Laser pressure catapulting followed by B actin gene identification in Japanese quail macrochromosomes and microchromosomes using teflon-coated coverslip slides.

Laser microdissection of individual mammalian chromosomes (> 2 microm) has been achieved though the use of a microscope slide coated with a polyethylene naphthalate (PEN) membrane. Although these slides have proved sufficient for larger chromosomes, they are insufficient for small chromosomes (< 1 microm). We have developed a new type of slide which allows laser microdissection of single Japanese quail microchromosomes (0.5 microm) and macrochromosomes (3-4 microm). To test the usefulness of these slides, a Japanese quail single nucleus, a macrochromosome, and a microchromosome were collected with Laser pressure catapulting, the B-actin gene was PCR amplified, and sequenced. The resulting PCR product was confirmed by nucleotide sequencing to be B-actin. These newly developed slides were shown to facilitate the laser microdissection of both Japanese quail macrochromosomes and microchromosomes.

Observations on the muscles of the eye of the bowhead whale, Balaena mysticetus.

The muscles of the eyelids and the extraocular muscles of mysticete whales are poorly described for a variety of reasons, including considerable difficulty in obtaining specimens. Our objective is to provide such a description for the bowhead whale, Balaena mysticetus. This study has examined the gross anatomy of the region in six specimens (five adults, one fetus) of the bowhead whale. Results show that the muscles associated with the eye are well developed and possess several distinctive features. For example, precise limits of each extraocular muscle are difficult to determine along their entire length because these muscles intermingle with one another near their insertion. Furthermore, some fibers from these muscles (except the retractor bulbi) also insert into the eyelids. Pulling on these muscles to simulate contraction results in movement of the eyelids and suggests a role for these muscles in palpebral retraction. Insertion of a large levator palpebrae superioris muscle into the upper eyelid further enhances opening of the palpebral fissure. Another unusual feature is the presence of tunnel-like structures that redirect the dorsal and ventral oblique muscles. The dorsal oblique muscle is redirected caudally about 90 degrees, then directed medially by another 90 degrees. These directional changes are accomplished via a connective tissue tunnel derived in part from the fibrous connective tissue of the dorsal rectus and the levator palpebrae superioris muscles. In most terrestrial mammals, a similar change in direction is accomplished by a cartilaginous trochlea. The ventral oblique muscle originates via a slender tendon from the frontal bone and undergoes a similar radical change in direction. Its tendon of insertion undergoes about a 90-degree change in direction that is accomplished through a tunnel-like structure derived from fibrous connective tissue of the ventral rectus muscle. Based on the morphology of the musculature presented, it is likely that the eyeballs and eyelids of the bowhead whale are quite mobile and appear capable of complex movement. The possibility of retraction and protrusion of the eyeball is discussed.

Effect of the antimicrobial peptide, D-hecate, on trichomonads.

Tritrichomonas foetus and Trichomonas vaginalis are protozoan parasites that cause sexually transmitted diseases in cattle and humans, respectively. There is a need for new antimicrobial agents to treat or prevent trichomoniasis because there are currently no approved chemotherapeutic agents against T. foetus and resistance of T. vaginalis to metronidazole does occur. Therefore, we evaluated the effect of a novel antimicrobial peptide, D-hecate, on the viability of 6 isolates of T. foetus and T. vaginalis in vitro. Tritrichomonas foetus and T. vaginalis were grown to mid log phase (24 hr) or late log/stationary phase (48 hr). Parasites at 10(6)/ml were mixed with equal volumes of D-hecate to final concentrations of 10 microM, 20 microM. and 40 microM of D-hecate. Controls had minimal essential medium (MEM) alone. The numbers of viable parasites were determined microscopically after 10, 20, and 30 min of incubation at 37 C with D-hecate or MEM. Our results show that D-hecate killed all 6 isolates of T. foetus and T. vaginalis evaluated. The killing effect was dependent on the concentration of the peptide, incubation time, and phase of growth of the parasites. Ultrastructural studies of parasites treated with 10 microM of D-hecate revealed extensive damage to the plasma membrane of most T. foetus and T. vaginalis cells, while a few cells were distorted but remained intact. D-Hecate may be a useful chemotherapeutic agent for the treatment of trichomoniasis.

