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Purpose: Erectile dysfunction (ED) is frequently underreported in men suffering from prolactinomas and can be challenging to manage. Both dopamine agonists (DAs) and transsphenoidal surgery (TSS) correct hyperprolactinemia and restore gonadal function. However, there is scarce data regarding their effectiveness in correcting ED over the long term., Methods: This study is a retrospective single-center comparative cohort study analyzing men diagnosed with prolactinomas, both with and without confirmed erectile dysfunction (ED) at diagnosis. Independent risk factors for persistent ED over the long term were examined using multivariate logistic regression., Results: Among the 39 men with lactotroph adenomas, ED was one of the presenting symptoms in 22 (56%). The mean age at diagnosis was 45 ± 12 years. Surgery was the primary treatment in 6 (27%) ED patients and 8 (47%) non-ED patients. After a mean follow-up of 74 ± 48 months, remission from hyperprolactinemia was achieved in the majority (76%) of men: 71% in the non-ED cohort and 81% in the ED group (p = 0.70), regardless of the primary treatment strategy (surgical 84% versus medical 72%, p = 0.46). Long-term remission of ED was noted in 16 (73%) patients. Interestingly, high baseline BMI levels emerged as potential risk factors for persistent ED over the long term (OR 1.4, 95%CI 1.0-1.9; p = 0.04), while neither the initial adenoma size nor the primary treatment strategy (i.e., TSS vs. DAs) reached statistical significance., Conclusions: Correcting hyperprolactinemia and its associated hypogonadism significantly improves ED in the majority of men with prolactinomas over the long term, regardless of the primary treatment strategy employed. In addition to addressing endocrine deficiencies, the early initiation of weight control programs may be considered for men with lactotroph adenomas and ED. Although our study suggests an association between BMI and the risk of persistent ED, further research is needed to establish any causal relationships., (© 2024. The Author(s).)

Background: In clinical practice, the size of adenomas is crucial for guiding prolactinoma patients towards the most suitable initial treatment. Consequently, establishing guidelines for serum prolactin level thresholds to assess prolactinoma size is essential. However, the potential impact of gender differences in prolactin levels on estimating adenoma size (micro- vs. macroadenoma) is not yet fully comprehended., Objective: To introduce a novel statistical method for deriving gender-specific prolactin thresholds to discriminate between micro- and macroadenomas and to assess their clinical utility., Methods: We present a novel, multilevel Bayesian logistic regression approach to compute observationally constrained gender-specific prolactin thresholds in a large cohort of prolactinoma patients ( N  = 133) with respect to dichotomized adenoma size. The robustness of the approach is examined with an ensemble machine learning approach (a so-called super learner), where the observed differences in prolactin and adenoma size between female and male patients are preserved and the initial sample size is artificially increased tenfold., Results: The framework results in a global prolactin threshold of 239.4 μg/L (95% credible interval: 44.0-451.2 μg/L) to discriminate between micro- and macroadenomas. We find evidence of gender-specific prolactin thresholds of 211.6 μg/L (95% credible interval: 29.0-426.2 μg/L) for women and 1,046.1 μg/L (95% credible interval: 582.2-2,325.9 μg/L) for men. Global (that is, gender-independent) thresholds result in a high sensitivity (0.97) and low specificity (0.57) when evaluated among men as most prolactin values are above the global threshold. Applying male-specific thresholds results in a slightly different scenario, with a high specificity (0.99) and moderate sensitivity (0.74). The male-dependent prolactin threshold shows large uncertainty and features some dependency on the choice of priors, in particular for small sample sizes. The augmented datasets demonstrate that future, larger cohorts are likely able to reduce the uncertainty range of the prolactin thresholds., Conclusions: The proposed framework represents a significant advancement in patient-centered care for treating prolactinoma patients by introducing gender-specific thresholds. These thresholds enable tailored treatment strategies by distinguishing between micro- and macroadenomas based on gender. Specifically, in men, a negative diagnosis using a universal prolactin threshold can effectively rule out a macroadenoma, while a positive diagnosis using a male-specific prolactin threshold can indicate its presence. However, the clinical utility of a female-specific prolactin threshold in our cohort is limited. This framework can be easily adapted to various biomedical settings with two subgroups having imbalanced average biomarkers and outcomes of interest. Using machine learning techniques to expand the dataset while preserving significant observed imbalances presents a valuable method for assessing the reliability of gender-specific threshold estimates. However, external cohorts are necessary to thoroughly validate our thresholds., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor BEG declared a past co-authorship with the authors LA, GS, CM, AT., (© 2024 Huber, Luedi, Schubert, Musahl, Tortora, Frey, Beck, Mariani, Christ and Andereggen.)

