Control of leucine transport in yeast by periplasmic binding proteins

The concentrative inward transport of leucine in Saccharomyces carlsbergensis involves two transport systems (S1 and S2); S1 is a system of high affinity and low translocation velocity, and S2 is a system of low affinity and high translocation velocity. The inward transport process of the amino acid is discriminated into two kinetically defined steps: first, binding to periplasmic proteins and second, translocation across the plasmalemma. When cells were incubated with glucose to increase the metabolic energy charge, we observed that JTmax (maximum flux that each system can exhibit for the translocation step) increased for both systems. This increase in JTmax is due to variations in the parameters defining the initial step (Ks (apparent dissociation constant) and N (concentration of binding sites)): for S1, N1 increases and for S2, KS2 diminishes. Dissipation of the electrochemical proton gradient produced an increase of KS1 and a decrease of N2, resulting in a decrease of JTmax in both systems. Instead, osmotic shock decreases N1 and N2, which suggests that periplasmic components were removed, resulting also in a decrease of JTmax in both systems. These results are consistent with the proposition that the total unidirectional flux of the amino acid proceeds by means of a system of multiple components, with the simultaneous operation of two independent transport processes. We propose that the initial interaction of leucine with components of the cellular envelope might be the essential step for the subsequent translocation of the amino acid across the permeability barrier.     

A mutation of beta -actin that alters depolymerization dynamics is associated with autosomal dominant developmental malformations, deafness, and dystonia

Actin, one of the major filamentous cytoskeletal molecules, is involved in a variety of cellular functions. Whereas an association between muscle actin mutations and skeletal and cardiac myopathies has been well documented, reports of human disease arising from mutations of nonmuscle actin genes have been rare. We have identified a missense point mutation in the gene coding for beta -actin that results in an arginine-to-tryptophan substitution at position 183. The disease phenotype includes developmental midline malformations, sensory hearing loss, and a delayed-onset generalized dystonia syndrome in monozygotic twins. Cellular studies of a lymphoblastoid cell line obtained from an affected patient demonstrated morphological abnormalities of the actin cytoskeleton and altered actin depolymerization dynamics in response to latrunculin A, an actin monomer-sequestering drug. Resistance to latrunculin A was also observed in NIH 3T3 cells expressing the mutant actin. These findings suggest that mutations in nonmuscle actins may be associated with a broad spectrum of developmental malformations and/or neurological abnormalities such as dystonia.     

Nerve growth factor expression in the developing hippocampus isolated in vitro.

Nerve growth factor (NGF) is synthesized in the hippocampus and neocortex and provides trophic support for afferent cholinergic neurons of the basal forebrain. To determine the capacity of the developing hippocampus to express NGF in the absence of NGF-responsive afferents, embryonic hippocampal cells isolated prior to septal innervation were studied in reaggregating cell culture. The expression of NGF protein in vitro was qualitatively and quantitatively similar to that observed in situ. The expression of NGF mRNA exhibited an initial increase in vitro but then plateaued and was maintained at a steady level. This latter finding was in contrast to the steady rise in NGF mRNA levels observed in situ. These data suggest that (i) intrinsic hippocampal interactions regulate the onset of NGF expression, but that (ii) additional extrinsic developmental signals may be required for proper regulation of hippocampal NGF expression during ontogeny.     

Stereochemical and structural effects of (2R,6R)-hydroxynorketamine on the mitochondrial metabolome in PC-12 cells

Background

Impairment in mitochondrial biogenesis and function plays a key role in depression and anxiety, both of which being associated with changes in fatty acid and phospholipid metabolism. The antidepressant effects of (R,S)-ketamine have been linked to its conversion into (2S,6S;2R,6R)-hydroxynorketamine (HNK); however, the connection between structure and stereochemistry of ketamine and HNK in the mitochondrial homeostatic response has not yet been fully elucidated at a metabolic level.

Determination of the anticancer agent CI-980 in plasma by achiral liquid chromatography on a Pirkle-type stationary phase

A high-performance liquid chromatographic assay has been developed and validated for the determination of the anticancer agent CI-980 in plasma samples from pediatric patients. After ether extraction from alkaline plasma, the CI-980 was chromatographed on a (R)-N-(3,5-dinitrobenzoyl)phenylglycine stationary phase using a mobile phase composed of hexane–isopropanol (70:30, v/v) modified with 1% acetonitrile. The method was linear over a 0.25 to 25.00 ng/ml range and the intra- and inter-day coefficients of variation (C.V.s) were less than 15%. The method was applied to the determination of the plasma concentration–time profile for a pediatric patient receiving 3.5 mg/m² of CI-980 per day as a continuous 72-h infusion.     

A non-peptide morphine-like compound from brain

A non-peptide morphine-like compound was extracted from calf brain and partially purified by immunoabsorption to antimorphine antibodies. The material was able to depress electrically stimulated contractions of the myenteric plexus-longitudinal muscle of the isolated guinea pig ileum. This effect could be prevented and reversed by naloxone and by antimorphine antibodies. Biological activity and morphine-like immunoreactivity were not affected by treatment with proteolytic enzymes. These results represent the first independent confirmation of the non-peptide morphine-like compound reported by Gintzler $\text{et al}$.     

