Stage-and tissue-specific expression of a Col2a1-Cre fusion gene in transgenic mice.

To achieve chondrocyte-specific deletion of floxed genes we generated a transgenic mouse line expressing the Cre recombinase under the control of the mouse type II collagen gene (Col2a1) regulatory regions. Northern and in situ hybridization analyses demonstrated the expression of the transgene (Col2a1-Cre) in cartilaginous tissues. To test the excision efficiency of Cre, the Col2a1-Cre strain was crossed with the ROSA26 reporter strain. LacZ staining of double transgenic mice revealed Cre activity in both chondrogenic and non-chondrogenic tissues. During early embryonic development (E9.5-11.5), LacZ expression was detected in tissues where the endogenous Col2a1 transcript is expressed such as the otic capsule, notochord, developing brain, sclerotome and mesenchymal condensations of future cartilage. At later stages, Cre activity was observed in all cartilaginous tissues with virtually 100% of chondrocytes being LacZ positive. These data suggest that the Col2a1-Cre mouse strain described here can be useful to achieve Cre-mediated recombination in Col2a1 expressing cells, especially in chondrocytes.

Antisense RNA inhibition of cathepsin L expression reduces tumorigenicity of malignant cells.

Several tumour-forming cell lines are known to overproduce the lysosomal cysteine peptidase cathepsin L. We have used an antisense approach to investigate whether inhibition of cathepsin L overexpression in two malignant cell lines (myeloma SP cells and L cells) reduces their tumorigenic potential. Two different cDNA fragments of murine cathepsin L were inserted in the antisense direction into the pcDNA3 vector, and SP and L cells were stably transfected with these plasmid constructs. Several of the selected clones expressing the antisense transcript showed specific reduction of the mRNA level and the intracellular activity of cathepsin L, and a greatly diminished amount of secreted procathepsin L. When tested in Balb/c nu/nu mice, the cell lines with low cathepsin L activity exhibited a significantly decreased potential for tumour growth when compared with control cells expressing wild-type levels of cathepsin L activity. This observation suggests that cathepsin L is a critical factor in tumour growth.

Complete genomic structure of the mouse cathepsin K gene (Ctsk) and its localization next to the Arnt gene on mouse chromosome 3.

In preparation for gene manipulative experiments in mice we have isolated genomic clones covering the entire murine cathepsin K gene (Ctsk). Sequence analysis revealed that the gene spans 10.1 kb and contains eight exons and seven introns. The sizes of the coding exons 2-7 as well as the pattern of intron sizes are conserved between the human and mouse cathepsin K genes. Genomic organization of the mouse cathepsin K gene also closely resembles those of cathepsins S and L. More than 26% of the Ctsk sequence consists of different repetitive elements. Detailed sequence analysis of 1.7 kb of Ctsk promoter revealed several putative binding sites for transcription factors and two stretches of 280-320 bp which were > 70% homologous with the human cathepsin K promoter. Analysis of genomic clones extending further upstream of the Ctsk gene identified the 3' end of the gene coding for arylhydrocarbon-receptor nuclear translocator (Arnt). This places Ctsk approximately 4.5 kb downstream of Arnt on mouse chromosome 3 at locus 47.9.

Immunoreactivity of recombinant human glandular kallikrein using monoclonal antibodies raised against prostate-specific antigen.

The gene encoding human glandular kallikrein (KLK2) was expressed in Escherichia coli, and the corresponding protein (hK2) was produced by fermentation. The hK2 was characterized by Western blotting and epitope map using monoclonal antibodies (MAbs) specific for another protease, prostate-specific antigen (PSA) with high structural identity (80%). MAbs that recognized three different epitopes were bound to hK2, representing 7 out of 23 MAbs tested. One epitope was localized to the sequence region around amino acid position 78, which is believed to be glycosylated in hK2. The affinities of MAbs recognizing hK2 were similar to those for PSA, suggesting that common epitopes seem to contain very conserved structures. The results may help in designing specific diagnostic assays for the assessment of prostate cancer.

Polymorphic changes of mannitol during freeze-drying: effect of surface-active agents.

The effect of polysorbate 80 on the crystalline properties of mannitol during freeze-drying was studied. Crystallinity and polymorphism play an important role in, e.g., the solubility and stability of freeze-dried products. Polysorbate 80 had an effect on both the polymorphism and the crystallinity of freeze-dried mannitol.

Immunofluorometric assay for sensitive and specific measurement of human prostatic glandular kallikrein (hK2) in serum.

