Incidence of Clostridium botulinum Type E in Salmon and Other Marine Fish in the Pacific Northwest

Salmon, sole, cod, oysters, clams, and crabs from ocean waters along the coast of Oregon and Washington were examined for the presence of Clostridium botulinum type E. The organism was detected by identification of the type E toxin in enrichment cultures of the viscera of individual fish. Of 369 salmon specimens, 48 yielded cultures containing toxin lethal to mice, and almost half of the toxic cultures were shown to contain botulinal toxin, chiefly type E. Eighteen of 113 sole and cod specimens, 4 of 22 Dungeness crab specimens, 5 of 16 oyster specimens, and 27 of 115 clam specimens gave rise to cultures containing botulinal toxin which was usually type E, although types A and B were occasionally encountered.     

The Activity of Collagenase-1 Is Required for Keratinocyte Migration on a Type I Collagen Matrix

We have shown in a variety of human wounds that collagenase-1 (MMP-1), a matrix metalloproteinase that cleaves fibrillar type I collagen, is invariably expressed by basal keratinocytes migrating across the dermal matrix. Furthermore, we have demonstrated that MMP-1 expression is induced in primary keratinocytes by contact with native type I collagen and not by basement membrane proteins or by other components of the dermal or provisional (wound) matrix. Based on these observations, we hypothesized that the catalytic activity of MMP-1 is necessary for keratinocyte migration on type I collagen. To test this idea, we assessed keratinocyte motility on type I collagen using colony dispersion and colloidal gold migration assays. In both assays, primary human keratinocytes migrated efficiently on collagen. The specificity of MMP-1 in promoting cell movement was demonstrated in four distinct experiments. One, keratinocyte migration was completely blocked by peptide hydroxymates, which are potent inhibitors of the catalytic activity of MMPs. Two, HaCaTs, a line of human keratinocytes that do not express MMP-1 in response to collagen, did not migrate on a type I collagen matrix but moved efficiently on denatured type I collagen (gelatin). EGF, which induces MMP-I production by HaCaT cells, resulted in the ability of these cells to migrate across a type I collagen matrix. Three, keratinocytes did not migrate on mutant type I collagen lacking the collagenase cleavage site, even though this substrate induced MMP-1 expression. Four, cell migration on collagen was completely blocked by recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1) and by affinity-purified anti–MMP-1 antiserum. In addition, the collagen-mediated induction of collagenase-1 and migration of primary keratinocytes on collagen was blocked by antibodies against the α2 integrin subunit but not by antibodies against the α1 or α3 subunits. We propose that interaction of the α2β1 integrin with dermal collagen mediates induction of collagenase-1 in keratinocytes at the onset of healing and that the activity of collagenase-1 is needed to initiate cell movement. Furthermore, we propose that cleavage of dermal collagen provides keratinocytes with a mechanism to maintain their directionality during reepithelialization.     

Stereospecific aversive property of narcotic antagonists in morphine-free rats

If endogenous, morphine-like substances have physiological functions, narcotic antagonists should have effects $\text{in vivo}$ even in the absence of exogenous, narcotic agonists. This hypothesis was supported by studies of taste aversions conditioned with narcotic antagonists; rats drank smaller amounts of distinctively flavoured solutions when their consumption on previous occasions preceded injections of naloxone (1–10 mg/kg), naltrexone (3.2 mg/kg), Mr 1452 (10 mg/kg) or (-)-BC-2860 (10 mg/kg). Stereoisomers (i.e. Mr 1453, (+)-BC-2860) which were inactive as narcotic antagonists did not induce significant taste aversions. It was suggested that the consistency and stereospecificity of aversion with the antagonists gave some support to interpretations in terms of antagonist actions at receptors for endogenous opioids.     

Biodeterioration of Mayan buildings at uxmal and tulum,Mexico

Uxmal and Tulum are two important Mayan sites in the Yucatan peninsula. The buildings are mainly composed of limestone and grey/black discoloration is seen on exposed walls and copious greenish biofilms on inner walls. The principal microorganisms detected on interior walls at both Uxmal and Tulum were cyanobacteria; heterotrophic bacteria and filamentous fungi were also present. A dark‐pigmented mitosporic fungus and Bacillus cereus, both isolated from Uxmal, were shown to be acidogenic in laboratory cultures. Cyanobacteria belonging to rock‐degrading genera Synechocystis and Gloeocapsa were identified at both sites. Surface analysis previously showed that calcium ions were present in the biofilms on buildings at Uxmal and Tulum, suggesting the deposition of biosolubilized stone. Apart from their potential to degrade the substrate, the coccoid cyanobacteria supply organic nutrients for bacteria and fungi, which can produce organic acids, further increasing stone degradation.     

