Skin-associated Corynebacterium amycolatum shares cobamides.

The underlying interactions that occur to maintain skin microbiome composition, function, and overall skin health are largely unknown. Often, these types of interactions are mediated by microbial metabolites. Cobamides, the vitamin B12 family of cofactors, are essential for metabolism in many bacteria, but are only synthesized by a small fraction of prokaryotes, including certain skin-associated species. Therefore, we hypothesize that cobamide sharing mediates skin community dynamics. Preliminary work predicts that several skin-associated Corynebacterium species encode de novo cobamide biosynthesis and that their abundance is associated with skin microbiome diversity. Here, we show that commensal Corynebacterium amycolatum produces cobamides and that this synthesis can be tuned by cobalt limitation. To demonstrate cobamide sharing by C. amycolatum, we employed a co-culture assay using an E. coli cobamide auxotroph and show that C. amycolatum produces sufficient cobamides to support E. coli growth, both in liquid co-culture and when separated spatially on solid medium. We also generated a C. amycolatum non-cobamide-producing strain (cob-) using UV mutagenesis that contains mutated cobamide biosynthesis genes cobK and cobO and confirm that disruption of cobamide biosynthesis abolishes support of E. coli growth through cobamide sharing. Our study provides a unique model to study metabolite sharing by microorganisms, which will be critical for understanding the fundamental interactions that occur within complex microbiomes and for developing approaches to target the human microbiota for health advances.

Impact of bariatric surgery on early-onset colorectal cancer risk: a systematic review and meta-analysis.

The aim of this meta-analysis is to determine the impact of bariatric surgery on the risk of early-onset colorectal neoplasia. This systematic review was conducted according to PRISMA recommendations. It was registered in the PROSPERO international database. A comprehensive search was conducted in electronic databases (MEDLINE, EMBASE, and Web of Science) for completed studies until May 2022. The Search was made using a mixture of indexed terms and title, abstract and keywords. The search included terms: obese, surgical weight loss intervention, colorectal cancer, and colorectal adenomas. Studies that included bariatric intervention patient's vs non-surgical obese patients younger than 50 years were considered. Inclusion criteria were patients with BMI more than 35 kg/m2 who underwent a colonoscopy. Studies with follow-up colonoscopy performed in less than 4 years after bariatric surgery and those that evaluated patients with a mean age difference of 5 or more years between groups were excluded. Outcomes analyzed in obese patients with surgical treatment vs control patients included colorectal cancer incidence. From 2008 to 2021, a total of 1536 records were identified. Five retrospective studies that included 48,916 patients were analyzed. Follow-up period ranged from 5 to 22.2 years. 20,663 (42.24%) patients underwent bariatric surgery and 28,253 (57.76%) were part of the control patients. Roux-en-Y gastric bypass was performed in 14,400 (69.7%) individuals. The intervention and control group were similar in age range, proportion of female participants and initial body mass index (35-48.3 vs 35-49.3, respectively). 126/20663 (0.61%) patients in the bariatric surgery group and 175/28253 (0.62%) individuals in the control group presented CRC. In this meta-analysis, we were unable to demonstrate a significant impact of the Bariatric Surgery on EOCRC risk. Prospective trials with longer follow-up periods should be done to prove the colorectal cancer risk reduction.

Cetylpyridinium Chloride Mouthwash to Reduce Shedding of Infectious SARS-CoV-2: A Double-Blind Randomized Clinical Trial.

The airborne transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via respiratory fluids and droplets suggests that mouthwashes containing substances with virucidal activity can help reduce viral spread. We conducted a multicenter, double-blind, placebo-controlled, randomized trial to assess the virucidal activity of cetylpyridinium chloride (CPC) mouthwashes. Outpatients who tested positive for SARS-CoV-2 infection with or without symptoms were randomized to perform washes and gargles for 1 min with 15 mL of either colored distilled water or 0.07% CPC (Vitis CPC Protect) mouthwash. The study outcomes were the SARS-CoV-2 log10 viral RNA load and the nucleocapsid protein levels, both in saliva at 1 and 3 h after the intervention. In total, 118 patients were enrolled and randomized (mean [SD], age 46 [14] y). Thirteen of 118 participants (11%) did not complete follow-up or had insufficient sample volume for testing and were excluded from the analysis. The assessment of the viral load showed no significant differences between groups at any of the investigated points. However, the levels of SARS-CoV-2 nucleocapsid protein of lysed viruses were significantly higher in the CPC group compared with the control group at 1 h (adjusted difference 269.3 pg/mL; 95% confidence interval [CI], 97.1-441.5) and at 3 h postintervention (561.1 pg/mL; 95% CI, 380.0-742.2). In nonhospitalized patients with asymptomatic or mild symptomatic SARS-CoV-2 infection, a 0.07% CPC mouthwash, compared to placebo, was associated with a significant increase of nucleocapsid protein levels in saliva, indicating enhanced disruption of viral particles.

