Microbiome-induced Increases and Decreases in Bone Tissue Strength can be Initiated After Skeletal Maturity.

Recent studies in mice have indicated that the gut microbiome can regulate bone tissue strength. However, prior work involved modifications to the gut microbiome in growing animals and it is unclear if the same changes in the microbiome, applied later in life, would change matrix strength. Here we changed the composition of the gut microbiome before and/or after skeletal maturity (16 weeks of age) using oral antibiotics (ampicillin + neomycin). Male and female mice (n=143 total, n=12-17/group/sex) were allocated into five study groups:1) Unaltered, 2) Continuous (dosing 4-24 weeks of age), 3) Delayed (dosing only 16-24 weeks of age), 4) Initial (dosing 4-16 weeks of age, suspended at 16 weeks), and 5) Reconstituted (dosing from 4-16 weeks following by fecal microbiota transplant from Unaltered donors). Animals were euthanized at 24 weeks of age. In males, bone matrix strength in the femur was 25-35% less than expected from geometry in mice from the Continuous (p= 0.001), Delayed (p= 0.005), and Initial (p=0.040) groups as compared to Unaltered. Reconstitution of the gut microbiota, however, led to a bone matrix strength similar to Unaltered animals (p=0.929). In females, microbiome-induced changes in bone matrix strength followed the same trend as males but were not significantly different, demonstrating sex-related differences in the response of bone matrix to the gut microbiota. Minor differences in chemical composition of bone matrix were observed (Raman spectroscopy). Our findings indicate that microbiome-induced impairment of bone matrix in males can be initiated and/or reversed after skeletal maturity. The portion of the femoral cortical bone formed after skeletal maturity (16 weeks) is small; however, this suggests that microbiome-induced changes in bone matrix occur without osteoblast/osteoclast turnover using an, as of yet unidentified mechanism. These findings add to evidence that the mechanical properties of bone matrix can be altered in the adult skeleton.

Novel esters and amides of nonsteroidal antiinflammatory carboxylic acids as antioxidants and antiproliferative agents.

A series of phenolic antioxidant ester and amide derivatives of the nonsteroidal antiinflammatory drug naproxen was designed to have both antiinflammatory and cytoprotective activity. Compounds were evaluated in vitro both for antioxidant activity, as assessed indirectly by thiobarbituric acid reactive substance (TBARS) formation in a membrane lipid peroxidation assay, and for antiproliferative activity, as indexed by the inhibition of DNA synthesis in cultured human vascular endothelial cells. Compounds of this series exhibited potent antioxidant activity, with IC50 values (1.6-11.63 microM) 2-6-fold lower than that of Trolox (6-hydroxy-2,5, 7,8-tetramethylchroman-2-carboxylic acid) and 400-1300-fold lower than that of vitamin E. Structural modifications of the ester or amide substructure (5a and 6a) did not affect antioxidant activity, but methylation of the 6-hydroxy substituent resulted in compound 6f which was devoid of antioxidant activity. Although indistinguishable in antioxidant activity, the amide derivatives tended to be more potent as antiproliferative agents than the corresponding esters. The IC50's for the amide derivatives (3, 5a-e, 8) ranged from 2 to 7 microM, while the IC50's for the structurally related esters (1, 2a-c, 6a-e) ranged from 9 to 22 microM. Moreover, studies with compound 6a indicate that the observed inhibition of DNA synthesis is reversible, suggesting that the antiproliferative activity is due to a cytostatic rather than cytotoxic activity of the compounds. Thus, the antioxidant-naproxen derivatives represent a novel series of agents that both protect against free-radical damage and possess cytostatic activity in vascular endothelial cells. Studies are in progress to assess the utility of these compounds as potential components of an ocular irrigating solution.

Elevated cutaneous leukocyte concentration in a rodent model of acute venous hypertension.

