Association of galectin-1 and galectin-3 with Gemin4 in complexes containing the SMN protein.

In previous studies we showed that galectin-1 and galectin-3 are factors required for the splicing of pre-mRNA, as assayed in a cell-free system. Using a yeast two-hybrid screen with galectin-1 as bait, Gemin4 was identified as a putative interacting protein. Gemin4 is one component of a macromolecular complex containing approximately 15 polypeptides, including SMN (survival of motor neuron) protein. Rabbit anti-galectin-1 co-immunoprecipitated from HeLa cell nuclear extracts, along with galectin-1, polypeptides identified to be in this complex: SMN, Gemin2 and the Sm polypeptides of snRNPs. Direct interaction between Gemin4 and galectin-1 was demonstrated in glutathione S-transferase (GST) pull-down assays. We also found that galectin-3 interacted with Gemin4 and that it constituted one component of the complex co-immunoprecipitated with galectin-1. Indeed, fragments of either Gemin4 or galectin-3 exhibited a dominant negative effect when added to a cell-free splicing assay. For example, a dose-dependent inhibition of splicing was observed in the presence of exogenously added N-terminal domain of galectin-3 polypeptide. In contrast, parallel addition of either the intact galectin-3 polypeptide or the C-terminal domain failed to yield the same effect. Using native gel electrophoresis to detect complexes formed by the splicing extract, we found that with addition of the N-terminal domain the predominant portion of the radiolabeled pre-mRNA was arrested at a position corresponding to the H-complex. Inasmuch as SMN-containing complexes have been implicated in the delivery of snRNPs to the H-complex, these results provide strong evidence that galectin-1 and galectin-3, by interacting with Gemin4, play a role in spliceosome assembly in vivo.

Intrauterine subdural hematoma.

A patient with neonatal macrocephaly due to bilateral chronic subdural hematoma is presented. There was no history of intrauterine trauma or coagulopathy. Such patients are apparently rare. The pathogenesis of intrauterine chronic subdural hematoma in such patients is unclear.

Galectin-3 expression and subcellular localization in senescent human fibroblasts.

Galectin-3 is a galactose/lactose-binding protein (M(r) approximately 30,000), identified as a required factor in the splicing of pre-mRNA. In the LG1 strain of human diploid fibroblasts, galectin-3 could be found in both the nucleus and the cytoplasm of young, proliferating cells. In contrast, the protein was predominantly cytoplasmic in senescent LG1 cells that have lost replicative competence through in vitro culture. Incubation of young cells with leptomycin B, a drug that disrupts the interaction between the leucine-rich nuclear export signal and its receptor, resulted in the accumulation of galectin-3 inside the nucleus. In senescent cells, galectin-3 staining remained cytoplasmic even in the presence of the drug, thus suggesting that the observed localization in the cytoplasm was due to a lack of nuclear import. In heterodikaryons derived from fusion of young and senescent LG1 cells, the predominant phenotype was galectin-3 in both nuclei. These results suggest that senescent LG1 cells might lack a factor(s) specifically required for galectin-3 nuclear import.

Export of galectin-3 from nuclei of digitonin-permeabilized mouse 3T3 fibroblasts.

Galectin-3 is a galactose-/lactose-binding protein (M(r) approximately 30,000), identified as a required factor in the splicing of pre-mRNA. Immunofluorescence staining revealed that galectin-3 distributes differentially between the nucleus and the cytoplasm, depending on the proliferative state of the cells under analysis. Using digitonin-permeabilized mouse 3T3 fibroblasts, we provide evidence that galectin-3 is rapidly and selectively exported from the nucleus. Although both phosphorylated and nonphosphorylated isoforms of galectin-3 are found in the nuclear fraction, only phosphorylated galectin-3 is identified in the exported fraction, implying that phosphorylation is important for the nuclear export of the protein. The rate of galectin-3 export is temperature dependent and is decreased by the addition of wheat germ agglutinin. More strikingly, galectin-3 export can be inhibited by the addition of leptomycin B, a drug that disrupts the interaction between the leucine-rich nuclear export signal and its receptor, CRM1 (chromosome maintenance region 1). Indeed, a putative leucine-rich nuclear export signal can be found in residues 241-249 of the murine galectin-3 sequence. Finally, gel filtration of the exported material showed that galectin-3 can be found in at least two high molecular weight complexes (approximately 650 and approximately 60 kDa), both of which can be disrupted by lactose.

