Biomedical subjects

M Kalina

Publications and source records attributed to M Kalina.

At least 37 records · Page 2Linked to original sources

PubMed1994

Secretion of cytokines by rat alveolar epithelial cells: possible regulatory role for SP-A.

Cultured alveolar type II cells and pulmonary epithelial (PE) cells in long-term culture were found to secrete colony-stimulating factors (CSF) into the medium in similar fashion to alveolar macrophages. CSF activity was determined by using the in vitro assay for myeloid progenitor cells [colony-forming units in culture (CFU-C)]. Both lipopolisaccharide (LPS) and interleukin-1 alpha (IL-1 alpha) were found to upregulate the secretion 6.5- to 8-fold from alveolar type II cells and macrophages. However, no stimulatory effect of these factors was observed in PE cells that release CSF into the medium constitutively, possibly due to the conditions of long-term culture. The CSF activity was partially neutralized (70% inhibition) by antibodies against murine granulocyte/macrophage (GM)-CSF and IL-3, thus indicating the presence of both GM-CSF and IL-3-like factors in the CSF. However, the presence of other cytokines in the CSF is highly probable. Surfactant-associated protein A (SP-A), which is known to play a central role in surfactant homeostasis and function, was also found to upregulate secretion of CSF (at concentrations of 0.1-5 micrograms/ml) from alveolar type II cells and macrophages. Control cells such as rat peritoneal macrophages, alveolar fibroblasts, and 3T3/NIH cell line could not be elicited by SP-A to release CSF. The results are discussed in relation to the possible participation of the alveolar epithelial cells in various intercellular signaling networks. Our studies suggest that alveolar type II cells and SP-A may play an important regulatory role in the modulation of immune and inflammatory effector cells within the alveolar space.

Animals↗

PubMed1993

Impact of on-site care, prehospital time, and level of in-hospital care on survival in severely injured patients.

A sample of 360 severely injured patients was selected from a cohort of 8007 trauma victims followed prospectively from the time of injury to death or discharge. A case referent study was used to test the association between on-site care, total prehospital time, and level of care at the receiving hospital with short-term survival. Multiple logistic regression analyses showed that use of Advanced Life Support (ALS) at the scene was not associated with survival, whereas treatment at a level I compatible hospital was associated with a 38% reduction in the odds of dying, which approached statistical significance. Total prehospital time over 60 minutes was associated with a statistically significant adjusted relative odds of dying (OR = 3.0). The results of this study support the need for regionalization of trauma care and fail to show a benefit associated with ALS.

Adult↗

PubMed1993

Internalization of surfactant protein A (SP-A) into lamellar bodies of rat alveolar type II cells in vitro.

Pulmonary surfactant is thought to be internalized and processed for reuse by alveolar Type II cells. In the present study we followed the internalization and intracellular trafficking of purified surfactant protein A (SP-A) by primary cultures of alveolar Type II cells. Internalization of native rat SP-A was compared with that of recombinant rat and human SP-A isolated from a patient with alveolar proteinosis. All SP-A species were conjugated with colloidal gold for visualization by electron microscopy. The gold conjugates were biologically active, as demonstrated by inhibition of phospholipid secretion from alveolar Type II cells. The SP-A-gold conjugates were internalized to lamellar bodies (LB) via the endosomal system, which included both electron-lucent and -dense multivesicular bodies. Labeling of LB was time dependent, and after 7 hr 30-40% of these organelles were labeled. Alkylation of SP-A greatly reduced internalization, as did an excess of non-conjugated SP-A. No qualitative differences in uptake were observed with the three forms of SP-A. The percent of labeled LB was similar (30-40%) after 7 hr of internalization with the three species of SP-A. The recombinant SP-A produced using a baculovirus vector lacked hydroxyproline and had an altered oligosaccharide, but these features did not affect its internalization or the rate of LB labeling. Internalization of the gold-conjugated SP-A and endocytosis of the fluid-phase marker Lucifer Yellow were related to the shape of Type II cells. Both uptake of SP-A, which is receptor mediated, and fluid-phase endocytosis were found to be less active in the flattened than in the rounded cells. Therefore, cell shape and hence cytoskeletal organization may play an important role in SP-A recycling. However, it is possible that both morphology and decreased endocytosis are independent manifestations related to the loss of differentiated function of cultured Type II cells.

