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Biomedical subjects

A W Lipkowski

Publications and source records attributed to A W Lipkowski.

At least 19 recordsLinked to original sources
PubMed2000

Modulation of glutamine uptake and phosphate-activated glutaminase activity in rat brain mitochondria by amino acids and their synthetic analogues.

Uptake of L-[14C]Gln and phosphate-activated glutaminase (PAG) activity were measured in nonsynaptic mitochondria isolated from rat cerebral hemispheres, in the presence of protein and nonprotein amino acids and their synthetic structural analogues and derivatives. The uptake was inhibited by > 50% in the presence of a 10-fold excess of His, homocysteine (Hcy), Trp, Leu, Tyr, Ile, Thr, Ala, Phe, Met, Ser, by > 20% in the presence of a 10-fold excess of Val, Arg, Glu, and was not affected by a 10-fold excess of Orn, alpha-ketoglutarate, Tau and Pro. Uptake of L-[14C] Leu differed from Gln uptake by its resistance to Arg, Glu, and a relatively high sensitivity to the reference inhibitor of the plasma membrane transport of large neutral amino acids (L-system)--BCH (2-aminobicyclo[2.2.1]heptane-2-carboxylic acid), and a number of natural L-system substrates. A newly synthesized alanine analogue, 2'-cyano-(biphenyl) alanine, referred to as MRC01, was the only compound tested that inhibited Gln uptake more strongly than Leu uptake. The strongest Gln uptake inhibitors: MRC01, His, Hcy and Leu, inhibited PAG activity by > 50% when added at the inhibitor/Gln concentration ratio of 1:2. PAG activity was not affected by Tau, Lys or Pro, compounds which did affect Gln uptake. The results suggest that a number of natural amino acids function as common endogenous modulators of cerebral mitochondrial Gln uptake and its degradation. MRC01, because of its inhibitory potency towards both mitochondrial Gln uptake and PAG activity, may become a convenient tool in studying the role of Gln transport in its mitochondrial metabolism in intact CNS cell and tissues.

Alanine↗
PubMed1999

Ischemic rats as a model in the study of the neurobiological role of human beta-amyloid peptide. Time-dependent disappearing diffuse amyloid plaques in brain.

Brains from patients with Alzheimer's disease contain diffuse and senile amyloid plaques. Using an experimental model, we have addressed the issue whether diffuse plaques of amyloid persist, develop with time, or both, in rats injected with human beta-amyloid-(1-42)-peptide for 3 and 12 mon after brain ischemia. Rats receiving beta-amyloid peptide for 3 months after brain ischemia demonstrated widespread diffuse amyloid plaques in hippocampus and cerebral cortex. Neuronal, glial, ependymal, endothelial and pericyte cell bodies were observed filled with beta-amyloid peptide. No staining was observed in control brains. In the group alive 1 year no deposition of human beta-amyloid peptide was observed, too. Direct evidence that diffuse amyloid plaques can disappear in the brain is thus provided for the first time.

Alzheimer Disease↗
PubMed1999

Biological activity of fragments and analogues of the potent dimeric opioid peptide, biphalin.

The synthesis and biological activity of two fragments of the very potent opioid peptide biphalin, showed that Tyr-D-Ala-Gly-Phe-NH-NH<-Phe is the minimal fragment necessary to express equal affinities and the same biological activity profile as the parent biphalin. The replacement of N'-Phe with other L- or D- lipophilic amino acids showed the possibility of modification of receptor efficacy of the analogues.

Amino Acid Sequence↗
PubMed1998

[125I-Tyr1]biphalin binding to opioid receptors of rat brain and NG108-15 cell membranes.

