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PubMed2003

[SARS-associated coronavirus gene fragments were detected from a suspected pediatric SARS patient].

UNLABELLED: A Special "Fever and Cough" Clinic was set up at the Children's Hospital Affiliated to Capital Institute of Pediatrics for children with symptoms of fever and cough in late April when the severe acute respiratory syndrome (SARS) epidemic was at its peak in Beijing to separate the children with fever from others during their visit to the Outpatient Department. OBJECTIVE: For patients with fever, normal or low count of white blood cell and with suspected pneumonia suggested by X-ray, it was urgent to determine the etiological agents of the diseases before they were admitted to the hospital. METHODS: Throat swabs or nasopharyngeal aspirate specimens were collected from those patients and common respiratory virus antigens including influenza virus A and B, respiratory syncytial virus, adenovirus, parainfluenza virus types I, II, and III were tested by indirect immunofluorescent assay. The patients with atypical pneumonia diagnosed by X-ray and evidences of common respiratory virus infection were admitted to the regular ward for children with respiratory diseases. Children with pneumonia demonstrated by X-ray and negative for common respiratory viruses were admitted to the isolated ward for suspected SARS patients for the first step and further viral etiological studies were requested. RT-PCR was performed for those patients to detect gene fragments of human metapneumovirus (HMPV), rhinovirus (RhV) and enterovirus (EV) in their specimens. Nested RT-PCR was also developed to detect SARS coronavirus gene fragment from the specimens. Primer sequences for SARS virus detection with the PCR were selected according to the primer sequences published online by WHO on April 18, 2003. All the primers derived from the sequence at the 1b frame of coronavirus replicase gene and products with a size of 368 or 348 bp were expected with 2 different primer pairs. RESULTS: Amplicons with the sizes of 368 bp and 348 bp were obtained from a throat swab specimen collected from a 17 years old girl, who was admitted to the isolated ward because of high fever (39.5 degrees C) for 7 days, cough for 2 days, low WBC count, and pneumonia shown by X-ray when she visited the "Fever and Cough" Clinic, and without known history of contact with probable SARS patient. Antigens for the common respiratory viruses were all negative, RT-PCR for HMPV, RhV and EV were also negative while RT-PCR with different primer pairs for SARS virus were all positive which indicated that SARS coronavirus gene fragments were amplified from the specimen from this girl. The amplified fragment with a size of 368 bp was sequenced and the sequence was compared with those in the GenBank. The sequence shared 100% homology with the sequences from 1b frame of replicase genes from all 17 of SARS coronaviruses published in the GenBank so far, and shared very low homology with 2 reference strains of human coronavirus as well as other animal coronaviruses. The serum collected before her discharge from the hospital (19 days after the onset of the disease) showed SARS specific IgM and IgG antibodies. CONCLUSION: These data indicate that the patient was a confirmed case of SARS. It is of great importance to differentiate SARS patients from those infected with common respiratory viruses during SARS epidemic, especially for pediatric patients, because most of the patients visiting the outpatient department present with the symptoms of fever, cough and normal WBC count. The data mentioned above indicate that antigen and gene detections for those common respiratory viruses are useful methods for the differentiation to avoid the spread of SARS.

Adolescent↗

PubMed2000

Time-resolved fluorescence imaging of slab gels for lifetime base-calling in DNA sequencing applications.

