Anatomical variations of the biceps brachii insertion: a proposal for a new classification.

BACKGROUND: The biceps brachii (BB) muscle is one of the three muscles located in the anterior compartment of the arm. Its insertion consists of two parts. The first part - main tendon - attached in the radial tuberosity and the second part - lacertus fibrosus (LF) - in the fascia of the forearm flexors. The intention of research was to reveal the morphological diversity of the insertion of this muscle. Thanks to the results of this work, have been created a classification of the distal attachment of BB. The results of that research can be used to further develop surgical procedures in the given region. MATERIALS AND METHODS: Eighty (40 left, and 40 right, 42 female, 38 male) upper limbs fixed in 10% formalin solution were examined. RESULTS: We observed three types of the insertion of the BB. Type I was characterised by a single tendon and occurred most frequently in 78.75% of the examined limbs. The second most common type was type II which was characterised by a double tendon and was observed in 13.75% of all the limbs. The last and least common was type III which was characterised by three tendons and occurred in 7.5% of the examined limbs. Additionally, the type of LF was analysed. In 8 (10%) specimens it was absent, i.e. in 2 specimens with type II insertion and 6 specimens with type III (p = 0.0001). Therefore, it may be deduced that type III BB insertion tendon predisposes to LF deficiency. CONCLUSIONS: The BB tendon is characterised by high morphological variability. The new classification proposes three types of distal attachment: type I - one tendon; type II - two separated band-shaped tendons; type III - three separated band-shaped tendons. The presence of type III BB tendon predisposes to a lack of LF.

A guideline to medical photography: a perspective on digital photography in an orthopaedic setting.

PURPOSE: Quality photographs are essential for clinical documentation, research, and publication in scientific journals and teaching. Oftentimes, non-ideal lighting and a sterile environment restrict the medical photographer, resulting in lower-quality photographs. This article aims to provide a clear and comprehensible guideline for medical photography in an orthopaedic setting. METHODS: This article is based on extensive photographic involvement in operating and laboratory settings, in close collaboration with medical professionals from the Steadman Clinic (Vail, Colorado, USA), Gothenburg University (Göteborg, Sweden) and Erasmus MC (Rotterdam, the Netherlands). Background literature was searched through Google Scholar and PubMed. RESULTS: Three relevant journal articles, and one book on medical photography, were used to write this paper. Seventeen Internet articles were used for background information. CONCLUSION: A relevant, up-to-date and comprehensive guideline to medical photography for medical professionals, with or without photographic experience, is provided. LEVEL OF EVIDENCE: Expert opinion, Level V.

Segmental expression of the bradykinin type 2 receptor in rat efferent ducts and epididymis and its role in the regulation of aquaporin 9.

Water and solute transport in the efferent ducts and epididymis are important for the establishment of the appropriate luminal environment for sperm maturation and storage. Aquaporin 9 (AQP9) is the main water channel in the epididymis, but its regulation is still poorly understood. Components of the kinin-kallikrein system (KKS), leading to the production of bradykinin (BK), are highly expressed in the lumen of the male reproductive tract. We report here that the epididymal luminal fluid contains a significant amount of BK (2 nM). RT-PCR performed on epididymal epithelial cells isolated by laser capture microdissection (LCM) showed abundant BK type 2 receptor (Bdkrb2) mRNA expression but no type 1 receptor (Bdkrb1). Double-immunofluorescence staining for BDKRB2 and the anion exchanger AE2 (a marker of efferent duct ciliated cells) or the V-ATPase E subunit, official symbol ATP6V1E1 (a marker of epididymal clear cells), showed that BDKRB2 is expressed in the apical pole of nonciliated cells (efferent ducts) and principal cells (epididymis). Triple labeling for BDKRB2, AQP9, and ATP6V1E1 showed that BDKRB2 and AQP9 colocalize in the apical stereocilia of principal cells in the cauda epididymidis. While uniform Bdkrb2 mRNA expression was detected in the efferent ducts and along the epididymal tubule, marked variations were detected at the protein level. BDKRB2 was highest in the efferent ducts and cauda epididymidis, intermediate in the distal initial segment, moderate in the corpus, and undetectable in the proximal initial segment and the caput. Functional assays on tubules isolated from the distal initial segments showed that BK significantly increased AQP9-dependent glycerol apical membrane permeability. This effect was inhibited by BAPTA-AM, demonstrating the participation of calcium in this process. This study, therefore, identifies BK as an important regulator of AQP9.

Differential effects of ionic strength, divalent cations and pH on the pore-forming activity of Bacillus thuringiensis insecticidal toxins.