Mono iodinated analogues of biphalin [(Tyr-D-Ala-Gly-Phe-NH-)2], both nonradioactive [I-Tyr1]biphalin and radioactive [125I-Tyr1]biphalin have been synthesized. The radioligand binding profiles of these compounds for two types of tissues, rat brain membranes, and NG108-15 cell membranes were identical to the parent biphalin. This is additional evidence for the hypothesis that biphalin behaves like a monomeric ligand and that only one intact tyrosine is necessary for high biological activity. The second tyrosine could be used for successful radioiodination which may greatly simplify biochemical and pharmacological studies of biphalin. The results of receptor binding studies show that the binding of both biphalin and [I-Tyr1]biphalin to the delta and mu opioid receptors are not independent. [125I-Tyr1]Biphalin binds to delta receptors as shown in NG108-15 cell membranes. Nevertheless, [125I]biphalin binding to delta receptors in rat brain membranes was hardly evident and mu receptor binding predominated or at least was much more readily detectable in this preparation.

Animals↗
PubMed1998

Inhibitory effect of biphalin and AZT on murine Friend leukemia virus infection in vitro.

Biphalin is a bivalent opioid analogue containing two tyrosine residues. We have examined the effect of biphalin's anti-retroviral potency in vitro using a murine model. Biphalin, in non-cytotoxic concentrations, suppressed in a dose-dependent fashion the replication of Friend leukemia virus (FLV) in Mus dunni cells as determined using a focus forming assay. FLV replication was substantially reduced by biphalin at 10(-4) M concentration. When biphalin was combined with 3'-azido-3'-deoxythymidine (AZT) the two acted synergistically in inhibiting FLV replication compared to either used alone. Using a reverse transcriptase (RT) assay, FLV RT levels also were noted to be reduced in the presence of biphalin. These observations indicate that biphalin possesses anti-retroviral activity in vitro, suggesting that this opioid peptide should be examined further in vivo to determine if it is a candidate for combined therapy with AZT and possibly other drugs for retrovirus infections including the human immunodeficiency virus (HIV).

Antiviral Agents↗
PubMed1998

Ultrastructural changes in the central and peripheral nervous system in the rat with experimental allergic encephalomyelitis.

The aim of the study was the evaluation of ultrastructural changes in rats central and peripheral nervous system after the introduction of experimental allergic encephalomyelitis (EAE) and after the treatment with spinal cord protein hydrolysate. Reduced structural disturbances in myelin were found after oral treatment with hydrolysate. In addition, the indications of remyelinization processes have been observed.

Animals↗
PubMed1998

Decreased hypotensive responsiveness to nitric oxide donor S-nitroso N-acetyl-DL-penicillamine (SNAP) in spontaneously hypertensive (SHR) rats.

The aim of the study was to compare hemodynamic effects of intravenously (i.v.) applied nitric oxide (NO) donor S-nitroso N-acetyl-DL-Penicillamine (SNAP) in conscious spontaneously hypertensive (SHR) to those observed in normotensive Wistar Kyoto (WKY) rats. The study was performed on 7 SHR and 8 WKY instrumented with polyethylene catheters inserted to the abdominal aorta and vena cava for blood pressure (MAP) and heart rate period (HRp) monitoring, and for i.v. administration of SNAP (0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 40.0 and 75.0 microM/kg of body weight). The following differences were found between SHR and WKY rats: 1) the threshold dose of SNAP, eliciting significant decrease of MAP was markedly higher in SHR (1.0 microM/kg b.w.) than in WKY (0.2 microM/kg b.w.), 2) SHR responded with significantly smaller maximum decreases of MAP to administration of 1.0, 2.0, 5.0 and 10.0 microM/kg b.w. of SNAP and with smaller heart rate acceleration to administration of 10.0, 40.0 and 75.0 microM/kg b.w. of SNAP, 3) in SHR MAP decreased progressively, the greatest decline being observed after administration of the highest dose (75 microM/kg b.w.) of SNAP while in WKY the log dose/delta MAP response curve reached plateau beginning with 2 microM/kg b.w. of SNAP, 4) the slopes and intercepts of the regression lines describing relationship between MAP and HRp after administration of SNAP were significantly different in SHR and WKY rats (P < 0.01). The results indicate that SHR are significantly less sensitive to hypotensive effects of NO generated from moderate doses of SNAP.

Animals↗
PubMed1998

Binding of SPXK- and APXK-peptide motifs to AT-rich DNA. Experimental and theoretical studies.

