Characterization of a newin vitro model for studies of reepithelialization in human partial thickness wounds

Summary Reepithelialization of artificial partial thickness wounds made in biopsies of human skin was determined after 3, 5, or 7 d of incubation, submerged or elevated to the air-liquid interface. The biopsies were reepithelialized within 5–7 d, with a more complete epidermal healing in wounds exposed to air. Both types of wounds showed similar time-course in deposition of basement membrane components, as detected by immunofluorescence labeling. Laminin and collagen type VII were deposited underneath the migrating tips, whereas collagen type IV was detected after reepithelialization. Markers of terminal differentiation showed a pattern close to normal in the air-liquid incubated wounds after reepithelialization. Involucrin was detected in the suprabasal regions of the migrating epidermis and thereafter in the upper half of neo-epidermis in the air-liquid incubated wound. Filaggrin could not be detected in the submerged wounds at any time during healing, whereas wounds exposed to air showed a well-differentiated epidermis by Day 7. Tritiated thymidine-incorporation indicated proliferation of epidermal and dermal cells during reepithelialization and a maintained viability, as shown by cultivation of endothelial- and fibroblast-like cells obtained from the dermis 7 d after wounding. Reepithelialization in this humanin vitro model is supported by a matrix close to normal with the possibility of extracellular influences and cell-cell interactions and, in addition, the technique is simple and reproducible. Therefore, we suggest this model for studies of regeneration in culture and as a complement toin vivo studies on epidermal healing.     

A rapid method to determine the mammalian cell cycle

A method was developed for determining the duration of the mammalian cell cycle and each of its major phases, mitosis, G1, DNA synthetic period, and G2. Mitotic time was determined by assessment of the mitotic index at intervals after cells collected in mitosis and stored at 4 °C were reincubated at 37 °C. The duration of the three remaining phases was derived from a graphic representation of the uptake of ³H-thymidine by a synchronous population of cells grown directly in scintillation vials. The scintillation counting method for determination of these parameters is advantageous over methods using autoradiography in that the investigator's bias in scoring cells is eliminated. Complex mathematical interpretations are unnecessary, and the data obtained from the scintillation counter are readily processed. Results from scintillation counting and autoradiographic methods are shown to be comparable.     

The effects of temperature and pH on the ethanol tolerance of the wine yeasts, Saccharomyces cerevisiae, Candida stellata and Kloeckera apiculata

The influences of temperature and pH on the survival and growth of Saccharomyces cerevisiae, Candida stellata and Kloeckera apiculata were examined in the presence of ethanol concentrations between 2.5 and 15% v/v. At 15°C, the maximum concentrations of ethanol permitting the growth of S. cerevisiae, C. stellata and K. apiculata were 15%, 11% and 9%, respectively. These maximum concentrations were decreased at 10°C and 30°C. Cells of S. cerevisiae showed no loss in viability when incubated for 12 d at 10°C or 15°C in the presence of 15% ethanol but showed some loss at 30°C. Cells of C. stellata were tolerant of 12.5% ethanol at 10°C and 15°C but not at 30°C. Cells of K. apiculata were tolerant of 10–12.5% ethanol at 15°C but not at 10°C or 30°C. Sensitivity of the yeast cells to ethanol was marginally increased on decreasing the pH from 6-0 to 3–0.     

Quantitative electron microscopic autoradiography on the mouse thyroid gland after administration of 3H-L-DOPA

Summary The cellular and subcellular distribution of radioactivity in the mouse thyroid gland different times (20 min — 8 hours) after intravenous administration of ³H-L-DOPA was studied by means of quantitative electron microscopic autoradiography.High concentrations of autoradiographic silver grains occur over parafollicular cells and adrenergic nerves while the labelling of follicular cells and lumina is low or absent and similar to the labelling of connective tissue cells at all observation times.Over the parafollicular cells high levels of radioactivity can be recorded already 20 min after administration of the labelled amino acid. The grain counts are highest at 1 hour and decrease then at 2.5 and 8 hours.The intracellular distribution of label is similar at all observation times; thus, the concentration of silver grains over the typical cytoplasmic granules of the parafollicular cells is 4–5 times higher compared to the concentration over the remainder of the cytoplasm and the nucleus.Treatment with a decarboxylase inhibitor prior to the injection of ³H-L-DOPA results in a low and uniform labelling of all thyroid cells. This finding, taken together with the observation that also pretreatment with reserpine abolishes the autoradiographic reaction over the cytoplasmic granules, gives strong support to the idea that the great majority of silver grains observed over parafollicular cells represents dopamine formed by decarboxylation of the labelled precursor.This study was supported by grant K71-12X-3352-01 from the Swedish Medical Research Council. The author wishes to express his gratitude to Mrs. Gunnel Bokhede and Miss Dala Sjögren for expert technical assistance.     

