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Objective: To determine the effects of various suture patterns on cutaneous blood flow (CBF) at the wound edge as increasing tension is applied through the suture., Methods: Four different suture patterns commonly used for wound closure (simple, vertical mattress, horizontal mattress, and Allgower-Donati) were placed individually after a full-thickness incision was made in an anesthetized pig. A laser Doppler flowmeter (LDF) was placed on the skin edge after the suture was passed. Baseline CBF was recorded. Increasing tension was applied to the wound edge via the suture through a tensionometer in 0.5-lb (0.23-kg) increments from 0 to 2.5 lb (1.13 kg). CBF was then recorded as a function of tension for each suture pattern., Results: The Allgower-Donati suture pattern affected CBF significantly less than the other three suture patterns did for all tensions from 0.5 to 2.0 lb (0-0.9 kg; P < 0.05). There were no significant differences between vertical mattress, horizontal mattress, and simple suture patterns., Conclusions: The Allgower-Donati suture pattern had the least effect on CBF with increasing tension in this model. Further study is warranted on the benefits of this suture pattern because it may decrease wound complications in traumatized tissues.

Calreticulin is a Ca(2+)-buffering ER chaperone that also modulates cell adhesiveness. In order to study the effect of calreticulin on the expression of adhesion-related genes, we created a calreticulin inducible Human Embryonic Kidney (HEK) 293 cell line. We found that fibronectin mRNA and both intra- and extra-cellular fibronectin protein levels increased following calreticulin induction. However, despite this increase in fibronectin, HEK293 cells did not assemble an extracellular fibrillar fibronectin matrix regardless of the level of calreticulin expression. Furthermore, HEK293 cells exhibited a poorly organized actin cytoskeleton, did not have clustered fibronectin receptors at the cell surface, and did not form focal contacts. This likely accounts for the lack of fibronectin matrix deposition by these cells regardless of calreticulin expression level. Vinculin abundance did not appreciably increase upon calreticulin induction and the level of active c-Src, a regulatory kinase of focal contacts, was found to be abundant and unregulated by calreticulin induction in these cells. The inability to form stable focal contacts and to commence fibronectin fibrillogenesis due to high c-Src activity may be responsible for the poor adhesive phenotype of HEK 293 cells. Thus, we show here that HEK293 cells are not suitable for microscopical studies of cell-substratum adhesions, but are best suited for biochemical studies.

To determine if cardiogenesis causes endoplasmic reticulum stress, we examined chaperone expression. Many cardiac pathologies cause activation of the fetal gene program, and we asked the reverse: could activation of the fetal gene program during development induce endoplasmic reticulum stress/chaperones? We found stress related chaperones were more abundant in embryonic compared to adult hearts, indicating endoplasmic reticulum stress during normal cardiac development. To determine the degree of stress, we investigated endoplasmic reticulum stress pathways during cardiogenesis. We detected higher levels of ATF6alpha, caspase 7 and 12 in adult hearts. Thus, during embryonic development, there is large protein synthetic load but there is no endoplasmic reticulum stress. In adult hearts, chaperones are less abundant but there are increased levels of ATF6alpha and ER stress-activated caspases. Thus, protein synthesis during embryonic development does not seem to be as intense a stress as is required for apoptosis that is found during postnatal remodelling.

The nucleus accumbens, a key structure of the limbic circuitry, is involved in the regulation of motivated behaviors. The accumbens performs its roles via interconnections with brain areas where glucose-monitoring neurons have been localized. To search for such integrative chemosensory cells here, extracellular single neuron activity was recorded in the nucleus accumbens of Wistar rats by means of multibarreled glass microelectrodes during microelectrophoretic administration of D-glucose and other chemicals. Every fourth neuron tested changed in activity in response to glucose. Accumbens cells also displayed distinct gamma-amino-n-butyric acid type sensitivity. It is suggested that differential distribution of the chemosensory units, demonstrated between subdivisions of the nucleus accumbens, has particular significance with respect to functional dichotomy of the shell and core subregions.

Calreticulin is an ER calcium-storage protein, which influences gene expression and cell adhesion. In this study, we analysed the differences in adhesive properties of calreticulin under- and overexpressing fibroblasts in relation to the calmodulin- and calcium/calmodulin-dependent kinase II (CaMK II)-dependent signalling pathways. Cells stably underexpressing calreticulin had elevated expression of calmodulin, activated CaMK II, activated ERK and activated c-src. Inhibition of calmodulin by W7, and CaMK II by KN-62, caused the otherwise weekly adhesive calreticulin underexpressing cells to behave like the overexpressing cells, via induction of increased cell spreading. Increased vinculin, activated paxillin, activated focal adhesion kinase and fibronectin levels were observed upon inhibition of either the calmodulin or the CaMK II signalling pathways, which was accompanied by an increase in cell spreading and focal contact formation. Both KN-62 and W7 treatment increased cell motility in underexpressing cells, but W7 treatment led to loss of directionality. Thus, both the calmodulin and CaMK II signalling pathways influence cellular spreading and motility, but subtle differences exist in their distal effects on motility effectors., ((c) 2007 Wiley-Liss, Inc.)