BACKGROUND: The pathophysiologic mechanism of the skin pathology in chronic venous insufficiency is venous hypertension (VHTN). Microvascular dysfunction involving leukocytes has recently been proposed as the primary mediator of tissue damage from VHTN. We developed a rodent model allowing the investigation of the effects of acute VHTN on tissue leukocyte concentration. MATERIALS AND METHODS: Under general anesthesia, adult male rats underwent transperitoneal isolation of the inferior vena cava and the common iliac veins and arteries. Bilateral thigh incisions allowed isolation of the common femoral veins and superficial epigastric veins (SEV: distal branch of the femoral vein in the thigh). Pressure in the SEV and flow in the iliac artery were measured before (T-Pre), immediately after (T-0), and for 135 min (T-1) after ligation of the cava, iliac, and femoral veins. Sham rats were identical except no venous ligation was performed. After the T-1 pressures were obtained, the distal hindlimb and forelimb skin was harvested and processed to measure myeloperoxidase (MPO) activity, an index of the number of tissue leukocytes. To evaluate the effect of arterial flow reduction known to occur with acute venous ligation, the above measurements were made in an Aortic group of rats in which the aorta was manually stenosed. RESULTS: This venous ligation technique resulted in a significant (P < 0.05) and sustained rise in venous pressure (T-Pre, 9.91 +/- 0.94 and T-1, 26.22 +/- 2.15). Hypertensive rats had significantly elevated hindlimb MPO activity (4.77 +/- 0.36) vs forelimb (0.60 +/- 0.39), Sham (hindlimb, 0.77 +/- 0.41; forelimb, 0.10 +/- 0.05), and Aortic (hindlimb, 0.96 +/- 0.38; forelimb, 0.58 +/- 0.11) controls. CONCLUSIONS: Acute VHTN was successfully created by venous ligation in this newly developed rat model. VHTN, but not arterial flow reduction, was associated with significantly elevated hindlimb skin MPO activity, suggesting that leukocytes may indeed be mediators of skin pathology in VHTN. This model will allow further investigation into the mechanisms of microvascular dysfunction in VHTN.

Nitric oxide maintains dilation of immature and mature collaterals in rat hindlimb.

The role of nitric oxide (NO) in maintaining collateral dilation was investigated in rats with acute and chronic superficial femoral artery occlusion. Collateral function was evaluated from arterial pressure measurements proximal (mean arterial pressure, MAP) and distal (PD) to the occlusion under resting and hyperemic conditions. For resting and hyperemic conditions, respectively, PD increased with the duration of occlusion from 34 +/- 2.1 and 14 +/- 0.8 mm Hg acutely postocclusion to 37 +/- 2.8 and 19 +/- 3.6, 42 +/- 4.9 and 25 +/- 1.5, and 49 +/- 5.6 and 25 +/- 2.1 mm Hg at 1, 4, and 12 weeks. After NO synthase inhibition with N omega-nitro-L-arginine methyl ester (L-NAME), PD in control limbs increased but in occluded limbs decreased by 20 +/- 2.0 mm Hg (acute) and 18 +/- 2.1, 15 +/- 4.4, and 20 +/- 8.1 mm Hg at 1, 4, and 12 weeks postligation, respectively. The percent of MAP (%MAP) dissipated by large arteries and collateral vessels decreased during collateral development (from 71 +/- 1.7% acutely to 57 +/- 2.2% at 12 weeks postligation) and was increased approximately 20% by L-NAME at each time point. The rise in PD and decrease in %MAP dissipated by collaterals following chronic occlusion indicates that significant collateral development occurred. The fall in PD and increase in %MAP dissipation after L-NAME administration suggest that NO-mediated vasodilatory tone is maintained throughout the period of collateral development.

Early collateral and microvascular adaptations to intestinal artery occlusion in rat.

The technique to repeatedly observe exactly the same vessels in the rat intestine was used to investigate vascular compensation during the 1st wk after abrupt arterial ligation. A collateral-dependent tissue region was created by ligation of three to four sequential intestinal arteries. At the center of the collateral-dependent region, arterial pressure decreased from 96 +/- 3.7 to 29 +/- 2.5 mmHg, and intestinal blood flow fell approximately 80% during maximal dilation initially postligation. One week later, pressure and blood flow at the center had increased 31 and 250%, respectively. Relative to preligation values, the only compensatory adaptation was an enlargement (31 +/- 11%) of the collateral arteries located between normal tissue and the center; no increase was observed in the diameter or numbers of arterioles or collateral arteries at the center. Wall shear rate was increased 173 +/- 35% initially postligation at the site where luminal enlargement occurred. The selective enlargement of collateral arteries away from the center region is consistent with the hypothesis that collateral enlargement is induced by chronic increases in wall shear rate and can occur independently of tissue ischemia.

