Kate M. Baldwin

Regulation of Midgut Growth, Development, and Metamorphosis

The insect midgut is an important site of entry for pathogens and insect control agents. This review focuses on recent information related to midgut epithelial growth, metamorphosis, and repair as a defense against pathogens. The roles of stem cell mitogens and differentiation factors are described. Included is a discussion of apoptosis and autophagy in the yellow body. Sloughing, also described, protects the midgut from virus infections and bacterial toxins through death and replacement of affected cells. The mechanisms by which the repair process reduces the effectiveness of pest control strategies are discussed. Primary tissue culture methods also are described, and their value in understanding the mechanisms by which biologically based insecticides work is discussed.

Identification of ten exon deleted ERβ mRNAs in human ovary, breast, uterus and bone tissues: alternate splicing pattern of estrogen receptor β mRNA is distinct from that of estrogen receptor α

Four different human tissues and breast cancer cell lines were screened to identify exon deletion variant transcripts of estrogen receptor β (ERβ) by reverse transcription‐polymerase chain reaction using the ‘splice targeted primer approach’ that amplifies each category of exon deleted variants as a separate gene population. A total of 10 different variant mRNAs that have deletions in various combination of exons were identified by sequence analysis. They were exon 2Δ; exons 2 and 5–6Δ; exon 3Δ; exon 4Δ; exon 5Δ; exons 5 and 2Δ; exon 6Δ; exons 6 and 2Δ; exons 6, 2–3Δ; and exons 5–6Δ. In some cases, deletion of an exon appears to be associated with a mutation of a specific base. Although ERα and ERβ are highly homologous, have identical exon and functional domain organization, exhibit similar ligand‐binding profiles and interact with identical DNA response elements, the sequence of exon skipping in ERβ pre‐mRNA appears to be distinct from that of ERα mRNA. Furthermore, results described here also suggest that alternate splicing of ERβ mRNA is tissue specific. The presence of a ERβ variant profile together with other ER isoforms in a tissue may have functional implications in binding and response to a particular ligand.

Estrogen receptors beta4 and beta5 are full length functionally distinct ERβ isoforms: cloning from human ovary and functional characterization

We describe here the cloning and functional characterization of two unique ER isoforms, ERβ4 and ERβ5. The full length ERβ4 and ERβ5 were identified by asymmetric PCR using human ovary cDNA, cloning, and sequence analyses. Both receptors share identical sequences with ERβ1 from exon 1 to exon 7. In the place of exon 8, ERβ4 has unique sequences arising from a region downstream of the ERβ gene and upstream of the SYNE2 gene. ERβ5 has sequences arising from retention of the 5′ end of the intron between exon 7 and 8. Both receptors bind promoter sequences on DNA but do not bind estrogen. They translocate to the nucleus and exhibit three to four times higher estrogen-independent transcriptional activity than ERβ1. When co-transfected with ERα, they predominantly form heterodimers and negatively regulate its transcriptional activity. Estrogen-independent transcriptional activity of ERβ5, but not ERβ4, was inhibited by ERα, demonstrating for the first time that ERα regulates ERβ. Tissue-specific expression of ERβ4 and ERβ5, together with their ligand-independent transcriptional properties and ERα modulating activities, could have a number of implications in seemingly unlinked biological processes regulated by estrogen.

The role of stem cells in midgut growth and regeneration

SummaryTheManduca sexta (L.) [Lepidoptera: Sphingidae] andHeliothis virescens (F.) [Lepidoptera: Noctuidae] midguts consist of a pseudostratified epithelium surrounded by striated muscle and tracheae. This epithelium contains goblet, columnar, and basal stem cells. The stem cells are critically important in that they are capable of massive proliferation and differentiation. This growth results in a fourfold enlargement of the midgut at each larval molt. The stem cells are also responsible for limited cell replacement during repair. While the characteristics of the stem cell population vary over the course of an instar, stem cells collected early in an instar and those collected late can start in vitro cultures. Cultures of larval stem, goblet, and columnar cells survive in vitro for several mo through proliferation and differentiation of the stem cells. One of the two polypeptide differentiation factors which have been identified and characterized from the culture medium has now been shown to be present in midgut in vivo. Thus the ability to examine lepidopteran midgut stem cell growth in vitro and in vivo is proving to be effective in determining the basic features of stem cell action and regulation.

Cell differentiation in the embryonic midgut of the tobacco hornworm, Manduca sexta

While the larval midgut of Manduca sexta has been intensively studied as a model for ion transport, the developmental origins of this organ are poorly understood. In our study we have used light and electron microscopy to investigate the process of midgut epithelial cell differentiation in the embryo. Our studies were confined to the period between 56 and 95 hr of embryonic development (hatching is at 101 hr at 25 degrees C), since preliminary studies indicated that all morphologically visible differentiation of the midgut epithelium occurs during this time. At 56 hr the midgut epithelium is organized into a ragged pseudostratified epithelium. Over the next 10 hr, the embryo molts and the midgut epithelium takes on a distinctive character in which the future goblet and columnar cells can be identified. With further differentiation, closed vesicles in the goblet cells expand and subsequently communicate to the outside by way of a valve. The columnar cells form numerous microvilli on their apical surfaces that extend over the goblet cells. Both cell types form basal folds from a series of plasmalemmal invaginations. Differentiation occurs concurrent with a six-fold elongation of these cells.

