Virologic risk factors for vertical transmission of HIV type 1 in Puerto Rico.

HIV-1 vertical transmission in Puerto Rico has decreased significantly due to the implementation of antiviral therapy. Several studies have shown that the phenotype of the HIV-1 isolates initially recovered from infected infants has generally been one that replicates rapidly, infects macrophages, and preferentially use the CCR5 coreceptor. Our hypothesis is that viral genotypic and phenotypic differences exist between HIV-1 nontransmitter and transmitter mothers. Viral DNA samples and virus isolates were analyzed from a Puerto Rican perinatal population. Heteroduplex tracking assay (HTA) was performed on DNA samples to detect env V3 evolutionary variants and the extent of heterogeneity within each sample. HIV-1 C2-V3 variants were cloned from each patient to study sequence variation among the groups. Differences in replication kinetics of viral isolates in macrophage and GHOST CCR5 cells were analyzed by use of repeated measures linear regression analysis. HTA analysis showed that only two nontransmitter patient samples showed the presence of evolutionary variants. Phylogenetic analysis between maternal-infant pairs showed that transmission of a single maternal variant occurred, with the exception of one sample pair. When evaluating amino acid sequences from cloned PCR products, nontransmitting mothers appear to have a higher number of distinct sequences than both the transmitting mothers (p = 0.0410) and the infected infants (p = 0.0315). Analysis of replication kinetics indicated that transmitters showed faster replication kinetics in GHOST CCR5 cell cultures at 12 days postinfection (p = 0.0434) and 15 days postinfection (p = 0.0181). In conclusion, viral homogeneity and rapid replication kinetics were correlated with vertical transmission.

Expression and purification of recombinant hemoglobin I from Lucina pectinata.

Hemoglobin I (HbI) from Lucina pectinata reacts with hydrogen sulfide to form the ferric sulfide complex needed to transport H2S to the bacterial endosymbiont. To further study HbI, expression studies of this protein were performed in Escherichia coli. This is the first time that the recombinant HbI was produced using a recombinant DNA expression system. Hemoglobin I cDNA was amplified and cloned into the TOPO-PBAD expression vector, which contains a fusion tag of six histidine residues (6XHis tag). Plasmid clone sequence analysis was carried out in order to ensure that the insert was in the correct reading frame for proper protein expression in E. coli. The expression of recombinant HbI was optimal when induced for 5 hr with 0.002% of L-arabinose as detected by Western blot analysis. The proto-porphyrin group was inserted into the recombinant HbI. Purification of the heme-bound recombinant protein was performed under native conditions by affinity chromatography using Ni-NTA and Probond resins. The sodium dithionite-reduced recombinant protein presented a shift from the Soret band at 413-435 nm, indicating the presence of the heme group in the adequate amino acid environment of HbI. These results indicate that recombinant HbI from Lucina pectinata can be successfully expressed in a prokaryotic system retaining its activity toward reduction, oxidation, and ligand binding.

Characterization of HIV isolates from Puerto Rican maternal-infant pairs reveal predominance of non-syncytium inducing (NSI) variants with CCR5 genotype.

In this study, HIV-1 variants from a cohort of forty-eight Puerto Rican pregnant women and their 50 infants (one had triplets), were isolated and characterized, in order to determine the type of HIV-1 variants that are predominantly transmitted. All were enrolled in the prenatal AIDS Clinical Trials Group (ACTG) and received anti-retroviral therapy. Fifteen of the 50 infants (30%) were positive by V3 PCR suggesting that they harbored a copy of the HIV envelope gene. Three of 50 infants (6%) were HIV-1 culture and PCR positive, indicating active infection. HIV-positive cultures were obtained from 32 of the 48 mothers. Sixty two percent of the isolates (20/32) were macrophage-tropic and non-syncytium inducing, three percent (1/32) had dual tropism, and thirty four percent (11/32) were non-syncytium inducing and did not grow in macrophages. Phenotype and genotype of the HIV variants from the three infected infants revealed the presence of macrophage-tropic and non-syncytium-inducing strains. Genotype analysis of the HIV env V3 loop revealed the presence of specific amino acids that are predictive of CCR5 usage. Sequence analysis of the HIV pol gene from the three infected infants indicated that vertical transmission was not caused by the presence of antiviral resistance mutations. These results indicate that mothers undergoing antiretroviral treatment at different stages of the disease and with different viral loads transmit predominantly macrophage-tropic/non-syncytium inducing/CCR5 variants to their infants.

