Evasion of a Human Cytomegalovirus Entry Inhibitor with Potent Cysteine Reactivity Is Concomitant with the Utilization of a Heparan Sulfate Proteoglycan-Independent Route of Entry.

The dependence of viruses on the host cell to complete their replicative cycle renders cellular functions potential targets for novel antivirals. We screened a panel of broadly acting cellular ion channel inhibitors for activity against human cytomegalovirus (HCMV) and identified the voltage-gated chloride ion channel inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) as a potent inhibitor of HCMV replication. Time-of-addition studies demonstrated that DIDS inhibited entry via direct interaction with the virion that impeded binding to the plasma membrane. Synthesis and analysis of pharmacological variants of DIDS suggested that intrinsic cysteine, and not lysine, reactivity was important for activity against HCMV. Although sequencing of DIDS-resistant HCMV revealed enrichment of a mutation within UL100 (encoding glycoprotein M) and a specific truncation of glycoprotein RL13, these did not explain the DIDS resistance phenotype. Specifically, only the introduction of the RL13 mutant partially phenocopied the DIDS resistance phenotype. Serendipitously, the entry of DIDS-resistant HCMV also became independent of heparan sulfate proteoglycans (HSPGs), suggesting that evasion of DIDS lowered dependence on an initial interaction with HSPGs. Intriguingly, the DIDS-resistant virus demonstrated increased sensitivity to antibody neutralization, which mapped, in part, to the presence of the gM mutation. Taken together the data characterize the antiviral activity of a novel HCMV inhibitor that drives HCMV infection to occur independently of HSPGs and the generation of increased sensitivity to humoral immunity. The data also demonstrate that compounds with cysteine reactivity have the potential to act as antiviral compounds against HCMV via direct engagement of virions.IMPORTANCE Human cytomegalovirus (HCMV) is major pathogen of nonimmunocompetent individuals that remains in need of new therapeutic options. Here, we identify a potent antiviral compound (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid [DIDS]), its mechanism of action, and the chemical properties required for its activity. In doing so, the data argue that cysteine-reactive compounds could have the capacity to be developed for anti-HCMV activity. Importantly, the data show that entry of DIDS-resistant virus became independent of heparan sulfate proteoglycans (HSPGs) but, concomitantly, became more sensitive to neutralizing antibody responses. This serendipitous observation suggests that retention of an interaction with HSPGs during the entry process in vivo may be evolutionarily advantageous through better evasion of humoral responses directed against HCMV virions.

Re-induction of obese body weight occurs more rapidly and at lower caloric intake in beagles.

For the purpose of investigating the mechanism of obesity-induction/re-induction including weight-cycling in beagles, a study was conducted using commercially available dog food combined with human food to mimic at home-snacking and diet-supplementation behaviours. Adult female beagles, which had free access to water and exercise, were used (n = 9). All dogs were initially offered two times their daily calculated number of calories using a dry extruded diet plus blend of canola and soybean oils and allowed to eat ad libitum. After 3 weeks, Pecan shortbread cookies were added to the diet mixture. Obesity was induced during a 19-week period with 1875-2250 kcal/day consumed, on average, during this period. The dogs were then subjected to a weight-loss regimen while consuming 490-730 kcal/day. After weight loss, a similar degree of obesity was re-induced for 17 weeks even though dogs consumed only 1125-1250 kcal/day. Body weight, body condition scores, kcal consumption and food efficiency were recorded. Results indicated that less time and fewer kcal were required to re-induce the same degree of obesity compared with the initial obesity induction. Human snack foods appeared to stimulate appetite and thus contribute to the obese state. Food efficiency was also increased during the obesity-reinduction period compared with the induction period. This information may help pet owners better understand the need to limit table scraps and human-type food snacks in dogs prone to obesity as well as weight maintenance after weight loss.

Maternal age and trisomy--a unifying mechanism of formation.

