Plasma levels of osteopontin before and 24 h after percutaneous coronary intervention.

OBJECTIVE: Previous studies demonstrated that osteopontin (OPN) was increased after vascular injury, such as atherosclerosis and restenosis following angioplasty. We sought to determine the effects of percutaneous coronary intervention (PCI) on plasma OPN levels compared with coronary arteriography (CA). METHODS: Plasma OPN levels were determined in 103 patients who underwent CA or PCI with stent implantation, at baseline and 24 h after the procedure. Patients were divided into three groups; group I: patients without significant coronary artery stenosis, group II: patients with coronary artery disease in whom only CA was performed, group III: patients with coronary artery disease who had PCI and stent implantation. RESULTS: Plasma OPN levels before the procedure were similar in all three groups. OPN levels 24 h after the procedure were significantly higher only in group III compared with baseline. Among three groups, the OPN levels observed in 24 h were significantly higher in group III compared with group I. Patients in group III had significantly higher OPN values after the procedure, depending on the number of stents implanted (p = 0.03). CONCLUSION: The increase in OPN levels after PCI suggests that vascular injury due to PCI is responsible for this phenomenon.

Relationship between plasma osteopontin and oxidative stress in patients with coronary artery disease.

BACKGROUND: It is known that oxidative stress plays an important role in the pathogenesis of atherosclerosis and that an association exists between osteopontin (OPN) and atherosclerosis. OBJECTIVES: It was proposed that malondialdehyde (MDA), a biomarker of lipid peroxidation and oxidative stress, would be related to plasma OPN levels in patients with coronary artery disease (CAD). METHODS/RESULTS: Plasma OPN and MDA levels were measured in 71 patients (60 males and 11 females; mean age 61.7 +/- 10 years). Fifty-eight patients had significant CAD (group I) and 13 patients were free of CAD as defined angiographically (group II). Plasma OPN was measured by enzyme-linked immunosorbent assay (ELISA), while MDA was determined spectrophotometrically. Multivariate regression analysis revealed that ln-transformed OPN levels were independently associated with MDA after adjustment for age, hypertension and diabetes mellitus (R(2) = 0.278, p = 0.0004 and beta regression coefficient = 0.252 [standard error = 0.0958], p = 0.011). OPN and MDA levels were higher in patients with diabetes (73.6 +/- 36.2 ng/ml versus 56.1 +/- 30.9 ng/ml, p = 0.02 and 2.5 +/- 0.5 microM versus 2.0 +/- 0.5 microM, p = 0.002, respectively). CONCLUSIONS: The association between OPN and MDA levels in patients with CAD suggests an interaction between OPN and oxidative stress. This interaction may play a role in the pathogenesis of atherosclerosis.

Blood vessels and desmin control the positioning of nuclei in skeletal muscle fibers.

Skeletal muscle fibers contain hundreds to thousands of nuclei which lie immediately under the plasmalemma and are spaced out along the fiber, except for a small cluster of specialized nuclei at the neuromuscular junction. How the nuclei attain their positions along the fiber is not understood. Here we show that the nuclei are preferentially localized near blood vessels (BV), particularly in slow-twitch, oxidative fibers. Thus, in rat soleus muscle fibers, 81% of the nuclei appear next to BV. Lack of desmin markedly perturbs the distribution of nuclei along the fibers but does not prevent their close association with BV. Consistent with a role for desmin in the spacing of nuclei, we show that denervation affects the organization of desmin filaments as well as the distribution of nuclei. During chronic stimulation of denervated muscles, new BV form, along which muscle nuclei align themselves. We conclude that the positioning of nuclei along muscle fibers is plastic and that BV and desmin intermediate filaments each play a distinct role in the control of this positioning.

Desmin cytoskeleton linked to muscle mitochondrial distribution and respiratory function.

