Haploinsufficiency of X-linked intellectual disability gene CASK induces post-transcriptional changes in synaptic and cellular metabolic pathways.

Heterozygous mutations in the X-linked gene CASK are associated with intellectual disability, microcephaly, pontocerebellar hypoplasia, optic nerve hypoplasia and partially penetrant seizures in girls. The Cask+/- heterozygous knockout female mouse phenocopies the human disorder and exhibits postnatal microencephaly, cerebellar hypoplasia and optic nerve hypoplasia. It is not known if Cask+/- mice also display seizures, nor is known the molecular mechanism by which CASK haploinsufficiency produces the numerous documented phenotypes. 24-h video electroencephalography demonstrates that despite sporadic seizure activity, the overall electrographic patterns remain unaltered in Cask+/- mice. Additionally, seizure threshold to the commonly used kindling agent, pentylenetetrazol, remains unaltered in Cask+/- mice, indicating that even in mice the seizure phenotype is only partially penetrant and may have an indirect mechanism. RNA sequencing experiments on Cask+/- mouse brain uncovers a very limited number of changes, with most differences arising in the transcripts of extracellular matrix proteins and the transcripts of a group of nuclear proteins. In contrast to limited changes at the transcript level, quantitative whole-brain proteomics using iTRAQ quantitative mass-spectrometry reveals major changes in synaptic, metabolic/mitochondrial, cytoskeletal, and protein metabolic pathways. Unbiased protein-protein interaction mapping using affinity chromatography demonstrates that CASK may form complexes with proteins belonging to the same functional groups in which altered protein levels are observed. We discuss the mechanism of the observed changes in the context of known molecular function/s of CASK. Overall, our data indicate that the phenotypic spectrum of female Cask+/- mice includes sporadic seizures and thus closely parallels that of CASK haploinsufficient girls; the Cask+/- mouse is thus a face-validated model for CASK-related pathologies. We therefore surmise that CASK haploinsufficiency is likely to affect brain structure and function due to dysregulation of several cellular pathways including synaptic signaling and cellular metabolism.

Chloroplast PNPase exists as a homo-multimer enzyme complex that is distinct from the Escherichia coli degradosome.

In Escherichia coli, the exoribonuclease polynucleotide phosphorylase (PNPase), the endoribonuclease RNase E, a DEAD-RNA helicase and the glycolytic enzyme enolase are associated with a high molecular weight complex, the degradosome. This complex has an important role in processing and degradation of RNA. Chloroplasts contain an exoribonuclease homologous to E. coli PNPase. Size exclusion chromatography revealed that chloroplast PNPase elutes as a 580-600 kDa complex, suggesting that it can form an enzyme complex similar to the E. coli degradosome. Biochemical and mass-spectrometric analysis showed, however, that PNPase is the only protein associated with the 580-600 kDa complex. Similarly, a purified recombinant chloroplast PNPase also eluted as a 580-600 kDa complex after gel filtration chromatography. These results suggest that chloroplast PNPase exists as a homo-multimer complex. No other chloroplast proteins were found to associate with chloroplast PNPase during affinity chromatography. Database analysis of proteins homologous to E. coli RNase E revealed that chloroplast and cyanobacterial proteins lack the C-terminal domain of the E. coli protein that is involved in assembly of the degradosome. Together, our results suggest that PNPase does not form a degradosome-like complex in the chloroplast. Thus, RNA processing and degradation in this organelle differ in several respects from those in E. coli.

A myosin I isoform in the nucleus.

A nuclear isoform of myosin I beta that contains a unique 16-amino acid amino-terminal extension has been identified. An affinity-purified antibody to the 16-amino acid peptide demonstrated nuclear staining. Confocal and electron microscopy revealed that nuclear myosin I beta colocalized with RNA polymerase II in an alpha-amanitin- and actinomycin D-sensitive manner. The antibody coimmunoprecipitated RNA polymerase II and blocked in vitro RNA synthesis. This isoform of myosin I beta appears to be in a complex with RNA polymerase II and may affect transcription.

Peripheral arterial obstruction: prospective study of treatment with a transluminally placed self-expanding stent-graft. International Trial Study Group.