Survival of Campylobacter jejuni in raw, pasteurized and ultra-heat-treated goat's milk stored at different temperatures

The survival of a human strain of Campylobacter jejuni in raw, pasteurized and ultra-heat-treated goat's milk stored at 5°, 10°, 15° and 20°C was studied. No viable units were detected in raw milk after 24 h at 20°C and 48 h at 15°C. None were detected in pasteurized milk after 48 h at 20°C. In all other samples, there was a decline in viable units in the first 24 h but very little decline in the next 24 h period. The organism survived best at 5° and 10° C.     

Relationship between Bacillus sphaericus spore heat resistance and sporulation temperature

Bacillus sphaericus 9602 was grown in batch culture at various temperatures. At 10°C and 12°C the maximum sporulation yield was <10%, while at 15°C, 20°C and 30°C, a sporulation yield of >95% was achieved. However at 40°C B. sphaericus grew only vegetatively. The heat resistances (D values at 90°C) of spores grown at 15°C and 20°C were significantly higher than those grown at 30°C.     

Influence of photoperiod on low temperature acclimation for cold-hardiness in Drosophila auraria

ABSTRACT. Imagines of Drosophila auraria Peng, a reproductive diapause species, developed cold-hardiness at low temperatures to a greater extent when exposed to a diapause-inducing photoperiod (LD10:14 h) than when exposed to a diapause-preventing photoperiod (LD 16:8h). Imagines kept at 18°C, which was the temperature at which they were reared to eclosion, did not survive a test exposure to -5°C for 8 days regardless of age or photoperiod. When transferred to 10 or 5°C, either from eclosion or from 8 days after eclosion, the survival rate, on testing, rose with time since transfer and rose faster and higher with a photoperiod of LD 10:14h than with LD16:8h. Flies transferred to 15°C only showed improved ability to survive the test if they were kept in LD 10:14h. When cultured at 18°C to the age of 8 days after eclosion, diapause was terminated in about 30% of females even at LD 10:14h. In these post-diapause females the ability to develop cold-hardiness at lower temperatures was somewhat less than in the diapausing females, but apparently greater than in the non-diapause females. These results suggest that the physiological mechanism which promotes cold-hardiness under a diapause-inducing photoperiod is not directly linked to the process causing reproductive diapause.
In Sapporo, flies from a natural population became tolerant to cold in October when they entered diapause and daily mean temperature fell below 15°C and the light/dark cycle fell below LD 12:12h.     

Re‐epithelialization of large wound in paedomorphic and metamorphic axolotls

Axolotls (Ambystoma mexicanum ) may heal their skin wounds scar‐free in both paedomorphs and metamorphs. In previous studies on small punch skin wounds, rapid re‐epithelialisation was noted in these two axolotl morphs. However, large wound size in mammals may affect wound healing. In this study, large circumferential full thickness excision wounds on the hind limbs were created on juvenile paedomorphic and metamorphic axolotls. The results showed re‐epithelialisation was more quickly initiated in paedomorphs than in metamorphs after wounding. The migrating rate of epidermis on the wound bed was faster in paedomorphs than in metamorphs and thus completion of re‐epithelialisation was faster in paedomorphs than in metamorphs. Within these re‐epithelialisation periods, neither basement membrane nor dermis was reformed. Epidermal cell proliferation was detected by EdU‐labelling technique. In the normal unwounded skin, epidermal proliferation rate was higher in paedomorphs than in metamorphs. After wounding, the epidermal proliferation rate was significantly lower in the migrating front on the wound bed than in the normal skin in paedomorphs. The EdU‐labelling rate between normal skin and migration front was not different in metamorphs. Lacking of more proliferating epidermal cells on the wound bed indicated that the new epidermis here derived rather from migrating epidermal cells than from cell proliferation in situ. In conclusion, re‐epithelialisation in the large wound might be fully completed in both morphs despite it was initiated earlier and with faster rate in paedomorphs than in metamorphs. The new epidermis on the wound bed derived mainly from cell migration than by cell proliferation in the re‐epithelialisation period. J. Morphol. 278:228–235, 2017. © 2016 Wiley Periodicals,Inc.     

