Retrograde tracing and toe spreading after experimental autologous nerve transplantation and crush injury of the sciatic nerve: a descriptive methodological study

ABSTRACT: Evaluation of functional and structural recovery after peripheral nerve injury is crucial to determine the therapeutic effect of a nerve repair strategy. In the present study, we examined the relationship between the structural evaluation of regeneration by means of retrograde tracing and the functional evaluation analysis of toe spreading. Two standardized rat sciatic nerve injury models were used to address this relationship. As such, animals received either a 2 cm sciatic nerve defect (neurotmesis) followed by autologous nerve transplantation (ANT animals) or a crush injury with spontaneous recovery (axonotmesis; CI animals). Functional recovery of toe spreading was observed over an observation period of 84 days. In contrast to CI animals, ANT animals did not reach pre-surgical levels of toe spreading. After the observation period, the lipophilic dye DiI was applied to label sensory and motor neurons in dorsal root ganglia (DRG; sensory neurons) and spinal cord (motor neurons), respectively. No statistical difference in motor or sensory neuron counts could be detected between ANT and CI animals. In the present study we could indicate that there was no direct relationship between functional recovery (toe spreading) measured by SSI and the number of labelled (motor and sensory) neurons evaluated by retrograde tracing. The present findings demonstrate that a multimodal approach with a variety of independent evaluation tools is essential to understand and estimate the therapeutic benefit of a nerve repair strategy.     

Kappa-chain constant-region gene sequences in genus Rattus: coding regions are diverging more rapidly than noncoding regions

We have determined the nucleotide sequence of a 1,200-base pair (bp) genomic fragment that includes the kappa-chain constant-region gene (C kappa) from two species of native Australian rodents, Rattus leucopus cooktownensis and Rattus colletti. Comparison of these sequences with each other and with other rodent C kappa genes shows three surprising features. First, the coding regions are diverging at a rate severalfold higher than that of the nearby noncoding regions. Second, replacement changes within the coding region are accumulating at a rate at least as great as that of silent changes. Third, most of the amino acid replacements are localized in one region of the C kappa domain--namely, the carboxy-terminal "bends" in the alpha-carbon backbone. These three features have previously been described from comparisons of the two allelic forms of C kappa genes in R. norvegicus. These data imply the existence of considerable evolutionary constraints on the noncoding regions (based on as yet undetermined functions) or powerful positive selection to diversify a portion of the constant-region domain (whose physiological significance is not known). These surprising features of C kappa evolution appear to be characteristic only of closely related C kappa genes, since comparison of rodent with human sequences shows the expected greater conservation of coding regions, as well as a predominance of silent nucleotide substitutions within the coding regions.      

A Hidden Markov Model approach to variation among sites in rate of evolution

The method of Hidden Markov Models is used to allow for unequal and unknown evolutionary rates at different sites in molecular sequences. Rates of evolution at different sites are assumed to be drawn from a set of possible rates, with a finite number of possibilities. The overall likelihood of phylogeny is calculated as a sum of terms, each term being the probability of the data given a particular assignment of rates to sites, times the prior probability of that particular combination of rates. The probabilities of different rate combinations are specified by a stationary Markov chain that assigns rate categories to sites. While there will be a very large number of possible ways of assigning rates to sites, a simple recursive algorithm allows the contributions to the likelihood from all possible combinations of rates to be summed, in a time proportional to the number of different rates at a single site. Thus with three rates, the effort involved is no greater than three times that for a single rate. This "Hidden Markov Model" method allows for rates to differ between sites and for correlations between the rates of neighboring sites. By summing over all possibilities it does not require us to know the rates at individual sites. However, it does not allow for correlation of rates at nonadjacent sites, nor does it allow for a continuous distribution of rates over sites. It is shown how to use the Newton-Raphson method to estimate branch lengths of a phylogeny and to infer from a phylogeny what assignment of rates to sites has the largest posterior probability. An example is given using beta-hemoglobin DNA sequences in eight mammal species; the regions of high and low evolutionary rates are inferred and also the average length of patches of similar rates.      

Yeast iso-1-cytochrome c. A 2.8 A resolution three-dimensional structure determination

A molecular replacement approach, augmented with the results of predictive modeling procedures, solvent accessibility studies, packing analyses and translational coefficient searches, has been used to elucidate the 2.8 A (1 A = 0.1 nm) resolution structure of yeast iso-1-cytochrome c. An examination of the polypeptide chain folding of this protein shows it to have unique conformations in three regions, upon comparison with the structures of other eukaryotic cytochromes c. These include: residues -5 to +1 at the N-terminal end of the polypeptide chain, which are in an extended conformation and project in large part off the surface of the protein; residues 19 to 26, which form a surface beta-loop on the His18 ligand side of the central heme group; and, the C-terminal end of the helical segment composed of residues 49 to 56, which serves to form a part of the heme pocket. Structural studies also show that the highly reactive sulfhydryl group of Cys102 is buried within a hydrophobic region in the monomer form of yeast iso-1-cytochrome c. Dimerization of yeast iso-1-cytochrome c through disulfide bond formation between two such residues would require a substantial conformational change in the C-terminal helix of this protein. Another unique structural feature, the trimethylated side-chain of Lys72, is located on the surface of yeast iso-1-cytochrome c near the solvent-exposed edge of the bound heme prosthetic group. On the basis of the results of these and other structural studies, an analysis of the spatial conservation of structural features in the heme pocket of eukaryotic cytochromes c has been conducted. It was found that the residues involved could be divided into three general classes. The current structural analyses and additional modeling studies have also been used to explain the altered functional properties observed for mutant yeast iso-1-cytochrome c proteins.     

