Interleukin 2 mediates an alteration in the T200 antigen expressed on activated B lymphocytes

We have examined the effect of exogenous IL 2 on cell surface antigen expression in LPS/dextran sulfate-activated murine B cells with the use of a panel of fluorescein-conjugated lectins. Elevated binding of the lectins PNA and SBA to activated B cells was found to be mediated by IL 2-containing supernatants from stimulated EL4 cells as well as by recombinant IL 2. These lectins have specificity for terminal beta-(1-3)-N-acetylgalactosaminyl residues; thus, the quantity or accessibility of these moieties is mediated by IL 2 in activated B lymphocytes. PNA binding in all strains tested, regardless of MHC or background genes, was found to be elevated fivefold to 15-fold by exogenous IL 2. To observe this effect, IL 2 must be added during the first 24 hr of culture. Based on anti-Thy-1 + complement depletion studies, T cells were not required, suggesting a direct effect of IL 2 on B cells. The glycoprotein responsible for this elevated binding of PNA has an Mr of approximately 220K and by immunodepletion was shown to belong to the T200 (Ly-5) family of cell surface antigens. These data demonstrate that exogenous IL 2 can mediate alterations in T200 expression on activated B cells that may be related to IL 2-driven modulation of B cell proliferation and/or differentiation.     

Calcium homeostasis in bovine somatotrophs: calcium oscillations and calcium regulation by growth hormone-releasing hormone and somatostatin.

The free intracellular calcium ion concentration ([Ca2+]i) was measured in single cells of a population containing 65-80% somatotrophs, using the fluorescent Ca(2+)-indicator Fura-2 and digital imaging microscopy. Spontaneous oscillations in [Ca2+]i ranging in frequency up to 1.5 oscillations per minute were observed in 30% of somatotrophs. These Ca2+ oscillations were blocked by the Ca2+ channel blocker CoCl2 and were thus proposed to be the result of influx of Ca2+ into the cell, possibly as the result of spontaneous electrical activity. GHRH (10-100 nM) increased [Ca2+]i in 61% of the cells studied, although the amplitude and dynamics of the response varied from cell to cell. Typically [Ca2+]i rose from 170 +/- 26 nM to 321 +/- 44 nM (n = 13) in response to a challenge with 66 nM GHRH. GHRH also increased the frequency of Ca2+ oscillations in a number of cells, and some previously quiescent cells showed Ca2+ oscillations following addition of GHRH. Forskolin, which raises cAMP levels in bovine anterior pituitary cells, also stimulated a sustained rise in [Ca2+]i in 10 out of 14 cells tested. Somatostatin (SS) (10-80 nM) rapidly reduced basal [Ca2+]i, blocked Ca2+ oscillations, and blocked the [Ca2+]i response to GHRH. The Ca2+ channel blocker CoCl2 (4 mM) had similar actions on [Ca2+]i to those of SS. These results suggest that GHRH and SS may regulate GH release by modulating Ca2+ entry into the cell through the cell membrane. The [Ca2+]i oscillations seen in a proportion of the somatotrophs were modulated in frequency by GHRH and SS, and are probably generated by influx of Ca2+ through channels in the cell membrane. Thus GH secretion may be regulated by changes in the mean level of [Ca2+]i, which in turn, may be influenced by the frequency of [Ca2+]i oscillations in bovine somatotrophs.     

Variation in heat-shock proteins among species of desert fishes (Poeciliidae, Poeciliopsis)

Analysis of the heat-shock proteins (hsps) of six closely related species of Poeciliopsis demonstrated the existence of biochemical diversity in the hsp100, hsp70, hsp60, and hsp30 protein families among species. Each species expressed five to seven hsp70-related isoforms. Constitutive 70-kD isoforms were identical among species, but four different patterns of heat-inducible isoforms were seen in these six species. Members of the hsp70 family of molecular chaperones are included among the most highly conserved proteins known, and the possibility of variation in hsp70 among closely related species has rarely been addressed. The hsp30 family is known to be less conserved than the hsp70 family, and, as expected, the Poeciliopsis hsp30 patterns showed more variation. Most of the hsp30 isoforms characteristic of a particular species were unique to that species. Hsp100 and hsp60 were identical in five of the species, but alternate isoforms were found in P. monacha. The small size and limited geographical distribution of the P. monacha population have probably contributed to the uniqueness of the monacha pattern. Two of the species were shown to acquire thermotolerance, the ability to withstand normally lethal temperatures when subjected to a gradual temperature increase. Rapid-heating protocols commonly used to establish critical thermal maxima of organisms do not include this inducible component of thermoresistance and therefore do not adequately assess an organism's capacity to withstand thermal stress.      

