Philip M. Grimley

Histology and ultrastructure of carotid body paragangliomas. Comparison with the normal gland

Several surgically extirpated human carotid bodies and three paragangliomas of the carotid body region were compared by means of light and electron microscopy. Chief cells, both normal and neoplastic, are generously endowed with “dense‐cored” cytoplasmic granules of the catecholamine type. The granules appear to develop in the Golgi region. In two of the tumors, an increased level of norepinephrine was detected by fluorimetric assay. Sustentacular (Schwannlike) cells are a constant parenchymal element in the normal glands and serve to convey unmyelinated nerve fibers into direct synapse with chief cells. All of the tumors lacked nerve endings, but sustentacular forms persisted as a prominent element in two cases. Evidence of neurosecretion in paragangliomas, despite lack of chromaffinity, indicates that the dichromate reaction is an inadequate substitute for biochemical and ultrastructural classification of neuroendocrine cells. The functional significance of secretion granules in normal chief cells remains to be elucidated.

Protein Synthesis: Differential Stimulation of Cell-Specific Proteins in Epithelial Cells of Chick Oviduct

Immunofluorescent and radioautographic studies demonstrate that ovalbumin and avidin are cell-specific proteins synthesized by different epithelial cells in the chick oviduct mucosa. The mechanism of the selective induction of ovalbumin synthesis by estrogen and of avidin synthesis by progesterone may be through stimulation of specific target cells by these hormones.

Preparation of large epoxy sections for light microscopy as an adjunct to fine-structure studies.

Tissue blocks 2 × 2 × 0.4 cm were fixed 6-24 hr in phosphate-buffered 5% glutaraldehyde then sliced to 2 × 2 × 0.1 cm and soaked in 0.1 phosphate-buffer (pH 7.3) for at least 12 hr. Fixation was continued for 2 hr in phosphate-buffered 1-2% OsO4. The slices were dehydrated, infiltrated with Araldite, and embedded in flat-bottomed plastic molds. Sectioning at 1-8 μ with a sliding microtome was facilitated by addition of 10% dibutylphthalate to the standard epoxy mixture. The sections were spread on warm 1% gelatin and attached to glass slides by drying, baking at 60 C, fixing in 10% formalin or 5% glutaraldehyde and baking again. Sections were mordanted in 5% KMnO4 (5 min), bleached with 5% oxalic acid (5 min) and neutralized in 1% Li2CO3 (1 min). Several stains could then be applied: azure B, toluidine blue, azure B-malachite green, Stirlings gentian violet, MacCallums stain (modified), tribasic stain (modified) and phosphotungstic acid-hematoxylin. Nuclei, mitochondria, specific granules, elastic tissu...

Selection for Electron Microscopy of Specific Areas in Large Epoxy Tissue Sections

Tissue blocks 2 × 2 × 0.4 cm were fixed 6-24 hr in phosphate-buffered 6% glutaraldehyde then sliced to 2 × 2 × 0.1 cm and rinsed in phosphate buffer for at least 12 hr. Fixation was continued for 2 hr in phosphate-buffered 1-2% OsO4. The slices were dehydrated, infiltrated with Araldite, and embedded in flat-bottomed plastic molds. Sectioning at 4-8 μ with a sliding microtome was facilitated by addition of 10% dibutylphalate to the standard epoxy mixture. The sections were spread on water and attached to coverslips by drying, then heating to 80 C for 1 min. Staining 2 min with 1-3% KMnO4 and temporary mounting in glycerol on a slide allowed the desired area for electron microscopy to be selected and marked. This area was then cemented to the facet of a conventional epoxy casting with a drop of epoxy resin (without added dibutylphthalate). After polymerization, the coverslip was removed by quick cooling leaving a flat re-embedded portion of the original section. This portion was viewed by transillumination...

Ultrastructure of the Human Carotid Body A Perspective on the Mode of Chemoreception

Surgically excised human carotid bodies have been analyzed by electron microscopy. Several ultrastructural features suggest that they are closely related to sympathetic neuroendocrine glands and autonomic ganglia: the chief cells synthesize intensely osmiophilic cytoplasmic granules, occasionally develop neuroid processes, and are intimately associated with neurilemmal sustentacular cells. Other lines of evidence indicate that secretion of the chief cell is norepinephrine or a closely related bioamine-neurotransmitter. The role of chief cells in mediating chemosensation may be explained by modulated release of the bioamine in response to local metabolic conditions. Human chief cells are richly innervated. Recognizable zones of synapse display an efferent type of polarity and sometimes appear adrenergic. Thus, reflexive sympathetic excitation of chief cells may be physiologically significant in man. The anatomic compartmentalization characteristic of carotid body chief cells is discussed in relation to a possible functional significance.

