Protein carboxyl methyltransferase activity specific for age-modified aspartyl residues in mouse testes and ovaries: Evidence for translation during spermiogenesis

An antiserum prepared against the purified protein carboxyl methltransferase (PCMT) from bovine brain has been used to compare testicular and ovarian levels of the enzyme and to study the regulation of PCMT concentrations during spermatogenesis. The PCMT, which specifically modifies age-damaged aspartyl residues, is present at a significantly higher concentration in mature mouse testis than in ovary. However, the PCMT is present at nearly equal concentrations in extracts of germ cell-deficient ovaries and testes obtained from mutant atrichosislatrichosis mice. In normal testis, the concentration of the PCMT increases severalfold during the first 4–5 weeks after birth, paralleling the appearance and maturation of testicular germ cells. Both immunochemical and enzymatic measurements of PCMT specific activities in purified spermatogenic cell preparations indicate that PCMT levels are twofold and 3.5-fold higher in round spermatids and residual bodies, respectively, than in pachytene spermatocytes. The results are consistent with the enhanced synthesis and/or stability of the PCMT in spermatogenic cells and with the continued translation of the PCMT during the haploid portion of spermatogenesis. The relatively high levels of PCMT in spermatogenic cells may be important for the extensive metabolism of proteins accompanying spermatid condensation or for the repair of damaged proteins in translationally inactive spermatozoa.     

A Critical Tryptophan and Ca2+ in Activation and Catalysis of TPPI,the Enzyme Deficient in Classic Late-Infantile Neuronal Ceroid Lipofuscinosis

Background

Tripeptidyl aminopeptidase I (TPPI) is a crucial lysosomal enzyme that is deficient in the fatal neurodegenerative disorder called classic late-infantile neuronal ceroid lipofuscinosis (LINCL). It is involved in the catabolism of proteins in the lysosomes. Recent X-ray crystallographic studies have provided insights into the structural/functional aspects of TPPI catalysis, and indicated presence of an octahedrally coordinated Ca²⁺.

Methodology

Purified precursor and mature TPPI were used to study inhibition by NBS and EDTA using biochemical and immunological approaches. Site-directed mutagenesis with confocal imaging technique identified a critical W residue in TPPI activity, and the processing of precursor into mature enzyme.

Principal Findings

NBS is a potent inhibitor of the purified TPPI. In mammalian TPPI, W542 is critical for tripeptidyl peptidase activity as well as autocatalysis. Transfection studies have indicated that mutants of the TPPI that harbor residues other than W at position 542 have delayed processing, and are retained in the ER rather than transported to lysosomes. EDTA inhibits the autocatalytic processing of the precursor TPPI.

Conclusions/Significance

We propose that W542 and Ca²⁺ are critical for maintaining the proper tertiary structure of the precursor proprotein as well as the mature TPPI. Additionally, Ca²⁺ is necessary for the autocatalytic processing of the precursor protein into the mature TPPI. We have identified NBS as a potent TPPI inhibitor, which led in delineating a critical role for W542 residue. Studies with such compounds will prove valuable in identifying the critical residues in the TPPI catalysis and its structure-function analysis.     

LRGUK-1 Is Required for Basal Body and Manchette Function during Spermatogenesis and Male Fertility

Male infertility affects at least 5% of reproductive age males. The most common pathology is a complex presentation of decreased sperm output and abnormal sperm shape and motility referred to as oligoasthenoteratospermia (OAT). For the majority of OAT men a precise diagnosis cannot be provided. Here we demonstrate that leucine-rich repeats and guanylate kinase-domain containing isoform 1 (LRGUK-1) is required for multiple aspects of sperm assembly, including acrosome attachment, sperm head shaping and the initiation of the axoneme growth to form the core of the sperm tail. Specifically, LRGUK-1 is required for basal body attachment to the plasma membrane, the appropriate formation of the sub-distal appendages, the extension of axoneme microtubules and for microtubule movement and organisation within the manchette. Manchette dysfunction leads to abnormal sperm head shaping. Several of these functions may be achieved in association with the LRGUK-1 binding partner HOOK2. Collectively, these data establish LRGUK-1 as a major determinant of microtubule structure within the male germ line.     

Safety and efficacy of botox injection in alleviating post-operative pain and improving quality of life in lower extremity limb lengthening and deformity correction

Background

Distraction osteogenesis is the standard treatment for the management of lower limb length discrepancy of more than 3 cm and bone loss secondary to congenital anomalies, trauma or infection. This technique consists of an osteotomy of the bone to be lengthened, application of an external fixator, followed by gradual and controlled distraction of the bone ends. Although limb lengthening using the Ilizarov distraction osteogenesis principle yields excellent results in most cases, the technique has numerous problems and is not well tolerated by many children. The objective of the current study is to determine if Botulinum Toxin A (BTX-A), which is known to possess both analgesic and paralytic actions, can be used to alleviate post-operative pain and improve the functional outcome of children undergoing distraction osteogenesis.

Methods/Design

The study design consists of a multi centre, randomized, double-blinded, placebo-controlled trial. Patients between ages 5–21 years requiring limb lengthening or deformity correction using distraction will be recruited from 6 different sites (Shriners Hospital for Children in Montreal, Honolulu, Philadelphia and Portland as well as DuPont Hospital for Children in Wilmington, Delaware and Hospital for Sick Children in Toronto, Ont). Approximately 150 subjects will be recruited over 2 years and will be randomized to either receive 10 units per Kg of BTX-A or normal saline (control group) intraoperatively following the surgery. Functional outcome effects will be assessed using pain scores, medication dosages, range of motion, flexibility, strength, mobility function and quality of life of the patient. IRB approval was obtained from all sites and adverse reactions will be monitored vigorously and reported to IRB, FDA and Health Canada.