Topographical rearrangement of a plasma membrane antigen during capacitation of rat spermatozoa in vitro

We have previously described an antigen (termed 2B1) on rat spermatozoa that is present on the plasma membrane overlying the tail domain. The antigen is mobile within the plane of the plasma membrane and a mAb to it blocks fertilization in vitro. In the present study we describe some dynamic properties of this antigen in relation to its topographical distribution. When spermatozoa were incubated in vitro in a capacitation medium and stained with 2B1 mAb/FITC-rabbit anti-mouse F(ab')2, strong fluorescence appeared over the acrosomal domain. Acute exposure of fresh spermatozoa to dissociating reagents (1 M NaCl or 5 mM 2-mercaptoethanol) or inducers of the acrosome reaction (lysolecithin + Ca2+ or A23187 + Ca2+) failed to mimic these effects. Spermatozoa prelabeled with FITC-2B1 IgG and then capacitated in the presence of excess "cold" 2B1 IgG also showed accumulation of fluorescence on the acrosomal domain, suggesting that the antigen had migrated from the tail. Migration was selective and Ca2(+)- and temperature-dependent but was not inhibited by metabolic poisons (NaF or NaN3). Motility was not obligatory for migration. Immunogold-labeling studies at the ultrastructural level showed that 2B1 antigen was restricted to the surface membrane over both the tail and the acrosomal domains and that during migration it did not change the type of membrane into which it was inserted. From a quantitative analysis of fluorescence on spermatozoa prelabeled with FITC-2B1 IgG and then capacitated, the amount of antigen that appeared on the acrosomal domain was approximately equivalent to that lost from the midpiece domain. The Mr of 2B1 antigen extracted from capacitated spermatozoa was 300-500 Da less than that extracted from noncapacitated cells, suggesting that the molecule had undergone processing concomitant with migration. These results are discussed in relation to mechanisms for targeting antigens to sites where they become physiologically active and are correctly positioned to participate in gamete recognition processes.     

Methods for Studying the Distribution of Pigment in Malpighian Tubules of Drosophila Melanogaster

The pigmented cells in Malpighian tubules of Drosophila melanogaster contain yellow globules which are blackened by osmium tetroxide. Permanent preparations of the tubules showing pigmentation can thus be made. Following prestaining acid hydrolysis, osmium-fixed tubules can be stained with orcein. For Observations on pigment distribution and Feulgen-DNA content in the same preparation, tubules arc fixed in mercuric chloride. Color remains visible for several hours in unfixed tubules mounted in modified buffered Ringer solution.     

The Use of Vacuum Erection Devices in Erectile Dysfunction After Radical Prostatectomy

The risk of postoperative erectile dysfunction (ED) following radical prostatectomy (RP) is reported to be between 14% and 89%. With an increase in the detection of prostate cancer in younger men, there is a greater emphasis on the appropriate management of ED following RP. A number of options are available to manage ED after RP, including phosphodiesterase-5 inhibitors, intracorporeal injections, intraurethral alprostadil, and vacuum erection devices (VEDs). Penile rehabilitation programs are increasingly used to facilitate the return of natural postoperative erections; the VED is an ideal therapy given that it increases blood flow and oxygenation to the corpora to reverse the changes that result in ED after RP.Key words: Erectile dysfunction, Radical prostatectomy, Vacuum erection device, Penile rehabilitationProstate cancer is the most common cancer in men over the age of 50 years.¹ When patients undergo a radical prostatectomy (RP), there is a risk of postoperative erectile dysfunction (ED). The incidence of ED following RP has been reported to be between 14% and 89%.² With an increase in the detection of prostate cancer in younger men, there is a greater emphasis on the appropriate management of ED after RP. With an early diagnosis of prostate cancer, there is an increase in the rate of RP in younger men and the importance of ED as a quality-of-life issue has subsequently increased.² There are a number of options available to manage ED after RP, including phosphodiesterase-5 (PDE-5) inhibitors, intracorporeal injections, intraurethral alprostadil, and vacuum erection devices (VEDs). Despite highly reported satisfaction and efficacy with VEDs, there is a move by some medical practitioners away from VEDs due to cost. But what evidence is there for VED success after prostatectomy and what role do VEDs have in penile rehabilitation after ED? We present current evidence and provide our recommendations based on the latest literature.     