Systematic development of a block copolymer adjuvant for trivalent influenza virus vaccine.

The current influenza virus vaccines induce systemic humoral immunity and short lived cellular immunity in young adults. Unfortunately these vaccines are only 50% efficacious in the elderly (> 65 years) and high risk groups of the very young. The use of a vaccine adjuvant to correct this deficit would therefore be very beneficial to these population groups. We have developed high molecular weight synthetic non-ionic block copolymers with adjuvant activity. These copolymers are compatible with, and active in, aqueous, physiological formulations in which they spontaneously assemble into 500-3000 nm particles. By varying both the molecular weight and the proportions of hydrophilic and hydrophobic components of the molecule, we have designed the optimal copolymer adjuvant for use with influenza hemagglutinin. This copolymer, termed CRL-1005, was investigated for its ability to augment the immune response of mice to the commercially-available human influenza vaccine, Fluogen. Co-formulation of CRL-1005 with the vaccine resulted in markedly increased antibody titres measured by both ELISA and the functional haemagglutination inhibition assay, indicating that critical immunogen epitopes were not destroyed. A single dose of copolymer and vaccine produced both long term rising antibody titres (six months) and primed for a potent secondary response. This high molecular weight copolymer is non-toxic and should therefore be well suited for widespread use.

Development of an adjuvant-active nonionic block copolymer for use in oil-free subunit vaccines formulations.

Nonionic block copolymers, synthesized from repeating units of oxypropylene and oxyethylene, can be designed so that individual copolymers have unique physical properties with differential levels of adjuvant activity. We have designed high molecular weight block copolymers that spontaneously assemble into 500 nm-3 mum particles when formulated with protein antigens in aqueous solutions at physiological pH. The adjuvant activity of one of these copolymers, termed CRL1005, was compared to selected research adjuvants using ovalbumin (OVA) as the prototype vaccine antigen. Suboptimal doses of OVA were formulated with complete and incomplete. Freund's adjuvant (CFA/IFA), alum Quil-A saponins Ribi Adjuvant System (RAS) or the CRL1005 copolymer and these formulations were used to immunize C57BL/6 mice. The CRL1005 copolymer appeared to be more potent than either Quil-A or alum and comparable to the RAS formulation, based on the numbers of responding mice and the OVA-specific antibody titers. Alum. RAS and Quil-A all augmented the production of IgG1 and IgG2l, similarly whereas only the CFA/IFA boosted IgG2a levels significantly. The effect of adjuvants on relative antibody affinity was more variable with the CRL1005 and CFA/IFA inducing antibodies with the highest affinity scores. This high molecular weight nonionic copolymer is nontoxic in aqueous formulations and should therefore be compatible with a wide variety of protein or polysaccharide vaccine antigens.

Observations on the external morphology and vasculature of a fetal heart of the bowhead whale, Balaena mysticetus.