Animals↗

PubMed1992

Standardized mortality ratio analysis on a sample of severely injured patients from a large Canadian city without regionalized trauma care.

Flora's Z statistic and standardized mortality ratios (SMRs) as indicators of excess mortality were calculated for a sample of 355 patients with major trauma. A statistically significant overall excess mortality was observed in this sample (Z = 6.77, SMR = 1.81, p less than 0.05). Advanced life support provided by physicians at the scene (MD-ALS) was not associated with reduced excess mortality. A significant trend toward lower excess mortality was associated with a higher level of trauma care at the receiving hospital (p less than 0.05). Total prehospital time over 60 minutes was associated with a significant increase in excess mortality (p less than 0.001). These results support regionalization of trauma care and failed to show any benefit associated with MD-ALS.

Adolescent↗

PubMed1992

Surfactant protein C is expressed in alveolar type II cells but not in Clara cells of rat lung.

The expression of the surfactant-associated proteins in bronchiolar cells remains to be defined. We used in situ hybridization to identify sites of message expression of the surfactant-associated proteins A, B, and C (SP-A, SP-B, and SP-C) in adult and fetal rat lung. The expression of these messages by in situ hybridization was also compared with the localization of SP-A by immunocytochemistry. The localization of SP-A was used to identify type II cells and nonciliated bronchiolar epithelial (Clara) cells in these sections. The cRNA antisense probes for SP-A, SP-B, and SP-C appeared to hybridize over type II cells. Sense probes showed no localization or apparent specific hybridization. Messages for both SP-A and SP-B were also found in nonciliated bronchiolar epithelial (Clara) cells. However, no message for SP-C was observed in these cells. Clara cells from terminal to large bronchioles lacked detectable mRNA for SP-C. Expression of surfactant protein mRNAs was not detectable in type I cells, alveolar macrophages, interstitial cells, or vascular cells. Similarly, in fetal rat lung the messages for SP-A and SP-B but not SP-C were detected in bronchiolar cells. We conclude that rat Clara cells do not express SP-C mRNA, and thus SP-C can be regarded as a specific marker for rat type II cells.

Animals↗

PubMed1992

Ca(2+)-independent induction of acrosome reaction by protein kinase C in human sperm.

We report that activated protein kinase C (PKC) can induce acrosome reaction independently of elevated Ca2+. Addition of 12-O-tetradecanoyl phorbol-13-acetate or the membrane-permeable diacylglycerol analog 1-oleoyl-2-acetylglycerol to ejaculated human sperm resulted in stimulation of acrosomal reaction (2- to 3-fold), provided the sperm underwent capacitation. Induction of acrosome reaction by 12-O-tetradecanoyl phorbol-13-acetate was blocked by the PKC inhibitor staurosporine or by down-regulation of endogenous PKC, but not by removal of extracellular Ca2+. Acrosome reaction was also enhanced by the Ca2+ ionophore ionomycin in a Ca(2+)-dependent, PKC-independent fashion. Immunohistochemical analysis with type-specific PKC antibodies revealed the presence of PKC alpha and PKC beta II in the equatorial segment, whereas PKC beta I and PKC epsilon staining was found in the principal piece of the tail. Acrosome reaction, thus far believed to be induced only by elevated Ca2+, can therefore be triggered by activated PKC in a Ca(2+)-independent fashion. The PKC subtypes potentially involved in acrosome reaction are most likely alpha and beta II, whereas the beta I- and epsilon-subspecies might be involved in regulation of flagellar motility of human sperm.

Acrosome↗

PubMed1991

Medial vs lateral frontal lobe lesions and differential impairment of central-gaze fixation maintenance in man.