The combined effects of ionic strength, divalent cations, pH and toxin concentration on the pore-forming activity of Cry1Ac and Cry1Ca were studied using membrane potential measurements in isolated midguts of Manduca sexta and a brush border membrane vesicle osmotic swelling assay. The effects of ionic strength and divalent cations were more pronounced at pH 10.5 than at pH 7.5. At the higher pH, lowering ionic strength in isolated midguts enhanced Cry1Ac activity but decreased considerably that of Cry1Ca. In vesicles, Cry1Ac had a stronger pore-forming ability than Cry1Ca at a relatively low ionic strength. Increasing ionic strength, however, decreased the rate of pore formation of Cry1Ac relative to that of Cry1Ca. The activity of Cry1Ca, which was small at the higher pH, was greatly increased by adding calcium or by increasing ionic strength. EDTA inhibited Cry1Ac activity at pH 10.5, but not at pH 7.5, indicating that trace amounts of divalent cations are necessary for Cry1Ac activity at the higher pH. These results, which clearly demonstrate a strong effect of ionic strength, divalent cations and pH on the pore-forming activity of Cry1Ac and Cry1Ca, stress the importance of electrostatic interactions in the mechanism of pore formation by B. thuringiensis toxins.

Na+/H+ exchangers in the human eccrine sweat duct.

Using an anti-NHE1 antibody, we demonstrate the presence of a Na+/H+ exchanger of isoform 1 (NHE1) in the human eccrine sweat duct. A strong staining was observed at the basolateral membrane of the outer cell layer (NHE1basal), at the junction between inner and outer cells layers (NHE1inter), and along the lateral membranes (NHE1later) of all cells of the duct. At the luminal membrane, no staining was demonstrated either for NHE1 or NHE3. To investigate Na+/H+ mediated proton transport, straight sweat duct portions were isolated and perfused in vitro under HCO3-free conditions. In the presence of basolateral 5-ethyl-N-isopropyl amiloride (EIPA), an acidification of 0.29 +/- 0.03 pH units was observed, whereas no effect was observed with luminal EIPA. Bath sodium removal generated a stronger acidification (0.41 +/- 0.09 pH units). Removal of luminal sodium (in the absence or presence of basolateral EIPA), or low luminal chloride, led to an alkalinization, presumably due to a decrease in intracellular sodium, strongly suggesting functional activity of NHE1inter. We therefore conclude that in the sweat duct, NHE1 plays a major role in intracellular pH regulation.

Analysis of the properties of Bacillus thuringiensis insecticidal toxins using a potential-sensitive fluorescent probe.

A potential-sensitive fluorescent probe, 3,3'-dipropylthiadicarbocyanine iodide, was used to analyze, at pH 7.5 and 10.5, the effects of Bacillus thuringiensis toxins on the membrane potential generated by the efflux of K(+) ions from brush border membrane vesicles purified from the midgut of the tobacco hornworm, Manduca sexta. Fluorescence levels were strongly influenced by the pH and ionic strength of the media. Therefore, characterization of the effects of the toxins was conducted at constant pH and ionic strength. Under these conditions, the toxins had little effect on the fluorescence levels measured in the presence or absence of ionic gradients, indicating that the ionic selectivity of their pores is similar to that of the intact membrane. Valinomycin greatly increased the potential generated by the diffusion of K(+) ions although membrane permeability to the other ions used to maintain the ionic strength constant also influenced fluorescence levels. In the presence of valinomycin, active toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1C and Cry1E) efficiently depolarized the membrane at pH 7.5 and 10.5.

V-type H+-ATPase in the human eccrine sweat duct: immunolocalization and functional demonstration.

We investigated for the presence of a vacuolar-type H+-ATPase (V-ATPase) in the human eccrine sweat duct (SD). With the use of immunocytochemistry, an anti-V- ATPase antibody showed a strong staining at the apical membrane and a weaker one in the cytoplasm. Cold preservation followed by rewarming did not alter this staining pattern. With the use of the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein on isolated and perfused straight SD under HCO-free conditions and in the absence of Na+, proton extrusion was determined from the recovery rate of intracellular pH (dpH(i)/dt) following an acid load. Oligomycin (25 microM), an inhibitor of F-type ATPases, decreased dpH(i)/dt by 88 +/- 6%, suggesting a role for an ATP-dependent process involved in pH(i) recovery. Moreover, dpH(i)/dt was inhibited at 95 +/- 3% by 100 nM luminal concanamycin A, a specific inhibitor of V-ATPases, whereas 10 microM bafilomycin A1, another specific inhibitor of V-ATPases, was required to decrease dpH(i)/dt by 73%. These results strongly suggest that a V-ATPase is involved in proton secretion in the human eccrine SD.

Unfolding affects insect cell permeabilization by Bacillus thuringiensis Cry1C toxin.

