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S R King

Publications and source records attributed to S R King.

At least 37 records · Page 2Linked to original sources
PubMed1998

Masoprocol (nordihydroguaiaretic acid): a new antihyperglycemic agent isolated from the creosote bush (Larrea tridentata).

An ethnomedically-driven approach was used to evaluate the ability of a pure compound isolated from the creosote bush (Larrea tridentata) to lower plasma glucose concentration in two mouse models of type 2 diabetes. The results indicated that plasma glucose concentration fell approximately 8 mmol/l in male C57BL/ks-db/db or C57BL/6J-ob/ob mice following the oral administration of masoprocol (nordihydroguaiaretic acid), a well known lipoxygenase inhibitor. The decline in plasma glucose concentration following masoprocol treatment in the mice was achieved without any change in plasma insulin concentration. In addition, oral glucose tolerance improved and the ability of insulin to lower plasma glucose concentrations was accentuated in masoprocol-treated db/db mice. These data raise the possibility that masoprocol, or other lipoxygenase inhibitors, represents a new approach to the pharmacological treatment of Type 2 diabetes.

Animals↗
PubMed1998

Ethnobotanical-directed discovery of the antihyperglycemic properties of cryptolepine: its isolation from Cryptolepis sanguinolenta, synthesis, and in vitro and in vivo activities.

Using an ethnobotanical approach in combination with in vivo-guided fractionation as a means for lead discovery, cryptolepine was isolated as an antihyperglycemic component of Cryptolepis sanguinolenta. Two syntheses of cryptolepine, including an unambiguous synthesis, are reported. The hydroiodide, hydrochloride, and hydrotrifluoromethanesulfonate (hydrotriflate) salts of cryptolepine were synthesized, and a comparison of their spectral properties and their in vitro activities in a 3T3-L1 glucose transport assay is made. Cryptolepine and its salt forms lower blood glucose in rodent models of type II diabetes. While a number of bioactivities have been reported for cryptolepine, this is the first report that cryptolepine possesses antihyperglycemic properties.

3T3 Cells↗
PubMed1998

Cryptolepis sanguinolenta: an ethnobotanical approach to drug discovery and the isolation of a potentially useful new antihyperglycaemic agent.

Evidence has been published that a wide array of plant-derived active principles, representing numerous classes of chemical compounds, demonstrate activity consistent with their possible use in the treatment of patients with Type 2 diabetes mellitus (DM). Despite these interesting observations, to date, metformin is the only ethical drug approved for treatment of Type 2 DM derived from a medicinal plant. Why is this so, given the fact that higher plants are such a potential source of new drugs? The answer to this rhetorical question may lie in the reliance of most pharmaceutical companies on random, in vitro, mechanism-based, high throughput screening in the initial phases of plant drug research. In this article we describe an alternative pathway to discovery of drugs for the treatment of Type 2 DM: on based on an ethnomedical approach, involving ethnobotany and traditional medicine. In particular, we present evidence that cryptolepine, an indoloquinolone alkaloid isolated from Cryptolepis sanguinolenta, significantly lowers glucose when given orally to a mouse model of diabetes. The antihyperglycaemic effect of cryptolepine leads to a significant decline in plasma insulin concentration, associated with evidence of an enhancement in insulin-mediated glucose disposal. Finally, cryptolepine increases glucose uptake by 3T3-L1 cells. These data permit us to conclude that an ethnobotanical approach to drug discovery can identify a potentially useful drug for the treatment of Type 2 DM.

Adipose Tissue↗
PubMed1998

Gene transfer into human umbilical cord blood-derived CD34+ cells by particle-mediated gene transfer.