Background Information: Our previous studies have shown that calreticulin, a Ca2+-binding chaperone located in the endoplasmic reticulum, affects cell-substratum adhesions via the induction of vinculin and N-cadherin. Cells overexpressing calreticulin contain more vinculin than low expressers and make abundant contacts with the substratum. However, cells that express low levels of calreticulin exhibit a weak adhesive phenotype and make few, if any, focal adhesions. To date, the identity of the types of focal adhesions made by calreticulin overexpressing and low expressing cells has not been dissected., Results: The results of the present study show that calreticulin affects fibronectin matrix assembly in L fibroblast cell lines that differentially express the protein, and that these cells also differ profoundly in focal adhesion formation. Although the calreticulin overexpressing cells generate numerous interference-reflection-microscopy-dark, vinculin- and paxillin-containing classical focal contacts, as well as some fibrillar adhesions, the cells expressing low levels of calreticulin generate only a few weak focal adhesions. The fibronectin receptor was found to be clustered in calreticulin overexpressing cells, but diffusely distributed over the cell surface in low expressing cells. Plating L fibroblasts on fibronectin-coated substrata induced extensive spreading in all cell lines tested. However, although calreticulin overexpressing cells were induced to form classical vinculin-rich focal contacts, the low calreticulin expressing cells overcame their weak adhesive phenotype by induction of many tensin-rich fibrillar adhesions, thus compensating for the low level of vinculin in these cells., Conclusions: We propose that calreticulin affects fibronectin production and, thereby, assembly, and it indirectly influences the formation and/or stability of focal contacts and fibrillar adhesions, both of which are instrumental in matrix assembly and remodelling.

Calreticulin is an endoplasmic reticulum Ca2+-storage protein, which influences gene expression and cell adhesion. In this study, we show that calreticulin induces fibronectin gene expression and matrix deposition, leading to differences in cell spreading and focal adhesion formation in cells differentially expressing calreticulin. We further show that these effects of calreticulin occur via a c-Src-regulated pathway and that c-Src activity is inversely related to calreticulin abundance. Since c-Src is an important regulator of focal contact turnover, we investigated the effect of c-Src inhibition on cells differentially expressing calreticulin. Inhibition of c-Src rescued the poorly adhesive phenotype of the calreticulin-underexpressing cells in that they became well spread, commenced formation of numerous focal contacts, and deposited a rich fibronectin matrix. Importantly, we show that c-Src activity is dependent on releasable Ca2+ from the endoplasmic reticulum, thus implicating Ca2+-sensitive pathways that are affected by calreticulin in cell-substratum adhesion. We propose that calreticulin affects fibronectin synthesis and matrix assembly via the regulation of fibronectin gene expression. In parallel, calcium-dependent effects of calreticulin on c-Src activity influence the formation and/or stability of focal contacts, which are instrumental in matrix assembly and remodeling.

Physical examination of the wrist requires knowledge of wrist anatomy and pathology to make a diagnosis or narrow the differential diagnosis. Symptoms are provoked by palpation and signs are produced by manipulation. Negative findings elsewhere in the wrist are important. Final diagnosis may require diagnostic imaging. By having all three methods of assessment agree one is assured of correct diagnosis. The physical examination of the wrist is not unlike that of other joints, in that a systematic approach includes observation, range of motion, palpation, and special tests.

Carpal bone fractures make up a significant proportion of injuries to the wrist. The complex bone shape and articulations make diagnosis more difficult and missed injuries more common. This article reviews carpal bone fractures excluding the scaphoid.

Background: Calreticulin is a Ca2+ binding chaperone of the endoplasmic reticulum which influences gene expression and cell adhesion. The levels of both vinculin and N-cadherin are induced by calreticulin expression, which play important roles in cell adhesiveness. Cardiac development is strictly dependent upon the ability of cells to adhere to their substratum and to communicate with their neighbours., Results: We show here that the levels of N-cadherin are downregulated in calreticulin-deficient mouse embryonic hearts, which may lead to the disarray and wavy appearance of myofibrils in these mice, which we detected at all investigated stages of cardiac development. Calreticulin wild type mice exhibited straight, thick and abundant myofibrils, which were in stark contrast to the thin, less numerous, disorganized myofibrils of the calreticulin-deficient hearts. Interestingly, these major differences were only detected in the developing ventricles while the atria of both calreticulin phenotypes were similar in appearance at all developmental stages. Glycogen also accumulated in the ventricles of calreticulin-deficient mice, indicating an abnormality in cardiomyocyte metabolism., Conclusion: Calreticulin is temporarily expressed during heart development where it is required for proper myofibrillogenesis. We postulate that calreticulin be considered as a novel cardiac fetal gene.

We report on the observation of ultracold heteronuclear Feshbach molecules. Starting with a 87Rb Bose-Einstein condensate and a cold atomic gas of 85Rb, we utilize previously unobserved interspecies Feshbach resonances to create up to 25,000 molecules. Even though the 85Rb gas is nondegenerate, we observe a large molecular conversion efficiency due to the presence of a quantum degenerate 87Rb gas; this represents a key feature of our system. We compare the molecule creation at two different Feshbach resonances with different magnetic-field widths. The two Feshbach resonances are located at 265.44+/-0.15 G and 372.4+/-1.3 G. We also directly measure the small binding energy of the molecules through resonant magnetic-field association.

