‘Gelatinase-like’ activity from articular chondrocytes in monolayer culture

In addition to releasing collagenase and proteoglycanase activity, rabbit articular chondrocytes in monolayer culture released into the culture medium, latent, neutral enzyme activity which when activated by p-aminophenylmercuric acetate degraded fluorescein-labeled polymeric rat tail tendon Type I collagen and the tropocollagen TC_A and TC_B fragments of human Type II collagen into smaller peptides at 37°C. Enzyme activity was abolished if p-aminophenylmercuric acetate-activated culture medium was preincubated with 1,10-phenanthroline, a metal chelator. Thus, articular chondrocytes in monolayer culture are capable of producing neutral proteinases which acting together can result in complete degradation of tendon and cartilage collagen to small peptides.     

Decreased GABA receptor in the cerebral cortex of epileptic rats: effect of Bacopa monnieri and Bacoside-A

Abstact

Background

Gamma amino butyric acid (GABA), the principal inhibitory neurotransmitter in the cerebral cortex, maintains the inhibitory tones that counter balances neuronal excitation. When this balance is perturbed, seizures may ensue.

Methods

In the present study, alterations of the general GABA, GABA_A and GABA_B receptors in the cerebral cortex of the epileptic rat and the therapeutic application of Bacopa monnieri were investigated.

Results

Scatchard analysis of [³H]GABA, [³H]bicuculline and [³H]baclofen in the cerebral cortex of the epileptic rat showed significant decrease in B_max (P < 0.001) compared to control. Real Time PCR amplification of GABA receptor subunits such as GABA_Aά1, GABA_Aγ, GABA_Aδ, GABA_B and GAD where down regulated (P < 0.001) in epileptic rats. GABA_Aά5 subunit and Cyclic AMP responsible element binding protein were up regulated. Confocal imaging study confirmed the decreased GABA receptors in epileptic rats. Epileptic rats have deficit in radial arm and Y maze performance.

Conclusions

Bacopa monnieri and Bacoside-A treatment reverses epilepsy associated changes to near control suggesting that decreased GABA receptors in the cerebral cortex have an important role in epileptic occurrence; Bacopa monnieri and Bacoside-A have therapeutic application in epilepsy management.     

Repression of cell-cell fusion by components of the C. elegans vacuolar ATPase complex

Cell-cell fusion initiates fertilization, sculpts tissues during animal development, reprograms stem cells to new differentiated states, and may be a key step in cancer progression. While cell fusion is tightly regulated, the mechanisms that limit fusion to appropriate partners are unknown. Here, we report that the fus-1 gene is essential to repress fusion of epidermal cells in C. elegans: in severe fus-1 mutants, all epidermal cells, except the lateral seam cells, inappropriately fuse into a single large syncytium. This hyperfusion requires EFF-1, an integral membrane protein essential for fusion of epidermal cells into discrete syncytia. FUS-1 is localized to the apical plasma membrane in all epidermal cells potentiated to undergo fusion, whereas it is virtually undetectable in nonfusing seam cells. fus-1 encodes the e subunit of the vacuolar H(+)-ATPase (V-ATPase), and loss of other V-ATPase subunits also causes widespread hyperfusion. These findings raise the possibility of manipulating cell fusion by altering V-ATPase activity.     