A compact time-resolved near-IR fluorescence imager was constructed to obtain lifetime and intensity images of DNA sequencing slab gels. The scanner consisted of a microscope body with f/1.2 relay optics onto which was mounted a pulsed diode laser (repetition rate 80 MHz, lasing wavelength 680 nm, average power 5 mW), filtering optics, and a large photoactive area (diameter 500 microns) single-photon avalanche diode that was actively quenched to provide a large dynamic operating range. The time-resolved data were processed using electronics configured in a conventional time-correlated single-photon-counting format with all of the counting hardware situated on a PC card resident on the computer bus. The microscope head produced a timing response of 450 ps (fwhm) in a scanning mode, allowing the measurement of subnano-second lifetimes. The time-resolved microscope head was placed in an automated DNA sequencer and translated across a 21-cm-wide gel plate in approximately 6 s (scan rate 3.5 cm/s) with an accumulation time per pixel of 10 ms. The sampling frequency was 0.17 Hz (duty cycle 0.0017), sufficient to prevent signal aliasing during the electrophoresis separation. Software (written in Visual Basic) allowed acquisition of both the intensity image and lifetime analysis of DNA bands migrating through the gel in real time. Using a dual-labeling (IRD700 and Cy5.5 labeling dyes)/two-lane sequencing strategy, we successfully read 670 bases of a control M13mp18 ssDNA template using lifetime identification. Comparison of the reconstructed sequence with the known sequence of the phage indicated the number of miscalls was only 2, producing an error rate of approximately 0.3% (identification accuracy 99.7%). The lifetimes were calculated using maximum likelihood estimators and allowed on-line determinations with high precision, even when short integration times were used to construct the decay profiles. Comparison of the lifetime base calling to a single-dye/four-lane sequencing strategy indicated similar results in terms of miscalls, but reduced insertion and deletion errors using lifetime identification methods, improving the overall read accuracy.

DNA↗

PubMed2001

Lack of evidence of a genetic origin in the impaired spermatogenesis of a patient cohort with low-grade varicocele.

Varicocele is the most common clinical finding in infertile men but controversy continues to surround the utility of its treatment. An increased response of FSH to gonadotrophin-releasing hormone testing has been described in patients with varicocele, while the co-influence of Yq chromosome microdeletions in the infertility associated to this pathology is still under investigation. We studied 30 patients with first- and second-grade varicocele, 15 idiopathic oligozoospermic men and 21 age-matched healthy controls. All subjects underwent testicular Doppler ultrasonography, semen analysis, gonadotrophin-releasing hormone testing and baseline blood sampling for total and free testosterone, PRL, 17beta-estradiol, SHBG evaluation and Yq chromosome analysis. Apart from FSH, no difference in baseline hormonal levels was found between the groups. The patients with varicocele showed both an increased basal (p=0.007) and GnRH-induced FSH response (peak and AUC) (p=0.004) in comparison with the controls, while the idiopathic oligozoospermic men had only higher GnRH-induced FSH AUC (p=0.04). In the varicocele group, FSH peaks after GnRH testing correlated positively with the grade of disease (r=0.42, p=0.02) and negatively with sperm count (r=-0.50, p=0.005) and bilateral testis volume (r=-0.52, p=0.005). Sperm count and sperm motility were similarly significantly reduced both in patients with varicocele and in patients with idiopathic oligozoospermia in comparison with healthy controls. Yq chromosome analysis by sequence-tagged site PCR revealed no microdeletion in the AZF regions in any subject studied. Given the quite small number of subjects studied, our overall findings can only prompt us to suggest a possible causal role of varicocele in the impairment of spermatogenesis in our patients. Furthermore, although a genetic co-influence (i.e. Yq microdeletions) does not seem to be involved in the pathogenesis of infertility in men with varicocele and mild to moderate oligozoospermia, genetic screening seems to be advisable, especially in those patients who present a severe impairment of sperm count, as has been suggested by recent literature data.

Adult↗

PubMed1994

Modulation of extracellular matrix gene expression in bovine high-density chondrocyte cultures by ascorbic acid and enzymatic resuspension.