Probable germline gene sequences from thousands of aligned mature Ab sequences are inferred using simple computational matching to known V(D)J genes. Comparison of the germline to mature sequences in a structural region-dependent fashion allows insights into the methods that nature uses to mature Abs during the somatic hypermutation process. Four factors determine the residue type mutation patterns: biases in the germline, accessibility from single base permutations, location of mutation hotspots, and functional pressures during selection. Germline repertoires at positions that commonly contact the Ag are biased with tyrosine, serine, and tryptophan. These residue types have a high tendency to be present in mutation hotspot motifs, and their abundance is decreased during maturation by a net conversion to other types. The heavy use of tyrosines on mature Ab interfaces is thus a reflection of the germline composition rather than being due to selection during maturation. Potentially stabilizing changes such as increased proline usage and a small number of double cysteine mutations capable of forming disulfide bonds are ascribed to somatic hypermutation. Histidine is the only residue type for which usage increases in each of the interface, core, and surface regions. The net overall effect is a conversion from residue types that could provide nonspecific initial binding into a diversity of types that improve affinity and stability. Average mutation probabilities are approximately 4% for core residues, approximately 5% for surface residues, and approximately 12% for residues in common Ag-contacting positions, excepting the those coded by the D gene.

Calreticulin is an endoplasmic reticulum (ER) luminal Ca(2+)-binding chaperone involved in folding of newly synthesized glycoproteins via the "calreticulin-calnexin cycle." We reconstituted ER of calreticulin-deficient cells with N-terminal histidine (His25, His82, His128, and His153) calreticulin mutants and carried out a functional analysis. In crt(-/-) cells bradykinin-dependent Ca2+ release is altered, and the reestablishment of bradykinin-dependent Ca2+ release was used as a marker for calreticulin function. Bradykinin-dependent Ca2+ release from the ER was rescued by wild type calreticulin and by the His25, His82, or His128 mutant but not by the His153 mutant. Wild type calreticulin and the His25, His82, and His128 mutants all prevented in vitro thermal aggregation of malate dehydrogenase and IgY, whereas the His153 mutant did not, indicating that His153 chaperone function was impaired. Biophysical analysis of His153 mutant revealed that conformation changes in calreticulin mutant may be responsible for the loss of its chaperone activity. We conclude that mutation of a single amino acid residue in calreticulin has devastating consequences for its chaperone function, indicating that mutations in chaperones may play a significant role in protein folding disorders.

Objectives: The eversion endarterectomy of the internal carotid artery was introduced in Hungary in 1991. The aim of this study was to define the long-term restenosis rate of this new method., Materials and Methods: Between 1991 and 1993, 171 operations were performed by one surgeon on 151 patients: 109 patients had enough compliance to take part in long-term follow-up, which included annual physical and ultrasound (Ultramark 9) investigations. Restenosis rate and plaque morphology was defined. Survival and patency rates were calculated by life-table method., Results: The perioperative combined stroke morbidity and mortality rate was 0.8%. The 5-year patient survival rate was 85%, the recurrent stenosis free rate was 88%/5 years, and 9% of the patients had restenosis greater than 70% in this period. The plaque morphology showed calcification in 1 case. Two patients needed 3 reoperations (2.4%). Plaque histology showed myointimal hyperplasia in every 3 cases., Conclusions: Comparing our results to the literature (2-34% restenosis rate) it seems to be acceptable and encouraging for the future. The ultrasound and histological findings suggest that arteriosclerosis does not play significant role in development of restenosis after the eversion method.

Calreticulin is a Ca2+-binding chaperone in the endoplasmic reticulum (ER), and calreticulin gene knockout is embryonic lethal. Here, we used calreticulin-deficient mouse embryonic fibroblasts to examine the function of calreticulin as a regulator of Ca2+ homeostasis. In cells without calreticulin, the ER has a lower capacity for Ca2+ storage, although the free ER luminal Ca2+ concentration is unchanged. Calreticulin-deficient cells show inhibited Ca2+ release in response to bradykinin, yet they release Ca2+ upon direct activation with the inositol 1,4,5-trisphosphate (InsP3). These cells fail to produce a measurable level of InsP3 upon stimulation with bradykinin, likely because the binding of bradykinin to its cell surface receptor is impaired. Bradykinin binding and bradykinin-induced Ca2+ release are both restored by expression of full-length calreticulin and the N + P domain of the protein. Expression of the P + C domain of calreticulin does not affect bradykinin-induced Ca2+ release but restores the ER Ca2+ storage capacity. Our results indicate that calreticulin may play a role in folding of the bradykinin receptor, which affects its ability to initiate InsP3-dependent Ca2+ release in calreticulin-deficient cells. We concluded that the C domain of calreticulin plays a role in Ca2+ storage and that the N domain may participate in its chaperone functions.

We have produced an interacting quantum degenerate Fermi gas of atoms composed of two spin states of magnetically trapped 40K. The relative Fermi energies are adjusted by controlling the population in each spin state. Thermodynamic measurements reveal a resulting imbalance in the mean energy per particle between the two species, which is a factor of 1.4 at our lowest temperature. This imbalance of energy comes from a suppression of collisions between atoms in the gas due to the Pauli exclusion principle. Through measurements of the thermal relaxation rate we have directly observed this Pauli blocking as a factor of 2 reduction in the effective collision cross section in the quantum degenerate regime.

