A novel hydrogel-collagen composite improves functionality of an injectable extracellular matrix.

Cellular transplantation is now closer to becoming a practical clinical strategy to repair, regenerate or restore the function of skin, muscle, nerves and pancreatic islets. In this study we sought to develop a simple injectable collagen matrix that would preserve the normal cellular organization of skin cells. Three different scaffolds were created and compared: collagen-glycosaminoglycan (GAG) scaffolds, crosslinked collagen-GAG scaffolds without polyvinyl alcohol (PVA) and crosslinked collagen-GAG scaffolds containing PVA hydrogel. Importantly, all scaffolds were found to be non-cytotoxic. PVA-containing gels exhibited a higher tensile strength (P<0.05), faster fibril formation (P<0.001) and reduced collagenase digestion (P<0.01) compared with other gels. Free floating fibroblast-populated, PVA-borate scaffolds resisted contraction over a 10 day period (P<0.001). The fibroblast-populated scaffolds containing PVA demonstrated a 3-fold reduction in cellularity over 10 days compared with the control gels (P<0.001). Multicellular skin substitutes containing PVA-borate networks display a linear cellular organization, reduced cellularity and the formation of a keratinized epidermis that resembles normal skin. In conclusion, these data underscore the multifunctionality of a simple PVA-borate-collagen matrix as an injectable composite for tissue engineering or cell transplantation.

Is exercise associated with primary dysmenorrhoea in young women?

Anecdotal beliefs that exercise is an effective treatment for primary dysmenorrhoea have prevailed for many years although evidence is contradictory. Previous studies have also contained a number of methodological inadequacies. A questionnaire that assessed menstrual pain and levels of exercise was administered to 654 university students. Attempts were made to blind the purpose of the study. A response rate of 91.3% (597/654) was obtained. Analyses showed no association between participation in exercise and primary dysmenorrhoea. Prospective studies would be useful in further research.

A direct relationship between the partitioning of the pathogenic prion protein and transmissible spongiform encephalopathy infectivity during the purification of plasma proteins.

BACKGROUND: Experimental evidence from rodent models indicates that blood can contain transmissible spongiform encephalopathy (TSE) infectivity, which suggests a potential risk for TSE transmission via proteins isolated from human plasma. Because methods that can reduce TSE infectivity typically are detrimental to protein function, infectivity must be removed to ensure the safety of these therapeutic proteins. Animal bioassays are conventionally used to detect infectivity, but the pathogenic form of the prion protein (PrP(Sc)) can serve as a marker for TSE infectivity. STUDY DESIGN AND METHODS: Seven plasma protein-purification steps were performed after the plasma intermediates were spiked with TSE-infected material. Resulting fractions were analyzed for PrP(Sc) by using a Western blot assay and for TSE infectivity by using an animal bioassay. Western blots were quantitated by an endpoint dilution analysis, and infectivity titers were calculated by the Spearman-Kärber method. RESULTS: PrP(Sc) partitioning paralleled TSE infectivity partitioning, regardless of the nature of the protein-purification step. The detection ranges for PrP(Sc) and infectivity were 0 to 5.3 log and 1.1 to 8.9 log median infectious dose per unit, respectively. Clearance of PrP(Sc) and infectivity ranged from 1.0 to 6.0 log. CONCLUSION: Purification steps for isolating therapeutic proteins from human plasma showed the removal of both PrP(Sc) and TSE infectivity. PrP(Sc) partitioning coincided with infectivity partitioning, which showed a close relationship between PrP(Sc) and TSE infectivity. By exploiting this association, the in vitro Western blot assay for PrP(Sc) was valuable for estimating the partitioning of TSE infectivity during plasma protein purification.

Monitoring plasma processing steps with a sensitive Western blot assay for the detection of the prion protein.

Determining the risk of transmissible spongiform encephalopathy (TSE) transmission by blood or plasma-derived products requires sensitive and specific assays for the detection of either infectivity or a reliable marker for infectivity. To this end, a Western blot assay that is both sensitive and reproducible for the detection of PrP(RES), a marker for TSE infectivity, was developed. Using the 263K strain of TSE as a model system, the Western blot assay proved to be sensitive, specific and quantitative over a 3-4 log dynamic range. Compared to the rodent bioassay, the assay was shown to detect PrP(RES) down to approximately 10(3.4) IU/ml which is approximately 5-10 pg of PrP or approximately 10-20 ng brain equivalents. The Western blot was applied to monitor the partitioning of spiked PrP(Sc) through three plasma fractionation steps, cryoprecipitation, fraction I and fraction III, that are common to the purification of several human plasma-derived therapeutic products including albumin and immunoglobulins. The results from these studies demonstrated 1 log, 1 log and 4 logs of PrP(Sc) partitioning away from the effluent fraction for the cryoprecipitation, fraction I and fraction III steps, respectively.

Inhibition of cross-bridge binding to actin by caldesmon fragments in skinned skeletal muscle fibers.

