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Biomedical subjects

S Akhtar

Publications and source records attributed to S Akhtar.

At least 19 recordsLinked to original sources
PubMed1997

Interaction of oligonucleotide-conjugates with the dipeptide transporter system in Caco-2 cells.

Oligonucleotide-based therapies represent novel strategies for manipulating the expression and function of target proteins and are undergoing clinical evaluation for the treatment of viral diseases and malignancies. However, poor biological stability and cellular delivery represent potential limitations to the therapeutic development of oligonucleotides. Conjugation of oligonucleotides to lipophilic groups can improve delivery to cells but the enhanced cellular binding may also facilitate nonspecific interactions. In this report, we show that phosphorothioate oligonucleotides conjugated to lipophilic groups, either tocopherol (Vitamin E) or 2-Di-O-hexadecyl-3-glycerol, can significantly inhibit the functioning of the dipeptide transporter system (DTS) in cultured Caco-2 intestinal cells. Because the DTS mediates the binding and absorption of nutrient peptides and important drugs, such as the cephalosporin and penicillin antibiotics, this finding has important implications in relation to the potential toxicity of lipophilic conjugates in vivo. It also suggests a potential drug interaction with lipophilic oligonucleotide-conjugates if they were to be delivered orally.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1997

Modification of rat brain Kv1.4 channel gating by association with accessory Kvbeta1.1 and beta2.1 subunits.

We have examined the effects of co-expression of Kvbeta1.1 and Kvbeta2.1 subunits on the gating of rat brain Kv1.4 channels, expressed in Xenopus oocytes. Expression of Kv1.4 subunits alone produced a rapidly inactivating "A" type current, which activated at potentials beyond -60 mV in a solution containing high levels of rubidium. Current activation curves obtained from tail current measurements were fitted with a Boltzmann function, with V1/2 = -47 mV and k = 10 mV. Neither the Kvbeta1.1 nor Kvbeta2.1 subunits altered the voltage dependence of activation. Both subunits accelerated the activation time constant of Kv1.4, without affecting its voltage dependence. Surprisingly, the Kvbeta2.1 subunit, which lacks an N-terminal inactivation domain, was almost as effective as the Kvbeta1.1 subunit in speeding up Kv1.4. Steady-state inactivation of Kv1.4 was unchanged upon co-expression with either Kvbeta1.1 or Kvbeta2.1 subunits. Kv1.4 recovered from inactivation with two time constants; apart from an approximately 50% lengthening of the slow time constant with a high Kvbeta2.1 injection ratio, neither time constant was altered by either the Kvbeta1.1 or Kvbeta2.1 subunits, suggesting little interaction with recovery from C-type inactivation. Clearly, beta subunits have the potential to modify the gating of Kv1.4 channels in the brain more subtly than has been suggested previously.

Animals↗
PubMed1997

Bluetongue virus seropositivity in sheep flocks in North West Frontier Province, Pakistan.

The objectives of this study were to describe the prevalence and distribution of serum antibodies to Bluetongue virus (BTV) in a sample of 38 sheep flocks in northern areas of the North West Frontier Province of Pakistan and to identify demographic and productivity variables that are associated with BTV seropositivity. Blood samples were taken from a random sample of ewes in each flock in April 1995. The owners of the flocks were interviewed regarding some demographic, husbandry and productivity variables of the flocks on the day of blood sampling. A competitive enzyme-linked immunosorbent assay was conducted to test the serum samples for BTV group-specific antibodies. BTV seropositive reactions were obtained in 184 (48.4%) out of 380 tested sera, and in 89.5% (34/38) of the flocks. In the 34 seropositive flocks, the prevalences ranged from 12.5 to 100% (median = 47). A multivariable logistic analysis was carried out to study the influence of demographic and productivity variables on the BTV serological status of the sheep flocks. Abortion risk in the previous lambing season was mildly associated with the serological status of the flock (adjusted odds ratio = 1.16, P = 0.07). For the seropositive flocks, a linear multiple regression showed that distance travelled by the flock during transhuman movement was significantly associated with percent seropositivity (partial regression coefficient (+/- SE) = -0.091 +/- 0.045).

Abortion, Veterinary↗
PubMed1997

A sero-epidemiological study of Haemophilus somnus infection in dairy cattle.