The paper gives a brief overview from the complexation of metals by different ligands in the natural environment and it shows by several examples how transport, biological availability and environmental influences of the metals are attached by these processes.

This study reports an anatomical investigation of the angular branch of the thoracodorsal artery, which can be used in reconstructive surgery to serve as a pedicle for gaining osseous material from the scapula. The study was performed on 135 samples and topography and morphometry of the vessel were investigated. It was found that this artery has five main types of origin. It courses in the fascial gliding layer between the serratus muscle, and the teres major and subscapular muscle to the inferior angle of the scapula. There it provides the bone with blood through periostal branches as part of the arterial rete on the costal and dorsal side of the scapula. The mean length of the angular branch and the thoracodorsal artery together was 148 mm, and when also adding the subscapular artery the overall length became 167 mm. It was found that the length of a pedicle consisting of the angular branch and the thoracodorsal artery is determined by the difference in origin of the vessels with minor influences of the body size. The internal diameters of unfilled fixated vessels measured 3.06 mm for the subscapular, 1.77 mm for the circumflex scapular, 1.30 mm for the thoracodorsal and 0. 64 mm for the angular branch. The exact knowledge of the microanatomy in this area is necessary to be able to use the osseous free flap more selectively and have less traumatization in the donor area.

Background: Glutathione S-transferases (GSTs) are a family of isozymes that catalyze the conjugation of glutathione to various electrophilic compounds. Recently, the Yf subunit of GST-P, found in high concentrations in the epididymis, has been immunolocalized within epithelial principal and basal cells of this tissue. In order to determine which factor(s) were involved in the regulation of expression of this protein, adult rats were orchidectomized and treated with or without testosterone implants, ligated or hypophysectomized., Methods: The epididymides were fixed by perfusion with Bouin's fixative and examined with an anti-Yf antibody using light microscope immunocytochemistry., Results: In normal untreated animals, principal cells were reactive in the distal initial segment, intermediate zone and caput epididymidis, but unreactive in all other epididymal regions. Ligation of the efferent ducts up to day 14 had no effect on expression of the Yf protein in principal cells of any region. The staining pattern was comparable to that observed in normal untreated animals suggesting that luminally derived testicular factors were not involved in regulation of Yf expression. Following orchidectomy, principal cells became unreactive by day 14 in all epididymal regions. However, administration of testosterone to orchidectomized animals prevented the loss of reactivity seen in principal cells of the distal initial segment, intermediate zone, and caput epididymidis, suggesting that expression of the Yf protein in principal cells of these regions is regulated by circulating testosterone. In contrast, basal cells after each experimental treatment including hypophysectomy maintained the exact staining pattern observed in normal untreated animals, i.e., they were intensely reactive in the proximal initial segment, distal caput, corpus, and proximal cauda regions, suggesting that expression of the Yf protein in these cells does not require factors derived from the testis or pituitary gland. However, a change in the shape and arrangement of basal cells was noted following each treatment. These cells transformed from flattened hemispherical cells showing thin elongated lateral processes to large bulbous, dome-shape cells closely packed together and showing only a few short lateral processes., Conclusions: In basal cells, expression of the Yf protein is not regulated by testicular or pituitary factors, whereas expression in principal cells is regulated by circulating testosterone. Due to the decrease in size of the epididymal tubules after each treatment, it is postulated that the shape and arrangement of basal cells in normal untreated animals are governed in part by the volume and pressure exerted upon the tubules by luminal fluids and spermatozoa derived from the testis.

Glutathione S-transferases (GSTs) are dimeric proteins that come from a multigene family. They can be grouped into five classes (alpha, mu, pi, sigma, theta) based on the degree of amino acid homology of their subunits. These GST isozymes are able to catalyze the conjugation of glutathione with a wide variety of electrophiles, thereby protecting important cellular constituents from electrophilic attack. In the present study, the distribution of the Ya and Yc subunits from the alpha family, as well as the Yb1 and Yb2 subunits from the mu gene family was examined using immunocytochemistry in the adult rat testis and epididymis. The results of these four GST subunits were also compared with two other subunits, the Yf and Yo proteins, which have already been investigated in our laboratory [Veri et al. (1993), J. Androl., 14:23-44; Veri et al. (in press), J. Androl.]. In the testis, Leydig cells were intensely stained for all six subunits. Within the seminiferous epithelium, Sertoli cells were reactive only for antibodies raised against the Ya, Yb1 and Yf subunits. Among germ cells, all spermatogonia, spermatocytes and step 1-15 spermatids were virtually unreactive for each of the six GSTs. However, moderate to intense staining was seen over steps 16-19 spermatids with the anti-Yo and anti-Ya antibodies, and intense staining over step 19 spermatids with the anti-Yb1 and anti-Yb2 antibodies. In the rete testis, Yf, Yo, Yb1, and Yb2 subunits were intensely reactive over the epithelial cells with weak staining for Yc and no staining for Ya antibodies. Interestingly, in the efferent ducts the Yc, Yb1, and Yf proteins were intensely reactive over ciliated cells, whereas only the Yc protein was intensely reactive over nonciliated cells. In the epididymis, immunoreactivity varied among the principal and basal cells of a given epididymal region for each GST antibody. In the case of principal cells, several of the GSTs showed a similar immunostaining pattern along the tubule. Although not identical in intensity of reaction, the Yc, Yb1, Ya and Yo proteins showed an increase in staining intensity from the proximal to distal segments of the epididymis. In contrast, the Yb2 protein was intensely expressed only in the distal caput with weak levels throughout the rest of the epididymis. The Yf reactivity was strongest from the distal initial segment to the distal caput and unreactive in the corpus and proximal cauda epididymidis.(ABSTRACT TRUNCATED AT 400 WORDS)

