TGFβ alters growth and differentiation related gene expression in proliferating osteoblasts in vitro,preventing development of the mature bone phenotype

This study examines the mechanism by which TGF-β1, an important mediator of cell growth and differentiation, blocks the differentiation of normal rat diploid fetal osteoblasts in vitro. We have established that the inability for pre-osteoblasts to differentiate is associated with changes in the expression of cell growth, matrix forming, and bone related genes. These include histone, jun B, c-fos, collagen, fibronectin, osteocalcin, alkaline phosphatase, and osteopontin. Morphologically, the TGF-β1-treated osteoblasts exhibit an elongated, spread shape as opposed to the characteristic cuboidal appearance during the early stages of growth. This is followed by a decrease in the number of bone nodules formed and the amount of calcium deposition. These effects on differentiation can occur without dramatic changes in cell growth if TGF-β1 is given for a short time early in the proliferative phase. However, continuous exposure to TGF-β1 leads to a bifunctional growth response from a negative effect during the proliferative phase to a positive growth effect during the later matrix maturation and mineralization phases of the osteoblast developmental sequence. Extracellular matrix genes, fibronectin, osteopontin and α1(I) collagen, are altered in their expression pattern which may provide an aberrant matrix environment for mineralization and osteoblast maturation and potentiate the TGF-β1 response throughout the course of osteoblast differentiation. The initiation of a TGF-β1 effect on cell growth and differentiation is restricted to the proliferative phase of the culture before the cells express the mature osteoblastic phenotype. Second passage cells that are accelerated to differentiate by the addition of dexamethasone or by seeding cultures at a high density are refractory to TGF-β1. These in vitro results indicate that TGF-β1 exerts irreversible effects at a specific stage of osteoblast phenotype development resulting in a potent inhibition of osteoblast differentiation at concentrations from 0.1 ng/ml. © 1994 Wiley-Liss, Inc.     

The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.     

Effect of fibronectin on early embryo development in cows.

Two-cell bovine embryos produced in vitro were cultured in serum-free medium containing the soluble glycoprotein fibronectin (50 micrograms ml-1) to study the function of the extracellular matrix in early development. Some of the embryos (48/164, 29.3%), developed beyond the 16-cell stage compared with none of the 179 controls. Fibronectin at lower (5 micrograms ml-1) or higher (300 micrograms ml-1) concentrations did not promote embryo development (0/89 and 0/82, respectively). Indirect immunofluorescence demonstrated the presence of both fibronectin and its receptor on the surface of eight-cell embryo blastomeres, and biotinylated fibronectin demonstrated that exogenous fibronectin could cross the zona pellucida. These results, demonstrating the successful culture of bovine embryos in serum-free medium, support the hypothesis that the extracellular matrix, specifically fibronectin, plays a role in early development of bovine embryos.     

啤酒酵母菌的细胞融合

啤酒多倍体酵母菌原生质体已成功地与单倍体原生质体进行融合。经细胞壁再生后,稳定的融合重组体被分离出来。这些融合体的基因分析表明,融合体中含有双亲的基因型。孢子形成良好,且每个子囊中含有四个孢子,每个孢子确实是二倍体。这样原生质体融合就提供了一个对啤酒酿造酵母进行遗传分析的方法。但是如果没有一个方便的杂交技术,这个方法将是很困难的。     

Inhibitors of RNA synthesis and passage of chick embryo fibroblasts through the G1 period

Events that are essential for progression through the G1 period begin immediately or shortly after resting chick embryo cells are given fresh medium with serum. The following observations support the contention that the critical events include the production of non-ribosomal RNAs: (1) Addition to the “shift-up” medium of either of two inhibitors of RNA formation, comptothecin or 5, 6-dichloro?1-β-D-ribofuranosylbenzimidazole, delays the onset of DNA replication by about the length of time the cells are exposed to the drugs. (2) Although entry into the S phase is delayed by the inhibitors, the slopes of the DNA response curves are identical to that of control cultures. (3) Neither drug reduces significantly the rate of overall protein synthesis. Observations (2) and (3) are taken to mean that expansion of the G1 period is not due to cell damage. (4) A third inhibitor of RNA synthesis, cordycepin, also delays passage of stimulated cells throgh the G1 phase, but, in this case, the length of the delay period is greater than that of the exposure period. (5) A low dose of actinomycin D does not impede movement towards the S phase, even though the synthesis of preribosomal RNA is considerably reduced. The possibility is considered that the essential G1 molecules are mRNAs.     

Decreased gene expression from T7 promoters may be due to impaired production of active T7 RNA polymerase

Background  

Protein expression vectors that utilize the bacteriophage T7 polymerase/promoter system are capable of very high levels of protein production. Frequently, however, expression from these vectors does not reliably achieve optimal levels of protein production. Strategies have been proposed previously that successfully maintain high expression levels, however we sought to determine the cause of induction failure.     