Five repeated cross-sectional serological surveys of 790 dairy cattle in 4 dairy herds between December 1985 and February 1987 provided an opportunity to study the changes in the seroprevalence of Haemophilus somnus across the 5 surveys and with respect to some demographic and disease variables. The demographic variables included were age (heifers or cows) and farm, representing two groups of herds (two herds in each group, located in the Central and Northern Valleys of California). The serological status of cattle as either negative or positive against H. somnus, Campylobacter fetus and Leptospira hardjo were determined with enzyme linked-immunosorbent assays. Logistic regression analysis was used to compute maximum likelihood estimates of adjusted odds ratios and their respective 95% confidence intervals. The baseline risk of being H. somnus seropositive in the cattle observed at first sampling did not vary significantly during the study period after adjustment for the effects of covariates. Only at first sampling were cows about twice as likely to be H. somnus seropositive than heifers. At samplings 1 and 3, but not at 2 and 5, being in the herds of the Central Valley appeared protective. In contrast, at sampling 4 the cattle in herds in the Central Valley were about 7 times more likely to be H. somnus seropositive. C. fetus-positive cattle were about 3 times more likely to be H. somnus seropositive at sampling 1 only. The relationship between H. somnus status and L. hardjo was not significant during the study period.

Animals↗
PubMed1997

Cellular uptake properties of a 2'-amino/2'-O-methyl-modified chimeric hammerhead ribozyme targeted to the epidermal growth factor receptor mRNA.

Catalytic RNA or ribozymes have important potential applications as molecular biological tools in the study of gene expression and as therapeutic inhibitors of disease-causing genes. Very little is known, however, about the cellular uptake mechanisms of exogenously delivered synthetic ribozymes. In this study, we have characterized the uptake properties of a synthetic, 2'-O-methyl-modified ribozyme containing U4/U7 amino groups within the catalytic core of the hammerhead motif. The cellular uptake of the internally [32P]-radiolabeled hammerhead ribozyme in U87-MG glioma cells was temperature, energy, and pH dependent and involved an active process that could be competed with cold ribozyme of the same chemistry and sequence, an all 2'-O-methyl-modified ribozyme of the same sequence, antisense PS-ODNs, and a variety of other polyanions (salmon sperm DNA, spermidine, dextran sulfate, and heparin). Subcellular distribution studies of fluorescently labeled ribozymes confirmed an extranuclear, punctate localization similar to that observed for an endosomal marker, dextran. Our study highlights that hammerhead ribozymes, despite exhibiting a defined secondary structure, enter cells by an endocytic mechanism that appears to be similar to that reported for a variety of antisense ODNs. These observations should facilitate the development of more efficient delivery systems.

Base Sequence↗
PubMed1997

Sero-epidemiological studies on Toxoplasma gondii infection in man and animals in Bangladesh.

Sera from randomly selected 49 professional blood donors, 617 pregnant women, 14 butchers, 528 slaughtered goats and 24 domestic cats in the district of Mymensingh were tested for the presence of T. gondii antibodies using a Latex agglutination test (LAT). Overall 12.4% blood donors, 11.18% pregnant women, 50.00% butchers, 12.88% slaughtered goats and 33.33% cats had diagnostically significant antibody titers (> or = 1:64) to T. gondii. Epidemiological studies on T. gondii infection with LAT were conducted in 25 family members with sero-positive cats and 9 family members with 2 sero-positive women without cats in the family. Significantly (p < 0.01) higher sero-positivity rate was recorded in the family members (24.00%) with positive cats in comparison to family members (11.11%) without cats. The epidemiologic study indicates that infected cats and goat meat might be significant sources of T. gondii infection for humans in Bangladesh.

Animals↗
PubMed1996

Direct inhibitory effects of an antisense oligodeoxynucleotide upon the volume-sensitive chloride current in rat osteoblast-like (ROS 17/2.8) cells.

The effects of a 15-mer antisense c-myc phosphorothioate modified oligodeoxynucleotide (OdN) upon the volume-sensitive Cl- current in ROS 17/2.8 cells were investigated using the whole-cell configuration of the patch clamp technique. At 5 microM, the OdN reversibly inhibited the current in a voltage- and time-dependent fashion. This was evident from the reduction in the peak current as assessed at the termination of each voltage pulse and an acceleration of the time-dependent inactivation present at strongly depolarised potentials. The kinetic modifications induced by the OdN suggest it may act by blocking the pore of open channels when the cell membrane potential is depolarised.

Animals↗
PubMed1996

Epidemiologic association between antibody titres against bovine virus diarrhoea virus, rinderpest disease virus and infectious bovine rhinotracheitis virus in a buffalo herd.

