Submicrometer axial resolution optical coherence tomography.

Optical coherence tomography (OCT) with unprecedented submicrometer axial resolution achieved by use of a photonic crystal fiber in combination with a compact sub-10-fs Ti:sapphire laser (Femtolasers Produktions) is demonstrated for what the authors believe is the first time. The emission spectrum ranges from 550 to 950 nm (lambda(c)=725 nm , P(out)=27 mW) , resulting in a free-space axial OCT resolution of ~0.75 mum , corresponding to ~0.5 mum in biological tissue. Submicrometer-resolution OCT is demonstrated in vitro on human colorectal adenocarcinoma cells HT-29. This novel light source has great potential for development of spectroscopic OCT because its spectrum covers the absorption bands of several biological chromophores.

Physician unions: organizing women in the year 2000.

Interest in physician unions is growing, but surprisingly little has been written about whether union membership addresses the particular needs and interests of women physicians. We begin by looking at the history of physician unions in the United States and then examine physician unions today, and how labor laws influence union membership of physicians. The third section looks at why women join unions, whether these reasons hold true for women physicians, and what role women are playing in physician unions. Finally, we give examples of union responses to gender discrimination and such issues as maternity leave, salary inequities, sexual harassment, and promotions. Since women are prominent as leaders in physician unions, these unions seem to be responsive to the needs of their women members.

Escherichia coli bacteriophage T1 DNA methyltransferase appears to interact with Escherichia coli enolase.

Infection of Escherichia coli cells with bacteriophage T1 induces synthesis of a bacteriophage-specific DNA methyltransferase (M.EcoT1, EC No: 2.1.1.72) with a specificity for adenine residues in the sequence 5'-GATC-3'. Purification of M.EcoT1 allowed the determination of the coding sequence of the gene (Schneider-Scherzer et al., 1990). The peptide of the entire coding sequence was over-expressed as a histidine-hexapeptide tagged protein in E. coli. Affinity purification using a Ni2+ chelating (Ni-NTA) resin yielded a recombinant enzyme with almost the same enzymatic properties as the protein purified from T1 infected E. coli cells. Interestingly, in both purification procedures, a protein with a molecular weight of 50000 was found to copurify with M.EcoT1. The N-terminal amino acid sequence identified these proteins in both cases as E. coli enolase (EC No: 4.2.1.11).

Biosynthesis of the unusual amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine of cyclosporin A: enzymatic analysis of the reaction sequence including identification of the methylation precursor in a polyketide pathway.

3(R)-Hydroxy-4(R)-methyl-6(E)-octenoic acid, the C9-backbone of the unusual amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine (Bmt), is biosynthesized as a coenzyme A thioester from acetyl-CoA, malonyl-CoA, NADPH, and S-adenosylmethionine via a polyketide pathway. Here we present detailed enzymatic studies about the basic assembly process. After attachment of the activated building units to Bmt polyketide synthase the intermediates remained enzyme-bound throughout the cycle. Premature cutoff of biosynthesis led to the release of the intermediates from the enzyme, either as coenzyme A thioesters or, in the case of reactive C8-intermediates, as lactones. Enzyme-bound 3-oxo-4-hexenoic acid, the condensation product of the second elongation cycle, could be identified as the exclusive substrate for the introduction of the methyl group. Part of the biosynthesis including the first elongation cycle, the second condensation reaction, and the methylation step was shown to follow a processive mechanism. All activated intermediates of this processive part could be introduced into the correct pathway at the respective steps, whereas 2-methyl-3-oxo-4-hexenoyl-CoA and all following methylated intermediates were not able to enter the cycle any more. Obviously, the region of Bmt polyketide synthase responsible for this latter part of the biosynthetic pathway is inaccessible for externally supplied coenzyme A thioesters. Butyryl-CoA was recognized by Bmt polyketide synthase with an efficiency comparable to that of crotonyl-CoA and processed to 3-hydroxy-4-methyloctanoyl-CoA, the saturated analog of the natural basic assembly product, indicating a relaxed specificity of Bmt polyketide synthase with respect to the starter unit.