Amino acid uptake by yeasts: IV. Effect of thiol reagents on l-leucine transport in Saccharomyces cerevisiae

(1) $\text{N-}\text{Ethylmaleimide}$ (a penetrating SH- reagent) inactivated l-[¹⁴C]leucine entrance (binding and translocation) into Saccharomyces cerevisiae, the extent of inhibition depending on the time of preincubation with $\text{N-}\text{ethylmaleimide}$, $\text{N-}\text{ethylmaleimide}$ concentration, the amino acid external and internal concentration, and the energization state of the yeast cells. With d-glucose-energized yeast, $\text{N-}\text{ethylmaleimide}$ inhibited l-[¹⁴C]leucine entrance in all the assayed experimental conditions, but with starved yeast and low (0.1 mM) amino acid concentration, it did not inhibit l-[¹⁴C]leucine binding, except when the cells were preincubated with l-leucine. With the rho^? respiratory-deficient mutant (energized cells), $\text{N-}\text{ethylmaleimide}$ inhibited l[¹⁴C]leucine entrance as with the energized wild-type, though to a lesser extent. (2) Analysis of the $\text{N-}\text{ethylmaleimide}$ effect as a function of l-[¹⁴C]leucine concentration showed a significant decrease of $\text{J}{}_{\mathrm{<mtext>max</mtext>}}$ values of the high- (S₁) and low- (S₂) affinity amino acid transport systems, but $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ values were not significantly modified. (3) When assayed in the presence of d-glucose, $\text{N-}\text{ethylmaleimide}$ inhibition of d-glucose uptake and respiration contributed significantly to inactivation of l-[¹⁴C]leucine entrance. Pretreatment of yeast cells with 2,4-dinitrophenol enhanced the effect of l-[¹⁴C]leucine binding and translocation. (4) Bromoacetylsulfanilic acid and bromoacetylaminoisophthalic acid, two non-penetrating SH- reagents, did not inactivate l-[¹⁴C]leucine entrance, while $\text{p-}\text{chloromercuribenzoate}$, a slowly penetrating SH- reagent, inactivated it to a limited extent. When compared with the effect of $\text{N-}\text{ethylmaleimide}$, these negative results indicate that thiol groups of the l-[¹⁴C]leucine carrier were not exposed on the outer surface of the yeast cell permeability barrier.     

Automated Multi-Lesion Detection for Referable Diabetic Retinopathy in Indigenous Health Care

Diabetic Retinopathy (DR) is a complication of diabetes mellitus that affects more than one-quarter of the population with diabetes, and can lead to blindness if not discovered in time. An automated screening enables the identification of patients who need further medical attention. This study aimed to classify retinal images of Aboriginal and Torres Strait Islander peoples utilizing an automated computer-based multi-lesion eye screening program for diabetic retinopathy. The multi-lesion classifier was trained on 1,014 images from the São Paulo Eye Hospital and tested on retinal images containing no DR-related lesion, single lesions, or multiple types of lesions from the Inala Aboriginal and Torres Strait Islander health care centre. The automated multi-lesion classifier has the potential to enhance the efficiency of clinical practice delivering diabetic retinopathy screening. Our program does not necessitate image samples for training from any specific ethnic group or population being assessed and is independent of image pre- or post-processing to identify retinal lesions. In this Aboriginal and Torres Strait Islander population, the program achieved 100% sensitivity and 88.9% specificity in identifying bright lesions, while detection of red lesions achieved a sensitivity of 67% and specificity of 95%. When both bright and red lesions were present, 100% sensitivity with 88.9% specificity was obtained. All results obtained with this automated screening program meet WHO standards for diabetic retinopathy screening.     

p16INK4A and CDH13 hypermethylation in tumor and serum of non-small cell lung cancer patients

Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the etiopathogenesis of lung cancer and is a promising tool for cancer detection. In the present study, promoter hypermethylation of p16INK4A and CDH13 genes was investigated in tumor tissue and in matched serum from 61 patients with histologically confirmed non-small cell lung cancer. Using a fluorescence-based method of methylation-specific PCR (F-MSP), methylation of p16INK4A and CDH13 was detected in 79% and 66% of tumors, respectively, and was not significantly related to conventional clinicopathological characteristics of patients or tumors. Methylation of both genes was observed in 52% of tumors and of at least one gene in 92% of lesions. In matched serum, hypermethylation of p16INK4A and CDH13 was observed in 26% and 23% of patients, respectively, but as they were not associated, the methylation of at least one gene was detected in 39% of patients. In conclusion, the frequency of p16INK4A or CDH13 hypermethylation in patient serum, together with evidence of their early occurrence in lung cancerogenesis and the total lack of methylation in serum from healthy individuals, offer a promising tool for non invasive early detection of lung cancer.