Revisión narrativa de instrumentos de autoevaluación vocal en Chile / Narrative review of vocal self-assessment instruments in Chile

Los sujetos disfónicos no siempre presentan una adecuada percepción de su voz o de cómo las alteraciones vocales afectan a su entorno, modificando su calidad de vida. Los protocolos de auto evaluación vocal ayudan al clínico a percibir si el paciente disfónico se siente afectado por sus problemas vocales o no. Existen numerosos protocolos de auto evaluación vocal, sin embargo, pocos de estos están actualmente validados en Chile. Este trabajo corresponde a una revisión narrativa respecto de la validación de instrumentos de autoevaluación vocal en Chile. Se llevó a cabo una búsqueda bibliográfica en: Pubmed, Scielo y Bireme. Fueron empleados los siguientes descriptores: Chile, voz, autoevaluación, calidad de vida, estudios de validación, comparación transcultural y sus respectivos términos en inglés: Chile, voice, validation study, self-assessment, quality of life, crosscultural comparision. Dos restricciones fueron empleadas: las investigaciones debían estar publicadas en inglés y/o español, con fecha de publicación entre 2008 y 2020. Se encontraron 41 artículos, eliminándose 24 duplicados y 13 de contenidos no relacionados, resultando en 4 artículos analizados correspondientes a 3 instrumentos traducidos al español. El uso de protocolos de autoevaluación vocal complementa la evaluación clínica y objetiva de la voz, contribuyendo con la percepción del paciente, relevante para el tratamiento fonoaudiológico, sin embargo, la cantidad de protocolos de autoevaluación vocal en Chile con traducción, adaptación cultural y validación estandarizada continúa siendo escasa.

Dysphonic subjects do not always present an adequate perception of their voice, or of how vocal alterations affect their environment, modifying their quality of life. Vocal self-as-sessment protocols help the clinician to perceive whether the dysphonic patient feels affected by his vocal problems or not. There are numerous protocols for vocal self-assessment, however, few of these are currently validated in Chile. This work corresponds to a narrative review regarding the validation of vocal self-assessment instruments in Chile. The bibliographic search was conducted in the search sites: Pubmed, Scielo and Bireme. The following descriptors were used: Chile, voice, validation studies, cross-cultural comparison. Two restrictions were used: the research had to be published in English and/or Spanish, with a publication date between 2008 and 2020. A total of 41 articles were found, eliminating 24 duplicates and 13 of unrelated content, resulting in 4 articles analyzed corresponding to 3 instruments translated into Spanish. The use of vocal self-assessment protocols complements the clinical and objective assessment of the voice, contributing to the perception of the patient, relevant for speech therapy, however, the number of vocal self-assessment protocols in Chile with translation, cultural adaptation and standardized validation remains sparse.

Chronic fatigue syndrome: Abnormally fast muscle fiber conduction in the membranes of motor units at low static force load.

OBJECTIVE: Chronic fatigue syndrome (CFS) and fibromyalgia (FM) are disorders of unknown etiology and unclear pathophysiology, with overlapping symptoms of - especially muscular -fatigue and pain. Studies have shown increased muscle fiber conduction velocity (CV) in the non-painful muscles of FM patients. We investigated whether CFS patients also show CV abnormalities. METHODS: Females with CFS (n = 25), with FM (n = 22), and healthy controls (n = 21) underwent surface electromyography of the biceps brachii, loaded up to 20% of maximum strength, during short static contractions. The mean CV and motor unit potential (MUP) velocities with their statistical distribution were measured. RESULTS: The CV changes with force differed between CFS-group and both FM-group and controls (P = 0.01). The CV of the CFS-group increased excessively with force (P < 0.001), whereas that of the controls increased only slightly and non-significantly, and that of the FM-group did not increase at all. In the CFS-group, the number of MUPs conveying very high conduction velocities increased abundantly with force and the MUPs narrowed. CONCLUSION: Our results suggest disturbed muscle membrane function in CFS patients, in their motor units involved in low force generation. Central neural deregulation may contribute to this disturbance. SIGNIFICANCE: These findings help to detangle the underlying mechanisms of CFS.

