Search PubMed⌕ Search

Biomedical subjects

J Alam

Publications and source records attributed to J Alam.

At least 73 records · Page 4Linked to original sources
PubMed1994

Calcium oxalate stone agglomeration reflects stone-forming activity: citrate inhibition depends on macromolecules larger than 30 kilodalton.

To evaluate the clinical utility of in vitro calcium oxalate monohydrate (COM) crystallization kinetics measurements and to determine the effect of quantitative removal of urinary Tamm-Horsfall glycoprotein on such measurements, we examined 24-hour, room temperature urine collections of patients from our Stone Clinic and of normal subjects from our research laboratories at Ochsner Medical Institutions in New Orleans, LA, and compared their COM kinetic parameters in vitro before and after urine ultrafiltration (30 kd). Data from 53 calcium oxalate stone-forming patients (26% women; mean age, 47 years) who demonstrated radiographic or other evidence of forming at least one stone were compared with data from 22 healthy volunteers (25% women; mean age, 40 years). Hypercalciuria (> 7.5 mm/24 hr), hyperoxaluria (> 0.5 mm/24 hr), and hypocitraturia (< 2.0 mm/24 hr) were present in 38%, 26%, and 26% of the patient population, respectively. Urinary creatinine, urate, calcium, citrate, phosphate, oxalate, pH, volume, total immunoreactive-disaggregated Tamm-Horsfall glycoprotein, and the urine's effects on COM solubility, percent crystal growth inhibition, and crystal agglomeration inhibition [tm] were determined. Calcium oxalate monohydrate agglomeration inhibition, [tm], was reduced in stone-forming patients. It decreased with increasing stone frequency, making [tm] a useful tool for measuring the risk of stone recurrence. Urinary Tamm-Horsfall glycoprotein and citrate concentrations were linearly related to COM agglomeration inhibition. Their effects were synergistic. Tamm-Horsfall glycoprotein removal from urine reduced COM agglomeration inhibition dramatically. Alkali therapy increased urinary citrate concentration and increased [tm].(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

The effects of high-dose recombinant soluble CD4 on human immunodeficiency virus type 1 viremia.

In vitro, low-passage clinical human immunodeficiency virus type 1 (HIV-1) isolates require up to 1000 times greater serum levels of recombinant soluble CD4 (rsCD4) than have ever been given. To determine if sufficient serum levels of rsCD4 provide in vivo inhibition of HIV-1, 4 HIV-1 plasma-viremic subjects were given single-dose boluses of 2, 4, 6, 8, and 10 mg/kg intravenous rsCD4. Plasma HIV-1 cultures were done after infusion. Three subjects demonstrated a dose-dependent reduction in plasma HIV-1 viremia. The inhibitory effect of rsCD4 on plasma HIV-1 viremia was associated with the in vitro ID90-95 of the isolate, not the ID50. These data demonstrate that extremely high doses of rsCD4 inactivate cell-free HIV-1 in vivo and suggest that high doses of rsCD4 may have some short-term therapeutic utility, such as with accidental or occupational HIV-1 exposure.

CD4 Antigens↗
PubMed1993

Regulation of heme oxygenase and metallothionein gene expression by the heme analogs, cobalt-, and tin-protoporphyrin.

