[Gudden's diagnosis on Louis II from a contemporary and current psychiatric perspective]. / Guddens Diagnose über Ludwig II. aus zeitgenössischer und heutiger psychiatrischer Sicht.

BACKGROUND: Not only the circumstances of the simultaneous death of King Louis II and his psychiatrist Bernhard von Gudden on Pentecost 1886 are still the subject of controversial discussion but also the nature of Louis' mental illness and the expert report that formed the basis for removing Louis from power. RESULTS: When one considers the psychiatric knowledge of the time, however, it becomes clearer how the four experts who assessed Louis reached a diagnosis of paranoia (madness). Gudden left behind no textbook. Nevertheless, a comparison of the structure and symptom weighting of the expert report with the classification system used in the Compendium, the first edition of the textbook published in 1883 by Gudden's long-time pupil Emil Kraepelin, provides insight into Gudden's school of thought. The experts' interpretation of Louis' illness is an outstanding document in the history of psychiatry. Even after the death of Louis and Gudden, the three remaining experts did not change their views before the parliamentary committees investigating the incident. CONCLUSION: If we use the knowledge of the time as the basis for our assessment, there is no justification for claiming that Gudden and his fellow experts wrongly diagnosed Louis.

[Robert Schumann in the psychiatric hospital in Endenich near Bonn]. / Robert Schumann in der Heil- und Pflegeanstalt Endenich bei Bonn.

BACKGROUND: The composer Robert Schumann (1810-1856) spent the last two-and-a-half years of his life in the private psychiatric hospital in Endenich. His medical records emerged in 1991 and were published by Appel in 2006. METHODS: Daily entries by the physicians were analyzed concerning psychopathology and organic signs as well as the illness-related correspondence of the people closest to Schumann. RESULTS: The numerous entries reveal the treatment typical at that time for what was at first considered to be "melancholy with delusions": shielding from stimuli, physical procedures, and a dietary regimen. The feared, actual diagnosis, a "general (incomplete) paralysis," becomes a certainty in the course of the paranoid-hallucinatory symptoms with cerebro-organic characteristics and agitated states, differences in pupil size, and increasing speech disturbances. CONCLUSION: In the medicine of the time, syphilis is just emerging as the suspected cause, and the term "progressive paralysis" is coined as typical for the course. Proof of Treponema pallidum infection was not available until 1905. Nevertheless, the clinical signs strongly refer to the course of neurosyphilis. People close to Robert, in particular his wife Clara and the circle of friends around Brahms and Joachim, cared intensively for him and suffered under the therapeutic isolation. The medical records and disease-related letters contradict the theory that Schumann was disposed of by being put into the psychiatric hospital; they show the concern of all during the unfavorable illness course.

Mucormycosis in a liver allograft: salvage re-transplantation and targeted immunosuppressive management.

Zygomycetes infection is associated with a high mortality in transplant populations. We describe a child with liver allograft Rhizopus oryzae infection who was salvaged by liver re-transplantation. A 10-year-old child presented with anastomotic bile leak that was repaired. A combined antibiotics and voriconazole regimen was introduced for Escherichia coli and Candida krusei growth in the peritoneal fluid. Despite broad antibiotic and antifungal coverage, the patient continued to have an ongoing infection. A follow-up computed tomography scan 8 weeks later showed 2 liver abscesses infiltrating the stomach and the diaphragm, with splenic infarcts and pericardial effusion. Aspirated samples from the liver abscess and the pericardial fluid revealed R. oryzae. Immunosuppression was discontinued and an antifungal regimen combining amphotericin B, posaconazole, and caspofungin was introduced. After 3 weeks of treatment with control of the systemic signs of infection, a positron emission tomography showed the fluorescence stain limited to the liver. With infection confined to the liver, the child underwent liver re-transplantation, splenectomy, and partial gastrectomy. Immunosuppression was reintroduced with recovery of the immune response observed by the CD4 cells adenosine triphophate release (Cylex(™) ImmuKnow(®) assay) and posaconazole was continued for another year. At 3-year follow-up, the child maintained normal graft function. We conclude that discontinuation of immunosuppression combined with a modern antifungal regimen may allow salvage re-transplantation in patients with liver mucormycosis.

[Responsible dealing with sexuality. Recommendations in a clinical institution]. / Verantwortlicher Umgang mit Sexualität. Empfehlungen in einer klinischen Einrichtung.