BACKGROUND: Specialized demands within the aquatic environment for over some 60 million years have shaped unique morphological expressions in the whales, dolphins, and porpoises (Cetacea). Detailed consideration of these features, particularly in the great whales, has often been constrained by difficulties in securing adequate specimens for study. We had the opportunity to examine external heart morphology in a rarely obtained and prepared specimen from the bowhead whale, Balaena mysticetus. METHODS: The external morphology and in situ relations of a formalin-perfused heart were examined grossly in a near-term bowhead fetus. Latex injections assisted visualization of coronary vasculature. Magnetic resonance imaging was used to clarify heart positioning within the thoracic cavity in two younger (early and mid-gestational) intact fetuses. RESULTS: The heart was globular in form, with a blunt apex and wide base; it was laterally broad relative to height yet considerably compressed between nearly planar atrial (diaphragmatic) and auricular (sternocostal) surfaces. The heart constituted 0.01 of body mass in the near-term fetus. Within the thoracic cavity, the heart tilted forward on its long axis, placing the great basal vessels in the region of the thoracic inlet. The aorta extended forward from mid-base in parallel with the pulmonary trunk, arched sharply to the left, producing in succession the brachiocephalic trunk, left common carotid artery, and the left subclavian artery. Bifurcation of the brachiocephalic trunk yielded the right common carotid and right subclavian arteries. The distal portion of the aortic arch was linked to the pulmonary trunk via the ductus arteriosus. The aorta then swung caudally over the heart base, descending beneath the bodies of the thoracic vertebrae. The ascending aorta featured three bulbous sinuses immediately distal to the three semilunar cusps of the aortic valve. Originating along the distal boundaries of the left and right sinuses were the left and right coronary arteries. The arteries were similar in size and, because each sent contributions along their respective coronary and interventricular grooves, the heart can be described as bilateral relative to arterial supply. Anastomoses were common within and between the two arteries. Venous return from the heart was comprised of the great, middle, and right cardiac veins, all three converging in the coronary sinus. The right cardiac vein also included tributaries that emptied directly into the right atrium. CONCLUSIONS: External heart morphology in the fetal bowhead whale examined was distinguished by a laterally broad conformation with significant compression between its cranial and caudal surfaces. Aortic bulb configuration in combination with an expandable aortic arch may support blood service to the heart during diastole. Vascular service to the heart featured a complex vessel network with extensive intraarterial and intravenous anastomoses that enable many alternate blood perfusion pathways and may be adaptive to water-column-pressure fluctuations experienced by a large diving mammal.

Light-microscopic studies of 3T3 cell plasma membrane alterations mediated by melittin.

Various light microscopic techniques were used to study the effect of melittin, a major toxic constituent of honey bee venom, on plasma membranes of 3T3 mouse fibroblasts. Bright-field light microscopy and Trypan Blue dye exclusion were used to demonstrate changes in membrane permeability after exposure to melittin. Differential interference contrast (DIC) microscopy showed that membrane vesiculation induced by melittin was dose dependent. Using both fluorescent lipid and glycoprotein markers, we found that membrane vesicles were primarily composed of lipids. A sequence of events associated with vesicle formation was depicted by DIC and fluorescence microscopy. Confocal laser scanning fluorescence microscopy demonstrated a translocation of membrane glycoproteins from the plasma membrane to the cytosol following melittin treatment. The significance of membrane vesiculation and translocation of membrane glycoproteins in damaged cells is discussed.

Common epidermal lesions of the bowhead whale, Balaena mysticetus.

Samples of common skin abnormalities from 23 subsistence-harvested bowhead whales (Balaena mysticetus) were examined. Most lesions fell into three broad classes: shallow lacerations, circular depressions, and epidermal sloughing. Both circular depression lesions and epidermal sloughing lesions may be divided into more than one subgroup based on morphological criteria. Examination of each of the subgroups using light and scanning electron microscopy suggests relationships among the subgroups of a class. These proposed relationships are discussed, as are some possible etiologies. Scanning electron microscopy reveals abundant bacteria and diatoms present in association with each lesion class but no evidence of a particular association characteristic of a lesion class. The microflora were especially abundant wherever the stratum spinosum was exposed at the skin surface.

The morphological effects of two antimicrobial peptides, hecate-1 and melittin, on Escherichia coli.

The effects of the 26 amino acid, cationic, amphipathic, antibacterial peptide melittin and hecate-1, a 23 amino acid analog of it, on the gram negative bacterium Escherichia coli were investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and freeze-fracture. Both peptides killed virtually all bacteria at the peptide concentration and cell density used. TEM and SEM revealed aggregates of bacteria entangled with material extruded from the bacterial surfaces. SEM revealed irregular bacterial surfaces with bleb-like projections. TEM and freeze-fracture indicate that the bacterial inner and outer membranes, as well as the peptidoglycan layer between, were extensively damaged. The cytoplasmic contents of the cells, however, did not appear radically disturbed, providing little evidence for osmotically induced cytolysis.