A reliable general purpose method for extracting genomic DNA from Dictyostelium cells

In this protocol, we present a standard method for extracting DNA from cells of the social amoeba Dictyostelium discoideum. While this procedure is similar to other phenol:chloroform-based purification methods, it is modified to account for the high level of carbohydrate and nucleases found in Dictyostelium cells. Genomic DNA can be isolated from wild-type and genetically modified cells using the described protocol, allowing molecular genetic analyses to be performed. Following cell lysis, nucleic acid extraction, and precipitation, the isolated DNA is suitable for digestion by restriction enzymes, amplification by PCR and Southern blotting. This procedure takes approximately 3 h to complete.     

A general purpose method for extracting RNA from Dictyostelium cells

Here we present a protocol for the extraction of RNA from Dictyostelium discoideum. Dictyostelium is a social amoeba that undergoes a basic developmental program, and therefore analysis of RNA levels over a time course is a commonly used technique. This procedure is similar to other guanidine thiocyanate-based methods; however, it has been adjusted because of the large quantities of carbohydrate and nucleases found in Dictyostelium cells. After cell lysis and phenol:chloroform extraction, the resulting high-quality RNA isolated with the described protocol allows the molecular genetic analysis of wild-type and genetically modified cells. The purified RNA can be used for analyses such as northern blotting, RT-PCR and microarrays. This procedure requires approximately 2 h to complete.     

Multimodal pressure-flow method to assess dynamics of cerebral autoregulation in stroke and hypertension

Background

This study evaluated the effects of stroke on regulation of cerebral blood flow in response to fluctuations in systemic blood pressure (BP). The autoregulatory dynamics are difficult to assess because of the nonstationarity and nonlinearity of the component signals.

Methods

We studied 15 normotensive, 20 hypertensive and 15 minor stroke subjects (48.0 ± 1.3 years). BP and blood flow velocities (BFV) from middle cerebral arteries (MCA) were measured during the Valsalva maneuver (VM) using transcranial Doppler ultrasound.

Results

A new technique, multimodal pressure-flow analysis (MMPF), was implemented to analyze these short, nonstationary signals. MMPF analysis decomposes complex BP and BFV signals into multiple empirical modes, representing their instantaneous frequency-amplitude modulation. The empirical mode corresponding to the VM BP profile was used to construct the continuous phase diagram and to identify the minimum and maximum values from the residual BP (BP_R) and BFV (BFV_R) signals. The BP-BFV phase shift was calculated as the difference between the phase corresponding to the BP_R and BFV_R minimum (maximum) values. BP-BFV phase shifts were significantly different between groups. In the normotensive group, the BFV_R minimum and maximum preceded the BP_R minimum and maximum, respectively, leading to large positive values of BP-BFV shifts.

Conclusion

In the stroke and hypertensive groups, the resulting BP-BFV phase shift was significantly smaller compared to the normotensive group. A standard autoregulation index did not differentiate the groups. The MMPF method enables evaluation of autoregulatory dynamics based on instantaneous BP-BFV phase analysis. Regulation of BP-BFV dynamics is altered with hypertension and after stroke, rendering blood flow dependent on blood pressure.

     

HIV antigens can induce TGF-beta(1)-producing immunoregulatory CD8+ T cells

HIV-infected individuals may progressively lose both HIV-specific and unrelated CTL responses despite the high number of circulating CD8+ T cells. In this study, we report that approximately 25% of HIV+ donors produced TGF-beta(1) in response to stimulation with HIV proteins or peptides. The production of TGF-beta(1) was sufficient to significantly reduce the IFN-gamma response of CD8+ cells to both HIV and vaccinia virus proteins. Ab to TGF-beta reversed the suppression. We found the source of the TGF-beta(1) to be predominantly CD8+ cells. Different peptide pools stimulated TGF-beta(1) and IFN-gamma in the same individual. The TGF-beta(1) secreting cells have distinct peptide specificity from the IFN-gamma producing cells. This represents an important mechanism by which an HIV-specific response can nonspecifically suppress both HIV-specific and unrelated immune responses.     

Efflux pumps expression and its association with porin down-regulation and β-lactamase production among <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> causing bloodstream infections in Brazil

Background  

Multi-drug efflux pumps have been increasingly recognized as a major component of resistance in P. aeruginosa. We have investigated the expression level of efflux systems among clinical isolates of P. aeruginosa, regardless of their antimicrobial susceptibility profile.