Sexuality is excluded in house regulations, guidelines, instructions and regulations in German hospitals. The English literature does not show much more, but more often the wish for clear guidelines is formulated. Under the guidance of the clinical Ethics Committee a paper with recommendations was prepared, which comprises regulations for responsible handling of sexuality in the Pfalzklinikum. This includes sexuality of acute patients in psychiatry, nursing home inhabitants, forensic patients and above all patients in the department of child and youth psychiatry. The right of self-realization on the one hand, the staff's responsibility for patients with limitations in the determination of one's intent on the other hand and the rules for staff members define the range of the paper.

[Bernhard von Gudden on the occasion of the 125th anniversary of his death]. / Bernhard von Gudden zum 125. Todestag.

Bernhard von Gudden died 125 years ago together with King Ludwig II of Bavaria, his royal patient. The prominence of the "Fairy Tale King", the circumstances surrounding the deprivation of his power and his psychiatric internment as well as the establishment of Luitpold's reign and above all the catastrophic ending of the Bavarian royal drama still outshine Gudden's importance for the scientific development of the new subject of Nervenheilkunde (psychiatry and neurology), particularly Bernhard von Gudden's importance and integrity as a physician. Not only was he a much sought-after academic teacher, but he was also a patient-focused advocate of the principle of no restraint. As director of mental institutions, Gudden gave vital impulses for the improvement of mental health treatment. For 14 years he treated Prince Otto, the mentally ill brother of Ludwig II. Gudden rendered an expert opinion together with three other Bavarian psychiatrists resulting in Ludwig's legal incapacitation. Concerning the justification for the King's ousting there have been very different and controversial arguments from the constitutional and psychiatric point of view even in recent times. There is, however, a growing conviction that Ludwig II was incapable of reigning, the deprivation of his power followed the path prescribed by the constitution, and Gudden and his colleagues carried out a reviewing procedure considered valid by today's standards and appropriate under the circumstances. The royal disaster ending with the patient's and reviewer's death, however, has to be attributed to a misjudgement by Gudden that is based on the role diffusion between reviewer and treating physician.

Intercostal chest drain fixation strength: comparison of techniques and sutures.

OBJECTIVE: The accidental removal of an intercostal chest drain (ICD) is common and may result in serious complications. A number of fixation techniques and suture material are in use, and the selection is often based on personal preferences and equipment availability. This study is designed to determine which of the common techniques provides the strongest ICD fixation. METHODS: This study compared the mechanical strength of eight different ICD fixation techniques (purse string, 'Roman sandal', 'Jo'burg' (JO) technique, a suture through the tube, one and two passes through a locking plastic tie, tape fixation and a commercial disposable drainage tube holder) and two silk suture sizes using porcine cadavers and a digital push-pull dynamometer to simulate accidental removal of an ICD. A total of 14 different experimental set-ups produced 280 measurements. RESULTS: Significant differences in ICD fixation strength were observed. A modified JO technique using a size 1 silk suture was nearly three times stronger than a purse-string fixation using a size 0 silk and 10 times stronger from a commercial, adhesive-based device (180, 70 and 22, respectively). CONCLUSION: In situations where the mechanical strength of ICD fixation is important, using a size 1 silk and a modified JO technique may provide the strongest fixation.

Pooled nasopharyngeal and oropharyngeal samples for the identification of respiratory viruses in adults.

A pooled sample of oropharyngeal swabs, nasopharyngeal swabs and nasopharyngeal washings, taken from each of 1,000 subjects, was compared to separate specimens from the same sampling. Multiplex real-time polymerase chain reaction (mqRT-PCR) was used to identify 12 respiratory viruses. Two hundred and forty-three (97%) of the 251 viruses identified in the separate samples were also identified in the mixed samples. The sensitivity rate was identical at 100% for all virus groups except coronaviruses. This sensitivity rate clearly justifies the use of pooled samples instead of separate samples for clinical and epidemiological purposes. The reduction in costs attained from the use of pooled samples may represent a critical advantage when considering its use in extensive clinical and epidemiological studies.

The efficacy and safety of valganciclovir vs. oral ganciclovir in the prevention of symptomatic CMV infection in children after solid organ transplantation.