Tissue section AFM: In situ ultrastructural imaging of native biomolecules

Conventional approaches for ultrastructural high-resolution imaging of biological specimens induce profound changes in bio-molecular structures. By combining tissue cryo-sectioning with non-destructive atomic force microscopy (AFM) imaging we have developed a methodology that may be applied by the non-specialist to both preserve and visualize bio-molecular structures (in particular extracellular matrix assemblies) in situ. This tissue section AFM technique is capable of: i) resolving nm–µm scale features of intra- and extracellular structures in tissue cryo-sections; ii) imaging the same tissue region before and after experimental interventions; iii) combining ultrastructural imaging with complimentary microscopical and micromechanical methods. Here, we employ this technique to: i) visualize the macro-molecular structures of unstained and unfixed fibrillar collagens (in skin, cartilage and intervertebral disc), elastic fibres (in aorta and lung), desmosomes (in nasal epithelium) and mitochondria (in heart); ii) quantify the ultrastructural effects of sequential collagenase digestion on a single elastic fibre; iii) correlate optical (auto fluorescent) with ultrastructural (AFM) images of aortic elastic lamellae.     

Improving the clinical assessment of consciousness with advances in electrophysiological and neuroimaging techniques

In clinical neurology, a comprehensive understanding of consciousness has been regarded as an abstract concept - best left to philosophers. However, times are changing and the need to clinically assess consciousness is increasingly becoming a real-world, practical challenge. Current methods for evaluating altered levels of consciousness are highly reliant on either behavioural measures or anatomical imaging. While these methods have some utility, estimates of misdiagnosis are worrisome (as high as 43%) - clearly this is a major clinical problem. The solution must involve objective, physiologically based measures that do not rely on behaviour. This paper reviews recent advances in physiologically based measures that enable better evaluation of consciousness states (coma, vegetative state, minimally conscious state, and locked in syndrome). Based on the evidence to-date, electroencephalographic and neuroimaging based assessments of consciousness provide valuable information for evaluation of residual function, formation of differential diagnoses, and estimation of prognosis.     

Expression of semaphorin 3A and its receptors in the human intervertebral disc: potential role in regulating neural ingrowth in the degenerate intervertebral disc

Introduction  

Intervertebral disc (IVD) degeneration is considered a major underlying factor in the pathogenesis of chronic low back pain. Although the healthy IVD is both avascular and aneural, during degeneration there is ingrowth of nociceptive nerve fibres and blood vessels into proximal regions of the IVD, which may contribute to the pain. The mechanisms underlying neural ingrowth are, however, not fully understood. Semaphorin 3A (sema3A) is an axonal guidance molecule with the ability to repel nerves seeking their synaptic target. This study aimed to identify whether members of the Class 3 semaphorins were expressed by chondrocyte-like cells of the IVD addressing the hypothesis that they may play a role in repelling axons surrounding the healthy disc, thus maintaining its aneural condition.     

Transcriptional profiling of bovine intervertebral disc cells: implications for identification of normal and degenerate human intervertebral disc cell phenotypes

Introduction  

Nucleus pulposus (NP) cells have a phenotype similar to articular cartilage (AC) cells. However, the matrix of the NP is clearly different to that of AC suggesting that specific cell phenotypes exist. The aim of this study was to identify novel genes that could be used to distinguish bovine NP cells from AC and annulus fibrosus (AF) cells, and to further determine their expression in normal and degenerate human intervertebral disc (IVD) cells.