Shunt-dependent hydrocephalus (HC) is a common sequela following aneurysmal subarachnoid hemorrhage (aSAH). However, there is still poor evidence regarding the optimal timing of ventriculoperitoneal shunt (VPS) placement, particularly in the context of early aSAH-associated complications such as delayed cerebral ischemia (DCI). The purpose of this study was to compare the impact of early (< 21 days after aSAH) versus late (≥ 21 days after aSAH) VPS placement on the functional clinical outcome. We retrospectively analyzed data from 82 patients with VPS placement after aSAH enrolled in our institutional database between 2011 and 2021. We compared two groups, early VPS placement (< 21 days after aSAH) versus late VPS placement (≥ 21 days after aSAH) in terms of demographics, SAH grading, radiological parameters, externalized cerebrospinal fluid diversions, DCI, VPS variables, and functional outcome. We identified 53 patients with early and 29 patients with late VPS implantation. Baseline variables, such as the modified Rankin Scale (mRS), the World Federation of Neurological Surgeons Scale, the Glasgow Coma Scale, and Fisher grade were not significantly different between the groups. Postoperatively, the mRS (p = 0.0037), the Glasgow Outcome Scale (p = 0.0037), and the extended Glasgow Outcome Scale (p = 0.0032) showed significantly better functional results in patients with early cerebrospinal fluid diversion. The rate of DCI did not differ significantly between the groups (p = 0.53). There was no difference in the rate of VPS placement associated complications (p = 0.44) or overall mortality (p = 0.39). Early shunt implantation, within 21 days after aSAH and therefore during the timeframe of possible DCI, might not be harmful in patients developing HC after aSAH., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objectives: Prolactinomas represent the most common type of secreting pituitary adenomas, yet are rarely encountered in adolescent-onset (AO; i.e. <18 years) or elderly-onset (EO; i.e. ≥65 years) cohorts. As a result, it is not clear whether long-term strategies should be focused differently at both age extremes when comparing their therapeutic outcomes. We aimed at investigating long-term endocrinological outcomes, looking for differences between the two cohorts and evaluating the dependence on continued dopamine agonist (DA) therapy., Methods: Retrospective cross-sectional comparative study analyzing prolactinoma patients with a follow-up of ≥4 years. Clinical, radiological and biochemical characteristics were assessed at diagnosis and last follow-up. Longitudinal endocrinological outcomes between groups of extreme ages (i.e. AO and EO) and middle age (i.e. ≥18 years to 65 years) were compared. Independent risk factors for long-term dependence on DAs were calculated., Results: Follow-up at ≥4 years was recorded for 108 prolactinoma patients; 10 patients with AO and 10 patients with EO. Compared to AO patients, EO patients were predominantly men ( p  = 0.003), and presented with significantly higher prolactin (PRL) levels ( p  = 0.05) and higher body mass index ( p  = 0.03). We noted a significant positive correlation between patients' PRL values and their age (r = 0.5, p  = 0.03) or BMI ( r  = 0.6, p  = 0.03). After a median follow-up of 115 months, remission was noted in 87 (83%) patients; 9 (90%) in AO patients, and 7 (70%) in EO patients ( p  = 0.58). Continuation of DAs was required in 4 patients (40%) with AO and 7 patients (70%) with EO ( p  = 0.37). Patients with elderly-onset were an independent predictor of long-term dependence on DAs (HR 2.8, 95% CI 1.1-7.2, p  = 0.03)., Conclusions: Long-term control of hyperprolactinemia and hypogonadism does not differ between members of the AO and EO cohorts, and can be attained by the majority of patients. However, adjuvant DAs are often required, independent of the age of onset. Considering the clinical significance of persistent DA therapy for the control of hyperprolactinemia in many patients at both extremes of age, long-term monitoring may become recommended, in particular in patients with elderly-onset., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Andereggen, Tortora, Schubert, Musahl, Frey, Luedi, Mariani, Beck and Christ.)