The peptide synthetase catalyzing cyclosporine production in Tolypocladium niveum is encoded by a giant 45.8-kilobase open reading frame.

Cyclosporin A, a potent and clinically-important immunosuppressive drug (SandimmunR), is synthesized from its precursor amino acids by cyclosporin synthetase, a single multi-functional enzyme. In this study we report the cloning of the corresponding coding region of this synthetase. It contains an open reading frame of 45.8 kb which encodes a peptide with a calculated M(r) of 1,689,243. The predicted gene product contains 11 amino-acid-activating domains that are very similar to one another and to the domains of other peptide synthetases. Seven of these domains harbour N-methyltransferase functions. This is the largest genomic ORF described so far.

Purification and characterization of eucaryotic alanine racemase acting as key enzyme in cyclosporin biosynthesis.

A specific alanine racemase, which is a key enzyme in the biosynthesis of the undecapeptide cyclosporin A, was purified to electrophoretic homogeneity from the fungus Tolypocladium niveum. This is the first enzyme of this kind isolated from a eucaryotic organism. The enzyme catalyzes the reversible racemization of alanine and requires pyridoxal phosphate as the exclusive cofactor. Km values for L- and D-alanine were found to be 38 and 2 mM, respectively. Maximal reaction velocity was observed at 42 degrees C and pH 8.8 for the L to D direction. Molecular mass determinations of the denatured enzyme by SDS-polyacrylamide gel electrophoresis gave a value of 37 kDa, whereas gel filtration calibration studies yielded a value between 120 and 150 kDa, indicating an oligomeric native structure.

[Legal problems in headache]. / Gutachtliche Probleme bei Kopfschmerz.

Problems in expert opinion on headache patients are encountered in particular with cases of so-called posttraumatic headache. Symptoms in the vegetative field due to a head injury are characterized by a close time relationship with the accident or trauma. So is genuine post-traumatic headache. Like the so-called postconcussional syndrome, post-traumatic headache is very vaguely defined. To verify the causal connection between headache and head injury an in-depth neurological analysis is necessary. Lesions of intra- and extra-cranial structures sensitive to pain are apt to bring about subjective complaints in the form of headache. Severe craniocerebral injuries with persisting headache may be suggestive of chronic disturbances in cerebrospinal fluid circulation. On the other hand, extensive compound skull fractures and large cranial trephination defects rarely give rise to headache. Cephalgia occurring after cerebral concussions and minor cerebral contusions subside within a short period of time. The evolution of migraine following a head injury is extremely unusual. However, severe subjective complaints may be caused by traumatic subarachnoidal hemorrhage. An exceptional situation is that of neuralgic pain after an accident with injury to the head, especially in the wake of trigeminal nerve lesions. It seems important to mention the possibility of the combination of organic and psychological factors for cephalgia following craniocerebral trauma. Symptomatic headache generally does not cause special difficulties for expert opinion. However, more problems are encountered in the evaluation and appraisal of persistent headache and other subjective complaints in conversion neurosis and psychogenic disorders. Pensions for headache should only be considered in the most severe cases.

Biosynthesis of the unusual amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine of cyclosporin A. Identification of 3(R)-hydroxy-4(R)-methyl-6(E)-octenoic acid as a key intermediate by enzymatic in vitro synthesis and by in vivo labeling techniques.

