Eleutherobin, a novel cytotoxic agent that induces tubulin polymerization, is similar to paclitaxel (Taxol).

Eleutherobin is a novel natural product isolated from a marine soft coral that is extremely potent for inducing tubulin polymerization in vitro and is cytotoxic for cancer cells with an IC50 similar to that of paclitaxel. This compound is cross-resistant along with other multidrug-resistant agents against P-glycoprotein-expressing cells and is cross-resistant with paclitaxel against a cell line that has altered tubulin. In mechanistic studies, eleutherobin shares with paclitaxel the ability to induce tubulin polymerization in vitro and is most likely cytotoxic by virtue of this mechanism. Human colon carcinoma cells exposed to eleutherobin contain multiple micronuclei and microtubule bundles, and they arrest in mitosis, depending on concentration, cell line, and length of exposure. These morphological abnormalities appearing in cultured cells are indistinguishable from those induced by paclitaxel. Electron microscopy reveals that eleutherobin induces homogeneous populations of long, rigid microtubules similar to those formed by paclitaxel. Thus, eleutherobin is a new chemotype with a mechanism of action similar to that of paclitaxel and, as such, has promising potential as a new anticancer agent.

Utilization of electron probe microanalysis in gadolinium-treated mice.

Gadolinium is used as a contrast media for magnetic resonance imaging and, experimentally, to block Kupffer cell phagocytosis. In this study, we utilize electron probe microanalysis to determine the subcellular localization of gadolinium chloride (GdCl3) administered to mice in a short-term toxicology study. Male CD-1 mice were administered 0.0, 2.5, or 8.0 mg/kg GdCl3 iv for 14 consecutive weekdays. Liver-associated enzymes were significantly elevated in high-dose animals only and correlated histologically with multifocal, hepatocellular degeneration associated with a neutrophilic infiltrate. Morphological investigations were performed on high-dose animals. Hepatocytes and Kupffer cells had morphologic features of cellular injury consisting of swollen mitochondria and vesiculated profiles of endoplasmic reticulum. Hepatocytes, Kupffer cells, bile canaliculi, and neutrophils in the liver contained discrete aggregates of electron-dense granular material, as did pulmonary interstitial macrophages, splenic macrophages, and mesangial cells of the renal glomerulus. The intracellular granular material in the liver, lung, spleen, and kidney was confirmed as gadolinium by qualitative electron probe microanalysis. These results document both hepatic and extra-hepatic accumulation of gadolinium in cells of the mononuclear phagocytic system and highlight the importance of electron probe microanalysis in toxicologic assessment.

Didanosine (ddl) and stavudine (d4T): absence of peripheral neurotoxicity in rabbits.

Some 20 male New Zealand White rabbits (five/group) were given either didanosine (ddl) or stavudine (d4T) at 750 and 1500 mg/kg body weight/day by oral intubation for 24 wk. An additional group was given 300 mg/kg body weight/day zidovudine (AZT) as a negative control. After 13 weeks the high dose of ddl was lowered from 1500 to 1000 mg/kg body weight/day following the death of one rabbit and continued inappetence in the dose group. The rabbits were observed daily, plasma drug levels were monitored, and electrophysiological measurements of peripheral nerve conduction were performed during the study. Additionally, body weight and food intake were recorded, and clinicopathological parameters were evaluated. Sections of selected peripheral nerves, and dorsal and ventral spinal nerve roots were examined by light and transmission electron microscopy. Although peripheral neuropathy has been reported in rabbits with the nucleoside analogue zalcitabine (ddC), based on clinical observations, electrophysiological measurements, and light and electron microscopy, no evidence of peripheral neurotoxicity was observed in rabbits given either ddl of d4T.

Comparison of a novel ETA receptor antagonist and phosphoramidon in renal ischemia.

Infusion (0.46 mumol/kg/min) of the endothelin (ET)-converting-enzyme inhibitor, phosphoramidon (P), protected function and structure after 30 min renal ischemia in rats more than treatment (5 mumol/kg/min) with the ETA receptor antagonist, BMS-182874 (B). The glomerular filtration rate (GFR; 0.7 +/- 0.12 ml/min) and renal plasma flow (RPF) decreased approximately 40% at 2 h reflow versus controls (C: 1.2 +/- 0.12). B weakly protected the GFR (0.8 +/- 0.07 ml/min); P restored it (1.1 +/- 0.05). Both compounds reduced tubular injury at 2 h reflow; P ameliorated glomerular changes. At 24 h the GFR (0.6 +/- 0.06 ml/min) and RPF decreased 67% versus C (1.8 +/- 0.08). B did not protect the GFR and RPF. P partially protected the GFR (0.9 +/- 0.07 ml/min) but not RPF, and reduced tubular injury. The results suggest that both ETA and non-ETA receptors mediate ET-induced changes in ischemic renal failure.