Poor patient outcome after aneurysmal subarachnoid haemorrhage (SAH) is due to a multifactorial process. Delayed cerebral vasospasm, ischemic neurological deficits, and infarction are the most feared acute sequelae triggered by enhanced synthesis of serotonin and endothelin-1 (ET-1). During the past decades, multiple drugs have been analysed for protective effects without resounding success. Therefore, the authors wanted to analyse the potential beneficial role of Losartan (LOS). Male Sprague Dawley rats were randomised into either a group receiving two injections of blood into the cisterna magna (SAH group) or a group receiving two injections of isotonic sodium chloride (sham group). The animals were culled on day five and basilar artery ring segments were used for in vitro tension studies. Sarafotoxin S6c caused a dose-dependent vasorelaxation in sham and SAH segments, which was more pronounced in sham segments. LOS, applied in a concentration of 10−3 M, was able to significantly reduce serotonin- (p < 0.01) and ET-1- (p < 0.05, p < 0.01) mediated vasoconstriction in sham segments. These findings, along with the well-known beneficial effects of LOS on restoring the impaired endothelin-B1-receptor function after SAH, as well as on the neuroprotectional and antiepileptogenic aspects, might be implemented in advancing tailored concepts to sufficiently ameliorate patients’ functional outcome after SAH.

Background: First-line surgery for prolactinomas has gained increasing acceptance, but the indication still remains controversial. Thus, accurate prediction of unfavorable outcomes after upfront surgery in prolactinoma patients is critical for the triage of therapy and for interdisciplinary decision-making., Objective: To evaluate whether contemporary machine learning (ML) methods can facilitate this crucial prediction task in a large cohort of prolactinoma patients with first-line surgery, we investigated the performance of various classes of supervised classification algorithms. The primary endpoint was ML-applied risk prediction of long-term dopamine agonist (DA) dependency. The secondary outcome was the prediction of the early and long-term control of hyperprolactinemia., Methods: By jointly examining two independent performance metrics - the area under the receiver operating characteristic (AUROC) and the Matthews correlation coefficient (MCC) - in combination with a stacked super learner , we present a novel perspective on how to assess and compare the discrimination capacity of a set of binary classifiers., Results: We demonstrate that for upfront surgery in prolactinoma patients there are not a one-algorithm-fits-all solution in outcome prediction: different algorithms perform best for different time points and different outcomes parameters. In addition, ML classifiers outperform logistic regression in both performance metrics in our cohort when predicting the primary outcome at long-term follow-up and secondary outcome at early follow-up, thus provide an added benefit in risk prediction modeling. In such a setting, the stacking framework of combining the predictions of individual base learners in a so-called super learner offers great potential: the super learner exhibits very good prediction skill for the primary outcome (AUROC: mean 0.9, 95% CI: 0.92 - 1.00; MCC: 0.85, 95% CI: 0.60 - 1.00). In contrast, predicting control of hyperprolactinemia is challenging, in particular in terms of early follow-up (AUROC: 0.69, 95% CI: 0.50 - 0.83) vs. long-term follow-up (AUROC: 0.80, 95% CI: 0.58 - 0.97). It is of clinical importance that baseline prolactin levels are by far the most important outcome predictor at early follow-up, whereas remissions at 30 days dominate the ML prediction skill for DA-dependency over the long-term., Conclusions: This study highlights the performance benefits of combining a diverse set of classification algorithms to predict the outcome of first-line surgery in prolactinoma patients. We demonstrate the added benefit of considering two performance metrics jointly to assess the discrimination capacity of a diverse set of classifiers., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Huber, Luedi, Schubert, Musahl, Tortora, Frey, Beck, Mariani, Christ and Andereggen.)