Background: Glutathione S-transferases (GSTs) are a family of isozymes that catalyze the conjugation of glutathione with various toxic electrophilic compounds. GSTs are composed of several classes based on the degree of sequence homology of their subunits. The Yo subunit, a member of the mu class, is expressed at high levels in the testis and epididymis. The purpose of this study was to immunolocalize the GST-Yo in these tissues during development., Methods: The testes and epididymides of rats aged 7, 15, 21, 28, 39, 42, 45, 49, and 56 days were fixed in Bouin's fixative, and immunostained for light microscopic analysis., Results: In the testis the cytoplasm of all germ cells was unreactive until day 39. At that time, step 18 spermatids appeared moderately reactive, while the few observed step 19 spermatids were intensely reactive as were their residual bodies. The presence of residual bodies indicates that spermiation takes place as early as day 39; however, the number of step 19 spermatids is low at this age. A progressive increase in the size of the tubule and number of elongating spermatids was seen between days 42 and 49. In addition, by day 49, a weak staining was observed in steps 12-15, moderate in steps 16-17, and intense in steps 18-19 spermatids. In terms of the intensity of staining, cell types stained, size of the tubules, and number of elongating spermatids, no difference was noted between day 49, 56, and adult animals. Thus Yo protein expression in germ cells reached maturity by day 49. The epithelial cells of the rete testis were intensely reactive at day 7 and remained so throughout development. In contrast, while the epithelial cells of the efferent ducts at day 7 were intensely reactive, they were weakly reactive by day 39 and remained so at later ages. Along the entire epididymis, the columnar epithelial cells showed a moderate apical/supranuclear reaction from day 7 to 28. By day 39 principal cells of the initial segment became weakly reactive, while those in the caput and corpus were moderately stained, a situation seen at later ages including adults. Only by day 49 did principal cells of the proximal cauda become moderately stained as seen in adult animals. Thus the expression of the Yo protein in the principal cells of the proximal cauda may be regulated by different factors than those of the caput and corpus epididymidis. Alternatively, the expression of the Yo subunit in principal cells of the proximal cauda may develop later since this region would be the last to receive luminally derived testicular products. In the initial segment, the decrease in staining of principal cells at day 39 may be due to an inhibiting factor emanating from the testis. Spermatozoa appeared in the lumen of each epididymal region well after the expression of Yo had reached its adult staining pattern indicating that they are not a factor., Conclusions: Overall these results suggest that the expression of GST-Yo in the various cells of the testis and epididymis are controlled by different factors during postnatal development.

Background: Glutathione S-transferases (GSTs) are a family of isozymes that catalyze the conjugation of the tripeptide, glutathione, to various electrophilic compounds. The major GST in the pi class is GST-P, a homodimer of the Yf subunit, also known as Yp or rat subunit 7. This subunit is found in high concentrations in the epididymis and has recently been immunolocalized within epithelial principal and basal cells of the epididymis., Methods: In the present study we examine in groups of animals fixed in Bouin's fixative for light microscopy and in 4% paraformaldehyde and 0.5% glutaraldehyde in phosphate buffer for electron microscopy, the pattern of immunostaining for the Yf subunit of GST-P in the testis, efferent ducts and epididymis at various ages after birth., Results: In the epididymis, on postnatal days 7 and 15, an immunoperoxidase reaction was localized exclusively to the apical and supranuclear regions of the undifferentiated columnar epithelial cells of the entire epididymis. By day 21, a dramatic change had taken place. In the initial segment, intermediate zone and proximal caput epididymidis, the columnar cells showed a distinct checkerboard-like staining pattern with cells ranging from being intensely reactive to unreactive. In contrast, principal cells of the distal caput, corpus, and proximal cauda epididymidis were weakly reactive. By day 28 the ratio of reactive to unreactive cells in the initial segment, intermediate zone, and proximal caput epididymidis was higher. By day 39, the differentiated columnar epithelial cells, referred to as principal cells, took on their adult staining pattern in the proximal and middle areas of the initial segment as well as the corpus and proximal cauda epididymidis where they were slightly reactive; in the distal initial segment they were strongly reactive. At day 49, principal cells in the intermediate zone and proximal caput became intensely reactive, while showing a distinct checkerboard-like staining pattern in the distal caput; similar observations were made for tissues taken from 56 and 90-day-old animals. Basal cells also showed a variable staining pattern in the different epididymal regions as a function of age. At day 21, when they first appeared, they were unreactive except for an occasional reactive cell in the corpus region. At day 28, only in the corpus epididymidis were many basal cells seen to be reactive. By day 39 the more numerous basal cells of the corpus and proximal cauda epididymidis were intensely reactive and remained so into adulthood. In these regions, basal cells appeared as dome-shaped cells (days 21, 28, 39), but then gradually flattened out and exhibited processes (days, 49, 56, adults) which collectively appeared to envelop the base of each tubule in a mesh-like network. The change in basal cell shape in each region coincided with the arrival of fluid and spermatozoa into the lumen (corpus day 49, proximal cauda day 56). In other epididymal regions, basal cells at day 28 were mostly unreactive. However, there was a gradual increase in the number of reactive basal cells of these regions between day 39 and 56., Conclusions: The present results thus demonstrate a dramatic change in the immunostaining pattern for the Yf subunit of GST-P during postnatal development for both principal and basal cells along the epididymis. Such results suggest that different factors play a role in the regulation of the expression of the Yf protein, not only in different epididymal regions, but also in different cell types during postnatal development.