A novel occluding junction which lacks membrane fusion in insect testis.

Spermatocysts develop within the lumina of the lepidopteran testis. Each spermatocyst contains a clone of maturing germ cells which are separated from the fluid in the testicular lumen by a layer of somatic envelope cells. A blood-testis barrier is located at the level of the somatic envelope cells. We used macromolecular tracers horseradish peroxidase (applied before fixation) and ruthenium red (applied during fixation) with thin sections and freeze-fracture replicas to study the nature of this barrier in spermatocysts of the tobacco budworm, Heliothis virescens. Movement of the tracers into the spermatocysts was blocked by a structure at the outer edge of the septate junctions which join the spermatocyst envelope cells. In freeze-fracture replicas there was a P-face ridge or an E-face groove in this location. The ridge/groove appeared similar to a single-stranded vertebrate tight junction. Unlike tight junctions, however, there was no fusion or even close apposition of adjacent cell membranes in this location. We conclude, therefore, that a novel type of occluding junction was the barrier to paracellular movement of macromolecules in Heliothis spermatocysts.

Change of form of septate and gap junctions during development of the insect midgut

In insects, smooth septate junctions join cells derived from the embryonic midgut, and pleated septate junctions are found in all other tissues. Relatively little is known about either type of septate junction or the relationship between them, but they have been treated as two different junctions in the literature. The gap junctions which are associated with these septate junctions also differ. Crystalline gap junctions are found in the midgut, associated with smooth septate junctions, and irregular gap junctions are found in tissues where pleated septate junctions are located. We have examined the development of smooth septate junctions and crystalline gap junctions and the relationship between them, by studying the embryogenesis of the midgut in Manduca sexta (tobacco hornworm). At 56 hr of development (hatching is at 104 hr) pleated septate junctions and irregular gap junctions joined the midgut epithelial cells. At 65 hr, the septate junctions had disappeared, but gap junctions persisted. At 70 hr, smooth septate junctions had replaced the earlier pleated septate junctions and gap junctions associated with these smooth septate junctions were often of the crystalline form. In later embryos, the smooth septate junctions matured and enlarged, while all gap junctions became crystalline in form.

Freeze-fracture analysis of gap and septate junctions in embryos of a moth.

Gap and septate junctions were examined in embryos of Manduca sexta (tobacco hornworm). The junctions observed were similar in structure to those reported for adult insect tissues. In the epidermis typical pleated septate junctions were found. Associated with the pleated septate junctions were inverted gap junctions which had irregularly arranged particles and pits. In the midgut typical smooth septate junctions were found. Associated with these septate junctions were gap junctions which had regular hexagonal packing pattern. This codistribution of gap and septate junctions types is discussed in light of current theories that the gap junction types are alternative forms of the same structure in different metabolic environments. In addition to these gap and septate junctions a new junction, perhaps a modified septate junction, is described.

Evidence for high molecular weight proteins in arthropod gap and smooth septate junctions

The proteins that make up arthropod gap and septate junctions have not been identified with any certainty. Several candidate proteins for both types of junctions have been proposed in the literature, but there has been no agreement on any of these. Arthropod gap junctions do not label with antibodies to vertebrate gap junction connexins; it thus appears that unrelated proteins form these rather similar structures. Gap junctions inManduca sextamidgut epithelium are unusual since they function only during the molt and are non-functioning during the larval instars. We have developed a preparation from this tissue that is highly enriched in both gap and smooth septate junctions when examined by electron microscopy. SDS-PAGE gels of this preparation have two major protein bands, at 75 and 90 kDa. The presence of gap junctions correlates best with the 75 kDa protein and smooth septate junctions with the 90 kDa protein. Further, the 75 kDa band is stained by an antibody to a putative gap junction protein fromC. elegans. We propose that the 75 kDa protein is a major structural component of gap junctions inManduca sextamidgut epithelium and that the 90 kDa protein forms the smooth septate junctions.

Isolation of N-Acety-β-Hexosaminidase from Acanthamoeba castellanii

ABSTRACT. The lysosomal enzyme N‐acetyl‐β‐hexosaminidase (βhex) has been purified from Acanthamoeba castellanii growth medium by a three step procedure. The enzyme was precipitated with ammonium sulfate, partially purified on a DE52 column and purified to homogeneity on an affinity column. The purified βhex appeared to be a monomer with a molecular mass of 58 kDa and a pI of approximately 5.8. The enzyme activity in growth medium at RT was stable for several months. The purified βhex was enzymatically deglycosylated and injected, into two rabbits to make polyclonal antibodies. One antiserum was specific for βhex, but the other stained many bands on immunoblots of whole cell preparations. Using fluorescently labelled secondary antibodies we have determined that both antisera stain digestive vacuoles in the Acanthamoeba cytoplasm, and do not stain the contractile vacuole. The multi‐specific antiserum had high avidity for βhex, but also stained the carbohydrate portion of other molecules. These other molecules may be lysosomal enzymes as well, since the activity of several other lysosomal enzymes was partially immunoprecipitable with the antiserum. We plan to use these antibodies to study traffic patterns among the variety of vacuolar structures in Acanthamoeba cytoplasm.