Biomedical subjects

B S Weeks

Publications and source records attributed to B S Weeks.

At least 37 records · Page 2Linked to original sources

PubMed1996

Laminin stimulates expression of two mitochondrial proteins during neurite outgrowth.

Differential hybridization was utilized with mRNA from NG108-15 cells cultured on either tissue culture plastic or laminin for 4 hr to identify genes whose mRNA was increased by laminin, a potent stimulator of neurite outgrowth. Two of the 16 laminin-induced clones, cytochrome b and chargerin II, are mitochondrial proteins. Northern blotting confirmed that laminin increased the mRNA levels of cytochrome b and chargerin II several fold. Antibody to chargerin II stained both processes and cell bodies of the cerebellar Purkinje cells and localized in the mitochondria of NG108-15 cells, which also showed increased protein levels in the presence of laminin. In addition, higher levels of chargerin II protein were detected in the newborn brain compared to the adult. However, inhibitors of mitochondrial protein synthesis did not affect laminin-mediated neurite outgrowth. These data suggest that the increased synthesis of mitochondrial enzymes observed with laminin treatment may not be necessary for the formation of neurites.

Animals↗

PubMed1995

The synthetic [Tyr5,12,Lys7]-polyphemusin II peptide (T22) binds to the CD4 cell surface molecule.

The [Tyr5,12,Lys7]-polyphemusin II peptide (T22) inhibits HIV-1 replication in lymphocytes. The mechanism of T22 inhibition of HIV-1 replication may involve T22 competition with HIV-1 for attachment sites on the plasma membrane of targeted cells. Here we find that the T22 peptide binds to the CD4 molecule in affinity columns. We also find that antiserum to CD4 inhibits cell attachment to T22. Further CD4+ transfected cells attach to T22 while their parental cells which do not express CD4 do not attach to T22. These data demonstrate that T22 binds to the CD4 molecule and supports the hypothesis that T22 inhibits HIV-1 replication by binding to the cell surface CD4 molecule and inhibiting uptake of the virus.

Amino Acid Sequence↗

PubMed1995

Neurotoxicity of the human immunodeficiency virus type 1 tat transactivator to PC12 cells requires the Tat amino acid 49-58 basic domain.

The acquired immunodeficiency syndrome (AIDS) frequently involves the central nervous system (CNS) and manifests as dementia due to encephalitis or diffuse neurodegeneration. Human immunodeficiency virus type 1 (HIV-1) proteins, potentially transported into the CNS by mononuclear inflammatory cells, have been implicated in the etiology of this HIV-1 associated neurological dysfunction. Here we investigate the neurotoxicity of the essential HIV-1 regulator protein Tat in vivo after microinfusion into the rat brain and in vitro using PC12, NG108-15, and GT17 neuronal cell lines. Infusion of either chemically synthesized Tat (Tat86) or recombinant Tat (rTat) into the striatal gray matter in Sprague-Dawley rats resulted in postural deviation ipsilateral to the infusion, a clinical presentation in rats associated with complete striatal dysfunction. Histologic examination 3 days after infusion revealed massive necrosis in the area of the distribution of the infusion. Infusion of heat denatured rTat, peptide Tat49-58, or peptide Tat57-86 did not result in clinically or histologically detectable brain damage. After 3 days incubation in vitro, the lethal dose for half (LD50) of PC12 cells due to rTat was 5 micrograms/ml. The LD50 for Tat86 under the same conditions was 10 micrograms/ml. Tat49-58 and Tat57-86 peptides were not toxic in vitro even at 10-fold higher doses. At 5 micrograms/ml, rTat was toxic to 100% of GT17 cells after 24 hr. At 5 micrograms/ml, Tat86 was toxic to 90% of the NG108-15 cells after 7 days of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Acquired Immunodeficiency Syndrome↗

PubMed1995

Reproduction and sera embryotoxicity after immunization of monkeys with the laminin peptides YIGSR, RGD, and IKVAV.