Prostate-specific antigen (PSA) and human prostatic glandular kallikrein (hK2) have 79% identity with the primary structure. When we used recombinant hK2 protein, only 7 of 23 monoclonal anti-PSA IgGs (monoclonal antibodies, MAbs) cross-reacted with hK2, which enabled us to design a novel immunofluorometric MAb-MAb assay for the specific detection of hK2. In the first incubation, an excess of MAb 2H11, which does not cross-react with hK2, is added to prevent both free and complexed PSA from reacting in subsequent immunoreactions. In the second incubation, biotinylated MAb H50, which cross-reacts with hK2 by an epitope overlapping with MAb 2H11, served to bind only hK2 to the microtitration wells coated with streptavidin. In the third step, Eu-labeled MAb H117, which cross-reacts with hK2, detected the immobilized hK2. The hK2 assay was calibrated with recombinant hK2. The detection limit of the assay was 0.1 microgram/L, and the cross-reactivity with recombinant PSA was < or = 0.7%. The concentration of hK2 was measured in serum samples from 334 males with total PSA concentrations ranging from 1 to 3400 microgram/L. Most of the samples (57%) had hK2 concentrations below the detection limit. The proportions of hK2 relative to total PSA were 0-2% in 79%, 2-5% in 14%, 5-10% in 4%, and >10% in 3% of the samples. Gel filtration of 10 serum samples with increased hK2 concentrations showed a single peak of hK2 immunoreactivity with an apparent molecular size of approximately 30 kDa, corresponding to that of recombinant hK2 and free PSA.

Expression of prostate specific antigen on the surface of a filamentous phage.

We have constructed two phagemid vectors containing the gene coding for human Prostate Specific Antigen (PSA) translationally fused to a pelB signal sequence and minor coat protein III of phage fd leading to phage particles that carry PSA on their tips. Phages were characterized by Western blotting and by panning, using biotinylated monoclonal anti-PSA antibodies immobilized onto streptavidin-coated microtitration wells. Binding of PSA-phages was 10(2)- to 5 x 10(3)-fold more effective than that of wild-type phages. Trypsin treatment of the phage abolished the specific binding. Competitive panning with different concentrations of added purified PSA demonstrated that binding was PSA specific. Finally, Western-blot analysis confirmed that full-length and shorter degradation products of the fusion between PSA and protein III were visible as probed with anti-PSA antibody.

[Serum thiopental values, spontaneous frontal muscle electromyography activity and compressed EEG amplitudes and frequency values in thiopental infusion anesthesia]. / Serum-Thiopentalwerte, spontane M.-frontalis-EMG-Aktivität und komprimierte EEG-Amplituden- und Frequenzwerte bei Thiopentalinfusions-anästhesie.

Serum thiopentone (S-Thiop) was estimated every 10 minutes using a new high performance liquid chromatographic method during fentanyl-bolus-complemented (0.01 mg) thiopentone infusion anaesthesia (bolus of 5 mg/kg, infusion dosis 23.7 mg/min) for laryngo-microscopic procedures. Before stopping the infusion after 18/17 min (SD 8/5) (male/female) S-Thiop was (mean (SD) range) 16.0/12.4 (4.6/5.7) 5.9-26.8/5.1-26.6 mg/l; after stop of the infusion 12.8/10.5 (4.5/5.3) 7.6-25.5/4.5-23.4 mg/l. Patients reacted to verbal command after 11.5 (10.3) 11-81 min. There were no clinically relevant correlations between S-Thiop and frontal EMG and compressed 1-channel-EEG (ABM, Datex, Helsinki), nor with data recorded during recovery. Reasons may be: the total depolarisation block caused by succinylcholine; the method of compressing the EEG; subjective influences on the recording of recovery data; sampling in intervals of 10 min instead of at certain points of the anaesthesia course. We still recommend this total i.v.-anaesthesia for the short time endolaryngeal procedures need; before using this method for longer periods additional pharmacokinetic studies should be performed.

Fluid resuscitation of hypotensive emergency patients with and without an algorithm.

Seventy-seven consecutive hypotensive (mean arterial pressure (MAP) less than 80 mmHg) surgical emergency patients were resuscitated according to either physicians' individual orders (38 patients) or an algorithm (39 patients). The shock was mainly caused by accidental injuries or acute gastrointestinal bleeding. The patients of the algorithm group were given more plasma expanders than the patients of the control group, while the total amount of fluids administered was similar in both groups. The primary goal of the resuscitation (MAP greater than 80 mmHg) was reached within 30 min in three cases in the control group and in seven cases in the algorithm group. The treatment times at the emergency department and the intensive care unit were similar for the groups. The number of severe and moderate pulmonary disturbances was the same, but mild disturbances were significantly more common in the control group. Renal failure was somewhat more common in the control group and the renal function disturbances were significantly more severe among the control patients. The results suggest that the physicians in some extent altered their practices in fluid resuscitation when the algorithm was put to use, and that this change, perhaps, produced the somewhat better outcome of the patients. The authors recommend the algorithm to be used as a basis of shock treatment and particularly in those emergency departments where the resuscitation of hypotensive patients is performed by junior or inexperienced physicians.