Ultrastructural studies have previously suggested potential association of intermediate filaments (IFs) with mitochondria. Thus, we have investigated mitochondrial distribution and function in muscle lacking the IF protein desmin. Immunostaining of skeletal muscle tissue sections, as well as histochemical staining for the mitochondrial marker enzymes cytochrome C oxidase and succinate dehydrogenase, demonstrate abnormal accumulation of subsarcolemmal clumps of mitochondria in predominantly slow twitch skeletal muscle of desmin-null mice. Ultrastructural observation of desmin-null cardiac muscle demonstrates in addition to clumping, extensive mitochondrial proliferation in a significant fraction of the myocytes, particularly after work overload. These alterations are frequently associated with swelling and degeneration of the mitochondrial matrix. Mitochondrial abnormalities can be detected very early, before other structural defects become obvious. To investigate related changes in mitochondrial function, we have analyzed ADP-stimulated respiration of isolated muscle mitochondria, and ADP-stimulated mitochondrial respiration in situ using saponin skinned muscle fibers. The in vitro maximal rates of respiration in isolated cardiac mitochondria from desmin-null and wild-type mice were similar. However, mitochondrial respiration in situ is significantly altered in desmin-null muscle. Both the maximal rate of ADP-stimulated oxygen consumption and the dissociation constant (K(m)) for ADP are significantly reduced in desmin-null cardiac and soleus muscle compared with controls. Respiratory parameters for desmin-null fast twitch gastrocnemius muscle were unaffected. Additionally, respiratory measurements in the presence of creatine indicate that coupling of creatine kinase and the adenine translocator is lost in desmin-null soleus muscle. This coupling is unaffected in cardiac muscle from desmin-null animals. All of these studies indicate that desmin IFs play a significant role in mitochondrial positioning and respiratory function in cardiac and skeletal muscle.

Cloning and characterization of CcEcR. An ecdysone receptor homolog from the mediterranean fruit fly ceratitis capitata.

In order to understand the role that 20-hydroxyecdysone plays during development of the Mediterranean fruit fly Ceratitis capitata (medfly), a major agricultural pest, we have cloned a Ceratitis ecdysone receptor (CcEcR) and studied its expression and its binding properties to an ecdysone response element. Using the conserved DNA binding region of the Drosophila melanogaster ecdysone receptor (DmEcR) B1 cDNA as a probe, we isolated a medfly cDNA clone containing the coding region, a part of the 5'-untranslated region and the complete 3'-untranslated region of a CcEcR. The deduced CcEcR polypeptide contained all five domains typical of a nuclear receptor. Alignment comparisons and phylogenetic analyses indicated that CcEcR most closely resembled the B1 isoform of DmEcR and Lucilia cuprina EcR homolog (LcEcR) relative to all other known ecdysone receptors. In situ hybridization analysis showed that the CcEcR gene is mapped in the region 53B of the 4R chromosome arm, while Northern hybridization analysis showed that CcEcR transcripts have a size of approximately 8 kb. Significant levels of CcEcR transcripts were detected in eggs, middle and late embryos, late third instar larvae and middle prepupae. The levels of the CcEcR transcripts during the other larval stages as well as during pupal and adult stages were much lower, while during the early stages of embryogenesis were very low. Electrophoretic mobility shift assays indicated that CcEcR binds specifically to the Drosophila hsp27 ecdysone response element as a heterodimer with Drosophila USP, the product of the ultraspiracle gene. Our structural and biochemical data suggest that CcEcR is the functional homolog of the B1 isoform of DmEcR.

Cloning and characterization of a cDNA encoding a male-specific serum protein of the Mediterranean fruit fly, Ceratitis capitata, with sequence similarity to odourant-binding proteins.

Male-specific serum proteins (MSSPs) are low molecular weight proteins which accumulate in high amounts in the haemolymph of adult males of the medfly Ceratitis capitata. By screening an expression library with anti-MSSP antibodies, we have isolated and determined the nucleotide sequence of a cDNA clone coding for one of the male-specific polypeptides (MSSP-alpha). The MSSP-alpha mRNA encodes a polypeptide of 144 amino acids with a secretory signal sequence of sixteen amino acids. Southern analysis indicated that there are multiple copies of MSSP genes in the medfly genome. Northern analysis showed that the MSSP mRNAs are synthesized only in adult males. The accumulation pattern of these mRNAs during development suggests that the expression of the MSSP genes is developmentally regulated at both transcriptional and translational levels. The predicted peptide sequence of MSSP-alpha shows significant similarity to a group of pheromone- and general odourant-binding proteins of insects.