Structure-activity analyses of beta-amyloid peptides: contributions of the beta 25-35 region to aggregation and neurotoxicity.

The neurodegeneration of Alzheimer's disease has been theorized to be mediated, at least in part, by insoluble aggregates of beta-amyloid protein that are widely distributed in the form of plaques throughout brain regions affected by the disease. Previous studies by our laboratory and others have demonstrated that the neurotoxicity of beta-amyloid in vitro is dependent upon its spontaneous adoption of an aggregated structure. In this study, we report extensive structure-activity analyses of a series of peptides derived from both the proposed active fragment of beta-amyloid, beta 25-35, and the full-length protein, beta 1-42. We examine the effects of amino acid residue deletions and substitutions on the ability of beta-amyloid peptides to both form sedimentable aggregates and induce toxicity in cultured hippocampal neurons. We observe that significant levels of peptide aggregation are always associated with significant beta-amyloid-induced neurotoxicity. Further, both N- and C-terminal regions of beta 25-35 appear to contribute to these processes. In particular, significant disruption of peptide aggregation and toxicity result from alterations in the beta 33-35 region. In beta 1-42 peptides, aggregation disruption is evidenced by changes in both electrophoresis profiles and fibril morphology visualized at the light and electron microscope levels. Using circular dichroism analysis in a subset of peptides, we observed classic features of beta-sheet secondary structure in aggregating, toxic beta-amyloid peptides but not in nonaggregating, nontoxic beta-amyloid peptides. Together, these data further define the primary and secondary structures of beta-amyloid that are involved in its in vitro assembly into neurotoxic peptide aggregates and may underlie both its pathological deposition and subsequent degenerative effects in Alzheimer's disease.

Ultrastructural analysis of beta-amyloid-induced apoptosis in cultured hippocampal neurons.

Following treatment with the beta-amyloid (A beta) 25-35 analog, scanning and transmission electron microscopy were used to investigate the morphological changes in cultured hippocampal neurons during the course of degeneration. Ultrastructural analysis revealed focal cell surface blebbing and rapid condensation of nuclear chromatin. Changes in cytoplasmic morphology included prominent vacuole formation, dispersal of polyribosome rosettes and the disappearance of the golgi complex, smooth endoplasmic reticulum and microtubules with increased cytoplasmic electron density. Mitochondria and limited rough endoplasmic reticulum remained intact throughout the process of cell death. These results provide additional evidence suggesting A beta-induced cell death in vitro occurs via an apoptotic mechanism.

Analysis of mammalian connective tissue: relationship between hierarchical structures and mechanical properties.

It is widely accepted that the mechanical properties of implants must match those of the surrounding connective tissue to prevent stress concentration and premature failure. The purpose of this paper is to review the structure-mechanical property relationships that exist for connective tissue. The mechanical properties of connective tissue depend on the content of collagen, elastic tissue, and proteoglycans, as well as the geometric arrangement of the fibrous components, age, and location of the specimen. To a first approximation the geometry and loading pattern of the collagen networks in these tissues dominate the mechanical response at high strains. The behavior of the elastic fiber networks dominate the low-strain mechanical response in tissues where energy and shape recovery are critical. Proteoglycans are involved in resisting tissue compressive forces. Since the stiffness of connective tissue increases with age, it is necessary to attempt to match this property by designing implants that have similar behaviors to insure that stress concentration does not occur at the interface between the implant and host.

Liver macrophage-mediated cytotoxicity toward mastocytoma cells involves phagocytosis of tumor targets.