Distribution and morphology of immunoreactive gonadotropin-releasing hormone (GnRH) neurons in the basal forebrain of ponies.

Recent reports have indicated that analysis of changes in the staining characteristics of gonadotropin-releasing hormone (GnRH) neurons and characterization of morphological plasticity of the related structural framework may help to elucidate the physiological mechanisms involved in neuroendocrine control of mammalian reproduction. Whether comparative studies will facilitate this process or simply elucidate species-specific mechanisms is not yet clear. The present study was performed in order to begin analysis of GnRH neurons in a seasonally breeding species that exhibits an unusually long ovulatory luteinizing hormone (LH) surge. To this end, light microscopy and image analysis were used to characterize distribution and morphology of GnRH neurons in 15 adult male and female ponies. Samples were collected in the middle of the normal ovulatory season. Unipolar, bipolar, and multipolar GnRH neurons were organized in a loosely defined continuum that extended from the medial septum to tuberoinfundibular areas in the medical basal hypothalamus (MBH). Most cells were bipolar, and the majority of neurons were located in the MBH. Fiber projections to the median eminence included presumptive pathways similar to those previously described in other species. Image analysis of cell size indicated that cells in the MBH were larger than those in preoptic areas and GnRH neurons in both of these locations were larger than neurons in rostral areas of the medial septum. Results from this experiment suggest that the large population of MBH GnRH neurons in the equine species is likely to be of primary importance to reproductive function, whereas cells in other areas are fewer and smaller. Further work is needed to characterize morphological characteristics that may be related to physiological fluctuations in reproductive function of the equine species.

Evaluation of Rhipicephalus sanguineus as a potential biologic vector of Ehrlichia platys.

The brown dog tick, Rhipicephalus sanguineus (Acari: Ixodidae), transmits several diseases among dogs including Ehrlichia canis infection. The role of Rhipicephalus sanguineus as a biologic vector for E platys, the rickettsial agent of infectious canine cyclic thrombocytopenia, was studied in dogs. Laboratory-cultured, pathogen-free nymph ticks were fed to repletion on dogs acutely infected with E platys. Tick engorgement coincided with the development of initial parasitemia and thrombocytopenia in the infected dogs. Following repletion, nymph ticks were allowed to molt under controlled conditions. One-month-old E platys-exposed adult ticks failed to infect naive dogs in animal transmission studies. The presence of E platys was not detected in midguts or salivary glands of similarly exposed adult ticks by use of light and transmission electron microscopy. These studies indicate that R sanguineus may not transmit E platys infection.

Bovine coronavirus antigen in the host cell plasmalemma.

Expression of bovine coronavirus (BCV) antigen in the plasmalemma of epithelioid human rectal tumor (HRT-18) and fibroblastic bovine fetal spleen (BFS) cell lines was traced by immunofluorescence and immunoelectron microscopy facilitated by colloidal gold. Cytoplasmic fluorescence was first observed at 12 hr postinfection (h.p.i) in infected HRT-18 cultures. This fluorescence coincided with the appearance of cell surface antigen reacting with colloidal gold-labeled antibodies to BCV antigens. At 24 h.p.i the amount of viral antigens at the surface of HRT-18 had increased, although cytoplasmic fluorescence remained constant. Infected BFS cells but not HRT-18 cells formed polykaryons when incubated in the presence of trypsin. One viral antigen in the plasma membrane of BFS cells was thus identified as the S glycoprotein with a fusion domain. In contrast to HRT-18 cells, the overall amount of BCV antigens at the surface of BFS cells remained constant after the onset of fusion. Analysis of the labeling characteristics established that the gold-marked-sites represented de novo expression of BCV antigen in the plasma membrane of infected cells.

Microanatomy of the lung of the bowhead whale Balaena mysticetus.