A comparative nuclear localization study of galectin-1 with other splicing components.

Using both conventional and laser confocal fluorescence microscopy, the intracellular distribution of galectin-1 in HeLa cells was analyzed and compared with the localization of previously documented markers of the nucleus and cytoplasm. The Sm epitopes of the small nuclear ribonucleoprotein complexes (snRNPs) and the non-snRNP splicing factor SC35 yielded only nuclear staining. On the other hand, the enzyme lactate dehydrogenase was cytoplasmic. In contrast to these patterns in which nuclear versus cytoplasmic localizations appeared to be mutually exclusive, galectin-1, as well as galectin-3, yielded simultaneous nuclear and cytoplasmic staining. Confocal microscopy showed galectin-1 fluorescence throughout most of the sections from the top of the cell to the bottom. Through the middle sections, as the plane of focus cuts through the nucleus, there was definite fluorescence staining in the nuclear compartment. This nuclear localization was critically dependent on the type of detergent used to permeabilize the cell: cells treated with saponin or digitonin yielded exclusively cytoplasmic staining while Triton X-100-treated cells showed nuclear as well as cytoplasmic labeling. Finally, double-immunofluorescence analysis showed that, within the nucleoplasm, the following pairs of nuclear antigens could be colocalized in certain speckled structures: (a) SC35 versus Sm; (b) galectin-1 versus Sm; (c) galectin-3 versus Sm; and (d) galectin-1 versus galectin-3. These results establish the presence of galectin-1 in the nuclei of HeLa cells, a conclusion consistent with the identification of the protein in nuclear extracts of the same cells and with its documentation as a factor in pre-mRNA splicing.

Atretic parietal cephaloceles revisited: an enlarging clinical and imaging spectrum?

PURPOSE: We describe imaging features that are clues to the diagnosis of atretic cephaloceles and discuss clinical findings and a possible mechanism by which these lesions develop. METHODS: Eight children (five girls and three boys) ranging in age from 1 day to 3 years 4 months with midline subscalp lesions underwent radiologic examination with CT or MR imaging. In all cases, the lesions were surgically excised and subjected to pathologic examination. Imaging studies and medical records were reviewed retrospectively. RESULTS: Six of eight children had vertical embryonic positioning of the straight sinus with a prominent superior cerebellar cistern. A "spinning-top" configuration of the tentorial incisura, a "cigar-shaped" CSF tract within the interhemispheric fissure, fenestration of the superior sagittal sinus, and "peaking" of the tentorium were associated findings helpful in making this diagnosis. Two of the eight children had findings indistinguishable from focal dermoid, six were developmentally normal, one had mild motor delay, and one died at the age of 3 years. Pathologic examination revealed glial, meningeal (arachnoid), fibrous, and dermal elements. CONCLUSION: Characteristic findings on MR images and CT scans provide clues to the diagnosis of atretic cephalocele. However, even in the presence of abnormal imaging findings, these children may be developmentally normal.

Evidence for a role for galectin-1 in pre-mRNA splicing.