Monkeys with excellent reproductive histories were immunized with the laminin peptides YIGSR, RGD, IKVAV, and YD, a control sequence with no known biological function. Sera from the YIGSR-immunized monkey became toxic, causing neural tube defects in whole rat embryo cultures, and this monkey experienced fetal loss after immunization. Sera from the RGD-immunized monkey also became embryotoxic in culture after immunization, but this monkey appeared to become infertile as she failed to initiate a pregnancy for at least 2 years after immunization. In contrast, embryos cultured on sera from the IKVAV- or YD-immunized monkeys were predominantly normal and both monkeys completed successful pregnancies. Antibody levels to the respective peptides or to laminin were not predictive of embryotoxicity, but antibody binding to homogenized yolk sacs as well as to yolk sacs of cultured embryos was associated with sera embryotoxicity and reproductive outcomes in vivo. These observations suggested that the laminin sequences YIGSR and RGD may play a role in immune-mediated reproductive failure by reacting directly with embryonic tissue and could provide a basis for identifying individuals at risk for both spontaneous abortion and infertility.

Amino Acid Sequence↗

PubMed1995

Structure-activity study of a laminin alpha 1 chain active peptide segment Ile-Lys-Val-Ala-Val (IKVAV).

The IKVAV sequence, one of the most potent active sites of laminin-1, has been shown to promote cell adhesion, neurite outgrowth, and tumor growth. Here we have determined the structural requirements of the IKVAV sequence for cell attachment and neurite outgrowth using various 12-mer synthetic peptide analogs. All-L- and all-D-IKVAV peptides showed cell attachment and neurite outgrowth activities. In contrast, all-L- and all-D-reverse-sequence peptides were not active. Some of the analogs, in which the lysine and isoleucine residues of the IKVAV peptide were substituted with different amino acids, promoted cell attachment, but none of the analog peptides showed neurite outgrowth activity comparable to that of the IKVAV peptide. These results suggest that the lysine and isoleucine residues are critical for the biological functions of the IKVAV peptide.

Amino Acid Sequence↗

PubMed1995

HIV-1 infection alters monocyte interactions with human microvascular endothelial cells.

HIV infection of monocytes resulted in twofold elevation of adhesion molecule LFA-1 (both alpha L/CD11a and beta 2/CD18 subunits) and LFA-3 (CD58), with no apparent increase in LFA-2 (CD2) or various beta 1-integrins. Homotypic aggregation of monocytes was evident 2 h after exposure to virus and was inhibited by mAbs to both the alpha L- and beta 2-subunits of LFA-1. HIV-infected monocytes also showed a marked increase in adherence to human capillary endothelial cell monolayers derived from brain, lung, and skin. This adherence was inhibited by mAb to either LFA-1 subunit and by mAb to the counter-receptor intercellular adhesion molecule-1. Cocultivation of HIV-infected monocytes with endothelial cells increased permeability of endothelial cell monolayers to 125I albumin in transwell assay systems. The increased endothelial permeability induced by HIV-infected monocytes was associated with a substantial disruption of the endothelial cell monolayer. Morphologic disruption was not a direct toxic effect on endothelial cells, but appeared to be secondary to changes in endothelial cell-cell or cell-matrix interactions. Northern blot analysis showed increased expression of gelatinase B (92-kDa gelatinase), tissue inhibitor of metalloproteinase TIMP-1, and TIMP-2 in the HIV-infected monocytes. Consistent with these Northern analyses, secretion of gelatinase activity in culture fluids of HIV-infected monocytes was also increased and was dependent on the stage of virus replication. Incubation of HIV-infected monocytes with the proteinase inhibitors TIMP-1 and TIMP-2 inhibited the increased permeability of endothelial cell monolayers to 125I albumin. These results suggest possible mechanisms for extravasation of HIV-infected monocytes through vascular endothelium into tissue in early stages of HIV disease.

Antigens, CD↗

PubMed1994

Lymphocytes and promonocytes attach to the synthetic [Tyr5,12, Lys7]- polyphemusin II peptide.

The [Tyr5,12,Lys7]-polyphemusin II peptide (T22) has been shown to inhibit HIV-1 replication in lymphocytes. The mechanism of T22 inhibition of HIV-1 replication is not known but may involve T22 competition with HIV-1 for attachment sites on the plasma membrane of targeted cells. Here we find that three human immunocyte cell lines (H9, Jurkat, and U-937) attach to T22. The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), has been shown to activate intracellular protein kinase C and to stimulate lymphocyte attachment to various substrates through specific cell surface receptors. Here we find that TPA treatment enhances attachment of the immunocytes to T22 by three- to four-fold. These data demonstrate that T22 binds to immunocyte cell surfaces and support the hypothesis that T22 may inhibit HIV-1 replication by competing with the virus for a common cell surface receptor(s).

