Distribution of pulmonary vascular pressure as a function of perinatal age in lambs

The distribution of pulmonary vascular resistance (PVR) with respect to compliance was determined using vascular occlusion in isolated lungs from lambs at five ages, from 2 wk before birth to 1 mo of age. The major change in PVR occurred in the pressure gradient across the middle compliant region (delta Pm), which dropped sharply at birth, remained low for 2 wk, and increased at 1 mo. Pulmonary vasoreactivity also varied with ages. Lungs at 0-4 days did not respond to hypoxia and responded poorly to prostaglandin F2 alpha (PGF2 alpha). In contrast, lungs at 13-33 days had significant increases in delta Pm and the gradient across relatively indistensible arterial vessels during hypoxia and increases in all gradients with PGF2 alpha. Ventilation of fetal lungs reduced PVR, mainly because of a 50% reduction in delta Pm. Our results demonstrate that the magnitude and distribution of PVR relative to compliance varied as a function of perinatal age and that pulmonary vasoreactivity depended on postnatal age. The major effect of ventilating fetal lungs was on the middle region.     

Isolation and characterization of Sertoli cell plasma membranes and associated plasminogen activator activity

Plasma membranes were isolated from the cultured Sertoli cells of 20-day-old rat testes by differential centrifugation and sucrose density fractionation. The distribution and purity of subcellular components was determined by marker enzyme analysis of gradient fractions. The plasma membrane fraction showed an enrichment in two plasma membrane marker enzymes, 5'-nucleotidase and ouabain-sensitive Na+/K+-ATPase-specific activities, of 9- and 23-fold, respectively. Forty-two percent and 52% of the total cellular 5'-nucleotidase and ouabain-sensitive Na+/K+-ATPase activities, respectively, were found in the membrane fraction. The protein yield of plasma membrane was approximately 6% of the total cellular protein. Two-dimensional polyacrylamide gel electrophoresis was used to compare [35S] methionine- and [3H] glucosamine-labeled membrane proteins. The incorporation of [35S] methionine and [3H] glucosamine was increased in several proteins when the cultured Sertoli cells were treated with follicle-stimulating hormone, insulin, retinol, and testosterone. Isolated Sertoli cell membranes contained a membrane-associated form of plasminogen activator. Analysis of this plasminogen activator demonstrated that the membrane-associated enzyme existed primarily as a single 38,000-40,000-Mr form.     

Localization of sulfated glycoprotein-2 (clusterin) on spermatozoa and in the reproductive tract of the male rat

Sulfated glycoprotein-2 (SGP-2) is one of the major proteins secreted by rat Sertoli cells and epididymal cells in culture. The disulfide-linked dimeric protein secreted by Sertoli cells and found in seminiferous tubule fluid is composed of monomers of Mr 47 000 and 34 000 whereas the epididymal protein exhibits monomers of Mr 40 000 and 29 000. When both forms were chemically or enzymatically deglycosylated, they yielded proteins of similar molecular weight. No modification of the higher molecular weight testicular form by epididymal cells or fluids could be detected in incubation media. SGP-2 mRNA was localized in epididymal epithelium by in situ hybridization. Northern blot analysis indicated the testicular and epididymal mRNAs were of similar size. These findings suggest that the two forms of the protein occur because of tissue-specific post-translational modifications. The detergent-extracted protein from washed testicular spermatozoa is of the higher molecular weight form while epididymal sperm carry the lower molecular weight form. Immunohistochemical evidence suggests that the testicular form is removed prior to the initial segment of the epididymis and the epididymal form is applied in the proximal caput epididymidis. SGP-2 was immunolocalized to the sperm membrane at the ultrastructural level and was distinctly different from the immunolocalization of outer dense fiber proteins and fibrous sheath proteins.     

Beyond ribosomal DNA: on towards the telomere

We have sequenced and analyzed 8.3 kb of sequence adjacent and distal to the human ribosomal DNA (rDNA); this distal sequence connects to the rDNA cluster just 4 kb upstream of the first promoter and is shared among the acrocentric chromosomes and, at least in part, it is also present in other primates. The sequence differs in character from that of the rDNA intergenic spacer (IGS) in that it does not contain long stretches of either polypyrimidine or polypurine. However, just like the IGS, it contains numerous repetitive elements, including retroposed fragments of 28S rRNA and large pieces of the IGS. In addition, we show that the rDNA clusters are not interrupted by other sequences and do not recombine with this distal segment. Received: 9 September 1996; in revised form: 4 February 1997 / Accepted: 24 February 1997     

Prohibitin as the Molecular Binding Switch in the Retinal Pigment Epithelium

Previously, our molecular binding study showed that prohibitin interacts with phospholipids, including phosphatidylinositide and cardiolipin. Under stress conditions, prohibitin interacts with cardiolipin as a retrograde response to activate mitochondrial proliferation. The lipid-binding switch mechanism of prohibitin with phosphatidylinositol-3,4,5-triphosphate and cardiolipin may suggest the role of prohibitin effects on energy metabolism and age-related diseases. The current study examined the region-specific expressions of prohibitin with respect to the retina and retinal pigment epithelium (RPE) in age-related macular degeneration (AMD). A detailed understanding of prohibitin binding with lipids, nucleotides, and proteins shown in the current study may suggest how molecular interactions control apoptosis and how we can intervene against the apoptotic pathway in AMD. Our data imply that decreased prohibitin in the peripheral RPE is a significant step leading to mitochondrial dysfunction that may promote AMD progression.     

New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein

Cytoplasmic Polyadenylation Element Binding proteins (CPEBs) are a family of polyadenylation factors interacting with 3’UTRs of mRNA and thereby regulating gene expression. Various functions of CPEBs in development, synaptic plasticity, and cellular senescence have been reported. Four CPEB family members of partially overlapping functions have been described to date, each containing a distinct alternatively spliced region. This region is highly conserved between CPEBs-2-4 and contains a putative phosphorylation consensus, overlapping with the exon seven of CPEB3. We previously found CPEBs-2-4 splice isoforms containing exon seven to be predominantly present in neurons, and the isoform expression pattern to be cell type-specific. Here, focusing on the alternatively spliced region of CPEB3, we determined that putative neuronal isoforms of CPEB3 are phosphorylated. Using a new phosphospecific antibody directed to the phosphorylation consensus we found Protein Kinase A and Calcium/Calmodulin-dependent Protein Kinase II to robustly phosphorylate CPEB3 in vitro and in primary hippocampal neurons. Interestingly, status epilepticus induced by systemic kainate injection in mice led to specific upregulation of the CPEB3 isoforms containing exon seven. Extensive analysis of CPEB3 phosphorylation in vitro revealed two other phosphorylation sites. In addition, we found plethora of potential kinases that might be targeting the alternatively spliced kinase consensus site of CPEB3. As this site is highly conserved between the CPEB family members, we suggest the existence of a splicing-based regulatory mechanism of CPEB function, and describe a robust phosphospecific antibody to study it in future.     

Ribosomal RNA gene sequences and hominoid phylogeny

Sequences totaling 3,500 bases from the 28S rRNA gene and from one of the ribosomal internal transcribed spacers (ITS1) have been determined for human, chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), and orangutan (Pongo pygmaeus). Analyses of the rRNA alignments show (1) a clustering of substitutions in the "variable regions" of the 28S gene, (2) a 1.5-3-fold increase in divergence in the transcribed spacer over that in the exon, and (3) that human and chimpanzee are the most closely related pair, in agreement with the results of Miyamoto et al., Sibley and Ahlquist, and Caccone and Powell.