Bruce F. Giffin

Hepatic lobular patterns of phosphoenolpyruvate carboxykinase, glycogen synthase, and glycogen phosphorylase in fasted and fed rats.

The goal of this study was to localize phosphoenolpyruvate carboxykinase (PEPCK), glycogen synthase (GS), and glycogen phosphorylase (GP) in the liver lobule by immunocytochemical techniques and to describe the effects of feeding and fasting on the distribution and quantity of these enzymes. Livers from ad lib fed and overnight fasted normal adult male rats were frozen in liquid nitrogen after transcardial perfusion with 30% sucrose. Serial cryostat sections of tissue were collected on slides, fixed by immersion in 4% paraformaldehyde, and incubated with antibodies against PEPCK, GS, and GP. Antibodies to these enzymes were visualized with a gold-conjugated secondary antibody and a silver enhancement technique. Fed animals demonstrated a periportal to pericentral gradient of PEPCK. Fasting increased the periportal content of PEPCK, induced the midlobular and centrilobular cells to express the enzyme, and steepened the periportal to pericentral gradient. The increase of PEPCK was confirmed by Western blot analysis. GS and GP were distributed throughout the lobule in the fed animal but often showed a centrilobular pattern, and fasting did not alter the lobular distribution of either enzyme. Western blot analysis revealed no changes in the amount of these enzymes in the fed or fasted state. The cellular distribution of the three enzymes is similar to that of hepatic glycogen, in that the immunoreactive material has a clumped appearance in the periportal hepatocytes and is more dispersed in the pericentral cells. On fasting the periportal hepatocytes lose the dense compact localization of the enzymes and the protein becomes more homogeneously distributed throughout the cytosol. Further studies are needed to elucidate the functional significance of the regional heterogeneity of the glycogen-metabolizing enzymes and the molecular mechanisms regulating their gene expression.

Gross anatomy of the head and neck and neuroscience in an integrated first‐year medical school curriculum

The curriculum for first year medical students at the University of Cincinnati has changed. Beginning in the fall of 1998, material in the first year was presented in an Integrated Educational Program. The goal of this program was to provide students with an understanding of the normal structure, function, and development of the human body. The purpose of this report is to discuss the unique integration that occurs in a block offered during the Spring Quarter. The two components of this block are Gross Anatomy of the Head and Neck and Brain and Behavior I. Brain and Behavior I is a new offering combining neuroanatomy, neurophysiology, neurology, and a psychiatry/behavioral component. The unique combinations offered in this block are logical, educationally sound, and have been enthusiastically received by both the students and faculty. Anat Rec (New Anat) 261:89–93, 2000.

Effect of putrescine on the respiration of Trypanosoma brucei brucei

When bloodstream forms of Trypanosoma brucei brucei were exposed to exogenous putrescine for 24 h during in vitro culture, the rate of O2 consumption increased significantly in a concentration-related and time-dependent manner. Trypanosomes cultured with 100 microM DL-alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, were depleted of intracellular putrescine, and the rate of O2 consumption decreased by more than 50%. This effect could be abrogated if 100 microM putrescine was also present. A similar pattern was observed in trypanosomes harvested from rats after 36 h of DFMO treatment. If such trypanosomes were placed in culture for 2 h with 100 microM putrescine, the rate of O2 consumption returned to that of controls. When an intraperitoneal injection of putrescine was given to infected rats 18 h after commencement of DFMO treatment, rates of O2 consumption in the trypanosomes were found to return to control values. The addition of putrescine, spermidine or Mg2+ did not affect rate of O2 consumption in enriched mitochondrial preparations. However, when putrescine was present throughout the preparation of mitochondrial fractions, there was an increase of 23% in O2 uptake, which was 23% higher than in the controls. Putrescine may modulate trypanosomal respiration by stabilizing mitochondrial membranes.