To define the influence of time in culture on gene expression for extracellular matrix proteins we have examined the progression of changes in gene expression for chondrocyte extracellular matrix proteins from the time the chondrocytes are initially isolated from their native cartilaginous matrix through 13 days of high-density culture. We have also determined the effect of matrix depletion and shape change by enzymatically resuspending cells after 6 days in culture and sampling the replated cells at intervals up to 13 days. Northern blots of chondrocyte RNA were hybridized with probes for collagen alpha 1(II), alpha 1(I), aggrecan, link protein, and decorin mRNA. The steady-state level of alpha 1(II) collagen mRNA dropped to 45% of the initial value within 24 h, with a further decrease to 21% by Day 3. A similar decline occurred, but less rapidly in ascorbate supplemented cultures with values of 78%, at 24 h and 62% at Day 3. Very low levels of alpha 1(I) collagen mRNA were detectible in cells maintained for 2 weeks without ascorbate supplementation. Type I collagen alpha 1(I) mRNA was not detected in freshly isolated chondrocytes or at the earliest times in culture but was increasingly abundant from Days 5-13 in the presence of ascorbate. Ascorbic acid supplementation altered the pattern of aggrecan expression over time. Without ascorbate there was an increase in steady-state aggrecan mRNA with time in culture, but in the presence of ascorbate, aggrecan mRNA levels peaked at early culture times and progressively diminished. Decorin steady-state mRNA levels in cultures not supplemented with ascorbic acid steadily increased over time in culture following a lag of several days. In cultures treated with ascorbate, however, there was a progressive increase in decorin steady-state mRNA levels from the first day in culture. Resuspending chondrocytes by digestion of the cell layer with pronase and collagenase at Day 6, which resulted in a transient shape change as well as matrix depletion, resulted in a greater than 2-fold increase in alpha 1(II) mRNA at Day 7 in ascorbate supplemented cultures. Only with ascorbate was there a small increase in decorin mRNA at Day 7, after resuspension. Aggrecan mRNA, however, showed a 3-fold increase without ascorbate and a 10-fold increase with ascorbate within 24 h of resuspension. Similarly, link protein steady-state mRNA showed an 8-fold increase without ascorbate and a 9-fold increase with ascorbate within 24 h after resuspension.

Aggrecans↗

PubMed1995

Identification of an upstream region that controls the transcription of the human autocrine motility factor receptor.

We have isolated from a human placenta cosmid library a 0.7 kb genomic clone that contains the 5' terminal portion of the autocrine motility factor receptor (hAMFR) coding region. Chloramphenicol Acetyl Transferase (CAT) reporter gene assays have identified this region as the promoter of the hAMFR gene. A single transcription initiation site (+1) has been mapped to 129 bp upstream of the ATG start codon by primer extension. DNA sequence analysis and CAT assay revealed a TATA element at the position -485/-468 which was able to conduct only a marginal transcription (less than 5% of the total activity). The majority of the hAMFR promoter's activity is contributed by a transcription initiator (Inr) element overlapping the initiation site (+1) which independently controls the transcription of the hAMFR gene. Gel mobility shift assays showed that DNA-binding proteins in HeLa cells nuclear extract can bind specifically to both promoter's elements. DNA-binding proteins were found to be differentially expressed by sparse and dense cultured normal fibroblasts. The nuclear-binding protein expressed by sparse NIH-3T3 cells induced a DNA mobility shift similarly to the nuclear protein of HeLa cells, while a different DNA-protein complex size was observed with nuclear proteins extracted from dense cultured NIH-3T3 cells. Also CAT-reporter gene analysis revealed a significant lower activity in dense NIH-3T3 cells as compared with the sparse-cultured counterparts. These results help to explain the previously observed cell-cell contact regulation of AMFR expression in normal cells and its consecutive expression in tumor cells.

3T3 Cells↗

PubMed1994

Subcellular localization of metallothionein IIA in human bladder tumor cells using a novel epitope-specific antiserum.

Human metallothioneins (hMTs) are a family of highly homologous proteins thought to be critical in cellular protection against toxins. The comparative function of individual isoforms is, however, obscure. Antibodies to individual MT isoforms might help clarify this issue but their generation has been challenging. We now describe a strategy that has successfully produced an epitope-specific antiserum to a major human isoform, hMT IIA. The immunogen, a conjugated tridecyl amino acid synthetic peptide unique to residues 8-20 in hMT IIA (AAGDSCTCAGSCK), yielded an antiserum (B2) that reacted specifically with hMT IIA in a concentration- and titer-dependent manner and showed no reactivity with human or rabbit liver MT I or horse MTs. This antiserum recognized rabbit liver MT II and, surprisingly, also reacted weakly with chicken MT. MT amino acid sequence comparisons and peptide blocking studies suggested aspartate-11 and threonine-14 are important antigenic determinants for B2. Using confocal immunofluorescence microscopy and B2 antiserum, we observed nuclear localization of hMT IIA with human bladder T24 tumor cells in exponential growth but more cytoplasmic localization at confluence. These results suggest the subcellular location of hMT IIA is a function of cell density in T24 bladder carcinoma cells. The general approach of epitope-specific antibodies may be useful for the generation of antibodies to other MT isoforms and for studying the role of individual MT isoforms in biology and toxicology.

