Biomedical subjects

N M Rao

Publications and source records attributed to N M Rao.

At least 19 recordsLinked to original sources

PubMed2001

Novel non-glycerol-based cytofectins with lactic acid-derived head groups.

We report herein the design, synthesis, and transfection biology of a novel series of non-glycerol-based cationic lipids with lactic acid-derived head groups The synthetic procedure adopted herein for preparing 1-hydroxy-prop-2-yl head-group-based monocationic transfection lipids 1-7 is fairly straightforward and potentially applicable in designing other cationic lipids with lactic acid-derived head groups. A striking anchor-length dependency was observed in NIH3T3 cells in the sense that except lipid 4, all the other lipids were essentially transfection-inefficient. Ethidium bromide assay for the lipid:DNA interactions is consistent with the general observation that significant lipid:DNA interactions do not guarantee on improved transfection efficiency cationic lipid mediated gene delivery. Given its remarkable transfection properties and low cellular toxicity, lipid 4 is likely to find future use in the area of liposomal gene delivery.

3T3 Cells↗

PubMed2001

Design, synthesis, and transfection biology of novel cationic glycolipids for use in liposomal gene delivery.

The molecular structure of the cationic lipids used in gene transfection strongly influences their transfection efficiency. High transfection efficiencies of non-glycerol-based simple monocationic transfection lipids with hydroxyethyl headgroups recently reported by us (Banerjee et al. J. Med. Chem. 1999, 42, 4292-4299) are consistent with the earlier observations that the presence of hydroxyl functionalities in the headgroup region of a cationic lipid contributes favorably in liposomal gene delivery. Using simple sugar molecules as the source of multiple hydroxyl functionalities in the headgroup region of the transfection lipids, we have synthesized four novel simple monocationic transfection lipids, namely, 1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-xylitol (1), 1-deoxy-1-[methyl(ditetradecyl)ammonio]-D-arabinitol (2), 1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-arabinitol (3) and 1-deoxy-1-[methyl(dioctadecyl)ammonio]-D-arabinitol (4), containing hydrophobic aliphatic tails and the hydrophilic arabinosyl or xylose sugar groups linked directly to the positively charged nitrogen atom. Syntheses, chemical characterizations, and the transfection biology of these novel transfection lipids 1-4 are described in this paper. Lipid 1, the xylosyl derivative, showed maximum transfection on COS-1 cells. All the lipids showed transfection with cholesterol as colipid and not with dioleoylphosphatidylethanolamine (DOPE). Radioactive quantitation of free and complexed DNA combined with ethidium bromide exclusion measurements suggest that though nearly 70% of the DNA exists as complexed DNA, the DNA may not have condensed as was observed with other cationic lipids. Presence of additional (more than two) hydroxyl functionalities in the headgroup of the cationic lipids appears to have improved the transfection efficiency and made these lipids less cytotoxic compared to two-hydroxyl derivatives.

Animals↗

PubMed1999

Novel series of non-glycerol-based cationic transfection lipids for use in liposomal gene delivery.

A novel series of nontoxic and non-glycerol-based simple monocationic transfection lipids containing one or two hydroxyethyl groups directly linked to the positively charged nitrogen atom were synthesized. The in vitro transfection efficiencies of these new liposomal gene delivery reagents were better than that of lipofectamine, a widely used transfection agent in cationic lipid-mediated gene transfer. The most efficient transfection formulation was observed to be a 1:1:0.3 mol ratio of DHDEAB (N, N-di-n-hexadecyl-N,N-dihydroxyethylammonium bromide):cholesterol:HDEAB (N-n-hexadecyl-N,N-dihydroxyethylammonium bromide) using a DHDEAB-to-DNA charge ratio (+/-) of 0.3:1. Observation of good transfection at charge ratios lower than 1 suggests that the amphiphile-DNA complex may have net negative charge. Our results reemphasize the important point that in cationic lipid-mediated gene delivery, the overall charge of the lipid-DNA complex need not always be positive. In addition, our transfection results also imply that favorable hydrogen-bonding interactions between the lipid headgroups and the cell surface of biological membranes may have some role for improving the transfection efficiency in cationic lipid-mediated gene delivery.

Animals↗

PubMed1999

Effect of banana on cold stress test & peak expiratory flow rate in healthy volunteers.

