Pneumatoceles in preterm infants-incidence and outcome in the post-surfactant era.

OBJECTIVE: Pneumatoceles are gas-filled cysts within the lung parenchyma resulting mostly from ventilator-induced lung injury and air-leak in premature infants with respiratory distress syndrome. The use of surfactant in the treatment of respiratory distress syndrome has resulted in a decrease in the incidence of air-leak disease. Our aim was to study the incidence and clinical course of pneumatoceles in the surfactant era. STUDY DESIGN: A retrospective study of infants born at < or =30 weeks gestational age was admitted to the University of Connecticut Health Center NICU from 1998 to 2007. Pneumatoceles and other intrathoracic air-leaks were identified and comparisons were made with infants without these conditions. RESULT: Pneumatoceles were identified in 19 preterm infants, born at gestational age < or =30 weeks, needing positive pressure ventilation for respiratory distress syndrome between the years 1998 to 2007. Pneumatoceles appeared early (median, 7th day of life; range, 1st to 28th day of life) and usually resolved with decrease in mean airway pressure (median, 4 days; range, 3 to 125 days). The majority of pneumatoceles were located in the right parahilar region (18/19). Associated intrathoracic air-leaks were pulmonary interstitial emphysema (5/19), pneumothorax (10/19), and pneumomediastinum (1/19). None of the infants required any invasive procedures to alleviate the pneumatoceles. In infants who survived, most pneumatoceles resolved with a decrease in mean airway pressure or extubation (14/15). One infant had a persistent pneumatocele for 125 days without any cardiopulmonary compromise and five infants died as a result air-leaks along with other complications of prematurity. CONCLUSION: Pneumatoceles are a manifestation of intrathoracic air-leaks of prematurity. They are markers for ventilator-induced lung injury and are associated with significant mortality similar to other intrathoracic air-leaks. However, conservative management with reduction in mean airway pressure is effective in the resolution of this condition and interventional decompression of the pneumatocele is generally not necessary.

Vasa homolog genes in mammalian germ cell development.

Many vasa homologue genes to Drosophila vasa have been isolated in various animal species. They provide specific molecular probes to analyze the establishment and the differentiation of germ cell lineage. In mammals, the expression of VASA protein becomes detectable in PGCs at the late migrating stage. Interestingly, during spermatogenesis the intracellular localization of VASA protein is closely associated with the chromatoid body.

Phylloid pattern of pigmentary disturbance in a case of complex mosaicism.

Among the various types of pigmentary disturbances associated with mosaicism, the phylloid pattern (Greek phyllon = leaf, eidos = form) is characterized by multiple leaf-like patches reminiscent of an art nouveau painting. The number of cases displaying this unusual pattern is so far limited. We describe a phylloid pattern of hypomelanosis in a 3-year-old girl with multiple congenital anomalies including microcephaly, midfacial hypoplasia, cleft lip, coloboma, posteriorly rotated ears, pectus carinatum, and pronounced mental and physical retardation. In addition, this child had oval or oblong patches of hyperpigmentation involving the trunk in a horizontal arrangement dissimilar from the phylloid hypomelanotic pattern. In peripheral blood lymphocytes a karyotype 46,XX,-13,+t(13q;13q) was consistently found, whereas cultured skin fibroblasts showed a complex form of mosaicism comprising three different abnormal cell lines (46,XX,-13,+t(13q;13q)/45,XX,-13/45,XX,-13,+frag). This case provides further evidence that the phylloid pattern represents a separate category of pigmentary disturbance to be distinguished from other types of cutaneous mosaicism such as the lines of Blaschko or the checkerboard arrangement.

Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells.

To obtain a reliable molecular probe to trace the origin of germ cell lineages in birds, we isolated a chicken homolog (Cvh) to vasa gene (vas), which plays an essential role in germline formation in Drosophila. We demonstrate the germline-specific expression of CVH protein throughout all stages of development. Immunohistochemical analyses using specific antibody raised against CVH protein indicated that CVH protein was localized in cytoplasm of germ cells ranging from presumptive primordial germ cells (PGCs) in uterine-stage embryos to spermatids and oocytes in adult gonads. During the early cleavages, CVH protein was restrictively localized in the basal portion of the cleavage furrow. About 30 CVH-expressing cells were scattered in the central zone of the area pellucida at stage X, later 45-60 cells were found in the hypoblast layer and subsequently 200-250 positive cells were found anteriorly in the germinal crescent due to morphogenetic movement. Furthermore, in the oocytes, CVH protein was predominantly localized in granulofibrillar structures surrounding the mitochondrial cloud and spectrin protein-enriched structure, indicating that the CVH-containing cytoplasmic structure is the precursory germ plasm in the chicken. These results strongly suggest that the chicken germline is determined by maternally inherited factors in the germ plasm.