Immunogold-silver staining and epipolarized light microscopic detection of phosphoenolpyruvate carboxykinase and glycogen phosphorylase in rat liver

The subcellular distribution of enzymes related to carbohydrate metabolism was determined in sections of paraformaldehyde fixed and polyethylene glycol-1540-embedded rat liver and in cryostat sections. For this purpose, goat anti-rat phosphoenolpyruvate carboxykinase (PEPCK) serum and rabbit anti-rat glycogen phosphorylase (GP) serum were used as primary antibodies to localize the corresponding antigens. The primary antibodies were localized by 5 nm colloidal gold labeled secondary antibodies (either rabbit anti-goat IgG for PEPCK or goat anti-rabbit IgG for GP), and the gold particles were enhanced by silver staining using appropriate development reagents. The silver enhanced gold particles were detected by epipolarized light microscopy. PEPCK and GP immunoreactive molecules were found only in glycogen-containing areas of the cytosome of hepatocytes, and not in other cells. No immunocytochemical staining of hepatocytes was found when normal serum replaced the primary antibody in the procedures. Visio-Bond semithin (0.35–1.0 μm) sections provided higher resolution for subcellular immunostaining of PEPCK and GP than cryosections of 10 μm. Epipolarized light microscopy provided detection at high sensitivity of the gold-labeled antibody, and combined with transmitted light, allowed simultaneous visualization of the tissue morphology.

Enhancement of antigenic site detection with gold labeled secondary and tertiary antibodies using the immunogold-silver staining method

We report a modification of the immunogold-silver staining method (IGSS) for localizing hepatic phosphoenolpyruvate carboxykinase (PEPCK) in tissue sections, and we compare the efficacy of localizing the primary antibody with either a 5 nm gold labeled secondary antibody or 5 nm gold labeled secondary and tertiary antibodies. Light microscope examination of 10 microns frozen sections demonstrated that the use of combined secondary and tertiary gold labeled antibodies was superior to using a secondary gold labeled antibody alone. The increased labeling density (number of colloidal gold particles/antigenic site/cell) achieved by combined gold labeled antibodies was confirmed by electron microscopy. The increased labeling density resulted in a two-thirds reduction in the time needed for the IGSS physical development of the silver shells and less background. We achieved intense specific staining of hepatocytes expressing PEPCK while minimizing background staining. The use of combined secondary and tertiary gold labeled antibodies enhances the signal-to-noise ratio, achieves high resolution and is a suitable method for use in both light and electron microscopy.

The Role of Polyamines in the Growth and Transformation of the African Trypanosome

The transformation from bloodstream to procyclic trypomastigote is a crucial step in the life cycle of the African trypanosome. The alteration in cellular architecture is accompanied by biochemical changes which permit survival of the bloodstream form trypanosome in the midgut of the insect vector. While a specific inducer of transformation has not yet been identified, recent studies have implicated the polyamines as having some role to play in this process.

Successful antidote of multiple lethal infections using sustained delivery of difluoromethylornithine by means of ceramic drug delivery devices