Two heme analogs, cobalt- and tin-protoporphyrin (CoPP and SnPP, respectively) have been used to probe the heme-hemopexin interaction, hemopexin receptor binding, and the mechanism of regulation of heme oxygenase (HO) and metallothionein-1 (MT-1) gene expression by hemopexin. Both CoPP and SnPP are HO inhibitors and hemopexin binds SnPP (Morgan, W. T., Alam, J., Deaciuc, V., Muster, P., Tatum, F. M., and Smith, A. (1988) J. Biol. Chem. 263, 8226-8231) and CoPP. The association of CoPP with hemopexin produces characteristic changes in the absorbance spectrum of CoPP and quenches the intrinsic fluorescence of hemopexin. Binding of CoPP is tight (Kd ca. 3 x 10(-7) M) although of lower affinity than heme itself (Kd < pM); and CoPP binding, like heme, produces conformational changes in hemopexin shown by an increase in the molar ellipticity at 233 nm and affords protection from proteolysis of the hinge region between the two structural domains of hemopexin. The coordination of the central cobalt atom is predicted to be similar to that of heme and to involve His56 and His127 of rabbit hemopexin. Furthermore, CoPP-hemopexin, like SnPP-hemopexin, binds to the hemopexin receptor as shown by competitive inhibition studies with radioactive heme-hemopexin. The effect of free heme analogs and their hemopexin complexes on HO and MT gene regulation was investigated and compared with the extent of induction by heme and heme-hemopexin. Free CoPP is a more effective inducer of HO steady state mRNA levels than free heme and produces a 5-fold increase within 1 h compared to only a 2-fold increase with heme, but free SnPP (up to 10 microM) produces no detectable increase in HO mRNA. In contrast, by 3 h heme-hemopexin and SnPP-hemopexin increase HO mRNA levels 11- and 6-fold, respectively; but the CoPP-hemopexin complex causes no detectable change in HO mRNA levels. The complexes of hemopexin with heme or either of the two heme analogs are effective inducers of metallothionein (MT) mRNA. Induction of MT mRNA by heme-hemopexin is rapid, increasing 4-fold within 1 h and 14-fold by 3-4 h. Strikingly, an even more rapid and slightly more extensive induction of MT mRNA is seen in response to either CoPP- or SnPP-hemopexin complexes, with MT mRNA rising 8-fold within 1 h. In contrast, free heme and the free analogs are far less effective inducers, increasing MT and mRNA levels and in vitro transcription rates only 3-4-fold and declining after 2-3 h.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Identification of the histidine residues of hemopexin that coordinate with heme-iron and of a receptor-binding region.

Rabbit hemopexin cDNA was cloned from a rabbit liver lambda gt11 cDNA expression library using a mixture of five monoclonal antibodies raised against rabbit hemopexin, and the entire rabbit hemopexin sequence was determined. The heme-binding domain I of rabbit hemopexin (Smith, A., and Morgan, W. T. (1984) J. Biol. Chem. 259, 12049-12053) contains only 4 histidine residues which are conserved in rabbit, human, rat, and mouse hemopexin. The 2 axial heme-iron coordinating histidine residues, identified by Edman microsequencing and amino acid analyses of chemically modified domain I and isolated fragments of domain I, are the conserved histidine residues at positions 56 and 127 of the mature rabbit protein. The epitope recognized by JEN-14 (a monoclonal antibody which specifically reacts with domain I and blocks the hemopexin-receptor interaction (Morgan, W. T., Muster, P., Tatum, F. M., McConnell, J., Conway, T. P., Hensley, P., and Smith, A. (1988) J. Biol. Chem. 263, 8220-8225) was shown to lie between residues 122 and 142 by Western blotting of protease-digested domain I and transposon-insertion mutants of domain I expressed in a plasmid vector system. The location of this epitope near the heme-binding histidine residue 127 is compatible with a transport mechanism in which the release of heme from hemopexin is accompanied by a concomitant transfer of heme to the hemopexin receptor or the membrane heme-binding protein (Smith, A., and Morgan, W. T. (1985) J. Biol. Chem. 260, 8325-8329).

Amino Acid Sequence↗
PubMed1993

Different ATP effects on natriuretic peptide receptor subtypes in LLC-PK1 and NIH-3T3 cells.

We have observed different ATP interactions in two guanylate cyclase (GC)-coupled natriuretic peptide (NP) receptor subtypes, designated NPR-A and NPR-B. The NPR-A is selectively expressed by LLC-PK1 epithelial cells and the NPR-B by NIH-3T3 fibroblast cells. In LLC-PK1 membranes, ATP-Mg2+ potentiated ANP-stimulated GC activity (ANP-s-GC). In contrast, in NIH-3T3 membranes, ATP-Mg2+ inhibited ANP-s-GC but enhanced CNP-stimulated GC activity (CNP-s GC). ATP in the presence of Mn2+ inhibited LLC-PK1 and NIH-3T3 membrane ANP-s-GC and CNP-s-GC. These are the first data suggesting that the ATP-Mg2+ produces different effects between membrane NPR-A and -B subtypes. We have also demonstrated that GC of NPR-B is sensitive to methylene blue.