The effect of banana on cold stress induced hypertension, peak expiratory flow rate and plasma ACE activity in healthy human volunteers was tested. Systolic blood pressure (P < 0.005), diastolic blood pressure (P < 0.025) and mean arterial blood pressure (P < 0.005) were significantly decreased during cold stress after banana treatment compared to controls subjected to cold stress. There was no significant changes in heart rate and peak expiratory flow rate but only significant decrease in plasma ACE activity after banana treatment. Banana decreased the rise of systolic blood pressure and diastolic blood pressure in healthy volunteers subjected to cold stress test without much effect on heart rate and peak expiratory flow rate.

Adult↗

PubMed1998

Conformation and lipid binding properties of four peptides derived from the membrane-binding domain of CTP:phosphocholine cytidylyltransferase.

We are probing the mechanism of the lipid selective membrane interactions of CTP:phosphocholine cytidylyltransferase (CT). We have proposed that the membrane binding domain of CT (domain M) consists of a continuous amphipathic alpha-helix between residues approximately 240-295 [Dunne, S. J., et al. (1996) Biochemistry 35, 11975-11984]. This study examined the secondary structure and membrane binding properties of synthetic peptides derived from domain M: a 62mer peptide encompassing the entire domain (Pep62), a 33mer corresponding to the N-terminal portion (PepNH1), and two 33mers corresponding to the three C-terminal 11mer repeats, one with the wild-type sequence (Pep33Ser), and one with the three serines in the nonpolar face substituted with alanine (Pep33Ala). Peptide secondary structure was analyzed by circular dichroism, and lipid interactions were analyzed by a direct vesicle binding assay, by effects of lipid vesicles on peptide tryptophan fluorescence, and by monolayer surface pressure changes. All peptides bound to vesicles as alpha-helices with selectivity for anionic lipids. Binding involved intercalation of the peptide tryptophan into the hydrophobic membrane core. PepNH1, the peptide with the highest positive charge density, showed strong selectivity for anionic lipids. PepNH1 and Pep33Ser did not bind to PC vesicles; however, the more hydrophobic peptides, Pep33Ala and Pep62, did bind to PC vesicles, with apparent partition coefficients for PC that were only approximately 1 order of magnitude lower than those for PC/PG (1/1). Our results suggest that the polar serines interrupting the nonpolar face of the amphipathic helix serve to lower the lipid affinity and thereby enhance selectivity for anionic lipids. Although diacylglycerol is an activator of the enzyme, none of the peptides responded differentially to PC/diacylglycerol vesicles versus pure PC vesicles, suggesting that domain M alone is not sufficient for the enzyme's response to diacylglycerol. Increases in surface pressure at an air-water interface indicated that the domain M peptides had strong surface-seeking tendencies. This supports a binding orientation for domain M parallel to the membrane surface. Binding of CT peptides to spread lipid monolayers caused surface pressure reductions, suggesting condensation of lipids in the formation of lipid-peptide complexes. At low monolayer surface pressures, Pep62 interacted equally with anionic and zwitterionic phospholipids. This suggests that one determinant of the selectivity for anionic lipids is the lipid packing density (area per molecule).

Amino Acid Sequence↗

PubMed1998

Reduced DNA repair capacity in breast cancer patients and unaffected individuals from breast cancer families.

It has been suggested that increased fragile site expression in lymphocyte cultures can be used as a marker for genetic predisposition to cancer. We wished to determine whether aphidicolin (APC), an inhibitor of the DNA repair enzyme DNA polymerase alpha, could be used as a reliable biomarker in identification of DNA repair capacity in unaffected individuals at high risk from breast cancer families. PHA-stimulated lymphocyte cultures, with and without APC, were set up in 65 individuals, of whom 14 were breast cancer patients, 26 were unaffected individuals from breast cancer families, and 25 were controls. A significant proportion of breast cancer patients and unaffected individuals from familial breast cancer (FBC) families exhibited premature separation of centromeres (PSC) and aneuploidy in the untreated cultures. In the APC treated cultures, almost all such individuals exhibited a marked depression of mitotic index and increased aneuploidy, as compared to controls. Our results indicate that these individuals have defective DNA repair capacity. Such individuals could thus have a much higher risk of cancer as compared to persons exhibiting PSC and aneuploidy or DNA repair defects alone. We propose that APC may be a valuable biomarker in identifying individuals with genetic predisposition to cancer from FBC families.