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1994

LZIP-1 and LZIP-2: two novel members of the bZIP family.

A large family of bZIP proteins, containing a basic DNA-binding domain and a leucine zipper, have been described that recognize the CRE and AP-1 elements. Here, we have identified two new members, designated LZIP-1 and LZIP-2. The murine cDNA for LZIP-1 coded for a 379-amino-acid (aa) residue protein containing several distinct domains, including a Ser-rich region, a basic DNA-binding region, and an unusually long leucine zipper. A second form, LZIP-2, contained an additional 25 aa in the N-terminal region. Western immunoblotting revealed that antibody raised against part of recombinant LZIP-1 detected both forms in a variety of tissues. Gel mobility shift assays demonstrated that the recombinant protein possessed specific DNA-binding activity for both the CRE AP-1 sites. The present identification of two more ubiquitous members of the bZIP family emphasizes the complex nature of transcription factor interactions at the CRE and AP-1 sites.

Animals↗

PubMed1994

12-O-tetradecanoylphorbol 13-acetate stimulates human T-lymphocyte adherence to the fibronectin RGD domain and the laminin IKVAV domain.

In order for T cells to exit the circulatory system, these cells must attach to extracellular matrix proteins. We have used 12-O-tetradecanoylphorbol 13-acetate (TPA) to study the ability of human T cells to adhere to fibronectin or laminin or to specific domains on these extracellular matrix proteins. Both primary human T-lymphocytes and a T-cell line (H-9) adhered and spread well on solid-phase fibronectin and laminin in the presence of TPA, with maximum activity at 3 hr of treatment. Furthermore, attachment of both cell populations to fibronectin was inhibited using a soluble RGD-containing synthetic peptide or by pretreating the fibronectin with antibodies that block the RGD domain. A synthetic peptide from the CSI alternatively spliced region of fibronectin did not inhibit attachment to fibronectin. The H-9 cells also attached to the laminin A chain IKVAV-containing synthetic peptide, but not to the laminin-derived YIGSR- or RGD-containing sequences. Immunoprecipitation of 32P-labeled H-9 cells with antibodies to the beta 1 integrin subunit demonstrated phosphorylation of an alpha integrin subunit after treatment with TPA. These data demonstrate that TPA activates T-cell adherence to laminin and to fibronectin via specific sites on each protein and that this adhesion may be associated with integrin phosphorylation.

Amino Acid Sequence↗

PubMed1994

Rat embryo development on human sera is related to numbers of previous spontaneous abortions and nutritional factors.

OBJECTIVES: The objectives were to determine (1) if sera from women with histories of spontaneous abortions were teratogenic to cultured embryos more often than were sera of nonaborters, (2) if the teratogenicity could be corrected by adding nutrients to the sera, and (3) if these findings were relevant to reproductive outcomes. STUDY DESIGN: Rat embryos were cultured for 48 hours on sera from 102 subjects who had experienced spontaneous abortions. Samples from 48 were retested with nutrients added and 10 took dietary supplements, were again tested with embryo cultures, and reported on their pregnancy outcomes. RESULTS: The frequencies of teratogenic sera increased with numbers of spontaneous abortions (0 to > or = 5) in a manner that did not deviate from linearity (27% to 89%) (chi 2 p > 0.957). Nutrient supplements were added to 48 samples, and 40 were corrected and 10 subjects were given dietary supplement. Sera from six showed improved embryo cultures, and these women completed their pregnancies. CONCLUSIONS: Rat embryo cultures may provide unique insights into the causes and treatment of spontaneous abortions.

Abortion, Habitual↗

PubMed1994

bFGF and its low affinity receptors in the pathogenesis of HIV-associated nephropathy in transgenic mice.

HIV-associated nephropathy is characterized by extensive tubulointerstitial disease with epithelial cell injury, microcystic proliferation, and tubular regeneration with glomerulosclerosis. To explore the role of bFGF as a mediator of HIV-induced interstitial disease, we utilized an HIV transgenic mouse model that manifests clinical and histological features observed in patients. In transgenic mice, simultaneous renal epithelial cell proliferation and injury were detected in vivo. In areas of microcystic proliferation, immunoreactive bFGF colocalized with extracellular matrix. Kidneys from transgenic mice had increased bFGF low affinity binding sites, particularly in the renal interstitium. In vitro, transgenic renal tubular epithelial cells proliferated more rapidly and generated tubular structures spontaneously, in marked contrast to nontransgenic renal cells where these pathologic features could be mimicked by exogenous bFGF. These studies suggest that renal bFGF and its receptors play an important role in the pathogenesis of HIV-associated nephropathy.