Delivery of genes into hematopoietic progenitor cells offers an attractive means for the introduction of corrective or protective genes into cells of both the myeloid and lymphoid lineage. Previously, investigators have often used murine retroviral vectors for gene delivery which require cells to be cycling for efficient delivery. We describe a nonviral method of gene delivery using particle-mediated gene transfer to obviate many disadvantages of viral vectors related to safety, production costs and the need for cell cycle proliferation. Using a CMV-CAT reporter plasmid, we show transfection of highly purified CD34+ cells isolated from umbilical cord blood. Effective gene transfer was shown in unstimulated and in growth-stimulated cells. Following transfection with a neomycin resistance gene, differentiation into cells of the myeloid lineage was observed, assayed by CFU-GM in the presence of G-418. Both unstimulated and stimulated cells gave rise to CFU-GM in the presence of G-418, indicating that stable expression of the neomycin resistance gene was maintained in early progenitors. These results demonstrate that particle-mediated gene transfer into human hematopoietic cells from umbilical cord blood can be achieved without affecting their CFU-GM differentiation potential. This gene transfer method offers an alternative approach to gene therapy studies involving human hematopoietic progenitor cells.

Anti-Bacterial Agents↗
PubMed1998

Fluconazole pharmacokinetics in burn patients.

The pharmacokinetics of fluconazole in nine adult patients with severe (30 to 95% total body surface area) burns were studied. There was no significant difference in half-life (t1/2), clearance (CL), or volume of distribution (V) over time in five patients on days 3 and 8 of the study (P > 0.05). Combined parameter estimates (means +/- standard deviations) for all nine patients for the two study periods were as follows: t1/2, 24.4 +/- 5.8 h; CL, 0.36 +/- 0.09 ml/min/kg; and V, 0.72 +/- 0.12 liters/kg. These estimates of t1/2 and CL in burn patients were approximately 13% shorter and 30% more rapid, respectively, than the most extreme estimates reported for other populations.

Adult↗
PubMed1998

Spatio-temporal expression patterns of steroidogenic acute regulatory protein (StAR) during follicular development in the rat ovary.

The steroidogenic acute regulatory protein (StAR) is a vital mitochondrial protein that is indispensable for the synthesis of steroid hormones in the steroidogenic cells of the adrenal cortex and the gonads. Recent studies have shown that StAR enhances the conversion of the substrate for all steroid hormones, cholesterol, into pregnenolone, probably by facilitating cholesterol entry into the inner compartment of the mitochondria where the steroidogenic cytochrome P450scc complex resides. To study the potential of StAR to affect ovarian steroidogenesis during follicular development, we examined the time-dependent expression of StAR protein and messenger RNA in PMSG/human CG (hCG)-treated immature rats. Western blot analyses and immunohistochemical and RT-PCR methodologies have revealed a biphasic expression of StAR in the ovaries responding to hormones. The first peak of StAR expression was generated by PMSG administration and lasted for 24 h. Furthermore, it was restricted to the entire network of the ovarian secondary interstitial tissue, as well as to a fewer scattered theca-interna cells. The second burst of StAR expression was observed in response to the LH surge, as simulated by hCG. This time, StAR was expressed in the entire theca-interna and interstitial tissue, as well as in those granulosa cells that were confined to periovulatory follicles. Immunoelectron microscopy studies revealed the over 90% of StAR antigenic sites are localized in the inner compartments of the mitochondrion, suggesting a rapid removal of StAR precursor from the mitochondrial surface, where it is believed to exert its activity. Altogether, our observations portray dynamic acute alterations of StAR expression during the process of follicular maturation in this animal model. Furthermore, if StAR indeed determines steroidogenic capacities in the ovary, our findings imply that, in immature rats undergoing hormonally induced first ovulation: 1) the early phases of follicular development are supported by androgen production originating from nonfollicular cells; 2) estrogen production in the granulosa cells of Graafian follicles is nourished by a submaximal androgenic output in the theca-interstitial compartments of the ovary.

Animals↗
PubMed1998

Electrical conduction within the cerebrovasculature of stroke-prone spontaneously hypertensive rats.