Adult↗

PubMed1998

Effect of chloride and diamide on angiotensin converting enzyme from sheep testis and epididymis.

Effect of chloride and diamide on testicular and epididymal angiotensin converting enzyme (ACE) activity was investigated using Hip-His-Leu as substrate in sheep. The chloride ions functioned as ACE activators, however, there was no linear correlation between the two. The optimum chloride concentrations were 500 mM for epididymal ACE and 900-1100 mM for testicular ACE. Further, optimum chloride concentration increased ACE activity of testis and epididymis 25.40- folds and 12.84- folds respectively of the activities at physiological chloride concentration. The differences found in the effect of chloride on testicular and epididymal ACE activity suggest dissimilar three dimensional structure of ACE in these tissues. Increased testicular and epididymal ACE activity on diamide pretreatment indicates that tissue oxidation may affect ACE activity.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1998

Natural killer cell function and genetic instability in unaffected individuals from breast cancer families.

Several recent reports highlight the importance of modifying factors in determining the risk for cancer of a person carrying a mutant allele of a tumour susceptibility gene. The study of two such risk modifying factors namely, natural killer (NK) cell function and constitutional cytogenetic anomalies in members of families with familial breast cancer is presented in this paper. We observed that, compared to healthy controls, a significant proportion of unaffected persons from breast cancer families not only display lower NK cell function or genetic instability alone, but also in conjunction. The significance of these observations is discussed. We propose that amongst the unaffected members, persons with lower NK cell function as well as constitutive cytogenetic anomalies may be at a higher risk for cancer. The need for a set of suitable biomarkers to identify individuals at high risk from familial breast cancer families has been recognized for many years. Constitutional cytogenetic anomalies, otherwise seen in breast tumours, have also been observed in lymphocyte cultures from unaffected persons from such families. Lowered NK cell function has previously been demonstrated in first degree relatives of cancer patients. Both these parameters have been implicated in determining the risk of developing malignancy. In the present study these aspects have been investigated simultaneously in order to assess their utility as potential biomarkers.

Adult↗

PubMed1997

Characterization of biomimetic surfaces formed from cell membranes.

A method for fabricating biomimetic surfaces from intact cell membranes is described. A monolayer of alkanethiol on gold is covered by a second layer derived from the components of erythrocyte membranes either by self-assembly or by Langmuir-Blodgett methods. The resulting asymmetric hybrid layer was characterized by ellipsometry, surface plasmon resonance (SPR), contact angle, capacitance, voltammetry, and electron and atomic force microscopy. The erythrocyte membrane layer was measured to be approximately 30-40 A in thickness. Using SPR, the presence of erythrocyte components on the surface was demonstrated by their selective removal by enzymatic action. The uniform deposition of membranous material on the substrate was shown by electron and atomic force microscopy. Demonstration of acetylcholinesterase (AChase) activity, a membrane-anchored enzyme, on the surface for at least 8 days, suggests that the outer leaflet of the erythrocyte membrane is present in its native form. Cyclic voltammetry demonstrates that enhanced electron transport from a solution redox species accompanies formation of the erythrocyte layer at the surface. This enhanced electron transport is blocked by 4,4'-diisothiocyanate stilbene-2,2'-disulfonic acid, a well known blocker of anion transport, suggesting that an erythrocyte anion transporter protein is incorporated into the surface layer in an active conformation.

Acetylcholinesterase↗

PubMed1997

Sheep testicular and epididymal angiotensin converting enzyme: inhibitions by captopril, lisinopril and enalapril.

Inhibition of angiotensin converting enzyme(ACE) in presence of captopril(C), lisinopril(L) and enalapril(E) were investigated in testis and epididymis of sheep using Hip-His-Leu as substrate. Captopril, lisinopril and enalapril were competitive inhibitors of the enzyme from both tissues. Differences in the I50 and Ki values using these three inhibitors reflects the affinities of these inhibitors for the ACE. In addition, the relative potencies of captopril, lisinopril and enalapril were different for testicular ACE(C > L > E) and epididymal ACE(L > C > E). This observation suggests differences between the active sites of the testicular and epididymal ACE which may reflect on their functions in vivo.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1997

Inhibition of angiotensin converting enzyme from sheep tissues by captopril, lisinopril and enalapril.