Macrophage-mediated cytotoxicity toward tumor cells usually involves extracellular lysis of the targets. In this study, we report that liver macrophages from rats treated with lipopolysaccharide (5 mg/kg, intravenous) also kill certain tumor cell targets by phagocytosis. Liver macrophages were coincubated with P815 mouse mastocytoma cells for 24 to 72 hr at an effector/target ratio of 10:1. Macrophage phagocytosis was characterized by flow cytometry and by light and electron microscopy. For flow-cytometric studies, P815 cells were prelabeled with the fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate. We found that coincubation of macrophages with labeled targets resulted in a time-dependent increase in macrophage-associated fluorescence, reaching a maximum at 72 hr. This correlated with light-microscopic observations of increased numbers of tumor cells in the macrophages and enhanced macrophage surface area and density. Electron microscopic studies revealed that the initial event in the phagocytic process involved the capture of P815 cells by the pseudopodia of the macrophages. Target cells were then surrounded by lamellipodia, internalized in phagosomes and destroyed. These data, together with previous studies, provide evidence for multiple mechanisms of cytotoxicity mediated by activated liver macrophages.

Differential production of interleukin 1 on the surface of biomaterials.

The production of cytokines on the surface of surgical biomaterials plays a major role in their biocompatibility. Membrane-associated interleukin 1 (mIL-1) is a cytokine found on the surface of macrophages activated by biomaterials. To better understand the host-foreign body interaction, we quantitated the production of mIL-1 on the surface of two materials commonly used in surgery, expanded polytef (ePTFE) and silicon elastomer (SE). The mean (+/- SD) level of mIL-1 produced by adherent cells to ePTFE significantly decreased from day 2 (13,746 +/- 3630 cpm per disk) compared with day 7 (2828 +/- 1304 cpm per disk). However, the level of mIL-1 produced by ePTFE-adherent cells was still markedly greater than the level of mIL-1 produced by cells adherent to SE (1877 +/- 1028 vs 1595 +/- 822 cpm per disk). These results indicate that ePTFE and SE elicit a differential host response in terms of cytokine production. This study may enhance our understanding of the cellular events on the surface of biomaterials that underlie clinical observations.

Achilles tendon replacement by a collagen fiber prosthesis: morphological evaluation of neotendon formation.

Reconstituted type I collagen was processed into fibers which were subsequently severely dehydrated and cyanamide cross-linked. Fibers prepared by this method were stronger and more resistant to degradation than uncrosslinked fibers. When used as a tendon replacement prosthesis, morphological events occurred which were observed by light, scanning, transmission electron microscopy and electron histochemistry. Resorption was the initial host response to the prosthesis and involved gradual biodegradation. Formation of a host-replacement tendon was the second response. Increased collagen fibril diameters and a transition in the proteoglycan/collagen fibril interactions occurred in the newly developing connective tissue between 3 and 10 weeks postimplantation. These extracellular matrix transitions were major events occurring during wound healing and led to the assembly of a mature connective tissue. When used as a tendon prosthesis, these collagen fibers rapidly resorb while allowing simultaneous formation of aligned connective tissue. The fibers may have other applications in the fields of Orthopaedic Surgery, Neurosurgery and Biomaterials Research.

Steady-state plasma levels and pharmacokinetics of guanfacine in patients with renal insufficiency.

The steady-state pharmacokinetics and renal clearance of guanfacine were studied in patients with renal insufficiency. Apparent total body and renal clearances of guanfacine, as well as apparent volume of distribution, decreased in parallel with the decline in renal function. Higher plasma levels of guanfacine were observed in patients with GFR less than 30 ml/min. The steady state t 1/2 of guanfacine in the presence of renal insufficiency was insignificantly prolonged relative to that observed in the presence of normal renal function and appeared to be independent of the degree of renal insufficiency.

Treatment of hypercholesterolemia in black patients.

The treatment of hypercholesterolemia in the black patient will be the next public health challenge facing physicians in the black community. Cost-effective care of hypercholesterolemia will be necessary and is possible, but it will require skill in the use of available therapies, extensive patient education, and excellent communication between patients and health care providers.

Clinical applications of electron microscopy in the analysis of collagenous biomaterials.

Scanning and transmission electron microscopy are of clinical value in assessing the interaction between biomaterials and ingrowing tissues. Ultrastructural information allows the clinician and biomaterials specialist to determine events occurring during wound healing and the biocompatibility of prosthetic devices. This paper reviews some of the experimental and clinical studies done in our laboratory on the use of natural and reconstituted collagen as replacements for connective tissues. Consideration is given to collagen flakes used for the treatment of dermal ulcers, a collagen fiber prosthesis used for tendon and ligament replacement, the effects of chemical preservatives on cartilage used for replacement of tissues during plastic surgery and the growth and orientation of nerve cells on reconstituted collagen fibers. Our results show that reconstituted collagen can be prepared into prosthetic devices which encourage cell attachment and orientation thereby facilitating healing of injured tissues. Furthermore chemical preservation of cartilagenous tissues kills chondrocytes resulting in eventual resorption by inflammatory cells.

