Severe CD4+ T-cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy

Gut-associated lymphoid tissue (GALT) harbors the majority of T lymphocytes in the body and is an important target for human immunodeficiency virus type 1 (HIV-1). We analyzed longitudinal jejunal biopsy samples from HIV-1-infected patients, during both primary and chronic stages of HIV-1 infection, prior to and following the initiation of highly active antiretroviral therapy (HAART) to determine the onset of CD4(+) T-cell depletion and the effect of HAART on the restoration of CD4(+) T cells in GALT. Severe depletion of intestinal CD4(+) T cells occurred during primary HIV-1 infection. Our results showed that the restoration of intestinal CD4(+) T cells following HAART in chronically HIV-1-infected patients was substantially delayed and incomplete. In contrast, initiation of HAART during early stages of infection resulted in near-complete restoration of intestinal CD4(+) T cells, despite the delay in comparison to peripheral blood CD4(+) T-cell recovery. DNA microarray analysis of gene expression profiles and flow-cytometric analysis of lymphocyte homing and cell proliferation markers demonstrated that cell trafficking to GALT and not local proliferation contributed to CD4(+) T-cell restoration. Evaluation of jejunal biopsy samples from long-term HIV-1-infected nonprogressors showed maintenance of normal CD4(+) T-cell levels in both GALT and peripheral blood. Our results demonstrate that near-complete restoration of mucosal immune system can be achieved by initiating HAART early in HIV-1 infection. Monitoring of the restoration and/or maintenance of CD4(+) T cells in GALT provides a more accurate assessment of the efficacy of antiviral host immune responses as well as HAART.     

Phosphorylation enhances mitochondrial targeting of GSTA4-4 through increased affinity for binding to cytoplasmic Hsp70

Recently we showed that three different isoforms of cytosolic glutathione S-transferases (GST), including GSTA4-4, are also localized in the mitochondrial compartment. In this study, we have investigated the mechanism of mouse GSTA4-4 targeting to mitochondria, using a combination of in vitro mitochondrial import assay and in vivo targeting in COS cells transfected with cDNA. Our results show that the mitochondrial GSTA4-4 is more heavily phosphorylated compared with its cytosolic counterpart. Protein kinase activators (cAMP, forskolin, or phorbol-12-myristate-13-acetate) markedly increased GSTA4-4 targeting to mitochondria, whereas kinase inhibitors caused its retention in the cytosol. Immunoinhibition and immunodepletion studies showed that the Hsp70 chaperone is required for the efficient translation of GSTA4-4 as well as its translocation to mitochondria. Co-immunoprecipitation studies showed that kinase inhibitors attenuate the affinity of GSTA4-4 for cytoplasmic Hsp70 suggesting the importance of phosphorylation for binding to the chaperone. Mutational analysis show that the putative mitochondrial targeting signal resides within the C-terminal 20 amino acid residues of the protein and that the targeting signal requires activation by phosphorylation at the C-terminal-most protein kinase A (PKA) site at Ser-189 or protein kinase C (PKC) site at Thr-193. We demonstrate for the first time that PKA and PKC modulate the cytoplasmic and mitochondrial pools of GSTA4-4.     

Surgical considerations in the management of malignant melanoma of the ear

Malignant melanomas of the external ear are rare and are difficult lesions to treat because of the cosmetic importance and the reconstructive difficulty of their location. The literature suggests that these lesions have a worse prognosis than melanomas occurring elsewhere and that radical resection is the "correct" treatment. To clarify this issue, we examined 21 consecutive patients (19 male, 2 female) with malignant melanoma of the ear seen at the Yale-New Haven Hospital over the last 10 years. Nineteen patients had a diagnosis of primary malignant melanoma of the ear, one had a local recurrence, and one had an in-transit melanoma from an unknown primary site. The mean thickness of the lesions was 2.7 mm. Two patients had palpable nodes, which in both cases turned out to be histologically positive for tumor. All patients underwent local excision and reconstruction using chondrocutaneous or fasciocutaneous flaps or skin grafts. There was one local recurrence (0.5 mm original thickness); there were two patients with regional recurrences, both of whom died within a year with disseminated disease. Forty-three percent have been followed for 5 or more years and all are alive and free of disease. This suggests that malignant melanoma of the ear may be safely treated by conservative excision and reconstruction.     