Routine prophylaxis for CMV with valganciclovir is common in adult recipients but data to support its use in children are scarce. The aim of this study was to compare the efficacy and safety of valganciclovir vs. ganciclovir in a pediatric cohort. We performed a retrospective analysis of 92 children after KTx and/or LTx. All children have received IV ganciclovir for two wk, and then oral ganciclovir (TID; n = 41) before 2004, or valganciclovir (OD; n = 51) thereafter. Treatment was given for three months in R+/D+ or R+/D- recipients and for six months in R-/D+. Patients were followed for one yr post transplant. Both groups were comparable in their demographic and transplant-related history. Symptomatic CMV infection/disease developed in 13.7% vs. 19.5% of valganciclovir and ganciclovir groups, respectively (P-NS). Time-to-onset of CMV infection was comparable in both groups (P-NS); rates of acute allograft rejection were similar in both groups (3.9% vs. 9.8%). Risk factors for CMV infection included young age, serostatus of R-/D+, and allograft from cadaver donor. No significant side effects were noted in both groups. As in adults, valganciclovir appears to be as efficacious and safe as oral ganciclovir. Valganciclovir should be considered as a possible prophylactic treatment for CMV in pediatric recipients of KTx or LTx.

Cyclic AMP-dependent phosphorylation of the precursor to beta subunit of mitochondrial F1-ATPase: a physiological mistake?

By using the purified rat liver protein for reference in electrophoresis and peptide mapping experiments, I have identified the beta subunit of mitochondrial F1-ATPase and its cytoplasmic precursor in two-dimensional gel patterns of proteins from S49 mouse lymphoma cells. The beta subunit precursor is a substrate for cAMP-dependent phosphorylation during its synthesis. Normally, both nonphosphorylated and phosphorylated forms of beta subunit precursor are processed rapidly to the smaller, more acidic forms of mature beta subunit. When processing is inhibited with valinomycin, both nonphosphorylated and phosphorylated forms of beta subunit precursor are stabilized. Nonphosphorylated beta subunit is one of the most stable of cellular proteins, but the phosphorylated form is eliminated within minutes of processing. This suggests that phosphorylated beta subunit is recognized as aberrant and excluded from assembly into the ATPase complex. These results argue that cAMP-dependent phosphorylation of the beta subunit precursor is a physiological mistake that is remedied after mitochondrial import and processing.

Sites of phosphorylation and mutation in regulatory subunit of cyclic AMP-dependent protein kinase from S49 mouse lymphoma cells: mapping to structural domains.

A novel peptide mapping approach has been used to map sites of charge modification to major structural domains of regulatory subunit (R) of type I cAMP-dependent protein kinase from S49 mouse lymphoma cells. Proteolytic fragments of crude, radiolabeled R were purified by cAMP affinity chromatography and displayed by two-dimensional polyacrylamide gel electrophoresis. [35S]methionine-labeled peptides containing sites of mutation or phosphorylation exhibited charge heterogeneity attributable to the modification. Phosphate-containing fragments were also labeled with [32P]orthophosphate to confirm their phosphorylation. Major fragments from [35S]methionine-labeled S49 cell R corresponded in size to carboxyterminal cAMP-binding fragments reported from proteolysis of purified type I Rs from various mammalian species; additional fragments were also visualized. End-specific markers in Rs from some mutant S49 sublines confirmed that cAMP-binding fragments extended to the carboxyterminus of R. Aminoterminal endpoints of fragments could be deduced, therefore, from peptide molecular weights. Clustering of proteolytic cleavage sites within the "hinge-region" separating aminoterminal and carboxyterminal domains of R permitted high resolution mapping in this region: the endogenous phosphate and a "phenotypically-silent" electrophoretic marker mutation fell within a 2.5-kdalton interval at its aminoterminal end. On the other hand, Ka mutations that increase the apparent constant for activation of kinase by cAMP mapped within the large cAMP-binding region of R. A map of charge density distribution within the hinge-region of R was constructed to facilitate structural comparisons between Rs from S49 cells and from other mammalian sources.

Role of commercial aircraft in global monitoring systems.