The biosynthesis of (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine (abbreviation: Bmt, systematic name: 2(S)-amino-3(R)-hydroxy-4(R)-methyl-6(E)-octenoic acid) is proposed to involve two principal phases: the formation of a polyketide backbone and a subsequent transformation process to the final product. Here we report on the identification of 3(R)-hydroxy-4(R)-methyl-6(E)-octenoic acid as the end product of the first phase. The primary indication of 3(R)-hydroxy-4(R)-methyl-6(E)-octenoic acid as the key intermediate in the proposed biosynthetic route came from in vivo labeling studies with [1-13C,18O2]acetate, demonstrating retention of 18O in the 3-hydroxy group. Final identification of this intermediate in in vitro polyketide assays with enriched enzyme fractions of Tolypocladium niveum was achieved after development of highly sensitive and specific detection methods and by use of synthetic reference substances. Two additional methylated in vitro products could be detected and characterized as 4(R)-methyl-(E,E)-2,6-octadienoic acid and 4(R)-methyl-6(E)-octenoic acid by liquid chromatography-mass spectrometry analysis and comparison with synthetic reference samples. Their relevance for Bmt biosynthesis is discussed. Bmt polyketide synthase shows optimal activity at substrate concentrations of 200 microM acetyl-CoA, 150 microM malonyl-CoA, and 200 microM S-adenosylmethionine, around pH 7 and at 35 degrees C. Interestingly the Bmt backbone is released from the enzyme as a coenzyme A thioester, suggesting that subsequent transformation to Bmt takes place upon this activated intermediate.

Identification of a structural glycoprotein of an RNA virus as a ribonuclease.

One of the three structural glycoproteins of classical swine fever virus (CSFV) is E0, a disulfide-bonded homodimer that induces virus-neutralizing antibodies and occurs in a virion-bound as well as a secreted form. E0 was shown to be similar to a family of fungal and plant ribonucleases. Purified E0 from CSFV-infected cells was a potent ribonuclease specific for uridine and inhibitable by zinc ions.

The human ribonuclease/angiogenin inhibitor is encoded by a gene mapped to chromosome 11p15.5, within 90 kb of the HRAS protooncogene.

Ribonuclease/angiogenin inhibitor (RAI) is a tight-binding inhibitor of ribonucleolytic and angiogenic activities involved in tumor progression. It is translated from various mRNAs differing in their 5 regions and originating from a single gene locus. Recently, this gene (RNH) has been assigned to 11p15.5, the terminal part of the short arm of chromosome 11. The regional chromosomal localization was confirmed by somatic cell and in situ hybridization and further refined by long-range restriction mapping. The data place RNH within 90 kb of the Harvey-ras protooncogene (HRAS), so far the most telomeric gene on 11p, in a region involved in growth regulation and tumor development.

Primary structure of a DNA (N6-adenine)-methyltransferase from Escherichia coli virus T1. DNA sequence, genomic organization, and comparative analysis.

Escherichia coli virus T1 encodes a DNA (N6-adenine)-methyltransferase (M.T1) with the same sequence specificity as the E. coli DNA (N6-adenine)-methyltransferase (M.Eco dam). This enzyme was purified to homogeneity and a partial amino acid sequence determined. Oligonucleotides were constructed and used not only as probes to map the gene on the T1 genome, but also as primers in sequencing reactions to establish the nucleotide sequence of the M.T1 locus by primer extension. These data represent the first analysis of the genomic organization of bacterial virus T1 on a molecular level. Significant homology to E. coli consensus transcription and translation-initiation signals suggest that the gene for M.T1 is most probably under control of its own promoter. It may be transcribed as a polycistronic mRNA, together with a downstream open reading frame which codes for a polypeptide containing 83 amino acids (HP 83). Both the deduced primary and the secondary structure of the M.T1 were compared to those of other known DNA methyltransferases, especially those recognizing the sequence, GATC; there is little similarity of the T1 enzyme to the other members of this family.

[Intracranial pressure and brain death]. / Hirndruck und Hirntod.

Cerebral death occurs during reanimation as an isolated destruction of the entire brain. It is the result of a malignant and irreversible increase of the intracranial pressure. Continuous registration of the intracranial and systemic blood pressures which is done as a routine monitoring procedure in the majority of deep coma patients, allows to identify the moment when cerebral perfusion has come to a complete standstill, and also allows to confirm its irreversibility. At the end of an ischaemic period of 8 to 10 minutes, absolutely lethal to brain tissue, cerebral death is completed. To be on the safe side, the expiration of a 15 to 20 minute period of complete circulatory arrest within the cranial cavity is recommended before further diagnostic measures, especially cerebral arteriography, are undertaken as final proof of dissociated brain death, permitting the explantation of vital organs for grafting. At present, due to possible technical difficulties, reliance upon epidural intracranial pressure measurement alone must still be discouraged. Nevertheless, this investigation method can be most useful in the early timing of the so-called terminal angiography in order not to delay the diagnosis of brain death and its medical consequences.