Background: Giant intracranial aneurysms (GIA) are often not eligible for direct clip occlusion. Surgical alternatives include partial clip occlusion or the placement of a cerebrovascular bypass or the combination of both. These alternative indirect strategies are expected to lead to a decrease in GIA volume over time rather than instantaneously. To examine whether this is the case, we analyzed follow-up imaging results 1 year after surgery., Methods: We retrospectively screened the prospective GIA Registry's imaging database for anterior circulation GIA treated by surgical strategies other than direct clipping. We measured pre- and 1-year post-treatment GIA volume, lateral ventricle volume (LVV), and mid-line shift (MLS) in 19 cases., Results: After a mean follow-up of 466 days (standard deviation ±171) GIA volumes decreased from 9.6 cm(3) (interquartile range (IQR) 6.1-14.1) to 4.3 cm(3) (IQR 2.9-5.7; p < 0.01). Ipsilateral LVV increased from 8.6 cm(3) (IQR 6.4-24.9) to 16.0 cm(3) (IQR 9.1-27.2; p < 0.01) while contralateral LVV increased from 10.3 cm(3) (IQR 7.3-20.1) to 11.7 cm(3) (IQR 8.2-19.4; p = 0.02). MLS changed from 0.1 mm (IQR -1.9 to 2.0) to -0.9 mm (IQR -1.8 to 0.4; p = 0.03). The decrease in GIA volume correlated with the increase in ipsilateral LVV (rs = 0.60; p = 0.01) but not with the changes in MLS (rs = 0.41; p = 0.08)., Conclusions: In our patient cohort, surgical strategies other that direct clipping for the treatment of anterior circulation GIA lead to a significant decrease in GIA volume over time. The resulting decrease in mass effect was more sensitively monitored by the measurement of changes in ipsilateral LVV than changes in MLS., Clinical Trial Registration-Url: http://www.clinicaltrials.gov . Unique identifier: NCT02066493.

Navigated transcranial magnetic stimulation (nTMS) is a novel tool for preoperative functional mapping. It detects eloquent cortical areas directly, comparable to intraoperative direct cortical stimulation (DCS). The aim of this study was to evaluate the advantage of nTMS in comparison with functional magnetic resonance imaging (fMRI) in the clinical setting. Special focus was placed on accuracy of motor cortex localization in patients with rolandic lesions. Thirty consecutive patients were enrolled in the study. All patients received an fMRI and nTMS examination preoperatively. Feasibility of the technique and spatial resolution of upper and lower extremity cortical mapping were compared with fMRI. Consistency of preoperative mapping with intraoperative DCS was assessed via the neuronavigation system. nTMS was feasible in all 30 patients. fMRI was impossible in 7 out of 30 patients with special clinical conditions, pediatric patients, central vascular lesions, or compliance issues. The mean accuracy to localize motor cortex of nTMS was higher than in fMRI. In the subgroup of intrinsic tumors, nTMS produced statistically significant higher accuracy scores of the lower extremity localization than fMRI. fMRI failed to localize hand or leg areas in 6 out of 23 cases. Using nTMS, a preoperative localization of the central sulcus was possible in all patients. Verification of nTMS motor cortex localization with DCS was achieved in all cases. The fMRI localization of the hand area proved to be postcentral in one case. nTMS has fewer restrictions for preoperative functional mapping than fMRI and requires only a limited level of compliance. nTMS scores higher on the accuracy scale than fMRI. nTMS represents a highly valuable supplement for the preoperative functional planning in the clinical routine.

