A method for marking histopathological specimens of neck--technical note.

The authors present a technical note for marking the location of lymph nodes of the neck for histopathological examination. A more precise histopathological report permits more effective overall management of patients with neoplastic disease of the head and neck.

Mediastinitis from odontogenic infection. A case report.

We report a case of mediastinitis complicating a dental infection in a 40-year-old male. Despite drainage of the localised neck abscess and the administration of systemic antibiotics, his submandibular abscess extended to involve the pericardial and pleural cavities. Drainage procedures and thoracotomies were required to treat the empyema and purulent pericarditis. Computed tomography was used to follow the progression of disease and assess the efficacy of treatment.

Dissolution test for silymarin tablets and capsules.

Silybine (SBN), isosilybine (ISBN), silycristine (SCN), silydianine (SDN), and taxifoline (TXF) are the main active flavonoids commonly found in the dried fruits of Silybum marianum, Gaertner (Compositae). Concentrations of these compounds, except TXF, are usually expressed together as silymarin content. This paper describes a simple dissolution test developed to estimate silymarin (Sl) in pharmaceutical formulations. Five commercial products were tested using this new method (including tablets, sugar tablets, and capsules): two from Argentina, one from Brazil, one from Spain, and one from Italy. Results demonstrated that, provided the dosage form disintegrates, amounts dissolved range from 50 to 90% of the labeled value. Products were analyzed by high performance liquid chromatography (HPLC) and UV spectrophotometry.

Purification of apolipoprotein H (beta 2-glycoprotein I)-like protein from human follicular fluid.

We purified a glycoprotein of molecular weight 50 kDa that has an N-terminal sequence similar to that of apolipoprotein H indicating that it is identical to or highly homologous to apolipoprotein H. There are indications that apolipoprotein H or its homologue may be involved in the fertilization process. Sperm motion was assessed employing computer-assisted semen analysis. The addition of the purified protein to prepared sperm samples from normospermic men increases significantly the straight line velocity (VSL) and the amplitude of lateral head displacement (ALH) but does not increase the number of progressively motile sperm.

Preliminary X-ray analysis of a C2-like domain from protein kinase C-delta.

C2 domains are intracellular modules of approximately 130 residues that are found in many proteins involved in membrane trafficking and signal transduction. They are known to serve a variety of roles including binding ligands such as calcium, phospholipids and inositol polyphos-phates as well as interacting with larger macromolecules. Although originally identified in the Ca2+-dependent protein kinase C isoforms (PKC), initially no C2 domain was evident within the Ca2+-independent isoenzymes. A recent study identified a divergent C2 domain in several novel, Ca2+-independent PKCs (delta, epsilon, eta and straight theta), located at their N-termini in a region previously referred to as a variable domain zero (Vo) [Ponting & Parker (1996). Protein Sci. 5, 2375-2390]. The functional importance of this domain in the context of the novel PKCs is at present not well understood though it has been implicated in substrate recognition. The expression, crystallization and preliminary crystallographic analysis of recombinant Vo domain (residues 1-123) from PKC-delta is reported here. Crystals were obtained from incomplete factorial screens after removal of the histidine tag used to aid purification. These crystals diffracted to Bragg spacings of approximately 3 A using a rotating-anode source and to 1.9 A using synchrotron radiation. The crystals have cell parameters of a = 60.7, b = 120.9 and c = 40.7 A and systematic absences consistent with the orthorhombic space group P212121. To facilitate structure determination we have prepared, characterized and crystallized selenomethionine-substituted material.

Crystal structure of the C2 domain from protein kinase C-delta.

