Immunohistochemical demonstration of carbonic anhydrase isoenzyme VI (CA VI) expression in rat lower airways and lung.

Carbonic anhydrase isoenzyme VI (CA VI), which is transported in high concentrations in saliva and milk into the alimentary tract, is an important element of mucosal protection in the upper alimentary tract. Like alimentary tract mucosa, the respiratory tract mucosa is also exposed to heavy microbial, physical, and chemical stress. The protective and renewal-promoting factors present in the surface mucus of the respiratory tract are mainly produced by the seromucous tracheobronchial glands. Here we studied the secretion of CA VI by these glands in adult and developing rats using immunohistochemical techniques. The serous acinar and duct cells of the tracheobronchial glands stained for CA VI. The presence of the enzyme also in the duct content indicates its active secretion into the surface mucus. CA VI was also visible in the secretory cells and at the base of the ciliated cells of the tracheobronchial surface epithelium. Moreover, the Clara cells of the bronchiolar surface epithelium stained for CA VI. These findings are consistent with the hypothesis that CA VI has a mucosa-protective role not only in the gastrointestinal tract but also in the respiratory tract, where CA VI may act as a pivotal pH neutralizer and growth factor.     

Needle ontogeny of mature Scots pines under enhanced UV-B radiation

The aim of this work was to test our hypothesis that pine needles protect themselves against UV-B radiation via anatomical changes in the epidermal layer. This could lead to needle growth reductions if large quantities of assimilates are allocated for the epidermal protective mechanisms at the expense of photosynthetic area. Effects of enhanced UV-B radiation on the needle ontogeny of mature Scots pines (Pinus sylvestris L.) were studied during the second season of a field experiment. Depending on the season and the time of the year (1996-1997), the enhanced UV-B irradiance varied from 0.92 to 5.09 kJ m^-2 day^-1 UV-B_BE compared to 0.54-2.44 kJ m^-2 day^-1 UV-B_BE of ambient radiation. It was found that UV-B treatment accelerated the early development of needles. In 6-day-old enhanced UV-B-treated needles, mesophyll and hypodermic cells were fully differentiated, whereas in ambient-treated needles, no lobate mesophyll cells were seen and hypodermic cells had not yet developed. In fully grown needles, no accelerated differentiation was seen, except that the epidermal cross-sectional area was smaller. The continuation of the experiment will show if such a significant difference only occurs irregularly and incidentally or if it is of consistent significance for needles.     

Expression of Six Peptidases from Lactobacillus helveticus in Lactococcus lactis

For development of novel starter strains with improved proteolytic properties, the ability of Lactococcus lactis to produce Lactobacillus helveticus aminopeptidase N (PepN), aminopeptidase C (PepC), X-prolyl dipeptidyl aminopeptidase (PepX), proline iminopeptidase (PepI), prolinase (PepR), and dipeptidase (PepD) was studied by introducing the genes encoding these enzymes into L. lactis MG1363 and its derivatives. According to Northern analyses and enzyme activity measurements, the L. helveticus aminopeptidase genes pepN, pepC, and pepX are expressed under the control of their own promoters in L. lactis. The highest expression level, using a low-copy-number vector, was obtained with the L. helveticus pepN gene, which resulted in a 25-fold increase in PepN activity compared to that of wild-type L. lactis. The L. helveticus pepI gene, residing as a third gene in an operon in its host, was expressed in L. lactis under the control of the L. helveticus pepX promoter. The genetic background of the L. lactis derivatives tested did not affect the expression level of any of the L. helveticus peptidases studied. However, the growth medium used affected both the recombinant peptidase profiles in transformant strains and the resident peptidase activities. The levels of expression of the L. helveticus pepD and pepR clones under the control of their own promoters were below the detection limit in L. lactis. However, substantial amounts of recombinant pepD and PepR activities were obtained in L. lactis when pepD and pepR were expressed under the control of the inducible lactococcal nisA promoter at an optimized nisin concentration.     

Flotillin microdomains interact with the cortical cytoskeleton to control uropod formation and neutrophil recruitment

We studied the function of plasma membrane microdomains defined by the proteins flotillin 1 and flotillin 2 in uropod formation and neutrophil chemotaxis. Flotillins become concentrated in the uropod of neutrophils after exposure to chemoattractants such as N-formyl-Met-Leu-Phe (fMLP). Here, we show that mice lacking flotillin 1 do not have flotillin microdomains, and that recruitment of neutrophils toward fMLP in vivo is reduced in these mice. Ex vivo, migration of neutrophils through a resistive matrix is reduced in the absence of flotillin microdomains, but the machinery required for sensing chemoattractant functions normally. Flotillin microdomains specifically associate with myosin IIa, and spectrins. Both uropod formation and myosin IIa activity are compromised in flotillin 1 knockout neutrophils. We conclude that the association between flotillin microdomains and cortical cytoskeleton has important functions during neutrophil migration, in uropod formation, and in the regulation of myosin IIa.     