3T3 Cells↗
PubMed1992

Distal AP-1 binding sites mediate basal level enhancement and TPA induction of the mouse heme oxygenase-1 gene.

Basal expression of a chimeric gene (pMHO4CAT) consisting of approximately 7 kilobase pairs (kbp) of the 5'-flanking region of the mouse heme oxygenase-1 (HO-1) gene fused to the bacterial chloramphenicol acetyltransferase gene is 2- to 10-fold greater than that of an analogous construct containing only 1287 bp of the 5'-flanking region (pMHO1CAT) in transiently transfected cultured cells. The enhancer activity has been localized to a 268-base pair (bp) fragment positioned approximately 4 kilobase pairs upstream of the transcription initiation site. This fragment contains two high affinity protein binding sites, regions A and B, as determined by DNase I protection assays using nuclear protein extracts from rat C6 glioma cells. Both sites include core sequence elements, TGAGTCA (region A) and TGTGTCA (region B), that resemble the consensus binding site, TGA(G/C)TCA, of the Jun/Fos (AP-1) family of transcription factors. Purified, bacterially expressed AP-1 (c-Jun homodimer) specifically binds to both elements, exhibiting greater affinity for the region A motif. The expression of pMHO4CAT, but not of pMHO1CAT, is stimulated by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), and the 268-bp enhancer fragment confers TPA inducibility and c-Jun/c-Fos transactivation to the heterologous SV40 promoter. These functions are mediated by the AP-1 binding sites as multiple copies of the region A motif also confer TPA induction and c-Jun/c-Fos transactivation upon a heterologous promoter.

Animals↗
PubMed1992

Heme-hemopexin-mediated induction of metallothionein gene expression.

Hemopexin-mediated heme transport into mouse hepatoma (Hepa) cells and human promyelocytic (HL-60) cells stimulates the expression of heme oxygenase via transcriptional activation (Alam, J., and Smith, A. (1989) J. Biol. Chem. 264, 17637-17640). Incubation of both these cell types in serum-free medium containing heme-hemopexin is shown here also to increase the steady-state level of metallothionein (MT) mRNA in a time- and dose-dependent manner. Heme-hemopexin is a far more effective inducer (12-fold) of the MT isozyme 1 (MT-1) in Hepa cells than nonprotein-bound heme (4-fold). Apohemopexin has no effect on MT-1 expression, and incubation with heme-hemopexin of mouse L fibroblasts that lack hemopexin receptors does not affect MT-1 expression. Thus, an interaction between the heme-hemopexin complex and its receptor is necessary for increased accumulation of MT-1 transcripts. In vitro nuclear "run-on" analysis indicates that the heme-hemopexin-mediated accumulation of MT-1 mRNA is regulated primarily at the level of initiation of transcription. A highly labile protein is required for constitutive MT-1 gene expression and acts to repress transcription. Transcriptional activation by heme or metals may require decreased concentrations or inactivation of the repressor as well as an additional inducer-specific trans-acting factor. Inhibition of protein synthesis augments the heme-hemopexin-mediated accumulation of MT-1 mRNA. Activation of heme oxygenase (HO) gene transcription by heme requires the synthesis of one (or more) heme-inducible proteins that are labile or become labile upon cycloheximide-sensitive processing or activation. Our comparison of MT and HO points to significant differences in the mechanisms of gene regulation by heme. The concomitant regulation of gene expression of MT-1 and HO in response to heme-hemopexin appears to be a concerted adaptive response of the cells, mediated at the level of the plasma membrane hemopexin receptor, and may relate to the proposed role of MT as an intracellular antioxidant or to a need to sequester zinc which otherwise would compete with iron and occupy sites on regulatory proteins such as the iron-responsive elements.