The lungs from six bowhead whales harvested by Alaskan Eskimos have been examined with light and electron microscopes. Airways ranging from 1 to 40 mm in luminal diameter are lined by a pseudostratified ciliated epithelium containing numerous mucus-secreting cells. The underlying lamina propria-tela submucosa of these airways contains tubuloalveolar glands, plasma cells, and lymphatic accumulations in addition to both elastic and collagenous fibrillar elements. Cartilage extends to the level of the respiratory airways, but smooth muscle is absent from airways larger than 3 mm, and tubuloalveolar glands are absent from airways smaller than 3 mm. Respiratory airways are lined by pseudostratified, simple cuboidal, and simple squamous epithelia. Alveolar ducts are lined by simple squamous epithelium exclusively. A connective tissue core composed mostly of elastic fibers supports the walls of the alveolar ducts. Neither smooth muscle nor cartilage has been observed in these structures. Alveoli contain the typeical cetacean double capillary bed separated by a thick septum composed mainly of collagenous connective tissue. Alveoli are lined by a simple squamous epithelium similar to that encountered in alveolar ducts and respiratory airways. This epithelium is composed of type I and II pneumocytes closely appressed to an underlying capillary network. The type II pneumocytes contain typical lamellar bodies and tubular myelin can be seen in the air spaces. The lung is surrounded by a thick (X = 2.5 mm) visceral pleura rich in blood vessels and elastic fibers.

Initial events in bovine coronavirus infection: analysis through immunogold probes and lysosomotropic inhibitors.

The early events in the infection of human rectal tumor cells by bovine coronavirus were investigated by colloidal gold-mediated immunoelectron microscopy and by analysis of the effect of lysosomotropic weak bases on virus yield. Electron microscopic studies revealed sites of fusion between the virus envelope and the plasmalemma but fusion events along intracellular membranes were not observed despite extensive searches. Virion-antibody-colloidal gold complexes were, in fact, endocytosed by synchronously infected cells. These complexes were apparently non-infectious, and they accumulated in vacuoles that resembled secondary lysosomes. Exposure of cells to ammonium chloride or to methylamine during the first hour of infection had little inhibitory effect on the production of infectious virus. Chloroquine treatments were inhibitory but this effect depended on relatively late events in the infectious process. The chloroquine inhibitory step blocked infection of virus absorbed to cells that were exposed to buffers in the pH range of 4.4 to 8.4. These findings indicate that BCV penetrates its host cell by direct fusion with the plasmalemma and does not require an acidic intracellular compartment for infectious entry.

Macroscopic, microscopic and ultrastructural findings in experimental haemarthrosis of rhesus monkeys.

The effects of a single episode of massive haemarthrosis in rhesus monkeys were studied morphologically. Autologous whole blood was injected into a femorotibial joint of 16 anaesthetized monkeys, equally divided into four groups and killed 7 days, 2, 3 and 6 months post injection (PI). Synovial membrane and articular cartilage were examined for macroscopic, microscopic and ultrastructural changes. Haemarthrosis was only evident in one monkey by 7 days PI. Slight yellow-brown discoloration of synovium and cartilage was evident in groups killed early after injection. Histologically, a hyperplastic and inflammatory reaction was present in the synovium at 7 days PI. Ultrastructural examination of synoviocytes in this group revealed numerous cytoplasmic vacuoles and prominent microplicae compatible with increased phagocytic activity. Erythrophagocytosis by synoviocytes was observed by light microscopy and confirmed by transmission EM. Results of scanning EM suggested that red cells might also pass through the synovial intima. Transmission EM also revealed mild degenerative changes in superficial chondrocytes. Rhesus monkeys reacted morphologically to haemarthrosis in the same way as dogs and rabbits, with mild morphological changes that resolved by 2 months PI.

The granulomatous inflammatory response in jirds, Meriones unguiculatus, to Brugia pahangi: an ultrastructural and histochemical comparison of the reaction in the lymphatics and peritoneal cavity.