Galectins are a family of beta-galactoside-binding proteins that contain characteristic amino acid sequences in the carbohydrate recognition domain (CRD) of the polypeptide. The polypeptide of galectin-1 contains a single domain, the CRD. The polypeptide of galectin-3 has two domains, a carboxyl-terminal CRD fused onto a proline- and glycine-rich amino-terminal domain. In previous studies, we showed that galectin-3 is a required factor in the splicing of nuclear pre-mRNA, assayed in a cell-free system. We now document that (i) nuclear extracts derived from HeLa cells contain both galectins-1 and -3; (ii) depletion of both galectins from the nuclear extract either by lactose affinity adsorption or by double-antibody adsorption results in a concomitant loss of splicing activity; (iii) depletion of either galectin-1 or galectin-3 by specific antibody adsorption fails to remove all of the splicing activity, and the residual splicing activity is still saccharide inhibitable; (iv) either galectin-1 or galectin-3 alone is sufficient to reconstitute, at least partially, the splicing activity of nuclear extracts depleted of both galectins; and (v) although the carbohydrate recognition domain of galectin-3 (or galectin-1) is sufficient to restore splicing activity to a galectin-depleted nuclear extract, the concentration required for reconstitution is greater than that of the full-length galectin-3 polypeptide. Consistent with these functional results, double-immunofluorescence analyses show that within the nucleus, galectin-3 colocalizes with the speckled structures observed with splicing factor SC35. Similar results are also obtained with galectin-1, although in this case, there are areas of galectin-1 devoid of SC35 and vice versa. Thus, nuclear galectins exhibit functional redundancy in their splicing activity and partition, at least partially, in the nucleoplasm with another known splicing factor.

Hypertensive encephalopathy in children.

We present five cases of hypertensive encephalopathy in children, three with MR imaging findings and two with CT findings alone. One of the five patients had MR perfusion imaging, which showed perfusion abnormalities that support the concept of vasodilation as the major contributor to the syndrome. Hypertensive encephalopathy is rarely reported in children, and its true prevalence may be underestimated. Characteristic lesions in the severely hypertensive child should be recognized as manifestations of hypertensive encephalopathy, and subsequent clinical management should focus on treatment of the hypertension and/or its underlying causes.

Adolescent parenting: outcomes and maternal perceptions.

OBJECTIVE: To describe selected outcomes and maternal perceptions of adolescent parenting. DESIGN: Qualitative and quantitative methods, interview, and two standardized instruments were combined in this follow-up study of adolescents who received perinatal services between 1985 and 1988. SETTING: Data were collected in the mothers' homes. PARTICIPANTS: Mothers who were randomly selected for an earlier chart outcome audit (N = 98) and could be located (n = 19). MAIN OUTCOME MEASURES: Subsequent pregnancies; school completion; children's development, indicated by the Developmental Profile II (DPII); parental attitudes, indicated by the Adult-Adolescent Parenting Inventory (AAPI); and maternal perceptions. RESULTS: Responses revealed irregular use of contraceptives as one reason for the initial pregnancy and for subsequent unplanned pregnancies. Sixteen mothers completed high school, and 18 intend to complete postsecondary programs. The DPII indicated age-appropriate development of the children. AAPI scores for 84% of the mothers indicated nonnurturing attitudes. Mothers described family support, motherhood, and their children. CONCLUSIONS: Research is needed with larger samples and to test interventions to promote regular use of contraception. Findings support the need for research-based programs to educate and promote the development of adolescent mothers and their children.

PIP: This study uses quantitative and qualitative methods for describing the nature of US adolescent mothers' pregnancies and parenting experiences and children's health and development. The sample was drawn from mothers who attended the Teen Obstetrical Perinatal and Parenting Service (TOPPS) clinic at the University of Arkansas for Medical Sciences during 1985-88 and were followed up later in their homes. Reference is made in the literature review to five child-rearing practices among adolescent mothers: insensitivity to infant cues, preference for physical punishment, a pattern of nonverbal interaction, lack of knowledge of child development, and an inadequate learning environment in the home. Descriptive statistics among the study population apply to pregnancy, education, developmental status of children, parenting attitudes, and maternal perceptions of family support, clinic advice, and the maternal role variables. Mothers reported on the status of child health and nutrition, positive and negative characteristics of their child, and discipline. In general, adolescent mothers were found to become pregnant largely due to misunderstandings about reproduction and birth control. Adolescent mothers continued to be at risk for subsequent unplanned pregnancies. Most adolescent mothers completed high school. Most children were developmentally on track for their age. Adolescent mothers were found to be at risk for non-nurturing behaviors, such as inappropriate expectations and reversal of parenting roles. Mothers could identify available support systems. Most would have postponed the pregnancy. Most had realistic perceptions of their children and provided basic health care. It is suggested that adolescent mothers should continue to receive developmentally appropriate services. Education about abstinence and contraception is needed as well as education aimed at promoting self-esteem, interpersonal skills, and age-appropriate development of adolescent parents and children.

