Insulin-responsive compartments containing GLUT4 in 3T3-L1 and CHO cells: regulation by amino acid concentrations.

In fat and muscle, insulin stimulates glucose uptake by rapidly mobilizing the GLUT4 glucose transporter from a specialized intracellular compartment to the plasma membrane. We describe a method to quantify the relative proportion of GLUT4 at the plasma membrane, using flow cytometry to measure a ratio of fluorescence intensities corresponding to the cell surface and total amounts of a tagged GLUT4 reporter in individual living cells. Using this assay, we demonstrate that both 3T3-L1 and CHO cells contain intracellular compartments from which GLUT4 is rapidly mobilized by insulin and that the initial magnitude and kinetics of redistribution to the plasma membrane are similar in these two cell types when they are cultured identically. Targeting of GLUT4 to a highly insulin-responsive compartment in CHO cells is modulated by culture conditions. In particular, we find that amino acids regulate distribution of GLUT4 to this kinetically defined compartment through a rapamycin-sensitive pathway. Amino acids also modulate the magnitude of insulin-stimulated translocation in 3T3-L1 adipocytes. Our results indicate a novel link between glucose and amino acid metabolism.

Mousepox outbreak in a laboratory mouse colony.

Mousepox was diagnosed in and eradicated from a laboratory mouse colony at the Naval Medical Research Institute. The outbreak began with increased mortality in a single room; subsequently, small numbers of animals in separate cages in other rooms were involved. Signs of disease were often mild, and overall mortality was low; BALB/cByJ mice were more severely affected, and many of them died spontaneously. Conjunctivitis was the most common clinical sign of disease in addition to occasional small, crusty scabs on sparsely haired or hairless areas of skin. Necropsy findings included conjunctivitis, enlarged spleen, and pale liver. Hemorrhage into the pyloric region of the stomach and proximal portion of the small intestine was observed in experimentally infected animals. In immune competent and immune deficient mice, the most common histologic finding was multifocal to coalescing splenic necrosis; necrosis was seen less frequently in liver, lymph nodes, and Peyer's patches. Necrosis was rarely observed in ovary, vagina, uterus, colon, or lung. Splenic necrosis often involved over 50% of the examined tissue, including white and red pulp. Hepatic necrosis was evident as either large, well-demarcated areas of coagulative necrosis or as multiple, random, interlacing bands of necrosis. Intracytoplasmic eosinophilic inclusion bodies were seen in conjunctival mucosae and haired palpebra. Ectromelia virus was confirmed as the causative agent of the epizootic by electron microscopy, immunohistochemistry, animal inoculations, serologic testing, virus isolation, and polymerase chain reaction. Serologic testing was of little value in the initial stages of the outbreak, although 6 weeks later, orthopoxvirus-specific antibody was detected in colony mice by indirect fluorescent antibody and enzyme-linked immunosorbent assay procedures. The outbreak originated from injection of mice with a contaminated, commercially produced, pooled mouse serum. The most relevant concern may be the unknown location of the source of the virus and the presence of a reservoir for this virus within the United States.

Effect of lactacidosis on pyridine nucleotide stability during ischemia in mouse brain.

Brain levels of NADH and NAD+ were measured in three models of cerebral ischemia to determine whether degradation of the pyridine nucleotides is enhanced in models that generate high concentrations of lactic acid. Complete ischemia (decapitation), in which lactate increased to 14 mmol/kg, caused a gradual decrease in the NAD pool to 50% of control by 2 h. During focal ischemia (occlusion of the middle cerebral artery), the decrease in the NAD pool was less pronounced (82% of control at 2 h) despite the accentuated accumulation of lactate to 33 mmol/kg. In a third model (unilateral hypoxia-ischemia), pretreatment of animals with glucose augmented the ischemic elevation of lactate from 30 mmol/kg to 40 mmol/kg and greatly impaired restoration of energy metabolites during recirculation. However, glucose pretreatment had no effect on the size of the NAD pool during ischemia or early recovery. These results, therefore, demonstrate that the pyridine nucleotide pool is not rapidly degraded during ischemic insults that accumulate high concentrations of lactic acid. The stability of the NAD pool may have been enhanced by the limited increase in brain levels of NADH that occurred in these models of incomplete ischemia.

Effect of glucose on recovery of energy metabolism following hypoxia-oligemia in mouse brain: dose-dependence and carbohydrate specificity.

