Intensive cataract training: a novel approach.

PURPOSE: To evaluate the safety of an intensive cataract surgery training programme. METHODS: An intensive cataract surgery training programme was implemented in August 2010 for year 3 ophthalmology trainees in the East Midlands Deanery North Rotation (United Kingdom). Trainees participated in extra-ocular surgery and 50 h of virtual reality cataract surgery simulator training over a 2-year period. Their third year comprised 6 months of intensive phacoemulsification training in a tertiary centre followed by a 6-month period of consolidation in a district general hospital. The complication rates and case numbers were evaluated after the first 2 years of implementation. RESULTS: At 2 years, three trainees had completed a full year of intensive training. In the first 6 months of training, Trainee 1 completed 156 cases, Trainee 2 completed 194 cases, and Trainee 3 completed 151 full cases as primary surgeons with an average rate of posterior capsule rupture (PCR) of 1%. At 12 months, Trainee 1 completed 291, Trainee 2 completed 318, and Trainee 3 completed 294 cases, with an average PCR rate of 0.66%. The trainees required 84 lists on average to complete 150 full cataract procedures. CONCLUSION: The combination of simulation and the new intensive training programme is safer than the traditional programme for cataract surgery training.

The tammar wallaby and fur seal: models to examine local control of lactation.

Mammary development and function are regulated by systemic endocrine factors and by autocrine mechanisms intrinsic to the mammary gland, both of which act concurrently. The composition of milk includes nutritional and developmental factors that are crucial to the development of the suckled young, but it is becoming increasingly apparent that milk also has a role in regulating mammary function. This review examines the option of exploiting the comparative biology of species with extreme adaptation to lactation to examine regulatory mechanisms that are present but not readily apparent in other laboratory and livestock species. The tammar wallaby has adopted a reproductive strategy that includes a short gestation (26 d), birth of an immature young, and a relatively long lactation (300 d). The composition of milk changes progressively during the lactation cycle, and this is controlled by the mother and not the sucking pattern of the young. Furthermore, the tammar can practice concurrent asynchronous lactation; the mother provides a concentrated milk high in protein and fat for an older animal that is out of the pouch and a dilute milk low in fat and protein but high in carbohydrates from an adjacent mammary gland for a newborn pouch young. This phenomenon suggests that the mammary gland is controlled locally. The second study species, the Cape fur seal, has a lactation characterized by a repeated cycle of long at-sea foraging trips (up to 28 d) alternating with short suckling periods of 2 to 3 d ashore. Lactation almost ceases while the seal is off shore, but the mammary gland does not progress to apoptosis and involution, most likely because of local control of the mammary gland. Our studies have exploited the comparative biology of these models to investigate how mammary function is regulated by endocrine factors, and particularly by milk. This review reports 3 major findings using these model animals. First, the mammary epithelial cell has an extraordinary intrinsic capacity for survival in our culture model, and the path to either function or death by apoptosis is actively driven. The second outcome is that the route to apoptosis is most likely regulated by specific milk factors. Finally, whey acidic protein, a major milk protein in some species, may play a role in normal mammary development, but that role in vivo may be limited to marsupials. Evolutionary pressure has led to changes in the structure of the protein with an accompanying change in function. Therefore, we propose that a loss of function of this protein in eutherians may relate to a reproductive strategy that is less dependent on lactation.

Chromatin proteins are determinants of centromere function.

Recent advances in the identification of molecular components of centromeres have demonstrated a crucial role for chromatin proteins in determining both centromere identity and the stability of kinetochore-microtubule attachments. Although we are far from a complete understanding of the establishment and propagation of centromeres, this review seeks to highlight the contribution of histones, histone deposition factors, histone modifying enzymes, and heterochromatin proteins to the assembly of this sophisticated, highly specialized chromatin structure. First, an overview of DNA sequence elements at centromeric regions will be presented. We will then discuss the contribution of chromatin to kinetochore function in budding yeast, and pericentric heterochromatin domains in other eukaryotic systems. We will conclude with discussion of specialized nucleosomes that direct kinetochore assembly and propagation of centromere-defining chromatin domains.