Distribution of motor unit potential velocities in short static and prolonged dynamic contractions at low forces: use of the within-subject's skewness and standard deviation variables.

Behaviour of motor unit potential (MUP) velocities in relation to (low) force and duration was investigated in biceps brachii muscle using a surface electrode array. Short static tests of 3.8 s (41 subjects) and prolonged dynamic tests (prolonged tests) of 4 min (30 subjects) were performed as position tasks, applying forces up to 20% of maximal voluntary contraction (MVC). Four variables, derived from the inter-peak latency technique, were used to describe changes in the surface electromyography signal: the mean muscle fibre conduction velocity (CV), the proportion between slow and fast MUPs expressed as the within-subject skewness of MUP velocities, the within-subject standard deviation of MUP velocities [SD-peak velocity (PV)], and the amount of MUPs per second (peak frequency=PF). In short static tests and the initial phase of prolonged tests, larger forces induced an increase of the CV and PF, accompanied with the shift of MUP velocities towards higher values, whereas the SD-PV did not change. During the first 1.5-2 min of the prolonged lower force levels tests (unloaded, and loaded 5 and 10% MVC) the CV and SD-PV slightly decreased and the MUP velocities shifted towards lower values; then the three variables stabilized. The PF values did not change in these tests. However, during the prolonged higher force (20% MVC) test, the CV decreased and MUP velocities shifted towards lower values without stabilization, while the SD-PV broadened and the PF decreased progressively. It is argued that these combined results reflect changes in both neural regulatory strategies and muscle membrane state.

Fibroblast-like synoviocytes from patients with rheumatoid arthritis are more sensitive to apoptosis induced by the viral protein, apoptin, than fibroblast-like synoviocytes from trauma patients.

OBJECTIVE: Fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) show characteristics of transformation. Because the chicken anemia virus protein, apoptin, induces apoptosis solely in transformed cells, it was investigated whether FLS from patients were more sensitive to apoptin-induced apoptosis than FLS from normal joints obtained from trauma patients. METHODS: FLS were transduced with maltose-binding protein (MBP)-apoptin recombinant protein or MBP as a control protein by microinjection. After 24 hours, cells were fixed and stained with immunofluorescence to detect apoptin or MBP and the number of dead cells was assessed. Furthermore, phosphorylation of apoptin was analysed in FLS from patients with RA and from trauma patients by in vitro kinase assay. RESULTS: FLS from patients with RA were significantly more sensitive to apoptin-induced apoptosis than FLS from trauma patients (p = 0.0263). Furthermore, MBP-apoptin induced more apoptosis than MBP in RA FLS (p = 0.004). No phosphorylation of apoptin was observed in FLS from patients with RA. DISCUSSION: FLS from patients with RA are more sensitive to apoptin-induced apoptosis than normal FLS, which is consistent with a transformed phenotype of these cells. However, given the lack of phosphorylation of apoptin in RA FLS the mechanism of action of apoptin seems to differ between tumour cells and RA FLS. This study indicates that apoptin may help to identify a new therapeutic pathway against hyperplasia of the synovium and joint destruction in RA.

Senescence in innate immune responses: reduced neutrophil phagocytic capacity and CD16 expression in elderly humans.

Elderly humans are more susceptible to bacterial infections because of declining immune status. We have investigated the effect of aging on neutrophil bactericidal responses, comparing neutrophil function in healthy, young (23-35 years) and elderly (>65 years) volunteers. Superoxide generation in response to fMLP was slightly increased in neutrophils from elderly donors, and serum from the elderly was able to opsonize E. coli efficiently. In contrast, phagocytic index was significantly lower in neutrophils from the elderly, compared with young donors (P<0.005). CD11a and CD11b expression was not affected by age, but CD16 was significantly reduced in neutrophils from elderly donors (P<0.0001). CD16 expression and phagocytic index were measured in the same neutrophils using FITC-labeled E. coli, PE-conjugated anti-CD16 antibody, and CD16 expression correlated with phagocytic index (r=0.83; P<0.05). In elderly patients with bacterial infection, CD16 expression remained low. We propose that reduced neutrophil CD16 expression and phagocytosis contribute to human immunesenescence.