Amino Acid Sequence↗

PubMedDate not supplied

Zidovudine resistance mutations and human immunodeficiency virus type 1 DNA burden: longitudinal evaluation of six patients under treatment.

Zidovudine (ZDV) is by far the most widely used drug to counteract human immunodeficiency virus type 1 (HIV-1) infection, both in monotherapy and in combination therapy regimens. However, the majority of patients under prolonged ZDV therapy have been shown to harbour HIV-1 mutant genomes displaying reduced sensitivity to the drug in vitro. In order to investigate the pathogenic role of in vitro resistance to ZDV, six HIV-1-infected ZDV-treated subjects were evaluated longitudinally (mean follow-up 28.5 months, range 12-39 months) for HIV-1 DNA load in peripheral blood mononuclear cells (PBMC) and for the presence of HIV-1 pol gene mutations responsible for ZDV resistance. Quantitation of HIV-1 DNA was performed by competitive polymerase chain reaction (cPCR) and the pol genotype was determined by direct sequencing of PCR products. All of the six patients developed one or more of the HIV-1 pol mutations known to confer resistance to ZDV in vitro (Met41-->Leu, Asp67-->Asn, Lys70-->Arg, Thr215-->Phe/Tyr, Lys219-->Gln/Glu). A temporal association was found between HIV-1 DNA burden and the level of ZDV resistance, as predicted on the basis of the pol genotype (genotypic resistance). Both virus load and ZDV resistance were inversely correlated with CD4+ cell counts. These results are compatible with a direct in vivo pathogenetic role for pol gene mutations shown to be involved in resistance to ZDV in vitro. Monitoring the degree of genotypic resistance to ZDV and to other antiretroviral drugs should be considered in designing protocols for the management of treated patients.

Adult↗

PubMed1990

Mouse epidermal cell renewal after topical treatment with different concentrations of the epidermal peptide pyroGlu-Glu-Asp-Ser-GlyOH.

Earlier work has shown that epidermal cells contain a peptide, pyroGlu-Glu-Asp-Ser-GlyOH, that induces a moderate but long-lasting inhibition of epidermal cell proliferation when given at low (picomol) doses ip in vivo and in vitro. In the present study, the epidermal pentapeptide was applied topically to the back skin of hairless mice at different concentrations and in a water-miscible cream. A single topical application of either high (0.25% wt/wt) or low (0.004% or 0.02% wt/wt) doses of the pentapeptide was followed by oscillations in epidermal DNA synthesis and G2-M cell flux (mitotic rate). In general, epidermal cell proliferation was inhibited during the first 10-day period after treatment with the two lower doses, while the highest concentration of pentapeptide (0.25%) stimulated epidermal cell proliferation. In spite of the effects on epidermal cell proliferation the size of the epidermal cell population in the treated area (number of nucleated cells and epidermal thickness) showed no corresponding alterations. The results could imply that the epidermal pentapeptide modifies epidermal cell proliferation and terminal differentiation in such a way that the two are balance with each other.

Administration, Topical↗

PubMed1995

Retinoids: effects on growth, differentiation, and nuclear receptor expression in human pancreatic carcinoma cell lines.

BACKGROUND & AIMS: Advanced pancreatic carcinoma has a dismal prognosis despite extensive chemotherapeutic trials. The aim of this study was to evaluate the role of retinoids as an experimental therapeutic approach for pancreatic cancer. METHODS: Four ductal and one acinar pancreatic tumor cell lines were investigated. Growth was determined by cell number and a human tumor clonogenic assay. In vivo growth was assessed by xenografts transplanted into nude mice. Differentiation was characterized by immunofluorescence microscopy and carbonic anhydrase II gene expression. Retinoid receptors were characterized by Northern blotting and reverse-transcriptase polymerase chain reaction. RESULTS: Retinoid treatment results in a time- and dose-dependent growth inhibition in vitro and in vivo of ductal but not acinar pancreatic tumor cells. Retinoid treatment induces a more differentiated phenotype in ductal tumor cells as shown by morphological criteria and increased expression of carbonic anhydrase II. All pancreatic tumor cell lines expressed a broad panel of cellular retinoid binding proteins and nuclear retinoid receptors. Retinoic acid receptor gamma and cellular retinoic acid binding protein II were found in all retinoid-sensitive ductal tumor cell lines but not in the retinoid-resistant acinar cell lines. CONCLUSIONS: Detailed knowledge of nuclear retinoid receptor expression may provide rational strategies for retinoid treatment of pancreatic cancer.