Discussion

BTX-A injection has been widely used world wide with no major side effects reported. However, to the best of our knowledge, this is the first time BTX-A is being used under the context of limb lengthening and deformity correction.

Trial Registration

NCT00412035     

Bmpr1a signaling plays critical roles in palatal shelf growth and palatal bone formation

Cleft palate, including submucous cleft palate, is among the most common birth defects in humans. While overt cleft palate results from defects in growth or fusion of the developing palatal shelves, submucous cleft palate is characterized by defects in palatal bones. In this report, we show that the Bmpr1a gene, encoding a type I receptor for bone morphogenetic proteins (Bmp), is preferentially expressed in the primary palate and anterior secondary palate during palatal outgrowth. Following palatal fusion, Bmpr1a mRNA expression was upregulated in the condensed mesenchyme progenitors of palatal bone. Tissue-specific inactivation of Bmpr1a in the developing palatal mesenchyme in mice caused reduced cell proliferation in the primary and anterior secondary palate, resulting in partial cleft of the anterior palate at birth. Expression of Msx1 and Fgf10 was downregulated in the anterior palate mesenchyme and expression of Shh was downregulated in the anterior palatal epithelium in the Bmpr1a conditional mutant embryos, indicating that Bmp signaling regulates mesenchymal-epithelial interactions during palatal outgrowth. In addition, formation of the palatal processes of the maxilla was blocked while formation of the palatal processes of the palatine was significantly delayed, resulting in submucous cleft of the hard palate in the mutant mice. Our data indicate that Bmp signaling plays critical roles in the regulation of palatal mesenchyme condensation and osteoblast differentiation during palatal bone formation.     

SynTReN: a generator of synthetic gene expression data for design and analysis of structure learning algorithms

Background  

The development of algorithms to infer the structure of gene regulatory networks based on expression data is an important subject in bioinformatics research. Validation of these algorithms requires benchmark data sets for which the underlying network is known. Since experimental data sets of the appropriate size and design are usually not available, there is a clear need to generate well-characterized synthetic data sets that allow thorough testing of learning algorithms in a fast and reproducible manner.     

Thrombospondin 1 mediates angiotensin II induction of TGF-beta activation by cardiac and renal cells under both high and low glucose conditions

Renal and cardiac fibrosis leading to organ failure are complications of both diabetes and hypertension. These disease processes, when combined, exacerbate development of fibrotic complications. Control of latent transforming growth factor (TGF)-β activation is a potential determinant of fibrotic progression. Both glucose and angiotensin II (Ang II) upregulate thrombospondin-1 (TSP1), a major activator of latent TGF-β, and stimulate increased TGF-β activity. We previously showed that high glucose stimulated TSP1-dependent TGF-β activation in rat mesangial cells (RMCs). In this paper, we examined whether Ang II similarly upregulates TSP1 production and TSP1-dependent TGF-β activation alone or in combination with high glucose concentrations. Ang II and high glucose stimulated increases in TSP1 protein levels in the conditioned media of both rat cardiac fibroblasts (RCFs) and rat mesangial cells (RMCs). Meanwhile, Ang II stimulated increases in both TGF-β activity and protein by RMCs, whereas, RCFs responded to both Ang II and high glucose with increased TGF-β activity in the absence of altered TGF-β protein levels. A combination of Ang II and high glucose induced synergistic TGF-β activation by RCFs. Moreover, Ang II induction of TSP1 and increased TGF-β activity were blocked by losartan, an antagonist of the Ang II type 1 (AT1) receptor. The increase in TSP1 expression leads to increased TGF-β activity upon Ang II and/or glucose treatment, since peptide antagonists of TSP1-mediated TGF-β activation blocked Ang II and glucose-induced TGF-β activation. Our data support a role for TSP1 in the development and progression of renal and cardiac fibrosis in hypertension and diabetes.     

Islet hypertrophy following pancreatic disruption of Smad4 signaling

To investigate the role of transforming growth factor (TGF)-beta family signaling in the adult pancreas, a transgenic mouse (E-dnSmad4) was created that expresses a dominant-negative Smad4 protein driven by a fragment of the elastase promoter. Although E-dnSmad4 mice have normal growth, pancreas weight, and pancreatic exocrine and ductal histology, beginning at 4-6 wk of age, E-dnSmad4 mice show an age-dependent increase in the size of islets. In parallel, an expanded population of replicating cells expressing the E-dnSmad4 transgene is found in the stroma between the enlarged islets and pancreatic ducts. Despite the marked enlargement, E-dnSmad4 islets contain normal ratios and spatial organization of endocrine cell subtypes and have normal glucose homeostasis. Replication of cells derived from primary duct cultures of wild-type mice, but not E-dnSmad4 mice, was inhibited by the addition of TGF-beta family proteins, demonstrating a cell-autonomous effect of the transgene. These data show that, in the adult pancreas, TGF-beta family signaling plays a role in islet size by regulating the growth of a pluripotent progenitor cell residing in the periductal stroma of the pancreas.