Bacillus thuringiensis Cry toxins are efficient, environment-friendly biological insecticides. Their molecular mode of action on target insect cells remains largely unknown. The aim of this study was to investigate the relation between the conformational state of the Cry1C toxin and its ionophoric activity on live Sf9 cells of Spodoptera frugiperda, a target insect for this protein. Potassium ion movement induced by Cry1C across the cell membrane was measured with a fluorescent assay developed previously and the conformation of the toxin was studied using tryptophan spectroscopy. Following treatment with 4 M guanidinium hydrochloride, which resulted in the unfolding of its N-terminal half, the toxin retained its full capacity to permeabilize the cells while the fully unfolded toxin did not induce potassium leakage. Therefore, permeabilization of Sf9 cells by Cry1C requires the integrity of the C-terminal half of the toxin and may depend on an initial unfolding step provided by the acidic environment of the cells.

Ion channels induced in planar lipid bilayers by the Bacillus thuringiensis toxin Cry1Aa in the presence of gypsy moth (Lymantria dispar) brush border membrane.

The apical brush border membrane, the main target site of Bacillus thuringiensis toxins, was isolated from gypsy moth (Lymantria dispar) larval midguts and fused to artificial planar lipid bilayer membranes. Under asymmetrical N-methyl-d-glucamine-HCl conditions (450 mm cis/150 mm trans, pH 9.0), which significantly reduce endogenous channel activity, trypsin-activated Cry1Aa, a B. thuringiensis insecticidal protein active against the gypsy moth in vivo, induced a large increase in bilayer membrane conductance at much lower concentrations (1.1-2.15 nm) than in receptor-free bilayer membranes. At least 5 main single-channel transitions with conductances ranging from 85 to 420 pS were resolved. These Cry1Aa channels share similar ionic selectivity with P(Cl)/P(NMDG) permeability ratios ranging from 4 to 8. They show no evidence of current rectification. Analysis of the macroscopic current flowing through the composite bilayer suggested voltage-dependence of several channels. In comparison, the conductance of the pores formed by 100-500 nm Cry1Aa in receptor-free bilayer membranes was significantly smaller (about 8-fold) and their P(Cl)/P(NMDG) permeability ratios were also reduced (2- to 4-fold). This study provides a detailed demonstration that the target insect midgut brush border membrane material promotes considerably pore formation by a B. thuringiensis Cry toxin and that this interaction results in altered channel properties.

Differential effects of pH on the pore-forming properties of Bacillus thuringiensis insecticidal crystal toxins.

The effect of pH on the pore-forming ability of two Bacillus thuringiensis toxins, Cry1Ac and Cry1C, was examined with midgut brush border membrane vesicles isolated from the tobacco hornworm, Manduca sexta, and a light-scattering assay. In the presence of Cry1Ac, membrane permeability remained high over the entire pH range tested (6.5 to 10.5) for KCl and tetramethylammonium chloride, but was much lower at pH 6.5 than at higher pHs for potassium gluconate, sucrose, and raffinose. On the other hand, the Cry1C-induced permeability to all substrates tested was much higher at pH 6.5, 7.5, and 8.5 than at pH 9.5 and 10.5. These results indicate that the pores formed by Cry1Ac are significantly smaller at pH 6.5 than under alkaline conditions, whereas the pore-forming ability of Cry1C decreases sharply above pH 8.5. The reduced activity of Cry1C at high pH correlates well with the fact that its toxicity for M. sexta is considerably weaker than that of Cry1Aa, Cry1Ab, and Cry1Ac. However, Cry1E, despite having a toxicity comparable to that of Cry1C, formed channels as efficiently as the Cry1A toxins at pH 10.5. These results strongly suggest that although pH can influence toxin activity, additional factors also modulate toxin potency in the insect midgut.

Effect of Bacillus thuringiensis Cry1 toxins in insect hemolymph and their neurotoxicity in brain cells of Lymantria dispar.

Little information is available on the systemic effects of Bacillus thuringiensis toxins in the hemocoel of insects. In order to test whether B. thuringiensis-activated toxins elicit a toxic response in the hemocoel, we measured the effect of intrahemocoelic injections of several Cry1 toxins on the food intake, growth, and survival of Lymantria dispar (Lepidoptera) and Neobellieria bullata (Diptera) larvae. Injection of Cry1C was highly toxic to the Lymantria larvae and resulted in the complete inhibition of food intake, growth arrest, and death in a dose-dependent manner. Cry1Aa and Cry1Ab (5 microg/0.2 g [fresh weight] [g fresh wt]) also affected growth and food intake but were less toxic than Cry1C (0.5 microg/0.2 g fresh wt). Cry1E and Cry1Ac (5 microg/0.2 g fresh wt) had no toxic effect upon injection. Cry1C was also highly toxic to N. bullata larvae upon injection. Injection of 5 microg/0.2 g fresh wt resulted in rapid paralysis, followed by hemocytic melanization and death. Lower concentrations delayed pupariation or gave rise to malformation of the puparium. Finally, Cry1C was toxic to brain cells of Lymantria in vitro. The addition of Cry1C (20 microg/ml) to primary cultures of Lymantria brain cells resulted in rapid lysis of the cultured neurons.