The binding properties of the SPXK- and APXK-type peptides to the AT-rich DNA fragments of different length were studied by measuring the competition of peptides with Hoechst 33258 dye for DNA binding and by the gel shift assay analysis. In parallel to the experimental studies, molecular modeling techniques were used to analyze possible binding modes of the SPXZ and APXK motifs to the AT-rich DNA. The results of the competition measurements and gel shift assays suggest that serine at the i-1 position (i is proline) can be replaced by alanine without affecting the binding properties of the motif. Thus, the presence of the conserved serine in this motif in many DNA-binding proteins is probably not dictated by structural requirements. Based on the results of molecular modeling studies we propose that the binding mode of the SPXK-type motifs to the AT-rich DNA resembles closely that between the N-terminal arm of the homeodomain and DNA. This model confirms that serine in the SPXK motifs is not essential for the DNA binding. The model also indicates that if X in the motif is glutamic acid, this residue is probably protonated in the complex with DNA.

Amino Acid Sequence↗
PubMed1997

Interaction of a highly potent dimeric enkephalin analog, biphalin, with model membranes.

Biphalin, (Tyr-D-Ala-Gly-Phe-NH)2, is a highly potent dimeric analog of enkephalin. Its analgesic efficacy is due in part to its ability to permeate the blood-brain barrier. To aid in understanding the mechanism of the transmembrane movement we determined and analyzed the permeability and partition coefficients of biphalin and a series of analogues where F, Cl, I, NO2, or NH2 were placed in the para position of the aromatic rings of Phe4,4'. Liposomes composed of neutral phospholipids and cholesterol were used as the model membrane. The overall good correlation between permeability and water-membrane partition coefficients suggests that the movement of biphalins across the model membrane is controlled by diffusion and depends on the water-membrane partition coefficient. To explain the observed correlation between permeability and the electron withdrawing/donating character of the substituents in the phenylalanine ring, we examined various folding patterns of Leu-enkephalin, an endogenous pentapeptide that exhibits affinities toward the same classes of opioid receptors (delta and mu). The observed permeabilities and partition coefficients of biphalin and analogues, as well as the tyrosine side chain accessibility, are consistent with the presence of the type of folding where the tyrosine and phenylalanine side chains are in a close contact. We propose that the aromatic ring interaction can promote the peptide permeability by stabilizing a more compact structure of biphalin that would minimize the number of hydrogen bonds with water and therefore enhances partitioning into the model membrane.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1997

Transport of human beta-amyloid peptide through the rat blood-brain barrier after global cerebral ischemia.

In an attempt to produce an animal model of the Alzheimer's disease (AD), beta-amyloid-(1-42)-peptide (beta A1-42) was injected into the femoral vein in rats after single and repeated cardiac arrest (CA). After survival of 3.5 months, the brains immunoreactivity was evaluated using light microscopic immunocytochemistry of monoclonal beta-amyloid peptide (beta A) antibody 4G8 (mAb 4G8). Rats receiving beta A1-42 after CA demonstrated multifocal and widespread extravasation of beta A1-42 in extra- and intracellular space. The permeability to beta A1-42 was significantly higher in rats after repeated cerebral ischemia. As in AD, there were irregular diffuse amyloid plaque-like deposits and neuronal loss with reactive gliosis. Our data in ischemic rats with beta A1-42 represent a novel animal model of Alzheimer's pathology.

Amyloid beta-Peptides↗
PubMed1997

Structure-activity relationship of biphalin. The synthesis and biological activities of new analogues with modifications in positions 3 and 4.