Inhibition of NO synthase prevents acute collateral artery dilation in the rat hindlimb.

The role of endothelium-derived nitric oxide (EDNO) in the initial opening and sustained dilation of collateral vessels in the peripheral circulation was investigated. Abrupt arterial occlusion was produced by clamping the rat superficial femoral artery (SFA). Arterial pressures were measured proximal (MAP) and distal (PD) to the occlusion site. In one group (INITIAL DILATION), the clamp was removed after PD reached a plateau, and then the nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME), was administered and the occlusion repeated in the same limb to evaluate the role of nitric oxide in the initial opening of collateral vessels. In another group (SUSTAINED DILATION), L-NAME was administered after acute compensation to SFA occlusion had occurred to determine if inhibition of nitric oxide synthase would reverse any acute compensation. Collateral dilation was evaluated by recovery in PD relative to MAP (PD-%MAP) and by the percent of total resistance in the collateral-dependent circuit due primarily to collaterals (%Rc). The acute compensation indicated by an increase in PD-%MAP (14 +/- 1.9% to 27 +/- 2.6%) and decrease in %Rc (86 +/- 1.9 to 73 +/- 2.6%) in the INITIAL DILATION group was prevented by L-NAME (P < 0.0005). L-NAME also reversed the compensation in the SUSTAINED DILATION group; PD-%MAP decreased from 28 +/- 2.3% to 11 +/- 1.9% and %Rc increased from 72 +/- 2.3% to 93 +/- 1.1% after administration of L-NAME. Although collateral flow was not measured, the results are consistent with the hypothesis that acute collateral dilation is mediated by increases in flow or shear stress which stimulate the release of EDNO.

Early adaptations in collateral and microvascular resistances after ligation of the rat femoral artery.

Collateral and microvascular (including feed artery) resistances in the rat hindlimb were determined immediately or 1 wk after ligation of the femoral artery. Collateral-to-microvascular resistance ratios were determined from in vivo pressure measurements proximal and distal to the ligation. Microvascular resistance was 32 +/- 2.5 and 41 +/- 1.5% of the total collateral-dependent vasculature in acutely and chronically ligated limbs, respectively, and decreased 20% in both groups during reactive hyperemia. Minimum resistances of collateral vessels and the microcirculation arising from arterial branches proximal and distal to the ligation were determined by using a modification of the standard hindquarter perfusion technique for determining maximum vascular conductance. One week postligation, minimum total hindquarter resistance was decreased by a reduction in the resistance of the collaterals (approximately 50%) and microcirculation (approximately 33%) proximal to the ligation. The results suggest that the microvasculature distal to the occlusion is able to increase flow by dilation both initially and at 1 wk postligation but that collateral adaptations are primarily responsible for decreases in the minimum total resistance of the collateral-dependent region.

Exercise training effects on collateral and microvascular resistances in rat model of arterial insufficiency.

Experiments were performed to determine if exercise training reduces collateral or microvascular resistances in the hindlimb of rats with arterial insufficiency. After right femoral arterial ligation (age 10 wk), rats were divided into sedentary (Sed) and treadmill-trained (Tr) groups (7-9 wk, final intensity: 27 m/min, 6 degree grade, 60 min/day). Minimal resistances (mmHg.ml-1.min.100 g) of the total limb (RT), collateral vessels (RC), and the microcirculations distal (Rfmc) and proximal (Rimc) to the ligation site were determined during pump perfusion of the hindlimbs. RT was lower in nonligated (open) and acutely ligated limbs of Tr than Sed rats (open: 0.69 +/- 0.011 vs. 0.93 +/- 0.071; acute: 0.92 +/- 0.028 vs. 1.18 +/- 0.070 mmHg.ml-1.min.100 g) but not in chronically ligated limbs (chronic: 0.88 +/- 0.072 vs. 1.00 +/- 0.046 mmHg.ml-1.min.100 g). RC was similar between the chronically ligated limbs of Sed and Tr rats (1.69 +/- 0.165 vs. 1.97 +/- 0.227 mmHg.ml-1.min.100 g) and was approximately 70% lower than in acutely ligated limbs of both groups. Rfmc and Rimc were not affected by arterial ligation, but Rimc was significantly lower in Tr than in Sed rats (acute: 1.05 +/- 0.026 vs. 1.54 +/- 0.163; chronic: 1.24 +/- 0.071 vs. 1.81 +/- 0.202 mmHg.ml-1.min.100 g). These results indicate that the primary site of vascular adaptations to chronic arterial ligation is in the collateral vessels. Exercise training does not significantly alter the collateralization process but may provide protection against acute arterial occlusion by stimulating microvascular growth.