Unilateral cerebral hypoxia-oligemia was produced in anesthetized mice using carotid artery occlusion combined with systemic hypoxia (10% O2). In the cerebral cortex ipsilateral to the carotid occlusion, ATP levels were depleted during a 30-min insult, but were restored to 64% of control during 60 min of recovery. Pretreatment of animals with glucose diminished the restoration of ATP in a dose-dependent manner. Thus, when blood glucose levels exceeded 12-13 mM (225 mg/dl), ATP recovery was greatly impaired. Neither galactose nor 3-O-methylglucose mimicked the detrimental effect of glucose. However, pretreatment with mannose, which is readily metabolized by brain, impaired restoration of ATP. The impairment, therefore, appears to be specific for substrates of cerebral metabolism. The ischemic accumulation of lactate in the ipsilateral cortex was augmented by only 30% at blood glucose levels well above the threshold for ATP recovery. Thus, unless recovery of energy metabolism is sensitive to small increments in brain lactate, it is difficult to explain the glucose-induced energy failure on the basis of enhanced lactic acidosis. Ipsilateral cerebral blood flow (CBF), measured with [14C]iodoantipyrine during hypoxia and recovery, was lower in glucose-pretreated than in saline-pretreated animals. However, the poor correlation between CBF and ATP, measured in the same tissue samples at 15 min recovery, failed to substantiate that regeneration of ATP was flow-limited early in recovery.

Immunoglobulin determinants on the surface of mouse splenic lymphocytes from Trypanosoma musculi-infected mice.

The splenic B-lymphocyte population of C57BL/6 mice was analyzed by flow microfluorometry to determine the relative density of surface immunoglobulin (sIg) during the course of infection with Trypanosoma musculi and after the injection of T. musculi derivatives. Cells were stained with fluorescent-conjugated antisera directed against 7S mouse immunoglobulins or the major sIg components, IgM and IgD. Evaluation of relative density of sIg was made through observation of histograms plotted by the Fluorescence Activated Cell Sorter (FACS). The FACS also provided the percentage of fluorescence-positive cells detected with each stain. The relative density of sIg was altered in all experimental groups as evidenced by abnormal histograms. These alterations in relative density of sIg persisted only on B cells of the trypanosome-infected and not the T. musculi-derivative-treated animals. The changes in sIg presumably resulted from sIgD modulation, because the histograms resulting from cells stained with anti-IgM remained unchanged. In the trypanosome-infected animals, there also was a reduction in the percentage of sIgD-positive cells near the end of the parasitemia. Observations of spleen sizes showed the same pattern of change with splenomegaly as with relative density of sIg. These data may be the result of the persistence of the parasite in host tissues after clearance of the blood infection.

Prostaglandin I2, indomethacin, and heparin promote postischemic neuronal recovery in dogs.

Forty-five conditioned male mongrel dogs were exposed to multifocal ischemia sufficient to maintain suppression for 60 minutes of the P1-N1 amplitude of the cortical sensory evoked response (CSER), a quantifiable index of neuronal function. Ischemia was induced and regulated by successive embolization of 20 to 50 microliters increments of air via the right internal carotid artery. Subsequently, the P1-N1 amplitude recovery of the CSER was followed for an additional 15, 60, or 120 minutes while the dogs were treated or left untreated. The combination of prostaglandin I2 (PGI2), indomethacin, and heparin promoted a statistically significant augmentation of return of CSER amplitude relative to no treatment, PGI2 alone, indomethacin alone, PGI2 and heparin, indomethacin and heparin, or PGI2 and indomethacin. After 60 minutes of recovery, animals receiving combined PGI2, indomethacin, and heparin achieved a 57% recovery of P1-N1 amplitude relative to baseline, while the corresponding recoveries in all other groups clustered around 20%. By 120 minutes of postischemic follow-up, the CSER recovery induced by PGI2, indomethacin, and heparin was 80% compared to 17% in untreated animals. By 15 minutes into the recovery period, the combination of the three agents had eliminated very low flows in the "neuron-disabling" range (defined as 0 to 15 ml/100 gm/min for gray matter and 0 to 6 ml/100 gm/min for white matter) in contrast to the relative inefficacy of no treatment or treatment with other than the triple combination of drugs. The study lends some support to a planned clinical trial of PGI2, indomethacin, and heparin in acute occlusive stroke in humans.

Preparation of microbiological specimens for scanning electron microscopy.

This tutorial paper describes techniques for processing microorganisms for scanning electron microscopy (SEM). For ease of discussion, the subject is divided into two sections: A. General Processing Requirements, and B. Specific Processing Techniques. The objective of the first section is to outline processing requirements, explain their purpose, and point out where variations are possible. The following basic steps are discussed: (1) specimen selection, (2) prefixation treatment, (3) chemical fixation, (4) dehydration, (5) critical-point drying and freeze-drying, (6) coating, and (7) viewing. The second section describes methods of manipulating microorganisms through the processing steps. Instructions will cover microorganisms processed for SEM (1) in suspension, (2) in tissues. (3) in tissue culture, and (4) on agar. We are relying heavily on our own experiences in the laboratory and the results are illustrated by use of bacteria, mycoplasmas, rickettsiae, fungi, free-living protozoa, and parasitic protozoa. This tutorial is intended to be a general reference for processing microorganisms for study with the scanning electron microscope. Although we have described basic requirements and several specific techniques, it must be emphasized that there is a wide range of preparation flexibility permissible, depending upon the objectives of the study.