Clinical and programmatic mismanagement rather than community outbreak as the cause of chronic, drug-resistant tuberculosis in Buenaventura, Colombia, 1998.

SETTING: Buenaventura, Colombia. OBJECTIVE: To assess whether antituberculosis drug resistance was generated by poor management or community transmission. DESIGN: Treatment-failure and new tuberculosis (TB) patients identified between May 1997 and June 1998 were interviewed and their treatment histories reviewed. Bacteriologic testing, including drug susceptibility profiles (DSP) and DNA fingerprinting by restriction fragment length polymorphism (RFLP), was performed and human immunodeficiency virus (HIV) testing was offered. RESULTS: DSP and RFLP fingerprints were obtained for isolates from 34 of 64 treatment-failure patients; 25 (74%) were resistant to > or = one drug. Fifteen of the 25 patients consented to HIV testing; none were positive. An average of 2.8 major treatment errors per patient was identified. RFLP from the treatment-failure patients revealed 20 unique isolates and six clusters (isolates with identical RFLP); 4/6 clusters contained isolates with different DSP. Analysis of the RFLP from both treatment-failure and new patients revealed that 44/111 (40%) isolates formed 18 clusters. Four of 47 (9%) new patients had multidrug-resistant TB (MDR-TB). Eleven isolates belonged to the Beijing family, related to the MDR strain W. CONCLUSION: Drug resistance in Buenaventura results from both poor management and community transmission. Dependence on DSP to identify TB transmission is inadequate when programmatic mismanagement is common.

Serine/threonine protein kinases and apoptosis.

Over the past decade, our understanding of apoptosis, or programmed cell death, has increased greatly, with the identification of some of the major components of the apoptotic programme and the processes regulating their activation. Although apoptosis is an intrinsic process present in all cells, it can be regulated by extrinsic factors, including hormones, growth factors, cell surface receptors, and cellular stress. The actions of both pro- and antiapoptotic factors are often affected by modulation of the phosphorylation status of key elements of the apoptotic process. This minireview will focus on the role of protein kinases in apoptosis. Apoptosis is a multistep process and protein kinases have been implicated both in the upstream induction phase of apoptosis and in the downstream execution stage, as the direct targets for caspases. Due to the space constraints of this review it is not possible to discuss all of the kinases involved in the apoptotic process and we have focused here on the role of the serine/threonine protein kinases. The kinases of this family that have been suggested to play a role in apoptosis are the mitogen-activated protein kinase (MAPK) family, specifically p42/44 ERK, p38 MAPK and c-Jun N-terminal kinase (JNK), cyclic AMP-dependent protein kinase (PKA), protein kinase B (PKB), or Akt and protein kinase C (PKC). We have also considered briefly the potential for the regulation of these kinases by tyrosine protein kinases, such as c-abl.

Differential responses to CD40 ligation among Burkitt lymphoma lines that are uniformly responsive to Epstein-Barr virus latent membrane protein 1.

Ligation of CD40 on the surface of B cells induces multiple phenotypic effects, many of which are mimicked by the EBV latent membrane protein 1 (LMP1) through its interaction with downstream components of the CD40 signaling pathway. Because the effects of LMP1 have been most closely studied in human Burkitt Lymphoma (BL) cell lines retaining a tumor biopsy-like phenotype in vitro, we have examined the response of a panel of such lines to CD40 ligation. Two distinct patterns of response were observed that were unrelated to the surface level of CD40 or to the EBV genome status of the lines. Following exposure to either CD40-specific mAbs or the soluble trimeric ligand (sCD40L), high responder (HR) lines showed rapid aggregation, activation of NF-kappa B, up-regulation of cell surface markers ICAM-1/CD54 and Fas/CD95, and growth inhibition. Aggregation was seen at lower doses than those required to elicit the other effects. By contrast, low responder (LR) lines showed no detectable response to CD40 mAbs, while their responses to sCD40L were limited to activation of NF-kappa B and up-regulation of CD95 only. However, in transfection experiments, LMP1 uniformly induced the full spectrum of phenotypic effects in both HR and LR lines. We conclude that some BL cell lines show a highly restricted response to CD40 ligation but remain fully susceptible to LMP1.

RGD peptides induce apoptosis by direct caspase-3 activation.