The ability to maintain central-gaze fixation was studied in 35 patients with unilateral frontal lobe lesions. The stability of central-gaze fixation was tested in 2 oculomotor tasks requiring the suppression of reflexive saccades triggered by the sudden appearance of novel, peripheral visual stimuli. In the first task ('Instructed'), maintenance of gaze fixation was based on verbal instruction, whereas, in the second task ('Non-instructed'), it was based upon a foveating mechanism maintained by sensory stimulation during the performance of a categorization task. Patients with frontal lobe lesions were classified into 3 groups: a ventrolateral (n = 18), a dorsolateral (n = 10) and a medial (n = 7) group. The control group consisted of 20 healthy subjects. In the Non-instructed task, no group differences were found in the rate of reflexive saccades. Thus, lesions of the frontal lobe did not affect reactivity to extraneous stimuli per se. The only exception to this rule was the group of patients with ventrolateral lesions in the left frontal lobe, in which a higher number of reflexive saccades directed to the contralateral hemifield was observed. In the Instructed task, 2 different patterns of deficit in suppression of reflexive saccades were found. First, a bidirectional deficit was noted in the ventrolateral group, which might be attributed to an impairment in either maintaining a selected program of behaviour 'on-line' or suppressing inappropriate alternatives interfering with that program. Second, a unidirectional deficit in voluntary suppression of reflexive saccades triggered from within the contralateral hemifield was observed in the medial group. It is suggested that this type of deficit may reflect malfunctioning of the supplementary motor area--anterior cingulate system involved in the suppression of externally triggered interfering programs. Finally, no significant impairment was found in the dorsolateral group.

Adult↗

PubMed1991

Morphometric analysis of gold particles in low-label cell compartments.

Quantitative estimation of the binding of gold-conjugated ligands to various cell organelles has become a commonly used method to quantify the amount of ligand-binding sites associated with those organelles. However, often a small percentage of organelles is labeled or the density of gold labeling is low. We have defined the "gold-labeled region" as a zone that has a boundary defined by the localization of the outermost gold particles. Such a phenomenon was recently observed in a study of the internalization of gold-labeled native surfactant into lamellar bodies of cultured pulmonary type II cells. We estimated the size and density of gold-labeled regions in lamelar bodies using a simple stereological approach and demonstrated that the low percentage of gold-labeled organelles can be explained as a result of the probability of random selecting through the labeled areas. Our method, which permits use of transmission electron microscopy to calculate the true parameters of gold-labeled regions, can significantly facilitate analyses of ligand binding to various cell compartments.

Animals↗

PubMed1991

Endocytosis in cultured rat alveolar type II cells: effect of lysosomotropic weak bases on the processes.

We investigated the uptake of Lucifer yellow and surfactant complexed with gold (S-G) by isolated alveolar Type II cells. The fluid phase marker Lucifer yellow did not reach lamellar bodies (LB) even after prolonged incubation time, whereas S-G was internalized and found in LB. Treatment of Type II cells with lysosomotropic weak bases (NH4Cl and chloroquine) resulted in dilation of endosomes, lysosomes, and LB. The effect of these agents on LB resulted in disappearance of their lamellar organization, as detected by polarized light and electron microscopy. After incubation in lysosomotropic agent-free medium, endocytosis of Lucifer yellow and S-G in treated cells was mainly directed towards large vacuoles resembling either multivesicular bodies (MVB) or lysosomes. The possible relationship between LB, MVB, and lysosomes in freshly isolated as well as cultured alveolar Type II cells is discussed.

Ammonium Chloride↗

PubMed1991

Chromogranins or chromogranin-like proteins are present in lamellar bodies and pulmonary surfactant of rat alveolar type II cells.

Rat alveolar Type II cells were immunostained with antibodies directed against chromogranin A (monoclonal, LK2H10) and chromogranins A and B (polyclonal, LKZM1U). The chromogranins or chromogranin-like proteins were identified in cells in lung tissue sections and isolated Type II cells at the light and electron microscopic levels. We used post-embedding immunoelectron microscopy, with immunogold, to detect the proteins' immunoreactivity in osmicated tissues. Gold particles were distributed over the phospholipid lamellae within the lamellar bodies of alveolar Type II cells and over the lattice structure of tubular myelin. Quantitative analysis of gold labeling densities in the various cell compartments indicated that only the latter two structures were specifically labeled. Controls, which included pre-absorption of both anti-chromogranin antibodies with excess chromogranin A or with native surfactant, resulted in a greater than 60% decrease in gold labeling. A possible role of chromogranins or chromogranin-like proteins as Ca2+ binding proteins in alveolar Type II cells is discussed.

Animals↗

PubMed1991

Immunocytochemical localization of protein kinase-C subtypes in anterior pituitary cells: colocalization in hormone-containing cells reveals heterogeneity.