Alterations in electrical conductivity between smooth muscle cells (SMCs) can alter the spread and effectiveness of electromechanical SMC contraction. We attempted to determine whether alterations in pressure-dependent constriction (PDC) occurring in relation to stroke development within the middle cerebral arteries (MCAs) of Wistar-Kyoto stroke-prone hypertensive rats (SHRsp) were associated with changes in electrical conductivity between the SMCs. Current was injected into nonpressurized MCAs, using a suction electrode. The conducting distance along the length of the MCA where the amplitude of the membrane potential deflection (electronic potential) produced by current injection declined to 1/e (length constant) was used to measure conductivity. PDC to a 100 mmHg pressure step was measured with a pressure myograph. A loss of PDC in the MCAs of SHRsp preceded stroke development. Heptanol (4 mM), a gap junction communication inhibitor, reversibly inhibited conductivity and PDC in the MCA of prestroke SHRsp. The ability of heptanol to reversibly inhibit PDC was likely not related to it's ability to alter electrical conduction. The length constant of electrical conduction in the MCAs was about 0.75 mm and didn't differ between MCA sampled from pre-versus post-stroke SHRsp or Sprague-Dawley rats. It was concluded that alterations in electrical conductivity along the MCA could modify the spread of PDC, but such changes do not contribute to the loss of PDC within the MCA of poststroke SHRsp.

Animals↗
PubMed1997

Phosphorylation of steroidogenic acute regulatory protein (StAR) modulates its steroidogenic activity.

Steroidogenic acute regulatory protein (StAR) plays a critical role in steroid hormone synthesis. StAR is thought to increase the delivery of cholesterol to the inner mitochondrial membrane where P450scc resides. Tropic hormones acting through the intermediacy of cAMP rapidly increase pregnenolone synthesis, and this rapid steroidogenic response is believed to be due to StAR's action. The StAR protein contains two consensus sequences for phosphorylation catalyzed by protein kinase A that are conserved across all species in which the amino acid sequence of the StAR protein has been determined. We demonstrated that human StAR expressed in COS-1 cells exists in at least four species detectable by two-dimensional gel electrophoresis followed by Western blotting. The two more acidic species disappeared after treatment of the cell extracts with alkaline phosphatase. 32P was incorporated into StAR protein immunoprecipitated from COS-1 cell extracts, and a 10-min treatment with 8-bromo-cAMP increased 32P incorporation into the StAR preprotein. StAR protein generated by in vitro transcription/translation was phosphorylated by the protein kinase A catalytic subunit in the presence of [gamma-32P]ATP. Mutation of potential sites for protein kinase A-mediated phosphorylation at serine 57 and serine 195 to alanines, individually, reduced 32P incorporation from labeled ATP into StAR preprotein produced by in vitro transcription/translation when incubated with protein kinase A catalytic subunit. 32P labeling of StAR protein expressed in COS-1 cells was also reduced when serine 57 or serine 195 were mutated to alanines. A double mutant in which both serine 57 and serine 195 were changed to alanines displayed markedly reduced 32P incorporation. To determine the functional significance of StAR phosphorylation, we tested the steroidogenic activity of the wild-type StAR and mutated StAR proteins in COS-1 cells expressing the human cholesterol side chain cleavage enzyme system. Mutation of the conserved protein kinase A phosphorylation site at serine 57 had no effect on pregnenolone synthesis. However, mutation of the serine residue at 195 resulted in an approximately 50% reduction in pregnenolone production. The S195A mutant construct did not yield the more acidic species of StAR detected in two-dimensional Western blots, indicating that the mutation affected the ability of the protein to be post-translationally modified. Mutation of the corresponding serine residues in murine StAR (Ser56 and Ser194) to alanines yielded results that were similar to those obtained with human StAR; the S56A mutant displayed a modest reduction in steroidogenic activity, whereas the S194A mutant had approximately 40% of the activity of murine wild-type StAR. In contrast to the human S195A mutation, conversion of serine 195 to an aspartic acid residue had no effect on steroidogenic activity, consistent with the idea that a negative charge at this site modulates StAR function. Our observations suggest that phosphorylation of serine 194/195 increases the biological activity of StAR and that this post- or co-translational event accounts, in part, for the immediate effects of cAMP on steroid production.