Repercussions of the COVID-19 pandemic on athletes: a cross-sectional study

The COVID-19 pandemic has presented significant challenges and implications for the sports community. Thus, this study aimed to describe the prevalence of COVID-19 in Brazilian athletes and identify the epidemiological, clinical, athletic, life and health factors associated with the disease in these individuals. A cross-sectional study was performed involving 414 athletes from 22 different sports using an online questionnaire from August to November 2020. The association between the athletes’ characteristics and COVID-19 was evaluated using a logistic regression model. The prevalence of COVID-19 was 8.5%, although only 40% of athletes reported having been tested. Being under 27 years of age (3-fold), having children (~5-fold), having a teammate test positive for COVID-19 (2.5-fold), and smoking (14-fold) were associated with a possible higher risk of disease. Almost 20% of athletes self-reported musculoskeletal injuries during the period of the pandemic that was studied. Athletes with a university education (P = 0.02), a profession other than sports (P < 0.001), those from a low-income family (P = 0.01), and public health system users (P = 0.04) were significantly less frequently tested for COVID-19, whereas international competitors, athletes who received a wage, and athletes who had a teammate who tested positive for COVID-19 were 2-, 3-, and 15-fold more likely to be tested for COVID-19, respectively. Approximately 26% of the athletes who tested negative or were untested reported more than three characteristic COVID-19 symptoms, and 11% of athletes who tested positive for COVID-19 were asymptomatic. The identification of modifiable (have children, smoking, and teammates positively tested) and non-modifiable (age under 27 years) factors related to COVID-19 in athletes can contribute to implementing surveillance programmes to decrease the incidence of COVID-19 in athletes and its negative impacts in sports.     

The Effect of Molecular Weight,PK, and Valency on Tumor Biodistribution and Efficacy of Antibody-Based Drugs

Poor drug delivery and penetration of antibody-mediated therapies pose significant obstacles to effective treatment of solid tumors. This study explored the role of pharmacokinetics, valency, and molecular weight in maximizing drug delivery. Biodistribution of a fibroblast growth factor receptor 4 (FGFR4) targeting CovX-body (an FGFR4-binding peptide covalently linked to a nontargeting IgG scaffold; 150 kDa) and enzymatically generated FGFR4 targeting F(ab)₂ (100 kDa) and Fab (50 kDa) fragments was measured. Peak tumor levels were achieved in 1 to 2 hours for Fab and F(ab)₂versus 8 hours for IgG, and the percentage injected dose in tumors was 0.45%, 0.5%, and 2.5%, respectively, compared to 0.3%, 2%, and 6% of their nontargeting controls. To explore the contribution of multivalent binding, homodimeric peptides were conjugated to the different sized scaffolds, creating FGFR4 targeting IgG and F(ab)₂ with four peptides and Fab with two peptides. Increased valency resulted in an increase in cell surface binding of the bivalent constructs. There was an inverse relationship between valency and intratumoral drug concentration, consistent with targeted consumption. Immunohistochemical analysis demonstrated increased size and increased cell binding decreased tumor penetration. The binding site barrier hypothesis suggests that limited tumor penetration, as a result of high-affinity binding, could result in decreased efficacy. In our studies, increased target binding translated into superior efficacy of the IgG instead, because of superior inhibition of FGFR4 proliferation pathways and dosing through the binding site barrier. Increasing valency is therefore an effective way to increase the efficacy of antibody-based drugs.     

Persistent nuclear ribosomal DNA sequence polymorphism in the Amelanchier agamic complex (Rosaceae)

Individual plants of several Amelanchier taxa contain many polymorphic nucleotide sites in the internal transcribed spacers (ITS) of nuclear ribosomal DNA (nrDNA). This polymorphism is unusual because it is not recent in origin and thus has resisted homogenization by concerted evolution. Amelanchier ITS sequence polymorphism is hypothesized to be the result of gene flow between two major North American clades resolved by phylogenetic analysis of ITS sequences. Western North American species plus A. humilis and A. sanguinea of eastern North America form one clade (A), and the remaining eastern North American Amelanchier make up clade B. Five eastern North American taxa are polymorphic at many of the nucleotide sites where clades A and B have diverged and are thought to be of hybrid origin, with A. humilis or A. sanguinea as one parent and various members of clade B as the other parent. Morphological evidence suggests that A. humilis is one of the parents of one of the polymorphic taxa, a microspecies that we refer to informally as A. "erecta." Sequences of 21 cloned copies of the ITS1- 5.8S gene-ITS2 region from one A. "erecta" individual are identical to A. humilis sequence or to the clade B consensus sequence, or they are apparent recombinants of A. humilis and clade B ITS repeats. Amelanchier "erecta" and another polymorphic taxon are suspected to be relatively old because both grow several hundred kilometers beyond the range of one of their parents. ITS sequence polymorphisms have apparently persisted in these two taxa perhaps because of polyploidy and/or agamospermy (asexual seed production), which are prevalent in the genus.      