The association between antibody titres against bovine virus diarrhoea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV) and rinderpest disease virus (RPDV) in buffaloes was investigated in a cross-sectional study. Thirty-six lactating buffaloes from a herd in Landhi Cattle Colony, Karachi were bled and serum samples subjected to a micro-neutralisation test at the Plum Island Animal Disease Center, USA, to categorise each buffalo either as positive or negative against each of 3 viral antigens. Log-linear analysis was used to evaluate the association among 3 categorical variables. The "best-fitting" log-linear model was the model of complete independence. This model includes all 3 main effects (BVDV, IBRV, RPDV), suggesting that the seropositivity of buffaloes against these viral antigens tended to occur independently. Estimates of parameters of the model showed that the proportion of buffaloes seropositive (97.2%) to RPDV antigen tended to be higher than expected, whereas the proportion of BVDV seropositive (30.6%) and IBRV seropositive (16.7%) buffaloes tended to be smaller than expected.

Animals↗
PubMed1996

Dynamic and steady state response of heart rate to orthostatic stress in normotensive and hypertensive pregnant women.

We determined the dynamic and steady state responses of heart rate (HR) to orthostatic stress (standing up) in normotensive and hypertensive pregnant women. Using a continuous recording with servo-photosphygmography, HR response to change in posture from left lateral recumbent position to standing was analysed. The subjects were divided into five groups comprising: Groups I, II and III: normotensive pregnant women in each of the three trimesters of pregnancy (total n = 77); Group IV: women with gestational proteinuric hypertension (GPH) in the third trimester (n = 16); Group V: age-matched non-pregnant normotensive controls (n = 15). The HR reacted with a typical overshoot response to this orthostatic change with HR rising to a peak and then settling to a new but higher steady state. Change in steady state HR from lying to standing (delta HR), rate of rise of HR in response to standing (i.e. the acceleration slope (HRon)), and rate of fall of HR after reaching the peak (i.e. deceleration slope (HRoff)) were evaluated from standing heart rate time curves. HRon in response to standing showed a downward trend with gestation (ANOVA, P < 0.05) in normotensive gravida. The deceleration slope (HRoff) showed a distinct gestational age-related decrease from first to third trimester in normotensive women (ANOVA, P < 0.01). The most striking observation was that the slope of HRoff for the GPH group was significantly steeper than that of normotensive women of comparable gestational age (unpaired t-test P < 0.01) and approximated to that of the non-pregnant group. The difference in HR response between normotensive women and those with GPH in the third trimester suggests it may have potential as a new marker for pre-eclampsia.

Adult↗
PubMed1996

Interactions of phosphodiester and phosphorothioate oligonucleotides with intestinal epithelial Caco-2 cells.

PURPOSE: Oral bioavailability for antisense oligonucleotides has recently been reported but the mechanistic details are not known. The proposed oral delivery of nucleic acids will, therefore, require an understanding of the membrane binding interactions, cell uptake and transport of oligonucleotides across the human gastro-intestinal epithelium. In this initial study, we report on the cell-surface interactions of oligonucleotides with human intestinal cells. METHODS: We have used the Caco-2 cell line as an in vitro model of the human intestinal epithelium to investigate the membrane binding interactions of 20-mer phosphodiester (PO) and phosphorothioate (PS) oligonucleotides. RESULTS: The cellular association of both an internally [3H]-labelled and a 5'end [32P]-labelled PS oligonucleotide (3.0% at 0.4 microM extracellular concentration) was similar and was an order of magnitude greater than that of the 5'end [32P]-labelled PO oligonucleotide (0.2%) after 15 minutes incubation in these intestinal cells. The cellular association of PS was highly saturable with association being reduced to 0.9% at 5 microM whereas that of PO was less susceptible to competition (0.2% at 5 microM, 0.1% at 200 microM). Differential temperature-dependence was demonstrated; PS interactions were temperature-independent whereas the cellular association of PO decreased by 75% from 37 degrees C to 17 degrees C. Cell association of oligonucleotides was length and pH-dependent. A decrease in pH from 7.2 to 5.0 resulted in a 2- to 3-fold increase in cell-association for both backbone types. This enhanced association was not due to changes in lipophilicity as the octanol:aqueous buffer distribution coefficients remained constant over this pH range. The ability of NaCl washes to remove surface-bound PS oligonucleotides in a concentration-dependent manner suggests their binding may involve ionic interactions at the cell surface. Cell-surface washing with the proteolytic enzyme, Pronase, removed approximately 50% of the cell-associated oligonucleotide for both backbone types. CONCLUSIONS: Binding to surface proteins seems a major pathway for binding and internalization for both oligonucleotide chemistries and appear consistent with receptor (binding protein)-mediated endocytosis. Whether this binding protein-mediated entry of oligonucleotides can result in efficient transepithelial transport, however, requires further study.

