HIV-associated neurodevelopmental delay: prevalence, predictors and persistence in relation to antiretroviral therapy initiation and viral suppression.

CONTEXT: HIV infection in infancy may influence the developing brain, leading to adverse neurodevelopmental consequences. OBJECTIVE: We aim to describe neurodevelopmental characteristics of a cohort of HIV-infected infants and young children prior to antiretroviral therapy (ART) initiation and after achieving viral suppression. METHODS: As part of the Neverest 2 trial, 195 HIV-infected children under 2 years of age were assessed using the Ages and Stages Questionnaire (ASQ) prior to ART initiation and at subsequent age-appropriate time points after ART had been started. The ASQ is a simple screening questionnaire used to identify children at risk of neurodevelopmental delays. Questionnaires completed by the parent/caregiver assess neurodevelopmental functioning in five domains: communication, gross motor, fine motor, problem solving and personal-social. RESULTS: Median age pre-ART was 8.8 months (range 2.2-24.9) and 53.9% were male. Mean time to viral suppression was 9.4 months (range 5.9-14.5). Compared with pre-ART better outcomes were reported at time of viral suppression with a lower proportion of children failing the gross motor (31.5% vs. 13%, p = 0.0002), fine motor (21.3% vs. 10.2%, p = 0.017), problem solving (26.9% vs. 9.3%, p = 0.0003) and personal-social (19.6% vs. 7.4%, p = 0.019) domains. However, there was no change in the communication domain (14.8% vs. 12.0%, p = 0.6072). CONCLUSION: Although achieving viral suppression on ART resulted in significant improvements in markers of neurodevelopmental function of young HIV-infected children, potential neurodevelopmental delays still persisted in a large proportion. Further interventions are needed to limit potential disabilities and maximize developmental outcomes.

HLA-G 14 bp deletion/insertion polymorphism and mother-to-child transmission of HIV.

The human leukocyte antigen HLA-G, highly expressed at the maternal-fetal interface, has a pivotal role in mediating immune tolerance. In this study we investigated the influence of HLA-G 14 bp insertion polymorphism in human immunodeficiency virus (HIV)-1 mother-to-child HIV-1 transmission. The 14 bp insertion polymorphism was analyzed among 99 HIV-1 positive mothers and 329 infants born to HIV-positive mothers in Zambia, among whom vertical transmission status and timing had been determined. HLA-G 14 bp insertion polymorphism was detected using a custom TaqMan single nucleotide polymorphisms (SNPs) genotyping assay. Logistic regression was conducted to examine the associations between HLA-G alleles and the risk of HIV transmission. The 14 bp insertion allele was more frequent in HIV exposed-uninfected (EU) infants than in infected infants, and was associated with reduced risk of both in utero (IU) and intrapartum (IP) HIV transmission, after adjusting for maternal cluster of differentiation 4 (CD4) cell count and plasma viral load. Maternal HLA-G 14 bp insertion genotype and HLA-G concordance between mother and child were not associated with the risk of perinatal HIV transmission. The presence of the 14 bp insertion associates with protection toward IU and IP HIV infection in children from Zambia, suggesting that HLA-G could be involved in the vertical transmission of HIV.

Antidepressant Exposure and DNA Methylation: Insights from a Methylome-Wide Association Study.