Insulin-induced gene 33 mRNA expression in Chinese hamster ovary cells is insulin receptor dependent.

Gene 33 (g33) is a non-tissue-specific gene regulated in rat liver and hepatoma cells by insulin and other agents. It is thought to participate in the transition from quiescence to proliferation in mitogen-treated cells. The mechanism(s) by which insulin exerts its action on g33 are not totally understood; it is unclear whether a functional insulin receptor is required for this action. In this study, we evaluate the mechanism for insulin induction of g33 mRNA in Chinese hamster ovary (CHO) cells transfected with the neomycin-resistant plasmid (CHONeoB), human insulin receptor (CHONewIRa), and a kinase-defective insulin receptor mutated at the ATP-binding site (CHOK1018A). Transfected cells had higher levels of insulin binding than that of CHONeoB cells; insulin-induced phosphorylation of the insulin receptor and its intracellular substrates were impaired in CHOK1018A cells. Maximal insulin induction of mRNA(g33) occurred 3 h after hormonal exposure in all cell lines. The degree of insulin stimulation of g33 mRNA levels was four- to sixfold higher in CHONewIRa than in CHONeoB or CHOK1018A cells, which had minimal levels of insulin-stimulated g33 mRNA levels. Half-maximal stimulation of g33 mRNA levels was observed at 0.06 +/- 0.01 nM in CHONewIRa cells, consistent with insulin interaction with its own receptor. Wortmannin, an inhibitor of phosphatidyl inositol 3-kinase (PI3K), had some effects on insulin stimulation of g33 mRNA in CHO NewIRa cells. PD98059, an inhibitor of mitogen-activated kinase kinase (MAPKK), and rapamycin, a p70 S6 kinase inhibitor, had minimal effect on insulin stimulation of g33 mRNA in all cells tested. By contrast, hydroxy-2-naphthalenylmethyl)phosphonic acid triacetoxymethyl ester (HNMPA(AM)(3), a selective inhibitor of the insulin receptor tyrosine kinase, caused complete inhibition of insulin stimulation of g33 mRNA levels. These data indicate that the insulin receptor with intact kinase activity is required for insulin stimulation of g33 mRNA levels. They also suggest that AKT, a PI 3-kinase downstream effector molecule, could mediate insulin stimulation of g33 mRNA. The mechanism(s) of insulin regulation of g33 expression downstream of receptor do not seem to rely entirely on the classic insulin receptor transduction pathway, as a minor effect was observed upon inhibition of MAPKK, suggesting that multiple pathways may be involved.

Gene 33/Mig-6, a transcriptionally inducible adapter protein that binds GTP-Cdc42 and activates SAPK/JNK. A potential marker transcript for chronic pathologic conditions, such as diabetic nephropathy. Possible role in the response to persistent stress.

Chronic stresses, including the mechanical strain caused by hypertension or excess pulmonary ventilation pressure, lead to important clinical consequences, including hypertrophy and acute respiratory distress syndrome. Pathologic hypertrophy contributes to decreased organ function and, ultimately, organ failure; and cardiac and diabetic renal hypertrophy are major causes of morbidity and morality in the developed world. Likewise, acute respiratory distress syndrome is a serious potential side effect of mechanical pulmonary ventilation. Whereas the deleterious effects of chronic stress are well established, the molecular mechanisms by which these stresses affect cell function are still poorly characterized. gene 33 (also called mitogen-inducible gene-6, mig-6) is an immediate early gene that is transcriptionally induced by a divergent array of extracellular stimuli. The physiologic function of Gene 33 is unknown. Here we show that gene 33 mRNA levels increase sharply in response to a set of commonly occurring chronic stress stimuli: mechanical strain, vasoactive peptides, and diabetic nephropathy. Induction of gene 33 requires the stress-activated protein kinases (SAPKs)/c-Jun NH(2)-terminal kinases. This expression pattern suggests that gene 33 is a potential marker for diabetic nephropathy and other pathologic responses to persistent sublethal stress. The structure of Gene 33 indicates an adapter protein capable of binding monomeric GTPases of the Rho subfamily. Consistent with this, Gene 33 interacts in vivo and, in a GTP-dependent manner, in vitro with Cdc42Hs; and transient expression of Gene 33 results in the selective activation of the SAPKs. These results imply a reciprocal, positive feedback relationship between Gene 33 expression and SAPK activation. Expression of Gene 33 at sufficient levels may enable a compensatory reprogramming of cellular function in response to chronic stress, which may have pathophysiological consequences.