The irradiation of skeletal muscle myosin subfragment 1 (S1) in the presence of 2,2,2-trichloroethanol (TCE) reduces S1 fluorescence intensity in two phases. In the first phase, there is an increase in MgATPase activity, and no significant change in the fluorescence intensity increase upon ATP binding. In the second phase, the activity remains elevated, but there is a complete loss of the ATP-induced intensity increase. Measurements on denatured S1 indicate that fluorescence intensity reductions of one fifth of the total occur during each of the two phases, consistent with the fluorescence intensity increase upon forming S1.MgADP.P(i) being due to one of the five heavy-chain tryptophans.

Skeletal muscle myosin is an enzyme that interacts allosterically with MgATP and actin to transduce the chemical energy from ATP hydrolysis into work. By modifying myosin structure, one can change this allosteric interaction and gain insight into its mechanism. Chemical cross-linking with N,N'-p-phenylenedimaleimide (pPDM) of Cys-697 to Cys-707 of the myosin-ADP complex eliminates activity and produces a species that resembles myosin with ATP bound (Burke et al., 1976). Nucleotide-free pPDM-modified myosin subfragment 1 (S1) was prepared, and its structural and allosteric properties were investigated by comparing the nucleotide and actin interactions of S1 to those of pPDM-S1. The structural properties of the nucleotide-free pPDM-S1 are different from those of S1 in several respects. pPDM-S1 intrinsic tryptophan fluorescence intensity is reduced 28%, indicating a large increase of an internal quenching reaction (the fluorescence intensity of the related vanadate complex of S1, S1-MgADP-Vi, is reduced by a similar degree). Tryptophan fluorescence anisotropy increases from 0.168 for S1 to 0.192 for pPDM-S1, indicating that the unquenched tryptophan population in pPDM-S1 has reduced local freedom of motion. The actin affinity of pPDM-S1 is over 6,000-fold lower than that of S1, and the absolute value of the product of the net effective electric charges at the acto-S1 interface is reduced from 8.1 esu2 for S1 to 1.6 esu2 for pPDM-S1. In spite of these changes, the structural response of pPDM-S1 to nucleotide and the allosteric communication between its ATP and actin sites remain intact. Compared to pPDM-S1, the fluorescence intensity of pPDM-S1 *MgADP is increased 50%(compared to 8 and 31% increases, respectively, for MgADP and MgATP binding to S1). Compared to acto-pPDM-S1, the absolute value of the product of the net effective electric charge at the actin binding interface of acto-pPDM-S1 *MgADP increases 7.3 esu2 (compared to a 0.9 esu2 decrease and an 11.0 esu2 increase, respectively, for MgADP and MgATP binding to acto-Sl).The interaction free energy for the ligands MgADP and actin, is -2.0 kcal/mol for pPDM-S1, compared to -1.2 kcal/mol for unmodified S1.

The effects of nucleotide binding and temperature on the internal structural dynamics of myosin subfragment 1 (S1) were monitored by intrinsic tryptophan phosphorescence lifetime and fluorescence anisotropy measurements. Changes in the global conformation of S1 were monitored by measuring its rate of rotational diffusion using transient electric birefringence techniques. At 5 degrees C, the binding of MgADP, MgADP,P and MgADP,V (vanadate) progressively reduce the rotational freedom of S1 tryptophans, producing what appear to be increasingly more rigidified S1-nucleotide structures. The changes in the luminescence properties of the tryptophans suggest that at least one is located at the interface of two S1 subdomains. Increasing the temperature from 0 to 25 degrees C increases the apparent internal mobility of S1 tryptophans in all cases and, in addition, a reversible temperature-dependent transition centered near 15 degrees C was observed for S1, S1-MgADP and S1-MgADP,P, but not for S1-MgADP,V. The rotational diffusion constants of S1 and S1-MgADP were measured at temperatures between 0 and 25 degrees C. After adjusting for the temperature and viscosity of the solvent, the data indicate that the thermally induced transition at 15 degrees C comprises local conformational changes, but no global conformational change. Structural features of S1-MgADP,P, which may relate to its role in force generation while bound to actin, are presented.

Cytochrome oxidase exhibits phosphorescence from tryptophan in aqueous solution in the absence of oxygen. The lifetime for the resting reduced enzyme suspended in Tween-20 is around 30 ms at pH 8. The lifetime is longest between pH 7 and 8 and decreases with lowering of pH. Oxygen quenches the phosphorescence with a Stern-Volmer quenching constant of approximately 5 x 10(7) M-1.s-1 at 5 degrees C whereas cytochrome c has no effect. We interpret these results to indicate that room temperature tryptophan phosphorescence arises from tryptophan(s) in structured region(s) remote from the hemes and that the protein does not impose a significant barrier for the diffusion of oxygen.