Importance: Understanding antidepressant mechanisms could help design more effective and tolerated treatments. Objective: Identify DNA methylation (DNAm) changes associated with antidepressant exposure. Design: Case-control methylome-wide association studies (MWAS) of antidepressant exposure were performed from blood samples collected between 2006-2011 in Generation Scotland (GS). The summary statistics were tested for enrichment in specific tissues, gene ontologies and an independent MWAS in the Netherlands Study of Depression and Anxiety (NESDA). A methylation profile score (MPS) was derived and tested for its association with antidepressant exposure in eight independent cohorts, alongside prospective data from GS. Setting: Cohorts; GS, NESDA, FTC, SHIP-Trend, FOR2107, LBC1936, MARS-UniDep, ALSPAC, E-Risk, and NTR. Participants: Participants with DNAm data and self-report/prescription derived antidepressant exposure. Main Outcomes and Measures: Whole-blood DNAm levels were assayed by the EPIC/450K Illumina array (9 studies, N exposed = 661, N unexposed = 9,575) alongside MBD-Seq in NESDA (N exposed = 398, N unexposed = 414). Antidepressant exposure was measured by self- report and/or antidepressant prescriptions. Results: The self-report MWAS (N = 16,536, N exposed = 1,508, mean age = 48, 59% female) and the prescription-derived MWAS (N = 7,951, N exposed = 861, mean age = 47, 59% female), found hypermethylation at seven and four DNAm sites (p < 9.42x10 -8 ), respectively. The top locus was cg26277237 ( KANK1, p self-report = 9.3x10 -13 , p prescription = 6.1x10 -3 ). The self-report MWAS found a differentially methylated region, mapping to DGUOK-AS1 ( p adj = 5.0x10 -3 ) alongside significant enrichment for genes expressed in the amygdala, the "synaptic vesicle membrane" gene ontology and the top 1% of CpGs from the NESDA MWAS (OR = 1.39, p < 0.042). The MPS was associated with antidepressant exposure in meta-analysed data from external cohorts (N studies = 9, N = 10,236, N exposed = 661, f3 = 0.196, p < 1x10 -4 ). Conclusions and Relevance: Antidepressant exposure is associated with changes in DNAm across different cohorts. Further investigation into these changes could inform on new targets for antidepressant treatments. 3 Key Points: Question: Is antidepressant exposure associated with differential whole blood DNA methylation?Findings: In this methylome-wide association study of 16,536 adults across Scotland, antidepressant exposure was significantly associated with hypermethylation at CpGs mapping to KANK1 and DGUOK-AS1. A methylation profile score trained on this sample was significantly associated with antidepressant exposure (pooled f3 [95%CI]=0.196 [0.105, 0.288], p < 1x10 -4 ) in a meta-analysis of external datasets. Meaning: Antidepressant exposure is associated with hypermethylation at KANK1 and DGUOK-AS1 , which have roles in mitochondrial metabolism and neurite outgrowth. If replicated in future studies, targeting these genes could inform the design of more effective and better tolerated treatments for depression.

Influence of intragenic CCL3 haplotypes and CCL3L copy number in HIV-1 infection in a sub-Saharan African population.

Two CCL3 haplotypes (HapA1 and Hap-A3) and two polymorphic positions shared by the haplotypes (Hap-2SNP (single nucleotide polymorphism)) were investigated together with CCL3L copy number (CN), for their role in HIV-1 disease. Hap-A1 was associated with protection from in utero HIV-1 infection: exposed uninfected (EU) infants had higher representation of wild type (WT)/Hap-A1 than infected infants (excluding intrapartum (IP)-infected infants), which maintained significance post maternal Nevirapine (mNVP) and viral load (MVL) correction (P=0.04; odds ratio (OR)=0.33). Mother-infant pair analyses showed the protective effect of Hap-A1 is dependent on its presence in the infant. Hap-A3 was associated with increased IP transmission: WT/Hap-A3 was increased in IP-transmitting vs non-transmitting (NT) mothers, and remained significant post mNVP and MVL correction (P=0.02; OR=3.50). This deleterious effect of Hap-A3 seemed dependent on its presence in the mother. Hap-2SNP was associated with lower CD4 count in the NT mothers (P=0.03). CCL3 Hap-A1 was associated with high CCL3L CN in total (P=0.001) and EU infants (P=0.006); the effect was not additive, however, having either Hap-A1 or high CCL3L CN was more significantly (P=0.0008) associated with protection from in utero infection than Hap-A1 (P=0.028) or high CCL3L CN (P=0.002) alone. Linkage disequilibrium between Hap-A1 and high CCL3L CN appears unlikely given that a Nigerian population showed an opposite relationship.