We have examined the distribution and colocalization of protein kinase-C (PKC) in cultured rat anterior pituitary cells by light microscopic immunocytochemistry using monoclonal antibodies to group A rat brain PKC subspecies. Type I (PKC gamma) was not detected in the cells, in line with the assertion that the gamma-enzyme is expressed specifically in central nervous tissues. The other subspecies recognized by the antibodies (PKC beta and PKC alpha) were present throughout the cytoplasm in a diffused pattern, while the nuclei were apparently unstained. The number of cells stained with the antibodies in juvenile animals (12 days old) increased rapidly with age and reached a plateau between adult (5-month-old) and older (1-yr-old) rats. Type II (PKC beta) was the predominant subspecies detected in anterior pituitary cells. Double immunofluorescence staining techniques enabled the colocalization of PKC with various anterior pituitary cell types. Surprisingly, not all of the hormone-producing cells were stained with the PKC antibodies. Moreover, within the different pituitary cell types, the percentage of PKC-stained cells varied, revealing heterogeneity among the various cell populations. Thus, among somatrophs, mammotrophs, thyrotrophs, ACTH-containing cells, and gonadotrophs, only 9%, 22%, 13%, 44%, and 26%, respectively, reacted with the PKC antibodies. We suggest that activation of pituitary PKC might mobilize only a fraction of the hormone-containing cells.

Animals↗

PubMed1990

Decrease in the basal levels of cytosolic free calcium in chondrocytes during aging in culture: possible role as differentiation-signal.

Cell- and matrix-related parameters, which characterize the aging and differentiation process of cartilage in vivo, were measured in cultured chick epiphyseal chondrocytes during maintenance in a suspension culture for 34 days. A gradual decrease in the rates of proliferation and an increase in the size of the cells were observed. Ultrastructural examination revealed increased vacuolization and appearance of glycogen-storing pools. The rate of proteoglycan synthesis gradually increased. Age-related changes in the composition of the proteoglycan consisted of an increase in the ratio of keratan sulfate/chondroitin sulfate. The results indicate that the process of aging in culture resembles maturation and differentiation of cartilage tissue in vivo. The levels of cytosolic free calcium ions ([Ca2+]i) were measured in fura-2-loaded cells during the course of aging in culture. A gradual decrease in [Ca2+]i was observed. In 5-day cultures, a value of 184 nM [Ca2+]i was measured; this value decreased to 61 nM in 34-day cultures. On the basis of the present data and the previous results, which showed that cartilage-derived growth factors caused a decrease in [Ca2+]i, concomitantly with enhancing differentiation, whereas factors which elevated [Ca2+]i caused an increase in proliferation and a decrease in proteoglycan synthesis, we suggest a model for control of chondrocyte differentiation and aging. The model suggests that the rate of differentiation may be paced by changes in steady-state levels of [Ca2+]i.

Animals↗

PubMed1990

Modulation of alkaline phosphatase activity in alveolar type II like cells.

Alveolar type II like cells (ALT II) represent a small subpopulation of alveolar type II cells, which is able to proliferate, can be passaged and possess many characteristics of differentiated adult type II cells. A correlation was found between the growth and development of ALT II cells in culture and their alkaline phosphatase activity. Unlike alveolar type II cells, which lose the activity in culture, ALT II cells regain the activity and maintain it for a long culture period. Quantitative histochemical analysis of the stained cells indicate that 80% of the cells at days 15-20 in culture are alkaline phosphatase positive. Inhibition studies indicate that alkaline phosphatase from ALT II cells and freshly isolated type II cells were similar. The inhibition of ALT II alkaline phosphatase by L-levamisole and its heat stability are similar to that of the bone enzyme and differ from the intestinal enzyme. Alkaline phosphatase expression is considered part of the differentiated phenotype of these cells. Therefore, the presence of this enzyme in ALT II cells adds support to the notion that these cells maintain many aspects of mature alveolar type II cells.

Alkaline Phosphatase↗

PubMed1990

Protein kinase C is present in human sperm: possible role in flagellar motility.

We report the presence of protein kinase C (PKC) in ejaculated human sperm as revealed by enzymatic activity assay and indirect immunohistochemistry. PKC is localized in the equatorial segment and in the principal piece of the tail. Addition of phorbol 12-myristate 13-acetate resulted in increased flagellar motility that was blocked by known PKC inhibitors such as sphingosine, staurosporine, and 1-(5-isoquinoylinylsulfonyl)-2-methylpiperazine. A very good correlation (r = 0.9, P less than 0.001) was found between the percentage of PKC-stained sperm cells and motility. We propose that PKC is involved in the regulation of flagellar motility in human sperm.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1990

Internalization of pulmonary surfactant into lamellar bodies of cultured rat pulmonary type II cells.