Acute compensation to abrupt occlusion of rat femoral artery is prevented by NO synthase inhibitors.

The hemodynamic significance of endothelium-derived relaxing factor (EDRF)-mediated mechanisms in vascular responses to abrupt rat femoral artery occlusion was investigated. Temporary arterial occlusion was produced before and after inhibition of nitric oxide synthase by N omega-nitro-L-arginine methyl ester (L-NAME) or NG-monomethyl-L-arginine (L-NMMA). Iliac artery blood flow and arterial pressures proximal and distal to the occlusion were measured. Normal vascular compensation included a return of resistance to preocclusion levels and a rise in distal pressure to a plateau within 5 min postocclusion. After treatment with L-NAME and L-NMMA, postocclusion resistance remained elevated by 53 and 36%, respectively. Collateral dilation after occlusion, as indicated by the rise in distal pressure, was prevented by L-NAME but not L-NMMA. Increases in adrenergic tone and mean arterial pressure by phenylephrine did not prevent compensation, suggesting the effects of L-NAME and L-NMMA did not result from elevated sympathetic activation or pressure. The results are consistent with the hypothesis that the stimulated release of endothelium-derived relaxing factor mediates the acute vascular compensation to abrupt arterial occlusion.

Evaluation of carbocyanine-labeled erythrocytes for microvascular measurements.

Red blood cells labeled with the carbocyanine dyes, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO), were evaluated for use in making microvascular measurements in rat small intestine and spinotrapezius muscle. We determined the minimum concentration of each dye which produced near maximal fluorescent intensity and labeled cell fraction. These dyes, which have excitation and emission spectra similar to fluorescein and rhodamine derivatives, have a number of advantages over the isothiocyanates: (1) the labeling procedure is quicker, easier, and less expensive; (2) the labeled cell fraction and the fluorescent intensity of DiI and DiO cells are stable for long periods of time in the rat circulation; and (3) DiI-labeled cells are brighter and transmit light through overlying erythrocytes better than rhodamine X isothiocyanate. However, in vitro and in vivo evaluations illustrate the potential limiting effects of vessel diameter and cell velocity on the accuracy of microvascular measurements made using this technique. In the small intestine and spinotrapezius muscle preparations, measurements of labeled cell flux were readily reproducible and could be partly automated with image analysis only in capillaries and small venules. Counting labeled cells in larger vessels by human observation or with automation was not reproducible, presumably due to absorption and dispersion of the fluorescent signal by overlying erythrocytes and smearing of the cell image at high cell velocities.

Improvement of flow through arterial stenoses by drag reducing agents.

The potential of drag reducing polymers (DRP) to selectively improve blood flow through clinically significant arterial stenoses was investigated. An artificial stenosis of the left common iliac artery in dogs decreased left femoral artery pressure by 25%. High-molecular-weight polyacrylamide (PA) or polyethylene oxide (PEO) were infused at a slow constant rate while we measured left and right common iliac artery blood flows and left and right femoral artery and vein pressures. As DRP were infused, left iliac artery flow (QL) increased early and then decreased to baseline values as flow began to increase in the right iliac artery. The peak increase in QL was 24 +/- 9% for PA and 46 +/- 19% for PEO and occurred before right iliac artery flow (QR) increased. As additional polymer was infused, QR increased to a maximum of 41 +/- 12 and 131 +/- 40% with PA and PEO, respectively. Femoral artery pressures and hindlimb resistances tended to decrease in both limbs but the only significant differences occurred in the right (nonstenosed) side when QR was elevated. This study provides the first evidence that low concentrations of DRP might be capable of improving blood flow through stenotic blood vessels without altering flow in normal vessels. Although DRP might represent a new class of compounds that could be utilized in the treatment of cardiovascular diseases, the degree of variation in individual responses is a concern, the exact mechanism of action is unclear, and information on pharmacodynamics is lacking.

Comparison of cadmium cytotoxicity in human versus rat nasal epithelial cells in vitro.