Transmission electron microscopy specimen preparation method for multiphase porous functional ceramics.

An optimum method is proposed to prepare thin foil transmission electron microscopy (TEM) lamellae of multiphase porous functional ceramics: prefilling the pore space of these materials with an epoxy resin prior to focused ion beam milling. Several advantages of epoxy impregnation are demonstrated by successful preparation of TEM specimens that maintain the structural integrity of the entire lamella. Feasibility of the TEM alignment procedure is demonstrated, and ideal TEM analyses are illustrated on solid oxide fuel cell and solid oxide electrolysis cell materials. Some potential drawbacks of the TEM specimen preparation method are listed for other samples.

[Useful knowledge regarding tattoos]. / Nützliches Wissen zu Tätowierungen.

The number of people with tattoos has continued to increase in recent years. In the USA about 23% and in Europe 9-12% of the population have tattoos. In the German media (2019) and by the infoportal Statista (2017), it is assumed that 21-25% of citizens have tattoos and that the trend is increasing (Statista 2018: 36%). Men and women wear tattoos equally. The age group 20-29 years dominates with almost 50% having tattoos. The following article describes the new regulations especially the REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals) regulation, legal basis, and governmental controls on the subject of "tattoos". The composition of tattooing agents and testing options relevant for the user before and for the performance of tattooing are presented. Dermatologically associated diseases and testing procedures are listed. Since 70% of the population denies knowledge of this information even when they have tattoos themselves, this update is written as an overview for treating physicians and users.

Physiology of the widespread pulsating soft coral Xenia umbellata is affected by food sources, but not by water flow.

Coral energy and nutrient acquisition strategies are complex and sensitive to environmental conditions such as water flow. While high water flow can enhance feeding in hard corals, knowledge about the effects of water flow on the feeding of soft corals, particularly those pulsating, is still limited. In this study, we thus investigated the effects of feeding and water flow on the physiology of the pulsating soft coral Xenia umbellata. We crossed three feeding treatments: (i) no feeding, (ii) particulate organic matter (POM) in the form of phytoplankton and (iii) dissolved organic carbon (DOC) in the form of glucose, with four water volume exchange rates (200, 350, 500 and 650 L h-1) over 15 days. Various ecophysiological parameters were assessed including pulsation rate, growth rate, isotopic and elemental ratios of carbon (C) and nitrogen (N) as well as photo-physiological parameters of the Symbiodiniaceae (cell density, chlorophyll-a and mitotic index). Water flow had no significant effect but feeding had a substantial impact on the physiology of the X. umbellata holobiont. In the absence of food, corals exhibited significantly lower pulsation rates, lower Symbiodiniaceae cell density and lower mitotic indices compared to the fed treatments, yet significantly higher chlorophyll-a per cell and total N content. Differences were also observed between the two feeding treatments, with significantly higher pulsation rates and lower chlorophyll-a per cell in the DOC treatment, but higher C and N content in the POM treatment. Our findings suggest that the X. umbellata holobiont can be viable under different trophic strategies, though favouring mixotrophy. Additionally, the physiology of the X. umbellata may be regulated through its own pulsating behaviour without any positive or negative effects from different water flow. Therefore, this study contributes to our understanding of soft coral ecology, particularly regarding the competitive success and widespread distribution of X. umbellata.

Twelve Weeks of Additional Fish Intake Improves the Cognition of Cognitively Intact, Resource-Limited Elderly People: A Randomized Control Trial.