BACKGROUND: The protein kinase C (PKC) family of lipid-dependent serine/theonine kinases plays a central role in many intracellular eukaryotic signalling events. Members of the novel (delta, epsilon, eta, theta) subclass of PKC isotypes lack the Ca2+ dependence of the conventional PKC isotypes and have an N-terminal C2 domain, originally defined as V0 (variable domain zero). Biochemical data suggest that this domain serves to translocate novel PKC family members to the plasma membrane and may influence binding of PKC activators. RESULTS: The crystal structure of PKC-delta C2 domain indicates an unusual variant of the C2 fold. Structural elements unique to this C2 domain include a helix and a protruding beta hairpin which may contribute basic sequences to a membrane-interaction site. The invariant C2 motif, Pro-X-Trp, where X is any amino acid, forms a short crossover loop, departing radically from its conformation in other C2 structures, and contains a tyrosine phosphorylation site unique to PKC-delta. This loop and two others adopt quite different conformations from the equivalent Ca(2+)-binding loops of phospholipase C-delta and synaptotagmin I, and lack sequences necessary for Ca2+ coordination. CONCLUSIONS: The N-terminal sequence of Ca(2+)-independent novel PKCs defines a divergent example of a C2 structure similar to that of phospholipase C-delta. The Ca(2+)-independent regulation of novel PKCs is explained by major structural and sequence differences resulting in three non-functional Ca(2+)-binding loops. The observed structural variation and position of a tyrosine-phosphorylation site suggest the existence of distinct subclasses of C2-like domains which may have evolved distinct functional roles and mechanisms to interact with lipid membranes.

Human leukocyte antigen system in clozapine-induced agranulocytosis.

Forty-three schizophrenic patients participating in this study were serotyped for human leukocyte antigens (HLA-A, -B, -C, -DR, -DQ antigens). Thirty-six of them were hospitalised in two state mental hospitals and 7 in our general hospital, psychiatric unit. The patients from our unit were typed for HLA before commencing clozapine treatment whereas the patients from state hospitals were typed after commencing treatment. Three out of 43 patients developed agranulocytosis. One had a combination of both 'high-risk' haplotypes (HLA-B16(38,39), DR4, DQ3 and HLA-DR2, DQ1), another had HLA-DR2, DQ1, whereas the last had a totally different haplotype. Between non-agranulocytic patients 1 was found to carry the HLA-B16(38,39), DR4, DQ3 haplotype and 14 (out of 40) had the HLA-DR2, DQ1. Taking into account other factors supposed to be involved (a noxious metabolite, and the presence of a humoral cytotoxic factor) we must admit that despite the finding of a high-risk haplotype in Jewish populations there are other aspects of this question awaiting clarification.

HLA phenotypes in healthy aged subjects.

One hundred and seventy-one unrelated elderly healthy subjects selected according to the Senieur protocol (57 men and 114 women), aged 75-104 years, and 405 healthy individuals (238 men and 167 women), aged 18-65 years, were typed for HLA-A, HLA-B, and HLA-DR antigens. The purpose of the study was to investigate a possible association between HLA antigens and longevity. In the total group of elderly, an increased frequency of HLA-B16 (11.11 vs. 5.43%) and HLA-DR7 (38.33 vs. 15.67%) and a decreased frequency of HLA-B15 (1.75 vs. 5.18%) and HLA-DR4 (11.66 vs. 24.15%) were observed. The HLA-B15DR4 haplotype was not represented (vs. 2.1%), HLA-A1B8 was found with a low frequency (2.9 vs. 4.4%), and HLA-B8DR3 was very rarely found (1.6 vs. 10.1%), whereas the HLA-B13DR7 haplotype was observed with an increased frequency (6.6 vs. 3.3%). These results are in agreement with other published data and suggest that longevity in humans may be influenced by the genetic background.

Incorporating anomalous scattering centres into macromolecules.

The widespread application of multiwavelength anomalous diffraction (MAD) for phase evaluation has been hampered in the past by the small selection of anomalous scattering centres that could be introduced into macromolecules. Recently, the use of chemical modification, protein engineering or biosynthetic labelling has provided suitable tools to overcome the previous limitations, thereby making most structural analyses amenable to a MAD approach.

Antimicrobial activity of 5,6-dihydrobenzo-[a]-carbazoles. Part II.

A new series of twenty-two 5,6-dihydrobenzo[a]carbazoles was synthesized, some showing good antibacterial activity. The presence and position of substituents seems to be critical for such activity.

Crystallization and preliminary crystallographic analysis of cytochrome c553 peroxidase from Nitrosomonas europaea.

The di-heme peroxidase (cytochrome c553 peroxidase) from Nitrosomonas europaea has been crystallized in a form suitable for high-resolution X-ray structure determination. A complete data set was obtained to 2.5A and the data were indexed in space group P2(1) with a = 88.79 A, b = 55.93 A, c = 144.37 A, beta = 103.87 degrees. The self-rotation function indicates one homodimer per asymmetric unit.