The mechanism of trisomy formation and its relationship to increased maternal age is not understood. Molecular analysis of the pattern of inheritance of DNA markers in trisomy families shows trisomies can be grouped according to whether the affected chromosomes inherited from their mothers are heterozygous or homozygous with respect to the centromeres. Furthermore, molecular analysis reveals that those that are heterozygous have fewer chiasmata, which are located more distally, while those that are homozygous have more chiasmata proximally located. Cytogenetic analysis of human oocytes shows that the kind of imbalance predicted by the classic hypothesis of nondisjunction, i.e. extra whole chromosomes at the second metaphase, is rarely found, whereas the common expression of imbalance is seen as single chromatids. We hypothesise that one mechanism links these data: the mechanism depends on the prediction from the cytogenetic data that cohesion within the bivalent complex is severely weakened during the extended dictyate stage in older women. Consequently, when meiosis resumes, at the time of ovulation, the bivalent emerges as four chromatids held together only by its chiasmata. In accordance with the rules of orientation on the spindle, the final balanced shape of the configuration achieved at metaphase I, in this case determined by the position of the chiasmata, will dictate whether the subsequent segregation of the chromatids will result in their heterozygosity or homozygosity. It follows that the concept of "first division" and "second division" errors, i.e. of nondisjunction originating at first or second meiotic division as defined by centromeric hetero- or homozygosity, may be erroneous.

Population-based guidelines for performance measurement: a preliminary report.

This paper describes the development of--and early efforts to validate--guidelines that indicate average amounts of service expected to be used by a population of patients with a given disorder who are served by a comprehensive mental health system. These guidelines address expected service use by individuals in 55 diagnostic groups. The purpose of these guidelines is to provide a gauge for evaluating the amounts of service being delivered by managed care organizations. Three population-based guidelines (for attention-deficit/hyperactivity disorder, major depressive disorder, and schizophrenia) are compared to actual amounts of service delivered to enrollees in large behavioral health care systems.

First-meiotic-division nondisjunction in human oocytes.

Reject oocytes from in vitro-fertilization patients are currently the only practical source of human oocyte material available for meiotic studies in women. Two hundred clearly analyzable second meiotic (MII) metaphase oocytes from 116 patients were examined for evidence of first meiotic (MI) division errors. The chromosome results, in which 67% of oocytes had a normal 23,X chromosome complement but none had an extra whole chromosome, cast doubt on the relevance, to human oocytes, of those theories of nondisjunction that propose that both chromosomes of the bivalent fail to disjoin at MI so that both move to one pole and result in an additional whole chromosome at MII metaphase. The only class of abnormality found in the MII oocytes had single chromatids (half-chromosomes) replacing whole chromosomes. Analysis of the chromosomally abnormal oocytes revealed an extremely close correlation with data on trisomies in spontaneous abortions, with respect to chromosome distribution, frequency, and maternal age, and indicated the likelihood of the chromatid abnormalities being the MI-division nondisjunction products that lead to trisomy formation after fertilization. The most likely derivation of the abnormalities is through a from of misdivision process usually associated with univalents, in which the centromeres divide precociously at MI, instead of MII, division. In the light of recent data that show that altered recombination patterns of the affected chromosomes are a key feature of most MI-division trisomies, the oocyte data imply that the vulnerable meiotic configurations arising from altered recombination patterns are processed as functional univalents in older women. Preliminary evidence from MI-metaphase oocytes supports this view.

Meiosis I in human oocytes.

Oocytes aspirated from women undergoing laparoscopic sterilisation were matured in vitro from the germinal vesicle stage. They showed that germinal vesicle breakdown spanned a period of about 12 h; the oocytes were at diakinesis by 12 h, and at metaphase I by 16 h where they remained for a further 10-12 h. Anaphase I took place after about 26 h. The meiotic preparations lacked the clarity of chromosome definition seen in mouse oocytes and accurate chiasma counts could not be made. However univalents at first meiotic metaphase could be detected without ambiguity and were found in women significantly older than those whose metaphase I oocytes contained bivalents only.

Prioritization of mental health services in Oregon.

The Oregon Health Plan is an approach to health care reform that increases access to mental health and chemical dependency services. A key feature is the integration of mental and physical health care. The mental health community had to educate policy makers about the importance of mental health and chemical dependency services. They constructed a prioritized list of mental health and chemical dependency services and interdigitated the list with the set of physical health services. The result is a unique attempt to develop a seamless health care system that minimizes discrimination against persons affected by mental illness. The opportunity to achieve parity for mental health must not be delayed or compromised; to do so would worsen the lives of persons already affected by the trauma and stigma associated with mental illness.

First meiotic division abnormalities in human oocytes: mechanism of trisomy formation.

Trisomy is the single most frequent type of chromosome abnormality in humans and has considerable impact on many aspects of human pathology. It arises most commonly through "nondisjunction" at maternal meiosis I, but the underlying mechanism of formation remains obscure. Analysis of 100 haploid oocytes at second meiotic metaphase shows that the only type of chromosome abnormality compatible with trisomy formation after fertilisation is the presence of single chromatids in addition to, or replacing, whole chromosomes. The mechanism resulting in the presence of single chromatids is considered to be precocious division of univalents or dyads at first meiotic anaphase.