A granulomatous inflammatory response develops in jirds with lymphatic or intraperitoneal infections of Brugia pahangi. Light, histochemical, and ultrastructural microscopy were used for comparative studies of the reactions in these 2 locations. The reactions observed were categorized into 3 types: (1) an initial response in which lymphocytes, monocytes, macrophages, and eosinophils were present; (2) an intermediate one which consisted of macrophages, epithelioid cells, lymphocytes, eosinophils, collagen, and mesothelial/endothelial cells with central areas of necrosis; and (3) a terminal reaction consisting of degenerating, necrotic cells. Microfilariae and adult worms were associated with these reactions. Macrophages were the predominant cell type in the lesion and were often found attached to the surface of the parasite. The inflammatory responses to B. pahangi in the lymphatics and in the peritoneal cavity appear to be similar, and thus, the peritoneal cavity may be useful in studying specific cell-parasite interactions to further define the pathogenesis of filarial disease.

3-Methylindole-induced nasal mucosal damage in mice.

3-Methylindole (3MI) damages nasal olfactory epithelium in mice. Lesions were studied histologically from 30 minutes to 28 days after intraperitoneal injection of 400 mg 3MI/kg. Cellular swelling was apparent in olfactory epithelium by 6 hours after injection of 3MI, while respiratory epithelium was normal. Necrosis of olfactory epithelium and subepithelial glands was diffuse by 48 hours. Subsequent ulceration resulted in epithelial hyperplasia, squamous metaplasia, fibroplasia, and ossification. Partially occlusive intranasal fibrous and osseous tissue persisted through 28 days after 3MI injection.

Comparative morphology of esophageal mucosa and submucosa in dogs from 1 to 337 days of age.

Scanning electron microscopy, light microscopy, and morphometric analyses were used to examine the morphology of the tunica mucosa-tela submucosa of the cervical, thoracic, and abdominal parts of the esophagus of healthy dogs from birth to 337 days of age from 3 litters of dogs whelped and reared under controlled conditions. Apart from the absence of a lamina muscularis mucosae from the cervical part of the esophagus of all dogs examined, little regional variation was detected. However, morphologic variation associated with age was observed. The number and complexity of microplicae on surface epithelial cells, as observed with the scanning electron microscope, increased with age, particularly between 1-day-old and 21-day-old dogs. Although scanning electron microscopy revealed typical duct openings from the submucosal glands in 1-day-old dogs, light microscopy revealed few functional glands. These glands gradually developed into a complex strata of tubules and acini during the time that secretory activity and the volume fraction of the tunica mucosa-tela submucosa occupied by glands increased with age. The presence of ciliated cells in the esophagus of 1-day-old dogs was apparent with scanning electron and light microscopes. Ciliated cells were always most abundant in the abdominal part of the esophagus. Finally, morphometric analyses revealed a significant increase in epithelial and connective tissue compartment thicknesses, as well as a marked increase in the volume fraction occupied by glands between 1 day and 161 days after dogs were whelped. A smaller decrease in all 3 measures occurred between 161 and 337 days after the dogs were whelped.

Chemical modulation of 3-methylindole toxicosis in mice: effect on bronchiolar and olfactory mucosal injury.

C57BL/6N mice were treated to induce tolerance, to modulate the mixed function oxidase system or to deplete glutathione (GSH) before injection with 400 mg 3-methylindole (3MI)/kg. Effect of pretreatment was determined by histologic comparison of pulmonary and nasal lesions 24 hours after 3MI. beta-Naphthoflavone and 3MI pretreatment significantly decreased 3MI-induced bronchiolar epithelial damage in male and female mice, while phenobarbital protection was significant only in female mice. Only beta-naphthoflavone decreased nasal olfactory epithelial damage. Pretreatment with piperonyl butoxide, SKF 525-A, or alpha-naphthoflavone had no significant effect on development of lesions. Diethylmaleate pretreatment significantly increased mortality and bronchiolar damage in both sexes. Significant differences between male and female mice were not detected in any group. The results suggest that pretreatment with low doses of 3MI or induction of cytochrome P-448 or P-450 protects against 3MI toxicosis while GSH depletion increases mortality and pulmonary lesions.