An engineered cation site in cytochrome c peroxidase alters the reactivity of the redox active tryptophan.

The crystal structures of cytochrome c peroxidase and ascorbate peroxidase are very similar, including the active site architecture. Both peroxidases have a tryptophan residue, designated the proximal Trp, located directly adjacent to the proximal histidine heme ligand. During the catalytic cycle, the proximal Trp in cytochrome c peroxidase is oxidized to a cation radical. However, in ascorbate peroxidase, the porphyrin is oxidized, not the proximal Trp, despite the close similarity between the two peroxidase active site structures. A cation located approximately 8 A from the proximal Trp in ascorbate peroxidase but absent in cytochrome c peroxidase is thought to be one reason why ascorbate peroxidase does not form a Trp radical. Site-directed mutagenesis has been used to introduce the ascorbate peroxidase cation binding site into cytochrome c peroxidase. Crystal structures show that mutants now bind a cation. Electron paramagnetic resonance spectroscopy shows that the cation-containing mutants of cytochrome c peroxidase no longer form a stable Trp radical. The activity of the cation mutants using ferrocytochrome c as a substrate is < 1% of wild type levels, while the activity toward a small molecule substrate, guaiacol, increases. These results demonstrate that long range electrostatic effects can control the reactivity of a redox active amino acid side chain and that oxidation/reduction of the proximal Trp is important in the oxidation of ferrocytochrome c.

Probing the cytochrome c peroxidase-cytochrome c electron transfer reaction using site specific cross-linking.

Engineered cysteine residues in yeast cytochrome c peroxidase (CCP) and yeast iso-1-cytochrome c have been used to generate site specifically cross-linked peroxidase-cytochrome c complexes for the purpose of probing interaction domains and the intramolecular electron transfer reaction. Complex 2 was designed earlier [Pappa, H.S., & Poulos, T.L. (1995) Biochemistry 34, 6573-6580] to mimic the known crystal structure of the peroxidase-cytochrome c noncovalent complex [Pelletier, H., & Kraut, J. (1992) Science 258, 1748-1755]. Complex 3 was designed such that cytochrome c is tethered to a region of the peroxidase near Asp148 which has been suggested to be a second site of interaction between the peroxidase and cytochrome c. Using stopped flow methods, the rate at which the ferrocytochrome c covalently attached to the peroxidase transfers an electron to peroxidase compound I is estimated to be approximately 0.5-1 s-1 in complex 3 and approximately 800 s-1 in complex 2. In both complexes the Trp191 radical and not the Fe4+=O oxyferryl center of compound I is reduced. Conversion of Trp191 to Phe slows electron transfer about 10(3) in complex 2. Steady state kinetic measurements show that complex 3 behaves like the wild type enzyme when either horse heart or yeast ferrocytochrome c is used as an exogenous substrate, indicating that the region blocked in complex 3 is not a functionally important interaction site. In contrast, complex 2 is inactive toward horse heart ferrocytochrome c at all ionic strengths tested and yeast ferrocytochrome c at high ionic strengths. Only at low ionic strengths and low concentrations of yeast ferrocytochrome c does complex 2 give wild type enzyme activity. This observation indicates that in complex 2 the primary site of interaction of CCP with horse heart and yeast ferrocytochrome c at high ionic strengths is blocked. The relevance of these results to the pathway versus distance models of electron transfer and to the interaction domains between peroxidase and cytochrome c is discussed.

Site-specific cross-linking as a method for studying intramolecular electron transfer.

Site-directed mutagenesis has been used to introduce cysteine residues into yeast cytochrome c peroxidase and yeast cytochrome c for the purpose of forming site-specific cross-linked intermolecular complexes. This enables the formation of well-defined homogeneous covalently linked complexes for the purpose of relating structure to intramolecular electron transfer. Two complexes have been prepared and analyzed. Complex I has an engineered cysteine at position 290 near the C-terminus of the peroxidase linked to the naturally occurring Cys102 near the C-terminus of yeast cytochrome c. This complex exhibits undetectable rates of intramolecular electron transfer. Complex II has Cys290 of the peroxidase linked to the engineered Cys73 of cyt c. This complex was designed to mimic the crystal structure of the peroxidase-cytochrome c noncovalent complex [Pelletier & Kraut (1992) Science 258, 1748-1755]. Stopped-flow studies show that complex II carries out intramolecular electron transfer from ferrocytochrome c to peroxidase compound I at a rate of approximately 500-800 s-1. This indicates that the binding orientation observed in the crystal structure is competent in rapid intramolecular electron transfer.

