Michael N. Horst

Adult reserve stem cells and their potential for tissue engineering

Tissue restoration is the process whereby multiple damaged cell types are replaced to restore the histoarchitecture and function to the tissue. Several theories, have been proposed to explain the phenomenon of tissue restoration in amphibians and in animals belonging to higher order. These theories include dedifferentiation of damaged tissues, transdifferentiation of lineage-committed progenitor cells, and activation of reserve, precursor cells. Studies by Young et al. and others demonstrated that connective tissue compartments throughout postnatal individuals contain reserve precursor cells. Subsequent repetitive single cell-cloning and cell-sorting studies revealed that these reserve precursor cells consisted of multiple populations of cells, including, tissue-specific progenitor cells, germ-layer lineage stem cells, and pluripotent stem cells. Tissue-specific progenitor cells display various capacities for differentiation, ranging from unipotency (forming a single cell type) to multipotency (forming multiple cell types). However, all progenitor cells demonstrate a finite life span of 50 to 70 population doublings before programmed cell senescence and cell death occurs. Germ-layer lineage stem cells can form a wider range of cell types than a progenitor cell. An individual germ-layer lineage stem cell can form all cells types within its respective germ-layer lineage (i.e., ectoderm, mesoderm, or endoderm). Pluripotent stem cells can form a wider range of cell types than a single germ-layer lineage stem cell. A single pluripotent stem cell can form cells belonging to all three germ layer lineages. Both germ-layer lineage stem cells and pluripotent stem cells exhibit extended capabilities for self-renewal, far surpassing the limited life span of progenitor cells (50–70 population doublings). The authors propose that the activation of quiescent tissue-specific progenitor cells, germ-layer lineage stem cells, and/or pluripotent stem cells may be a potential explanation, along with dedifferentiation and transdifferentiation, for the process of tissue restoration. Several model systems are currently being investigated to determine the possibilities of using these adult quiescent reserve precursor cells for tissue engineering.

Bioaccumulation and Metabolic Effects of the Endocrine Disruptor Methoprene in the Lobster, Homarus americanus

Abstract Methoprene is a pesticide that acts as a juvenile hormone agonist. Although developed initially against insects, it has since been shown to have toxic effects on larval and adult crustaceans. Methoprene was one of several pesticides applied to the Western Long Island Sound (WLIS) watershed area during the summer of 1999; the other pesticides were malathion, resmethrin, and sumethrin. These pesticides were applied as part of a county-by-county effort to control the mosquito vector of West Nile Virus. Subsequently, the seasonal lobster catches from the WLIS have decreased dramatically. The lethality of the pesticides to lobsters had been unknown. We studied the effects of methoprene while other investigators studied effects of the other pesticides. We questioned whether methoprene, through its effects on larvae, adults or both, could have contributed to this decline. We found that low levels of methoprene had adverse effects on lobster larvae. It was toxic to stage II larvae at 1 ppb. Stage IV larvae were more resistant, but did exhibit significant increases in molt frequency beginning at exposures of 5 ppb. Juvenile lobsters exhibited variations in tissue susceptibility to methoprene: hepatopancreas appeared to be the most vulnerable, reflected by environmental concentrations of methoprene inhibiting almost all protein synthesis in this organ. Our results indicated that methoprene concentrates in the hepatopancreas, nervous tissue and epidermal cells of the adult lobster. Methoprene altered the synthesis and incorporation of chitoproteins (cuticle proteins) into adult postmolt lobster explant shells. SDS PAGE analyses of adult post–molt shell extracts revealed changes in the synthesis of chitoproteins in the methoprene-treated specimens, suggesting that methoprene affects the normal pathway of lobster cuticle synthesis and the quality of the post-molt shell. Although it is likely that a combination of factors led to the reduced lobster population in WLIS, methoprene may have contributed both by direct toxic effects and by disrupting homeostatic events under endocrine control.

