Bruce F. Cameron

MORPHOLOGICAL EVIDENCE FOR THE PARTICIPATION OF MICROTUBULES IN AXONAL TRANSPORT

Fine structural studies have shown a uniform pattern of neuronal organization, in that the complex intracellular environment of the perikaryon is separated from morphologically well-characterized and often distant synaptic foci, by a tract of axoplasm that is structurally simple in its variety of components and notably possesses longitudinally oriented neurofilaments and microtubules set in an electron-lucent matrix. Studies on living neurones have indicated that the axoplasm is not only secreted within the cell body and passed slowly outwards, but that it is, in addition, the site of the varied traffic in which structural and molecular components are translocated at a range of speeds in excess of that of bulk centrifugal flow. Intracellular translocation of particulates is a widespread cytoplasmic activity, and the conservatism of evolution and specialization emerging from studies on subcellular structure and function prompts a search for a common mechanism underlying this activity. Experimental evidence from neurones and other cell types suggests that a salient function of cytoplasmic microtubules is the control and/or mediation of intracellular transport. The premise of this account is that functional coupling between translocated particulates and any axoplasmic component responsible for the movement and preserved as an identifiable system by present preparative techniques is likely to involve close physical juxtaposition. Accordingly, the distribution of membrane-limited inclusions with respect to neurofilaments and microtubules has been examined in detail, in thin sections of central axons of ammocoete larvae of the lamprey (Petromyzon marinus), to augment previous studies by a number of investigators on the nervous system of other animals. Subjective examination of electron micrographs may leave open the possibility of apparent association of axoplasmic components by fortuitous pairing in a randomly dispersed system, and the distribution of axoplasmic mitochondria was selected for more quantitative analysis by comparison between data obtained from micrographs and appropriately constructed computed “model axons.” It is suggested that the axoplasm is morphologically more organized than is sometimes supposed, and that this should be taken into account in attempts to identify the structural basis of neuronal function and especially of the logistic details of the physiological rapport between the cell body and its extensions.

Resting peripheral blood lactate elevation in survivors of prehospital cardiac arrest: Correlation with hemodynamic, electrophysiologic and oxyhemoglobin dissociation indexes☆

Thirteen patients who were survivors of sudden unexpected cardiac arrest in the community were followed up for up to 3 years. All showed an anomalous relation between erythrocyte levels of oxygen dissociation (P50) and 2,3-diphosphoglyceric acid (2,3-DPG). This could not be explained by hemoglobinopathy, carbon monoxide or methemoglobinemias. Because lactate accumulation in red blood cells may alter oxygen dissociation, whole blood and red blood cell lactate levels were measured. An average of 4.4 measurements per patient were obtained over a mean time of 5.6 months of the post-hospital phase of the follow-up period, which had a total mean duration of 22 months. The patients did not have overt congestive heart failure and were not acidotic (mean venous pH = 7.35). Lactate levels were elevated (mean = 15.1 mg/100 ml ± 0.8 mg/100 [standard error of the mean], compared with normal values of 7.6 mg/100 ml ± 1.4 mg/100 ml; P < 0.01). When lactate was plotted against red blood cell 2,3-diphosphoglycerate, a positive curvilinear relation was found (r2 = 0.12, P < 0.05). The production of lactate in chronic ischemia may increase red blood cell 2,3-diphosphoglycerate through glycolysis. The expected effect on oxygen dissociation of this increase in 2,3-diphosphoglycerate is offset by a counterbalancing leftward shift of the oxyhemoglobin dissociation curve by an increase in red blood cell lactic acid. When lactate was compared with left ventricular ejection fraction, there was a significant negative correlation (r = 0.86, P < 0.01). Serial 24 hour ambulatory electrocardiograms (mean 4 per patient) were analyzed for changes in quantity and severity of ventricular arrhythmia at the time of lactate determinations. Six patients had lactate level variation of more than 30 percent, and five of these six patients had an increase in quantity and severity of ventricular ectopic activity when their lactate levels were in the higher range. We conclude that elevated resting lactate levels correlate with impaired ventricular function, and fluctuations in a given patient may identify changes in clinical and electrophysiologic status.

Separation of irreversibly sickled cells (ISC) from blood of sickle cell anemia patients.

The irreversibly sickled cell (ISC) fraction in sickle cell anemia (homozygous hemoglobin S) was separated from the bulk of erythrocytes by performing a neutral density separation in bovine serum albumin (BSA), ϱ = 1.1 g cm−3, pH = 6.8. The ISC and non-ISC fractions were identified by both light microscopy and scanning electron microscopy. Electronic cell volume analysis confirmed the separation of these cell fractions and provided evidence for the origin of the polydispersity of cell volumes in unfractionated sickle cell blood. Normal ISC proportions range from 0 to 35% in sickle cell anemia patients.