PURPOSE: To evaluate the safety and efficacy of an endoluminal prosthesis for treatment of peripheral arterial occlusive disease (PAOD). MATERIALS AND METHODS: A self-expanding endoprosthesis with an expanded polytetrafluoroethylene tube inside a nitinol support structure was implanted in 127 patients with symptomatic PAOD in the iliac (61 limbs) and femoral arteries (80 limbs). Clinical category status, ankle-brachial index, and color duplex flow imaging results were recorded before treatment, at discharge, and at 1, 3, 6, and 12 months after treatment. Aspirin was administered throughout the study, and heparin was administered during and for 2 days after the procedure. RESULTS: Endoprosthesis deployment was technically successful in all patients. Complications occurred in 24 of 141 procedures and included three major complications. Early thrombosis (within 30 days) occurred in one iliac and three femoral arteries. Late restenosis or reocclusion was observed in five iliac and 14 femoral arteries within the 1st year. Primary patency rates in iliac arteries were 98% +/- 3% (standard error) and 91% +/- 4%, respectively, at 6 and 12 months after treatment. Primary patency rates in femoral arteries were 90% +/- 3% and 79% +/- 5%, respectively, at 6 and 12 months. Secondary patency rates were 95% and 93% for iliac and femoral arteries, respectively, at 12 months after treatment. CONCLUSION: The device used in this study can be implanted without additional risks to the patient and provided encouraging patency rates up to 1 year.

Immunodominance among EBV-derived epitopes restricted by HLA-B27 does not correlate with epitope abundance in EBV-transformed B-lymphoblastoid cell lines.

Using synthetic peptides, the HLA-B27-restricted CTL response to EBV in asymptomatic virus carriers has been mapped to four epitope regions in EBV latent cycle Ags. One of these peptide-defined epitopes (RRIYDLIEL) tends to be immunodominant and is recognized in the context of all three B27 subtypes studied, B*2702, B*2704, and B*2705. The other peptide-defined epitopes induce responses only in the context of one subtype, the immunogenic combinations being RRARSLSAERY/B*2702, RRRWRRLTV/B*2704, and FRKAQIQGL/B*2705. We used immunoaffinity chromatography to isolate the naturally presented viral peptides associated with these MHC class I molecules on the surface of EBV-transformed B-LCL. Using CTL reconstitution assays in conjunction with mass spectrometry, we established that the naturally processed and presented peptides are identical with the previously identified synthetic sequences. Despite the subtype-specific immunogenicity of three of the four epitopes, all four epitope peptides were found in association with each of the three different HLA-B27 subtypes. Indeed, those peptides that failed to induce a response in the context of a particular HLA-B27 subtype were frequently presented at greater abundance by that subtype than were the immunogenic peptides. Furthermore, among the peptides that did induce a response, immunodominance did not correlate with epitope abundance; in fact the immunodominant RRIYDLIEL epitope was least abundant, being present at less than one copy per cell. The relationship of this unexpected finding to the persistence of EBV is discussed.

Biochemical identification of a mutated human melanoma antigen recognized by CD4(+) T cells.

CD4(+) T cells play a critical role in generating and maintaining immune responses against pathogens and alloantigens, and evidence suggests an important role for them in antitumor immunity as well. Although major histocompatibility complex class II-restricted human CD4(+) T cells with specific antitumor reactivities have been described, no standard method exists for cloning the recognized tumor-associated antigen (Ag). In this study, biochemical protein purification methods were used in conjunction with novel mass spectrometry sequencing techniques and molecular cloning to isolate a unique melanoma Ag recognized by a CD4(+) tumor-infiltrating lymphocyte (TIL) line. The HLA-DRbeta1*0101-restricted Ag was determined to be a mutated glycolytic enzyme, triosephosphate isomerase (TPI). A C to T mutation identified by cDNA sequencing caused a Thr to Ile conversion in TPI, which could be detected in a tryptic digest of tumor-derived TPI by mass spectrometry. The Thr to Ile conversion created a neoepitope whose T cell stimulatory activity was enhanced at least 5 logs compared with the wild-type peptide. Analysis of T cell recognition of serially truncated peptides suggested that the mutated amino acid residue was a T cell receptor contact. Defining human tumor Ag recognized by T helper cells may provide important clues to designing more effective immunotherapies for cancer.

Massive fetomaternal hemorrhage and fetal death: are they predictable?

OBJECTIVE: To report the incidence of massive fetomaternal hemorrhage (FMH) associated with fetal death and to test the hypothesis that FMH is more likely to occur in those with risk factors for FMH. STUDY DESIGN: All cases of fetal death of infants weighing > 500 gm between January 1, 1990 and December 31, 1994 were reviewed for evidence of massive FMH (> or = 2% fetal cells in the maternal circulation as measured by the Betke-Kleihauer test). Women with risk factors were compared with those without risk factors with respect to the occurrence of massive FMH. RESULTS: The prevalence of massive FMH was 14 of 319 (4.4%) cases, occurring in 4 of 102 (3.9%) of those with risk factors and 10 of 217 (4.6%) of patients without risk factors (p = 0.78). Otherwise unexplained fetal death was associated with massive FMH in 5 of 141 (3.5%). Major fetal anomalies were present in 5 of 14 (35.7%) cases of massive FMH. CONCLUSION: Clinical risk factors do not predict an increased likelihood of massive FMH. Massive FMH is associated with fetal anomalies. Betke-Kleihauer testing should be performed in all cases of fetal death, including those with anomalies regardless of the presence or absence of risk factors for FMH.