EFFECTS OF THE PESTICIDE METHOPRENE ON MORPHOGENESIS AND SHELL FORMATION IN THE BLUE CRAB CALLINECTES SAPIDUS

The juvenile hormone analog methoprene causes both cytologic and biochemical alterations in larval and adult stages of the blue crab Callinectes sapidus. This insect growth regulator, used for mosquito control, caused (at a concentration of 10 gM) profound ultrastructural changes in the cuticular epithelial cells of postmolt adult blue crabs studied in vitro; these changes included loss of secretory organelles as well as distention and blebbing of the outer membrane of the nuclear envelope. Biochemically, 10 gM methoprene caused decreased deposition of extracellular cuticular chitin and protein, as well as a remarkable intracellular accumulation of chitoprotein precursors. These findings suggest that methoprene alters exocytosis and deposition of cuticular components. In vivo studies indicated that 5-10 gM methoprene is able to penetrate the embryonic investment coat to localize in lipovitellin. Exposure to methoprene at environmental concentrations (2-10 giM) produced morbidity and mortality in the form of an overall reduction in the number of successful hatching and lethargic behavior exhibited by the surviving zoeae. Methoprene exposure (3.3 gM) was also toxic to megalopae, delaying the molt to the first crab form and resulting in death of 80% of larvae after 10 days. The blue crab Callinectes sapidus Rathbun is a crustacean of both ecologic and economic importance to the coastal zones of the eastern and southern United States. Its life cycle and feeding habits bring it into estuarine environments subjected to pesticide treatments for mosquito control. It is logical to assume that the blue crab might be impacted by compounds active in other arthropods. The pesticide methoprene, a compound frequently applied to wetlands and salt marshes, belongs to the group of pesticides known as insect growth regulators (IGRs), which, in general, exert their toxic effects by disrupting insect development and/or reproduction. We offer in this report evidence that methoprene can penetrate the embryonic cuticle and can produce morbidity and mortality in larval forms of the blue crab. We also present in vitro findings indicating that methoprene may be toxic to the adult blue crab as well by altering the exocytosis of cuticular material.

Characterization of a major outer-membrane antigen of Edwardsiella ictaluri

Abstract Edwardsiella ictaluri is the cause of enteric septicemia of catfish. A monoclonal antibody (MAb AA224) was used to identify a specific and predominant outer-membrane antigen of E. ictaluri. The MAb AA224 was produced by conventional cell fusion technology with spleen cells from mice immunized with an affinity-purified antigen. The affinity-purified antigen was obtained by immunoaffinity chromatography of an E. ictaluri extract with immunoaffinity purified immunoglobin from sera of channel catfish Ictalurus punctatus immune to E. ictaluri as a result of natural infection. The immunoaffinity-purified antigen was used for immunization and identification of the hybridoma producing MAb AA224 by enzyme-linked immunosorbent assay. The predominant antigen was purified by immunoaffinity chromatography with MAb AA224 as the immunoadsorbent. Immunoblotting and high-pressure liquid chromatography were used to determine that the relative sizes of the predominant antigens are 60 and 36 kilodaltons. Immunoelect...

METABOLIC EFFECTS OF ACUTE EXPOSURE TO METHOPRENE IN THE AMERICAN LOBSTER, HOMARUS AMERICANUS

Abstract Methoprene was a constituent of the pesticide cocktail applied to the Western Long Island Sound (WLIS) watershed area during the summer of 1999. Subsequently, the seasonal lobster catches from the WLIS have decreased dramatically. We have been engaged in ongoing studies of the effects of methoprene on larval, juvenile and adult lobsters. Most recently, we found that Stage IV larvae exposed to 50 ppb methoprene experience >90% mortality rate after 3 days. Bioaccumulation studies on adult lobsters showed that methoprene concentrated against the gradient of the surrounding seawater (50 ppb) in hepatopancreas (1.55 ppm), gonad (5.18 ppm), epithelial tissue (6.17 ppm) and, most significantly, the eyestalks (28.83 ppm). Exposure to methoprene altered the expression of the stress proteins and the pattern of ubiquitinylation of cytosolic proteins by Day 1 Stage I larvae and by epithelial tissue of postmolt juvenile lobsters. Postmolt juvenile animals also demonstrated an altered pattern of protein phosphorylation in their epithelial tissues following methoprene exposure, indicating that it may interfere with cell signaling pathways. Increasing concentrations of methoprene were associated with increasing chitoproteins in the microsomal fractions of Day 1 Stage I larvae, suggesting that methoprene may compromise the exocytosis of shell matrix precursors from the epithelial cells. Methoprene did not, however, alter the activity of chitin synthase in these larvae. Although it is likely that a combination of harmful events and exposures led to the reduced lobster population in WLIS, methoprene may have contributed to the decline both by direct toxic effects and by disrupting homeostatic processes.

Morphologic effects of in vivo acute exposure to the pesticide methoprene on the hepatopancreas of a non-target organism, Homarus americanus.

Methoprene is a pesticide widely used for mosquito control. It is an endocrine disruptor, acting as an analog of juvenile hormone. While targeting insect larvae, it also impacts non-target animals including crustaceans. Anecdotal reports suggested that methoprene has unintended effects on adult arthropods. Earlier, we documented effects in adult lobsters at the metabolic and gene expression levels. In this study we have documented morphologic corollaries to our prior observations. We examined the light and electron microscopic changes in the hepatopancreas of adult lobsters following in vivo acute exposure to methoprene. Changes by light and electron microscopy levels were evident following exposure to sub-lethal concentrations of methoprene for 24h. Tissue from exposed animals showed the formation of extensive cytoplasmic spaces (vesiculation) with disruption and loss of specific subcellular organelles. The findings provide morphologic correlates to the metabolic and genomic alterations we have observed in previous investigations.