The role of commercial aircraft in monitoring meteorological parameters and atmospheric constituents has been limited in the former case and virtually nonexistent in the latter. I have tried to point out that this situation can and should be changed now. The new family of wide-bodied jets such as the 747, DC-10, and L-1011 aircraft can be used to supply important global atmospheric and tropical meteorological data for which there is a pressing need. While scientists are not in total agreement on the magnitude of the effect of particulates and gases on the atmosphere, there is almost unanimous concurrence that we are severely limited in information, and that global baseline concentrations must be established for particulates and gases in the troposphere and lower stratosphere as soon as possible. Also, more synoptic meteorological information from the tropical troposphere is highly desirable. In the final analysis, commercial aircraft may offer the most inexpensive way to monitor our atmosphere in the near future. Much of the instrumentation technology is here and the rest is certainly within our grasp. The fact of the matter is that there are now over 220 Boeing 747's and Douglas DC-10's in service, flying an average of 10 hours a day. Long-range flights, such as those from Tokyo to Anchorage to London in the Northern Hemisphere and from Hawaii to Pago Pago to Sydney in the Southern Hemisphere, are commonplace. These aircraft are equipped with inertial navigation systems and central air data computers coupled to advanced data storage systems which can readily be interrogated by satellite. This means that there is now a large amount of snyoptic weather information which can be obtained with a minimum of effort and cost. Likewise, a start at obtaining measurements of atmospheric constituents on a global basis can be made now. All we need to do is make the effort.

Nucleon stability: a geochemical test independent of decay mode.

By analyzing published geochemical data on xenon isotopes measured in a 2.46 x 10(9)-year-old telluride ore, a lower limit of 1.6 x 10(25) years has been obtained for the mean lifetime of the nucleons in the tellurium-130 nucleus. This result is insensitive to the particular mode by which the nucleons decay and therefore provides a rigorous limit on possible baryon number nonconservation. The new limit is about two orders of magnitude better than the previous rigorous limit on nucleon stability.

Technical note: A new and cost-effective method for detection of the bovine acyl-CoA:diacylglycerol acyltransferase 1 K232A polymorphism in cattle.

A new, quick, and inexpensive method for detecting the bovine acyl-CoA:diacylglycerol acyltransferase1 (DGAT1) polymorphism (K232A) through tetra-primer amplification refractory mutation system by PCR (ARMS-PCR) was developed in the present investigation. The DGAT1 gene was recently identified as underlying variation in milk production traits. To date, PCR techniques such as PCR-RFLP have been used for detecting the DGAT1 K232A polymorphism, despite being expensive and laborious. The method proposed here, a tetra-primer ARMS-PCR, showed 100% sensitivity and specificity when compared with PCR-RFLP results obtained in a sample of 80 animals tested in a double-blind system. Our results suggest that the use of tetra-primer ARMS-PCR for DGAT1 K232A polymorphism genotyping could greatly reduce costs providing information for both research purposes and for dairy cattle breeders who perform DGAT1 genotyping for gene-assisted selection.

Effects of the beta3-adrenoceptor (Adrb3) agonist SR58611A (amibegron) on serotonergic and noradrenergic transmission in the rodent: relevance to its antidepressant/anxiolytic-like profile.

SR58611A is a selective beta(3)-adrenoceptor (Adrb3) agonist which has demonstrated antidepressant and anxiolytic properties in rodents. The present study confirmed the detection of Adrb3 mRNA transcript in rodent brain sub-regions and evaluated the effect of SR58611A on serotonergic and noradrenergic transmission in rats and mice in an attempt to elucidate the mechanism(s) underlying these properties. SR58611A (3 and 10 mg/kg, p.o.) increased the synthesis of 5-HT and tryptophan (Trp) levels in several rodent brain areas (cortex, hippocampus, hypothalamus, striatum). Moreover, SR58611A (10 mg/kg, p.o.) increased the release of 5-HT assessed by in vivo microdialysis in rat prefrontal cortex. Systemic (3 mg/kg, i.v.) or chronic administration of SR58611A (10 mg/kg, p.o.), in contrast to fluoxetine (15 mg/kg, p.o.), did not modify the activity of serotonergic neurons in the rat dorsal raphe nucleus. The increase in 5-HT synthesis induced by SR58611A was not observed in Adrb3s knockout mice, suggesting a selective involvement of Adrb3s in this effect. SR58611A (3 and 10 mg/kg, p.o.) did not modify norepinephrine synthesis and metabolism but increased its release in rat brain. Repeated administration of SR58611A (10 mg/kg, p.o.) did not modify basal norepinephrine release in rat prefrontal cortex whereas it prevented its tail-pinch stress-induced enhancement similarly to reboxetine (15 mg/kg, p.o.). Finally SR58611A increased the firing rate of noradrenergic neurons in the rat locus coeruleus following systemic (3 mg/kg, i.v.) or local (0.01 and 1 microM) but not chronic (10 mg/kg, p.o.) administration. These results suggest that the anxiolytic- and antidepressant-like activities of SR58611A involve an increase of brain serotonergic and noradrenergic neurotransmissions, triggered by activation of Adrb3s.