Animals↗
PubMed1991

Characterization of the IS895 family of insertion sequences from the cyanobacterium Anabaena sp. strain PCC 7120.

A family of repetitive elements from the cyanobacterium Anabaena sp. strain PCC 7120 was identified through the proximity of one element to the psbAI gene. Four members of this seven-member family were isolated and shown to have structures characteristic of bacterial insertion sequences. Each element is approximately 1,200 bp in length, is delimited by a 30-bp inverted repeat, and contains two open reading frames in tandem on the same DNA strand. The four copies differ from each other by small insertions or deletions, some of which alter the open reading frames. By using a system designed to trap insertion elements, one of the elements, denoted IS895, was shown to be mobile. The target site was not duplicated upon insertion of the element. Two other filamentous cyanobacterial strains were also found to contain sequences homologous to IS895.

Amino Acid Sequence↗
PubMed1991

Reduced chloramphenicol acetyltransferase activity observed with vectors containing an upstream SphI recognition sequence.

Chloramphenicol acetyltransferase (CAT) is the most commonly used reporter gene for studying the regulation of mammalian gene transcription. Some of the currently available CAT vectors contain the recognition sequence for the restriction endonuclease SphI within the multiple cloning site. This sequence introduces an ATG triplet that is out of frame with the initiation codon of the CAT gene. Transient expression of CAT fusion genes, constructed using three different cellular promoters, demonstrates that the presence of the upstream AUG triplet in the CAT transcript reduces CAT activity, presumably by interfering with the translation of the coding sequence. Deletion of the SphI site from each of the plasmids increased CAT activity between 4-fold and 5-fold. From these results, we conclude that upstream, out-of-frame ATG triplets must be avoided in order to achieve maximum expression of the reporter gene.

Base Sequence↗
PubMed1989

Receptor-mediated transport of heme by hemopexin regulates gene expression in mammalian cells.

Hemopexin (HPX) transports heme to liver parenchymal cells, undergoes receptor-mediated endocytosis, and recycles intact. Incubation of mouse hepatoma (Hepa) cells with heme-HPX causes a rapid dose- and time-dependent increase in the steady-state level of heme oxygenase (HO) mRNA. A maximum induction of 20-25-fold is achieved within 3 h after incubation with 10 microM heme-HPX. This accumulation of HO mRNA results primarily from increased transcription of the HO gene as judged by in vitro nuclear run-on assays. In addition, receptor-mediated transport of heme into Hepa cells significantly decreases the steady-state level of transferrin receptor (TfR) mRNA. While a 25-30-fold decrease in the amount of TfR mRNA is observed within 3 h of incubation of Hepa cells with 10 microM heme-HPX, no significant change in the rate of TfR gene transcription was detected. These regulatory effects of heme-HPX are not restricted to hepatic cells but are also observed in human promyelocytic HL-60 cells. This is the first direct demonstration of receptor-mediated transport of heme by hemopexin regulating gene expression in mammalian cells.

Animals↗
PubMed1989

Transcriptional activation of the heme oxygenase gene by heme and cadmium in mouse hepatoma cells.

Treatment of mouse hepatoma (Hepa) cells with heme or cadmium chloride in serum-free medium causes a rapid increase in the steady-state level of heme oxygenase (HO) messenger RNA. This increase is both dose- and time-dependent. Maximum accumulation of HO mRNA is observed 3 h after addition of either agent. Treatment of Hepa cells with heme or CdCl2 also stimulates the transcription of the HO gene, as judged by in vitro nuclear transcription run-on assays. The maximum rate of HO gene transcription occurs 2 h after treatment with either agent. Comparison of the relative increase in the rate of HO gene transcription with the relative increase in the level of HO mRNA demonstrates that transcriptional activation is the primary mechanism by which heme and cadmium produce the accumulation of HO mRNA in Hepa cells. Cadmium may also influence other processes involved in the expression of HO, since the time course of mRNA accumulation diverges from that of gene transcription. However, neither heme nor cadmium alters the rate of HO mRNA degradation. Cobalt chloride and heat shock, which are potent inducers of HO mRNA in rat liver and rat C6 glioma cells, respectively, have only a small effect on the level of HO mRNA in mouse hepatoma cells.