Fungal Enolase, β-Tubulin, and Chitin Are Detected in Brain Tissue from Alzheimer's Disease Patients.

Recent findings provide evidence that fungal structures can be detected in brain tissue from Alzheimer’s disease (AD) patients using rabbit polyclonal antibodies raised against whole fungal cells. In the present work, we have developed and tested specific antibodies that recognize the fungal proteins, enolase and β-tubulin, and an antibody that recognizes the fungal polysaccharide chitin. Consistent with our previous studies, a number of rounded yeast-like and hyphal structures were detected using these antibodies in brain sections from AD patients. Some of these structures were intracellular and, strikingly, some were found to be located inside nuclei from neurons, whereas other fungal structures were detected extracellularly. Corporya amylacea from AD patients also contained enolase and β-tubulin as revealed by these selective antibodies, but were devoid of fungal chitin. Importantly, brain sections from control subjects were usually negative for staining with the three antibodies. However, a few fungal structures can be observed in some control individuals. Collectively, these findings indicate the presence of two fungal proteins, enolase and β-tubulin, and the polysaccharide chitin, in CNS tissue from AD patients. These findings are consistent with our hypothesis that AD is caused by disseminated fungal infection.

Molecular and Cellular Aspects of Chitin Synthesis in Larval Artemia

The cuticle of Artemia and other crustaceans contains chitin which is covalently attached to protein[1–3]. While the role of such proteins in structural support of the cuticle seems clear, the possible functions of the proteins in the biosynthesis of the cuticle are not obvious. Recent work in my laboratory has indicated that crustaceans synthesize a chitin oligosaccharide via a dolichol-linked intermediate[4]. Subsequent transfer of the oligosaccharides to either endogenous polypeptides or synthetic peptide acceptors[5] yields a chitoprotein which may serve as a substrate for chitin synthetase, the enzyme responsible for polymerization of macromolecular chitin[6]. However, the pathway of synthesis for this material within the crustacean epithelial cell is not clear; one notion is that the chitoprotein is synthesized in the rough endoplasmic reticulum (RER), moves to the Golgi apparatus (where chitin synthetase is presumably located) and serves as a primer molecule for chitin synthetase, yielding a chitin-protein complex. The mechanism whereby this product is exported to the outside of the apical membrane and incorporated into the growing cuticle is not known.

Pesticide induced alterations in gene expression in the lobster, Homarus americanus.

Using subtractive hybridization, we have identified 17 genes that are either up- or down-regulated in the hepatopancreas (Hp) of the lobster, Homarus americanus, by acute exposure to the juvenile hormone analog methoprene. The expression of some of the genes obtained from the subtraction libraries was confirmed by real time Q-PCR experiments. These genes encode several different classes of proteins including: structural, enzymatic and regulatory polypeptides. Enzymes represent the predominant genes up-regulated by methoprene. Included in this group are betaine-homocysteine S-methyltransferase (BHMT) and two other enzymes of the methionine cycle. Increased expression of a translation factor (eIF2), as well as of cytosolic (aldose reductase), structural (beta-tubulin, L5A) and plasma membrane (CD42d) proteins was observed. In addition, a major feature of altered gene expression in methoprene treated Hp was increased levels of enzymes associated with protein turnover, including trypsin, ubiquitin conjugating enzyme and ubiquitin carboxyl terminal hydrolase. Down-regulation of the members of the hemocyanin family was observed. Assays confirmed elevated levels of trypsin in the Hp of lobsters after 24 h exposure to methoprene. Our findings suggest a wide variety of cellular targets are altered by methoprene.

Biochemical Effects of Diflubenzuron on Chitin Synthesis in the Postmolt Blue Crab Callinectes Sapidus

ABSTRACT In vivo and in vitro metabolic studies were conducted on the effects of the insect growth regulator diflubenzuron (DFB) on chitin synthesis in the postmolt blue crab Callinectes sapidus. The effects of the pesticide on the incorporation of either 3H-glucosamine or 3H-N-acetylglucosamine into sodium dodecyl sulfate (SDS) insoluble cuticular residue were examined. The radiolabeled product formed was identified as chitin by chemical and enzymatic criteria. 3HN-acetylglucosamine was found to be incorporated to a greater extent than 3H-glucosamine during metabolic studies. During in vitro explant culture experiments, the highest concentration of DFB tested (1 ppm) caused 98% inhibition of chitin synthesis; 64% inhibition was observed at concentrations as low as 0.7 parts per billion. The results indicate that diflubenzuron inhibits the incorporation of 3H-N-acetylglucosamine into cuticular chitin in postmolt blue crabs. The data are consistent with our previous autoradiographic and ultrastructural studies on the effects of DFB in the blue crab; i.e., that the Golgi complex is disrupted by DFB treatment.