Behaviour of fibroblasts and epidermal cells cultivated on analogues of extracellular matrix.

A porous collagen sponge can be used for supporting epidermal cells and fibroblasts in order to manufacture an artificial skin. Fibroblasts were grown on analogues of extracellular matrix containing collagen and glycosaminoglycans and/or glycoproteins. Cell replication, and also infiltration of fibroblasts, were enhanced by the presence of hyaluronic acid and/or fibronectin. Epidermal cells grown on a collagen sponge have been characterized by microscopic observations. Epidermal cells on the surface of the sponge showed an incomplete differentiation in comparison to normal skin; clumps of epidermal cells were found in the interior of the sponge. Epidermal cell replication was enhanced in the presence of collagen sponge seeded with fibroblasts.

Differential sensitivity of tumor targets to liver macrophage-mediated cytotoxicity.

Liver macrophages activated in vivo with bacterially derived lipopolysaccharide (LPS) display enhanced chemotaxis, phagocytosis, and oxidative metabolism. To determine if LPS also activates these mononuclear phagocytes for tumor cell killing, we compared the cytotoxic activity of macrophages from livers of rats treated with LPS (5 mg/kg, i.v.) with resident Kupffer cells. We found that both macrophage cell types displayed cytotoxicity towards rat N1S1 hepatoma and RBL-1 basophilic leukemia cells. Cytotoxicity of resident and LPS-activated liver macrophages towards these targets increased with cocultivation time, was dependent on the effector:target cell ratio, and appeared to involve extracellular lysis. No direct correlation between macrophage activation and cytotoxicity was observed towards these targets. While liver macrophages from LPS treated rats were more cytotoxic towards N1S1 cells, resident Kupffer cells were more cytotoxic towards RBL-1 cells. In further studies, resident Kupffer cells were also found to display extracellular cytolytic activity towards mouse P815 mastocytoma cells. In contrast, LPS-activated liver macrophage-mediated killing of these targets involved phagocytosis of intact tumor cells, as evidenced by light and electron microscopy and by uptake of 51Cr-labeled cells. These results suggest that cytotoxicity mediated by liver macrophages depends on the type of macrophage and the nature of the tumor cell target. In addition, cytotoxicity towards tumor targets appears to involve at least two different mechanisms including extracellular cytolysis and phagocytosis.

Calcium-sensitive cellular and subcellular transport of sodium, potassium, magnesium, and calcium in sodium-loaded vascular smooth muscle. Electron probe analysis.

Electron probe x-ray microanalysis of the composition of rabbit portal anterior mesenteric vein smooth muscle was performed following sodium loading and washout into sodium-free lithium solutions. Sodium and lithium were also measured with atomic absorption spectrophotometry. Cellular uptake of sodium and loss of potassium during sodium loading were much faster at high (37 degrees C) than at low (2 degrees C) temperature, as was the passive ouabain-resistant uptake of potassium during lithium washout. The loss of sodium at 2 degrees C into lithium solution consisted of two components: a rapid efflux that was complete by 30 minutes, and a slow component that required at least 24 hours for completion. The amount of sodium lost through the first component (approximately 200-300 mmol/kg dry weight) was relatively independent of the amount of sodium loading. The loss of cellular sodium at 2 degrees C, after 30 minutes, was accompanied by a gain of cellular lithium. Ouabain-resistant sodium loss and lithium and potassium uptake were markedly accelerated at 37 degrees C; sodium loss was complete (1200 mmol sodium/kg dry weight lost) by 30 minutes of washout. Sodium-loaded cells also lost chloride ion and gained magnesium during sodium efflux at 37 degrees C. Mitochondrial and nuclear sodium and potassium were correlated with the respective cytoplasmic concentrations during both sodium loading and sodium washout, indicating the relatively rapid equilibration of the monovalent ions between the cytoplasm and organelles. Calcium-free solutions markedly inhibited the ouabain-resistant sodium and chloride ion effluxes and potassium influx in muscles incubated, after sodium loading, in lithium solutions at 37 degrees C. These fluxes could be restored to near normal values by 0.2 mM calcium. The calcium sensitivity of the ouabain-resistant sodium, potassium, and chloride ion fluxes observed in this and other studies raises the possibility that some abnormalities of monovalent ion transport observed in cells of hypertensives are secondary to changes in cellular calcium.