Synthetic peptides containing the arginine-glycine-aspartate (RGD) motif have been used extensively as inhibitors of integrin-ligand interactions in studies of cell adhesion, migration, growth and differentiation, because the RGD motif is an integrin-recognition motif found in many ligands. Here we report that RGD-containing peptides are able to directly induce apoptosis without any requirement for integrin-mediated cell clustering or signals. We show that RGD-containing peptides enter cells and directly induce autoprocessing and enzymatic activity of procaspase-3, a pro-apoptotic protein. Using the breast carcinoma cell line MCF-7, which has a functional deletion of the caspase-3 gene, we confirm that caspase-3 is required for RGD-mediated cell death. In addition to an RGD motif, pro-caspase-3 also contains a potential RGD-binding motif, aspartate-aspartate-methionine (DDM), near the site of processing to produce the p12 and p17 subunits. On the basis of the ability of RGD-DDX interactions to trigger integrin activation, we suggest that RGD peptides induce apoptosis by triggering conformational changes that promote pro-caspase-3 autoprocessing and activation. These findings provide an alternative molecular explanation for the potent proapoptotic properties of RGD peptides in models of angiogenesis, inflammation and cancer metastasis.

Corn Stunt Complex Mollicutes in Belize.

Successive plantings of sweet corn in Orange Walk District, Belize (<200 m ASL) were observed to be performing poorly. Plants were stunted with shortened upper internodes, over-production (proliferation) of ears, and chlorosis of ears and leaf bases. Plants of hybrid white corn in Cayo District (<200 m ASL) had leaf-base chlorosis, mid-vein reddening, chlorotic bands on the leaves, and die-back of leaf tips: symptoms attributed to infection by the corn stunt complex (CSC) pathogens. Spiroplasma kunkelii was detected in symptom-bearing leaf-base samples of white corn but not sweet corn, using a specific F(ab')2 protein-A enzyme-linked immunosorbent assay (ELISA; D. Gordon, Ohio). Polymerase chain reactions with maize bushy stunt (MBS) phytoplasma-specific primers (1) resulted in amplification products of the expected size (740 bp) when DNA extracts from either sample type were used as template. DNAs from apparently healthy sweet or white corn from the field, or from glasshouse-grown sweet corn, did not yield this product. MBS and S. kunkelii are transmitted by leafhoppers of the genus Dalbulus, often simultaneously with maize rayado fino virus, the other CSC component (not tested for in this study). All the sweet corn varieties examined had a high incidence of the symptoms, suggesting that they are highly susceptible to one or both of the CSC mollicutes. With the increase in area dedicated to maize production and successive year-round plantings, the potential for spread and increased incidence of MBS or CSC in Belize is considerable. Reference: (1) N. A. Harrison et al. Plant Dis. 80:263, 1996.

Antigen receptor-mediated transmembrane signaling in Wiskott-Aldrich syndrome.

The X-linked immunodeficiency Wiskott-Aldrich syndrome (WAS) is a condition that includes a deficient anti-polysaccharide Ab response. Recently, it has been suggested that B cells from patients with WAS show a defective calcium mobilization response upon engagement of sIgM. Because primarily EBV-transformed cells were used in these studies, we tested freshly isolated blood B cells for their calcium mobilization capability upon engagement of sIg and CD19. No significant differences in the calcium mobilization capability of CD20+ B cells of four individual WAS patients compared with capability in normal controls were found. Receptor desensitization as assessed by calcium mobilization inhibition also seemed to be intact. T cells were tested for their anti-CD3-induced calcium flux and, again, no abnormalities could be observed when compared with T cells from healthy individuals. We conclude that WAS B and T cells can be stimulated into a normal calcium mobilization response when their AgRs are cross-linked. It is highly improbable that the immune dysfunction observed in WAS patients is related to a direct disorder of their B and/or T cell AgRs.

Functional analysis of human Fc gamma RII (CD32) isoforms expressed in B lymphocytes.