AIDS-Associated Nephropathy↗

PubMed1993

beta-Amyloid precursor protein binds to the neurite-promoting IKVAV site of laminin.

We previously characterized a 110-kDa membrane-associated laminin-binding protein (LBP110) from brain which binds the laminin A chain -Ile-Lys-Val-Ala-Val-(IKVAV) site and increases in injury. Here we demonstrate that antisera directed against different epitopes of beta-amyloid precursor protein (APP) recognize LBP110 and that APP is recognized by LBP110 antiserum. APP specifically binds IKVAV and not another biologically active laminin-derived peptide containing the amino acid sequence -Tyr-Ile-Gly-Ser-Arg-. PC-12 cells transfected with antisense APP RNA produce less APP and LBP110, and they form fewer processes when cultured on either laminin or the IKVAV peptide. Thus, LBP110 is a member of the APP family and a function for APP in neurite outgrowth is now defined.

Amino Acid Sequence↗

PubMed1993

Transforming growth factor beta enhances integrin expression and type IV collagenase secretion in human monocytes.

Transforming growth factor beta (TGF-beta), secreted within an inflammatory site or injected locally, induces leukocyte margination, chemotaxis, and accumulation. In addition to its potent direct chemotactic activity, TGF-beta may promote this leukocyte response by influencing cell surface integrin expression. At picomolar concentrations, TGF-beta increases steady-state mRNA levels for both the alpha 5 and the beta 1 chain of the fibronectin receptor in human blood monocytes. This increase in gene expression is reflected by selectively enhanced expression of alpha 5 (CDw49e), beta 1 (CDw29), and also alpha 3 (CDw49c) adhesion molecules on the cell surface. Functionally, TGF-beta promotes, in a dose- and time-dependent fashion, monocyte adhesion to type IV collagen, laminin, and fibronectin. Potentially facilitating the movement of monocytes through the extracellular matrix, TGF-beta triggers transcriptional and posttranscriptional regulation of both the 92-kDa and the 72-kDa gelatinase/type IV collagenase. Thus, TGF-beta may play a pivotal role in the early phases of inflammation and repair through its ability to mediate monocyte adhesion, chemotaxis, and enzymatic digestion of extracellular matrix, whereas in chronic lesions, excess TGF-beta may contribute to persistent leukocyte accumulation.

Amino Acid Sequence↗

PubMed1993

Identification of a novel cell attachment domain in the HIV-1 Tat protein and its 90-kDa cell surface binding protein.

The HIV-1 transactivator protein Tat is essential for viral gene expression and replication. Tat is taken up by cells and transactivates the HIV-LTR promoter in the cell nucleus. The present studies show that cells adhere to both synthetic and recombinant Tat, and, using synthetic peptides, we localize the binding site to a region spanning amino acid residues 49-57 (peptide Tat49-57). Tat49-57 also inhibited cell attachment to solid phase full-length Tat peptide and to recombinant Tat protein. Using Tat peptide affinity chromatography, we identified a 90-kDa cell surface protein that binds to Tat. The 90-kDa protein could be eluted from the Tat column using the Tat49-57 peptide. A 90-kDa cell surface Tat binding protein was also identified by coprecipitation with Tat after incubation with radiolabeled cell membrane preparations. Co-precipitation of the 90-kDa protein was inhibited by competition with a Tat49-65 peptide, but not with Tat55-86. Our findings suggest that cellular attachment to Tat is mediated through a 90-kDa cell surface protein that binds to a Tat domain between amino acids 49 and 57.

Amino Acid Sequence↗

PubMed1993

Human T lymphocytes synthesize the 92 kDa type IV collagenase (gelatinase B).