Background: Cerebral vasospasm (CV) is a potentially disastrous consequence of subarachnoid hemorrhage despite medical treatment. Nimodipine is a potent drug for vessel relaxation, but side effects may preclude a sufficient dose., Objective: To explore whether continuous local intra-arterial nimodipine administration (CLINA) can reverse vasospasm and prevent delayed ischemic neurological deficit., Methods: Six consecutive subarachnoid hemorrhage patients (5 women; mean age, 47.2 years) with severe CV despite maximum medical therapy underwent CLINA within 2 hours after the onset of clinical symptoms. After anticoagulation, microcatheters were inserted distally in the concerning supra-aortic vessels. Glyceryl trinitrate injection (2 mg) was followed by CLINA (nimodipine 0.4 mg/h for 70-147 hours). Duration of CLINA was determined by neurological status, transcranial Doppler sonography, and partial tissue oxygen pressure values., Results: In all patients, neurological deficits improved or partial tissue oxygen pressure values returned to normal and transcranial Doppler sonography confirmed a reduced blood flow velocity within 12 hours. Magnetic resonance imaging showed no ischemic lesion caused by CV. Neurological outcome was good (modified Rankin Scale score, 0-2) in 3 patients, whereas 1 patient had a moderate clinical outcome (modified Rankin Scale score, 3-4) and 2 patients had a poor outcome (modified Rankin Scale score, 5) because of the SAH., Conclusion: Preliminary data show that CLINA is a straightforward, effective, and safe option for patients with severe CV refractory to medical therapy. Dilation of spastic arteries starts within a few hours and is lasting. Indication for CLINA is peripheral and diffuse CV at any location., (Copyright © 2011 by the Congress of Neurological Surgeons)

Background: Operating on tumors with close margins to the primary motor cortex requires a precise preoperative planning. Transcranial magnetic stimulation (TMS) is the only preoperative technique for detecting eloquent cortical regions that is directly comparable to direct cortical stimulation (DCS). Combining this well established method in neurological diagnostics with a non-invasive navigation system using the patient's preoperative MRI scans in the NEXTSTIM system might be a promising tool in preoperative planning., Case Report: We used preoperative navigated TMS to localize the motor strip in a 75-year-old female patient with a central lesion suspected to be a meningeoma. The elicited data were fused to the navigation MRI in the Brainlab IPLAN system. Following the preoperatively acquired MEP data, a posterior approach to the tumor was planned. After craniotomy, DCS and phase reversal were performed to verify the preoperative results. The motor strip could be located in the same cortical area where the motor response was elicited preoperatively., Conclusion: Navigated transcranial cortical stimulation via the NEXTSTIM system is a helpful tool for preoperative planning., (© Georg Thieme Verlag KG Stuttgart · New York.)

Objective: Intracranial hypotension is a frequently misdiagnosed syndrome which is caused by reduced intracranial cerebrospinal fluid (CSF) pressure due to spontaneous spinal CSF leakage. We present our series of intracranial hypotension regarding especially the required diagnostic imaging and the treatment., Methods: A retrospective analysis was performed on 8 patients (5 males, 3 females, mean age 49 years) with postural and non-postural headache due to spinal CSF collection., Results: Cranial MRI showed diffuse pachymeningeal gadolinium enhancement in all cases. CSF leakage detected by gadolinium-enhanced MR cisternography could be either diffuse (n=5) or precisely located around a dural tear (n=3). All but one leakages were located at the thoracic spine. In 6 patients 40-65 mL of blood were injected through epidurally placed drainages. In 1 patient, a dural tear was sealed with fibrin glue and fat. One patient refused surgical intervention. One epidural haematoma had to be revised. 5 of 7 patients showed excellent results., Conclusion: Gadolinium-enhanced MR cisternography best revealed CSF leaks. In the majority of patients with spontaneous intracranial hypotension, complete recovery may be achieved via a midthoracic epidural blood patch with minimal complications., ((c) Georg Thieme Verlag KG Stuttgart . New York.)

Vacuum extraction in nonprogressive labor is a relatively safe procedure. Only a few major complications have been reported in the literature. The authors present a case of severe brain damage with rupture of the sinus after vacuum extraction delivery for which surgical repair of the dural tear and brain prolapse was required.