Influence of water temperature and feeding regime on otolith growth in Anguilla japonica glass eels and elvers: does otolith growth cease at low temperatures?

The influences of water temperature and feeding regime on otolith growth in Anguilla japonica glass eels and elvers were investigated using individuals reared at 5, 10, 15, 20, 25 and 30° C and in fed or unfed conditions at salinity 32 after their otoliths were marked with alizarin complexone (ALC). To eliminate the difficulty of observing the edges of otoliths with optical (OM) or scanning electron (SEM) microscopes, three to 10 individuals were sampled from each tank at 10, 20 and 30 days during the experiment and reared for an additional 10 days at 25° C after their otoliths were marked a second time. Otolith growth and the number of increments were measured using both OM and SEM. Most A. japonica commenced feeding after 10 days at 20–30° C or after 20 days at 15° C, but no feeding occurred at 5 and 10° C. No otolith growth occurred at 5 and 10° C except in two individuals with minimal increment deposition at 10° C. Otolith growth was proportional to water temperature within 15–25° C and not different between 25 and 30° C. At 15, 25 and 30° C, the mean otolith growth rate in fed conditions was higher than in unfed conditions. The number of increments per day was significantly different among water temperatures (0·00–0·01 day⁻¹ at 5 and 10° C, 0·43–0·48 day⁻¹ at 15° C and 0·94–1·07 day⁻¹ at 20–30° C). These results indicated that otolith growth in A. japonica glass eels and elvers was affected by temperature and ceased at ≤10° C under experimental conditions. Hence, future studies analysing the otoliths of wild-caught A. japonica glass eels and elvers need to carefully consider the water temperatures potentially experienced by the juveniles in the wild.     

Induction of embryos in pollen cultures of Nicotiana sylvestris

Induction of embryos in pollen cultures was possible when young buds from plants induced to flower at 15°C were given a cold treatment at 10°C for 15 days. Following selective isolation of embryogenic pollen the cultures raised were given cold treatment at 10°C for 12 or more days. In such cultures the embryos appeared at a frequency of 1.2% of the cultured pollen on mineral-sucrose medium at pH 6.8.     

Heat resistance of Listeria monocytogenes in dairy products as affected by the growth medium

Listeria monocytogenes strains 1151 and Scott A were grown in broth at 30 °C and transferred to half cream, double cream and butter stored at 5 °C to determine the influence of dairy product composition on heat resistance at 52, 56, 60, 64 and 68 °C. Strain 1151 showed a higher heat resistance than strain Scott A. The heat resistance of both strains was higher in the dairy products than in broth, particularly at lower temperatures. A significant difference was observed between log 10 of the D -values in the different dairy products. The D -values obtained for both strains resuspended in all the dairy products would result in efficient elimination of the pathogen at 72·7 °C for 15 s. The highest D -value was 11·30 s at 68 °C and by using a z -value of 6·71 °C it can be determined that at 72·7 °C the D -value would be 1·5 s. The 15 s process would therefore achieve 10 log reductions. The effect of growth conditions on the heat resistance at 60 °C of L. monocytogenes Scott A was also investigated. When the cells were grown in the dairy products themselves, and particularly butter, the heat resistance of Scott A was enhanced; for example, the D -values were 7·15 times higher than in broth. Further studies are required to investigate if this protection against heating exists at higher temperatures, in which case the efficiency of pasteurization treatments or other heat treatments would be considerably lowered.     

Cold-acclimation induced changes in freezing tolerance and translatable RNA content in Citrus grandis and Poncirus trifoliata

Cold-acclimation-induced changes in freezing tolerance and translatable RNA content were compared in seedlings of a relatively cold sensitive citrus species, Citrus grandis L. Osb. cv. Thong Dee (pummelo), and the cold-hardy citrus relative, Poncirus trifoliata L. Raf. cv. Pomeroy (trifoliate orange). Cold acclimation of pummelo (10 days at 15°C followed by 4 weeks at 10°/5°C, day/night) resulted in a decrease in LT₅₀ from −6 to −8°C, while in trifoliate orange (acclimated for 7 weeks at 5°C), the LT₅₀ decreased from −9 to −18°C. Qualitative changes in the in vitro translation profile, revealed by two-dimensional gel electrophoresis, were observed following cold acclimation in both species. An mRNA for a large polypeptide (ca 160 kDa) was detected following cold acclimation of trifoliate orange. A similar change was not observed in pummelo following cold acclimation.     

