 10 μmol/kg. The LED for BSF and BTS was 50 μmol/kg, DMS and DES were mutagenic in doses of 2500 μmol/kg, while ESF was only weakly mutagenic with a LED of 5000 μmol/kg.The dose-response studies in the host-mediated assay and the results obtained in the in vitro spot test demonstrate similarities in the mutagenic action of the cyclic sulfates PSF and BSF and the respective sulfones, while the stronger alkylating compound ESF was a weak mutagen both in vitro and in vivo.     

Assessment of chemically-induced changes in the neuromuscular function of rats using a new recording grip meter

The effects of several psychoactive drugs on the grip strength of rats were evaluated by a new recording grip meter technique that provides graded or continuous data. The same subjects were also tested by an inclined screen procedure, which yields quantal or all-or-none data. The lowest doses (lowest effective dose; LED) required to produce a significant decrease in grip meter scores by chlorpromazine (CPZ), chlordiazepoxide (CDZ), and phenobarbital (PHEN) were 5, 9, and 20 mg/kg respectively. $\text{d}$-Amphetamine (0.5– 8 mg/kg) resulted in a significant, dose-related increase in grip meter scores. The LEDs for CPZ and PHEN using the inclined screen test were 10 and 40 mg/kg, respectively. These values are 2 times the corresponding figures obtained in the grip meter test. The LED for chlordiazepoxide was 9 mg/kg in the inclined screen, which is the same as that in the grip meter test, while $\text{d}$-amphetamine had no effect on neuromuscular function measured by the inclined screen.The results of these experiments indicate that the grip method is a useful technique capable of measuring both increases and decreases in the grip strength of rats. Relative to the inclined screen procedure, which measures a combination of factors, the grip meter technique appears to be a sensitive procedure capable of measuring forelimb grip strength selectively.     

Histochemical reactions of gastrointestinal mucosubstances with orcein, high iron diamine and Alcian blue after prior oxidation of tissue sections.

The histochemical orcein reaction (orc) for mucosubstances in tissue samples from the human gastrointestinal tract was compared with PAS, high iron diamine (HID) and Alcian blue reactions at pH 1.0 or 2.5 (AB 1 and AB 2.5). Orc, HID and AB 1 reactions were performed also with prior oxidation of the tissue sections with potassium permanganate or performic acid (ox-orc, ox-HID and ox-AB reactions, respectively). Orc reaction stained mucosubstances similarly to HID and AB 1; only the brush border and goblet cells in the colon were stained. The reactions of the mucosubstances obtained with ox-orc differed from those with PAS, HID, AB 1 or AB 2.5 but were similar to those with ox-HID or ox-AB; the mucosubstances in the brush border and the goblet cells in the colon and small bowel and in the foveolar epithelium of the stomach were strongly stained. Pyloric and cardiac glands were stained faintly with ox-orc but not with ox-HID or ox-AB. Brunner's glands were negative with ox-orc, ox-HID and ox-AB reactions. It was assumed that the orc reaction stains, like HID or AB 1, sulphate groups in epithelial mucosubstances, and that sulphonic acid residues, resulting from oxidation of disulphide groups in the protein core of mucus glycoproteins, are responsible for the ox-orc as well as for the ox-HID and ox-AB reactions.     

Inhibitor of Apoptosis Proteins Physically Interact with and Block Apoptosis Induced by Drosophila Proteins HID and GRIM

Reaper (RPR), HID, and GRIM activate apoptosis in cells programmed to die during Drosophila development. We have previously shown that transient overexpression of RPR in the lepidopteran SF-21 cell line induces apoptosis and that members of the inhibitor of apoptosis (IAP) family of antiapoptotic proteins can inhibit RPR-induced apoptosis and physically interact with RPR through their BIR motifs (D. Vucic, W. J. Kaiser, A. J. Harvey, and L. K. Miller, Proc. Natl. Acad. Sci. USA 94:10183–10188, 1997). In this study, we found that transient overexpression of HID and GRIM also induced apoptosis in the SF-21 cell line. Baculovirus and Drosophila IAPs blocked HID- and GRIM-induced apoptosis and also physically interacted with them through the BIR motifs of the IAPs. The region of sequence similarity shared by RPR, HID, and GRIM, the N-terminal 14 amino acids of each protein, was required for the induction of apoptosis by HID and its binding to IAPs. When stably overexpressed by fusion to an unrelated, nonapoptotic polypeptide, the N-terminal 37 amino acids of HID and GRIM were sufficient to induce apoptosis and confer IAP binding activity. However, GRIM was more complex than HID since the C-terminal 124 amino acids of GRIM retained apoptosis-inducing and IAP binding activity, suggesting the presence of two independent apoptotic motifs within GRIM. Coexpression of IAPs with HID stabilized HID levels and resulted in the accumulation of HID in punctate perinuclear locations which coincided with IAP localization. The physical interaction of IAPs with RPR, HID, and GRIM provides a common molecular mechanism for IAP inhibition of these Drosophila proapoptotic proteins.     