Yeast histone deposition protein Asf1p requires Hir proteins and PCNA for heterochromatic silencing.

BACKGROUND: Position-dependent gene silencing in yeast involves many factors, including the four HIR genes and nucleosome assembly proteins Asf1p and chromatin assembly factor I (CAF-I, encoded by the CAC1-3 genes). Both cac Delta asfl Delta and cac Delta hir Delta double mutants display synergistic reductions in heterochromatic gene silencing. However, the relationship between the contributions of HIR genes and ASF1 to silencing has not previously been explored. RESULTS: Our biochemical and genetic studies of yeast Asf1p revealed links to Hir protein function. In vitro, an active histone deposition complex was formed from recombinant yeast Asf1p and histones H3 and H4 that lack a newly synthesized acetylation pattern. This Asf1p/H3/H4 complex generated micrococcal nuclease--resistant DNA in the absence of DNA replication and stimulated nucleosome assembly activity by recombinant yeast CAF-I during DNA synthesis. Also, Asf1p bound to the Hir1p and Hir2p proteins in vitro and in cell extracts. In vivo, the HIR1 and ASF1 genes contributed to silencing the heterochromatic HML locus via the same genetic pathway. Deletion of either HIR1 or ASF1 eliminated telomeric gene silencing in combination with pol30--8, encoding an altered form of the DNA polymerase processivity factor PCNA that prevents CAF-I from contributing to silencing. Conversely, other pol30 alleles prevented Asf1/Hir proteins from contributing to silencing. CONCLUSIONS: Yeast CAF-I and Asf1p cooperate to form nucleosomes in vitro. In vivo, Asf1p and Hir proteins physically interact and together promote heterochromatic gene silencing in a manner requiring PCNA. This Asf1/Hir silencing pathway functionally overlaps with CAF-I activity.

Medicine information help lines: a survey of hospital pharmacy-based services in the UK and their conformity with guidelines.

OBJECTIVE: To describe the prevalence and nature of hospital pharmacy-based medicine help lines for consumers in the UK and to compare service provision with published guidelines. BACKGROUND: Since 1992, telephone help lines for patients have proliferated in hospital pharmacies in the UK. There is no common template for such services with variations in target group, number and type of calls, and arrangements for training and audit. Data on these factors will help guide further development of such services. METHODS: All medicine help lines operating from hospital pharmacies in the UK were identified through the national Drug Information Pharmacists network. They were sent a piloted questionnaire covering many aspects of help line operation, including funding, method of advertisement, procedures, target group, number and nature of calls, and audit procedures. RESULTS: Eighty-two help lines were identified in England, Scotland, Wales, and Northern Ireland. Completed responses were received from 69 help lines (84% response rate). The pharmacy drug information center was the help line site in 57% of hospitals; all other help lines were located in the dispensary. In 55% of cases, help lines were open only to patients of the hospital. In the remainder of help lines, calls from the public were answered (although the majority of help lines only advertised to hospital patients). Calls were answered by pharmacists only in 45% of services, and additional staff training had been provided in 43%. Only 48% of services had written procedures or guidelines for operation of the help line. Forty-six percent of the services received fewer than five calls per week, 31% received between five and 10 calls per week, and 22% received 11 or more calls per week. In 59% of the sites, calls took an average of 10 minutes or less to answer; it took 11-15 minutes in 32% of the sites and >15 minutes in 9% of the sites. The most common queries related to adverse effects, dosage and administration, and interactions (including alcohol). Only 33% of help lines had any auditing or monitoring of the service in place. CONCLUSIONS: The increasing use of the telephone to provide services directly to consumers is reflected in the growth of hospital-based medicine help lines in the UK. The telephone route is likely to become more important as patients' needs for information about their medicines increase. However, the rate of calls is low when compared with the number of patients issued prescriptions; further research is needed to investigate the reasons for this low response. There is currently reason for concern because most help lines lack not only professional training in telephone counseling, but also proper documentation, monitoring, and audit procedures.