Effect of temperature in the regulation of DNA synthesis in synchronous cultures of Chlorella

The influence of temperature on DNA replication of Chlorella pyrenoidosa grown at 30 °C was studied in synchronous cultures. Final DNA content and cell counts/ml were 30 to 40% less than controls after a 4 h exposure to 15 °C, but reached normal levels after being kept temporarily at 10 °C. The rates of DNA synthesis at these two temperatures were, respectively, one-sixth and one-tenth of the rate at 30 °C, but during the 15 °C treatment the cells lost the ability to enter a further replication cycle more rapidly than at 10 °C. This loss of capacity to initiate further replication cycles was prevented by inhibiting protein synthesis with cycloheximide.     

Epithelial locomotion and differentiation in frog skin cultures

Small explants from the medioventral skin of the green frog were maintained in culture for 5 days. During the first hours, a rapid outgrowth of the stratum germinativum was observed at the periphery of the fragment (2 X 3 X 0.75 mm). The Malpighian cells stretched and emitted long lamellipodia following the cut edges of the dermis. These cells acquired a fibroblastic shape and were covered by other flattened cells from the stratum spinosum and even from the stratum granulosum. This progression of cells simulated an epiboly; cell divisions occurred and were revealed by autoradiography. The underlying dermis could be totally covered after 3 days. An increase in the number of cellular layers was observed. The migrating cells redifferentiated, in particular at the lower side of the dermis, which provided a suitable substratum for the newly formed epidermis. A new basal lamina was formed. Some exfoliations of keratinized layers were seen. After 5 or 6 days of culture, epiboly was complete, the epithelium formed a closed system, and degenerative processes occurred, probably by non-penetration of the nutritive medium into the deeper regions of the explant.     

Effects of Temperature and Leaf Weness on the Latent Period of Rbynchosporium Secalis (Leaf Blotch) on Leaves of Winter Barley

Effects of temperature and leaf wetness on the latent period of Rhynchosporium secaits (leaf blotch) on winter barley were examined in controlled environment experiments. At 100% relative humidity (continuous leaf wetness) the mean length of the latent period was c.24 days at 5°C, c. 19 days at 10°C, c. 16 days at l5°C and c. 13 days at 20°C. The mean number of days between the appearance of the first and the last lesions was c. 13 days at 5°C, c. 6 days at 10°C, c. 5 days at 15°C and c. 3 days at 20°C. A negative curvilinear regression of latent period on temperature accounted for 99% of the variance. The mean area of lesions per leaf was 38 mm² at 5°C, 46 mm² at 10°C, 24 mm² at 15°C and 24 mm² at 20°C. At 10°C, after a 48 h wet infection period, the interruption of leaf wetness for 5 or more days at any time during the next 15 days of the latent period did not decrease subsequent lesion area. However, absence of leaf wetness after these 15 days, at the onset of sporuiation, did decrease the area of lesions which developed.     

DNA Synthesis, Mitosis and Ploidy of Dividing Cells in Early Crown Gall

DNA synthesis, mitosis and ploidy of dividing cells were studied during 30 h after wounding around wounds inoculated with Agrobacterium tumefaciens, around sterile wounds and in control stems of Vicia faba. DNA synthesis was examined by counting nuclei labelled with ³H-thymidine in slides in which mitoses had been counted and analyzed before autoradiography. The main results were that around inoculated wounds, DNA synthesis and the number of mitoses showed a peak between 12 and 22.5 h. Both types of wounding induced mitoses, many of them polyploid (DNA content higher than 4C), both in the pith and the cortex, whereas in the control stems only diploid mitoses, mostly in the stelar area, were seen. The first polyploid (8C) mitoses around the inoculated wounds took place at 12 h and at 15.5 h 32C mitoses were seen; around the sterile wounds the first 8C divisions occurred at 26 h. The frequency of polyploid mitoses and their degree of ploidy continued to be considerably higher around the crown gall than around the control wounds. When a cell with a higher than 4C content is induced to divide, the 12 chromosomes, as a rule, consist of four, eight, 16 or 32 chromatids, instead of the normal two. The early division of highly polyploid cells around the inoculated wounds is obviously caused by growth factors which are known to be produced by the bacteria. It appears possible that this ability to synthesize excessive amounts of growth factors is subsequently transferred to the host cells through bacterial DNA.     