Induction of apoptosis by Drosophila reaper, hid and grim through inhibition of IAP function

Induction of apoptosis in Drosophila requires the activity of three closely linked genes, reaper, hid and grim. Here we show that the proteins encoded by reaper, hid and grim activate cell death by inhibiting the anti-apoptotic activity of the Drosophila IAP1 (diap1) protein. In a genetic modifier screen, both loss-of-function and gain-of-function alleles in the endogenous diap1 gene were obtained, and the mutant proteins were functionally and biochemically characterized. Gain-of-function mutations in diap1 strongly suppressed reaper-, hid- and grim-induced apoptosis. Sequence analysis of these alleles revealed that they were caused by single amino acid changes in the baculovirus IAP repeat domains of diap1, a domain implicated in binding REAPER, HID and GRIM. Significantly, the corresponding mutant DIAP1 proteins displayed greatly reduced binding of REAPER, HID and GRIM, indicating that REAPER, HID and GRIM kill by forming a complex with DIAP1. These data provide strong in vivo evidence for a previously published model of cell death regulation in Drosophila.     

Reevaluation of the 9 compounds reported conclusive positive in yeast Saccharomyces cerevisiae aneuploidy test systems by the Gene-Tox Program using strain D61.M of Saccharomyces cerevisiae

The state of aneuploidy test methodology was appraised by the U.S. Environmental Protection Agency in 1986 in analyzing published data. In Saccharomyces cerevisiae 9 chemicals were reported to be conclusive positive for aneuploidy induction in either mitotic or meiotic cells. We reevaluated these 9 chemicals using Saccharomyces cerevisiae D61.M, a strain that detects mitotic chromosome malsegregation. Acetone (lowest effective dose (LED): 40 microliters/ml), bavistan (LED: 5 micrograms/ml), benomyl (LED: 30 micrograms/ml) and oncodazole (LED: 4 micrograms/ml) induced a dose-dependent increase in the frequencies of chromosomal malsegregation. Ethyl methanesulfonate (EMS; highest tested dose (HTD): 1000 micrograms/ml) and methyl methanesulfonate (MMS; HTD: 100 micrograms/ml) did not induce malsegregation but were both potent inducers of other genetic events, detected by an increase in the frequencies of cyhR cells. No increases in both endpoints (malsegregation and other genetic events) were observed after treatment of S. cerevisiae D61.M with cyclophosphamide (CP; HTD: 16 mg/ml) in the absence of S9, p-D,L-fluorophenylalanine (p-FPA; HTD: 250 micrograms/ml) and phorbol-12-myristate-13-acetate (TPA; HTD: 50 micrograms/ml). A marginal increase in the frequency of mitotic chromosome malsegregation was obtained with cyclophosphamide in the presence of S9. Thus our test results largely disagree with those previously published by various authors and taken as conclusive by EPA. We interpret the discrepancies to be due to lack of properly controlled testing (e.g., no check for multiple mutational events). Only with a careful test design it is possible to discriminate between chemicals inducing only chromosome loss and no other genetic effects (e.g., acetone, oncodazole), chemicals inducing a variety of genetic damage but no chromosome loss (e.g., EMS, MMS) and chemicals inducing neither chromosome loss nor other genetic events in yeast (e.g., TPA, p-FPA).     

Design and synthesis of novel imidazoline derivatives with potent antihyperglycemic activity in a rat model of type 2 diabetes

Imidazoline derivatives have been reported to show antihyperglycemic activity in vivo. In the present study, we first showed that there was no correlation between the in vivo antidiabetic activity and the in vitro affinities for the I1/I2 binding sites for several substituted aryl imidazolines. Among these compounds, 2-(alpha-cyclohexyl-benzyl)-4,5-dihydro-1H-imidazole 2 exhibited potent antihyperglycemic properties. It was then chosen as lead compound. Thirty-six new derivatives were synthesized by replacing the cyclohexyl/benzyl group by various cyclic systems or the imidazoline ring by isosteric heterocycles. These compounds were evaluated in vivo for their antihyperglycemic activity using an oral glucose tolerance test (OGTT) in a rat model of type-2 diabetes obtained by giving a single intravenous (iv) injection of a low dose of streptozotocin to rats (STZ rats) and in normal rats. Nine compounds with an imidazoline moiety, possibly substituted by a methyl group, had a potent effect on the glucose tolerance in normal or STZ-diabetic rats, after an oral (po) administration of the test compound at a dose of 30 or 10 mg kg(-1), without any hypoglycemia. Replacement of the imidazoline ring by isosteric heterocycles resulted in a total loss of activity.     