INTRODUCTION: Dietary omega 3 polyunsaturated fatty acids (PUFA) may reduce the risk of dementia. Many studies have investigated PUFA supplementation in high-income countries, yet food-based randomized control trials using omega 3 PUFA rich fish in lower to middle income countries, are lacking. OBJECTIVE: To determine the effect on cognition of adding either fish or non-fish foods for twelve weeks to an enhanced diet of cognitively intact, independently living, resource-limited elderly people. DESIGN: Randomized control trial (National Health Trial register: DOH-27-061-6026). SETTING: Retirement center in urban South Africa. PARTICIPANTS: Fifty-seven (74% female, mean age: 72±7 years) elderly participants with cognitive function exceeding 22 on the Mini Mental State Examination were randomized into an intervention (n=31) and control (n=26) group. INTERVENTION: The usual diets of both groups were enhanced with context-appropriate foods to mimic elements of the Mediterranean-DASH Intervention for Neurodegenerative Delay (MIND) diet. The intervention group additionally received canned pilchards and fish spread every week amounting to an additional (theoretical) intake of 2.2g omega 3 PUFA daily. The control group received canned meatballs and texturized soya every week. MEASUREMENTS: Cognition was measured twice before and once after the intervention phase using the Cognitive Abilities Screening Instrument (CASI). Adherence was assessed by a study-specific food frequency questionnaire and red blood cell (RBC) PUFA biomarkers. Data were analyzed using a non-parametric analysis of covariance (ANCOVA) with, and without, bootstrap imputation. RESULTS: Participants in the intervention group had a significantly higher post intervention (P=0.036) CASI score than the control group, when the model was fitted with imputation and controlled for baseline scores. Participants in the intervention group also had a significantly higher intake of calculated dietary omega 3 PUFA and higher levels of RBC eicosapentaenoic acid and docosapentaenoic acid content than the control group (P < 0.05). CONCLUSION: Twelve weeks of fish intake in the context of a modified MIND diet may improve the cognition of cognitively intact, resource-limited elderly people.

The sourcing and use of high physical resemblance personal protective equipment to train healthcare workers, improve confidence and conserve medical-grade equipment.

Personal protective equipment (PPE) is essential for healthcare worker (HCW) safety. Conservation of PPE for clinical use during the COVID-19 pandemic reduced its availability for training, necessitating an innovative approach to sourcing high physical resemblance PPE (HPR-PPE). We present a case study of crowd-sourcing of HPR-PPE to train HCWs. Survey results indicated that HPR-PPE enabled high-fidelity practise of PPE application and removal, aided procedure recall, improved user confidence and was sufficiently similar to medical-grade PPE. HPR-PPE provided a novel and cost-effective alternative. We also demonstrated that medical-grade PPE can be sourced from non-medical institutions and businesses during a pandemic.

Peyer's patches: immunologic studies.

The immune capabilities of the Peyer's patches have been investigated by the use of an in vitro system. Despite our failure to stimulate Peyer's patch lymphocytes in vivo it appears that Peyer's patches behave immunologically as peripheral lymphoid tissues. Cultures prepared from the dissociated Peyer's patches of normal rabbits respond to sheep erythrocytes. The response is comparable to that obtained with spleen cultures from the same animals and is not dependent on the presence of the epithelial cells which line the lumen. Similar thymic cultures do not respond. Our experiments with cultures prepared from rabbits which have received one or two injections of SRC show that the Peyer's patches contain both IgM and IgG "memory" cells which have migrated from the spleen. The concentration of these cells in the spleen remains several hundredfold higher.

Teaching old precursors new tricks: Fast room temperature synthesis of surfactant-free colloidal platinum nanoparticles.

A fast, simple, instrument-free room temperature synthesis of stable electroactive surfactant-free colloidal Pt nanoparticles in alkaline methanol and methanol-water mixtures is presented. Pair distribution function (PDF) analysis suggests that methoxy substitution of chloride ligands from H2PtCl6 occurs in methanol. X-ray absorption spectroscopy (XAS) studies and UV-vis measurements show that solutions of H2PtCl6 in methanol age and are reduced to Pt(II) species over time. These species are ideal precursors to significantly reduce the induction period typically observed in colloidal Pt nanoparticle syntheses as well as the temperature needed to form nanoparticles. The room temperature synthesis presented here allows designing simple in situ studies of the nanoparticle formation. In situ infra-red spectroscopy gives insight into the formation and stabilization mechanism of surfactant-free nanoparticles by CO surface groups. Finally, the surfactant-free nanoparticles ca. 2-3 nm in diameter obtained are shown to be readily active electrocatalysts e.g. for methanol oxidation. The synthesis approach presented bears several advantages to design new studies and new syntheses of surfactant-free colloidal nanomaterials.