Cell Budding from Normal Appearing Epithelia: A Predictor of Colorectal Cancer Metastasis?

Background: Colorectal carcinogenesis is believed to be a multi-stage process that originates with a localized adenoma, which linearly progresses to an intra-mucosal carcinoma, to an invasive lesion, and finally to metastatic cancer. This progression model is supported by tissue culture and animal model studies, but it is difficult to reconcile with several well-established observations, principally among these are that up to 25% of early stage (Stage I/II), node-negative colorectal cancer (CRC) develop distant metastasis, and that circulating CRC cells are undetectable in peripheral blood samples of up to 50% of patients with confirmed metastasis, but more than 30% of patients with no detectable metastasis exhibit such cells. The mechanism responsible for this diverse behavior is unknown, and there are no effective means to identify patients with pending, or who are at high risk for, developing metastatic CRC.Novel findings: Our previous studies of human breast and prostate cancer have shown that cancer invasion arises from the convergence of a tissue injury, the innate immune response to that injury, and the presence of tumor stem cells within tumor capsules at the site of the injury. Focal degeneration of a capsule due to age or disease attracts lymphocyte infiltration that degrades the degenerating capsules resulting in the formation of a focal disruption in the capsule, which selectively favors proliferating or “budding” of the underlying tumor stem cells. Our recent studies suggest that lymphocyte infiltration also triggers metastasis by disrupting the intercellular junctions and surface adhesion molecules within the proliferating cell buds causing their dissociation. Then, lymphocytes and tumor cells are conjoined through membrane fusion to form tumor-lymphocyte chimeras (TLCs) that allows the tumor stem cell to avail itself of the lymphocyte''s natural ability to migrate and breach cell barriers in order to intravasate and to travel to distant organs. Our most recent studies of human CRC have detected nearly identical focal capsule disruptions, lymphocyte infiltration, budding cells, and the formation of TLCs. Our studies have further shown that age- and type-matched node-positive and -negative CRC have a significantly different morphological and immunohistochemical profile and that the majority of lymphatic ducts with disseminated cells are located within the mucosa adjacent to morphologically normal appearing epithelial structures that express a stem cell-related marker.New hypothesis: Based on these findings and the growth patterns of budding cells revealed by double immunohistochemistry, we further hypothesize that metastatic spread is an early event of carcinogenesis and that budding cells overlying focal capsule disruptions represent invasion- and metastasis-initiating cells that follow one of four pathways to progress: (1) to undergo extensive in situ proliferation leading to the formation of tumor nests that subsequently invade the submucosa, (2) to migrate with associated lymphocytes functioning as “seeds” to grow in new sites, (3) to migrate and intravasate into pre-existing vascular structures by forming TLCs, or (4) to intravasate into vascular structures that are generated by the budding cells themselves. We also propose that only node-positive cases harbor stem cells with the potential for multi-lineage differentiation and unique surface markers that permit intravasation.     

Studies on the turnover of plasma membranes in cultured mammalian cells.: II. Demonstration of heterogeneous rates of turnover for plasma membrane proteins and glycoproteins

The relative rate of turnover of individual membrane proteins and glycoproteins in exponentially growing and contact-inhibited MK₂ cells was investigated. Plasma membranes were isolated from cells that had been sequentially labelled with ¹⁴C and ³H isotopes of leucine and glucosamine. The membranes were then solubilized in sodium dodecylsulfate and their polypeptides separated by acrylamide gel electrophoresis. The ³H/¹⁴C ratios of the individual polypeptides reflected their relative rates of turnover. The proteins and glycoproteins of the exponentially growing cells exhibited markedly heterogeneous rates of turnover. In contrast, polypeptides in membranes of contact-inhibited cells exhibited a lesser degree of heterogeneity of turnover. In both exponential and contacted cell membranes a glycoprotein with a high apparent molecular weight exhibited the fastest rate of turnover.