Calcium and sodium distribution and movements in smooth muscle.

Electron probe microanalysis (EPMA) has been used to study the subcellular distribution of Ca, Na, K, Cl, and Mg in smooth muscle. The EPMA results indicate that the sarcoplasmic reticulum (SR) is the major intracellular source and sink of activator Ca: norepinephrine decreases the Ca content of the junctional SR in portal vein smooth muscle. Mitochondria do not play a significant role in regulating cytoplasmic free Ca2+, but mitochondrial Ca content can be altered to a degree compatible with suggestions that fluctuations in matrix Ca contribute to the control of mitochondrial metabolism. The rise in total cytoplasmic Ca during a maintained, maximal contraction is very much greater than the rise in free Ca2+, and is probably in excess of the known binding sites available on calmodulin and myosin. Cell Ca is not increased in normal cells that are Na-loaded. The non-Donnan distribution of Cl is not due to compartmentalization, but reflects high cytoplasmic Cl. Na-loading of smooth muscle in K-free solutions is temperature dependent, and may exhibit cellular heterogeneity undetected by conventional techniques. The total cell Mg is equivalent to approximately 12 mM, and less than 50% of it can be accounted for by binding to ATP and to actin. Mitochondrial monovalent cations in smooth muscle are relatively rapidly exchangeable.

Central and peripheral neurosecretory pathways to an insect flight motor nerve.

Ultrastructural examination of the IIN1b nerve to the dorsal longitudinal flight muscle of Manduca sexta L. verified the presence of neurosecretory processes. Subspherical and irregular vesicles were found where the nerve enters the muscle, while spherical vesicles were found in the proximal region only. A dorsal unpaired median (DUM) cell, the median nervous system, and two or more peripheral cells are the sources of these neurosecretory inclusions. Light the electron microscopy CoCl2 backfills of the transverse nerve produced intensification of a peripheral neuron (#1) and processes in nerves IIN1a and IIN1b. Similar backfills of nerve IIN1b produced intensification of a DUM cell, a second peripheral neuron (#2), and processes in the transverse nerve and nerve IIN1a. Neuron #1 contained large spherical electron-dense vesicles while neuron #2 contained smaller subspherical vesicles. These cells were situated upon the link and/or transverse nerves. Based on these results, we suspect central and peripheral neurosecretory processes reach nerve IIN1b as follows: the link nerve projects prothoracic median nervous system and neuron #2 processes, nerve IIN1a projects neuron #1 processes, and nerve IIN1 projects mesothoracic DUM cell processes, although this latter pathway was less clear.

Vertical distribution of elements in cells and matrix of epiphyseal growth plate cartilage determined by quantitative electron probe analysis.

Quantitative electron probe analysis was performed on chick epiphyseal growth cartilage prepared by two anhydrous methods, ultrathin cryosections and freeze-dried epoxy-embedded tissue. Levels of Na, Mg, P, S, Cl, K, and Ca were determined in cytoplasm, mitochondria, extracellular matrix, matrix vesicles, and mineral nodules in four zones of the cartilage--proliferative, prehypertrophic, early hypertrophic, and early calcification. The exceptionally high levels of Na and K (up to 550 and 200 mmol/kg wet wt, respectively) found in the matrix are believed to be largely bound to fixed anions. Within cells, Na was higher than K (140 versus 20-34 mmol/kg wet wt), a condition that may reflect hypoxia. Ca and P were low in cells and unmineralized matrix. Ca and P were high in mitochondrial granules of the early hypertrophic zone and diminished in amount in the calcifying zone; the converse occurred in matrix vesicles. Mg was low to undetectable except in heavily mineralized structures (i.e., mitochondrial granules, matrix vesicles, and mineral nodules). S levels were high in matrix (approximately 400 mmol/kg wet wt) and increased slightly with maturation. The amount of S present greatly exceeds Ca levels and implies that sulfate, the predominant form of sulfur in proteoglycans, may serve as an ion-exchange mechanism for the passage of Ca through the matrix to sites where Ca and phosphate are precipitated.