The mouse homolog of Drosophila Vasa is required for the development of male germ cells.

Restricted expression of a mouse Vasa homolog gene (Mvh) expression is first detected in primordial germ cells (PGCs) after colonization of the genital ridges. Subsequently, Mvh is maintained until postmeiotic germ cells are formed. Here, we demonstrate that male mice homozygous for a targeted mutation of Mvh exhibit a reproductive deficiency. Male homozygotes produce no sperm in the testes, where premeiotic germ cells cease differentiation by the zygotene stage and undergo apoptotic death. In addition, the proliferation of PGCs that colonize homozygous male gonads is significantly hampered, and OCT-3/4 expression appears to be reduced. These results indicate that the loss of Mvh function causes a deficiency in the proliferation and differentiation of mouse male germ cells.

Expression and intracellular localization of mouse Vasa-homologue protein during germ cell development.

To demonstrate the cellular and subcellular localization of mouse vasa homologue protein during germ cell development, specific antibody was raised against the full-length MVH protein. The immunohistochemical analyses demonstrated that MVH protein was exclusively expressed in primordial germ cells just after their colonization of embryonic gonads and in germ cells undergoing gametogenic processes until the post-meiotic stage in both males and females. The co-culture of EG cells with gonadal somatic cells indicated inductive MVH expression caused by an intercellular interaction with gonadal somatic cells. In adult testis, MVH protein was localized in the cytoplasm of spermatogenic cells, including chromatoid bodies in spermatids, known to be a perinuclear nuage structure which includes polar granules that contain VASA protein in Drosophila.

In vivo gene transfer to mouse spermatogenic cells by deoxyribonucleic acid injection into seminiferous tubules and subsequent electroporation.

An in vivo gene transfer technique for living mouse testes was used to develop a novel transient expression assay system for transcriptional regulatory elements of spermatogenic specific genes. The combination of DNA injection into seminiferous tubules and subsequent in vivo electroporation resulted in an efficient and convenient assay system for gene expression during spermatogenesis. The transfer of the firefly luciferase reporting gene driven by the Protamine-1 (Prm-1) enhancer region revealed a significant increase in the activity of the reporter enzyme. Histochemical studies of the transfer of the lacZ gene driven by the Prm-1 enhancer showed specific lacZ expression only in haploid spermatid cells in adult testes, corresponding with the expression pattern of endogenous Prm-1. We were able to detect long-lasting transgene expression in the transfected spermatogenic cells. A group of spermatogenic differentiating cells maintained the transfected lacZ expression after more than 2 mo of transfection, suggesting that spermatogenic stem cells and/or spermatogonia could also incorporate foreign DNA and that the transgene could be transmitted to the progenitor cells derived from a transfected proliferating germ cell.

Enhanced expression in seminoma of human zinc finger genes located on chromosome 19.

Six Krüppel-type zinc finger (ZF) genes were cloned from a seminoma cDNA library. One, ZFS-1, showed high sequence homology to the ZNF91 KRAB (Krüppel-associated box) ZF gene family and also the same chromosomal assignment. Interestingly, Northern blot analyses using ZFS-1 and ZNF91 revealed that multiple ZF genes on chromosome 19 were predominantly expressed in seminomas. In addition, the testis and the seminoma showed specific expression of 2.3 kb transcript. Our results suggest that ZF genes on chromosome 19 may be implicated in the development and/or growth of seminomas.

Identification of two Sox17 messenger RNA isoforms, with and without the high mobility group box region, and their differential expression in mouse spermatogenesis.