Prion diseases are characterized by the deposition of PrP(Sc), an abnormal form of the cellular prion protein PrP(C). A growing body of evidence suggests that antibodies to PrP(C) can antagonize deposition of PrP(Sc). However, host tolerance hampers the induction of immune responses to PrP(C), and cross-linking of PrP(C) by bivalent anti-PrP antibodies is neurotoxic. In order to obviate these problems, we explored the antiprion potential of recombinant single-chain antibody (scFv) fragments. scFv fragments derived from monoclonal anti-PrP antibody 6H4, flagged with c-myc and His6 tags, were correctly processed and secreted by mammalian RD-4 rhabdomyosarcoma cells. When cocultured with cells secreting anti-PrP scFv, chronically prion-infected neuroblastoma cells ceased to produce PrP(Sc), even if antibody-producing cells were physically separated from target cells in transwell cultures. Expression of scFv with irrelevant specificity, or of similarly tagged molecules, was not curative. Therefore, eukaryotically expressed scFv exerts a paracrine antiprion activity. The effector functions encoded by immunoglobulin constant domains are unnecessary for this effect. Because of their small size and their monovalent binding, scFv fragments may represent candidates for gene transfer-based immunotherapy of prion diseases.

Variant Creutzfeldt-Jakob disease and bovine spongiform encephalopathy are initiated by extracerebral exposure to prions. Although prion transmission from extracerebral sites to the brain represents a potential target for prophylaxis, attempts at vaccination have been limited by the poor immunogenicity of prion proteins. To circumvent this, we expressed an anti-prion protein (anti-PrP) mu chain in Prnp(o/o) mice. Transgenic mice developed sustained anti-PrP titers, which were not suppressed by introduction of Prnp+ alleles. Transgene expression prevented pathogenesis of prions introduced by intraperitoneal injection in the spleen and brain. Expression of endogenous PrP (PrP(C)) in the spleen and brain was unaffected, suggesting that immunity was responsible for protection. This indicates the feasibility of immunological inhibition of prion disease in vivo.

Monoclonal antibody MIB-1 is a reliable tool for determining proliferating cells in human tissues, but does not react with the homologous mouse antigen and is therefore useless in experimental pathology using mice as model systems. Standard method for assessment of cellular proliferation in formalin-fixed, paraffin-embedded murine tissues is immunohistochemical detection of DNA synthesis using antibodies against exogenously injected 5-bromodeoxyuridine (BrdU), which is a tedious procedure and not useful for routine investigations. We tested monoclonal antibody MIB-5 and monoclonal and polyclonal anti-MCM3 antibodies as immunohistochemical proliferation markers for paraffin-embedded nonneoplastic and neoplastic tissues of wild-type and transgenic mice, compared to anti-BrdU immunostaining. Percentage of proliferating cells was determined with continuously decreasing antibody dilutions. Percentages of MIB-5 and anti-BrdU immunostained cells correlated strongly, as well as percentage of MIB-5-decorated cells and frequency of mitotic figures. Anti-MCM3 antibodies labeled significantly higher percentages of cells than anti-BrdU or MIB-5, and showed a linear decrease with increasing antibody dilutions. We conclude that MIB-5 detects reliably the cell growth fraction in formalin fixed, paraffin-embedded murine tissues, bypassing methodological drawbacks of BrdU. Anti-MCM3 antibodies are less useful for determination of proliferating cells although they might detect the fraction of cells remaining competent for proliferation.

Since the discovery of the prion protein (PrP) gene more than a decade ago, transgenetic investigations on the PrP gene have shaped the field of prion biology in an unprecedented way. Many questions regarding the role of PrP in susceptibility of an organism exposed to prions have been elucidated. For example mice with a targeted disruption of the PrP gene have allowed the demonstration that an organism that lacks PrPc is resistant to infection by prions. Reconstitution of these mice with mutant PrP genes allowed investigations on the structure-activity relationship of the PrP gene with regard to scrapie susceptibility. Unexpectedly, transgenic mice expressing PrP with specific amino-proximal truncations spontaneously develop a neurologic syndrome presenting with ataxia and cerebellar lesions. A distinct spontaneous neurologic phenotype was observed in mice with internal deletions in PrP. Using ectopic expression of PrP in PrP knockout mice has turned out to be a valuable approach towards the identification of host cells that are capable of replicating prions. Transgenic mice have also contributed to our understanding of the molecular basis of the species barrier for prions. Finally, the availability of PrP knockout mice and transgenic mice overexpressing PrP allows selective reconstitution experiments aimed at expressing PrP in neurografts or in specific populations of hemato- and lymphopoietic cells. Such studies have shed new light onto the mechanisms of prion spread and disease pathogenesis.