Nisin induces changes in membrane fatty acid composition of Listeria monocytogenes nisin-resistant strains at 10°C and 30°C

Listeria monocytogenes isolates resistant to 10⁵ IU ml^-1 nisin were obtained at 30°C (NR30) and at 10°C (NR10). Nisin prolonged the lag phase of isolate NR30 at 10°C. Isolates NR30 and NR10 did not produce a nisinase. Protoplasts of isolate NR30 were unaffected by exposure to nisin. The fatty acid composition from the wild-type strain and NR isolates was determined. As expected, temperature-induced differences in the C15/C17 fatty acid ratios were found. Growth of the NR strains in the presence of nisin resulted in significantly different C15/C17 ratios and a significant increase in the percentage of C16:0, C16: 1, C18:0 and C18: 1 fatty acids at 10°C and 30°C. Both the NR10 and NR30 isolates had similar growth rates at low temperatures, but these were slower than the wild-type strain. These results indicate that 'nisin resistance'is an environmentally defined phenotype and that nisin induces changes in the fatty acid composition of the membrane in L. monocytogenes nisin-resistant isolates regardless of the growth temperature.     

A comparison of the effects of temperature on wound healing in a tropical and a temperate teleost

The rate of healing of a surgical wound was studied in two teleost fish, one with a tropical, and the other with a temperate temperature range. Comparisons were made of both the rate and qualitative nature of wound healing within and between species at temperatures of 30,23,10 and 5° C. The rate of wound healing was found to be proportional to temperature and temperature stress had little effect on healing rates. The findings were related to reported rates of wound healing in man. In general the wounds studied healed at a rate comparable to those reported for the healing of superficial skin wounds in man and other mammals despite the fact that the fish wounds were not merely superficial but involved integument and muscle.     

An investigation of photoperiod and temperature in relation to the life cycles of Mnium hornum Hedw. and M. undulatum Sw. (Musci) with reference to their histology

Antheridia and archegonia of Mnium hornum are produced according to an endogenous rhythm that approximates to an annual cycle. Short days appear to have a delaying effect on gametangial production, but continuous exposure to 7.25-hour days at both 10° and 20°C is incapable of causing complete suppression. The cycle shows some tendency to precede spring conditions and it is suggested that the effect of short days in winter is to preserve its co-ordination with the seasons.
Archegonial induction in M. undulatum takes place in response to long day treatment, although an alternative stimulus can be provided by a rise in temperature, the critical day length being between 7.25 and 12 hours at 10° C. Male M. undulatum is also a long day plant, but a diurnal fluctuation in temperature is essential to inflorescence production. Temperature ranges of 5.5°-11° C and 10°-20°C are suitable for antheridial induction in 15-hour days.     

Isolation and preliminary characterisation of twenty-five temperature-sensitive mutants of mouse cytomegalovirus

Abstract To study the pathogenicity of mouse cytomegalovirus (MCMV) and to identify virulence determinants, we have isolated and phenotypically characterised 25 temperature-sensitive ( ts ) mutants. Six of these ( tsm 9, tsm 13, tsm 20, tsm 22, tsm 28 and tsm 30) failed to replicate in mice and were avirulent. Five mutants ( tsm 14, tsm 18, tsm 19, tsm 25 and tsm27 ) were to similar virulence to the parenthal wild-type ( wt ) virus, five ( tsm 7, tsm 15, tsm 24, tsm31 ) were 12–100 fold less virulent, five ( tsm 8, tsm 12, tsm 16, tsm 23 and tsm 29) were 150–1500 fold less virulent and four ( tsm 10, tsm 11, tsm 17 and tsm 21) were between 2,000 and 85,000 fold less virulent than wt . One mutant ( tsm 28) did not plaque or replicate at 39°C while 5 other mutants ( tsm 7, tsm 9, tsm 23, tsm 24 and tsm 27) also failed to plaque at 39°C but only failed to replicate or replicated poorly at 40°C. A further two mutants ( tsm 10 and tsm 13) were able to plaque and replicate at 39°C but not 40°C. Six other mutants ( tsm 14, tsm 15, tsm 16, tsm21 , tsm 22 and tsm 30) failed to form plaques at 40°C and were severely restricted in their replication at 40°C. The remaining 11 mutants exhibited varying degrees of restriction in ability to plaque and/or replicate at non-permissive temperatures. These 25 mutants, together with 6 isolated previously, comprise at least 24 complementation groups.