Differences in the sensitivity of human and Fischer 344 rat tissues to cadmium sulphate (CdSO(4)) toxicity were investigated in an in vitro model using human and rat nasal turbinate epithelial (NTE) cells. Both rat and human NTE cells were obtained from fresh, normal tissue. Methods were developed for isolating and culturing NTE cells from rat and human tissue using identical procedures, and for measuring the cellular nucleotides by high-performance liquid chromatography. Changes in adenylate energy charge and nucleotide levels were used as toxicity endpoints. Cellular Cd levels were measured by graphite-furnace atomic absorption spectrometry and expressed per unit DNA. Cd uptake was significantly greater in human NTE cells than in rat cells, particularly at the highest exposure concentration (4.8 mm-CdSO(4)). The effects of CdSO(4) on the adenylate energy charge of human and rat NTE cells were similar except at high exposure concentrations and after long exposure times; after a 2-4-hr exposure to 4.8 mm CdSO(4) the adenylate energy charge of human cells was significantly less than that of the rat cells.

Acellular vascular matrix: a natural endothelial cell substrate.

A preliminary assessment was made of the acellular vascular matrix graft as a substrate for endothelial cell seeding, with respect to surface pretreatment (none versus fibronectin and/or serum) and presence of exogenous growth factor. Arteries were harvested from greyhounds and exposed to a sequential detergent extraction process to produce the acellular vascular matrix. Human umbilical vein endothelial cells were grown in tissue culture, harvested in first passage, then seeded at 10(5) cells/cm2 on sections of acellular vascular matrix and on gel-coated polystyrene positive controls. After 18 hour incubation, endothelial cell-seeded acellular matrices were fixed and processed for histologic and planimetric analysis; control wells were fixed and endothelial cells were counted by planimetry. Pretreatment of the acellular vascular matrix was found to have no effect on the percentage of endothelial cell coverage of the matrix. There was significantly better endothelial cell coverage of the acellular matrix than on matched gel-treated polystyrene control wells. Withdrawal of growth factor resulted in a significant reduction in endothelial cell coverage for all acellular vascular matrix groups. Growth factor withdrawal also significantly reduced attachment of endothelial cells on gel-treated polystyrene. Cell surface area was significantly smaller when growth factor was withdrawn from all groups except from the acellular vascular matrix without pretreatment. We conclude that: (1) the acellular vascular matrix is conductive to endothelial cell adherence and spreading even without pretreatment; and (2) sudden withdrawal of exogenous growth factor may impair early coverage of substrates by endothelial cells due to an effect on their adherence or spreading.

Elastase inhibitor. Characterization of the human elastase inhibitor molecule associated with monocytes, macrophages, and neutrophils.

A fast-acting inhibitor of serine elastase has been detected at high levels in human neutrophils, fresh monocytes, matured monocytes, and macrophages. The elastase inhibitor was isolated from large scale cultures of the monocyte-like cell line U937 by DNase chromatography, disulfide exchange, Phenyl-Sepharose, Red A-agarose, and DEAE HPLC chromatography with an average yield of 480 micrograms from 1.8 x 10(10) cells. The isolated polypeptide was verified as elastase inhibitor by its ability to (a) form a covalent complex with elastase; and (b) inhibit the elastinolytic activity of elastase. The purified elastase inhibitor molecule is unique, i.e., physiochemical and/or functional properties distinguish it from all other serine proteinase inhibitors. Treatment with iodoacetamide abrogates the ability of the molecule to form a complex with elastase, thereby providing evidence for the presence of an essential cysteine residue. Based on functional criteria, this elastase inhibitor has been grouped with the proteinase inhibitors of the serpin superfamily. The purified elastase inhibitor is a single polypeptide of Mr approximately 42,000. The NH2 terminus appears to be blocked. Compositional analyses indicates five cysteine residues per molecule of approximately 360 amino acid residues. Negligible levels of carbohydrate were detected on gas-liquid chromatography. This finding and the insensitivity of the molecule to peptide N-glycosidase F treatment strongly indicate that the elastase inhibitor is a nonglycosylated protein.

Cost reductions from a smoking policy.