Analysis of 13 800 subclavian vein catheterizations.

During the period 1974-1983, 13 857 subclavian or internal jugular vein catheterizations were analysed in the same hospital. The data on the catheterizations have been collected prospectively in the hospital computer. In 93% of the cases, catheterization was subclavian vein cannulation performed through the infraclavicular route. In 15% of the cases the catheter position was not ideal, verified with x-ray; e.g. the catheter tip was turned into the internal jugular vein. Of the catheters 70% were kept in place for less than 7 days, and only 6% for over 2 weeks. More than one catheterization was required in 26% of the cases during the same hospitalization. Recatheterization is an important prophylactic measure for preventing septic complications, if suspicion of infection arises. In 5% of the cases, some complications occurred, but they were mostly minor, such as haematoma at the puncture site. More serious complications were 19 pneumothoraces (0.1%). The results suggest that subclavian vein catheterization is a fairly safe method for large-scale use in a hospital, if only a limited group of physicians perform it. Certain precautions should, however, be taken.

The antagonistic effect of pentazocine on fentanyl induced respiratory depression compared with nalorphine and naloxone.

The effect of pentazocine, a strong analgesic with a weak opiate antagonistic activity, on fentanyl-induced respiratory depression was studied after anaesthesia in patients undergoing gynaecological laparotomy. Pentazocine (1 mg/kg) was given intravenously at the end of operation. The postoperative respiratory depression in these patients was compared with that in patients who received strong opiate antagonists, nalorphine and naloxone, or no opiate antagonists. Respiratory depression was evaluated by measuring respiratory rate, respiratory minute volume and blood gases. The results show that pentazocine has a clear antagonistic effect on fentanyl-induced respiratory depression but the effect of 1 mg/kg is weaker and shorter than that produced by 5 mg of nalorphine or 0.4 mg of naloxone. Postoperative analgesia in patients who received pentazocine was not longer than that in patients who received no opiate antagonists at the end of the operation.

[Documentation of central venous catheterization using a computer laboratory system (author's transl)]. / Dokumentationskontrolle zentraler Venenkatheter mit Hilfe eines EDV-Laborsystems.

Full documentation of every case of central venous catheterization is a prerequisite for reducing the incidence of failures and complications. A laboratory computer program already in use was modified for this purpose: the start of the operation is put in in "real time", abandoned or failed procedures or incomplete data are detected, and can be queried on the day of patient is discharged from hospital. Adverse effects are shown up at an early stage by the quarterly print-out. The program involves less work than a conventional system without computer. The experiences gained with this modified system in the course of one year (including the run-in period) are reviewed.

Risk factors in surgical patients with verified preoperative myocardial infarction.

A series of 89 surgical patients (111 operations) with preoperative myocardial infarction (MI) was analysed for factors predisposing to the development of a postoperative reinfarction. Six of them suffered postoperative MI, and three of these patients died. In the statistical analysis the following risk factors emerged: age over 60 years, anaemia, hypertension, and the fact that the previous MI had been posterior. Abdominal operations were more dangerous concerning reinfarction than other operations. In the other series of 11 deceased patients with postoperative reinfarction collected from the autopsy material, about the same risk factors were found. The most important factor seemed to be hypotension, which complicated the surgery. All 11 patients had arrhythmias in their preoperative electrocardiogram. Previously treated heart failure was present in five of these patients. Postoperative symptoms analysed in the first series suggest that if a patient with preoperative MI has arrhythmias, hypotension, dyspnoea, diffuse unlocalized pain or chest pain after surgery, he is very likely to have a reinfarction.

Epidural anaesthesia with mixtures of bupivacaine-lidocaine and etidocaine-lidocaine.

