Study of thiol proteases of normal human skin fibroblasts

The protease activity of cultured normal human skin fibroblasts was studied using the synthetic fluorigenic peptides, the modified protein 4-methylumbelliferyl-casein, the thiol inhibitors and the affinity for concanavalin A-Sepharose. The majority of the activity to N-benzyloxycarbonyl-L-phenylalanyl-L-arginyl-7-amido-4-methyl-coumarin and N-a-benzyloxycarbonyl-L-arginyl-arginyl-7-amido-4-methylcoumarin had a pH optimum of 6.0, and was thiol-dependent and inhibited by leupeptin and antipain. The activity toward N-benzyloxycarbonyl-L-phenylalanyl-L-arginyl-7-amido-4-methylcoumarin represents both cathepsin B and cathepsin L, whereas the activity towards 4-methylumbelliferyl-casein represent only cathepsin L. Cathepsin H could not be detected when assayed with L-arginine-7-amido-4-methylcoumarin substrate. Cathepsin D was present in comparatively small amounts when assayed with 4-methylumbelliferyl-casein. Activity towards 4-methylumbelliferyl-casein had pH optima at 3 and 6 and was stimulated by dithiothreitol. A proportion of the activity at pH 6.0 was not dependent on thiols and not inhibited by leupeptin, and had the general characteristics of a carboxyl proteinase. Over 70 per cent of the activity was in the lysosomal fraction and showed structure-linked latency. All the detectable protein emerged from the immobilized concanavalin A column and the fractions eluted by alpha-methyl-D-mannoside were significantly hydrolysed the synthetic peptides. Only that fraction which bound to concanavalin A was active towards 4-methylumbelliferyl-casein. Cathepsin B had no affinity for concanavalin A-Sepharose due to the absence of glycoprotein content, unlike cathepsin L which showed a strong affinity for concanavalin A-Sepharose.     

A mannose-specific lectin from Vicia villosa seeds

A lectin specific to mannose has been purified from Vicia villosa seed by (NH₄)₂SO₄ fractionation, GalNAc-Sepharose and Man-Sepharose affinity chromatography. It was defined as VVL_M, which showed a single band on an acidic-PAGE stained with Coosmassie brilliant blue. The molecular weight of VVL_M was 50 kDa as determined by gel filtration on Biogel P-100 column. The VVL_M molecule consists of 2 distinct subunits with apparent molecular weight of 30 kDa and 22kDa determined by SDS-PAGE. VVL_M has at least four isolectins with similar haemagglutinating activity. Its extinction coefficient is calculated as A_1cm¹ = 16.4 at 280 nm. Sugars could not be detected phenol-sulfuric acid method. The circular dichroism analysis at far UV indicated that VVL_M was a β-sheet-rich protein, and gave no α-helix, 69% β-sheet, 14% β-turn by Provencher and Glockner method. The lectin was inhibited by α-methyl-d-mannose at 12.5 mM and glucose or GlcNAc at 50 mM. The carbohydrate binding specificity of VVL_M was investigated by using affinity chromatography on a VVL_M-Sepharose column. Among various Asn-linked oligosaccharides, core structure Manα1→3(Manα1→6)Manβ1→4GlcNAcβ1→4GlcNAc_OT were found to have high affinity for VVL_M-Sepharose. The antisera of VVL_M did not produce precipitin line with VVL_G in agar double diffusion plate indicating so serological relationship between VVL_M and VVL_G. However VVL_M showed similar immunodeterminants of some other lectins of mannose specificity such as Con A, PSL, LCA and VFL.     

The Detection of Hamster Connexins: A Comparison of Expression Profiles with Wild-Type Mouse and the Cancer-Prone Min Mouse

The open reading frames of 17 connexins from Syrian hamster (using tissues) and 16 connexins from the Chinese hamster cell line V79, were fully (Cx30, Cx31, Cx37, Cx43 and Cx45) or partially sequenced. We have also detected, and partially sequenced, seven rat connexins that previously were unavailable. The expression of connexin genes was examined in some hamster organs and cultured hamster cells, and compared with wild-type mouse and the cancer-prone Min mouse. Although the expression patterns were similar for most organs and connexins in hamster and mouse, there were also some prominent differences (Cx29 and 30.3 in testis; Cx31.1 and 32 in eye; Cx46 in brain, kidney and testis; Cx47 in kidney). This suggests that some connexins have species-specific expression profiles. In contrast, there were minimal differences in expression profiles between wild type and Min mice. Species-specific expression profiles should be considered in attempts to make animal models of human connexin-associated diseases.     