A Tribasic Stain for Thin Sections of Plastic-Embedded, OsO4-Fixed Tissues

Thin (0.5–1 μ) sections of plastic-embedded, OsO4-fixed tissues were attached to glass slides by heating to 70 C for 1 min. A saturated solution combining toluidine blue and malachite green was prepared in ethanol (8% of each dye) or water (4% of each dye). Methacrylate or epoxy sections were stained in the ethanol solution for 2–5 min. The water solution was more effective for some epoxy sections (10–80 min). Epoxy sections could be mordanted by 2% KMnO4, in acetone (1 min) before use of the aqueous dye, reducing staining time to 5–10 min and improving contrast. Aqueous basic fuchsin (4%) was used as the counter-stain in all cases; staining time varied from 1–30 min depending upon the embedding medium and desired effects, methacrylate sections requiring the least time. In the completed stain, nuclei were blue to violet; erythrocytes and mitochondria, green; collagen and elastic tissue, magenta; and much and cartilage, bright cherry red. Sections were coated with an acrylic resin spray and examined or pho...

Assembly of virus particles during mixed infection with wild-type vaccinia and a rifampicin-resistant mutant

Abstract A rifampicin-resistant vaccinia virus mutant was isolated from wild-type virus stocks. The mutant was purified by sucrose density gradient centrifugation and had an infectivity, electron microscopic appearance, and polypeptide composition that was similar to the wild-type virus. Growth of the mutant was inhibited only slightly at concentrations of rifampicin which completely prevented the growth of drug-sensitive virus, and at no concentration tested was there a detectable effect on viral envelope formation. The mutant virus was capable of rescuing wild-type during mixed infection in the presence of rifampicin; after simultaneous infection the ratio of genomes in the progeny reflected that of the original inoculum. Superinfection experiments demonstrated that beat-inactivated mutant was capable of efficiently rescuing wild-type virus and that extensive replication of the mutant genome was not required for this. Characteristic rifampicin blocked viral forms, normal maturing forms, and intermediate structures were seen in the same ultrathin cell sections after simultaneous infection with low multiplicities of mutant and wild-type virus. In cells simultaneously infected with high multiplicities of mutant and wild-type virus, rifampicin had no detectable effect on viral envelope formation. No difference was found in pulse-labeled polypeptides synthesized in rifampicin-treated cells infected with either mutant virus, wild-type virus, or both when analyzed by SDS-polyacrylamide gel electrophoresis. After a 4-hr chase, cleavage of the high molecular weight precursors of structural polypeptides was inhibited in cells infected with wild-type virus but occurred to similar extents in cells infected with mutant alone and cells simultaneously infected with mutant and wild-type virus.

Toxoplasma gondii and cytomegalovirus: mixed infection by a parasite and a virus.

Human fibroblasts infected in vitro with cytomegalovirus are relatively resistant to infection by Toxoplasma gondii during the first 4 days of virus infection. After 5 days, however, the cytomegalovirus-infected cells become susceptible to the parasites. The toxoplasmas replicate in paracentral rosettes surrounded by host cell mitochondria. This growth configuration differs from that seen in human fibroblasts infected in vitro with toxoplasma only but resembles the pattern seen in doubly infected cells found in human necropsy tissue.

Assembly of vaccinia virus particles from polypeptides made in the presence of rifampicin

Abstract Vaccinia viral polypeptides, labeled in the presence of rifampicin, were incorporated into virus particles after rifampicin was removed. All structural polypeptides, resolved by polyacrylamide gel electrophoresis, were labeled in the presence of rifampicin. The specific effect of rifampicin on the formation of the vaccinia viral envelope was reversed within 10 min even in the presence of NaF or inhibitors of protein or nucleic acid synthesis. Further maturation of some virus particles, which were characterized by their ultrastructure and isolated by sucrose gradient sedimentation, proceeded in the presence of inhibitors of protein synthesis. In contrast, addition of NaF or actinomycin D almost completely blocked the development of mature virus particles. Rifampicin acted specifically at the stage of envelope formation and did not prevent later steps in maturation.

Ultrastructure of a rat cytomegalovirus

Abstract A cytomegalovirus isolated from Panamanian rats was examined by electron microscopy. Capsid structure of the virions in negatively stained preparations appeared identical to human cytomegalovirus and other viruses of the herpes family. In thin sections, the naked nucleocapsid measured slightly larger (138 nm) than herpes-type and cytomegaloviruses previously reported. Development of the virus was observed in rat kidney and hamster kidney cell cultures. Periodic fibrils were observed in the nuclei of infected rat kidney cells, and virus particles accumulated in unusually large cytoplasmic aggregates.