Bovine seminal plasma proteins PDC-109, BSP-A3, and BSP-30-kDa share functional roles in storing sperm in the oviduct

On ejaculation, sperm become coated with proteins secreted by the male accessory sex glands. In the bull, these proteins consist predominantly of the bovine seminal plasma family of proteins (BSPs): PDC-109 (BSP-A1/-A2), BSP-A3, and BSP-30-kDa. PDC-109 plays a role in forming an oviductal sperm reservoir by enabling sperm to bind to oviductal epithelium. Because PDC-109 has high sequence identity with the other BSPs, we tested BSP-A3 and BSP-30-kDa for the capacity to bind sperm to oviductal epithelium. BSP-A3 and BSP-30-kDa each increased binding of epididymal sperm to epithelium and were as effective as PDC-109 in competitively inhibiting binding of ejaculated sperm. Because binding extends the motile life of sperm, BSPs were tested for the ability to maintain sperm motility. BSP-treated epididymal sperm incubated with plasma membrane vesicles from bovine oviductal epithelium maintained progressive motility longer than untreated sperm. To our knowledge, this is the first report of this protective effect of BSPs. Similarities in function among the BSPs were reflected in their three-dimensional structure, whereas surface maps of electrostatic potential indicated differences in binding affinities and kinetics. Such differences may provide sperm with greater adaptability to variations among females. Altogether, these results indicate that BSPs play a crucial role in fertilization by maintaining sperm motility during storage.     

Sex differences in the rate of fatigue development and recovery

Background

Many musculoskeltal injuries in the workplace have been attributed to the repetitive loading of muscle and soft tissues. It is not disputed that muscular fatigue is a risk factor for musculoskeltal injury, however the disparity between gender with respect to muscular fatigability and rate of recovery is not well understood. Current health and safety guidelines do not account for sex differences in fatiguability and may be predisposing one gender to greater risk. The purpose of this study was to quantify the sex differences in fatigue development and recovery rate of lower and upper body musculature after repeated bouts of sustained isometric contractions.

Methods

Twenty-seven healthy males (n = 12) and females (n = 15) underwent bilateral localized fatigue of either the knee extensors (male: n = 8; female: n = 8), elbow flexors (male: n = 8; female: n = 10), or both muscle groups. The fatigue protocol consisted of ten 30-second sub-maximal isometric contractions. The changes in maximum voluntary contraction (MVC), electrically evoked twitches, and motor unit activation (MUA) were assessed along with the ability to control the sustained contractions (SLP) during the fatigue protocol using a mixed four-factor repeated measures ANOVA (gender × side × muscle × time) design with significance set at p < 0.05.

Results

There was a significant loss of MVC, MUA, and evoked twitch amplitude from pre- to post-fatigue in both the arms and legs. Males had greater relative loss of isometric force, a higher rate of fatigue development, and were less capable of maintaining the fatiguing contractions in the legs when compared to the females.

Conclusion

The nature of the induced fatigue was a combination of central and peripheral fatigue that did not fully recover over a 45-minute period. The results appear to reflect sex differences that are peripheral, and partially support the muscle mass hypothesis for explaining differences in muscular fatigue.

     

Is low dose inhaled corticosteroid therapy as effective for inflammation and remodeling in asthma? A randomized,parallel group study

Background

While most of the clinical benefits of inhaled corticosteroid (ICS) therapy may occur at low doses, results of dose-ranging studies are inconsistent. Although symptom/lung function response to low and high dose ICS medication is comparable, it is uncertain whether low dose ICSs are as effective as high dose in the treatment of inflammation and remodeling.

Methods

22 mild or moderate asthmatic adult subjects (corticosteroid free for > 2 months) participated in a randomized, parallel group study to compare effects of fluticasone propionate (FP) 200 mcg/day and 1000 mcg/day. Alveolar macrophage (AM)-derived cytokines and basement membrane thickness (BMT) were measured at baseline and after 7 weeks treatment while symptoms, spirometry, exhaled nitric oxide (eNO) and airway hyperresponsiveness (AHR) to mannitol at baseline and 6 weeks.

Results

FP improved spirometry, eNO, symptoms and AHR with no difference between low and high dose FP. Both high and low dose FP reduced GM-CSF, TNF-alpha and IL-1ra, with no change in BMT and with no differences between low and high dose FP.

Conclusions

200 μg/day of FP was as effective as 1000 μg/day in improving asthma control, airway inflammation, lung function and AHR in adults in the short term. Future studies should examine potential differential effects between low and high dose combination therapy (ICS/long acting beta agonist) on inflammation and airway remodeling over longer treatment periods.     

Influence of the annual flood-pulse on catch per unit effort,condition and reproduction of Clarias gariepinus from the upper Okavango Delta,Botswana

Catch per unit effort (CPUE), length, weight and maturity data for Clarias gariepinus were collected during monthly gillnet surveys in the upper Okavango Delta between 2001 and 2009 to investigate their relationship with the annual flood-pulse. CPUE, condition factor (K) and the proportion of ripe-running fish (P_RR) in the population followed a unimodal annual cycle that could be modelled using water temperature and flood-pulse hydrology. Increased CPUE during declining water levels was most likely a result of feeding migrations and aggregation behaviour. The observed increase in K during low floods in October and November preceded the increase in P_RR, which increased mainly with increasing temperature but appeared less dependent on flow. This study provided quantitative evidence that the biology of fish in the Okavango Delta is mainly dependent on the annual flood regime and, therefore, that conservation efforts should be focused on maintaining natural flow patterns in the face of climate change and potential water extraction schemes upstream.