Azides↗
PubMed1996

The influence of polarized epithelial (Caco-2) cell differentiation on the cellular binding of phosphodiester and phosphorothioate oligonucleotides.

Cell aging and the degree of cellular differentiation are thought to be important variables governing uptake of oligonucleotides but remain poorly understood. The Caco-2 colon carcinoma cell line has the ability to spontaneously differentiate into enterocytes in vitro and serves as a useful model to further investigate the effect of differentiation on oligonucleotide binding and uptake. In this study, we report that the extent of oligonucleotide association and the expression of cell surface binding proteins are governed by the age and thus the degree of differentiation of Caco-2 epithelial cells in culture. Cellular association (normalized for cell number) of an all phosphodiester (PO), all phosphorothioate (PS), and a phosphodiester oligonucleotide containing two terminal phosphorothioate internucleotide linkages at the 3' end (EC-PO) gradually increased from day 3 to around day 17 of the culture, followed by a plateau, or slight decrease, up to day 21 of the cell aging study. Overall, a threefold to fourfold increase in binding was observed from day 3 to day 17. Oligonucleotide binding was temperature and pH dependent, but the magnitude of the effect was influenced by cell aging and the degree of differentiation. PS oligonucleotides exhibited greater binding (up to threefold) at the basolateral surface compared with the apical surface within the pH range 5-7. These findings could be directly correlated with the expression levels of cell surface oligonucleotide binding proteins during the aging study. A Caco-2 cell surface protein binding complex of around 46 kDa was identified as the major site of binding for both PO and PS oligonucleotides, although the latter also bound to several other proteins, especially at low pH.

Caco-2 Cells↗
PubMed1996

Antisense therapy.

The sequence specificity of the antisense technique makes it an attractive basis for novel molecular therapeutics. Inhibition of gene expression by antisense in cell culture models has provided a strong rationale for identification and validation of disease targets. Analogues modified from normal phosphodiester oligodeoxynucleotides have entered clinical trials of diseases including AIDS, cancer, and inflammation. It is becoming increasingly apparent that these drugs act by means of a complex mechanism of action, and some of their effects can be sequence independent. Nevertheless, these oligodeoxynucleotides offer considerable promise as novel molecular drugs. Harnessing the therapeutic potential of this powerful technique depends on elucidation of the complex mechanism of action so that effective and meaningful therapeutic modalities can be realized.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Blood cultures in adult patients released from an urban emergency department: a 15-month experience.

OBJECTIVE: To determine the frequency of positive blood cultures obtained from adult patients with potential occult bacteremia released from an urban ED and how often these positive cultures alter the subsequent patient course or management. METHODS: This retrospective case series study was conducted at the ED of a large, urban teaching hospital. The study population consisted of a convenience sample of adult patients who presented to the ED with evidence of fever or other clinical conditions suggesting the possibility of bacteremia. The records of all patients who had blood cultures done and who were not admitted to an inpatient service were reviewed. Follow-up was obtained for all patients for whom culture results were positive. A substantial influence on the medical management or clinical course by a (noncontaminant) positive blood culture result was defined as a positive result that directly led to: further diagnostic testing, hospital admission, initiation or alteration of antibiotic therapy, or a different diagnosis. Culture-positive patients who were noncompliant with requested ED follow-up were included in this estimate. An estimate of the laboratory charges per diagnosis of bacteremia also was derived. RESULTS: Only 24 of 1,350 patients (1.8% of the study population; 95% CI 1.1-2.5%) had true-positive blood cultures. Only 7 patients (0.52% of the population; 95% CI 0.14-0.90%) potentially had their medical management affected by the positive blood culture results. Based on the laboratory charges associated with all blood cultures for this patient group, the cost per clinically significant positive blood culture result was $ 11,570. CONCLUSIONS: The prevalence of bacteremia was 1.8% among the released patients who had blood cultures obtained in the ED. Furthermore, only 0.52% of the patients had positive blood cultures that potentially affected their medical management. Further study is warranted to identify specific criteria for selecting ambulatory patients for whom the use of blood cultures may be cost-effective.

Adult↗