Mcm proteins are abundant nuclear proteins involved in the regulation of genome replication. Previous experiments had shown that levels of Mcm-specific mRNAs increase at the G1/S phase transition of the cell cycle, but that the amounts of Mcm proteins do not change much during the cell cycle. To learn more about the stability of an Mcm protein we performed experiments which showed that: (i) more than 60% of [35S]methionine pulse-labeled Mcm3 protein appears to be degraded during a 24-h chase in HeLa cells; (ii) the amount of Mcm3 protein significantly decreases during the differentiation of HL60 cells in vitro (whereas another replication-initiation protein, hOrc2, remains fairly constant); and (iii) according to immunohistochemical staining, Mcm3 protein is present in nuclei of cells in the proliferating zone of human epidermal tissue, but in decreasing amounts in nuclei of differentiating cells of the upper cell layers. Our interpretation is that Mcm3 protein is no longer synthesized after initiation of differentiation and slowly disappears at a half-life of approximately 24 h.

A yeast two-hybrid screen was employed to identify human proteins that specifically bind the amino-terminal 400 amino acids of the retinoblastoma (Rb) protein. Two independent cDNAs resulting from this screen were found to encode the carboxy-terminal 137 amino acids of MCM7, a member of a family of proteins that comprise replication licensing factor. Full-length Rb and MCM7 form protein complexes in vitro, and the amino termini of two Rb-related proteins, p107 and p130, also bind MCM7. Protein complexes between Rb and MCM7 were also detected in anti-Rb immunoprecipitates prepared from human cells. The amino-termini of Rb and p130 strongly inhibited DNA replication in an MCM7-dependent fashion in a Xenopus in vitro DNA replication assay system. These data provide the first evidence that Rb and Rb-related proteins can directly regulate DNA replication and that components of licensing factor are targets of the products of tumor suppressor genes.

For the study of prion neurotoxicity, we used neural-grafting techniques: mice devoid of the normal host prion protein (Prnp% mice) received a neural graft and were intracerebrally infected with mouse prions. The growth and differentiation properties of neural grafts were defined. Growth of embryonic neuroectodermal tissue was optimal at gestational days 12.5-13.5. The blood-brain barrier is reconstituted after 7 weeks in most animals. Scrapie-infected PrPC-expressing grafts develop a severe spongiform encephalopathy and contain proteinase-resistant protein and infectivity. Infected grafts deliver high amounts of prions to the host brain without eliciting disease. Infected grafts show a progressive disruption of the blood-brain barrier. Following intraocular prion inoculation of a transplanted Prnp% mouse, prions do not reach the intracerebral graft, indicating that PrP expression is required for propagation along the optic tract.

Recently we identified a cDNA fragment encoding a conserved part of a new human minichromosome maintenance (Mcm) protein, provisionally termed P1.1Mcm3. Here, we report that the protein is most highly related to a yeast cell-division-cycle protein, Cdc47, encoded by the open reading frame YBR1441 on chromosome 11 of Saccharomyces cerevisiae. The human protein migrates on a polyacrylamide gel with an apparent molecular mass of 85 kDa and shares areas of significant similarity with the Mcm family of replication proteins. It is, therefore, designated as p85Mcm. Microscopic immuno-fluorescence studies revealed that protein p85Mcm is located in the nuclei of interphase cells, but is evenly distributed throughout the cell during mitosis. The amounts of p85Mcm do not significantly change during the cell cycle, but mRNA levels rise with the beginning of the S phase. However, in vitro differentiation of HL60 cells results in a striking decrease of both p85Mcm mRNA and protein levels, suggesting a role for p85Mcm in proliferating, but not in differentiated cells. Under physiological salt conditions, p85Mcm is a component of a high molecular-mass complex including other Mcm proteins. The complex dissociates at high ionic strength giving rise to stable subcomplexes, one of which contains protein p85Mcm together with Mcm proteins hCdc21 and p1O5Mcm.