The heat shock 70 gene family in the Mediterranean fruit fly Ceratitis capitata.

The cloning and the characterization of the heat shock 70 (hsp70) genes of the medfly C. capitata, a major agricultural pest, are presented. Six genomic clones were isolated by screening a medfly genomic library with an hsp70 genomic fragment of Drosophila melanogaster. They form two 30 kb contigs, both of which map cytogenetically in a single major heat shock puff (3L:24C) of the salivary gland polytene chromosomes. Restriction mapping and blot hybridization indicated the presence of six putative hsp70 genes in these two closely linked regions. The sequence of one of these genes suggests that it is a heat-inducible hsp70 gene. The 638-codon open reading frame shows 84% identity at the amino acid level (73.5% at the nucleotide level), relative to corresponding D. melanogaster sequences. The 5' untranslated leader sequence, approximately 200 bp long, is not interrupted by introns and is very rich (48%) in adenine residues, resembling Drosophila heat-inducible hsp70 genes. Furthermore, the promoter of this gene contains two characteristic heat shock elements close upstream from the TATA box. The levels of the hsp70 transcripts are very low at 25-30 degrees C, increase significantly at 33 degrees C and reach maximum at 39 degrees C.

Isolation, primary structure, and evolution of the third component of chicken complement and evidence for a new member of the alpha 2-macroglobulin family.

Although the third component of complement, C3, has been isolated and its primary structure determined from most living classes of vertebrate, limited information is available on its structure and function for aves, which represent a significant stage in complement evolution. In this study, we present the complete cDNA sequence of chicken C3, the cDNA sequences of the thioester region for two chicken alpha 2-macroglobulin (alpha 2M)-related proteins, a simplified method for purifying chicken C3, and an analysis of the C3 convertase and factor I-mediated cleavages in chicken C3. Using the reverse-transcriptase PCR, with degenerate oligonucleotide primers derived from two conserved C3 sequences (GCGEQN/TM, TWLTAY/FV) and liver mRNA as template, we isolated three distinct 220-bp PCR products, one with a high degree of sequence similarity to C3 and two to alpha 2M and pregnancy zone protein from other species. The complete cDNA sequence of chicken C3 was obtained by screening a chicken liver lambda gt10 library with the C3 PCR product and probes from the 5' end of the partial-length C3 clones. The obtained sequence is in complete agreement with the protein sequence of several tryptic peptides of purified chicken C3. Chicken pro-C3 consists of an 18-residue putative signal peptide, a 640-residue beta-chain (70 kDa), a 989-residue alpha-chain (111 kDa), and an RKRR linker region. It contains an internal thioester and three potential N-glycosylation sites, all in the alpha-chain. The convertase cleavage site, predicted to be Arg-Ser, was confirmed by sequencing the zymosan-bound C3 fragments generated upon complement activation. NH2-terminal sequencing of the purified C3 chains showed that 1) pro-C3 is indeed cleaved at the RKRR linker sequence to generate the mature two-chain molecule, and 2) the beta-chain of chicken C3 is blocked. The deduced amino acid sequence shows 54, 54, 54, 53, 52, 57, and 55% amino acid identities to human, mouse, rat, guinea pig, rabbit, cobra, and Xenopus C3, respectively, and an identity of 44, 31, and 33% to trout, hagfish, and lamprey C3, respectively. The identities to human C4, C5, and alpha 2M are 31, 29 and 23%, respectively. A phylogenetic tree for C3, C4, C5, and alpha 2M-related proteins was constructed based on the sequence data and is discussed.

The third component of Xenopus complement: cDNA cloning, structural and functional analysis, and evidence for an alternate C3 transcript.