Social cognitive impairment and autism: what are we trying to explain?

Early psychological theories of autism explained the clinical features of this condition in terms of perceptual and sensory processing impairments. The arrival of domain-specific social cognitive theories changed this focus, postulating a ‘primary’ and specific psychological impairment of social cognition. Across the years, evidence has been growing in support of social cognitive and social attention explanations in autism. However, there has also been evidence for general non-social cognitive impairments in representational understanding, attention allocation and sensory processing. Here, I review recent findings and consider the case for the specificity and primacy of the social cognitive impairment, proposing that we should focus more explicitly on clinically valid features for insights on the integration of ‘social’ and ‘non-social’ cognition.     

Characterization of human-dog social interaction using owner report

Dog owners were surveyed for observations of social behaviors in their dogs, using questions adapted from the human Autism Diagnostic Observation Schedule (ADOS) pre-verbal module. Using 939 responses for purebred and mixed-breed dogs, three factors were identified: initiation of reciprocal social behaviors (INIT), response to social interactions (RSPNS), and communication (COMM). There were small or no effects of sex, age, breed group or training. For six breeds with more than 35 responses (Border Collie, Rough Collie, German Shepherd, Golden Retriever, Labrador Retriever, Standard Poodle), the behaviors eye contact with humans, enjoyment in interactions with human interaction, and name recognition demonstrated little variability across breeds, while asking for objects, giving/showing objects to humans, and attempts to direct humans’ attention showed higher variability across these breeds. Breeds with genetically similar backgrounds had similar response distributions for owner reports of dog response to pointing. When considering these breeds according to the broad categories of “herders” and “retrievers,” owners reported that the “herders” used more eye contact and vocalization, while the “retrievers” used more body contact. Information regarding social cognitive abilities in dogs provided by owner report suggest that there is variability across many social cognitive abilities in dogs and offers direction for further experimental investigations.     

Molecular Characteristics and Peptide Specificity of Vasoactive Intestinal Peptide Receptors from Rat Cerebral Cortex

Vasoactive intestinal peptide (VIP) receptors have been identified in CNS by their chemical specificity and molecular size. Using synaptosomes isolated from rat cerebral cortex, it was shown that central VIP receptors discriminated among natural and synthetic VIP-related peptides, because half-maximal inhibition of [125I]VIP binding to synaptosomes was obtained for 0.6 nM VIP, 9 nM peptide histidine isoleucineamide (PHI), 50 nM VIP 2-28, 70 nM secretin, 100 nM rat growth hormone-releasing factor (GRF), and 350 nM human GRF. Other peptides of the VIP family, such as glucagon and gastric inhibitory polypeptide, did not interact with cortical VIP receptors. The molecular components of VIP receptors in rat cerebral cortex were identified after [125I]VIP cross-linking to synaptosomes using the cross-linker dithiobis(succinimidyl propionate). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of synaptosomal proteins revealed two major [125I]VIP-protein complexes of Mr 49,000 and 18,000. The labeling of the Mr 49,000 component was specific, because it was abolished by native VIP, whereas the labeling of the Mr 18,000 component was not. Natural VIP agonists reduced the labeling of the Mr 49,000 component with the following order of potency: VIP greater than PHI greater than secretin approximately equal to rat GRF. In contrast, glucagon and octapeptide of cholecystokinin were without effect, a result indicating its peptide specificity. Densitometric scanning of autoradiographs showed that the labeling of the Mr 49,000 component was inhibited by low VIP concentrations between 10(-10) and 10(-6) M (IC50 = 0.8 nM), a result indicating the component's high affinity for VIP.(ABSTRACT TRUNCATED AT 250 WORDS)