Inhibition of angiotensin converting enzyme(EC 3.4,15.1, ACE) in presence of captopril, lisinopril and enalapril were investigated in kidney, lung and serum of sheep using Hip-His-Leu(HHL) as substrate. The activity in kidney, lung and serum was inhibited at HHL concentration above 5 mM. The inhibitory constants (IC50) ranged between 5.6 nM for serum ACE with lisinopril and 70000 nM for renal ACE with enalapril while Ki ranged from 1.0 nM for serum ACE with lisinopril to 12000 nM for kidney ACE with enalapril. Differences in inhibition observed in different tissues suggest that the inhibitors may block function(s) of ACE to varying degrees in each tissue.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1996

Role of the intersubunit disulfide bond in the unfolding pathway of dimeric red kidney bean purple acid phosphatase.

Quantitative equilibrium denaturation studies on oligomeric proteins have the potential to provide information on the role of subunit interactions in protein function and structure. We studied the equilibrium denaturation of red kidney bean purple acid phosphatase (KBPAP), a homodimer with a single disulfide bond between the two subunits, with an objective to understand the role of the intersubunit disulfide bond in KBPAP structure. Binding of 8-anilino-1-naphthalenesulfonic acid, enzymatic activity, size-exclusion chromatography, tryptophan fluorescence and circular dichroism studies revealed that the protein undergoes unfolding through at least three intermediates. Susceptibility of KBPAP for denaturation increases on reduction of the disulfide and aggregation was the predominant product of denaturation. In terms of stability, an intersubunit disulfide bond contributes to 25% of the overall stability of the dimer.

Acid Phosphatase↗

PubMed1996

Unfolding pathway in red kidney bean acid phosphatase is dependent on ligand binding.

Structural basis for ligand-induced protein stabilization was investigated in the case of an acid phosphatase (red kidney bean purple acid phosphatase (KBPAP)) from red kidney bean. Phosphate, a physiological ligand, increases the stability against solvent denaturation by 3.5 kcal/mol. Generality of phosphate stabilization was shown by similar effects with other KBPAP ligands viz. adenosine 5'-O-(thiotriphosphate), a nonhydrolyzable ligand, and arsenate, an inhibitor. The dissociation constant of phosphate obtained from denaturation curves matches with the dissociation constant estimated by conventional methods. The guanidinium chloride-mediated denaturation of KBPAP was monitored by several structural and functional parameters viz. activity, tryptophan fluorescence, 8-anilinonaphthalene 1-sulfonic acid binding, circular dichroism, and size exclusion chromatography, in the presence and absence of 10 mm phosphate. In the presence of phosphate, profiles of all the parameters shift to a higher guanidinium chloride concentration. Noncoincidence of these profiles in the absence of phosphate indicates multistate unfolding pathway for KBPAP; however, in the presence of phosphate, KBPAP unfolds with a single intermediate. Based on the crystal structure, we propose that the Arg258 may have an important role to play in stabilization mediated by phosphate.

Acid Phosphatase↗

PubMed1996

Premature separation of centromere and aneuploidy: an indicator of high risk in unaffected individuals from familial breast cancer families?

It is estimated that one in every four women with a first-degree relative affected by breast cancer will develop the disease. Recent evidence suggests that susceptibility to breast cancer can be inherited. We have carried out cytogenetic analysis on PHA-stimulated lymphocyte cultures of breast cancer patients (familial and sporadic), patients with benign breast lesions, unaffected individuals from families with a history of breast cancer and healthy controls. A high incidence of premature separation of centromere (PSC) and aneuploidy was observed in a significant proportion of familial breast cancer patients and patients with fibrocystic disease as well as in some unaffected individuals from breast cancer families. These observations are also supported by cytogenetic analysis of EBV-transformed lymphoblastoid cell lines established from some of these individuals. No such aberrations were detected in the controls. Further, most of the affected and unaffected individuals with these two anomalies also exhibited structural chromosomal aberrations of 1q, 6q, 7q, 16q, 18q, or Xq. Based on these observations, we propose that the presence of both PSC and aneuploidy in lymphocyte cultures of unaffected individuals from breast cancer families can be used as an important predictive parameter to determine the risk of developing cancer.