The low affinity IgG receptor Fc gamma RII (CD32) represents the most widely distributed class of human Fc gamma R. To analyze the biologic functions of different Fc gamma RII isoforms, we stably transfected Fc gamma RIIb1, IIb1*, IIb2, IIa, and a IIa tail- mutant to the mouse IIA1.6 B lymphoma cell line. Of these, Fc gamma RIIb1* represents a receptor variant that is identical to IIb1 except for a single amino acid difference in the cytoplasmic tail (amino acid position 11) where a tyrosine (IIb1) is replaced by an aspartic acid (IIb1*). Evaluation of capping ability showed the Fc gamma RIIb1 molecules to cap effectively, which was even more apparent with IIb1*. None of the Fc gamma RIIa, IIa tail-, or IIb2 isoforms capped significantly. Internalization of Fc gamma R-antibody complexes proved very efficient for both the Fc gamma RIIa and IIb2 isoforms, whereas the IIb1 molecules internalized moderately compared with IIb1*, which internalized less efficiently. Notably, human IgG aggregates were internalized effectively by Fc gamma RIIa and moderately by IIb2. Neither Fc gamma RIIb1 nor IIb1* proved capable of internalizing such IgG aggregates. Cross-linking of the different Fc gamma R molecules showed Fc gamma RIIa capable of triggering increases in [Ca2+]i. Fc gamma R expressed on B cells were able to down-regulate [Ca2+]i on co-cross-linking with slgG. Notably, all three Fc gamma RIIb receptors proved active in this respect, in contrast to Fc gamma RIIa. The cell distribution of these Fc gamma RII isoforms was analyzed in a panel of human B cell lines to complement the IIA1.6 B cell model. Fc gamma RIIa was found expressed both at message and protein levels in all tested human B cell lines. In the pre-B cell lines evaluated, no Fc gamma RIIb molecules were detectable, whereas both Fc gamma RIIb1 and IIb2 molecules were found present in more mature B cell lines. These data support both a complex expression pattern of Fc gamma RII isoforms in B cell lines and functional differences between these B cell molecules.

Intracellular magnesium movements and lymphocyte activation.

Activation of lymphocytes through ligation of the antigen receptor complex initiates activation of phospholipase C-gamma (PLC). Activated PLC hydrolyses phosphatidylinositol-4,5-bisphosphate into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (InsP3). InsP3 mediates the release of Ca2+ from intracellular stores into the cytoplasm, while InsP4 and InsP3 mobilize extracellular Ca2+. Both processes contribute to the temporary increase in [Ca2+]i that is observed after lymphocyte activation. Because of the availability of Mg(2+)-sensitive and specific fluorochromes like Mag-indo-1 it is now possible to monitor potential changes in [Mg2+]i. In lymphocytes that have responded to receptor activation with high [Ca2+]i, an increase in [Mg2+]i can be found. The [Mg2+]i is in the range that enables it to modulate the activity of a number of cellular enzymes, including key enzymes in the PLC transmembrane signalling pathway. It can be speculated that a differential Mg2+ mobilization response will have consequences for the ultimate cellular response to receptor activation.

[Paratyphoid B fever in 2 areas of Santiago. An analysis of the epidemiological significance]. / Fiebre paratifoidea B en dos áreas de Santiago. Un análisis de su significación epidemiológica.

Salmonella isolates from 3920 patients with typhoid fever from 2 areas in Santiago were analyzed to determine the frequency of association with S paratyphi B infection. This was demonstrated in 18.8% of subjects in both areas, a figure significantly higher than the 8-10% previously reported. The association with S paratyphi B was higher for females, especially for the younger age group. These findings suggest an infectious agent-gender-age interaction which may explain the discrepancy with previously reported rates of infection. Their possible relation to the chronic salmonella carrier state and association with biliary tract lithiasis and cancer is discussed.

Evolution of the human alpha-amylase multigene family through unequal, homologous, and inter- and intrachromosomal crossovers.

Human amylase haplotypes differ from each other by different numbers of a long direct repeat unit of approximately 100 kb, encompassing two complete salivary amylase genes and one amylase pseudogene lacking the first three exons. The two salivary genes are part of a 75-kb-long inverted repeat. Two short sequences, hybridizing with a probe containing exons 1-3, were found in the central part of the inverted repeat. Sequencing showed that these fragments, designated r, contain exon 3 sequences. We present evidence that these r-fragments and the pseudogene most likely are remnants of the same ancestral pancreatic gene. We determined the orientation of the exon 3 sequences present in the r-fragment and show that an inversion can explain their origination. Hybridization studies, using random fragments from the intergenic region of the AMY gene cluster as probes, enabled us to detect more extended homologous regions in this cluster than were found previously on the basis of restriction maps only. Together, these results allow us to present a model for the evolution of the human amylase multigene family by a number of consecutive events involving inter- and intrachromosomal crossovers.