P-glycoprotein Restricts Ocular Penetration of Loperamide across the Blood-Ocular Barriers: a Comparative Study in Mdr1a Knock-out and Wild Type Sprague Dawley Rats

The current research was undertaken to determine the existence and magnitude of P-glycoprotein (P-gp) expression on the blood-ocular barriers by studying the ocular penetration of loperamide, a specific P-gp substrate, in P-gp (Mdr1a) knock-out (KO) and wild type (WT) Sprague Dawley rats. A clear, stable, sterile solution of loperamide (1 mg/mL), for intravenous administration, was formulated and evaluated. Ocular distribution was studied in P-gp KO and WT rats following intravenous administration of loperamide (at two doses). The drug levels in plasma, aqueous humor (AH), and vitreous humor (VH) samples were determined with the aid of UHPLC-Q-TOF-MS/MS, and the AH/plasma (D _AH ) and VH/plasma (D _VH ) distribution ratios were estimated. Electroretinography (ERG), ultrastructural analyses, and histology studies were carried out, in both KO and WT rats, to detect any drug-induced functional and/or structural alterations in the retina. Dose-related loperamide levels were observed in the plasma of both WT and KO rats. The loperamide concentrations in the AH and VH of KO rats were significantly higher compared to that observed in the WT rats, at the lower dose. However, a marked increase in the D _AH and D _VH was noted in the KO rats. ERG, ultrastructure, and histology studies did not indicate any drug-induced toxic effects in the retina under the test conditions. The results from these studies demonstrate that P-gp blocks the penetration of loperamide into the ocular tissues from the systemic circulation and that the effect is more pronounced at lower plasma loperamide concentrations.     

Banded krait minor-satellite (Bkm)-associated Y chromosome-specific repetitive DNA in mouse.

The mouse Y chromosome remains highly condensed in all somatic tissues but decondenses extensively in testis. We have isolated a mouse Y chromosome-specific repeat M34 (11.5 kb) and shown that this is distributed along the Y chromosome except the sex-determining region (the Y short arm) in which GATA repeats are predominantly concentrated. It has 32 copies of GATA repeats in a 2.7 kb fragment. About 200-300 copies of M34 on the Y chromosome are interspersed among other sequences. A 1.2 kb fragment (p3) of M34, containing GATA repeats, also has scaffold attachment region (SAR) motifs which bind to nuclear matrices. A strong affinity of histone H1 to SAR motifs is implicated in maintaining the condensed state of the Y chromosome in somatic tissues. The probable significance of molecular organization of the Y chromosome is discussed.     

Behaviour of microchromosome-associated satellite DNA in the banded krait,Bungarus fasciatus (Ophidia,Elapidae)

Banded krait(Bungarus fasciatus) major satellite DNA (p = 1.700 g/cm³) is mainly localized in the C-band-positive regions of all the microchromosomes. Our study of the behaviour of this satellite DNA byin situ hybridization has revealed a striking polarization of this DNA in the follicular epithelial cells of the ovary during oogenesis and in the spermatids during spermiogenesis. The major satellite DNA is localized at the point of the subsequent protrusion of the acrosomal pole of the round spermatid nuclei and remains in close contact with the developing sperm tip during the process of spermiogenesis. There appears to be an attraction between a specific region of the nuclear membrane and satellite-rich chromatin of the microchromosomes that brings about their polarization. We discuss possible functions of such extreme polarization of microchromosomes in specific cell types during oogenesis and spermiogenesis.     

Molecular cloning and characterization of cDNA for androgen-repressible rat liver protein, SMP-2

SMP-2 is a rat liver protein whose synthesis is influenced by both androgens and aging. The steady-state level of its mRNA is repressed by the androgen. Compared to the adult male, SMP-2 mRNA is found in higher amounts in the prepubertal and senescent male rat livers which show relative androgen insensitivity. A cDNA library in the plasmid pBR322 was constructed from the female rat liver which contains a high level of SMP-2 mRNAs. Recombinant plasmids were screened by differential colony hybridization to 32P-labeled single-stranded cDNAs from adult female and adult male hepatic poly(A)+ RNAs. From a total of 3500 recombinant clones, 11 highly female specific clones were identified. From these female specific colonies the SMP-2 cDNA-containing plasmid (pSP11) was identified by its ability to select an mRNA species whose translation product is immunochemically and electrophoretically indistinguishable from SMP-2. This insert represents a 571-base pair portion of the SMP-2 cDNA. Rescreening of the library at a high colony density using the 32P-labeled cDNA insert of pSP11 identified several positive clones with larger inserts. Hybrid-selected mRNA translation again confirmed these clones to carry SMP-2 cDNA sequences. The plasmid pSP4a containing a 1040-base pair cDNA insert of SMP-2 was characterized by DNA sequence analysis. The size of the cDNA insert of pSP4a is close to the estimated size of the SMP-2 mRNA. The cDNA sequence provides an open reading frame of 282 amino acid residues. A comparison of the translated amino acid sequence with the protein sequences of NBRF-PIR, PSQNEW, and LOSALA data bases did not establish any sequence homology with known proteins. Northern blot analysis using the 32P-labeled cDNA insert of pSP4a confirms the androgenic repression of the SMP-2 mRNA.     

Spontaneous cholesterol movement between lipid vesicles and monkey small intestinal brush border membrane

[14C]Cholesterol movement between egg phosphatidylcholine-cholesterol lipid vesicles and vesicles prepared from monkey small intestinal brush border membrane (BBMV) was studied in physiological buffer at 37 degrees C. The rate of cholesterol transfer from sonicated unilamellar vesicles (ULV) to BBMV follows apparently first-order kinetics. Intermembrane cholesterol movement was strikingly similar in both the directions. However, from BBMV to ULV, the transfer rate was three times faster than that of ULV to brush border membrane (BBM). Similarity in the rate constant was observed when cholesterol transfer was studied using either large multilamellar lipid vesicles or ULV as the donor and BBMV as the acceptor membrane. Rate constant was also the same when the acceptor membrane used was either intact BBMV or ULV prepared from BBM lipids. The rate of transfer of label was not affected even when the acceptor vesicle concentration was increased over fivefold, indicating the first-order nature of the reaction. Transfer of cholesterol from ULV to BBMV was accelerated by the presence of acetone, dimethyl sulfoxide (DMSO), deoxycholate, and papain. Partially purified nonspecific lipid-exchange protein increased the rate of cholesterol transfer by about threefold. Reduction in BBM cholesterol and phospholipid content was noted by DMSO, acetone, and deoxycholate, while papain caused a small depletion of membrane protein. Cholesterol transfer is temperature dependent with an activation energy of 31 kJ X mol-1, which is almost identical in the presence or absence of nonspecific lipid-exchange protein. The molecular mechanism of intermembrane cholesterol movement is discussed in view of the kinetic data obtained.