Adult↗

PubMed1996

A simple technique for improving chromosome spreads from clumped metaphases.

A method has been described by which clumped metaphases either due to inadequate hypotonic KCl treatment or prolonged storage at 4 degrees C can be rescued. The cell pellet obtained from cell suspension following centrifugation was resuspended in freshly prepared Carnoy's fixative (1:3, acetic acid: methanol) at room temperature by vortexing. Twenty microliters of Triton X-100 at a concentration of 0.5% was added drop by drop while vortexing. Three changes with fixative containing 0.5% Triton X-100 were optimal for obtaining good metaphase spreads with complete removal of the cytoplasmic background. The advantage of this technique is that important patients' samples having clumped metaphases otherwise not useful for G-banding can be rescued and karyotyped by this method.

Chromosome Banding↗

PubMed1993

Sensitivity of phospholipase C (Bacillus cereus) activity to lipid packing in sonicated lipid mixtures.

We have recently demonstrated that phospholipase C (PLC) activity on membranes decreases in the presence of membrane-active peptides such as alamethicin, gramicidin S, and melittin [Rao, N. M. (1992) Biochem. Biophys. Res. Commun. 182, 682-688]. Since these peptides affect lipid packing in the membrane and induce nonbilayer phases depending on the lipid composition, we tested for the sensitivity of PLC activity to lipid packing. We monitored PLC activities on four lipid systems which demonstrate a transition from the bilayer to the nonbilayer phase as a function of one of the components. The four model systems are (1) dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE); (2) DOPE, DOPC, and cholesterol; (3) DOPE and lysophosphatidylcholine; and (4) DOPC and gramicidin D. On all four lipid systems, the PLC activity was high for lipid in the bilayer phase and decreased as the phase changed to the nonbilayer phase. The phase changes were also monitored in PLC assay conditions on the four model systems by 31P NMR to confirm the observations made with PLC. These results suggest that the lipid in bilayer and nonbilayer phases was differentially susceptible to PLC; hence, PLC activity may be used to monitor isothermal phase transitions at physiological conditions.

Bacillus cereus↗

PubMed1993

Interaction of wild-type signal sequences and their charged variants with model and natural membranes.

The interaction of synthetic peptides corresponding to wild-type signal sequences, and their mutants having charged amino acids in the hydrophobic region, with model and natural membranes has been studied. At high peptide concentrations, i.e. low lipid/peptide ratios, the signal peptides cause release of carboxyfluorescein (CF) from model membranes with lipid compositions corresponding to those of translocation-competent as well as translocation-incompetent membranes. Interestingly, mutant sequences, which were non-functional in vivo, caused considerable release of CF compared with the wild-type sequences. Both wild-type and mutant signal sequences perturb model membranes even at lipid/peptide ratios of 1000:1, as indicated by the activities of phospholipases A2, C and D. These studies indicate that such mutant signals are non-functional not because of their inability to interact with membranes, but due to defective targeting to the membrane. The signal peptides inhibit phospholipase C activity in microsomes, uncouple oxidative phosphorylation in mitochondria and increase K+ efflux from erythrocytes, and one of the mutant sequences is a potent degranulator of the mast cells. Both wild-type and mutant signal sequences have the ability to perturb vesicles of various lipid compositions. With respect to natural membranes, the peptides do not show any bias towards translocation-competent membranes.

Amino Acid Sequence↗

PubMed1993

Distribution and some properties of sheep (Ovis aries) angiotension converting enzyme.

Angiotensin converting enzyme (ACE) was measured in 15 sheep tissues by spectrophotometric assay with hippuryl-L-histidyl-L-leucine as substrate. Captopril inhibited the ACE activities of all the tissues. The ACE activity was highest in caput epididymidis, corpus epididymidis, cauda epididymidis, kidney and testis. The ACE activity was moderate in retina, little in cornea and lowest in lens. The greater increase in epididymal ACE activity than that of testicular ACE activity on maturation indicates that epididymal ACE may be highly sensitive to hormones. Chloride functioned as non-essential activator of corneal and retinal ACE. The ACE activities of sheep tissues are compared with those found in other species and probable role of ACE in different tissues is discussed.

Aging↗