The different mRNA isoforms of the mouse Sox17 gene were isolated from adult mouse testis cDNAs. One form (referred to as form Sox17) encodes an Sry-related protein of 419 amino acids containing a single high mobility group box near the NH2-terminus, while the other form (referred to as form t-Sox17) shows a unique mRNA isoform of the Sox17 gene with a partial deletion of the HMG box region. Analysis of genomic DNA revealed that these two isoforms were produced at least by alternative splicing of the exon corresponding to the 5' untranslated region and NH2-terminal 102 amino acids. RNA analyses in the testis revealed that form Sox17 began at the pachytene spermatocyte stage and was highly accumulated in round spermatids. Protein analyses revealed that t-Sox17 isoforms, as well as Sox17 isoforms, were translated into the protein products in the testis, although the amount of t-Sox17 products is lower in comparison to the high accumulation of t-Sox17 mRNA. By the electrophoretic mobility-shift assay and the random selection assay using recombinant Sox17 and t-Sox17 proteins, Sox17 protein is a DNA-binding protein with a similar sequence specificity to Sry and the other members of Sox family proteins, while t-Sox17 shows no apparent DNA-binding activity. Moreover, by a cotransfection experiment using a luciferase reporter gene, Sox17 could stimulate transcription through its binding site, but t-Sox17 had little effect on reporter gene expression. Thus, these findings suggest that Sox17 may function as a transcriptional activator in the premeiotic germ cells, and that a splicing switch into t-Sox17 may lead to the loss of its function in the postmeiotic germ cells.

Congenital shortening of the anterior lamella of all eyelids: the so-called ablepharon macrostomia syndrome.

The so-called ablepharon macrostomia syndrome is an extremely rate congenital condition that includes abnormal ears, an enlarged, fishlike mouth, absence of lanugo, redundant skin, and vertical shortening of all eyelids. Only four cases have been described so far. In these cases the nature of the eyelid anomalies has not been clearly defined. We report one more case showing that the condition is better described as a severe microblepharon because only the anterior lamella of the eyelids is shortened. The literature about this condition is reviewed, and oculoplastic treatment is discussed.

Isolation of a DEAD-family protein gene that encodes a murine homolog of Drosophila vasa and its specific expression in germ cell lineage.

In an effort to study the molecular basis of the determination processes of the mammalian germ cell lineage, we have tried to isolate a mouse gene homolog to vasa, which plays an essential role as a maternal determining factor for the formation of Drosophila germ cell precursors. By reverse transcriptase PCRs of mouse primordial germ cell cDNAs using family-specific primers, we obtained a gene (Mvh) encoding a DEAD-family protein that showed a much higher degree of similarity with the product of the Drosophila vasa gene (vas) than previously reported mouse genes. In adult tissues, Mvh transcripts were exclusively detected in testicular germ cells, in which Mvh protein was found to be localized in cytoplasm of spermatocytes and round spermatids including a perinuclear granule. The protein was also expressed in germ cells colonized in embryonic gonads but was not detected in pluripotential embryonic cells such as stem cells and germ cells. These results suggest the possibility that the Mvh protein may play an important role in the determination events of mouse germ cells as in the case of Drosophila vasa.

A novel murine zinc finger gene mapped within the tw18 deletion region expresses in germ cells and embryonic nervous system.

A novel zinc finger gene, designated NT fin12, belonging to the C2H2-Krüppel-type gene family was isolated from a newborn mouse testis cDNA library by using zinc finger consensus motif probes. Northern blot analyses showed that NT fin12 mRNA was expressed during the meiotic prophase of spermatogenesis and in embryogenesis. Transcripts were localized by in situ hybridization in spermatogonia and in early spermatocytes, and in testis cords in the genital ridge as well as in oocytes and follicle cells in the ovary. In midgestational embryos at 8.5-13.5 days postcoitum, transcripts were present in the neuroectoderm, and they were progressively restricted to peripheral ganglia derived from neural crest cells and neural placodes and to the motor nerve cells in the central nervous system. Taken together these results indicate that NT fin12 functions during germ cell development and also plays a role in the specification of a subpopulation of neuroectodermal cells. Genetic linkage analyses revealed that the NT fin12 locus mapped to the deletion region of the tw18 haplotype on mouse chromosome 17.

Expression of a mouse zinc finger protein gene in both spermatocytes and oocytes during meiosis.