Insulin and phorbol ester regulation of gene 33 expression in CHO cells.

Rat gene 33 (g33) mRNA has a widespread tissue distribution. Insulin and various agents such as glucocorticoids, phorbol esters and plant lectins regulate G33 expression in rat hepatoma cells. The regulation of g33 by insulin and a phorbol ester was examined in two Chinese Hamster ovary (CHO) cell lines, CHO-T cells (which overexpress human insulin receptors (hIR)) and wild type CHOwt cells. These cell lines were used to determine how expression of the hIR influences the capacity of g33 to respond to insulin and phorbol myristate acetate (PMA). Treatment of CHOwt and CHO-T cells with insulin increased mRNAg33 levels three to four-fold, with a maximum effect reached after three hours of treatment. PMA treatment of CHOwt and CHO-T cells caused a similar elevation of mRNAg33 levels after three hours. Insulin had no effect on mRNAg33 stability in both CHO cell lines. Additionally, the effects of insulin and PMA on mRNAg33 levels were additive only in CHO-T cells. Insulin or PMA-pretreated CHO-T cells were able to respond to both agents, but elevation of mRNAg33 levels was submaximal. In contrast, when insulin and/or PMA-pretreated CHOwt cells were exposed to insulin or PMA, g33 was able to respond maximally. These results suggest that insulin and phorbol esters act through different signaling mechanisms in CHOwt cells. Additionally, insulin's ability to stimulate g33 expression in CHOwt cells suggests that this insulin effect may be independent of the insulin receptor. There are differences in the regulation pattern of g33 by insulin and PMA in rat hepatoma and among the two CHO cell lines used in this study.

The cDNA-derived amino acid sequence of hemoglobin I from Lucina pectinata.

The tropical clam Lucina pectinata contains a unique hemoglobin (HbI) which serves to transport H2S to autotrophic bacteria. The cDNA-derived amino acid sequence was obtained from overlapping clones containing the cDNA that codes for HbI. The reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods were employed to synthesize the cDNA fragments. An initial 354-bp cDNA clone encoding 118 amino acid residues of HbI was amplified from total RNA by RT-PCR using degenerate oligonucleotides. Gene-specific primers derived from the HbI-partial cDNA sequence were used for obtaining the 5' and 3' ends of the cDNA by RACE. The length of the HbI cDNA, estimated from sequence analysis of overlapping clones, was 1322 bp for the full-length cDNA. The coding region of the full-length cDNA codes for 143 amino acid residues. The most conserved amino acid residues in HbI from Lucina pectinata were identified by a multiple alignment with nonvertebrate globin sequences.

Characterization of hemoglobin Hotel Dieu in a Puerto Rican adolescent.

PURPOSE: Hemoglobin Hotel Dieu (HbHD) is a high-oxygen affinity variant of HbA never before reported in a Hispanic patient. This Hb variant was first reported in 1981 by Blouquit et al. in a white person with erythrocytosis with a substitution in the beta 99 aspartic acid residue by glycine. METHODS: A 13-year-old Puerto Rican boy had pain in his chest, headaches, easy fatigability, and high Hb (as high as 19.1 g/dl). Protein analysis was performed by cellulose acetate, citrate agar, and isoelectric focusing electrophoresis and high-pressure liquid chromatography (HPLC), polymerase chain reaction (PCR) amplification, and DNA sequencing of the second exon of the beta gene in samples obtained from the mother, father, and the patient, and DNA fingerprinting to determine paternity. RESULTS: The variant found in the patient migrated on cellulose acetate electrophoresis to a cathodic position relative to HbF, and a band cathodal to HbA and close to HbF on isoelectric focusing electrophoresis. The patient showed an abnormal well-resolved peak on HPLC with a retention time slightly shorter than that for HbS. DNA analysis by direct sequencing of the PCR product demonstrated heterozygosity for codon 99 (GAT-->GGT) in the patient but not in either parent. DNA fingerprinting by multiplex PCR amplification of three simple tandem repeat loci showed that the patient shared alleles in all three loci with both parents, ruling out nonpaternity. CONCLUSIONS: The protein and DNA analysis indicate that the erythrocytosis is caused by the presence of HbHD in this Hispanic adolescent.