Cardiac myocyte adenosine A2a receptor activation fails to alter cAMP or contractility: role of receptor localization

Adenosine A2a receptors are found in coronary vascular tissue although, their presence in myocardium is subject to investigation. Although there have been numerous studies on adenosine A2a receptor agonist effects on contractility and cAMP levels in ventricular myocytes, these have yielded conflicting results. Negative pharmacological studies have even led to the conclusion that A2a receptors are not present in cardiac myocytes. The purpose of this study was to determine whether A2a receptors are expressed in rat ventricular myocytes and what physiological effects are mediated via activation of these receptors. Western blot analysis with a polyclonal antibody raised against a peptide sequence specific to the carboxy terminus of the A2a receptor revealed the presence of a band at approximately 45 kDa. However, the immunoreactivity was located in the nonmembrane fraction of the cell lysate. The membrane fraction only exhibited an immunoreactive band > or = 50 kDa. Treatment of isolated myocytes with the adenosine A2a agonist 2-[4-[(2-carboxyethyl)-phenyl]ethylamino]-5'-N-ethylcarboxamidoadenosine (CGS-21680) exerted no effects on cAMP levels or myocyte twitch amplitude. These results indicate that although rat ventricular myocytes appear to express adenosine A2a receptors, stimulation with an A2a agonist exerts no functional effects, possibly because of the subcellular localization of the A2a receptor.     

T7 DNA helicase: a molecular motor that processively and unidirectionally translocates along single-stranded DNA

DNA helicases are molecular motors that use the energy from NTP hydrolysis to drive the process of duplex DNA strand separation. Here, we measure the translocation and energy coupling efficiency of a replicative DNA helicase from bacteriophage T7 that is a member of a class of helicases that assembles into ring-shaped hexamers. Presteady state kinetics of DNA-stimulated dTTP hydrolysis activity of T7 helicase were measured using a real time assay as a function of ssDNA length, which provided evidence for unidirectional translocation of T7 helicase along ssDNA. Global fitting of the kinetic data provided an average translocation rate of 132 bases per second per hexamer at 18 degrees C. While translocating along ssDNA, T7 helicase hydrolyzes dTTP at a rate of 49 dTTP per second per hexamer, which indicates that the energy from hydrolysis of one dTTP drives unidirectional movement of T7 helicase along two to three bases of ssDNA. One of the features that distinguishes this ring helicase is its processivity, which was determined to be 0.99996, which indicated that T7 helicase travels on an average about 75kb of ssDNA before dissociating. We propose that the ability of T7 helicase to translocate unidirectionally along ssDNA in an efficient manner plays a crucial role in DNA unwinding.     

SN2 DNA-alkylating agent-induced phosphorylation of p53 and activation of p21 gene expression

p53 is an important player in the cellular response to genotoxic stress whose functions are regulated by phosphorylation of a number of serine and threonine residues. Phosphorylation of p53 influences its DNA-binding and gene regulation activities. This study examines p53 phosphorylation in HCT-116 (MMR-deficient) and HCT-116+ch3 (MMR-proficient) human colon cancer cells treated with a S(N)2 DNA-alkylating agent, methylmethane sulfonate (MMS). MMS induces phosphorylation of p53 on Ser15 and Ser392 in a dose- and time-dependent manner. MMS-induced p53 phosphorylation is independent of DNA mismatch repair (MMR) activity. Nuclear extracts from MMS-treated HCT-116 cells had higher p21WAF1/Cip1 (p21) promoter DNA-binding activity in vitro opposed to untreated cells. After MMS treatment, the activation of the cloned p21 promoter in a transient transfection assay and endogenous p21 mRNA levels in HCT-116(p53+/+) versus HCT-116(p53-/-) cells increased, which correlates with an increased levels of phospho-p53(Ser15) and phospho-p53(Ser392). These results suggest that SN2 DNA-alkylating agent-induced phosphorylation of p53 on Ser15 and Ser392 increases its DNA-binding properties to cause an increased expression of p21 that may play a role in cell cycle arrest and/or apoptosis of HCT-116 cells.     