Animals↗

PubMed2004

Enhancement of immunogenicity of an HIV Env DNA vaccine by mutation of the Tyr-based endocytosis motif in the cytoplasmic domain.

We investigated the effect of the conserved tyrosine-based endocytosis motif (YXXPhi) in the cytoplasmic domain of the human immunodeficiency viruses (HIV) envelope protein (Env) on its immunogenicity. Genes with codons optimized for mammalian expression were synthesized for the HIV 89.6 Env with a truncated cytoplasmic domain and a mutant Env in which the tyrosine residue in the YXXPhi motif was changed into a serine. Mutation of the Tyr residue enhanced surface expression of the Env protein. Analysis of immune responses induced by DNA immunization of mice showed that the DNA construct for the Tyr mutant Env induced moderately higher levels of T cell responses. More interestingly, the DNA construct for the mutant Env induced significantly higher levels of antibody responses against the Env protein in comparison to the construct for the wild type Env. Our results suggest that the YXXPhi motif in the HIV Env cytoplasmic domain may play a role in virus evasion of host immune responses through affecting its immunogenicity.

AIDS Vaccines↗

PubMed2004

Peripheral benzodiazepine receptor antisense knockout increases tumorigenicity of MA-10 Leydig cells in vivo and in vitro.

Peripheral benzodiazepine receptors (PBR), first described more than 20 years ago, have been attributed with many putative functions including ones in cellular proliferation and cellular respiration. Hence, it is quite conceivable that deregulation of this receptor could lead to pathology. We and others have reported the existence of PBR overexpression in different human and nonhuman malignancies, but it has never been made clear whether this aberrant malignant PBR expression is a cause or consequence of the cancer. In the current study we induced PBR underexpression by downregulating one critical subunit of the PBR complex, the isoquinoline-binding protein (IBP), using the stable antisense knockout approach, in the MA-10 Leydig cell line. Resultant clones, showing PBR deregulation, also demonstrated increased tumorigenicity, using both in vitro (loss of contact inhibition and growth in soft agar) and in vivo (increased mortality on grafting back into isogenic mice) assays. We suggest that this type of deregulation could be a later event in natural tumor progression. Consequently, PBR deregulation should be more closely studied in human malignancy.

Animals↗

PubMed1998

Delineation of the Cdc42/Rac-binding domain of p21-activated kinase.

p21-activated kinases (PAKs) serve as effector proteins for the GTP-binding proteins Cdc42 and Rac. They are serine/threonine kinases containing the Cdc42/Rac interactive binding (CRIB) motif. The main aim of this study was to define the minimal domain of alphaPAK required for Cdc42/Rac binding. Eight stable PAK fragments of varying lengths, each containing the CRIB motif (residues 75-88), were expressed in Escherichia coli, and their ability to interact with Cdc42 and Rac was assessed using scintillation proximity assays, isothermal titration calorimetry, and fluorescence techniques. The shortest fragments examined (residues 70-94 and 75-94) bound only weakly to either Cdc42 or Rac. A longer fragment starting at residue 75 and ending at residue 105 showed binding to Q61L Rac.GTP with Kd = 1.9 microM. Highest affinity binding (Kd approximately 0.05 microM) was seen with longer fragments ending at residue 118 or 132. A small increase in affinity was seen with those fragments starting at residue 70 rather than residue 75. PAK fragments bound with approximately 3-10-fold higher affinity to Cdc42 than to Rac and bound Q61L variants with 5-10-fold higher affinity than wild type. The dissociation rates of Q61L Rac.mant-GTP and of Q61L Cdc42. mant-GTP from PAK fragment residues 70-132 were measured to be 0.66 and 0.25 min-1, respectively, which are 100-fold lower than dissociation rates for Ras:Ras-effector domains, although their affinities are similar. Calorimetric measurements revealed that binding was associated with a relatively slow heat change. It is suggested that these PAK fragments (in the absence of Cdc42 or Rac) might exist predominantly in an inactive conformation that slowly interconverts with an active conformation and/or a slow conformational change may occur upon binding to Cdc42/Rac. In conclusion, the PAK CRIB motif itself is insufficient for high-affinity binding to Cdc42/Rac, but a 30 amino acid region of PAK (residues 75-105), containing this motif, is sufficient.

