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M C Reddy

Publications and source records attributed to M C Reddy.

18 recordsLinked to original sources
PubMed2000

Evidence for a Menkes-like protein with a nuclear targeting sequence.

Extracts from three human cell lines were found to contain abridged Menkes disease gene transcripts with novel insertion sequences. The transcript variant that is the focus of the present study codes for a 103-residue protein containing the first heavy-metal-binding domain (Hmb1) of ATP7A, the Cu-ATPase associated with Menkes disease. This transcript variant has a 45-bp nucleotide insert interposed between exons 1 and 2 of ATP7A that starts with a 5' ATG that is in-frame with the downstream ATG translation start site of ATP7A. We report here that the 66-bp nucleotides positioned between the upstream and downstream ATG sites encode 22 amino acid residues whose primary structure in part meets the criteria for a nuclear-localization sequence (NLS). We have referred to the transcript as nuclear Menkes-like (NML) 45. A green fluorescent protein (GFP) construct with NML45 when transfected in Chinese hamster ovary cells localized to the cell nucleus. A similar construct without the 66-bp segment exhibited a random dispersed fluorescent pattern in the cytosol. GFP constructs encoding ATP7A exons likewise failed to direct GFP into the cell nucleus, suggesting the nuclear determinant is not in an internal domain of the protein. The data suggest that the 22-residue segment contains an NLS for an 11.2-kDa protein with one Cu-binding site that may function as a chaperone to transport Cu into the nucleus of mammalian cells.

Adenosine Triphosphatases↗
PubMed1999

Multiple forms of the Menkes Cu-ATPase.

The 5' region of MNK cDNAs has a 45 bp insert terminating at the 5'end with an AGATG sequence. The ATG in the sequence is in-frame with the ATG downstream identified by Vulpe et al (1993) as a translation start site for MNK mRNA. Inserts of 192 bp and 45 bp have been found in the 5' region of MNK mRNAs from BeWo cells, Caco-2 cells and normal human fibroblasts. Extensions to the 5' end of these mRNAs could foretell a modified N-termini in certain forms of the Menkes Cu-ATPase. These modified H2N-terminal extensions are postulated to be targeting signals for post-translational processing and cellular localization. In this report, we provide evidence that the primary Menkes transcript in non-Menkes cells undergoes post-transcriptional splicing that gives rise to multiple transcripts. The data suggest that the Menkes gene is a copper locus that codes for more than one form of the Menkes Cu-ATPase and one of these forms could be a small Cu transport protein.

Adenosine Triphosphatases↗
PubMed1998

Multiple transcripts coding for the menkes gene: evidence for alternative splicing of Menkes mRNA.

We isolated cDNA fragments from four human cell lines that had sequences for the Menkes Cu-transporting ATPase (ATP7A). Primers designed to generate a 4.8 kb cDNA with the complete open reading frame generated a 1.9 kb cDNA in addition to the expected 4.8 kb product. Sequence analysis revealed that the 1.9 kb cDNA encoded one of the six Cu-binding sites and two of the eight transmembrane domains of ATP7A. Stop and start codons were also present. More striking, however, was an unusual union between exons 2 and 16 that retained an in-frame reference to exon 23. The 1.9 kb cDNA thus appeared to be a truncated Menkes mRNA that coded for an ATP7A variant that lacked exons 3-15. A 530 bp probe specific for exon 23 that avoided sequences in the exon 3-15 region hybridized to a 5.5 kb band on Northern blot analysis. Western blotting provided immunochemical evidence for the presence of both a 170 kDa and a 57 kDa protein with ATP7A sequences in detergent extracts of Caco-2 and induced BeWo cells. Extracts from non-induced BeWo cells, which lack the capacity to express the Menkes gene (MNK), showed neither protein. In a cell-free reticulocyte lysate, a plasmid containing the 1.9 kb cDNA insert directed the synthesis of a 59 kDa protein with antigenic properties of ATP7A. These studies provide evidence that non-Menkes cells have the capacity to synthesize more than one MNK mRNA. The one characterized in this report codes for a 57-59 kDa protein that lacks the core structure of the ATP7A protein. The smaller variant could be an alternative spliced form of MNK mRNA.

Adenosine Triphosphatases↗
PubMed1998

Functional analysis of copper homeostasis in cell culture models: a new perspective on internal copper transport.