Automated data processing system at the National Center for Blood Transfusion

1) Data processing of blood unit test result on Groupamatic with manual labelling listings:--according to unit numbers;--according to blood results. 2) Blood donor file (updating):--114 473 donors;--150 characters/standard donors;--250 characters/precious donors. 3) Automatic print-out of:--call-ups;--donor data cards;--national blood donor cards:--diplomas;--red cards of badges;--particular listings of blood donors for medical checking, calling up, selecting units with special characteristics;--general listings. 4) Automatic labelling of blood units, by reading the unit identification number, and by printing out the corresponding labels.     

LED Light-Induced ROS Differentially Regulates Focal Adhesion Kinase Activity in HaCaT Cell Viability

In this study, changes in cell signaling mechanisms in skin cells induced by various wavelengths and intensities of light-emitting diodes (LED) were investigated, focusing on the activity of focal adhesion kinase (FAK) in particular. We examined the effect of LED irradiation on cell survival, the generation of intracellular reactive oxygen species (ROS), and the activity of various cell-signaling proteins. Red LED light increased cell viability at all intensities, whereas strong green and blue LED light reduced cell viability, and this effect was reversed by NAC or DPI treatment. Red LED light caused an increase in ROS formation according to the increase in the intensity of the LED light, and green and blue LED lights led to sharp increases in ROS formation. In the initial reaction to LEDs, red LED light only increased the phosphorylation of FAK and extracellular-signal regulated protein kinase (ERK), whereas green and blue LED lights increased the phosphorylation of inhibitory-κB Kinase α (IKKα), c-jun N-terminal kinase (JNK), and p38. The phosphorylation of these intracellular proteins was reduced via FAK inhibitor, NAC, and DPI treatments. Even after 24 h of LED irradiation, the activity of FAK and ERK appeared in cells treated with red LED light but did not appear in cells treated with green and blue LED lights. Furthermore, the activity of caspase-3 was confirmed along with cell detachment. Therefore, our results suggest that red LED light induced mitogenic effects via low levels of ROS–FAK–ERK, while green and blue LED lights induced cytotoxic effects via cellular stress and apoptosis signaling resulting from high levels of ROS.     

尿激酶抗人交联纤维蛋白-D-二聚体单抗化学偶合体的制备

利用双功能试剂Ｎ－琥珀酰亚胺－３（２－二硫吡啶）丙酸酯（ＳＰＤＰ）作交联剂,合成了尿激酶（ＵＫ）－抗人交联纤维蛋白降解物Ｄ－二聚体单抗（ＭＡ－ＨＩＤ１）化学偶合体（ＵＫＭＡ－ＨＩＤ１）,并用苯甲脒－Ｓｅｐｈａｒｏｓｅ６Ｂ及人交联纤维蛋白降解物Ｄ－二聚体－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和柱纯化,获得偶合体产物．ＳＤＳ－ＰＡＧＥ呈现一条带,其分子量约为２０００００．纤维蛋白平板法测活结果显示,偶合体中酶比活为５３０００ＩＵ／ｍｇ尿激酶蛋白,与偶联前的５４３００ＩＵ／ｍｇ蛋白相仿．ＥＬＩＳＡ测试显示,偶合体对人交联纤维蛋白降解物Ｄ－二聚体有免疫反应性,并且与偶联前的抗Ｄ－二聚体单抗对此抗原的反应性相当     

Cytochemical visualization of anions in collagenous and elastic fiber-associated connective tissue matrix in neonatal and adult rat lungs using iron-containing stains

Summary The cytochemical reactivity of pulmonary connective tissue matrix components in neonatal and adult rat was evaluated using high iron diamine (HID) to detect sulfate ester end groups and dialyzed iron (DI) to detect sulfated and carboxylated end groups of complex carbohydrates, including glycoproteins and glycosaminoglycans at the ultrastructural level. The HID reaction product, in the form of discrete 5–12 nm silver particles following appropriate intensification with thiocarbohydrazide-silver proteinate, was found associated with cell surfaces, the elastin component of elastic fibers, and at regular intervals along the length of collagen fibers in large airways and deep lung interstitium. Staining was similar in adult and neonatal rats, except in areas where connective tissues were presumably still rapidly developing in the neonatal animals. Here large gaps or spaces cointaining reactive filamentous structures were observed between collagen and elastic fibers. The distribution of DI-reactive sites was similar to that seen with HID with the exception of elastic fibers in which only the microfibrillar portion stained. The collagen-associated reaction was not regularly disposed like that stained with HID, but rather it formed a tight continuous density around the fiber. These results indicated the presence and location of glycoproteins and glycosaminoglycans in connective tissue ground substance regions prior to the full development of elastic and collagenous elements in neonatal pulmonary airways and parenchyma. They also demonstrate cytochemically the presence of a sulfate ester-containing complex sugar found associated with the elastin component of elastic fibers in the lung.Supported by Public Health Servece Grant HL 24748