Stillbirth evaluation: what tests are needed?

OBJECTIVE: We sought to investigate what aspects of the stillbirth evaluation are considered to be essential and what tests can potentially be eliminated. STUDY DESIGN: A retrospective analysis of 745 stillbirths occurring from January 1990 to December 1994 was conducted. A stillbirth was defined by an estimated gestational age >20 weeks' gestational age or fetal weight >500 gm. We attempted to arrive at an apparent cause for each stillbirth after evaluation of genetic or chromosomal abnormalities, obstetric history, maternal medical illnesses, laboratory tests, autopsy findings, and placental pathologic conditions. RESULTS: We found that the most important aspects of stillbirth evaluation were placental pathologic conditions and autopsy. When the placenta was examined, a significant abnormality was detected in 30% (160 of 529) of the cases. When autopsy was performed, only 31% of fetal deaths (142 of 462) were unexplained; however, when no autopsy was performed, 44% (125 of 283) were unexplained (p = 0.0002). The following laboratory evaluations that were routinely performed were found to yield little definitive information: antinuclear antibody testing, Kleihauer-Betke test, and screening for congenital infections (toxoplasmosis, other viruses, rubella, cytomegalovirus, and herpes simplex virus). Overall, 36% (267 of 745) of stillbirths still remained unexplained despite a thorough evaluation in most cases. CONCLUSION: The causes of stillbirth are many and varied, with a large proportion having no obvious cause. As this study demonstrates, certain laboratory tests can be eliminated in the workup of fetal death. In the evaluation of stillbirth a complete systematic method that incorporates placental pathologic conditions, as well as autopsy findings, should prove to be beneficial.

Incidence of premature birth and neonatal respiratory disease in infants of HIV-positive mothers. The Pediatric Pulmonary and Cardiovascular Complications of Vertically Transmitted Human Immunodeficiency Virus Infection Study Group.

OBJECTIVE: We sought to determine the prematurity rate in infants of HIV-positive mothers and to characterize the incidence and severity of neonatal respiratory disease in this population. STUDY DESIGN: From 1990 to 1994, 600 live-born infants of HIV-infected mothers were enrolled prenatally (73%) or postnatally (27%) from five U.S. centers. Logistic regression was used to determine the association of HIV status in the infant with prematurity (< or = 37 weeks), low birth weight (< or = 2.5 kg), and very low birth weight (< or = 1.5 kg) rates. The incidence of respiratory distress syndrome (RDS), bronchopulmonary dysplasia, meconium aspiration syndrome, and neonatal pneumonia was compared with anticipated rates for gestational age and birth weight. RESULTS: Very high rates of prematurity (19%), low birth weight (18.3%), and very low birth weight (3.3%) were found in the infants of HIV-positive mothers; and HIV infection in the infant was associated with younger gestational age. The overall incidence of RDS was 3% (17/600), which coincided with the anticipated rate, after adjusting for prematurity and birth weight. Only five infants (all < or = 1.5 kg) had bronchopulmonary dysplasia, and none required assisted ventilation beyond 14 days. Three term infants had mild meconium aspiration syndrome, and there were no cases of documented neonatal pneumonia. CONCLUSION: Infants born to HIV-positive mothers exhibited high prematurity and low birth weight rates, and the odds of prematurity were higher in infants who were infected with HIV. Despite the high incidence of prematurity and perinatal risk of this population, incidence and severity of neonatal respiratory disease were not higher than would be expected from available neonatal data in populations not exposed to HIV.

Human immunodeficiency virus-1 seroprevalence among parturients in Los Angeles County public hospitals.

To estimate the prevalence of human immunodeficiency virus type 1 (HIV-1) among parturients in an area with a high cumulative incidence of AIDS, an HIV seroprevalence study was conducted in 1988 in Los Angeles County. Test results were available from 8485 (86.1%) of the 9860 women delivering at four public hospitals. The test results were linked to demographic and medical information available from routinely collected delivery records. Three specimens were HIV-positive, for a seroprevalence of 3.5 per 10,000. The seropositive women were all Latina. The prevalence of HIV among women delivering at all hospitals in Los Angeles County has been shown to be relatively low compared with that of other metropolitan areas with a high number of AIDS cases, such as New York city, but appears to be even lower among women delivering at public hospitals during this time period. This low prevalence is attributed to the predominance of Latinas, who may have lower levels of infection, among public hospital parturients. The relatively low prevalence of HIV among injection drug users in Los Angeles County may contribute to the overall lower prevalence among women in Los Angeles County compared with those in New York city. This study supplements county-specific data obtained from statewide blinded neonatal testing by providing details on HIV seroprevalence among the catchment populations of public hospitals in Los Angeles County.

Mother-child HIV disease: a model for collaborative care.