Microscopic and ultrastructural characteristics of Kudoa infection in a butterfly fish (Chaetodon).

Myxosporidiosis of the skeletal muscle was diagnosed in a pearl scale butterfly fish (Chaetodon). On the basis of light and electron microscopy, the infectious agent was thought to be a Kudoa. The muscle had fusiform cysts containing myxosporidian organisms within hypertrophied fibers. Ultrastructural features of the kudoa organisms were four external shell valves joined by sutural planes. Internally, four pyriform polar capsules with polar filaments were anterior to the sporoplasm.

Heterakidosis and nodular granulomas caused by Heterakis isolonche in the ceca of gallinaceous birds.

Five pheasants and two ocellated turkeys from the San Diego Zoological Garden had multiple cecal nodules. These nodules were composed mostly of granulomas and fibrous hyperplastic tissue associated with Heterakis isolonche.

Amebiasis in goldfish.

Five cases of amebiasis were diagnosed in goldfish (Carassius auratus) from home aquariums and from a laboratory aquarium. Granulomas containing amoebae were in many organs but were most numerous in kidneys. Because there were pseudopods, food vacuoles, vesicular nucleoli and other ultrastructural characteristics of the organisms, we identified the organisms as amoebae. On the basis of mitotic stages it is possible they belong in the family Hartmannellidae.

Twenty-four hour membrane oxygenation in dogs without anticoagulation.

Is systemic heparinization or heparin-bonded circuitry better than no anticoagulation during 24 hours of cardiopulmonary bypass? We compared blood pressure, coagulation state, oxygenator function, and scanning electron microscopic appearance of the circuits. There were three groups of five dogs each: Group I had no anticoagulants; Group II received systemic heparization; Group III perfusions utilized heparin-bonded circuits. Group I animals all survived, whereas 80 percent (four fifths) in Group II and 20 percent (one fifth) in Group III survived. Arterial pressures were better maintained in Group I as compared to Groups II and III. The coagulation parameters were similar in all groups. Oxygenator function was maintained at normal in all groups. No thrombi were present in any of the circuits following perfusion. The surfaces in Group I had less debris on them compared to Groups II and III. Animals that died had fibrin thrombi present in tissues examined histologically. Systemic heparinization had no advantage over no heparin in this study. The striking similarity of the coagulation state between Groups I and II and better preservation of the surfaces in Group I were unexpected. Heparin-bonded circuits were unsatisfactory when compared to no anticoagulation and systemic heparinization. Additional experiments with various species with and without anticoagulation must be done to determine the best guidlines for human cardiopulmonary bypass.

The relationship of circulating endogenous endotoxin to hemorrhagic shock in the baboon.

Experiments were carried out to test the hypothesis that during hemorrhagic shock endotoxin enters the circulation from ischemic bowel by way of the portal venous system and is then associated with irreversibility of the hemorrhagic shock state. After placement of sampling catheters in the portal vein, right atrium, and aorta, 14 awake, restrained baboons were subjected to 1 hour of hemorrhagic shock at a mean arterial pressure (MAP) of 60 torr followed by a second hour at 40 torr MAP. Six animals were resuscitated with Ringers lactate and their shed blood; 8 were maintained hypotensive until death. Serial blood samples were analyzed for the presence of endotoxin. Endotoxemia was found infrequently, with no greater incidence (p > 0.6) in portal venous samples than in systemic blood, so these data were pooled for further analysis. Furthermore, endotoxemia was no more frequent (p > 0.6) late in shock than it was in early shock or during the baseline period. Autopsy showed no evidence of ischemic damage to the splanchnic viscera. It was concluded that spontaneous endogenous entotoxemia is not a common feature of hemorrhagic shock in baboons and is not related to the duration or degree of severity of hemorrhagic shock in this subhuman primate species.

Ultrastructure of Ehrlichia canis.

The ultrastructure of Ehrlichia canis was examined in both pulmonary mononuclear cells and in monocytes cultured from an infected dog. The cytoplasmic inclusions, or morulae, of E. canis consisted of a membrane-lined vacuole-containing elementary bodies which varied in size and number. The elementary bodies were bound by two trilamellar membranes. The organism shared morphological properties of both the genus Rickettsia and genus Chlamydia.