Role of interdomain salt bridges in the pore-forming ability of the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac.

The four salt bridges (Asp(222)-Arg(281), Arg(233)-Glu(288), Arg(234)-Glu(274), and Asp(242)-Arg(265)) linking domains I and II in Cry1Aa were abolished individually in alpha-helix 7 mutants D222A, R233A, R234A, and D242A. Two additional mutants targeting the fourth salt bridge (R265A) and the double mutant (D242A/R265A) were rapidly degraded during trypsin activation. Mutations were also introduced in the corresponding Cry1Ac salt bridge (D242E, D242K, D242N, and D242P), but only D242N and D242P could be produced. All toxins tested, except D242A, were shown by light-scattering experiments to permeabilize Manduca sexta larval midgut brush border membrane vesicles. The three active Cry1Aa mutants at pH 10.5, as well as D222A at pH 7.5, demonstrated a faster rate of pore formation than Cry1Aa, suggesting that increases in molecular flexibility due to the removal of a salt bridge facilitated toxin insertion into the membrane. However, all mutants were considerably less toxic to M. sexta larvae than to the respective parental toxins, suggesting that increased flexibility made the toxins more susceptible to proteolysis in the insect midgut. Interdomain salt bridges, especially the Asp(242)-Arg(265) bridge, therefore contribute greatly to the stability of the protein in the larval midgut, whereas their role in intrinsic pore-forming ability is relatively less important.

Exchange of domain I from Bacillus thuringiensis Cry1 Toxins Influences protoxin stability and crystal formation.

Influence of domain I exchange on the stability and production of Bacillus thuringiensis Cry1 protoxins as well as on the shape of inclusion and toxicity to Spodoptera exigua and Plutella xylostella larvae was investigated. Chimeric genes were prepared by exchanging the regions coding for domain I between Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, and Cry1E. The AcCC chimera accumulated into bipyramidal inclusion bodies, whereas CEE produced round-shaped inclusion bodies, and ECC and AaEE protoxins produced small granules. AbEE and EAaAa did not produce any inclusion body and were visualized by immunodetection only. AcCC, CEE, ECC, and AaEE were stable to trypsin, whereas AbEE and EAaAa were not. Bioassays showed that the chimeras were not toxic in vivo. However, S. exigua larvae fed with the activated AcCC toxin displayed a lower growth rate.

Activation of an ATP-dependent K(+) conductance in Xenopus oocytes by expression of adenylate kinase cloned from renal proximal tubules.

In rabbit proximal convoluted tubules, an ATP-sensitive K(+) (K(ATP)) channel has been shown to be involved in membrane cross-talk, i.e. the coupling (most likely mediated through intracellular ATP) between transepithelial Na(+) transport and basolateral K(+) conductance. This K(+) conductance is inhibited by taurine. We sought to isolate this K(+) channel by expression cloning in Xenopus oocytes. Injection of renal cortex mRNA into oocytes induced a K(+) conductance, largely inhibited by extracellular Ba(2+) and intracellular taurine. Using this functional test, we isolated from our proximal tubule cDNA library a unique clone, which induced a large K(+) current which was Ba(2+)-, taurine- and glibenclamide-sensitive. Surprisingly, this clone is not a K(+) channel but an adenylate kinase protein (AK3), known to convert NTP+AMP into NDP+ADP (N could be G, I or A). AK3 expression resulted in a large ATP decrease and activation of the whole-cell currents including a previously unknown, endogenous K(+) current. To verify whether ATP decrease was responsible for the current activation, we demonstrated that inhibition of glycolysis greatly reduces oocyte ATP levels and increases an inwardly rectifying K(+) current. The possible involvement of AK in the K(ATP) channel's regulation provides a means of explaining their observed activity in cytosolic environments characterized by high ATP concentrations.

Lipid-induced pore formation of the Bacillus thuringiensis Cry1Aa insecticidal toxin.

After activation, Bacillus thuringiensis (Bt) insecticidal toxin forms pores in larval midgut epithelial cell membranes, leading to host death. Although the crystal structure of the soluble form of Cry1Aa has been determined, the conformation of the pores and the mechanism of toxin interaction with and insertion into membranes are still not clear. Here we show that Cry1Aa spontaneously inserts into lipid mono- and bilayer membranes of appropriate compositions. Fourier Transform InfraRed spectroscopy (FTIR) indicates that insertion is accompanied by conformational changes characterized mainly by an unfolding of the beta-sheet domains. Moreover, Atomic Force Microscopy (AFM) imaging strongly suggests that the pores are composed of four subunits surrounding a 1.5 nm diameter central depression.