The movement of copper ions across membrane barriers of vital organs and tissues is a priority topic in nutrition and one for which there continues to be little understanding of the mechanism. Reports of membrane-bound, copper-transporting adenosine triphosphatases (Cu-ATPases) selective for copper ions have brought new focus to the problem and prompted fresh ideas. Using a cell culture model approach, we attempted to learn whether transport into and out of cells depends on a Cu-ATPase. Measurement of transport kinetics in fibroblasts, brain glial cells, neuroblastoma cells, and placental cells showed differences in the rates of copper uptake and response to sulfhydryl reagents. BeWo cells, a human choriocarcinoma placental cell line, behaved as did Menkes fibroblasts by avidly absorbing copper but not releasing copper to the immediate environment. Further tests showed that BeWo cells did not express the transcript for the membrane-bound Cu-ATPase that has been identified with Menkes syndrome. Transcript induction, however, was achieved by growing BeWo cells on porous filters that allowed apical and basolateral surfaces to form. With transcript expression, the cells showed a capacity to release copper into the medium. BeWo cells also synthesized a form of ceruloplasmin whose structure differed from that of the plasma protein and hence may be a product of a different gene. BeWo cells may also express the gene for Wilson disease, thus linking Menkes and Wilson proteins to maternal delivery of copper. We constructed a model in which both ATPases work in concert in a vesicle-based transport mechanism. The vesicle model may help us understand the transport of copper across the placenta and all cells in general.

Adenosine Triphosphatases↗
PubMed1998

Genes regulating copper metabolism.

The metabolism of Cu is intimately linked with its nutrition. From gut to enzymes, Cu bioavailability to key enzymes and other components operates through a complex mechanism that uses transport proteins as well as small molecular weight ligands. Steps in Cu transport through the blood, absorption by cells, and incorporation into enzymes are slowly being understood. Cloning and sequencing of the genes for Menkes disease and Wilson disease has shown that membrane-bound enzymes analogous to Cu-ATPases in prokaryotes are equally important to Cu transport and homeostasis in mammalian cells. The primary structure of the mammalian Cu-ATPases has been deduced from cDNAs from tissues and organs. It now appears that mammalian Cu-ATPase have tissue and developmental specificity. In this review, we will focus on the Cu-ATPase that has been identified with Menkes disease. An emphasis will be placed on the existence of multiple forms of the ATPase and some indication as to how the different isoforms befit their role in the normal physiology of copper, specifically transmembrane transport and maintenance of a favorable internal cellular environment.

Adenosine Triphosphatases↗
PubMed1996

31P nuclear magnetic resonance study of phospholipids in ischemia/reperfusion injury in a rat fatty liver model.

Obese Zucker rats are susceptible to increased hepatic ischemia/reperfusion (I/RP) injury. Increased lipid peroxidation occurs in this model with warm ischemia. We hypothesized that a severe depletion of phospholipids (PL) occurs with warm I/RP in fatty livers. Obese (Ob) and lean (Ln) Zucker rats were subjected to 90 min of in vivo partial hepatic warm I followed by RP. Total lipids extracted from one gm of liver (median lobe) taken at the end of 1, 2 and 6 hr of RP and sham (Sh) surgery (n=5 Ln & Ob) were analyzed by 202.3 MHz 31P NMR, which provided good resolution of individual PL. Obese (Sh) rats contained 22% more PL than Ln (P= < 0.01). Ischemia caused similar decreases in PL in both Ob (to 67% Sh) and Ln rats (62%). Following 2 hr RP, PL in Ob rats decreased further (46% Sh) and recovered only marginally at 6 hr (53%), in marked contrast to the rapid recovery in Ln to preischemic levels (110% Sh at both 2 and 6 hr; P=<0.001). Mole percents of individual PL did not change significantly except for lysophosphatidylcholine, which increased from 0.43 to 1.3% (Sh vs. 6 hr RP) in the Ob, but decreased from 0.98 to 0.52% in Ln animals (P = <0.001). Fatty livers thus are more vulnerable to phospholipid depletion in response to warm ischemia/reperfusion than normal livers.

Animals↗
PubMed1996

Coincident expression of Menkes gene with copper efflux in human placental cells.

