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To explore the antitumor effect of insulin-like growth factor 1 (IGF-I) antisense RNA and the interaction of IGF-I with insulin-like growth factor-binding proteins (IGFBPs) in glioma cells, a recombinant retrovirus expressing IGF-I antisense RNA was constructed and introduced into C6 glioma cells. IGF-I antisense RNA reverses the transformed phenotype in glioma cells and inhibits glioma cell growth by blocking overexpression of endogenous IGF-I. Expression of IGFBP-2 is increased in glioma cells as compared with normal adult glial cells. IGF-I antisense RNA also inhibits expression of IGFBP-2 in glioma cells, but does not influence expression of the other IGFBPs. Although IGFBP-2 in conditioned medium from wild-type C6 cell cultures itself does not directly influence glioma cell growth, it synergistically enhances exogenous IGF-I-mediated DNA synthesis in IGF-I-negative C6 cells. These findings indicate the inhibitory effect of IGF-I antisense RNA on growth and development of glioma cells. IGF-I-dependent glioma cell growth may, in some circumstances, require IGFBP-2 as a cofactor. The antitumor effect of IGF-I antisense RNA is also associated with inhibition of IGFBP-2 expression.

We have proposed that glutamine serves in a facilitated diffusion process, mediated by the enzyme gamma-glutamyl transferase (gamma-glutamyl transpeptidase; gamma GT) and that it leaves the brain in exchange for entering amino acids. Glutamine is also a precursor of gamma-aminobutyric acid (GABA). Thus, providing an alternate substrate for gamma GT should spare brain glutamine, raise GABA, and cause an anticonvulsant effect. We have found that glycylglycine, the best-known substrate for gamma GT, and delta-aminovaleric acid (DAVA), a structural analog, have anticonvulsant activity in DBA/2J mice. Both compounds can decrease the incidence and severity of seizures induced by L-methionine-RS-sulfoximine or electroconvulsive shock. DAVA was also tested and found to be active against seizures caused by pentylenetetrazol or picrotoxin. [14C]DAVA entered the brain at the rate of 18.7 nmol/g/min. The activity of DAVA as a substrate of gamma GT was intermediate to that of glycylglycine and glutamine. Preliminary studies have shown that brain glutamine and perhaps GABA are elevated 3 h after administration of DAVA (7.5 mmol/kg). These findings support the theory that glutamine exchange plays a role in amino acid transport across the blood-brain barrier and suggests a new concept in anticonvulsant therapy.

In 1981 we (1) presented evidence that the blood-brain barrier consisted of (at least) two compartments . Subsequently, Pardridge (2) took issue with our data and with our interpretation. In response, we have thoroughly reexamined our findings as well as related work in the literature (2-5). From this we have confirmed that the blood-brain barrier does consist of (at least) two compartments . We have also discovered that one of the equations in our article was in error and, consequently, that some of our comments were erroneous and require correction. In our method (6, 7), the transport rate is mathematically defined by the slope of the straight line relating the molar accumulation of the tracer amino acid, in the brain, to the time elapsed since its injection. We found the blood-brain transport rate of tyrosine (Tyr) to be constant from about 2-15 min. After injection of the enzyme inhibitor methionine sulfoximine (MSO), however , the slope of the " t ranspor t l ine" was seen to decrease by 30-40% along with a marked increase in the value of the intercept at zero time. In our paper (I) we sought to explain how the intercept of the " t ranspor t l ine" could be anything other than zero at zero time since our method measures a randomly chosen segment of a constant, ongoing process. Focusing on the data from the MSO treated animals, we concluded that there were (at least) two linear processes: one, feeding a relatively small tissue comparment whose specific activity came to equilibrium with that of the plasma earlier than 2 rain and, because of the equilibrium, appeared to drop out. The second process was assumed to feed a larger tissue compartment that did not come to equilibrium with the plasma over the

DBA/2J mice were exposed in utero, between days 15-18 of gestation, to either of two enzyme inhibitors, previously shown to decrease blood-brain, large-neutral amino acid transport in adults: L-methionine-RS-sulfoximine and 2-imidazolidone-4-carboxylic acid. The young mice demonstrated persistently altered motor behavior relative to saline controls when 40-42 days old and evidence of differences in the entry and incorporation of 14C-valine in brain at up to 80 days of age. The findings suggest that interference with blood-brain amino acid transport in utero has long term consequences. This may be related to some human conditions such as maternal phenylketonuria.