Ten proteins that span a wide range of phosphorescence lifetimes were examined for sensitivity to quenching by four agents of disparate chemical nature. The results show that quenching efficiency is relatively independent of the quencher and is highly correlated with depth of burial of the phosphorescent tryptophan. The bimolecular quenching rate constants (kq) measured for the different proteins, spanning 5 orders of magnitude in kq, are found to decrease exponentially with the distance (r) of the tryptophan in angstroms from the protein surface--i.e., kq = Aexp(-r/rho), where A contains a geometrical factor dependent on tryptophan burial and surface geometry [corrected]. Theoretical analysis shows that this behavior can be expected for an electron-exchange reaction between the buried tryptophans and quenchers in solution in the rapid diffusion limit. Therefore, the results obtained provide evidence for an exponential dependence of electron-transfer rate on distance in a protein environment and evaluate the distance parameter, rho, for electron transfer through the general protein matrix at 1.0 A. For a unimolecular donor-acceptor pair with ket = koexp(-r/rho), ko approximately 10(9) sec-1.

The triplet state absorption and phosphorescence of Zn and Pd derivatives of myoglobin were compared. Both metal derivatives exhibit long triplet state lifetimes at room temperature, but whereas the Pd derivative showed exponential decay and an isosbestic point in the transient absorption spectra, the decay of the Zn derivative was nonsingle exponential and the transient absorption spectra showed evidence of more than one excited state species. No difference was seen in triplet quenching by oxygen for either derivative, indicating that differences in the polypeptide chain between the two derivatives are not large enough to affect oxygen penetrability. Quenching was also observed by anthraquinone sulfonate. In this case, the possibility of long-range transfer by an exchange mechanism is considered.

During to the peculiar morophology and symptomatology of the haemodynamic disturbance following occlusion of the innominate artery, the anonyma syndrome ought to be treated as an independent type of the cerebro-vascular syndrome. The anonyma syndrome is generally a part-symptom of obliterating arteriosclerosis of multiple localization and has therefore an unfavourable prognosis. After a brief survey of the literature the clinical data of patients operated for innominate artery occlusion are reported together with the surgical results.

Fluorescence and phosphorescence resemble each other and in many ways can give the same type of information. Both originate from a dipolar interaction between light and the molecule. In this regard, both are polarized and subject to the same type of quenching phenomena. In other respects the information which they divulge are complementary. The fluorescence quantum yield is higher for exposed tryptophans and this is expressed in longer lifetime (Grinvald and Steinberg, 1976); in contrast long lifetime of phosphorescence appears to correlate with burial. Phosphorescence, spin-disallowed, is much longer lived than fluorescence. This allows the structural/dynamic characterization of proteins to be studied on a new time regime. A really remarkable finding of studies of protein phosphorescence is that there is such variability both in phosphorescence lifetime and quenchability. We would interpret this to indicate that the tryptophan environment can range from essentially a crystal, almost comparable in rigidity as found at 77 K, to tryptophans in a flexible environment, almost as flexible as free in solution. An interesting task will be to examine the relationship between the yield and lifetime of phosphorescence and details of the tryptophan environment in terms of rigidity and adjacent amino acids among the proteins with known three dimensional structure.

The development and treatment of false aneurysm after cardiovascular intervention is discussed. At the Cardiovascular Surgical Clinic of Semmelweis Medical University (Budapest) 6900 reconstruction operations were performed. Ninety-five false aneurysms were operated on. According to the cause leading to the complication the patients are classified into 8 groups. To treat the false aneurysm 14 types of operation were carried out. Operative mortality was 12.6 per cent, but the outcome was successful in 70 per cent of the patients. The technique of surgical treatment is described. A review of the literature is presented.

The late results of the first 30 crural bypass operations are presented. The longest follow-up period was 6, the shortest 1 year. At the time of check-ups 14 of the bypasses were penetrable, and 16 were occluded. On 9 of the latter 16 amputation of the thigh had to be performed. Flow conditions and the problems of indication and contra-indication are discussed.

The dynamics and structuredness of the pyridoxal 5'-phosphate-binding region in glycogen phosphorylase b (EC 2.4.1.1) has been investigated with different techniques of fluorescence spectroscopy. Fluorescence polarization data of the thermal Perrin plot indicate some mobility in the cofactor binding site, while the isothermic measurements (at 20 degrees C, in high-viscosity solvents) demonstrate that the mobile unit carrying the emission oscillator is practically insensitive to the external viscosity. Characteristics of the thermal Perrin plots obtained for both native and reduced phosphorylase b can be interpreted either as a freely moving cofactor in a medium of high viscosity (0.3 P) or as the motion of a unit larger than a lysine-bonded pyridoxal 5'-phosphate in a medium with the viscosity of water. Data for acrylamide quenching and time-resolved fluorescence measurements suggest that the latter interpretation should valid. These data also suggest a tightly packed microenvironment around the pyridoxal moiety.