Identification of galectin-3 as a factor in pre-mRNA splicing.

Galectin-3 (M(r) approximately 35,000) is a galactose/lactose-specific lectin found in association with ribonucleoprotein complexes in many animal cells. Cell-free-splicing assays have been carried out to study the requirement for galectin-3 in RNA processing by HeLa cell nuclear extracts by using 32P-labeled MINX as the pre-mRNA substrate. Addition of saccharides that bind galectin-3 with high affinity inhibited product formation in the splicing assay, while addition of carbohydrates that do not bind to the lectin did not inhibit product formation. Nuclear extracts depleted of galectin-3 by affinity adsorption on a lactose-agarose column were deficient in splicing activity. Extracts subjected to parallel adsorption on control cellobiose-agarose retained splicing activity. The activity of the galectin-3-depleted extract could be reconstituted by the addition of purified recombinant galectin-3, whereas the addition of other lectins, either with a similar saccharide binding specificity (soybean agglutinin) or with a different specificity (wheat germ agglutinin), did not restore splicing activity. The formation of splicing complexes was also sensitive to galectin-3 depletion and reconstitution. Together, these results define a requirement for galectin-3 in pre-mRNA splicing and identify it as a splicing factor.

Evaluation of a clinic for pregnant adolescents.

A random retrospective review of the Teen Obstetrical Parenting Perinatal Services (TOPPS) clinic medical records for 1985 to 1989 was completed on 120 adolescent mothers (30 charts for each year). The purpose of this study was to evaluate maternal and infant outcomes related to the goals of the TOPPS clinic located at University Hospital on the UAMS campus in Little Rock, Arkansas. The clinic was cofounded by Lee Lee Doyle, Ph.D., who is now professor of obstetrics/gynecology at UAMS, and Betty Rouse, R.N., M.N.Sc., who is a clinical associate professor at UAMS, College of Nursing. The outcomes measured were nutritional status as measured by maternal weight gain, infant birth weight, gestational age and Apgar scores. Referrals to appropriate agencies during pregnancy were also reviewed. Analysis of the data revealed that 31% of clients received documented nutritional counseling, 60.2% of the babies were healthy (88% term and 87% appropriate for gestational age), and documented referrals (i.e. WIC, AFDC, Medicaid, etc.) were made in 32% of the cases. Conclusions were that both mothers and infants had positive outcomes. Documentation of referrals needs to be improved or rationale stated for non-referral.

Phenotypic conversion of TK-deficient cells following electroporation of functional TK enzyme.

The ability to phenotypically rescue a mutant (Rat-3, thymidine kinase-deficient) cell line by electroporation of functional TK enzyme has been investigated. Extracts of electroporated cells showed a 35-fold increase in TK enzyme levels under conditions where greater than 90% of the cells remained viable. The electroporated enzyme was intracellular, as demonstrated by the fact that cells were able to utilize exogenous [3H]thymidine for DNA synthesis. By in situ autoradiography, 82% of electroporated cells contained functional enzyme and incorporated [3H]thymidine into DNA. Thus, this technique can efficiently provide a missing metabolic function to cultured mammalian cells.

The toddler's cuboid fracture.

The authors describe two proved and two presumed cases of cuboid fractures in toddlers. These children were seen because of their inability to bear weight on the affected foot following a fall. Initial radiographs were normal; however, early scintigraphy revealed focal uptake in the cuboid. Follow-up radiographs demonstrated characteristic sclerosis of the base of the cuboid. Cuboid fractures are another example of a toddler's injury that may be difficult to diagnose at initial physical and radiographic examination.