Ideally, the material in a spot on a two-dimensional chromatogram forms a bivariate normal distribution in which the base of the figure corresponds to the dimensions of the circular spot and the height of the curve is a function of the maximum absorbance. Using a paraboloid as an approximation of the Gaussian figure, it has been possible to construct a mathematical model of a series of chromatographic standards over a five hundred-fold range of values and to define mathematically deviant “spots” which were more compact or more diffuse than the standard series. The model has been used to evaluate the various empirical techniques of direct chromatographic analysis including: spot length, area, maximum absorbance, (area) (maximum absorbance), total absorbance and slit scanning with fixed and fixed-ratio slit length. It was found that slit scanning where the length of the slit is a constant fraction of the spot diameter is probably the best technique for mono-dimensional chromatograms while for two-dimensional chromatograms the product of (spot area) × (maximum absorbance) appears to be the best method.

The exponential plasma specific activity curve 2.5 to 12.5 min after injection (sc) of [14C]tyrosine was integrated and divided by time to obtain the mathematical relationship between the average equivalent specific activity S and the measured specific activity S in any individual animal. S is the constant, average value of S that is equivalent to the curvllinearly varying quantity that the body tissues are actually exposed to. Dividing the total brain radioactivity by S gave the tissue Tyr uptake U. The function dU dt is linear from 2.5 to 12.5 min and represents the rate of uptake of the amino acid. Incorporation into protein was similarly measured. Brain uptake of Tyr averaged 7.06, and the apparent protein incorporation was 1.99 nmol/g of brain per min. The γ-glutamyl cycle inhibitor l -methionine-RS-sulfoximine reduced total brain uptake of tyrosine by 42.8% and the apparent rate of protein incorporation by 39.0%.

Blood-to-brain amino acid transport consists of at least two components: 1. a fast rate or early process, commonly measured by the intra-carotid bolus injection method and attributed to transport across the capillary endothelium and entry into the astrocytes, and, 2. a slow rate or later component measured over 2 to 15 minutes probably associated with exit from the astrocytes and entry into the neurons. Incorporation into brain protein is temporally related to the second process. In the present study the slow and fast rate transport components and the incorporation into brain protein of tyrosine (Tyr) and Valine (Val) was measured in young adult and aged male C57BL/6 mice. The results indicate that the fast rate transport component is unaffected by age while the rates of the slow process and protein turnover show an exponential decline most marked between 3 and 8 months of age. Changes in the relative incorporation of Tyr and Val suggest that brain protein metabolism is altered qualitatively as well as quantitatively in aging, in these animals.

Hereditary defects in amino acid metabolism are involved in several neurological disorders, 1,5,7,14,17,22 and the urinary excretion pattern has been shown to be a useful diagnostic aid. 9 Qualification of the amino acid content of a sample can best be done on an automatic analyzer. However, this technique requires 24 16 to 48 hours 19 for each assay for physiological fluids, with only one sample being examined at a time. This approach is unsuitable for the large-scale study of clinical populations. A screening procedure is necessary so that large numbers of patients can be routinely examined and those with a frankly normal distribution pattern eliminated without time-consuming quantitative analysis. Rapidity and small expense have encouraged the use of circular paper chromatography as a screening technique, 6 but this approach sacrifices resolution for speed. The "group" separation that is achieved is often inadequate. Thin-layer chromatography, 20 as a routine tool

Characteristic membranous bodies have been previously reported in the cytoplasm of the cerebral neurons in Tay-Sachs disease (1) (Fig. 1). Subsequently (2) these lamellated particles were isolated from homogenized autopsy tissue by differential and density gradient centrifugation and subjected to extensive chemical analysis. The membranous cytoplasmic bodies (MCB) were found to consist of about 50 per cent (dry weight) gangliosides and could account for the accumulation of this complex sphingolipid which characterizes this lipid-storage disease. The appearance and composition of the MCB suggested: (1) that they might arise from an alteration of cytoplasmic organelles, or (2) that they were formed spontaneously by interaction of the component lipids in a fashion analogous to myelin figure formation (3). Chemicals approximating the known composition of the MCB were combined in proper proportion in vitro as a test of this latter hypothesis. The resultant aggregates were examined with light and electron microscopy.