Much evidence, on both theoretical and experimental sides, indicates the importance of local fluctuations (in energy levels, conformational substates, etc.) of the macromolecular matrix in the biological activity of proteins. We describe here a novel application of the Förster-type energy-transfer process capable of monitoring changes both in local fluctuations and in conformational states of macromolecules. A new energy-transfer parameter, f, is defined as an average transfer efficiency, [E], normalized by the actual average quantum efficiency of the donor fluorescence, [phi D]. A simple oscillator model (for a one donor-one acceptor system) is presented to show the sensitivity of this parameter to changes in amplitudes of local fluctuations. The different modes of averaging (static, dynamic, and intermediate cases) occurring for a given value of the average transfer rate, [kt], and the experimental requirements as well as limitations of the method are also discussed. The experimental tests were performed on the ribonuclease T1-pyridoxamine 5'-phosphate conjugate (a one donor-one acceptor system) by studying the change of the f parameter with temperature, an environmental parameter expectedly perturbing local fluctuations of proteins. The parameter f increased with increasing temperature as expected on the basis of the oscillator model, suggesting that it really reflects changes of fluctuation amplitudes (significant changes in the orientation factor, k2, as well as in the spectral properties of the fluorophores can be excluded by anisotropy measurements and spectral investigations). Possibilities of the general applicability of the method are also discussed.

The early and late complications of vascular grafts in the Department of Vascular Surgery of Semmelweis University Medical School are discussed on the basis of a five year post mortem material. The mortality of the operations was 11.5%. Haemorrhage and infection are considered the most serious complications. The cause of the complications are discussed.

The late results of 171 thoracal sympathectomies performed on 142 patients in the 20-year period 1952 to 1971 are described. Favourable results were achieved in Raynaud's disease, obliterating endarteritis and other occlusive vascular diseases, and also in hyperhidrosis.

A novel experimental method, suitable for separate analysis of the quenching parameters characteristic of solvent-exposed and solvent-masked fluorophors of macromolecules, is described. The method is based on the modified Stern-Volmer analysis and requires simultaneous application of two kinds of quencher: one that can selectively quench the emission of exposed fluorophors (e.g., ionic quenchers) and another that is nonselective (e.g., oxygen or, in many cases, acrylamide), capable of quenching the fluorescence of both exposed and masked groups. In order to examine the accuracy of the model, a computer simulation was performed. The results showed that the errors are comparable to those arising from the conventional quenching experiments. The method is applicable to phosphorescence quenching as well and is extendable to time-resolved measurements (by replacing fluorescence intensities with lifetimes). The method was applied to resolve the quenching parameters of lysozyme fluorescence by the use of iodide as selective and acrylamide as nonselective quenchers. The determination of the acrylamide quenching constant associated with the internal fluorophor, Trp-108 (Kq = 3.5 M-1), permits specific studies on the dynamics of internal regions of the protein. The quenching constant determined for the more exposed residue Trp-62 (Kq = 1.6 M-1) provides local information about the surface independent of the electrostatic effects observed when an ionic quencher is used.

The quenching of coenzyme fluorescence in glycogen phosphorylase b is reinvestigated. Data with anionic quenchers show deviations from the original Stern-Volmer kinetics. A kinetic analysis based on measured lifetime data indicates a collisional quenching process, which is, however, not diffusion-controlled. It is proposed, that the quenching takes place primarily by enzyme-bound quencher species. The observed inhibition of the enzyme reaction by I- and IO-3 is consistent with this hypothesis. The inhibition pattern and spectral investigation refer to a true competition with the substrate, glucose-1-phosphate. So, this dynamic quenching can be regarded as an indicator of rapid conformational fluctuations which bring the two important active-site groups in contact. Effect of ligand binding on the quenching of coenzyme fluorescence should also be revaluated according to these results.

Sarcoplasmic reticulum vesicles isolated by conventional techniques usually contain, in addition to the recognized sarcoplasmic reticulum components, several other proteins (phosphorylase, myosin, glyceraldehyde-3-phosphate dehydrogenase, etc.) in variable amounts; these proteins complicate the interpretation of chemical modification data. Incubation of sarcoplasmic reticulum vesicles with Affi-Gel blue particles for 1-4 h at 2 degrees C, followed by sedimentation of the Affi-Gel in a clinical centrifuge, simplifies the protein composition by selective adsorption of the accessory proteins, and improves the consistency of the preparations. The Affi-Gel blue treatment is recommended as part of the standard procedure for the isolation of sarcoplasmic reticulum vesicles.

Phosphorescence of protein tryptophan was analyzed in sarcoplasmic reticulum vesicles, and in the purified Ca2+ transport ATPase in deoxygenated aqueous solutions at room temperature. Upon excitation with light of 295 nm wavelength, the emission maxima of fluorescence and phosphorescence were at 330 nm and at 445 nm, respectively. The phosphorescence decay was multiexponential; the lifetime of the long-lived component of phosphorescence was approximately equal to 22 ms. ATP and vandate significantly reduced the phosphorescence in the presence of either Ca2+ or EGTA; ADP was less effective, while AMP was without effect. The quenching by ATP showed saturation consistent with the idea that the ATP-enzyme complex had a lower phosphorescence yield. Upon exhaustion of ATP, the phosphorescence returned to starting level. Significant quenching of phosphorescence with a decrease in phosphorescence lifetime was also caused by NaNO2, methylvinyl ketone and trichloroacetate, without effect on ATPase activity; this quenching did not show saturation and was therefore probably collisional in nature.