The health of its workforce is of paramount importance to a business. The loss of even one key person to heart and lung disease can have a detrimental effect on organizational efficiency. Provided strides similar to those made in the last five years continue, perhaps within the next five years major employers will have all work areas smoke free. In large cities considerable help in implementing a nonsmoking policy is available to employers. Individual companies need to consider the merits of each type of program and then decide which best fits the organization. Compared with a nonsmoker, the average employed smoker in the United States 1. Takes approximately 50% more sick leave 2. Uses the health care system at least 50% more 3. Suffers over twice the mortality rate during working years 4. Spends 6% of working hours smoking 5. Causes indoor work facilities to expend six times the capital and operating outlays to maintain air circulation rates that meet building code specifications 6. Imposes a much greater maintenance burden for cleaning, repairing, repainting and replacing furnishings and equipment 7. Imposes unnecessary costly liability on employers for damages sustained by co-workers who are forced to work in a smoky environment 8. Exacts a high price in employee morale because of the irritation and discomfort suffered by fellow workers.

Role of platelet-derived growth factor in wound healing: synergistic effects with other growth factors.

Platelet-derived growth factor (PDGF) in vitro stimulates DNA synthesis and chemotaxis of fibroblasts and smooth muscle cells and stimulates collagen, glycosaminoglycan, and collagenase production by fibroblasts. These in vitro properties suggest that PDGF, delivered by platelets to the site of injury in vivo, may play an important role in the initiation of the wound repair process. Studies presented here show that the addition of pure PDGF to a wound site involving the epidermis and dermis has little effect on the morphology or biochemistry of the healing wound. In contrast, the addition of partially purified PDGF resulted in significant dose-dependent increases in the width of the newly synthesized connective tissue and epidermal layers. Autoradiography using [3H]thymidine revealed increased numbers of labeled cells in the new connective tissue and epithelial layers. Furthermore, addition of partially purified PDGF resulted in significant increases in the rate of protein and DNA synthesis and the total content of these components in biopsies taken from the wound site. Similar effects were obtained when insulin-like growth factor I was added in combination with pure PDGF. This combination of factors caused a 2.4-fold increase in the width of the newly formed connective tissue layer and a 95% increase in epidermal thickness compared with controls. Insulin-like growth factor I alone caused no significant morphologic changes. Epidermal growth factor alone or in combination with PDGF resulted in a thickening only of the epidermis. These results indicate that the synergistic actions of other factors with PDGF are important in the modulation of the wound healing process.

Effect of dietary ascorbic acid on the incidence of spontaneous mammary tumors in RIII mice.

A study of the effect of different amounts of L-ascorbic acid (vitamin C), between 0.076% and 8.3%, contained in the food has been carried out with ten groups of RIII mice (seven ascorbic acid and three control groups), with 50 mice in each group. With an increase in the amount of ascorbic acid there is a highly significant decrease in the first-order rate constant for appearance of the first spontaneous mammary tumor after the lag time to detection by palpation. There is also an increase in the lag time. The mean body weight and mean food intake were not significantly different for the seven ascorbic acid groups. Striking differences were observed between the 0.076% ascorbic acid and the control groups (which synthesize the vitamin): smaller food intake, decreased lag time, and increased rate constant of appearance of the first mammary tumor. This comparison cannot be made experimentally for guinea pigs and primates because the control groups would develop scurvy.

Endogenous effector of the benzodiazepine binding site: purification and characterization.

A protein has been isolated from the small intestine and bile duct which inhibits the binding of [3H]diazepam to specific benzodiazepine binding sites on synaptosomal membranes. When ion-exchange chromatography and gel filtration chromatography are used, this protein has been purified to apparent homogeneity. "Nepenthin" has been chosen as a name for this protein, which has an approximate molecular weight of 16 000, as determined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography. Purified nepenthin is a competitive inhibitor of [3H]diazepam binding with a Ki = 4.6 X 10(-8) M. It does not inhibit the binding of specific ligands to the enkephalin, beta-adrenergic, gamma-aminobutyrate, or dopamine binding sites in the CNS. Neither gamma-aminobutyric acid nor glycine alters the inhibition of [3H]diazepam binding by this protein. Nepenthin can be extensively treated with proteases (trypsin, chymotrypsin, and Pronase), and inhibition of diazepam binding remains stable, indicating that a lower molecular weight fragment retains activity. Antibodies raised against this purified effector have been used in in situ double antibody labeling studies with rat brain slices. These studies indicate that cells containing an immunologically similar material are present in the deep cortical region of the forebrain.