Rates of tyrosine and lysine transport and incorporation into protein were measured in control and undernourished weanling rats. Undernutrition was induced by feeding lactating dams a low protein diet (12 percent casein) from birth to day 21. At weaning, body and brain weights of undernourished rats were 50 percent and 88 percent, respectively, of control values. Lysine and tyrosine transport rates into skeletal muscle were reduced by over 75 percent, more than twice the reduction seen in brain. Rates of amino acid incorporation into muscle protein were reduced by approximately 50 percent; the change in rate of incorporation into brain protein was not statistically significant. These data indicate that, in spite of marked retardation of amino acid transport into brain, the brain seems fully capable of maintaining normal rates of protein synthesis.

The γ-glutamyl cycle has been proposed by Meister (1973) as one possible mechanism for the mediation of amino acid transport. The high energy requirement of the pathway, the very low specificity of γ-glutamyl transpeptidase and the inability to account for trans membrane stimulation of amino acid entry are but three criticisms of this hypothesis. It is proposed that the various objections can be overcome by postulating that the soluble form of γ-glutamyl transpeptidase transfers the γ-glutamyl moiety from gluthathione to glutamine (in the case of brain) and that the membrane sequestered form of this enzyme catalyzes the exchange of the γ-glutamyl group between γ-glutamyl glutamine and an entering neutral amino acid. The released glutamine leaves the cell. The γ-glutamyl amino acid then passes into the cytoplasm where it is acted upon by either γ-glutamyl cyclotransferase or the soluble γ-glutamyl transpeptidase which transfers the γ-glutamyl group to another molecule of glutamine. It is postulated that access to the membrane-bound enzyme is dependent on the relative lipophilia of the entering large-neutral amino acids. The available data support this mechanism. By regeneration of γ-glutamyl glutamine, a low expenditure of energy is required for the transport process. Specificity of transpeptidation is attained by the constraints of access to the membrane bound enzyme site.

Under steady-state conditions, the transport rates for amino acids from blood to brain have been found to be about half that seen using the intraarterial injection technique. Using a method that mathematically mimics the constant infusion procedure, we were able to reconcile this apparent discrepancy. At less than 1 min after subcutaneous injection of [14C]tyrosine in mice, we have observed a rate of entry into brain of 19.7 nmol/g/min, while from 1-15 min we have measured the rate at 6.4 nmol/g/min. Using methionine sulfoximine as an inhibitor of the gamma-glutamyl cycle, the early rate was reduced to 10.0 nmol/g/min and the later rate to 3.7 nmol/g/min. These data are consistent with a two-compartment system regulating amino acid transport into the neurons. A mathematical model fit to these data indicates that the first compartment contains 8.3 nanomoles of tyrosine per gram brain or about 6.7% of the brain total. It is speculated that the first compartment consists primarily of the astrocytes.

Two inhibitors of the gamma-glutamyl cycle, methionine sulfoximine (MSO) and 2-imidazolidone-4-carboxylic acid (ICA) were administered to C57LB/6J mice. Both agents resulted in a reduced rate of transport of tyrosine from blood to brain and a decreased rate of incorporation of tyrosine from plasma into brain protein. MSO administration also diminished to concentrations of brain tyrosine, dopamine, and norepinephrine. MSO decreased the transport rate of valine by brain as well as the rate of its incorporation into protein when expressed in relation to the plasma specific activity. The results demonstrate a significant role for the gamma-glutamyl cycle in the transport of large neutral amino acids from blood to brain.

In vivo inhibition of uptake 14 C-L-valine by brain following subcutaneous administration of either of two γ-glutamyl cycle enzyme inhibitors, 2-imidazolidone-4-carboxylic acid (ICA), or, L-methionine-S-sulfoximine (MSO) is documented in C57BL/6J mice. Dose related decrease in exploratory activity, impairment of memory for foot shock, and reduced operant responding for food reinforcement parallers the time course for interference with uptake of a large neutral amino acid by these two compounds previously shown to inhibit different enzymes in the γ-glutamyl cycle subserving active amino acid transport.