Longitudinal studies on maternal HIV-1 variants by biological phenotyping, sequence analysis and viral load.

In this study, the HIV-1 variant viruses from ten pregnant women and their infants were isolated and characterized longitudinally in order to determine the role that viral envelope (gp120-V3 loop) gene variation and viral tropism play in vertical transmission. Biological phenotyping of each HIV variant was accomplished by growth in MT-2, and macrophages from healthy and non-HIV-infected donors. Genetic characterization of the variants was accomplished by DNA sequence analysis. All the women enrolled in this study received ZDV therapy. Virus was cultured from eight out of ten env V3-PCR positive mothers. HIV-1 isolates were all non-syncitium inducing variants. None of the mothers were found to transmit HIV, as determined by DNA PCR and quantitative co-cultures on their infants which were seronegative for HIV-1 through one year after birth. Viral cultures from infant blood samples were negative and infants were all healthy. However, nested env V3-PCR detected proviral DNA in five out of ten infants. In contrast, conventional gag-PCR was negative in the same five infants. Sequences of the five maternal-infant pairs were different, suggesting unique infant HIV-1 variants. The three highest maternal viral load values corresponded to infants that were env V3-PCR positive. These results suggest that HIV-1 particles are transmitted from ZDV-treated mothers to infants. Infant follow up is recommended to determine if HIV-1 has been inhibited by the immune system of the infants.

Sequence analysis of the V1-V2 hypervariable region of the HIV-1 envelope gene from pediatric patients.

The presence of HIV-1 DNA sequence variants of pediatric AIDS patients was investigated in a two-stage polymerase chain reaction (PCR) using nested primers for the first and second (V1-V2) hypervariable regions of the proviral envelope (gp 120) gene (env1). Gel electrophoresis analysis yielded amplified DNA bands which exhibited length variations which were characteristic for each child. The PCR products were cloned and the resulting clones demonstrated inserts of different lengths in a patient and between patients. Analysis of five clones from two different patients at the level of DNA sequencing indicates an extreme heterogeneity in the V1-V2 region. DNA maximum similarity between all of the isolated clones ranged between 69 to 96%. Comparison between DNA sequences of the isolated clones and HIV-1 strains of other parts of the world was also performed. The highest percentage of similarity that was found with known HIV variants included the following strains: HIVADA, HIVJFL, HIVSW42, HIVSWB83, HIVTRA2, HIVWMJ22. The sequences also showed a high degree of similarity to the clade B virus HIVSF162. The analyzed HIV-1 sequences demonstrated the expected high degree of variation in the V1-V2 region of the envelope gene and in some cases that the variation between isolates from the same patient may exceed the variation between the individual clones and the reference HIV-1 strains.

Sequence analysis of the V1-V2 hypervariable region of the HIV-1 envelope gene from pediatric patients

The presence of HIV-1 DNA sequence variants of pediatric AIDS patients was investigated in a two-stage polymerase chain reaction (PCR) using nested primers for the first and second (V1-V2) hypervariable regions of the proviral envelope (gp 120) gene (env1). Gel electrophoresis analysis yielded amplified DNA bands which exhibited length variations which were characteristic for each child. The PCR products were cloned and the resulting clones demonstrated inserts of different lengths in a patient and between patients. Analysis of five clones from two different patients at the level of DNA sequencing indicates an extreme heterogeneity in the V1-V2 region. DNA maximum similarity between all of the isolated clones ranged between 69 to 96 percent. Comparison between DNA sequences of the isolated clones and HIV-1 strains of other parts of the world was also performed. The highest percentage of similarity that was found with known HIV variants included the following strains: HIVADA, HIVJFL, HIVSW42, HIVSWB83, HIVTRA2, HIVWMJ22. The sequences also showed a high degree of similarity to the clade B virus HIVSF162. The analyzed HIV-1 sequences demonstrated the expected high degree of variation in the V1-V2 region of the envelope gene and in some cases that the variation between isolates from the same patient may exceed the variation between the individual clones and the reference HIV-1 strains

An H1-like protein from the macronucleus of Euplotes eurystomus.