Amino Acid Sequence↗
PubMed1997

Inhibition of human immunodeficiency virus replication by the herpes simplex virus virion host shutoff protein.

The herpes simplex virus (HSV) virion host shutoff gene (vhs) encodes a protein which nonspecifically accelerates the degradation of mRNA molecules, leading to inhibition of protein synthesis. This ability to inhibit a critical cellular function suggested that vhs could be used as a suicide gene in certain gene therapy applications. To investigate whether vhs might be useful for treatment of AIDS, we tested the ability of both HSV type 1 (HSV-1) and HSV-2 vhs to inhibit replication of human immunodeficiency virus (HIV). Replication of HIV was substantially inhibited when an infectious HIV proviral clone was cotransfected into HeLa cells together with vhs under the control of the cytomegalovirus (CMV) immediate-early promoter. HSV-2 vhs was more active than HSV-1 vhs in these experiments, consistent with previously published studies on these genes. Since expression of vhs from the CMV promoter is essentially unregulated, we also tested the ability of vhs expressed from the HIV long terminal repeat (LTR) promoter to inhibit HIV replication. Wild-type HSV-1 vhs inhibited HIV replication more than 44,000-fold in comparison to a mutant vhs gene encoding a nonfunctional form of the Vhs protein. Production of Vhs in transfected cells was verified by Western blot assays. A larger amount of Vhs was observed in cells transfected with plasmids expressing vhs from the HIV LTR than from the CMV promoter, consistent with the greater inhibition of HIV replication observed with these constructs. Mutant forms of Vhs were expressed at higher levels than wild-type Vhs, most likely due to the ability of wild-type Vhs to degrade its own mRNA. The strong inhibitory activity of the vhs gene and its unique biological properties make vhs an interesting candidate for use as a suicide gene for HIV gene therapy.

Anti-HIV Agents↗
PubMed1996

Biological diversity, indigenous knowledge, drug discovery and intellectual property rights: creating reciprocity and maintaining relationships.

When new plant-derived therapeutics based on indigenous knowledge are being explored, it is important that the pharmaceutical companies return benefits to the native populations and the local governments from which the research material was obtained. When a potentially marketable plant product is being developed, it is essential that equitable agreements have already been established between the pharmaceutical companies and the people and/or countries from which this indigenous knowledge was acquired. Equally important is the commitment to provide immediate reciprocity that will enhance the welfare, the biocultural diversity and the well-being of the forest peoples. These measures should commence when a research project begins and continue during its duration. The development of these measures must be based upon the expressed needs of the indigenous communities. The relationship between the stability of the rain forest biocultural diversity, the creation and development of agro-forest resources and the long term benefits to the forest people is highlighted. Examples of initiatives taken by Shaman Pharmaceuticals Inc. and the Healing Forest Conservancy are described and discussed in the context of exploring appropriate use of intellectual property law to address the ethical issues facing all business and research groups working in the tropics.

Awards and Prizes↗
PubMed1996

ATP and a mitochondrial electrochemical gradient are required for functional activity of the steroidogenic acute regulatory (StAR) protein in isolated mitochondria.

The Steroidogenic Acute Regulatory (StAR) protein has been put forth as the rapidly synthesized, cycloheximide-sensitive protein that is required for the transport of cholesterol to the inner mitochondrial membrane and the P450scc enzyme and thereby acutely regulates steroidogenesis in steroidogenic tissues. In this study, several of the factors that may be required for StAR activity were examined using an in vitro system. Lysates from StAR-transfected COS-1 cells were added to mitochondria isolated from MA-10 Leydig tumor cells. Results obtained demonstrated that StAR-containing cell lysate increased steroidogenesis in isolated mitochondria, but failed to do so in the presence of m-CCCP, apyrase, or AMP-PNP, suggesting that StAR function requires ATP hydrolysis as well as an electrochemical gradient for maximal steroidogenic activity.

Adenosine Triphosphate↗
PubMed1996

College students' views on suicide.