Several regions within the 35-kDa COOH-terminal portion of caldesmon have been implicated in the ability of caldesmon to inhibit actin-activated myosin ATPase activity. To further define the functional regions of caldesmon, we have studied the effects of three chymotryptic fragments, one fragment produced by CNBr digestion and two fragments produced by digestion with submaxillaris arginase C protease, on the relaxed stiffness and active force of rabbit psoas fibers. Each of the regions of caldesmon studied had either direct or indirect effects on single-fiber mechanics. The 35-kDa and 20-kDa fragments of caldesmon, like intact caldesmon, were effective inhibitors of fiber stiffness, a measure of cross-bridge attachment. The 7.3-kDa and 10-kDa fragments, which constitute the NH2 and COOH halves of the 20-kDa fragment, inhibited both relaxed fiber stiffness and active force production, but with a reduced efficacy compared to the 20-kDa fragment. These results suggest that several regions within the 35-kDa COOH-terminal region of caldesmon are required for optimum function of caldesmon and that function includes inhibition of weak cross-bridge attachment and force production.

Covariability of visually evoked potentials and simple motor reaction times.

Visually evoked potentials (VEPs) were recorded during simple motor reaction time (RT) experiments using spatially localized, contrast modulated visual stimuli. VEPs were averaged in groups, called "subensembles," depending on the speed of the subject's motor response. To a specific visual stimulus, the VEP response time of a subensemble covaries with motor response latency according to the same linear relationship found to describe conventionally averaged VEPs and RTs over a wide range of stimuli.

Evoked potentials and simple motor reaction times to localized visual patterns.

Spatially localized, contrast modulated visual stimuli evoke a monophasic, inion negative scalp potential (VEP) which can be recorded simultaneously with motor responses in simple reaction time experiments. VEP response time and median motor response latency are linearly related over a wide range of stimuli. We interpret our results to suggest that VEPs and motor response latencies arise from a common sensory system with different thresholds for detection and reaction. A model is introduced to predict detection thresholds from VEP response times which are usually more robust measures than VEP amplitudes.

The complete amino acid sequence of a hirudin variant from the leech Hirudinaria manillensis.

Unlike the European leech Hirudo medicinalis, the Asian jawed leech Hirudinaria manillensis is specialized for feeding on mammalian blood. In the salivary glands of both these leeches, there is a potent inhibitor of thrombin, called hirudin, which acts as an anticoagulant. We have reported previously the isolation and purification of a variant of hirudin, called bufrudin, from the head portions of Hirudinaria. In the present study, the complete amino acid sequence of bufrudin was determined by automated Edman degradation of peptide fragments generated after cleavage of protein with trypsin or thermolysin. Comparison of the primary structure of bufrudin, with hirudin HV1, show about 70% sequence identity with deletion of two amino acids, but the key amino acids at the C-terminus, involved in the inhibition of thrombin, are conserved. However, similar sequence comparison of bufrudin with hirullin P18, a hirudin variant isolated from the same leech species but from whole leech, instead of heads, reveals even less sequence identity of about 60%. From the amino acid sequence, it is suggested that the conformation of the C-terminal portion of bufrudin may be significantly different from hirullin P18, but similar to hirudin HV1, upon its interaction with thrombin. These results indicate that, as with Hirudo leech, various isoforms of hirudin also exist in Hirudinaria leech, with a significant change occurring in the structure of the molecule during the evolution of leeches.

Mannitol, intracranial pressure, and vasogenic edema.

The effect of a 2 g/kg intravenous bolus of mannitol on intracranial pressure (ICP) and white matter water content was determined by following the time course of ICP and spatial white matter water content after administration of mannitol in a hemispheric cold lesion model of vasogenic edema in the cat. After mannitol infusion, maximal ICP reduction occurred at approximately 20 minutes and began to return to baseline after 26 minutes. White matter water content in the control hemisphere began to decrease much later, reaching a minimum at 60 minutes. White matter water content in the lesioned hemisphere was not changed significantly after mannitol infusion. We conclude that the rapid reduction of ICP after intravenous administration of mannitol is not solely due to the dehydration of white matter.

Amplified expression and large-scale purification of protein G'.

PCR was used to isolate the gene fragment coding for Protein G' (SpG'), a truncated bacterial cell surface protein from Streptococcus G148 which binds to the Fc region of IgG and expressed in E. coli [Goward et al. (1990) Biochem. J. 267: 171-177]. The PCR primer was designed to change the TTG initiation triplet to ATG and to incorporate it into an NdeI restriction site (CATATG), allowing the gene to be cloned in frame into an NdeI restriction site immediately downstream of a trp promoter. Expression of SpG' was estimated as about 30% total soluble cell protein which compares very favourably to the less than 1% total soluble cell protein obtained from the original system [Goward, et al. (1990) Biochem. J. 267: 171-177]. Homogeneous SpG' was recovered by a single anion-exchange chromatography step on Q-Sepharose FF in a process which avoided use of an affinity adsorbent. Even though SpG' consists of almost identical repetitive domains from amino acid sequence analysis, different proteolytic sensitivity of each domain was observed indicating their structural dissimilarity.