Animals↗
PubMed1989

The amino acid sequence of low-potential cytochrome c550 from the cyanobacterium Microcystis aeruginosa.

The low-potential cytochrome c550 has been purified from the cyanobacterium Microcystis aeruginosa and its amino acid sequence has been determined. The protein contains 135 amino acid residues with the Cys-X-X-Cys-His heme binding site at residues 37 to 41. The sequence from residue 28 to 45 shows similarity to cytochrome c553 residues 1 to 18 when the heme binding sites are aligned. Another region of similarity is in the carboxyl-terminal regions of these two proteins. The two aligning regions of cytochrome c553 correspond to helical segments in other related cytochromes. A partial sequence of cytochrome c550 from Aphanizomenon flos-aquae was obtained and showed a 48% identity to the sequence of the M. aeruginosa cytochrome. The single methionine residue in cytochrome c550 of M. aeruginosa occurs at position 119 but there is no methionine in this region in the A. flos-aquae cytochrome, indicating that methionine is not the sixth ligand to the heme iron atom. Histidine 92 is a possible sixth ligand in M. aeruginosa cytochrome c550. The far-uv circular dichroism spectrum indicates that this protein is approximately 17% alpha helix, 42% beta-pleated sheet, and 41% random coil.

Amino Acid Sequence↗
PubMed1989

Isolation, sequence, and expression in Escherichia coli of an unusual thioredoxin gene from the cyanobacterium Anabaena sp. strain PCC 7120.

Two sequences with homology to a thioredoxin oligonucleotide probe were detected by Southern blot analysis of Anabaena sp. strain PCC 7120 genomic DNA. One of the sequences was shown to code for a protein with 37% amino acid identity to thioredoxins from Escherichia coli and Anabaena sp. strain PCC 7119. This is in contrast to the usual 50% homology observed among most procaryotic thioredoxins. One gene was identified in a library and was subcloned into a pUC vector and used to transform E. coli strains lacking functional thioredoxin. The Anabaena strain 7120 thioredoxin gene did not complement the trxA mutation in E. coli. Transformed cells were not able to use methionine sulfoxide as a methionine source or support replication of T7 bacteriophage or the filamentous viruses M13 and f1. Sequence analysis of a 720-base-pair TaqI fragment indicated an open reading frame of 115 amino acids. The Anabaena strain 7120 thioredoxin gene was expressed in E. coli, and the protein was purified by assaying for protein disulfide reductase activity, using insulin as a substrate. The Anabaena strain 7120 thioredoxin exhibited the properties of a conventional thioredoxin. It is a small heat-stable redox protein and an efficient protein disulfide reductase. It is not a substrate for E. coli thioredoxin reductase. Chemically reduced Anabaena strain 7120 thioredoxin was able to serve as reducing agent for both E. coli and Anabaena strain 7119 ribonucleotide reductases, although with less efficiency than the homologous counterparts. The Anabaena strain 7120 thioredoxin cross-reacted with polyclonal antibodies to Anabaena strain 7119 thioredoxin. However, this unusual thioredoxin was not detected in extracts of Anabaena strain 7120, and its physiological function is unknown.

Amino Acid Sequence↗
PubMed1988

Interaction of hemopexin with Sn-protoporphyrin IX, an inhibitor of heme oxygenase. Role for hemopexin in hepatic uptake of Sn-protoporphyrin IX and induction of mRNA for heme oxygenase.