Amino Acid Sequence↗

PubMed2001

Structure, activity and evolution of the group I thiolactone peptide quorum-sensing system of Staphylococcus aureus.

In Staphylococcus aureus, the agr locus is responsible for controlling virulence gene expression via quorum sensing. As the blockade of quorum sensing offers a novel strategy for attenuating infection, we sought to gain novel insights into the structure, activity and turnover of the secreted staphylococcal autoinducing peptide (AIP) signal molecules. A series of analogues (including the L-alanine and D-amino acid scanned peptides) was synthesized to determine the functionally critical residues within the S. aureus group I AIP. As a consequence, we established that (i) the group I AIP is inactivated in culture supernatants by the formation of the corresponding methionyl sulphoxide; and (ii) the group I AIP lactam analogue retains the capacity to activate agr, suggesting that covalent modification of the AgrC receptor is not a necessary prerequisite for agr activation. Although each of the D-amino acid scanned AIP analogues retained activity, replacement of the endocyclic amino acid residue (aspartate) located C-terminally to the central cysteine with alanine converted the group I AIP from an activator to a potent inhibitor. The screening of clinical S. aureus isolates for novel AIP groups revealed a variant that differed from the group I AIP by a single amino acid residue (aspartate to tyrosine) in the same position defined as critical by alanine scanning. Although this AIP inhibits group I S. aureus strains, the producer strains possess a functional agr locus dependent on the endogenous peptide and, as such, constitute a fourth S. aureus AIP pheromone group (group IV). The addition of exogenous synthetic AIPs to S. aureus inhibited the production of toxic shock syndrome toxin (TSST-1) and enterotoxin C3, confirming the potential of quorum-sensing blockade as a therapeutic strategy.

Bacterial Proteins↗

PubMed1978

Hemoglobin Lincoln Park: a betadelta fusion (anti-Lepore) variant with an amino acid deletion in the delta chain-derived segment.

An electrophoretically slow-moving hemoglobin variant was identified in three members of a family originating from Southern Mexico. The variant, Hb Lincoln Park, made up approximately 14% of the total hemoglobin and appeared to have normal stability and functional properties. None of the individuals in whom the abnormal hemoglobin was present was anemic, but each had a mildly elevated reticulocyte count. Structural data suggest that the non-alpha chain of Hb Lincoln Park represents a betadelta gene-fusion product, with normal beta chain structure of the amino-terminal portion of the chain and delta sequences subsequently, the crossover point occurring between animo acid residues 22 and 50. An additional abnormality is the deletion of valine-137, a component of the delta gene-derived segment of the betadelta chain. To account for the development of this abnormal globin chain, a series of intergenic crossovers is proposed; the first, a nonhomologous crossover between the beta and delta genes, presumably gave rise to the betadelta fusion gene; two additional crossovers, one of them unequal, may then have occurred between the same beta and delta genes to produce the amino acid deletion.

Adult↗

PubMed1996

Colony growth of human hematopoietic progenitor cells in the absence of serum is supported by a proteinase inhibitor identified as antileukoproteinase.