Although the third component of complement has been purified from two amphibian species, Xenopus laevis and the axolotl, only limited information is available about its primary structure in these species. We now present (a) 95% of the cDNA sequence encoding C3 from a Xenopus laevis/Xenopus gilli (Xenopus LG) hybrid (b) an analysis of the C3 convertase and factor I cleavage sites in Xenopus C3, and (c) evidence for an alternative form of C3. The Xenopus LG sequence has a 57% nucleotide and 52% amino acid sequence identity to human C3 and contains one potential N-glycosylation site in the beta-chain. The deduced amino acid sequence showed that the C3 convertase and factor I cleavage sites (Arg-Ser) are conserved in Xenopus C3 and protein sequencing of Xenopus C3 fragments fixed on zymosan during complement activation demonstrated that Xenopus C3 is indeed cleaved by C3 convertase and factor I at these sites. Our screening of a liver cDNA library identified an unusual C3 clone with a deletion of 2502 bp, suggesting the presence of a novel C3 transcript in Xenopus LG liver. The presence of this C3 transcript was confirmed by reverse transcription polymerase chain reaction using Xenopus LG liver mRNA and specific oligonucleotide probes. This transcript encoded a putative 102-kDa protein comprising the beta-chain of C3, together with the first 59 residues and the last 103 residues of the alpha-chain; it would therefore lack many of the ligand binding sites found in the intact alpha-chain. However, the molecule may be an analog of a truncated C3 molecule that is found in the serum of allergic dermatitis patients and acts as an inhibitor of eosinophil cytotoxicity and neutrophil adherence.

Overexpression, purification, and characterization of third component of complement.

The third component of complement (C3) plays a critical role in both pathways of complement activation by interacting with numerous other complement proteins. To elucidate the molecular features of C3 that relate to the functional activities of the molecule, we expressed the cDNA of human complement component C3 in cultured insect cells using a baculovirus expression vector system derived from the baculovirus Autographa california nuclear polyhedrosis virus (AcNPV). The expression of C3 was controlled by the promoter of the polyhedrin gene and, when recombinant baculovirus infected insect cells were cultured in serum-free medium, C3 was detected at a level of 10 micrograms/ml of culture medium. Characterization of the recombinant C3 (rC3) by SDS-PAGE revealed that the C3 gene product was translated as a 188 kDa protein comprised of two chains of 115 kDa and 73 kDa analogous to the alpha and beta chains of serum-derived human C3 (sC3). An analysis of the glycosylation pattern of purified rC3 revealed that, whereas both the alpha and beta chains were glycosylated as in sC3, the proC3 moiety of rC3 also was glycosylated. When rC3 was produced in the High Five cell line of insect cells and evaluated for reactivity with a panel of anti-C3 monoclonal antibodies (MoAb), the results suggested that the conformation of the baculovirus expressed C3 was similar to that of native C3. When the rC3 was purified by anion exchange column chromatography, it was able to react with several C3-binding proteins (CR1, P and H), reconstitute C3-deficient serum and support the activation of both complement pathways thus demonstrating that a baculovirus-expressed C3 can participate in the formation of and can be cleaved by both the classical and alternative pathway convertases. Incubation of rC3 with factor I and H revealed that both C3 and proC3 are susceptible to cleavage by factor I.

Fluphenazine activity and antipsychotic response.

Plasma fluphenazine levels and plasma total neuroleptic activity (as quantitated by the neuroleptic receptor binding assay) were related to therapeutic response in 15 DSM-III schizophrenic patients who received a predetermined, fixed dose of fluphenazine for 14 days. Mean neuroleptic activity of the plasma was 84% greater than can be accounted for by the parent fluphenazine alone, and varied widely between patients. A sigmoidal relationship between total neuroleptic activity of plasma and response was found, with a continued plateau of response at higher total neuroleptic levels. Furthermore, the RBA data suggested (P less than 0.002) that two populations of drug-responsive schizophrenics exist which may be discriminated by the total D2 binding activity of plasma required for response.

Fluphenazine plasma levels and clinical response.