College students were asked to rate the acceptability of suicide for themselves and for others in various circumstances. It was hypothesized that acceptability would vary as a function of circumstance of the suicide, the students' religious affiliation, history of past attempts, and whether the suicide was contemplated for oneself or another. The authors found that the highest acceptability for suicide was in the circumstances of terminal or chronic illness and depression. Students affiliated with organized religion were less accepting of suicide than were the individuals without such an affiliation. Previous suicide attempts were associated with greater acceptance of suicide for oneself or for others. In general, participants were more likely to accept suicide for others than for themselves, but the individuals who had previously attempted suicide displayed the opposite pattern under the circumstance of depression. Implications of these findings and directions for further research are discussed.

Adolescent↗
PubMed1995

Selective loss of H-2Ds antigen on a murine B lymphoma due to a post-transcriptional block in expression.

The major histocompatibility (MHC) class I antigens are coordinately expressed in most cells. However, some tumors or virus-infected cells lack expression of one MHC class I antigen, while expression of the other MHC class I antigens is unaffected. We previously described the selective expression of MHC class I antigens on a B-cell lymphoma from SJL/J mice called RCS5. This tumor expresses H-2Ks, but has lost cell surface expression of H-2Ds. To understand the mechanism responsible for the selective loss of H-2Ds on the cell surface, we analysed H-2Ds mRNA and protein in the RCS5 tumor. Here we report that H-2Ds mRNA was expressed in RCS5, but H-2Ds protein was not detected in cell lysates. To determine whether the H-2Ds mRNA from RCS5 was able to direct the synthesis of H-2Ds protein, we performed cDNA cloning, in vitro translation and gene transfer experiments using a cell line related to RCS5 (cRCS-X). Our results indicated that the inhibition of H-2Ds expression in cRCS-X occurred after transcription of a non-defective H-2Ds mRNA. Furthermore, H-2Ds antigen expression was restored in cRCS-X using a retroviral vector to express the recombinant H-2Ds cDNA. These results indicate that the inhibition of H-2Ds expression could be overcome either by out competing an inhibitor that functions in trans or by removing cis-acting regulatory sequences from the endogenous H-2Ds mRNA.

Amino Acid Sequence↗
PubMed1995

Purification and characterization of a Shiga toxin A subunit-CD4 fusion protein cytotoxic to human immunodeficiency virus-infected cells.

In a previous paper, we reported that a chimeric toxin composed of the enzymatic domain of the Shiga toxin A polypeptide (StxA1) genetically fused to the human CD4 (hCD4) molecule selectively kills cells infected with human immunodeficiency virus type 1 (HIV-1). Although other hCD4-containing chimeras cytotoxic to HIV-infected cells have been developed, there is limited information regarding their receptor binding and internalization. Therefore, the goals of this study were to purify the StxA1-hCD4 fusion protein, identify the receptor(s), and investigate the cytosolic trafficking route used by the chimeric toxin. Sufficient quantities of the StxA1-hCD4 hybrid were isolated for this investigation by using the pET expression and purification system. Cos-1 cells were rendered sensitive to the StxA1-hCD4 chimera by transfection with the env gene, which encodes HIV-1 envelope glycoproteins. The entry and translocation pathway used by the StxA1-hCD4 hybrid toxin was investigated by assessing the protective capacities of chemical reagents which interfere with microfilament movement, acidification of endosomes, and the integrity of the Golgi apparatus. Our findings indicated that the chimera uses HIV-1 glycoprotein gp120, and perhaps gp41, as a receptor which directs its entry through receptor cycling. Uptake is pH independent, and the StxA1-hCD4 hybrid is apparently translocated to the Golgi complex as with other bipartite toxins.

Ammonium Chloride↗
PubMed1995

Steroid production after in vitro transcription, translation, and mitochondrial processing of protein products of complementary deoxyribonucleic acid for steroidogenic acute regulatory protein.