δ-Aminolevulinic acid and porphobilinogen were identified in urine from a patient with acute intermittent porphyria on routine amino acid chromatography. The compounds were well and rapidly separated by a two-dimensional combination of high-voltage, thin-layer electrophoresis and chromatography. Both compounds gave a yellowish color with ninhydrin; the porphobilinogen gave a strong red color when Ehrlich’s reagent was then applied. Such a pattern has not been observed in any blood and urinary amino acid chromatograms from over 900 patients with other conditions, who were receiving a variety of drugs. We consider this a rapid, specific diagnostic aid in cases of acute intermittent porphyria.

A variety of compounds have been quantified after thin-layer electrophoresis and/or thin-layer chromatography on microplates by means of the triple product of the largest and smallest diameters and the maximum absorbance of each spot. The diameters were measured with a graduated magnifier and spot absorbance was determined by means of a fiber optic light pipe fitted to a standard spectrophotometer. Precision and accuracy of ± 5% or less were obtained using amino acids, glucose, and cholesterol in the range of 10 −11 −10 −9 moles. A total of six different reagents were used on silica gel and cellulose, with glass plates and Mylar film supports after both mono-dimensional and two-dimensional separations. Except for the lipids which were applied with a modified syringe, all solutions were spotted by means of calibrated Nichrome wire loops delivering 36–126 nl. The procedure was found to be simple and economical and reliable in routine use.

A modified thionin staining procedure was used to identify the pathologic cytoplasmic bodies of Tay-Sachs disease in situ and in cell-free preparations. This technic was used to follow these particles through an isolation procedure. Unfixed smears of tissue suspensions, or sections of plastic-embedded tissue or tissue fractions, were stained directly. The slide was flooded with Laskey's thionin solution and placed on a hot plate. The dye was allowed to steam for about 15 sec, the excess drained off, and the slide immersed in distilled water. After about a minute's washing, the slide was dried (on the hot plate) and the tissue covered with glycerol and a cover slip. In phase contrast, the cytoplasmic bodies were seen as discrete blue-green particles. Only nucleoli gave the same staining reaction. More general applicability of this technic was indicated.

Simple, inexpensive applicators for thin-layer chromatography can be easily constructed to reproducibly deliver 40 nl or less. They are appropriate for quantitative work in the 10−10 molar range with a coefficient of variation of less than 5%. Although on calibration the variability appears to be much greater, in incremental spotting the deviations from the mean are additive and the pluses and minuses cancel out. Two spotter designs are described one of which is made from Nichrome wire and the other from ordinary sewing needles.

Determinations were made of carbohydrates in hemolymph collected from adult female mosquitoes (Anopheles stephensi). First the hemolymph was fractionated by extraction and precipitation procedures, after which qualitative and quantitative determinations of carbohydrates were made by thin layer chromatography. The most abundant sugars found in the hemolymph were glucose and trehalose, though maltose, glucuronic acid, and inositol could be found after the mosquitoes took blood meals. After the mosquitoes ingested a noninfected blood meal, their hemolymph sugar levels rose almost 4-fold. There was less of an increase following a blood meal infected with the rodent malaria parasite, Plasmodium berghei. Depletion of sugars in the hemolymph of infected mosquitoes may result from direct utilization of sugar by the malaria parasite developing within the mosquito.

Determinations were made of free amino acids in hemolymph collected from adult female Anopheles stephensi mosquitoes. The hemolymph first was fractionated by extraction and precipitation procedures, after which qualitative determinations of free amino acids were made by high voltage thin layer electrophoresis, and thin layer chromatography. Subsequent quantitative determinations were made with an automatic amino acid analyzer. The concentration of total free amino acids in the hemolymph rose 60--70% after the mosquito took a blood meal, and remained relatively constant thereafter. When mosquitoes took a blood meal infected with the rodent malaria parasite Plasmodium berghei, the rise in total free amino acids was only 15--25%. The chief differences that occurred with individual free amino acids was that infected mosquitoes had greater increases in arginine, greater decreases in valine and histidine, and a total loss of detectable methionine.

A simple, inexpensive sprayer for chromatographic use that can be constructed by a student as a laboratory project.