Tumor cells are the source of osteopontin and bone sialoprotein expression in human breast cancer.

Bone sialoprotein (BSP) and osteopontin (OPN) are secreted glycoproteins with a conserved Arg-Gly-Asp (RGD) integrin-binding motif and are expressed predominantly in bone. The RGD tripeptide is commonly present in extracellular attachment proteins and has been shown to mediate the attachment of osteosarcoma cells and osteoclasts. To determine the origin and incidence of BSP and OPN mRNA expression in primary tumor, a cohort of archival, primary invasive breast carcinoma specimens was analyzed. BSP transcripts were detected in 65% and OPN transcripts in 77% of breast cancers examined. In general, BSP and OPN transcripts were detected in both invasive and in situ carcinoma components. The transcripts were not detected in surrounding stromal cells or in peritumoral macrophages. Despite its abundance in carcinomas, BSP expression was not detected in a panel of 11 human breast cancer cell lines (MCF-7, T47D, SK-Br-3, MDA-MB-453, MDA-MB-231, MDA-MB-436, BT549, MCF-7ADR, Hs578T, MDA-MB-435, and LCC15-MB) and OPN expression was detected only in two of these (MDA-MB-435 and LCC15-MB). To examine the possibility that expression of these genes was down-regulated in cell culture, several cell lines were grown as nude mouse xenografts in vivo; however, these tumors also failed to express BSP. OPN expression was identified in all cell lines grown as nude mouse xenografts. Our data suggest that in human primary breast tumors, the origin of BSP and OPN mRNA is predominantly the breast cancer cells and that expression of these transcripts is influenced by the tumor environment.

Purification and characterization of the Sgs1 DNA helicase activity of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae Sgs1 protein is a member of a family of DNA helicases that include the Escherichia coli RecQ protein and the products of human Bloom's syndrome and Werner's syndrome genes. To study the enzymatic characteristics of the protein, a recombinant Sgs1 fragment (amino acids 400-1268 of the 1447-amino acid full-length protein) was overexpressed in yeast and purified to near homogeneity. The purified protein exhibits an ATPase activity in the presence of single- or double-stranded DNA. In the presence of ATP or dATP, unwinding of duplex DNA or a DNA-RNA heteroduplex by the recombinant Sgs1 fragment was readily observed. Similar to the E. coli RecQ helicase, displacement of the DNA strand occurs in the 3' to 5' direction with respect to the single-stranded DNA flanking the duplex. The efficiency of unwinding was found to correlate inversely with the length of the duplex region and was enhanced by the presence of E. coli single-stranded DNA-binding protein. In addition, the recombinant Sgs1 fragment was found to bind more tightly to a forked DNA substrate than to either single- or double-stranded DNA.

Characterization of the Aspergillus nidulans nmrA gene involved in nitrogen metabolite repression.

The gene nmrA of Aspergillus nidulans has been isolated and found to be a homolog of the Neurospora crassa gene nmr-1, involved in nitrogen metabolite repression. Deletion of nmrA results in partial derepression of activities subject to nitrogen repression similar to phenotypes observed for certain mutations in the positively acting areA gene.

The acetate regulatory gene facB of Aspergillus nidulans encodes a Zn(II)2Cys6 transcriptional activator.

Genetic studies have indicated that the facB gene of Aspergillus nidulans is a major regulatory gene involved in acetamide and acetate utilisation. Sequencing of the facB gene revealed that it encodes a protein that contains an N-terminal GAL4-like Zn(II)2Cys6 (or C6 zinc) binuclear cluster for DNA binding, leucine zipper-like heptad repeat motifs and central and C-terminal acidic alpha-helical regions, consistent with a function as a DNA-binding transcriptional activator. The Zn(II)2Cys6 cluster shows strong similarity with those of the Saccharomyces cerevisiae carbon metabolism regulatory proteins CAT8 and SIP4. A significant level of similarity with CAT8 is found throughout the length of the protein, suggesting at least partial functional homology. The facB genes of Aspergillus oryzae and Aspergillus niger were also sequenced and found to be highly conserved. Deletion of the facB gene confirmed that it is required for growth on acetate as a sole carbon source. Functional dissection using deletion and fusion constructs and in vitro mutagenesis indicated that the Zn(II)2Cys6 cluster and the C-terminal end of the protein are required for function.