Physical inactivity and obesity: a vicious circle

Objective: Physical activity (PA) begins to decline in adolescence with a concomitant increase in weight. We hypothesized that a vicious circle may arise between decreasing PA and weight gain from adolescence to early adulthood. Methods and Procedures: PA and self‐perceived physical fitness assessed in adolescents (16–18 years of age) were used to predict the development of obesity (BMI ≥30 kg/m²) and abdominal obesity (waist ≥88 cm in females and ≥102 cm in males) at age 25 in 4,240 twin individuals (90% of twins born in Finland, 1975–1979). Ten 25‐year‐old monozygotic (MZ) twin pairs who were discordant for obesity (with a 16 kg weight difference) were then carefully evaluated for current PA (using a triaxial accelerometer), total energy expenditure (TEE, assessed by means of the doubly labeled water (DLW) method), and basal metabolic rate (BMR, assessed by indirect calorimetry). Results: Physical inactivity in adolescence strongly predicted the risk for obesity (odds ratio (OR) 3.9, 95% confidence interval (CI) 1.4–10.9) and abdominal obesity (4.8, 1.9–12.0) at age 25, even after adjusting for baseline and current BMI. Poor physical fitness in adolescence also increased the risk for overall obesity (5.1, 2.0–12.7) and abdominal obesity (3.2, 1.5–6.7) in adulthood. Physical inactivity was both causative and secondary to the development of obesity discordance in the MZ pairs. TEE did not differ between the MZ co‐twins. PA was lower whereas BMR was higher in the obese co‐twins. Discussion: Physical inactivity in adolescence strongly and independently predicts total (and especially) abdominal obesity in young adulthood, favoring the development of a self‐perpetuating vicious circle of obesity and physical inactivity. Physical activity should therefore be seriously recommended for obesity prevention in the young.     

COX-2 inhibitors and genetic background reduce mammary tumorigenesis in cyclooxygenase-2 transgenic mice

Cyclooxygenase-2 (COX-2) overexpression is a widely recognized feature of human breast cancer and inhibitors of the enzyme have antitumor effects in a subset of tumor settings. Previously, we demonstrated that direct overexpression of COX-2 under control of the mammary-specific MMTV promoter/enhancer, was itself oncogenic and lead to the induction of mammary tumors in multiparous, outbred CD1 mice. In the present study, we provide evidence that COX-2 dependent tumor progression can also be studied in FVB/N, an inbred strain widely used for analysis of breast cancer progression. In these mice, the human COX-2 transgene was strongly induced during pregnancy/lactation and mammary tumors developed after multiple pregnancies. However, crossing the COX-2 FVB/N mice with the C57BL6 strain resulted in loss of the mammary tumorigenic phenotype despite the fact that the human COX-2 gene was induced. Treatment of the COX-2 transgenic mice in the FVB/N strain with celecoxib (1600 ppm), a COX-2 selective inhibitor, resulted significant reduction in tumor size and multiplicity when compared to transgenic mice fed with control chow. SC-560 (20 ppm), a COX-1 selective inhibitor did not have significant effect on tumorigenesis. These studies suggest that FVB/N is a susceptible mouse strain well suited to the study of COX-2 mediated tumor progression and may provide a tool for the identification of interacting genes and therapeutic treatments for this clinically important target.     

Acquired obesity is associated with increased liver fat, intra-abdominal fat, and insulin resistance in young adult monozygotic twins

We determined whether acquired obesity is associated with increases in liver or intra-abdominal fat or impaired insulin sensitivity by studying monozygotic (MZ) twin pairs discordant and concordant for obesity. We studied nineteen 24- to 27-yr-old MZ twin pairs, with intrapair differences in body weight ranging from 0.1 to 24.7 kg [body mass index (BMI) range 20.0-33.9 kg/m2], identified from a population-based FinnTwin16 sample. Fat distribution was determined by magnetic resonance imaging, percent body fat by dual-energy X-ray absorptiometry, liver fat by proton spectroscopy, insulin sensitivity by measuring the fasting insulin concentration, and whole body insulin sensitivity by the euglycemic insulin clamp technique. Intrapair differences in BMI were significantly correlated with those in intra-abdominal fat (r = 0.82, P < 0.001) and liver fat (r = 0.57, P = 0.010). Intrapair differences in fasting insulin correlated with those in subcutaneous abdominal (r = 0.60, P = 0.008), intra-abdominal (r = 0.75, P = 0.0001) and liver (r = 0.49, P = 0.048) fat. Intrapair differences in whole body insulin sensitivity correlated with those in subcutaneous abdominal (r = -0.72, P = 0.001) and intra-abdominal (r = -0.55, P = 0.015) but not liver (r = -0.20, P = 0.20) fat. We conclude that acquired obesity is associated with increases in intra-abdominal and liver fat and insulin resistance, independent of genetic factors.     

Plasma membrane residence of hyaluronan synthase is coupled to its enzymatic activity

Hyaluronan is a multifunctional glycosaminoglycan up to 10(7) Da molecular mass produced by the integral membrane glycosyltransferase, hyaluronan synthase (HAS). When expressed in keratinocytes, N-terminally tagged green fluorescent protein-HAS2 and -HAS3 isoenzymes were found to travel through endoplasmic reticulum (ER), Golgi, plasma membrane, and endocytic vesicles. A distinct enrichment of plasma membrane HAS was found in cell protrusions. The total turnover time of HAS3 was 4-5 h as judged by the green fluorescent protein signal decay and hyaluronan synthesis inhibition in cycloheximide-treated cells. The transfer from ER to Golgi took about 1 h, and the dwell time on the plasma membrane was less than 2 h in experiments with a relief and introduction, respectively, of brefeldin A. Constructs of HAS3 with 16- and 45-amino-acid C-terminal deletions mostly stayed within the ER, whereas a D216A missense mutant was localized within the Golgi complex but not the plasma membrane. Both types of mutations were almost or completely inactive, similar to the wild type enzyme that had its entry to the plasma membrane experimentally blocked by brefeldin A. Inhibition of hyaluronan synthesis by UDP-glucuronic acid starvation using 4-methyl-umbelliferone also prevented HAS access to the plasma membrane. The results demonstrate that 1) a latent pool of HAS exists within the ER-Golgi pathway; 2) this pool can be rapidly mobilized and activated by insertion into the plasma membrane; and 3) inhibition of HAS activity through mutation or substrate starvation results in exclusion of HAS from the plasma membrane.