New analogues of biphalin [(Tyr-D-Ala-Gly-Phe-NH-)2] with modifications of amino acid residues in positions 3,3' and 4,4' have been synthesized. The potency and selectivity of these analogues were evaluated by competitive radioreceptor binding assay in the rat brain using [3H]CTOP (mu ligand) and [3H][p-Cl-Phe4]DPDPE (delta ligand) as ligands, and by bioassay in the mouse vas deferens (MVD, delta receptor assay) and guinea pig ileum (GPI, mu receptor assay). The symmetrical substitution of phenylalanine in positions 4 and 4' with p-fluorophenylalanine or p-nitrophenylalanine resulted in an enhancement of the affinity at both delta and mu receptors, with some increase of the selectivity for delta opioid receptors. The analogue containing p-chlorophenylalanine in positions 4 and 4' is the most selective to the delta receptors in this series, with a selectivity ratio about 5. The symmetrical substitution of the glycine-3 residue with phenylalanine resulted in a decrease of binding affinities and biological potencies at both mu & delta receptors.

Analgesics↗
PubMed1996

Evidence of blood-brain barrier permeability/leakage for circulating human Alzheimer's beta-amyloid-(1-42)-peptide.

Brains from patients with Alzheimer's disease contain amyloid plaques which are composed of beta-amyloid peptide and are considered to play a causal role in the neuropathology of this disease. The origin of beta-amyloid peptide in brain parenchyma and vessels of Alzheimer's disease patients is not known. This study examined the permeability of the blood-brain barrier to beta-amyloid peptide in rats subjected to single or repeated episodes of global cerebral ischaemia followed by i.v. injections of human synthetic beta-amyloid-(1-42)-peptide. Rats receiving beta-amyloid peptide after ischaemia demonstrated multifocal and widespread accumulation of beta-amyloid peptide in hippocampus, cerebral cortex and occasionally in white matter. beta-Amyloid peptide penetration involved arterioles, veins and venules. Neuronal, glial and pericyte bodies were observed filled with beta-amyloid peptide. Direct evidence that soluble human beta-amyloid-(1-42)-peptide crosses the blood-brain barrier and enters the brain from the circulation is thus provided for the first time.

Amyloid beta-Peptides↗
PubMed1996

Synthesis and biological properties of gamma-glutamyl-dermorphin, a prodrug.

The possibility of using the gamma-glutamyl-transpeptidase system for transformation of inactive propeptide, gamma-glutamyl-neuropeptides into active neuropeptides has been tested on dermorphin and its gamma-glutamyl analogue. Gamma-glutamyl-dermorphin 2 showed little affinity for opioid receptors. Nonetheless, systemic (intraperitoneal (i.p.), or intravenous (i.v.)) application of this compound induced significant antinociceptive effects, although ten to twenty-fold higher doses were required compared to the parent dermorphin 1. On the other hand, the analogue 2 showed high, antinociceptive activity when injected intrathecally (i.t.). When compared to dermorphin, 2 was one third as potent, but did show a significant prolonged duration of the effect. These results suggest that in the periphery, the peptidase metabolism which results in degradation of bioactivity, is offset by gamma-glutamyl-transpeptidase (GGTP) activity that liberates bioactive peptide 2. On the other hand, in the central nervous system, the activity of gamma-glutamyl-transpeptidase system seems to be more effective than other peptidase systems, resulting in formation of active peptide 2 in a significant amount. These data suggests that gamma-glutamyl analogues of neuropeptides can be considered as potential prodrugs, especially for synthetic analogues which themselves are resistant to peptidase action.

Analgesics, Opioid↗
PubMed1996

Lipid membrane permeability of modified c[D-Pen2, D-Pen5]enkephalin peptides.

Permeability coefficients of a series of analogues of a potent opioid peptide, c[D-Pen2, D-Pen5]enkephalin, were measured in a model membrane system. The analogues included hydrophobic amino acid substitutions on position 3. Liposomes of a mixed composition consisting of zwitterionic lipids and cholesterol served as the model membranes. The obtained permeability coefficients range between 0.38 x 10(-12) and 2.9 x 10(-12) cm/s. These data were correlated with the hydrophobicity scale of Nozaki and Tanford (J. Biol. Chem. 246, 1971, 2211-2217) (correlation coefficient = 0.9933) and with determinations of lipid order perturbation by differential scanning calorimetry (correlation coefficient = -0.9779). The reasonably good correlation obtained within the family of analogues substituted on position 3 (Gly, Ala, Leu, Phe) indicates that changes in permeabilities are primarily related to increases in the partition coefficient of the peptide. However, Phe residue added on the N-terminal end of the peptide (position 0) does not appear to follow the observed trend, showing stronger lipid perturbation and lower permeability compared to the Phe3 analog. This observation demonstrates that each class of peptide modifications requires a new basis of permeability analysis and predictions.