Ethics and value strategies used in prioritizing mental health services in Oregon.

The authors describe the ethical considerations underlying the inclusion of mental health services into a prioritized health care system. The Oregon Health Plan is a process for defining and delivering basic health services to an entire state. As the plan was developed, the mental health community needed to decide whether or not to participate in the process and, if so, how. Lengthy discussions among mental health consumers, family members, and providers led to a strategy that emphasized the integration of mental health and chemical dependency services into a comprehensive and universal health care program. This approach appears to have achieved relative parity for mental health.

Chromosome anomalies in human oocytes in relation to age.

Cytogenetic analysis of oocytes remaining unfertilized after in-vitro fertilization showed that the source of data obtained could be divided into degenerating and 'healthy' oocytes. The degenerating oocytes, which showed different degrees of chromosome breakage, accounted for a quarter of the total. They were found in older patients with a mean age of 35.0 years. The healthy oocytes without chromosome breaks were mostly haploid and fell into two main groups, those with a normal MII,23,X chromosome complement, and those abnormal in which single chromatids replaced a whole chromosome. No oocytes hyperhaploid for an extra whole chromosome were found. We hypothesize that the single chromatids at second meiotic metaphase arise by precocious division of chromosome univalents at anaphase I (predivision) and that this may be the major mechanism for trisomy formation in man, rather than the non-disjunction of whole bivalents as generally assumed.

Cytogenetic analysis of unfertilized human oocytes.

Cytogenetic studies were carried out on 180 oocytes that appeared unfertilized after in-vitro fertilization. The majority of the 135 that were informative had grossly haploid second meiotic metaphases, two were grossly diploid, and five had a variety of different abnormalities. Twenty-one oocytes were abnormally fertilized and included prematurely condensed sperm chromosomes. The frequency of this phenomenon varied according to the stimulation protocol, those oocytes maturing longer in vivo showing less propensity to abnormal fertilizations. Thirteen per cent of the analysable haploid metaphases were hyperhaploid but none contained extra whole chromosomes. The extra components were a single chromatid (one case), or two single chromatids replacing a whole chromosome (four cases). The data suggest that the chromatids arose as a result of premature centromere division at meiosis I, and that this may be a major mechanism for trisomy formation rather than non-disjunction of whole bivalents at meiosis I, as generally believed.

Predivision in human oocytes at meiosis I: a mechanism for trisomy formation in man.

Cytogenetic preparations from oocytes remaining unfertilised after in vitro fertilisation revealed single chromatids (as opposed to whole chromosomes) in 4 out of 38 meiosis II metaphases. In one oocyte, a single chromatid was present in addition to the normal 23,X complement, and in three oocytes, two identical but separate chromatids replaced one whole chromosome within the complement. The data indicate that the chromatids have arisen as a result of premature division of the centromeres at meiosis I ("predivision"). None of the oocytes were hyperhaploid with an extra whole chromosome. These findings are at variance with the general belief that trisomy in man is largely a consequence of nondisjunction of whole bivalents at meiosis I and they suggest that predivision resulting in separate chromatids may be a significant mechanism for human trisomy.

Effects of a high-voltage direct-current transmission line on beef cattle production.

Two herds of beef cattle were maintained beneath a +/- 500 kV direct-current transmission line during a 30-month period, and were compared with two similar herds maintained away from the transmission line. Exposures of animals under the line were five to 30 times greater than those of control animals, depending on the parameter of interest, with average exposure magnitudes of 5.6 kV/m, 4.1 nA/m2, and 13 k ions/cm3, respectively, for electric field, ion current, and density of ions. Productivity and health status of cows and calves were similar between lines and control treatments. Mean body mass of cows increased with maturity, from 438 kg in 1985 to 496 kg in 1987. Calf gain averaged 0.93 kg per head per day. No unusual sources of mortality were observed. Based on this confinement study, beef cattle permitted to graze in the vicinity of a high-voltage, direct-current transmission lines are not expected to experience any decrease in frequency of conception, calving, growth rate, or survival.

Treatment of Tourette's syndrome with transdermal clonidine: a pilot study.

Nine patients with mild Tourette's syndrome completed a cross-over, placebo-controlled study of clonidine administered transdermally. Although the subjects showed no improvement on objective measures of symptom severity, most subjects felt they had improved. Seven subjects chose to continue taking clonidine. Larger, blinded studies of transdermal clonidine are indicated.