The objectives of this study were (1) to cure multiple infections of trypanosomiasis in rats by the sustained release of DFMO from biodegradable tricalcium phosphate (TCP) and aluminum-calcium-phosphorous oxide (ALCAP) delivery systems, and (2) to determine if the side effects associated with oral administration of DFMO can be avoided by using TCP and ALCAP capsules. Sixty-eight SD male albino rats (235-270 g) were divided randomly into five groups. Each rat in group I (n = 16) was implanted subcutaneously (s.c.) with four TCP capsules (two large TCP (L-TCP), one PLA-impregnated large TCP (IL-TCP) and one thin TCP capsule (TN-TCP)). Rats in group II (n = 16) were implanted s.c. with four ALCAP ceramics (two large ALCAP (L-ALCAP), one PLA-impregnated large ALCAP (IL-ALCAP) and one thin ALCAP capsule (TN-ALCAP)). Rats in groups III (n = 16), IV (n = 4) and V (n = 16) were left without implants. Rats in group III (n = 16) were given 4% (w/v) DFMO (pH 7) in drinking water at the day of inoculation and continued up to 7 days postinoculation. Rats in group IV (n = 4) served as a nontreated group. Rats in group V (n = 16) served as normal controls. The results showed that all rats implanted with with TCP or ALCAP implants had no intoxications symptoms or side effects such as diarrhea during the treatment period. In contrast, rats given DFMO in drinking water exhibited foul-smelling diarrhea during the treatment period. Microscopic evaluation of blood smears collected from rats receiving DFMO chemotherapy showed an occasional or limited number of stumpy shape (SS) trypanosomes. This study suggests that (1) ceramic drug delivery systems are capable of delivering DFMO in a sustained manner for two months, and were able to cure repeated infections of trypanosomiasis; (2) the use of ceramic implants avoided widely fluctuating, irregular levels of DFMO in the body by keeping sustained levels above minimal effective concentrations; (3) ceramic drug delivery systems provide a pharmacological potentiality for drugs such as DFMO which have been withheld from the market because of severe side effects when administered using conventional methods of drug administration; and (4) DFMO-filled ceramic devices can be implanted subcutaneously in animals that face a threat of lethal protozoal infections in highly infested areas of the world.

Altered intracellular polyamines in bloodstream form Trypanosoma brucei brucei: transformation to procyclic trypomastigotes

A monomorphic strain of Trypanosoma brucei brucei (EATRO 110) was cultured as long slender bloodstream forms in vitro for 24 h with 100 microM Eflornithine HCl. This resulted in depletion of intracellular putrescine, a greater than 50% decrease in spermidine, and a cessation of cell division. These Eflornithine treated trypanosomes were stimulated to synchronously transform to procyclic trypomastigotes by transfer into SDM-79 medium containing the citric acid cycle intermediates citrate and cis-aconitate (CCA). Within 48 h morphological transformation was complete and occurred without any increase in cell numbers. The coadministration of 100 microM putrescine with Eflornithine prevented the depletion of putrescine and abrogated the cytostatic effect of Eflornithine alone, but did not prevent the transformation to procyclic trypomastigotes. Eflornithine-induced short stumpy form trypanosomes resulting from 4 days of culture with 100 microM Eflornithine did not transform to procyclic trypomastigotes in SDM-79 medium unless it contained CCA. We conclude that the high concentration of CCA can trigger transformation in all morphological types of bloodstream form trypanosomes, regardless of their threshold of sensitivity for the stimulus. Furthermore, the CCA-stimulated transformation is not dependent on putrescine and can occur independently of cell division.

Optimal visualization of immunogold-silver staining of hepatic PEPCK with epipolarized light microscopy.

We used immunogold-silver staining to localize phosphoenolpyruvate carboxykinase in 10 microns cryosections of 4% paraformaldehyde perfusion-fixed normal male rat liver. The resolution and sensitivity of detection were improved by epipolarized light microscopy of 0.5 microns semi-thin plastic sections prepared from these pre-embedding immunogold-silver-enhanced 10-microns thick cryosections. Epipolarized light combined with transmitted light simultaneously demonstrated antigenic sites (visualized with epipolarized light illumination) and tissue morphology (revealed by transmitted light). To optimize the conditions for high resolution, an oil immersion objective lens (x 100) with adjustable iris diaphragm was used with different intensity settings for both light sources. Our observations indicate that if the intensity of the transmitted light is too high, the visibility of the gold-cored silver grains by epipolarized illumination is decreased; if the intensity of epipolarized light is too strong, haloes appear around the gold-cored silver particles. By adjusting the aperture in the objective lens and the neutral density filter in the transmitted light pathway to balance the intensities of transmitted and epipolarized light, an optimal image is obtained that shows the maximal number of antigenic sites and excellent morphology.