[Monitoring infection at the intensive care unit--a multicenter pilot study]. / Infektionsüberwachung an Intensivstationen--eine multizentrische Pilotstudie.

During a period of 3 months an infection survey was carried out in 4 intensive care units (ICUs), 2 in Vienna, Austria, and one each in Ulm and Münster, Federal Republic of Germany, using a common protocol. A total of 329 patients was monitored prospectively. This pilot study was performed to evaluate the usefulness of parameters included in the monitoring form. It was attempted to characterize the patient populations of the four units. Mean duration of stay (1-12 days), mortality (8-26%), leading diagnosis upon admission, intubation rate (41-91%) and use of pulmonary artery catheter (12-35%) were distinctly different. The rate of patients admitted already with an infection was 9-43%, septicemia was diagnosed in up to 27% of the diseased. The rate of infection acquired in the unit was between 12 and 37%, the most frequent types were bronchopneumonia, septicemia and urinary tract infection. When septicemia patients were compared to non-septicemia patients who had been admitted for more than 3 days, it appeared that the latter stayed significantly shorter at the ICU and showed less frequently bronchopneumonia or urinary tract infection at the time of admission. Septicemia patients acquired more frequently additional infections like broncho-pneumonia or urinary tract infection while staying at the ICU. The median day of onset of septicemia was the fifth day and only in a quarter of cases diagnosis could be supported by a positive blood culture. The use of antibiotics in the 4 ICUs is compared and shows marked differences. Based upon experience with this type of infection survey a new modified protocol is introduced, which displays the time course of documented events.

The primary structure of human ribonuclease/angiogenin inhibitor (RAI) discloses a novel highly diversified protein superfamily with a common repetitive module.

Immunological screening of a lambda gt11 library, constructed from HeLa mRNA, yielded several ribonuclease/angiogenin inhibitor (RAI) cDNA clones containing 900-bp inserts. Northern blot analysis revealed that the length of the RAI mRNA is approximately 1.9 kb. Construction and screening of a eukaryotic cDNA expression library (HeLa) containing preferentially complete cDNA inserts led to the isolation of a full length clone. The complete nucleotide sequence was determined. The C-terminal amino acid sequence deduced from the cDNA is identical to the peptide sequence obtained from a CNBr fragment of RAI, confirming the identity of the clone. The deduced primary structure of RAI consists of eight homologous tandem repeats with remarkable periodicity of leucine and cysteine residues. Each repeat is derived from the duplication of a leucine-rich 28-amino-acid module. This prototype module is closely related to a repetitive 24-amino-acid motif of unclear function, previously found in proteins involved in important biological processes such as blood coagulation, embryonic development, cell morphogenesis and signal transduction. Although homologous, the RAI modules show distinct differences in length and amino acid composition to the modules of this group of proteins, demonstrating their high potential of variability, necessary for adaptation to very diverse roles. Based on our results we propose that these repetitive modules are a common structural feature of a novel protein superfamily whose members exert their function by highly specific protein-protein interactions.

Identification, purification, and characterization of Escherichia coli virus T1 DNA methyltransferase.

An Escherichia coli virus T1-induced DNA methyltransferase was identified by activity gel analysis in homogenates of infected E. coli DNA-adenine-methylation-deficient strains. Although the Mr of this protein (31,000) is in the same range as that of the E. coli DNA adenine methyltransferase, the two proteins are not closely related; the E. coli dam gene does not hybridize with T1 DNA. Selective conditions for measurement of the T1 activity were developed, and the enzyme was purified to functional homogeneity, as shown by activity analysis in polyacrylamide gels. Requirements for optimal activity of the viral enzyme were determined to be pH 6.9, ionic strengths below 0.1 M KCl, and a temperature between 40 and 43 degrees C. The Km for S-adenosyl-L-methionine is 4.9 microM. The purified T1 DNA methyltransferase is capable of methylating adenine in 5'-GATC-3' sites in vitro.