Photo-oxidation of bovine oxymyoglobin in frozen solutions. The effect of redox active inorganic elements in muscle extracts.

Abstract 1. 1. Photocatalyzed autoxidation of MbO 2 →MMB in frozen extracts of bovine muscle was found to obey first order kineticsas found for MbO 2 of sperm-whale and horse. Formation of MMb-OH in crystallized beef MMb was studied. Its pK of ionization 8·90 is closely similar to that of sperm-whale and horse and differs from that of camel (8·53). 2. 2. Among the unbound elements examined in beef muscle extracts, iron is most responsible for photo-oxidation of MbO 2 . 3. 3. EDTA+iron caused further increase in oxidation. EDTA alone however, caused protection. 4. 4. Addition of ashed diffusates to beef extracts caused complete inhibition of MMb and resulted in a red pigment with absorption maxima at 580, 542 and 412 nm. 5. 5. A metmyoglobin cationic reducing agent was isolated from deproteinized horse and bovine muscle extracts by ion-exchange chromatography.

Depression of intramyocardial oxyhemoglobin dissociation by angiographic contrast media

The effect of the addition of radiographic contrast material (Renografin) to blood on the oxyhemoglobin dissociation curve and P50 was measured by a metabolic deoxygenation technique in a strongly buffered red cell suspension. With incubation time constant, increasing doses produced progressive decreases in P50. With incubation time varied at a constant dose, a decrease in P50 was seen after only one minute. In addition, in vivo studies were performed on 11 patients undergoing cardiac catheterization. Simultaneous proximal coronary sinus and aortic samples were drawn as controls, and then at one minute and five minutes after injection of the left coronary artery. In eight patients studies were performed after, and in three prior to left ventriculography. At one minute after left coronary injection there was a significant decrease of coronary sinus as compared to aortic P50 (p less than .10) (only when left ventriculography was performed prior to coronary arteriography). The magnitude of these effects in vivo is unknown, but they would be expected to be more severe in areas distal to a critical coronary lesion due to stasis of blood flow and ischemic metabolic changes.

Biphasic blood oxygen dissociation curves in hemoglobin S hemoglobinopathies. Sickle cell heterozygotes

Abstract The oxygen binding curves of whole cells of heterozygous sickle cell hemoglobinopathies were measured. Under the conditions of measurement (T=25°, pH=6.4, pCO2=0) the curves are biphasic. Samples in which 2,3-diphosphoglycerate (2,3-DPG) is very low or absent are monophasic, suggesting that there is a differential binding of 2,3-DPG to S and non-S hemoglobin in cells of heterozygotes. The biphasic curve has been confirmed in a hemoglobin SC patient, under physiologic conditions (T=37°, pH=7.4, pCO2=44 mm).

OXYGEN BINDING TO HUMAN ERYTHROCYTE CELL POPULATIONS

When preparation for this conference was in its early stages, the picture with respect t o hemoglobin oxygenation in cells seemed reasonably clear in its overall characteristics. Two lines of research, one concerned with oxygenation of sickle cell bloods and one with techniques for cell separation, seemed to converge nicely, and t o allow an evaluation of the “life history” of an erythrocyte with respect t o its oxygen transport function. In the succeeding months, however, this picture became increasingly complex as our knowledge increased of the properties of individual erythrocytes and the heterogeniety of erythrocytes in whole blood, not only morphologically but biochemically and physiologically. Thus this contribution is really in the nature of a progress report, of where we were, of the problems now apparent, and of the directions in which answers are beginning to emerge.

Gel Permeation Analysis of Macromolecular Association by an Equilibrium Method

Abstract An equilibrium method for determining macromolecular association by gel permeation is described. A thermodynamic description of the simplest case, dimerization, is presented in terms of the equilibrium constant for association, and equilibrium partition coefficients for species present. The theoretical analysis yields an equation relating the equivalents of monomer in the external phase to total equivalents, from which the equilibrium constant for association may be obtained. Those parameters critical in experimental design, gel swelling time, and centrifugal collapse point are determined for Sephadex gels of porosity G-25 to G-200. Application of the method to human hemoglobin gives a value of K′ α = 5 × 10−6 for dissociation of oxyhemoglobin at pH 7.0 in 0.1 M buffer.

Computer Analysis of Automatically Recorded Oxygen Dissociation Curves

The physiologic function of the erythrocyte is the transport of oxygen from lungs to tissue. The chemistry of this process is represented by a non-linear functional relationship between oxygen tension and oxygen saturation of the contained hemoglobin. This curve is usually characterized by two parameters, P50 (the oxygen tension at half-saturation) and the Hill constant, n (a measure of sigmoidicity).