Studies on bacteria-like particles sampled from the stratosphere

Bacteria-like particles recovered from the stratosphere and deposited on cellulose acetate membranes have been analysed to confirm their bacterial nature. One particle appeared to be attached to an inorganic particle apparently by mucoid material typically produced by bacteria. A filamentous structure, morphologically similar to a fungal hypha, was also observed. EDS analysis showed that the particles were all non-mineral and therefore could be biological in nature. However, the composition of several clumps of nanobacteria-sized particles were found, by SIMS analysis, to be inconsistent with that of bacteria. The results show that it is dangerous to assume that bacteria-like particles seen under scanning electron microscopy are necessarily bacteria.     

Invasive aspergillosis in a "healthy" patient.

A case of invasive aspergillosis complicated by the formation of an aspergilloma is described. The patient, a 48-year-old man, was apparently healthy except for mild alcoholic steatosis of the liver. A review of the literature revealed that 5 of the 14 previously reported cases of invasive aspergillosis in seemingly immunocompetent hosts were associated with liver disease. Immunologic investigation in this case revealed transient cutaneous anergy during the acute illness and normal lymphocyte function. Assessment of polymorphonuclear leukocyte function, however, showed abnormalities of phagocytosis as well as impairment of intracellular bactericidal activity. These abnormalities may have contributed to a relative immunodeficiency. Impairment of immune function may play a role in the pathogenesis of invasive aspergillosis in some apparently healthy patients.     

Lead-phase and red-stripe color morphs of red-backed salamanders Plethodon cinereus differ in hematological stress indices: A consequence of differential predation pressure?

Throughout the animal kingdom there are species that have two or more phenotypic forms or 'morphs', and many of these are amphibians. In North America, the red-backed salamander Plethodon cinereus can have either a red dorsal stripe or no dorsal stripe (lead-phase form), and evidence to date indicates the lead-phase form incurs a greater number of attacks from predators. In a recent collection of 51 P. Cinereus, blood smears of both color morphs (35 red-stripe, 16 lead-phase) were examined to obtain numbers of circulating leukocytes (via light microscopy), which can be used to indirectly estimate levels of stress hormones in vertebrates via a 'hematological stress index', which is the ratio between the number of two leukocyte types (neutrophils and lymphoeytes). Our results showed that lead-phase salamanders tended to have greater numbers of circulating neutrophils and lower numbers of circulating iymphocytes than red-stripe morphs, leading to higher average neutrophil-lymphocyte ratios in lead-phase individuals. Since the salamanders were held (refrigerated) for 7 days before sampling, we cannot be certain if this effect is a stress reaction to the captivity or the normal level for this morph. However comparison with two sets of related salamanders that were captured and sampled immediately indicates the red-stripe salamanders were either not stressed from the captivity at all, or their white blood cell distributions had returned to normal after 7 days of captivity. Taken together, our results indicate that lead-phase forms of P. Cinereus have higher stress levels than the red-stripe forms, which may be a consequence of their higher exposure to, and/or attacks from, predators. They may also indicate that the lead-phase form is less-suited to captivity than the red-stripe form of this species.     

Studies on bacteria-like particles sampled from the stratosphere

Bacteria-like particles recovered from the stratosphere and deposited on cellulose acetate membranes have been analysed to confirm their bacterial nature. One particle appeared to be attached to an inorganic particle apparently by mucoid material typically produced by bacteria. A filamentous structure, morphologically similar to a fungal hypha, was also observed. EDS analysis showed that the particles were all non-mineral and therefore could be biological in nature. However, the composition of several clumps of nanobacteria-sized particles were found, by SIMS analysis, to be inconsistent with that of bacteria. The results show that it is dangerous to assume that bacteria-like particles seen under scanning electron microscopy are necessarily bacteria.     

Climate change hastens the turnover of stream fish assemblages

Stream fish are expected to be significantly influenced by climate change, as they are ectothermic animals whose dispersal is limited within hydrographic networks. Nonetheless, they are also controlled by other physical factors that may prevent them moving to new thermally suitable sites. Using presence–absence records in 655 sites widespread throughout nine French river units, we predicted the potential future distribution of 30 common stream fish species facing temperature warming and change in precipitation regime. We also assessed the potential impacts on fish assemblages' structure and diversity. Only cold-water species, whose diversity is very low in French streams, were predicted to experience a strong reduction in the number of suitable sites. In contrast, most cool-water and warm-water fish species were projected to colonize many newly suitable sites. Considering that cold headwater streams are the most numerous on the Earth's surface, our results suggested that headwater species would undergo a deleterious effect of climate change, whereas downstream species would expand their range by migrating to sites located in intermediate streams or upstream. As a result, local species richness was forecasted to increase greatly and high turnover rates indicated future fundamental changes in assemblages' structure. Changes in assemblage composition were also positively related to the intensity of warming. Overall, these results (1) stressed the importance of accounting for both climatic and topographic factors when assessing the future distribution of riverine fish species and (2) may be viewed as a first estimation of climate change impacts on European freshwater fish assemblages.