Antimicrobial activity of 5,6-dihydrobenzo-[a]-carbazoles.

A new series of 5,6-dihydrobenzo[a]carbazoles was synthesized, some showing good antibacterial activity. The presence of a dialkylamino ethyl chain on the 2-, 3- or 4-O-substituent seems to be critical for such activity.

Increased frequency of HLA B17 antigen in girls with Turner syndrome and their fathers.

HLA-A, -B and -DR antigen distribution was studied in 49 girls with Turner Syndrome (TS), in 43 of their parents, as well as in 433 controls. No increased frequency of DR3, DR4 was found in our group. However, an increased frequency of HLA B17 antigen was disclosed (18.3% in TS versus 6.4% in the controls, p < 0.001 and pc < 0.01). Furthermore, the HLA B17 antigen was of paternal origin in 77.7% of the cases. The interpretation of the present findings is quite difficult. Most likely, the findings are related to the chromosomal abnormality rather than to autoimmunity. It is quite possible that genes within the region of class I genes create unfavorable circumstances leading to the loss of the sex chromosome or, alternatively, genes in this region confer protection and prevent miscarriage of the affected fetus.

A step towards understanding the folding mechanism of horseradish peroxidase. Tryptophan fluorescence and circular dichroism equilibrium studies.

The guanidinium chloride denaturation/renaturation of the holo- and apo-horseradish peroxidase isoenzyme c (HRP) has been studied by fluorescence and circular dichroism spectroscopies. A distinct equilibrium intermediate of the apoprotein could be detected at low concentrations of guanidinium chloride (0.5 M). This intermediate has a secondary structure content like that of the native protein but a poorly defined tertiary structure. Renaturation of the apo-HRP is reversible and 100% activity could be obtained after addition of a twofold excess of free haem. The denaturation of the holo-HRP is more complex and occurs in two distinct steps; unfolding of the protein backbone and loss of the haem. The denatured protein folds back to its native conformation but the incorporation of the haem occurs only after the secondary structure is formed. Ca2+ appears to be important for the stability of the protein as the apo-HRP is more resistant to denaturation in the presence of Ca2+. The free-energy change during unfolding of the apo-HRP was determined in the absence and presence of Ca2+ and found to be 9.2 kJ/mol and 16.7 kJ/mol, respectively. The importance of Ca2+ to the protein stability was also supported by studies on the loss of the haem from the protoporphyrin-IX-apo-HRP complex.

A study of beta-cell function after glucagon stimulation in thalassaemia major treated by high transfusion programme.

An increased incidence of diabetes mellitus and glucose intolerance has been reported in thalassaemia major treated with a high transfusion programme (HTP). To investigate beta-cell function, serum immunoreactive insulin (IRI), C-peptide (CP) and glucose were measured fasting and at 3, 6 and 10 min after i.v. administration of 1 mg glucagon in 20 thalassaemia patients treated by many transfusions and in nine healthy control subjects. Fasting C-peptide concentrations (mean +/- SEM) were higher in the thalassaemic group (2.15 +/- 0.17 ng/ml) than in the controls (1.41 +/- 0.13 ng/ml). After stimulation with glucagon, C-peptide concentrations were consistently higher (P less than 0.01) by approximately 50% in the thalassaemic than in the control group (5.29 +/- 0.31 vs 3.36 +/- 0.21 ng/ml, at 3 min; 5.22 +/- 0.30 vs 3.53 +/- 0.21 ng/ml at 6 min and 4.69 +/- 0.27 vs 3.30 +/- 0.17 ng/ml after 10 min). Plasma IRI concentrations increased in both groups after glucagon stimulation but were not significantly different. The glucose values were approximately 15% higher at each sampling time in the thalassaemic group than those of the normal subjects. It is concluded that disturbances in carbohydrate metabolism in thalassaemia major treated with HTP are the consequence of hepatic cirrhosis which accompanies secondary haemosiderosis, and possibly iron deposition in the beta-cells of the pancreas.