We have previously demonstrated that steroidogenic acute regulatory protein (StAR) is essential for the rate-limiting step in the acute regulation of steroidogenesis, which is the transport of cholesterol from the outer to the inner mitochondrial membrane. We have hypothesized that this transport occurs as the 37-kilodalton (kDa) precursor form of StAR is imported into the mitochondria and processed to its 30-kDa mature forms. Using an in vitro transcription and translation system in the presence of mitochondria isolated from unstimulated mouse MA-10 Leydig tumor cells, we now directly show that the 37-kDa form is indeed the cytosolic precursor of StAR and can be processed by mitochondria to all four 30-kDa mature forms. To determine the subcellular location of StAR in steroidogenic cells, ultrastructural immunocytochemistry was performed in adrenal zona fasciculata cells using the protein A-gold technique. We show that StAR is associated exclusively with the mitochondria. There, StAR is primarily localized in the intermembrane space and the intermembrane space side of the cristae membrane. StAR was shown to induce steroid production in isolated mitochondria. StAR protein was expressed in COS1 cells and the cell lysate, which was shown to contain abundant levels of StAR by Western blot analysis, was incubated with mitochondria isolated from unstimulated MA-10 cells. In these experiments, StAR increased steroid production by at least 4-fold over control mock-transfected lysate, and this increase was time and dose dependent. Furthermore, the increase in steroid production induced by StAR-containing lysate was not observed when COS1 lysate containing high levels of another mitochondrially imported protein, adrenodoxin, was used. We conclude from these results that in response to tropic hormone stimulation of steroidogenic cells, StAR is synthesized as a 37-kDa precursor, imported into the mitochondria, processed to its 30-kDa mature forms, and localized to the intermembrane space. During import and processing in vitro, StAR induces steroid production in isolated mitochondria in a specific manner.

Animals↗
PubMed1994

The purification, cloning, and expression of a novel luteinizing hormone-induced mitochondrial protein in MA-10 mouse Leydig tumor cells. Characterization of the steroidogenic acute regulatory protein (StAR).

The acute response of steroidogenic cells to trophic hormone stimulation is the mobilization of cholesterol from cellular stores to the mitochondrial outer membrane and the transfer of this cholesterol to the mitochondrial inner membrane where the first enzymatic step in steroidogenesis occurs. The transfer of cholesterol across the mitochondrial membranes is dependent upon de novo protein synthesis, and this is the regulated step in the process. Although the newly synthesized regulatory protein(s) have yet to be identified, we previously have proposed a candidate protein which we identified in MA-10 cells that is synthesized in response to luteinizing hormone stimulation and that is localized to the mitochondria. In the present study, we report the isolation of a cDNA that encodes this luteinizing hormone-induced protein. Analysis of the cDNA and protein sequences reveals this is a novel protein. Importantly, we demonstrate for the first time that expression of the protein in MA-10 cells in the absence of hormone stimulation is sufficient to induce steroid production. We conclude that this protein is required in the acute regulation of steroidogenesis and propose to call this protein the Steroidogenic Acute Regulatory protein (StAR).

Amino Acid Sequence↗
PubMed1994

From shaman to human clinical trials: the role of industry in ethnobotany, conservation and community reciprocity.

Shaman Pharmaceuticals is a development-stage company engaged in developing traditional pharmaceuticals identified through a discovery process focused on isolating active compounds from tropical plants with a history of medicinal use. This process has resulted in two products in clinical trials: Provir, an oral product for the treatment of respiratory viral infections, and Virend, a topical antiviral product for the treatment of herpes. Shaman's drug-discovery process targets specific plants that have been used for medicinal purposes by native peoples. By integrating the sciences of ethnobotany, medicine and plant natural product chemistry, Shaman has been able to achieve time and cost savings for the identification of active compounds and preclinical development of its initial products. Numerous drugs have entered the international pharmacopoeia via the study of ethnobotany and traditional medicine. Two important elements of this approach to drug discovery are the percentage of plants that show activity against specific viral pathogens and the correlation between the folk therapeutic classification of plants used and the percentage of those plants that have shown activity in our antiviral screens. Conservation and direct reciprocity to indigenous communities are important features of Shaman Pharmaceuticals' drug-discovery process.

Clinical Trials as Topic↗