The hapC gene of Aspergillus nidulans is involved in the expression of CCAAT-containing promoters.

The 5' regulatory region of the amdS gene of Aspergillus nidulans, which encodes an acetamidase required for growth on acetamide as a carbon and nitrogen source, contains a CCAAT sequence which is required for setting the basal level of amdS expression. Mobility shift studies have identified a factor in A. nidulans nuclear extracts which binds to this CCAAT sequence. In Saccharomyces cerevisiae the HAP3 gene encodes one component of a multisubunit complex that binds CCAAT sequences. A search of the EMBL and SwissProt databases has revealed an A. nidulans sequence with significant homology to the HAP3 gene adjacent to the previously cloned regulatory gene amdR. Sequencing of the remainder of this region has confirmed the presence of a gene, designated hapC, with extensive homology to HAP3. The predicted amino acid sequence of HapC shows extensive identity to HAP3 in the central conserved domain, but shows little conservation in the flanking sequences. A haploid carrying a hapC deletion has been created and is viable, but grows poorly on all media tested. This null mutant grows especially slowly on acetamide as a sole carbon and nitrogen source, indicating that hapC plays a role in amdS expression. In agreement with this notion, it has been shown that the hapC deletion results in reduced levels of expression of an amdS::lacZ reporter gene and this effect is particularly evident under conditions of carbon limitation. Nuclear extracts prepared from the hapC deletion mutant show no CCAAT binding activity to the amdS or gatA promoters, indicating that hapC may encode a component of the complex binding at this sequence.

Visual orienting deficits in high-functioning people with autism.

There has been renewed interest in the idea that attentional dysfunction may underlie autistic symptomatology (e.g., Bryson, Wainwright-Sharp, & Smith, 1990; Dawson & Lewy, 1989a, 1989b). Existing research indicates problems with overfocused attention (Lovaas et al., 1971; Rincover & Ducharme, 1987), and with shifting attention between sensory modalities (Courchesne et al., 1990). These phenomena were examined further by using Posner's (1978) visual orienting task with a group of high-functioning autistic adolescents and adults, and matched normal controls. Our results indicate that autistic people have difficulty processing briefly presented cue information. Evidence of problems disengaging and shifting attention within the visual modality was also provided. The findings can be seen as consistent with previous behavioral, autonomic, and electrophysiological research which has revealed impairments in the registration, processing, and response to external stimuli.

Delay and number of food reinforcers: Effects on choice and latencies.

Pigeons were given a choice between two identical-duration situations (terminal links of chain schedules). One terminal link of the choice pair provided two food deliveries, and the other provided five. The exact times of these food deliveries differed between the terminal links and were varied over conditions. A single response during the initial link gave immediate access to the corresponding terminal link. Forced trials, during which only one of the initial-link keys was lighted, were interspersed with choice trials during which both initial-link keys were lighted. Choice tended to favor whichever terminal link was correlated with the higher sum of the immediacies (i.e., the sum of the reciprocals of the delays to each of the reinforcers following the choice, with all delays measured from the choice). Latencies on forced trials and on choice trials also were related (negatively) to the sum of the immediacies. This correlation among response measures (choice and latencies) suggests that both measures are manifestations of the effect of conditioned reinforcement on response tendencies.

The rho-115 mutation in transcription termination factor rho affects its primary polynucleotide binding site.