An H1-like protein has been purified from the macronucleus (MAC) of the hypotrichous ciliated protozoan, Euplotes eurystomus. It is present in amounts comparable to the inner histones and is extracted by treatment with 5% perchloric acid or 0.65 M NaCl, but not by 0.35 M NaCl. Treatment of soluble MAC chromatin with the ionic exchange resin AG 50W-X2 in 80 mM NaCl removes MAC H1 and yields H1-depleted chromatin. Mac H1 is lysine-rich and deficient in acidic amino acids. The stoichiometry of the H1 protein is reduced in mononucleosome preparations, consistent with its postulated interaction with linker DNA regions. Thermal denaturation and circular dichroism studies reveal that H1-depleted chromatin contains a larger portion of destabilized DNA than control chromatin. The molecular weight of Euplotes MAC H1 is significantly smaller than most reported H1 proteins. Comparisons are made with extracts of macronuclei from other hypotrichous ciliated protozoa and published reports of other lower eukaryotes.

Subfractionation of soluble macronuclear chromatin and enrichment of specific genes as chromatin from Euplotes eurystomus.

Euplotes eurystomus is a hypotrichous ciliated protozoan possessing within one cytoplasm a transcriptionally-inactive micronucleus with chromosomal-size DNA and a transcriptionally active macronucleus with "gene-size" DNA fragments. The chromatin in the macronucleus can be isolated in a soluble form without prior treatment by nucleases. In this study, macronuclear, soluble chromatin was subfractionated using isokinetic sucrose density gradient ultracentrifugation in a buffer consisting of 50 mM NaCl, 1 mM Na2 EDTA, 1 mM TEA HCl, pH 7.0, 0.1 mM TLCK and 0.1 mM PMSF. Fractions were collected and analyzed by DNA and protein gel electrophoresis, dot blot hybridization with specific gene probes, and modified Miller chromatin spreads. Analysis of the chromatin spreads showed that the sizes of the chromatin fragments in the various fractions correlate with the DNA size of the fragments. When dot blots of the fractions were hybridized with 5S rRNA, tubulin and rRNA gene probes we obtained about a 6 to 14-fold enrichment of hybridizable sequences in individual fractions. There appear to be differences in the non-histones present on each fraction as well as some overall similarities in histone and non-histone proteins.

Preparation and characterization of soluble macronuclear chromatin from the hypotrich Euplotes eurystomus.

Euplotes eurystomus is a hypotrichous ciliate containing a transcriptionally active macronucleus (MAC) and a transcriptionally inactive micronucleus. Soluble MAC chromatin contains a normal complement of inner histones, an H1-like protein which is very sensitive to proteolysis, and a considerable proportion of non-histone proteins. A combination of N-Tosyl lysine chloromethyl ketone (TLCK) and PMSF was found to be most effective in preventing proteolysis. Microccocal nuclease digestion yielded an average nucleosome repeat length of 187 +/- 25 bp for soluble chromatin; and 190 +/- 9 bp for isolated macronuclei. Thermal denaturation profiles of MAC chromatin in 0.25 mM EDTA display two main transitions at about 76 and 83 degrees C, resembling the melting of soluble chicken erythrocyte chromatin. Circular dichroic spectra of MAC chromatin were compared to soluble chicken erythrocyte chromatin under the same ionic strength conditions and were found to be very similar. 2D chromatin/DNA agarose gel electrophoresis resulted in a diagonal line of DNA staining, which establishes a strict correlation between DNA and chromatin electrophoretic mobility.

Physical structure of gene-sized chromatin from the protozoan Oxytricha.

Oxytricha nova is a hypotrichous ciliate containing a transcriptionally active macronucleus and a transcriptionally inactive micronucleus. Two-dimensional gel electrophoresis shows that macronuclei contain a normal complement of inner histones. However, despite extensive efforts, no classical H1-like protein has been detected. Micrococcal nuclease digestion indicates a nucleosome repeat length of approximately 220 bp for macronuclear chromatin. Thermal denaturation profiles of macronuclear chromatin in 0.2 mM EDTA display four transitions at about 46, 57, 64, and 79 degrees C. The lowest of these shifts to higher temperature as the ionic strength is raised to 3-5 mM Na phosphate. These results are consistent with the absence of H1 and a nucleosome repeat of 220-230 bp. Circular dichroism (CD) results agree with these findings. By contrast, micronuclear chromatin displays a much smaller premelt and a more suppressed DNA CD signal at 285 nm, consistent with a micronuclear chromatin repeat of 165-185 bp as determined by micrococcal nuclease digestion.