Plasma levels of fluphenazine and clinical response were examined in 19 inpatient schizophrenics (DSM-III diagnoses) using a constant dose, steady-state methodology. A significant curvilinear correlation was demonstrated between clinical response and steady-state plasma levels of fluphenazine (p less than .05). A therapeutic range of plasma fluphenazine is suggested in the range of .13-.70 ng/ml. The lowest plasma level detected (.13 ng/ml) appeared to be well within the therapeutic range. The 9 patients with plasma fluphenazine levels in this range demonstrated a mean clinical improvement of 59% compared to 34% for patients with plasma levels above .70 ng/ml (p less than .01).

Haloperidol plasma and red blood cell levels and clinical antipsychotic response.

Two investigators have recently suggested therapeutic ranges for plasma haloperidol in the treatment of schizophrenia. An apparent optimal therapeutic range of red blood cell haloperidol as early as day 7 of the drug trial is described in this article. With continued treatment, an optimal plasma haloperidol range for response could be observed by day 14 of treatment. The previously described correlation between response at day 7 and plasma/red blood cell haloperidol ratio was confirmed but was found not to predict response at day 14 of drug treatment in this cohort of DSM-III schizophrenic patients.

Clinical relevance of thiothixene plasma levels.

The authors examined plasma levels of thiothixene and clinical response in 19 DSM-III diagnosed inpatient schizophrenics, using an improved methodology. A significant curvilinear correlation was demonstrated between clinical response and plasma levels for thiothixene (p less than 0.02). Optimal clinical response to thiothixene appears to be associated with plasma levels from 2.0 to 15.0 ng/ml (p less than 0.05). These findings suggest that laboratory measurement of thiothixene levels may assist in determining the minimum effective dose for individual patients.

Dopamine and non-dopamine psychoses.

The time course of antipsychotic response following the initiation of an antipsychotic drug and functional dopamine receptor sensitivity were explored in a cohort of recently admitted psychotic (mood-incongruent) patients. The distribution of the latencies of antipsychotic response suggested at least two populations. Rapid responders (RRs) had 60% reduction of baseline psychotic symptoms by a mean of 5.5 days of drug treatment. Delayed/nonresponders required 2-7 weeks for a similar reduction of psychotic symptoms. The sensitivity of postsynaptic dopamine receptors was explored using a neuroendocrine probe: growth hormone response to the dopamine agonist, apomorphine (AP). RRs had an exaggerated growth hormone response to AP in comparison to delayed/nonresponders (P less than 0.05). Exaggerated sensitivity of postsynaptic dopamine receptors and rapid antipsychotic response following dopamine receptor blockade in RRs suggest a true functional dopamine hypersensitivity disorder in the RR group. In contrast, lower postsynaptic receptor sensitivity (as reflected by lower growth hormone response to AP) and failure of early response following dopamine receptor blockade focus attention away from dopamine hyperactivity as a relevant etiologic mechanism in delayed/nonresponders. Response rates to neuroleptic drugs and neuroendocrine probes of receptor sensitivity may separate two or more etiologically distinct diseases with schizophrenic-like symptoms.

Adrenergic-receptor desensitization and course of clinical improvement with desipramine treatment.

alpha 2-Adrenergic receptor sensitivity was assessed during desipramine treatment by carefully monitoring clonidine-induced blood pressure changes, as well as symptomatology, in three patients with major depressive disorder. The data appear to be consistent with the hypothesis that the antidepressant action of desipramine is mediated by alpha 2-autoreceptor desensitization.

Clinical response and plasma haloperidol levels in schizophrenia.

Plasma haloperidol levels were compared to clinical response during the first 14 days of drug treatment in 14 DSM III-diagnosed inpatient schizophrenic patients using an improved methodology, which utilized predetermined constant dosages and derived a therapeutic range of plasma haloperidol levels from a curvilinear regression analysis. An inverted U-shaped relationship was found which reflected a significant curvilinear correlation (r = 0.66, P less than 0.02) between plasma levels (as assayed by gas chromatography) and improvement on the Serial New Haven Schizophrenia Index. A therapeutic window was suggested by the present study, with optimum patient response associated with plasma haloperidol levels of 4.2-11.0 ng/ml for the first 2 weeks of treatment.