Folding kinetics of the lipoic acid-bearing domain of human mitochondrial branched chain alpha-ketoacid dehydrogenase complex

Intracellular calcium is a second messenger involved in several processes in yeast, such as mating, nutrient sensing, stress response and cell cycle events. It was reported that glucose addition stimulates a rapid increase in free calcium level in yeast. To investigate the calcium level variations induced by different stimuli we used a reporter system based on the photoprotein aequorin. Glucose addition (50 mM) to nutrient-starved cells induced an increase in free intracellular calcium concentration, mainly due to an influx from external medium. The increase of calcium reached its maximum 100–120 s after the stimulus. A concentration of about 20 mM glucose was required for a 50% increase in intracellular calcium. This response was completely abolished in strain plc1Δ and in the isogenic wild-type strain treated with 3-nitrocoumarin, a phosphatidylinositol-specific phospholipase C inhibitor, suggesting that Plc1p is essential for glucose-induced calcium increase. This suggests that Plc1p should have a significant role in transducing glucose signal. The calcium influx induced by addition of high glucose on cells previously stimulated with low glucose levels was inhibited in strains with a deletion in the GPR1 or GPA2 genes, which suggests that glucose would be detected through the Gpr1p/Gpa2p receptor/G protein-coupled (GPCR) complex. Moreover, the signal was completely abolished in a strain unable to phosphorylate glucose, which is consistent with the reported requirement of glucose phosphorylation for GPCR complex activation.     

Yeast Rev1 protein is a G template-specific DNA polymerase

Rev1 protein of Saccharomyces cerevisiae functions with DNA polymerase zeta in mutagenic trans-lesion synthesis. Because of the reported preferential incorporation of a C residue opposite an abasic site, Rev1 has been referred to as a deoxycytidyltransferase. Here, we use steady-state kinetics to examine nucleotide incorporation by Rev1 opposite undamaged and damaged template residues. We show that Rev1 specifically inserts a C residue opposite template G, and it is approximately 25-, 40-, and 400-fold less efficient at inserting a C residue opposite an abasic site, an O(6)-methylguanine, and an 8-oxoguanine lesion, respectively. Rev1 misincorporates G, A, and T residues opposite template G with a frequency of approximately 10(-3) to 10(-4). Consistent with this finding, Rev1 replicates DNA containing a string of Gs in a template-specific manner, but it has a low processivity incorporating 1.6 nucleotides per DNA binding event on the average. From these observations, we infer that Rev1 is a G template-specific DNA polymerase.     

Postnatal growth,age estimation and development of foraging behaviour in the fulvous fruit bat<Emphasis Type="Italic">Rousettus leschenaulti</Emphasis>

This study documents the postnatal growth, age estimation and development of the foraging behaviour of the fulvous fruit batRousettus leschenaulti under captive conditions. At birth, the young were naked and pink with closed eyes and folded pinnae. By day four of age, their eyes had opened and the pups began to move. The mean length of forearm in 5-day-old pups was 24.9 mm and body mass was 10.8 g, equivalent to 32.3% and 14.2% of the values from postpartum females. The length of forearm and body mass increased linearly until 45 and 50 days, respectively, and thereafter maintained an apparent stability. The epiphyseal gap of the fourth metacarpal-phalangeal joint increased until 15 days, then decreased linearly until 75 days and thereafter closed. Age was estimated quantitatively, based on linear changes observed in the length of the forearm and epiphyseal gap. Pups began to roost separately, but adjacent to their mothers when 30 days old and flew clumsily when they were about 40 days old. After attaining clumsy flight, the young bats made independent foraging attempts feebly by biting and licking small fruit pieces. Young bats were engaged in suckling as well as ingesting fruits when they were about 50 days old. Between 55 and 65 days, they flew well and fed on fruits. At the age of 75 days, the young bats were completely weaned and at two months, their foraging behaviour was similar to that of their mothers. There was no significant difference in the growth pattern of the young maintained in captivity compared with those under natural conditions.     

Characterization of rhodamine conjugated Agiotensin II peptide: synthesis,analysis and receptor binding and internalization

The results in this study show that the rhodamine fluorophore can be specifically conjugated to Angiotensin II at Lys3 residue (substituted for a Val) without altering the biological activity of the parent compound. The conjugated peptide was characterized using HPLC, mass spectrometry, and N-terminal sequencing. The rhodamine-Angiotensin II binds effectively to AT1 receptor and gets internalized in clathrin coated vesicles by endocytosis. These results clearly suggest the usefulness of fluorophore-conjugated peptides in studies such as, ligand-receptor binding, and ligand-receptor complex internalization, for drug delivery using cell receptors and as an alternative to peptide hormone radioimmunoassays.