BeWo cells, a human choriocarcinoma cell line, have a high-affinity system for transporting copper ions into the cell (Km = 0.21 microM) but are sluggish in releasing copper back into the medium from preloaded cells. The slow efflux rate has recently been shown to correlate with a failure of BeWo cells to express the Menkes transcript [Y. Qian, E. Tiffany-Castiglioni, and E. D. Harris. Am. J. Physiol. 271 (Cell Physiol. 40). In press]. We have now determined that only when BeWo cells were grown on plastic surfaces such as petri dishes or flasks did they display negligible release and enhanced retention of 67Cu. Reverse transcriptase-polymerase chain reaction with the use of primers selective for the Menkes gene failed to show any evidence of a Menkes transcript in cells cultured on plastic surfaces. In contrast, cells grown on porous filters previously shown to allow apical and basolateral surfaces to develop did display the transcript and showed significant copper release with normal retention. Release of copper from filter-grown cells was blocked with p-chloromercuribenzoate, thus confirming sulfhydryl group involvement. Absorption of the 67Cu, either as a free ion or bound to ceruloplasmin, was unaffected by the different culture conditions. The data link the Menkes gene product with the ability of cells to release copper ions. They also suggest that the expression of the Menkes gene may be regulated by the development of polarized cell membranes.

Base Sequence↗
PubMed1995

Studies of hepatic warm ischemia in the obese Zucker rat.

The effects of warm ischemia were investigated in obese Zucker rats with severe hepatic steatosis in order to develop a nontransplant fatty liver ischemia model. Obese (Ob) and lean (Ln) Zucker rats were subjected to in vivo partial hepatic warm ischemia of 45 or 90 min. Injury was assessed by serum alanine aminotransferase, animal survival, and liver histology. Liver lipids were quantified in control animals. After 90-min ischemia and 2-hr reperfusion, liver malondialdehyde was measured and neutrophils in 12 microscopic fields were counted after esterase staining. After 45 and 90 min of ischemia, Ob animals had significantly higher alanine aminotransferase at 1-hr and 24-hr reperfusion, compared with Ln animals (P < 0.01). After 90 min of ischemia, none of the Ln and 8/9 Ob animals died within 48 hr (P < 0.01). Histologically, Ob animals had more hepatocyte necrosis than did Ln animals. Hepatic neutral and phospholipid content (mg/g) in Ob versus Ln animals was 45.2 +/- 2.6 versus 8.2 +/- 0.7 (P < 0.01) and 36.2 +/- 1.9 versus 27 +/- 2.2 (P < 0.05), respectively. After reperfusion, liver malondialdehyde content increased significantly in Ob animals (8.5 +/- 0.4 vs. 12.3 +/- 0.8 pM/mg protein; P < 0.05), but not in Ln animals. Neutrophils, scant in control livers, increased significantly (P < 0.01) after ischemia/RP, but it increased to a similar degree in Ob and Ln animals. Obese Zucker rats with hepatic steatosis are more susceptible to warm ischemia/reperfusion injury than lean animals, and lipid peroxidation may be an important contributory mechanism. Further studies in this model might help to investigate the human problem.

Alanine Transaminase↗
PubMed1995

Interaction of ATP and lens alpha crystallin characterized by equilibrium binding studies and intrinsic tryptophan fluorescence spectroscopy.

alpha-Crystallin, the most prevalent protein in vertebrate lenses, is a high molecular weight aggregate composed of alpha A and alpha B subunits. Evidence is presented that ATP, a major phosphorus metabolite of the lens binds to alpha-crystallin extracted from calf lenses. The following parameters were obtained from equilibrium binding studies conducted at 37 degrees C: binding sites per 400 kDa aggregate = 10 and Ka = 8.1 x 10(3) M-1; and an essentially identical Ka of 7.84 x 10(3) M-1 and 22 binding sites were determined for a 850 kDa aggregate. The cooperativity parameter, alpha H, approximates unity which denotes that the binding of ligand is at independent sites. Binding was not significant at 22 degrees C and was absent at 4 degrees C. The specificity of the binding site for ATP was established by intrinsic tryptophan fluorescence spectroscopy. In the presence of increasing concentrations of ATP (0.05-0.3 mM), tryptophan fluorescence decreases in a concentration dependent manner to a minimum of 0.2 mM above which there is a non-linear response. Quenching of fluorescence was not evident with P(i), AMP or ADP. GTP elicited a minimal quenching of fluorescence only at the highest concentration (0.30 mM). Modulation of both supramolecular organization and lens metabolism is predicted as a consequence of ATP/alpha-crystallin binding.

Adenosine Triphosphate↗
PubMed1994

Light scattering for young human and cataractous lenses.

The Rayleigh ratio for young human and cataractous lenses, having spherical particles with constant diameter embedded in a medium with a different refractive index, has been calculated as a function of concentration (volume fraction) using the theory of the small-q behaviour of the static structure factor, S(q). It involves treating the long-range forces between particles in the gel in the random phase approximation, dividing the pairwise interaction potential into a reference part and a perturbed part based on WCA model, and applying the perturbation approach of WCA to lenses obeying van der Waals' potential as a perturbative attraction over the Percus-Yevick (PY) hard sphere model. The calculated Rayleigh ratios are found in excellent agreement with experimental values, thereby showing that the present model works well for young human and cataractous lenses.