Women Connected and the Maternal Child Clinic are two unique dyad-centered programs for providing quality care to HIV-infected women and their children. Each is a collaborative effort to maintain standard of care for these patients within a large county facility. Women Connected facilitates their inpatient treatment and care, while the Maternal Child HIV Clinic provides follow-up outpatient health and social services.

Maternal factors associated with delivery of infants with birthweight less than 2000 grams in a low socioeconomic population.

Women of low socioeconomic status (SES) are known to have a higher incidence of low birthweight (LBW) infants who are a major source of neonatal mortality and morbidity. SES, however, does not define a homogeneous population. If prenatal programs could be oriented specifically toward women of higher risk within the low SES population, they should be more effective. Within our uniformly low SES population, we compared mothers who had given birth to infants weighing less than 2000 g with mothers of infants weighing over 3000 g in order to identify factors associated with a LBW birth. We found medical factors, including hypertension and infection during pregnancy, demographic factors, including race, marital status, and lack of prenatal care, and lifestyle factors, including drug and alcohol abuse, to be more common among women giving birth to infants weighing less than 2000 g. These factors can be used to develop more effective intervention programs for low SES populations.

Vitamin B-6 nutriture and plasma diamine oxidase activity in pregnant Hispanic teenagers.

Vitamin B-6 status was assessed by measuring erythrocyte glutamic-pyruvic transaminase (EGPT) indices in 122 pregnant Hispanic teenagers. Seventeen percent were vitamin B-6 deficient (EGPT indices greater than 1.25) at the initial interview (first or second trimester). A daily supplement of 5 mg vitamin B-6, beginning at initial interview, did not reduce prevalence of vitamin B-6 deficiency at final interview (third trimester). No association was found between EGPT indices greater than 1.25 and the outcome of pregnancy. The activity of diamine oxidase (DAO), a vitamin B-6-dependent enzyme produced by the placental decidua, was measured in maternal plasma. At initial and final interviews, plasma-DAO activity was increased by in vitro addition of pyridoxal-5'-phosphate. The activity in early pregnancy was positively associated with dietary vitamin B-6 intake and was lower in teenagers with EGPT indices greater than 1.25 at the final interview. Findings suggest that plasma-DAO activity is influenced by vitamin B-6 status.

Zinc supplementation during pregnancy in low-income teenagers of Mexican descent: effects on selected blood constituents and on progress and outcome of pregnancy.

As a follow-up of our study of pregnant women, we report effects of zinc supplementation during pregnancy in another population of 138 Hispanic teenagers in Los Angeles. Teenagers were randomized (double-blind) to a control or zinc-supplemented group and received similar daily vitamin and mineral supplements except for 20 mg zinc added to the zinc-supplemented group's capsules. Initially, mean dietary zinc intakes of both groups were about 50% of the Recommended Dietary Allowance and their mean serum zinc levels did not differ significantly (69.8 +/- 11.2 micrograms/dl in control and 69.0 +/- 11.4 micrograms/dl in zinc-supplemented group). Zinc supplementation did not maintain mean serum zinc levels during pregnancy but, as in our earlier study, it reduced (p = 0.018) the number of low serum zinc values (less than or equal to 53 micrograms/dl) in late pregnancy. Zinc supplementation did not affect outcome of pregnancy but serum zinc levels were lower (p = 0.038) in teenagers with pregnancy-induced hypertension than in normotensives.

Birth control and the private physician: The view from Los Angeles.

PIP: In an effort to elucidate how physicians in the Los Angeles area regard family planning services and how they actually make them available to their patients, a mail survey of a sample of physicians was undertaken in the summer of 1972. Of the 992 questionnaires sent out, 628 usable questionnaires were returned. 70% of the physicians indicated that they believe birth control provision should be a standard service. The highest level of support came from ob-gyns, 85% of whom said they believe it should be standard. This shows a rise since 1957 when 1 study reported that only 29% of nonCatholic physicians and 4% of Catholic physicians believed that birth control provision should be a standard medical service. A study by Silver in 1971 found that 62% of those sampled said it should be standard. The Los Angeles physicians showed some reluctance in initiating discussion of birth control. If requested, 80% would provide help to female minors with parental consent, compared with 54% who would initiate advice to unwed sexually active minors. More than 80% of the internists and more than 90% of the other specialists indicated that they have assisted patients by referral or otherwise in receiving sterilization. About 90% of the ob-gyns and GPs and about 2/3 of the internists and urologists believed that they were adequately qualified to provide birth control services. 5 questions probing the physicians' knowledge of the relative safety, effectiveness, and legality of various birth control methods raised some question as to whether the doctors did not overestimate their qualifications. 1 example of this was that when asked to rank the theoretical effectiveness of oral contraceptives, diaphragm with jelly, vaginal foam, and postcoital douche, almost 1/4 of the specialists failed to give the correct response.^ieng