We have investigated the effect of the rho-115 mutation on the catalytic properties of the Escherichia coli termination protein, rho. Comparison of the primary and secondary polynucleotide binding sites activities reveals dramatic differences between the mutant and wild-type molecules. Wild-type rho must bind single-stranded polynucleotides to activate its nucleotide triphosphatase (NTPase) activity, and either poly(C), or poly(dC) plus oligo(C), will suffice. In contrast, attempted activation of the rho-115 NTPase with poly(C) in the presence of poly(dC) showed the latter to be a potent inhibitor. Inclusion of small oligonucleotides such as oligo(C) in the activation assay does not inhibit the poly(C)-induced NTPase reaction of either wild-type rho or rho-115. This would indicate, in the two polynucleotide binding site model for rho proposed by Richardson (Richardson, J.P. (1982) J. Biol. Chem. 251, 5760-5766), that the mutation in rho-115 affects the primary polynucleotide binding site. Transcription termination in vitro at the rho-dependent site trp t' showed dramatically reduced termination with rho-115 protein compared to wild-type rho. In the presence of rho-115, the transcript is longer and termination occurs over a narrower range of nucleotides than with wild-type rho. This suggests that the primary polynucleotide binding site is important not only for efficient termination of transcription but may also be involved in determining the terminal end point of the transcript itself.

Erythrocyte adenosine transport: effects of Ca2+ channel antagonists and ions.

On the basis of observations of adenosine-Ca2+ competition, we assessed the effects on erythrocyte adenosine transport of Ca2+ channel antagonists, mono- and divalent cations, and Cl- and Cl- transport inhibitors. The Ca2+ channel antagonists, diltiazem and verapamil, competitively inhibited adenosine influx (Ki = 158 +/- 17.4 and 13.5 +/- 1.3 microM at 10 microM adenosine, respectively), despite no apparent effect on transport by Ca2+, Mg2+, Na+, or K+. Verapamil also inhibited uridine efflux (Ki = 1.7 +/- 0.3 microM at 84-100 microM intracellular uridine). The absence of Cl- decreased adenosine influx rates from 0.615 +/- 0.013 to 0.386 +/- 0.008 nmol X s-1 X ml intracellular H2O-1. The Cl- transport inhibitors, diisothiocyanostilbene disulfonate (10 microM), furosemide (1 mM), and NO-3 (145 mM), decreased adenosine influx rates to 0.301 +/- 0.008, 0.325 +/- 0.013, and 0.430 +/- 0.009 nmol X s-1 X ml intracellular H2O-1, respectively. These studies indicate that the Ca2+ channel antagonists inhibit adenosine release and uptake and therefore may modulate adenosine-mediated events. Additionally, they suggest that adenosine and anion transport systems are linked or share common features.

Choline transport across a carbon tetrachloride phase containing a chloroform-methanol extract of brain.

The presence of a chloroform-methanol extract of cat brain in a carbon tetrachloride phase separating two aqueous phases resulted in an increased passage of [3H]choline across the organic phase which was inhibited by the choline transport inhibitor hemicholinium-3 and by high concentrations of non-radioactive choline. In the absence of cat brain extract, [3H]choline passage across carbon tetrachloride was neither inhibited by hemicholinium-3, nor by non-radioactive choline.

Pausing of RNA polymerase during in vitro transcription through the ilvB and ilvGEDA attenuator regions of Escherichia coli K12.

Synchronized single-round transcriptions, in vitro, from templates encoding the leader RNA of the ilvB and ilvGEDA operons result in the accumulation of a transcript consistent with RNA polymerase pausing after the 1:2 stem that could form in each of the leader RNAs. Addition of L-factor or guanosine 5'-diphosphate,3-diphosphate extended the pause half-life obtained with the ilvB template; addition of L-factor and guanosine 5'-diphosphate,3'-diphosphate together had an additive effect on the pause half-life. L-factor also extended the pause half-life of the pause obtained with the ilvGEDA template; however, addition of guanosine 5'-diphosphate,3'-diphosphate did not. The results obtained are consistent with the model for attenuation proposed by Yanofsky et al. (Yanofsky, C., Das, A., Fisher, R., Kolter, R., and Berlin, V. (1984) UCLA Symposium of Gene Expression (Hamer, D., and Rosenberg, M., eds) pp. 295-310, Alan R. Liss, Inc., New York) in which transcriptional pausing after the synthesis of the 1:2 stem-loop closely couples transcription and translation of the leader region.