We investigated the uptake of surfactant by isolated alveolar type II cells by using native pulmonary surfactant complexed with colloidal gold. Internalization to lamellar bodies (LB) occurred via vesicles (mainly coated) and the endosomal system. The highest labeling density was found in the endosomal system: vacuoles and the electron-lucent multivesicular bodies (MVB), which were labeled within 10 min. The labeling of electron-dense MVB (D-MVB) and LB was time dependent, reaching a plateau after 120 min, at which time approximately 30% and 70% of the LB and D-MVB were labeled, respectively. Internalization of surfactant-gold was inhibited by the addition of native surfactant or treatment of the gold complex with antibody against surfactant apoproteins. The internalization pathway of lectin from Macula pomifera (MPA) complexed with gold was compared to that of surfactant. Both pathways were found to be similar, except that mainly smooth vesicles rather than coated ones were involved in the process of MPA-G internalization. The partial labeling of the LB, the possible routing to lysosomes, and the endosomes as junction between the biosynthetic and endocytic pathways are discussed.

Animals↗

PubMed1990

Further studies on the involvement of protein kinase C in human sperm flagellar motility.

Addition of the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) or the membrane-permeable diacylglycerol analog, 1-oleoyl-2-acetylglycerol to human sperm resulted in increased motility. The biologically inactive 4 alpha-phorbol 12,13 didecanoate had no effect on flagellar motility. Basal motility was markedly reduced in the absence of Ca2+ in the incubation medium, but TPA-induced sperm motility persisted even in the absence of Ca2+. Sperm motility was also enhanced by the Ca2+ ionophore ionomycin in a Ca2(+)-dependent, protein kinase c (PKC)-independent fashion. Although all stimulants examined here reached maximal response at about 15 min of incubation, nevertheless whereas the effect of TPA and 1-oleoyl-2-acetylglycerol declined at 60 min of incubation, that of ionomycin still persisted. Human sperm PKC activity is extremely low and represents only about 20% and 25% of the specific activity recovered from PC-12 and rat pituitary cells, respectively. Immunohistochemical analysis using various type-specific PKC antibodies revealed staining only in the equatorial segment and the principal piece of the tail. Thus, PKC is present in human ejaculated sperm and is involved in flagellar motility.

Adult↗

PubMed1989

Insulin and C-peptide (proinsulin) packaging: a quantitative immunocytochemical approach.

C-peptide and insulin antigenic sites in rat pancreatic beta cells were both labeled by the protein A and immunogold techniques. Gold bead density in the secretory granules was correlated with granule size distribution. Our results demonstrate that the distribution patterns of antigenic sites for C-peptide (which also represents proinsulin sites) and insulin are similar when granule size distribution is correlated to the density of antigenic sites. It was found that the ratio of C-peptide to insulin in granules is independent of granule size. Examination of frequency histograms by a Kolmogorov-Smirnov analysis excluded the possibility that the antigenic sites of C-peptide and insulin are distributed in the same manner. Mean gold bead density for insulin antigen (97 +/- 24) was slightly higher than C-peptide density (64 +/- 18). The efficiency of labeling was found to be very low; less than 0.1% of the theoretically estimated antigenic sites were labeled.

Animals↗

PubMed1989

Variability in gold bead density in cells. Quantitative immunocytochemistry.

Variability in gold bead distribution between individual cells was demonstrated in both pituitary melanotrophic cells immunocytochemically reacted for ACTH and in neurohypophysial terminals reacted for oxytocin-neurophysin. Gold beads were confined to the secretory granules compartment of both tissues. Density of gold beads in melanotrophic cells reacted for ACTH varied from 100-480 gold beads/microns 2. A much narrower range of gold beads distribution (460-900 gold beads/microns 2) was observed in axons of the neurohypophysis reacted with anti-oxytocin-neurophysin. These results indicate that the labeling density varies from cell to cell (as well as axon terminals) within morphologically homogeneous populations. Thus, it may reflect certain physiological differences between cells. A suggestion is being made that mean gold bead density coefficient of variation should be calculated by comparison between individual cells.

Adrenocorticotropic Hormone↗