Cataract↗
PubMed1993

Effect of catalytically active recruited crystallins on lens metabolism.

The impact on duck lens metabolism of recruited epsilon-crystallin/LDH-B4 and tau-crystallin/enolase was investigated by NMR spectroscopy. A comparison of the duck lens metabolite profile with that of the calf in which recruited crystallins are absent revealed significant increases in ATP, alpha-glycerophosphate (alpha-GP) and pyridine dinucleotide concentrations. The alterations in the concentrations of ATP and alpha-GP appear to be related to both the high concentration of NAD and the elevated reduced to oxidized NADH/NAD+ ratio.

Adenosine Triphosphate↗
PubMed1992

31P NMR studies of the ATP/alpha-crystallin complex: functional implications.

Evidence is presented for the binding of ATP to alpha-crystallin in the lens by 31P NMR spectroscopic measurements. The chemical shift data as well as the T1 and T2 values indicate that P beta and P gamma of ATP are of prime importance in binding. In addition, it is demonstrated that the association of alpha-crystallin with purified fiber cell membranes is significantly enhanced by the addition of ATP. These results suggest that ATP modulates the functional behavior of alpha-crystallin.

Adenosine Triphosphate↗
PubMed1992

Investigation of lens glycolytic enzymes: species distribution and interaction with supramolecular order.

Distribution of several glycolytic enzymes in the lenses of different vertebrate species and their organization in the calf lenses were studied. Though no general pattern of enzyme activities in different species is discernible, high activities of TPI followed, in decreasing order, by GAPDH, enolase, PK, LDH and aldolase appear to be more common. Our observation on the unusually high activities of aldolase in the pig, enolase in the sheep and LDH in the duck lens are interesting in view of the already known dual function of LDH as an enzyme and a structural protein (epsilon-crystallin) in duck. Controlled treatment with detergents Brij-58 and Triton X-100 caused distinctly differential purturbations in the lens cells. In spite of fiber membrane disruption and partial actin dissolution by Brij-58, no significant increase in the release of glycolytic enzymes compared to control was observed. This suggests that none of the enzymes existed as a completely soluble and freely diffusible fraction. But treatment with a strong detergent (Triton X-100) caused the release of higher amounts of enzymes suggesting either a direct or indirect interaction with the cytomatrix components. Aldolase appears to be maximally bound in the cytosol followed by TPI, GAPDH, LDH and PK in decreasing order. Although thin lens slices were incubated with the detergents for a total period of 40 min and the loss of fiber architecture and organization confirmed by light microscopy, in the Triton X-100 treated tissues less than 25% of the total activity of any enzyme except TPI appeared in the bathing medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1970

Ester production by Pseudomonas fragi. IV. Demonstration of esterase activity.

Assay of the esterase activity of sonically treated cell-free extracts, whole cell suspensions, and supernatant fluid of Pseudomonas fragi cultures with a differential respirometer revealed that the esterases were intracellular. Polyacrylamide-gel electrophoresis demonstrated six bands of esterase activity, which revealed substrate specificity differences. Band 1 exhibited slow mobility, bands 2, 3, and 4 moderate mobility, and bands 5 and 6 rapid mobility. Six bands were active with alpha-naphthyl acetate, four bands with alpha-naphthyl propionate, and 5 bands with alphanaphthyl butyrate. These esterases appeared to be more active with aromatic esters than with aliphatic esters.

Acetates↗
PubMed1969

Ester production by Pseudomonas fragi. II. Factors influencing ester levels in milk cultures.

Cultures of Pseudomonas fragi were grown at 21 C in sterile homogenized milk and reconstituted skim milk media supplemented with ethyl alcohol. Quantitative determinations of ethyl butyrate and ethyl hexanoate by gas-liquid chromatography showed definite increases in the concentrations of the two esters produced in these media in comparison to media not supplemented with ethyl alcohol. Supplementation with butyric acid in addition to ethyl alcohol generally elevated the ethyl butyrate concentration and usually depressed the cell count slightly. Aeration of any of the media during growth tended to reduce the cell population slightly. A relationship between increase in cell number and increase in concentration of esters during the growth of the culture was observed. Media containing high concentrations of ethyl alcohol plus milk fat or low-molecular-weight fatty acids were conducive to the production of a fruity aroma by P. fragi.

Animals↗