In order for T cells to exit the circulatory system, traverse the endothelial basement membrane, and arrive in target tissues, these cells must attach to and degrade basement membrane proteins. 12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown to stimulate lymphoid cell adhesion to basement membrane components. We have used TPA to study the ability of human lymphoid cells to secrete type IV collagenases, enzymes capable of degrading basement membrane proteins. Here, we found that human primary T cells and H-9 lymphoid cells synthesize the 92 kDa type IV collagenase (gelatinase B) and TPA stimulates the synthesis and secretion of this protease. Peak TPA-stimulated gelatinase B secretion and mRNA accumulation were observed 9 hours after TPA treatment, while the peak adhesion to type IV collagen was observed only 3 hours after TPA treatment. The protein kinase C inhibitor, H-7, inhibited TPA-stimulated gelatinase B secretion. Both the primary T cells and H-9 lymphoid cells also expressed the mRNA for the tissue inhibitor of metalloproteinase-1 (TIMP-1). These data demonstrate that TPA-stimulated lymphoid cells adhere to type IV collagen and subsequently synthesize and secrete gelatinase B and TIMP-1. We conclude that lymphoid cell extravasation may involve cellular employment of adhesion mechanisms prior to degradation of the matrix, which is similar to the process of extravasation used by metastatic cells.

Animals↗

PubMed1993

Increased expression of alpha 4 beta 1 and alpha 5 beta 1 integrins on HTLV-I-infected lymphocytes.

T cells interact with the extracellular matrix via integrin receptors and these interactions affect both cellular localization and proliferation. The importance of these interactions in retrovirus-induced diseases, however, remains less clear. In the present study, we investigated changes in T cell adhesion to extracellular matrix proteins by HTLV-I expressing cell lines and human peripheral blood lymphocytes infected with HTLV-I by cocultivation. Cell lines and acutely infected primary peripheral blood lymphocytes demonstrated enhanced adhesion to fibronectin. Acute infection of peripheral blood lymphocytes increased the expression of alpha 5 beta 1 and alpha 4 beta 1 integrins. Antibodies to the alpha 4, alpha 5, and beta 1 subunits inhibited attachment of infected cells to fibronectin. We conclude that HTLV-I infection is associated with an increase in the expression of both the classical fibronectin receptor and the receptor for the alternatively spliced domain of fibronectin on peripheral blood lymphocytes. HTLV-I-related alterations in cell surface adhesion molecules may contribute to the abnormal proliferation of T cells in adult T cell leukemia (ATL) or to the abnormal localization of activated or infected T cells to the central nervous system of patients with tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM).

Cell Adhesion↗

PubMed1993

The laminins: a family of basement membrane glycoproteins important in cell differentiation and tumor metastases.

Laminins are a family of basement membrane-derived glycoproteins that are very biologically active with a number of diverse cell types. The response of the cells is dependent on the cell type and various cell-specific intracellular events are activated. Multiple active sites on laminin and cellular receptors have been described. Both laminin and the synthetic peptides that define the active sites may have important clinical uses. For example, the neurite-promoting peptides may be useful in vivo in regeneration studies because of their potent activity with neural cells and their lack of antigenicity. Also, peptides, such as YIGSR, that inhibit angiogenesis are potentially useful for treating the vascularization of the eye that occurs in conditions such as diabetes mellitus. Likewise, the angiogenic peptide SIKVAV, because of its role in endothelial cell block vessel formation, may be useful for treating ischemia. The recent progress that has been made in characterizing basic mechanisms of action of laminin has laid the groundwork for more direct studies of its clinical relevance.

Amino Acid Sequence↗

PubMed1993

HIV-1 infection stimulates T cell invasiveness and synthesis of the 92-kDa type IV collagenase.

Tissue-specific localization of HIV-1-infected lymphoid cells may contribute to clinical manifestations of AIDS. Therefore we investigated the effect of HIV-1 infection on mechanisms of T lymphocyte invasion, a process required for movement of cells into and out of the circulation. In the present study, we demonstrate that HIV-1-infected human lymphocytes secrete increased amounts of the human 92-kDa type IV collagenase when compared to uninfected lymphocytes. Furthermore, HIV-1-infected lymphocytes degrade the extracellular matrix proteins collagen IV and fibronectin, and they are more invasive through a reconstituted basement membrane when compared to uninfected cells. The addition of either antibody to the 92-kDa collagenase or TIMP-2, a type IV collagenase inhibitor, abolishes invasive activity. These data suggest that HIV-1-infected lymphocytes express phenotypic characteristics that are consistent with an enhanced ability to leave the circulation and to localize in target tissues. Local viral infection or the release of viral proteins, cytokines, or proteolytic enzymes in tissues may contribute to pathogenesis.

Cell Movement↗