Chest radiographs in the evaluation of the febrile infant.

Chest radiographs are often considered an essential part of the workup of the febrile infant. Anteroposterior and lateral radiographs of the chest are frequently obtained in this group of patients, irrespective of respiratory tract symptoms and/or signs. A total of 226 children (less than or equal to 2 years old) with and without signs and symptoms of lower respiratory tract infections were examined to assess the yield of chest radiographs. The radiograph was considered positive only if a focal parenchymal infiltrate was present. Hyperinflation or bronchial thickening was not included as a positive finding because these children usually do not receive antibiotics despite the fact that viral illness or reactive airway disease may be present. In a retrospective study of 105 infants, confidence intervals for yield were established for children with (95% Cl = 12%, 32%) and without (95% Cl = 0%, 14%) symptoms or signs of lower respiratory tract infection. In a prospective study of 121 infants without chest symptoms or signs, confidence levels for positive yield were better defined (95% Cl = 0%, 3%). The data suggest that obtaining chest radiographs to look for parenchymal infiltrates treatable with antibiotics in infants less than 2 years old is necessary only in those infants who have clinical evidence of lower respiratory tract illness.

RNA metabolism in nuclei: adenovirus and heat shock alter intranuclear RNA compartmentalization.

Nuclear RNA compartmentalization has received little attention as an area where regulation of gene expression could occur. RNA transcription and processing occur in association with the nuclear matrix, a salt-insoluble proteinaceous network that fills the nuclear space and is contiguous with the peripheral lamina and pore complexes. Described here are experiments that determine the fate of nuclear RNA after it has completed these matrix-associated maturation steps. Continuous label experiments indicate that after nuclear RNA is processed it changes its state of attachment in the nucleus so that it is now removed from the nucleus in the high salt extraction step of matrix isolation. It is this salt-extractable RNA that will be transported to the cytoplasm. Late in adenovirus infection and following heat shock, when transport of cellular RNA is decreased, cellular transcripts do not make the transition from the matrix-associated to the salt-extractable nuclear pool. The implication of these data for the regulation of gene expression is discussed.

Reconstitution of the U1 small nuclear ribonucleoprotein particle.

Although the U1 small nuclear ribonucleoprotein particle (snRNP) was the first mRNA-splicing cofactor to be identified, the manner in which it functions in splicing is not precisely understood. Among the information required to understand how U1 snRNP participates in splicing, it will be necessary to know its structure. Here we describe the in vitro reconstitution of a particle that possesses the properties of native U1 snRNP. 32P-labeled U1 RNA was transcribed from an SP6 promoter-human U1 gene clone and incubated in a HeLa S100 fraction. A U1 particle formed which displayed the same sedimentation coefficient (approximately 10S) and buoyant density (1.40 g/cm3) as native U1 snRNP. The latter value reflects the ability to withstand isopycnic banding in Cs2SO4 without prior fixation, a property shared by native U1 snRNP. The reconstituted U1 particle reacted with both the Sm and RNP monoclonal antibodies, showing that these two classes of snRNP proteins were present. Moreover, the reconstituted U1 snRNP particle was found to display the characteristic Mg2+ switch of nuclease sensitivity previously described for native U1 snRNP: an open, nuclease-sensitive conformation at a low Mg2+ concentration (3 mM) and a more compact, nuclease-resistant organization at a higher concentration (15 mM). The majority of the U1 RNA in the reconstituted particle did not contain hypermethylated caps, pseudouridine, or ribose 2-O-methylation, showing that these enigmatic posttranscriptional modifications are not essential for reconstitution of the U1 snRNP particle. The extreme 3' end (18 nucleotides) of U1 RNA was required for reconstitution, but loop II (nucleotides 64 to 77) was not. Interestingly, the 5' end (15 nucleotides) of U1 RNA that recognizes pre-mRNA 5' splice sites was not required for U1 snRNP reconstruction.