In order to identify genes regulating meiosis, a mouse spermatocyte cDNA library was screened for sequences encoding proteins with C2H2-type zinc finger motifs which are typically expressed by the Drosophila Krüppel gene. Three new cDNAs were isolated, and they were designated CTfin33, CTfin51, and CTfin92. Among them, CTfin51 was selected for further study. The deduced amino acid sequence revealed seven zinc finger motifs in its C-terminal region. Northern blot and in situ hybridization showed CTfin51 mRNA expression in spermatocytes after the pachytene stage and in early stage round spermatids of prepuberal and adult males. Immunocytochemical staining with an antiserum against beta-gal-CTfin51 fusion protein was localized within nuclei of spermatocytes and spermatids. Oocyte nuclei after the pachytene stage also were immunoreactive for CTfin51 protein. Immunoblots revealed a band at M(r) 75,000 in protein extracts from the testis and the ovary. These results suggest that the CTfin51 gene encodes a DNA-binding regulatory protein functionally associated with meiosis in both male and female gametogenesis.

Isolation and characterization of cDNA encoding a spicule matrix protein in Hemicentrotus pulcherrimus micromeres.

A cDNA clone, termed pHPSMC, was obtained from the Japanese sea urchin Hemicentrotus pulcherrimus, and it was found to be highly homologous in sequence to the spicule matrix protein cDNA of Strongylocentrotus purpuratus (Sucov et al., Dev. Biol. 120: 507-519, 1987). During early embryogenesis, mRNA complementary to pHPSMC appeared in gastrulae and remained at a similar level until the pluteus stage. In situ hybridization revealed that the mRNA was localized exclusively in primary mesenchyme cells in gastrulae. pHPSMC mRNA was detected in micromeres in vitro after 48 h of culture, but it was not found in blastomeres immediately after isolation. These features suggested that pHPSMC represents the spicule matrix protein cDNA cognate in Hemicentrotus pulcherrimus. In the derived polypeptide, we detected a domain containing a tandemly repeated 13-amino acid sequence as did Sucov et al. (1987). Unexpectedly, the sequence of the repeated element was completely different from that originally reported for Strongylocentrotus purpuratus, but it was very similar to the corrected sequence that appeared recently.

Molecular cloning and nucleotide sequence analysis of the putative cDNA for the precursor molecule of the chicken LH-beta subunit.

A cDNA expression library was constructed from poly(A)+ RNA of broiler chicken adenohypophyses using lambda gt11 as a vector. After screening with a rabbit antiserum against chicken LH, a cDNA clone (L12) containing a 436 bp insert was obtained. Using a subclone of L12 in pUC19 (pL12) as the hybridization probe, another cDNA clone (LF127) with a 533 bp insert was isolated. The LF127 contained the full-length cDNA encoding the putative chicken LH-beta subunit precursor molecule. Hybridization of the pL12 cDNA insert to adenohypophysial RNA showed that chicken and Japanese quail adenohypophyses contained RNA species of about 0.8 and 1.0 kb respectively. The amount of this RNA species was ten times higher in adult male quails kept under long days at room temperature than in those kept under short days at 7 degrees C. In situ hybridization experiments showed the exclusive distribution of the signal in the LH cells of the adenohypophysis. The similarity of the nucleotide sequence of the apoprotein-coding region of LH-beta cDNA of the chicken to that of mammals is lower than that among mammals. The deduced amino acid sequence of the chicken LH-beta subunit supports the hypothesis that the number of proline residues increases in the LH-beta subunit the closer phylogenetically the vertebrate is to mammals.

Characterization of an unusual cAMP receptor and its related polypeptides in Dictyostelium discoideum.

Several lines of evidence indicate that cAMP modulates developmental gene activity via cell-surface receptors. We describe here a novel cAMP receptor, CABP1, whose properties are consistent with the idea that this protein is involved in gene regulation. Firstly, immunological techniques using anti-CABP1 antibodies as probes showed that this cAMP receptor can be detected on the surface of developing cells. Secondly, there is a steady migration of CABP1 to the nucleus during development. Thirdly, some genetic variants exhibiting an altered pattern of development are found to possess modified CABP1. We also showed that CABP1 co-purifies with at least seven other polypeptides which share common epitopes with CABP1. Interestingly, four of the CABP1-related polypeptides can be detected on the cell surface as well as in the nucleus.