Genetic polymorphism of the kappa-casein gene in Brazilian cattle.

Frequencies of kappa-casein gene alleles were determined in 1316 animals from the Brazilian Bos indicus genetic groups (Sindhi cows, Gyr sires, Gyr cows, Guzerat sires, Guzerat cows, Nellore sires, and Gyr x Holstein crossbreds) by means of polymerase chain reaction-restriction fragment length polymorphism analysis using two independent restriction nucleases (Hinf I and HaeIII). The genotyping of kappa-casein alleles (A and B) is of practical importance, since the B allele is found to correlate with commercially valuable parameters of cheese yielding efficiency. The frequencies of the B allele of kappa-casein among breeds ranged from 0.01 to 0.30. The Sindhi breed had the highest frequency for the B allele (0.30), while the frequencies of this allele in other breeds ranged from 0.01 to 0.18. A wide variation in the B allele frequency among B. indicus breeds was found suggesting that molecular selection for animals carrying the B allele could impact breeding programs for dairy production.

Fine-structure mapping of charge-shift mutations in regulatory subunit of type I cyclic AMP-dependent protein kinase.

A variety of structural mutations that alter functional properties of regulatory subunit (R) of type I cyclic AMP-dependent protein kinase are available in the cultured S49 mouse lymphoma cell system. Many of these mutations also alter the electrostatic charge of R by about 1 or 2 units. By a novel peptide mapping procedure, a number of these "charge-shift" structural mutations were localized to small regions within the R polypeptide. The procedure employed two-dimensional polyacrylamide gel electrophoresis to separate large overlapping fragments generated from denatured, affinity-purified R by limited digestion with papain. Mutations were mapped to intervals between the endpoints of these fragments. The position of one mutation was confirmed by mapping a new site for cleavage by Staphylococcus aureus V8 protease. Six different Ka mutations, which increase the concentrations of cyclic AMP required for kinase activation, mapped to three clusters in the carboxy-terminal half of R. Second-site mutations that cause phenotypic reversion of a single Ka mutant strain mapped to either side of the original mutation. By using charge-shift mutations for calibration, a map of charge density distribution was constructed for the R polypeptide. This map allowed tentative assignment of mutational lesions to portions of the R amino acid sequence implicated in cyclic AMP binding.

A kinase-negative mutant of S49 mouse lymphoma cells is defective in posttranslational maturation of catalytic subunit of cyclic AMP-dependent protein kinase.

Kinase-negative mutants of S49 mouse lymphoma cells, which lack detectable catalytic (C) subunit of cyclic AMP-dependent protein kinase, nevertheless contain cytoplasmic mRNAs for the two major forms of C subunit, C alpha and C beta. Investigation of the metabolism of C subunits in wild-type and mutant cells was undertaken to identify the step(s) at which C subunit expression was defective in kinase-negative cells. [35S]methionine-labeled C subunits from cytosolic fractions of wild-type S49 cells or C subunit-overexpressing cell lines were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after purification by either affinity chromatography using a peptide inhibitor of C subunit as the ligand or immunoadsorption with an anti-C subunit antiserum. Immunoadsorption revealed electrophoretic forms of C alpha and C beta subunits that migrated faster than those detected in affinity-purified samples; this unexpected heterogeneity suggested that functional activation of C subunit may require posttranslational modification. Immunoadsorption of cytosolic fractions from wild-type cells labeled for various times with [35S]methionine revealed an additional posttranslational maturation step. The bulk of immunoadsorbable C subunit label in cells pulse-labeled for 5 min or less was in an insoluble fraction from which it could be solubilized with a detergent-containing buffer; solubilization of the newly synthesized material proceeded over an incubation period of about 10 min. The primary defect in kinase-negative cells appeared to be in this solubilization step, since about equal C subunit radioactivity was found in detergent extracts of wild-type and kinase-negative cells but very little was found in mutant cytosols. I speculate that an accessory factor required for proper folding of newly synthesized C subunit in defective in the kinase-negative cells.