Export of mRNA from microinjected nuclei of Xenopus laevis oocytes.

Export of mRNA from the nucleus to the cytoplasm was studied in mature Xenopus laevis oocytes. In vitro transcribed, capped 32P-labeled mRNA was microinjected into nuclei, and its appearance in the cytoplasm measured by counting radioactivity or by RNA extraction and gel electrophoresis. Both for a 5.0-kb transferrin receptor mRNA and a 2.0-kb 4F2 antigen heavy chain mRNA we found saturable transport with an apparent Km of 3.6 x 10(8) molecules per oocyte nucleus. Under non-saturating conditions the half-time for mRNA export from the nucleus was approximately 2 min at 20 degrees C. At higher concentrations of injected mRNA this half-time was prolonged, and the maximal transport rate was reached at approximately 1.6 x 10(8) molecules/min. mRNA transport showed properties of an energy-dependent mechanism, since it was inhibited at 4 degrees C or by ATP depletion. Co-injection of the cap dinucleotide m7GpppG blocked the export effectively, suggesting a role for the cap in this process. The export was also inhibited by the pre-injection of wheat germ agglutinin. The effect of the lectin was specific and abolished by co-injection of N-acetylglucosamine. Finally, we found significant competitive inhibition in mRNA export by the presence of tRNA. Our results suggest that mRNA transport is a facilitated process which may share common steps with tRNA transport. Preliminary gel retardation experiments show that injected mRNA associates with endogenous nuclear proteins and suggest an exchange of some of the bound components during the transport to the cytoplasm.

Biological autoimmunity screening in hepatitis C patients by anti-HepG2 lysate and anti-heat shock protein 70.1 autoantibodies.

Viruses require viral and cellular chaperones during their life cycle and interactions of these molecules with the immune system are probable during the infection. Thus, an anti-chaperone antibody response has been firstly investigated in hepatitis C patients in this paper. A HepG2-lysate antigen (90, 79, 72, 70, 62, 54 and 48 kDa) was assayed in sera from 59 (19F/40M) chronic hepatitis C patients without cirrhosis before therapy. Forty of them were positive for anti-HepG2 lysate antigen antibodies and this test may evaluate biological autoimmunity. Hsp70.1, Hsp90 and calreticulin levels were significantly higher in this antigen than in a control HepG2 antigen. Secondly, Hsp70.1 was identified as Hsp 70 kDa protein-1 by proteomic analysis and studied as a possible antibody target. Fourteen out of 59 patients were positive for anti-Hsp70.1 antibodies that were inversely correlated with alanine aminotransferase levels, the Metavir activity index and viraemia. Finally, for comparative purposes, 50 sera from systemic lupus erythematosus (SLE) patients have been tested: eight and 41 of them were positive for anti-Hsp70.1 and anti-HepG2 lysate antigen antibodies, respectively. Therefore, anti-Hsp70.1 autoantibodies may be produced and can partially lead to biological autoimmunity in chronic hepatitis C patients.

Identification of new variants within the two functional genes CCL3 and CCL3L encoding the CCL3 (MIP-1alpha) chemokine: implications for HIV-1 infection.