The mixtures of 0.5% bupivacaine- 1% lidocaine and 1% etidocaine- 1% lidocaine were used in a double-blind manner for lumbar epidural anaesthesia in 48 patients undergoing prostatectomy in order to study whether these mixtures speed up the onset of analgesia, shorten the long motor block, motor block, or improve the quality of analgesia caused by bupivacaine or etidocaine alone. The mixture of bupivacaine-lidocaine caused an analgesia with a somewhat faster onset, similar duration and markedly shorter motor block than bupivacaine alone. The sensory block produced by the mixture of etidocaine-lidocaine tended to begin more slowly and had a distinctly shorter duration, as was the motor block, as compared to the block caused by etidocaine alone. The analgesia was complete in all 12 patients who received bupivacaine and in 11/12 patients who received the mixture of bupivacaine-lidocaine, but 6/12 patients anaesthetized with etidocaine and 5/12 patients with the mixture of etidocaine-lidocaine experienced considerable visceral pain during the operation. The results suggest that the mixture of bupivacaine-lidocaine produces a block with somewhat better properties than bupivacaine alone, but the mixture of etidocaine-lidocaine does not offer any advantages as compared to etidocaine alone, except the shorter motor block.

Positive end expiratory pressure ventilation, renal function and renin.

Positive end expiratory pressure (PEEP) during respirator therapy can impair renal function by altering renal haemodynamics or by increasing the secretion of the antidiuretic hormone. In the present study, the effect of the commonly used 10 cm H2O PEEP for two hours on renal function and on plasma renin activity was studied in eleven intensive care patients. During the examination period, the patients received analgesic, sedative, and muscle relaxant drugs, but no diuretics. PEEP decreased the mean urinary output by 21%. Urinary specific gravity and osmolality increased. Urinary sodium excretion decreased along with urinary volume. The creatinine clearance decreased slightly, but free water clearance became less negative suggesting reduced ability of tubules to concentrate urine during PEEP. The plasma renin activity was not altered significnalty by PEEP, nor did the urinary sodium/potassium ratio change. This may indicate that the water retention induced by PEEP is not caused by the increased secretion of aldosterone. The results suggest that 10 cm H2O PEEP impairs renal function in critically ill patients and causes mainly water retention.

Preoperative and postoperative use of clonidine with neurolept anaesthesia.

The abrupt cessation of clonidine therapy can induce a withdrawal syndrome. This may also appear immediately after anaesthesia if clonidine medication is discontinued during the operation day. We studied (1) whether the withdrawal syndrome occurs postoperatively as often as otherwise when clonidine therapy is discontinued, and (2) the action of this clonidine withdrawal on the circulation as compared to patients who received clonidine without interruption during the operation day. During the recovery period in 2 out of 10 patients in whom clonidine medication was discontinued, a hypertensive crisis occurred, which was relieved by clonidine readministration. Clonidine given with premedication eliminated high rises in blood pressure during anaesthesia. During the recovery period, the regular intramuscular administration of the same dose of clonidine as the patients had used orally decreased blood pressure to almost normotensive levels. The slightly increased urinary catecholamine excretion and plasma renin activity in these patients might, however, indicate that the circulatory homeostasis was disturbed in some degree. The results suggest that it is important to give clonidine continuously, even during the operation day. The dose may be the same as, or preferably somewhat smaller than that which the patients have previously received orally.

Controlled hypotension and moderate haemodilution in major hip surgery.

This study presents a series of 38 patients in whom total hip replacement was performed by using a hypotensive anaesthetic technique with moderate haemodilution. 17 patients operated on during normotension without haemodilution served as controls. Halothane, d-tubocurarine and pentolinium tartrate were the agents used. During the operation circulation was monitored carefully. Hypotension with haemodilution decreased the amount of the transfused blood to 1/3, compared with the normotensive group. The bloodless field during the hypotension also shortened the duration of the operations. No complications attributable to the hypotensive technique occurred. The results suggest that the hypotensive anaesthetic technique with haemodilution reduces the need for blood transfusion in major surgery. However, only patients in good cardiovascular status are suitable for this technique, and careful monitoring of the circulation is mandatory during the anaesthesia.

A comparison of carticaine and lidocaine in spinal anaesthesia.

The effects of Lidocaine and the new local anaesthetic carticaine in spinal anaesthesia were compared in a double blind study in 120 elderly male patients scheduled for small urological procedures. The variables investigated were: analgesia examined by pin prick method, loss of tactile sensation, motor block, and skin temperature on the big toe. Both local anaesthetics seem to act similarly, but the loss of tactile sensation and motor block began somewhat earlier with carticaine. The differences have hardly any clinical significance. The incidence of hypotension and postanaesthetic headache was similar in each group.

A comparative study of carticaine and prilocaine in epidural anaesthesia.

Carticaine and prilocaine (without adrenaline) in epidural anaesthesia were compared in a double blind study. Carticaine and prilocaine were used in 2 % concentration without adrenaline. The latency time was not significantly different, but carticaine was a shorter acting local anaesthetic. The motor block in both series was similar. Carticaine is a good and reliable analgesic in short operations but in operations longer than one and a half hours catheter technique, adrenaline addition or local anaesthetics of longer duration ought to be used.