Linked spontaneous CG----TA mutations at CpG sites in the gene for protein kinase regulatory subunit.

CG----TA transitions at CpG sequences account for many human point mutations and are thought to result from hydrolytic deamination of 5-methylcytosine residues in these sites. The gene for regulatory subunit of murine cyclic AMP-dependent protein kinase has two closely linked CpG sites, one of which is a strong hotspot for spontaneous CG----TA mutations leading to cyclic AMP resistance in S49 mouse lymphoma cells. About 5% of mutants with a spontaneous mutation at this CpG site had also acquired a second CG----TA mutation at the nearby CpG site. The two mutations were always at first positions of the Arg codons in which they occurred, and they were always together in a single regulatory subunit allele. Their linked appearance could be attributed to neither the selection conditions nor the preexistence of one mutation in the target cells. The high frequency of these double mutants suggests that their lesions result not from hydrolytic deamination but rather from an endogenous enzymatic mechanism.

Physiological phosphorylation of protein kinase A at Thr-197 is by a protein kinase A kinase.

Phosphorylation of the catalytic subunit of cyclic AMP-dependent protein kinase, or protein kinase A, on Thr-197 is required for optimal enzyme activity, and enzyme isolated from either animal sources or bacterial expression strains is found phosphorylated at this site. Autophosphorylation of Thr-197 occurs in Escherichia coli and in vitro but is an inefficient intermolecular reaction catalyzed primarily by active, previously phosphorylated molecules. In contrast, the Thr-197 phosphorylation of newly synthesized protein kinase A in intact S49 mouse lymphoma cells is both efficient and insensitive to activators or inhibitors of intracellular protein kinase A. Using [35S]methionine-labeled, nonphosphorylated, recombinant catalytic subunit as the substrate in a gel mobility shift assay, we have identified an activity in extracts of protein kinase A-deficient S49 cells that phosphorylates catalytic subunit on Thr-197. The protein kinase A kinase activity partially purified by anion-exchange and hydroxylapatite chromatography is an efficient catalyst of protein kinase A phosphorylation in terms of both a low Km for ATP and a rapid time course. Phosphorylation of wild-type catalytic subunit by the kinase kinase activates the subunit for binding to a pseudosubstrate peptide inhibitor of protein kinase A. By both the gel shift assay and a [gamma-32P]ATP incorporation assay, the enzyme is active on wild-type catalytic subunit and on an inactive mutant with Met substituted for Lys-72 but inactive on a mutant with Ala substituted for Thr-197. Combined with the results from mutant subunits, phosphoamino acid analysis suggests that the enzyme is specific for phosphorylation of Thr-197.

Autoactivation of catalytic (C alpha) subunit of cyclic AMP-dependent protein kinase by phosphorylation of threonine 197.

We recently found, using cultured mouse cell systems, that newly synthesized catalytic (C) subunits of cyclic AMP-dependent protein kinase undergo a posttranslational modification that reduces their electrophoretic mobilities in sodium dodecyl sulfate (SDS)-polyacrylamide gels and activates them for binding to a Sepharose-conjugated inhibitor peptide. Using an Escherichia coli expression system, we now show that recombinant murine C alpha subunit undergoes a similar modification and that the modification results in a large increase in protein kinase activity. Threonine phosphorylation appears to be responsible for both the enzymatic activation and the electrophoretic mobility shift. The phosphothreonine-deficient form of C subunit had reduced affinities for the ATP analogs p-fluorosulfonyl-[14C]benzoyl 5'-adenosine and adenosine 5'-O-(3-thiotriphosphate) as well as for the Sepharose-conjugated inhibitor peptide; it also had markedly elevated Kms for both ATP and peptide substrates. Autophosphorylation of C-subunit preparations enriched for this phosphothreonine-deficient form reproduced the changes in enzyme activity and SDS-gel mobility that occur in intact cells. A mutant form of the recombinant C subunit with Ala substituted for Thr-197 (the only C-subunit threonine residue known to be phosphorylated in mammalian cells) was similar in SDS-polyacrylamide gel electrophoresis mobility and activity to the phosphothreonine-deficient form of wild-type C subunit. In contrast to the wild-type subunit, however, the Ala-197 mutant form could not be shifted or activated by incubation with the phosphothreonine-containing wild-type form. We conclude that posttranslational autophosphorylation of Thr-197 is a critical step in intracellular expression of active C subunit.