Background: A complex of MCM proteins is implicated in ensuring that DNA replicates only once in each cell cycle, by 'replication licensing'. The nuclear membrane is also implicated in replication licensing, but the relationship between the MCM proteins and the nuclear membrane is unclear. Here, we investigate the relationship between XMCM3 (a component of the Xenopus MCM complex), nuclear envelope permeability and the initiation of DNA replication once per cell cycle., Results: Our results show that the nuclear envelope does not prevent the entry of XMCM3 into the nucleus, but that it does prevent the binding of XMCM3 to chromatin. We have also identified another component of the Xenopus MCM complex as a homologue of the Schizosaccharomyces pombe protein Cdc21. XMCM3 does not preferentially co-localize with sites of DNA replication. Instead, it is almost uniformly distributed on chromatin and is suddenly lost during replication. XMCM3 crosses intact nuclear membranes of G2-phase HeLa cells but cannot then bind to chromatin. Permeabilization of the nuclear envelope allows the binding of XMCM3 to G2-phase chromatin. We have therefore resolved replication licensing into two stages. The first requires the entry of a cytosolic 'loading factor' that is excluded by the nuclear membrane; subsequently, MCM3 can bind to chromatin in the presence or absence of a nuclear membrane, but only if the loading factor has gained access in the absence of the membrane., Conclusions: The Xenopus MCM complex contains homologues of yeast MCM2, MCM3, MCM5 and Cdc21 proteins. XMCM3 is displaced from chromatin during replication. The nuclear envelope allows entry of XMCM3 into the nucleus, but regulates its binding to chromatin; binding requires a loading factor which cannot cross the nuclear envelope. Based on these results we present a two-stage model for replication licensing.

We present the amino acid sequence of the human homologue of the yeast replication protein Cdc21, a member of the Mcm family of nuclear proteins. Specific antibodies, raised against protein hCdc21, were used to investigate the expression of the protein through the cell cycle. The protein is highly phosphorylated in mitotic cells. The phosphorylated form of protein hCdc21 appears to be less tightly bound to nuclear structures than the underphosphorylated form suggesting that phosphorylation/dephosphorylation reactions may determine the nuclear distribution of the protein. Protein hCdc21 forms a stable trimeric complex with two novel human Mcm proteins, p85Mcm and p105Mcm. Protein BM28/Mcm2 is more loosely associated with the trimeric hCdc21 complex.

Human nuclear proteins P1Mcm3 and P1Cdc46 have high sequence similarities with the corresponding yeast proteins known to be required for the initiation of genome replication. Nuclei of proliferating HeLa cells contain relatively high amounts of P1Mcm3 (about 10(6) molecules/nucleus) of which only a small fraction is bound to a nuclear structure, most probably chromatin. At 0.5 M NaCl, the structure-bound nuclear protein can be partially solubilized as a dimer composed of P1Mcm3 and the related protein P1Cdc46. However, most protein P1Mcm3 is not bound to a nuclear structure and appears in the nucleoplasm. About 10% of protein P1Mcm3 in the soluble fraction is free and uncomplexed, and the remaining P1Mcm3 forms stable complexes with protein P1Cdc46. These P1Mcm3/Cdc46 complexes occur as dimers and in high-molecular-mass complexes (approximately 500 kDa). The high-molecular-mass complexes dissociate in 0.5 M NaCl and release P1Mcm3/Cdc46 dimers. It has frequently been proposed that the Mcm proteins may function as licensing factors for genome replication. Our data imply that the active form of an Mcm protein is not a monomer, but a protein complex that includes an Mcm3/Cdc46 dimer. DNA polymerase alpha is not a component of this complex.