The CC chemokine CCL3 is encoded by two functional genes, namely CCL3 and CCL3L, and has been identified as a key chemokine in HIV-1 susceptibility and disease progression. The complete CCL3 and CCL3L genes and core promoters of 43 African mother-infant pairs (86 samples) and 28 Caucasian adults in South Africa were sequenced and extensively analysed for genetic variations. Africans were found to be more polymorphic in both genes with 25 single nucleotide polymorphisms (SNPs) in the CCL3 gene and 14 gene copy number single nucleotide polymorphisms (gcnSNPs) in the CCL3L gene, compared to nine CCL3 SNPs and eight CCL3L gcnSNPs in Caucasians. A total of 14 polymorphisms across the two genes were newly identified in this study, most (12/14) of which were exclusive to the African population. In addition, two indels were identified and characterized in the CCL3 and CCL3L genes of a small number of individuals. Of the numerous unique intragenic haplotypes found in the two genes, none were shared by the two population groups. A newly identified five-SNP CCL3 haplotype (Hap-C1) found in a high frequency in Caucasians, however, seems to be evolutionarily related to the most prevalent newly identified African seven-SNP CCL3 haplotype (Hap-A1). Hap-A1 also includes an SNP in the core promoter region and previous CCL3 haplotypes that have been reported to be associated with HIV-1 infection appear to be smaller haplotypes within Hap-A1. We thus propose Hap-A1 as a likely candidate for influencing levels of CCL3 production and in turn outcomes of HIV-1 infection.

Diversity of risk of mother-to-child HIV-1 transmission according to feeding practices, CD4 cell count, and haemoglobin concentration in a South African cohort.

OBJECTIVE: To estimate the probabilities of intrapartum and postpartum HIV-1 mother-to-child-transmission according to various feeding practices (formula feeding, exclusive breastfeeding, or mixed-feeding) and to other mother and infant covariates. METHODS: We used the promotion time model extended to multiple exposures to study the probability of infection attributable to each transmission occasion. Blood samples from 551 infants from Durban (South Africa) born to HIV-1 positive untreated mothers between 1995 and 1998 were sequentially tested until 15 months. RESULTS: The probability of infection attributable to in utero and intrapartum transmission was 21.88% (18.71-25.20) and was not significantly associated with the feeding practice. The probability of infection attributable to postnatal transmission through exclusive breastfeeding was negligible -0.7% (0-2.5) for 6 months of exclusive breastfeeding-- in comparison with that observed with mixed-feeding -6.15% (3.16-9.57) for 6 months of mixed-feeding. Maternal CD4 cell count and gestational age were significant predictors of intrapartum transmission probability while maternal CD4 cell count and maternal haemoglobin concentration were significant predictors of postpartum transmission probability. CONCLUSION: Decisions about appropriate infant feeding practices should take into account the difference in postpartum transmission risk between exclusive and mixed-feeding. Mixed-feeding should be all the more avoided that mothers have poor immunological statuses and low haemoglobin concentrations.

Identification of potential small-molecule protein-protein inhibitors of cancer metastasis by 3D epitope-based computational screening.

In cancer cells exposed to extracellular pressure or shear stress, AKT1-FAK interaction drives focal adhesion kinase (FAK) phosphorylation, leading to force-activated cancer cell adhesion and metastasis. Blocking the AKT1-FAK interaction is therefore an attractive target for cancer therapy, avoiding the side effects of global FAK inhibition. Starting with our previous identification of a short FAK peptide that binds AKT1, we identified a series of small-molecule inhibitor candidates using a novel approach for inhibiting protein-protein interactions. Using a 3D structural fragment of the FAK peptide as the query, millions of drug-like, commercially available molecules were screened to identify a subset mimicking the volume and chemistry of the FAK fragment to test for their ability to block pressure-sensitive FAK phosphorylation by AKT1. Two compounds reduced the stimulation of FAK phosphorylation in response to extracellular pressure in human SW620 colon cancer cells without affecting basal FAK phosphorylation. Thus, using a 3D protein interaction epitope as a novel query for ligand-based virtual screening can successfully identify small-molecules that show promise in modulating cancer cell adhesion and metastasis.

South African women's perspectives on self-sampling for cervical cancer screening: A mixed-methods study.