Serum contains many growth factors and nutrients that stimulate colony formation of hematopoietic progenitor cells (HPC) in semisolid cultures. In the absence of serum, no proliferation of HPCs could be obtained in semisolid medium cultures of partially purified bone marrow cells in the presence of multiple hematopoietic growth factors, insulin, cholesterol, and purified clinical-grade human albumin. This appeared to be due to a suppressive activity induced by monocyte- and T lymphocyte-depleted accessory cells on CD34+ HPCs. Serum-free conditioned medium from the bladder carcinoma cellline 5637 could replace serum to support the growth of HPCs in these cultures. After gel filtration and reverse-phase high-performance liquid chromatography of 5637 supernatants, this activity could be attributed to a 15-kD protein that was further identified by NH2-terminal sequence analysis as the serine proteinase inhibitor antileukoproteinase (ALP). The growth-supportive activity from the 5637 conditioned medium and the (partially) purified fractions could be completely neutralized by a polyclonal rabbit IgG antibody against human ALP (huALP). Similar supportive effects on the growth of HPC could be obtained in the presence of recombinant huALP. We demonstrated that the COOH-terminal domain of ALP containing the proteinase inhibitory activity was responsible for this effect. alpha-1 proteinase inhibitor was capable of similar support of in vitro HPC growth. These results illustrate that proteinase inhibitors play an important role in the in vitro growth of hematopoietic cells by the neutralization of proteinases produced by bone marrow accessory cells. This may be of particular relevance for in vitro expansion of human hematopoietic stem cells in serum-free media.

Amino Acid Sequence↗

PubMed2002

Characterization of pol, vif, vpr, and vpu genes of HIV type 1 in AIDS patients with high viral load and stable CD4+ T cell counts on combination therapy.

The success of combination therapy has also led to AIDS patients who exhibit elevated viral load without a corresponding decline in CD4+ T cells. In this study, we characterized changes in the pol gene and accessory genes vif, vpr, and vpu of HIV-1 isolated from the plasma of patients receiving highly active antiretroviral therapy. From each patient three sequences were obtained and compared with the sequence of HIV-1 from nontreated patients, revealing many substitutions that were similar in most cases. Protease and reverse transcriptase genes showed many mutations that were due to antiviral drugs. Premature termination was observed in the vif gene of one patient, leading to a protein truncated after 187 amino acids. In another patient the entire vpr open reading frame was missing, with no synthesis of Vpu protein because the 5' end of the gene was missing, including the start codon. In the same patient, the Vif protein was also truncated because of the deletion of 100 nucleotides at the 3' end of the vif gene.

Amino Acid Sequence↗

PubMed2004

Failure to detect human immunodeficiency virus type 1 superinfection in 28 HIV-seroconcordant individuals with high risk of reexposure to the virus.

We have examined the occurrence of HIV-1 superinfection in 14 HIV-seroconcordant couples (i.e., partners were independently infected with different HIV-1 strains) with high risk of reexposure to the virus. Phylogenetic analyses based on pol and env global sequences obtained from more than 100 longitudinal plasma samples (corresponding to a period of 1-4 years) failed to detect HIV-1 superinfection in this cohort of patients. Our results suggest that despite recent reports of HIV-1 reinfection, chronic HIV infection seems to confer protection against superinfection with a second HIV-1 strain.

CD4 Lymphocyte Count↗

PubMed2005

Antimicrobial resistance in isolates of Salmonella spp. from pigs and the characterization of an S. Infantis gene cassette.

One hundred and seven Salmonella isolates of various serotypes were investigated for resistance to a panel of nine antimicrobial agents by standardized methods. Thirty four isolates were susceptible to the selected antimicrobial agents. Thirty-six (of 107) were resistant to three or more antimicrobial agents and defined as multidrug resistant (MDR). Salmonella Typhimurium was the most resistant serotype. All resistant isolates were examined for the presence of class 1 integrons. Thirty-two integron-associated gene cassettes (of varying sizes) were identified. A 1,000-bp amplicon similar to that flanking the distal region of the Salmonella Genomic Island (SGI)-1 in Salmonella Typhimurium was detected in a majority of the S. Typhimurium isolates in this study. In contrast, a 1,800-bp amplicon was identified in all Salmonella Infantis isolates. This amplicon was completely characterized in one isolate. The presence of class 1 integrons in Salmonella spp. in pigs may be important if these zoonotic pathogens were to enter the food chain.

Abattoirs↗