Sn-protoporphyrin IX (SnPP), an inhibitor of heme oxygenase and a potential therapeutic agent for neonatal hyperbilirubinemia, is bound tightly by hemopexin. The apparent dissociation constant (Kd) at pH 7.4 is 0.25 +/- 0.15 microM, but estimation of the Kd for the SnPP-hemopexin complex is hampered by the fact that at physiological pH SnPP exists as monomers and dimers, both of which are bound by hemopexin. SnPP is readily displaced from hemopexin by heme (Kd less than 1 pM). The hemopexin-SnPP interaction, like that of heme-hemopexin, is dependent on the histidine residues of hemopexin. However, as expected from the differences in the coordination chemistries of tin and iron, the stability of the histidyl-metalloporphyrin complex is lower for SnPP-hemopexin than for mesoheme-hemopexin. Nevertheless, when SnPP binds to hemopexin, certain of the ligand-induced changes in the conformation of hemopexin which increase the affinity of the protein for its receptor are produced. Binding of SnPP produces the conformational change in hemopexin which protects the hinge region of hemopexin from proteolysis, but SnPP does not produce the characteristic increase in the ellipticity of hemopexin at 231 nm that heme does. Competition experiments confirmed that human serum albumin (apparent Kd = 4 +/- 2 microM) has a significantly lower affinity for SnPP than does hemopexin. Appreciable amounts of SnPP (up to 35% in adults and 20% in neonates) would be bound by hemopexin in the circulation, and the remainder of SnPP would be associated with albumin due to the latter's high concentration in serum. Essentially no non-protein-bound SnPP is present. Importantly, SnPP-hemopexin binds to the hemopexin receptor on mouse hepatoma cells with an affinity comparable to that of heme-hemopexin and treatment of the hepatoma cells with SnPP-hemopexin causes a rapid increase in the steady state level of heme oxygenase messenger RNA. These results show that hemopexin participates in the transport of SnPP to heme oxygenase and in its regulation by SnPP.

Animals↗
PubMed1988

Genes encoding the alpha, gamma, delta, and four F0 subunits of ATP synthase constitute an operon in the cyanobacterium Anabaena sp. strain PCC 7120.

A cluster of genes encoding subunits of ATP synthase of Anabaena sp. strain PCC 7120 was cloned, and the nucleotide sequences of the genes were determined. This cluster, denoted atp1, consists of four F0 genes and three F1 genes encoding the subunits a (atpI), c (atpH), b' (atpG), b (atpF), delta (atpD), alpha (aptA), and gamma (atpC) in that order. Closely linked upstream of the ATP synthase subunit genes is an open reading frame denoted gene 1, which is equivalent to the uncI gene of Escherichia coli. The atp1 gene cluster is at least 10 kilobase pairs distant in the genome from apt2, a cluster of genes encoding the beta (atpB) and epsilon (atpE) subunits of the ATP synthase. This two-clustered ATP synthase gene arrangement is intermediate between those found in chloroplasts and E. coli. A unique feature of the Anabaena atp1 cluster is overlap between the coding regions for atpF and atpD. The atp1 cluster is transcribed as a single 7-kilobase polycistronic mRNA that initiates 140 base pairs upstream of gene 1. The deduced translation products for the Anabaena sp. strain PCC 7120 subunit genes are more similar to chloroplast ATP synthase subunits than to those of E. coli.

Amino Acid Sequence↗
PubMed1987

Somatotopic alignment between climbing fiber input and nuclear output of the cat intermediate cerebellum.

The rostral dorsal accessory olive (rDAO) contains a detailed somatosensory map of the entire contralateral body surface. The rDAO projects to the anterior interpositus nucleus (NIA) directly as well as indirectly by way of Purkinje cells in cerebellar cortex. NIA maintains a topographic relation to different levels of the spinal cord through a relay in the magnocellular red nucleus (RNm) and, thus, contains a motor somatotopy. By using bidirectional transport of WGA-HRP, we demonstrate that the sensory somatotopy of rDAO aligns with the motor somatotopy of NIA. It is likely that rDAO information supplied to the cerebellum from a specific part of the body is used to influence movements restricted to that same body part.

Animals↗