BACKGROUND: Self-sampling as a method of screening for cervical cancer and its precursors is an attractive option for low-resource settings. However, to allow successful integration of self-sampling into national screening programmes, it is necessary to understand women's perceptions and beliefs surrounding this method of sampling the cervix. OBJECTIVES: To explore women's attitudes to self-collection of samples for cervical screening in a low-resource setting in South Africa (SA). METHODS: Mixed methods were used to meet the study objectives. We recruited women aged 30 - 65 years into a study in Cape Town, SA, to participate in a cross-sectional survey. All women collected a vaginal self-sample, and underwent visual inspection with acetic acid, colposcopy, and collection of cervical samples and appropriate histology specimens by a doctor. Women had a quantitative questionnaire-based exit interview. A subset of these women participated in focus group discussions (FGDs). RESULTS: A total of 822 women answered the exit survey questionnaire and 41 women participated in the FGDs. Most women from the survey had a positive perception of self-sampling, with 93.6% of the women reporting not feeling embarrassed and 89.4% reporting experiencing no discomfort at all when taking a self-sample. This was corroborated by the FGD participants, who found self-sampling easier, more comfortable and less embarrassing than clinician sampling. However, many women (64.7%) felt more confident when the sample was taken by a clinician, despite having a positive attitude towards self-sampling. In most cases this was because they thought that the clinician would take a better sample, as explained by the FGD participants. Although 93.9% of the women were willing to collect a self-sample, the women in the FGDs expressed a preference for doing so at the health facility rather than at home. There were many reasons for this, including the cost of returning to the clinic with the sample. CONCLUSIONS: Attitudes regarding self-sample collection were positive in this study population. Participants were willing to perform self-sampling, but expressed concerns regarding the quality of the specimen and the financial implications of returning to the clinic with it. Pilot implementation studies will be useful before this method of sampling is adopted and integrated into screening programmes.

Possible mechanism of phorbol diester-induced maturation of human promyelocytic leukemia cells.

The phorbol diesters are the most potent inducers of differentiation of the promyelocytic leukemia cell line, HL-60. Soluble phorbol diester receptors from HL-60 cells were obtained from the cytosolic fraction and from the particulate fraction by either divalent ion chelation or detergent extraction. The partially purified soluble phorbol diester receptors required exogenous Ca2+ and phospholipid for maximal binding and displayed a dissociation constant (KD) of 8.1 nM for [3H]phorbol 12,13-dibutyrate. Phorbol diester analogues inhibited [3H]phorbol 12,13-dibutyrate binding in a stereospecific manner consistent with their biologic potency. The soluble phorbol diester receptors prepared by all three methods copurified in a constant ratio with the Ca2+/phospholipid-dependent protein kinase C through ammonium sulfate precipitation, DEAE ion exchange, and gel filtration chromatography. Partially purified protein kinase C was directly activated by the phorbol diesters even in the absence of exogenous Ca2+. The ability of a series of phorbol analogues to activate the kinase correlated with their known activity as inducers of cell differentiation. In addition, phorbol diester stimulation altered the phosphate acceptor substrate profile of protein kinase C, at least in part, by alteration of the Michaelis constant (Km). These data suggest that protein kinase C is the phorbol diester receptor and that phorbol diester-induced macrophage maturation of HL-60 cells may be mediated by activation of intracellular protein kinase C.

Luminal heterodimeric amino acid transporter defective in cystinuria.

Mutations of the glycoprotein rBAT cause cystinuria type I, an autosomal recessive failure of dibasic amino acid transport (b(0,+) type) across luminal membranes of intestine and kidney cells. Here we identify the permease-like protein b(0,+)AT as the catalytic subunit that associates by a disulfide bond with rBAT to form a hetero-oligomeric b(0,+) amino acid transporter complex. We demonstrate its b(0,+)-type amino acid transport kinetics using a heterodimeric fusion construct and show its luminal brush border localization in kidney proximal tubule. These biochemical, transport, and localization characteristics as well as the chromosomal localization on 19q support the notion that the b(0,+)AT protein is the product of the gene defective in non-type I cystinuria.