Calorimetry, Differential Scanning↗
PubMed1996

Enkephalin analog prodrugs: assessment of in vitro conversion, enzyme cleavage characterization and blood-brain barrier permeability.

To improve the blood-brain barrier penetration of the delta-opioid receptor peptides [D-Pen2, D-Pen5]enkephalin (DPDPE) and [D-Pen2, L-Cys5]enkephalin (DPLCE), various prodrug forms were synthesized to increase lipophilicity and drug delivery to the brain. The aims of this study were 3-fold, 1) to assess the metabolic conversion of various DPDPE and DPLCE prodrugs in vitro using mouse brain homogenate and mouse serum, 2)to characterize the proteolytic enzymes responsible for cleaving prodrugs to the parent compounds using select peptidase inhibitors and 3)to assess the blood-brain barrier permeability of prodrugs, compared with their parent compounds, using the in vitro bovine brain microvessel endothelial cell culture model. The prodrugs with carboxyl-terminal phenylalanine residues (DPDPE-Phe and DPLCE-Phe) had significantly longer metabolic conversion times in both mouse serum and brain homogenates than did the prodrugs with amino-terminal phenylalanine residues. Inhibition of leucine aminopeptidase with bestatin in the serum increased the conversion time of Phe0-DPDPE from 6.8 min to 92.2 min. Inhibition of aminopeptidase M with amastatin in the brain homogenate increased the conversion time of Phe0-DPDPE from 3.9 min to > 450 min. The long half-life of DPLCE-Arg-Pro-Ala in serum (317 min) vs. brain (9.2 min) can be explained by the high levels of the degradative endopeptidase 24.15 (EC 3.4.24.15) in the central nervous system but not in plasma. The data also showed that, for specific prodrugs of DPDPE such as Phe0-DPDPE and DPDPE-Arg-Gly, the prodrug shows a significant improvement in permeability, compared with the parent compound. Therefore, these data provide evidence that prodrugs or prodrug-enzyme inhibitor combinations may optimize the delivery of peptide and/or protein drugs to the central nervous system.

Analgesics↗
PubMed1996

Blood-to-central nervous system entry and stability of biphalin, a unique double-enkephalin analog, and its halogenated derivatives.

Biphalin (Tyr-D-Ala-Gly-Phe-NH)2 is a unique opioid peptide analog that contains two active enkephalin pharmacophores and is more potent than morphine and etorphine in eliciting analgesia after intrathecal administration. After systemic administration, only a small amount was detected in the brain, but analgesia was observed. Because halogenation of enkephalin analogs has been shown to increase the brain uptake after systemic administration, our research group synthesized both p-[Cl-Phe4,4']biphalin and p-[F-Phe4,4']biphalin. The aim of the present study was to characterize and compare the blood-to-central nervous system (CNS) pharmacokinetics and biological stability of biphalin and related halogenated analogs. The initial screening used an in vitro blood-brain barrier model and identified p-[Cl-Phe4,4')biphalin as the enkephalin analog with the best potential for greater CNS entry. The CNS uptake and stability of biphalin and p-[Cl-Phe4,4']biphalin was examined further using an in situ brain perfusion technique coupled to high-performance liquid chromatography analysis. Both biphalin and its chlorohalogenated analog, were found to significantly enter the CNS through both the blood-brain and blood-cerebrospinal fluid barriers. Chlorohalogenation of biphalin was shown to both improve CNS entry, most likely through an enhancement in lipophilicity, and increase biological stability. This study suggests that incorporation of chlorohalogens at the p-Phe4,4' position is a promising structural modification in the development of biphalin as a successful opioid drug for the clinic.

Analgesics↗